Pharmaceutical info

Ask the Facilities Guy: How do I produce a Disaster Recovery Plan?

2012-01-20T22:25:00.000-08:00

By Richard Bilodeau, PEA question has arisen about terminology. Whether called a “disaster recovery plan” or a “crisis plan” or a “business continuity plan,” the end game is the same. Your goal is to resume manufacturing operations and fiscal stability, as quickly as possible, after a disruptive event. Some facilities and operations professionals believe that abandoning the term “disaster recovery plan” in favor of “business continuity plan” is more effective in procuring funding and support from senior management, and more palatable to the investment community. While the substance remains the same, the impression created by an undertaking titled “business continuity plan” is a focus on the future, generating product and profits. There is also a move afoot in some sectors to use the label BC/DR (business continuity / disaster recovery), suggesting the dual purpose of the process. You’ll need to decide what’s best for your organization, and facilitating your end goals.
This month in Round 2, I’ll guide you through some of the specifics of producing a disaster recovery plan tailored to the requirements of your manufacturing needs, including content. Pour that cup of Joe and settle in—those of us in facilities like nothing better than to write a plan, or read about writing one. But the pain you’ll feel today is miniscule compared to the pain of being unprepared for an unexpected disaster that impairs your manufacturing.
THE BEGINNING
You’ve completed the spadework: management is on board and has funded the effort; risk assessments are completed and prioritized regarding both probability of occurrence and damage potential; key team members are identified and recruited. Now what?
Before drafting the plan, make sure that all key departments of the organization are surveyed to a level appropriate to both their risk exposure and the potential financial impact a business disruption would produce. Rank and prioritize the information you glean and build it into your plan.
Following are some key components of the plan that will ensure its functionality. Consider using graphs, flow charts, and bold/bulleted text to convey the information as concisely as possible.

• Executive summary: not a bad place to outline the plan’s objectives, assumptions used in the process, and the scope of the plan.
• Statement of purpose: Make sure everyone reading the document understands what it is, and what it is not.
o Outline the conditions under which the plan will be implemented and clear chains of responsibility to make the call declaring an emergency and invoking the plan.
o Clearly outline notification triggers, processes/procedures, and recording requirements.
• Clearly delineate the names, contact information, and roles of the disaster recovery team members. Make sure each person has one, if not two, designated backups— depending upon your company’s travel schedules. Back-ups must be qualified individuals who are fully trained and can command the role for which they’re designated. It’s critical that this section of the plan be scrupulously updated.
o For companies with multiple locations, consider developing both a corporate team and site specific teams at each of your locations. Work hard to develop a sense of camaraderie and teamwork.
o Depending on the risks contemplated by the plan and your company’s organization structure, you will likely have several or all of the following teams: Emergency Management Team (including management, finance, logistics, procurement etc.), Crisis Communications Team, Incident Response Team and/or Emergency Response Team, Technical Services Team, Services Restoration Team. Some plans roll each of these groups under the Disaster Recovery Team mantle, with sub-groups with specific responsibilities selected from the list above.
o Make sure applicable outside emergency response and governmental agencies are listed, including a lead contact and their back-up. It’s critical that the emergency management team develop strong relationships, and undertake joint training, with local government officials and emergency response personnel.
• Keep a clear record of revisions chart near the front of the document. Make sure the plan is reviewed frequently, updated, and the new information is distributed to all team members. Do NOT wait for a scheduled review to update business changes, new personnel, or new risks. Ensure that any revisions are integrated in all copies of the plan.
• Extended team member roles, responsibilities, and authority: the heat of a crisis is no place for turf wars or lack of clarity in execution.
• Communications authority guidelines: Clearly designate who has the authority to speak with the press or other outside parties or governmental agencies, your investors (if applicable), customers, suppliers, and employees.
o Develop a strong communications protocol guide. Remember, in worse case scenarios, loose lips sink ships; in even the best cases they create confusion, conflicting information, and reputation damage.
• Emergency management standards and procedures: this is the heart of your plan. Many companies categorize potential disasters and develop specific protocol for each type, or for individual scenarios within these categories. Some broad categories include: natural disasters, workplace violence, key employee deaths or resignations, IT systems crises, chemical spills and release issues, supply chain disruption, fire, strikes, accidents, acts of terrorism, and criminal investigations.
o For each scenario, outline trigger thresholds for invoking the plan, escalation factors, notification requirements, and procedures.
• Checklists: Develop checklists that your teams can use to ensure a complete response and to organize actions during a crisis.

Focus on developing a draft document—don’t get caught up in the pursuit of perfection. Once you’ve developed a draft, make sure it’s thoroughly vetted— preferably in workshop forums set up with operations groups and members of the team. Encourage an open, constructive review format—your goal is not to collect kudos but to be sure everything is covered, clear, and structured in a way that will be most efficient in a crisis.
Scrupulously record the comments and suggestions and resolve any edits. Go back for a rewrite and then distribute the second draft for individual review and comment with a firm deadline for replies. Integrate any corrections into the final draft—the emphasis here is on the word “draft.” Business recovery plans are organic documents that must be constantly updated and amended to align with changing information and business conditions.

Pharma Facility Quality Audits: A Primer for Design Teams

2012-01-20T22:24:00.000-08:00

By Scott Overton

Here is a familiar demand: “Show me where in the regulations it says to do that!” If you’ve spent any time in Quality, working on a facility or equipment project in the pharmaceutical industry, you have certainly had that put to you at some point. And why not?
Far and away, what most people (including our industry engineers, technicians, and operators) think about when they think “Quality” is “Compliance with Regulations.” Compliance is certainly an issue of vital importance, but Quality does not stop there, and there is no time when that is more clear than during a Quality Audit. This article will show that understanding the potential audit outcomes in the future can serve as powerful design criteria today.
For many companies today, the majority of their external quality audits will not be performed by regulatory agencies, but by other companies. This company auditor has a day or two to assess the overall “quality culture” of a potential supplier or contractor. Unlike the FDA that has a free pass into all FDA-regulated facilities in the U.S. at any time, a company auditor performs the audit at the discretion of the company being audited. After this audit is complete, and provided that the auditor says everything is okay to link his company to the manufacturing processes of the other, the auditor’s company will have little to no direct experience with day-to-day processing. In short, that’s a couple of days to make a full assessment of all quality systems in a unique facility that will have to be valid for a year or more. This is a challenge for non-sterile raw material suppliers, which only grows more challenging as cleanrooms and sterility claims are added to the mix.
With so little time to assess a facility and a manufacturing process (or many of them!), how is it that auditors can effectively and thoroughly measure the strength of all Quality Systems? It is here that a large part of non-Quality personnel would answer something along the lines of “directly measure the facility against the regulations using a checklist or other tool.” Though compliance with regulations is absolutely required, measuring against regulations is a very small part of an audit. Furthermore, it is difficult to do so “directly.” How would one measure “Appropriate measures should be established and implemented to prevent cross-contamination from personnel and materials moving from one dedicated area to another”¹ directly? And how would it be done for every possible combination of personnel, materials, and products in a contract formulation and sterile liquid filling company, for example? In short, it can’t happen. Alternatively, there must be a way to achieve solid and defensible results.
The alternative is this: in order to be effective, auditors focus on two things:

How is the system controlled?
How is the system monitored?

Superficially, these two questions appear to provide a very narrow perspective. On the contrary, they comprise a far more expansive view of facilities and processing than most, including the normal view of design, for example, which is to design for a specific production volume. The first clear outcome of designing a facility or process with these two questions in mind is that design decisions will be made knowing that the final design must be auditable. A simple example would be to imagine an airlock that does not have any sort of air pressure gauges. The original design intent may be to have 10 Pa over each door, but without data provided by monitoring devices, how could it be proven that the airlock is behaving as designed? Without data, how could adjustments to controls be justified to an auditor? The fact that an audit trail must be available may be very influential, which will be seen through some examples below.
Now it is great that we understand that systems of control and monitoring will be the way an auditor approaches the facility once it is built and the process is running, and it’s also great that we understand that the final facility or process must be auditable. But these facts are not very useful to design engineers and architects if they cannot be leveraged to inform design of projects in specific ways up front. Having a holistic, “eyes of the auditor” type quality influence in the design phase of a project would be incredibly valuable since all projects in the pharmaceutical industry will eventually be production facilities or manufacturing systems, which will go for Pre-Approval Inspection and then biennial inspections by the FDA and international regulatory bodies, and also countless supplier audits for as long as that that facility or process exists.
Some people may balk at the notion of using the potential results of future audits as design criteria as it is commonly held that “regulations keep changing.” I deny that. What happens most often is that over time, the rest of society continually develops more sophisticated technologies like computers, advanced materials, and precision equipment. As the rest of society keeps progressing (or in production lingo, “continuously improving”), our industry is expected to do the same. Thirty years ago, a daily monitoring of room pressure on a written log was the norm. Today, it is very common to see Building Management Systems monitoring room pressures constantly. Physics didn’t change. Air itself didn’t change. Over time, technology advanced and the Pharmaceutical Industry just kept up with it.
Furthering that idea, better data monitoring reduces business and quality risk overall, so these advancing technologies are implemented as companies’ assessment of risk changes. The written regulations also change—usually pretty slowly—to keep up with changing technology and risk perceptions. As an example, consider a smartphone. These devices have incredibly simple user interfaces and are powerful enough to let the user do almost everything a laptop does. With that level of simplistic technology available to anyone, why should a confusing Operator Interface on a process skid be acceptable?
What does it really mean to focus on systems of control and monitoring? Said another way, it means an auditor will focus on systems that will assure product will continuously meet specifications, that any issues with that production are identifiable, and that all activity is traceable. So the auditor focus on control and monitoring systems is truly a focus on “assurance” and “reliability.” Here, it seems that the goal of the design team and the auditor are the same. In project development, no design team aims to create a facility or a process that is unreliable; an auditor merely seeks to validate those efforts. Approaching design from this perspective of “assurance” and “reliability” is obviously constructive and helps projects in our life-saving industry come to the best decisions for our products and our patients. The area where this is most clear is cleanroom design, which will be the focus of the examples below.
Consider a situation in two different ways. In each of these scenarios, the product has some specific requirements, such as sterility throughout the process as the product cannot undergo any downstream sterilization steps. Also, for clarity, these processes are in a single-product facility:
SCENARIO 1: AN OPEN PROCESS
In an open process for a sterile product, the space required is a Grade A with a Grade B background. Personnel in that space are fully gowned. In Grade A, there is continuous particulate monitoring, minimal per-process microbial monitoring, and a lot of air being pumped into the space (laminar in Grade A to boot!).
SCENARIO 2: A CLOSED PROCESS
In a closed process for a sterile product, operations are in Grade C, D, or maybe even unclassified (depending on risk and the way the process is set-up). Personnel are wearing dedicated clean-area clothes and shoes, EM is radically reduced from an aseptic area, and the air change rate is maybe half of that in aseptic areas.
Now, both of these process scenarios have the potential to be completely compliant. For one, though, compliance is far more costly, challenging, time consuming, labor-intensive, and complex, which means that there are many more tools required to control the environment, many more items to monitor, and far more opportunities for failure. Each of these additional failure modes adds another parameter to the process that must be auditable and increases the number of people required to audit. There is nothing in the regulations that would state one of these approaches is completely wrong. And maybe with more information available, the system with fewer parameters to control and monitor is actually the best choice for the production material. What if the product in question here is experimental, unproven, and therefore in need of the developmental flexibility that comes with multi-use Grade A areas? What if product volumes are so vanishingly small that product hold-up (potentially greater in the closed system) is critical? This may come into greater relief with another example:
POTENTIAL SYSTEM A:
The production system is fully stainless steel. Everything was engineered, with measured and exact slopes for all piping. In between uses, all parts must be washed and, before that, the cleaning cycles had to be validated. Their control is automatic and also validated, so repeatability is almost certainly assured.
POTENTIAL SYSTEM B:
This is a completely disposable system. The disposable components can be purchased from multiple vendors (some of which are not as reliable as others), have some potential variability in tubing length, and since they come coiled in sterilized bags, the tubing often sags when outstretched, leading to variability in holdup in the system. The disposable components need no cleaning validation or cleaning cycles. These systems are sterilized by an outside vendor, but are never put through a validated cleaning cycle to remove any particles introduced during assembly at the vendor.
Knowing that outcomes like purity and yield are critical parameters, which system should be selected? What if the product is moderately sensitive to materials used in a disposable system? What if cleaning chemical residues have an exponential negative effect on potency? Again, understanding the way the entire process will be audited quickly digs into many details not always at the forefront of a facility design.
Similarly, across the industry, room air quality must be monitored. But monitoring a Grade A space means continuous particle monitoring, whereas Grade C monitoring may be only weekly or even monthly. For a critical product-contact utility, an auditor will review every document from Design Qualification through routine monitoring over the previous year or two in detail. For a Grade C EM program, the procedure will be reviewed and enough data to show compliance will be reviewed.
In all of these examples, different teams will come to different final selections and it is highly unlikely that any selection will be “wrong.” The unfortunate potential is that for either path taken, the selection will be made, designed to, and built from a single perspective: the perspective that comes with budgets and schedules, that moves in one direction from original concept to construction and validation, and that is ultimately inflexible because it was scoped and designed to meet a specific need (throughput or production volumes, for example) without including the need to control or monitor the process and be fully transparent in an audit, regardless of the volumes.
This is perhaps the best case for viewing projects from an auditor’s perspective. Ten years from the initial build, an auditor will be in that facility. In those ten years, dozens of changes will have happened, production volumes would have increased (hopefully), and systems would have been updated. Cleanrooms designed to hold one production lot at a time might now hold multiple lots. It is here where controlling and monitoring systems for multiple quality systems come together to form an overall quality assurance program. It is here where an auditor could look at the initial design and aid in recognizing how, over the long term, systems to control and monitor different aspects of production can be stressed to the point of failure.
At a minimum, the products of the Pharmaceutical Industry improve the quality of life and, in many cases, save lives or prevent disease entirely. Competition in this field is inevitable and tenacious. Increasing production and lowering cost cannot result in products of lower quality with unreasonable risk to patients.² For successful products, processes and facilities are asked to increase production over time as demand increases and lower costs as patents expire and competitors come to market. These pressures are as inevitable for a production facility as is the fact that it will be audited dozens of times. We would all do well to employ the tools of an auditor in the design of facilities and processes.
References:

FDA Guidance for Industry / ICH Q7A Good Manufacturing Practice Guidance for Active Pharmaceutical Ingredients, Section D(4.4), August 2001
See Eudralex, Annex 11, Principle section

Why Static-Control Flooring Is So Important - And How to Find Solutions to Keep You Grounded

2012-01-20T22:22:00.000-08:00

By Dave Long

Selecting the right kind of ESD (electrostatic discharge) flooring is always a challenge, and in controlled environments, the stakes are particularly high.
While cleanroom environments are known for the exacting standards used to control contaminants, it’s ironic that their anti-static flooring doesn’t always meet industry specifications. This is a critical concern on several levels:

First, the ESD problem is intensifying as electronic devices continue to become smaller and more powerful. Miniaturization, also known as device scaling, reduces the room for on-chip protection, increasing vulnerability to ESD and accenting the need for static-control, fault tolerant flooring.
In addition, recent and proposed changes to ESD standards, including ANSI (American National Standards Institute), increases challenges to manufacturing facilities seeking ISO certification. These changes address the need to comply with revised static-prevention performance parameters, with failure to do so exposing companies to potential lost business due to non-compliance.
Floors installed in cleanrooms have enormous bottom-line implications if you consider the potential costs of installing a new, correctly specified, floor after your facility is operational.

In other words, it is paramount to get your ESD flooring right the first time, and there should be no room for compromising within the precious real estate of cleanrooms. Yet, problems persist. Why?
INITIAL CONSIDERATIONS
Understandably, many engineers and facility managers are frustrated and confused when it comes to selecting static-control floors for their factories. They typically don’t have the time nor the expertise needed to deal with electrical specifications and standards. Selecting a floor includes considerations like maintenance, durability, ergonomics, safety, installation procedures, and, most importantly, how the floor controls static charges on people based on their footwear.
In cleanroom environments, the process often requires the assistance of outside experts who specialize in ESD, contamination control, ion chromatography, material out-gassing tests, and particle analysis.
Overall, depending on the application and site considerations, ESD floors can be installed over old floors, over bare concrete, or on top of raised access-flooring panels. However, due to contamination and particle control considerations, only three forms of ESD flooring are generally considered suitable for cleanroom environments: rubber, vinyl, and epoxy.
With this as the backdrop, the following will focus on key factors to consider in the evaluation and selection process, including electrical resistance, footwear, cleanliness, mechanical properties, and ergonomics.
ELECTRICAL RESISTANCE
Rubber, vinyl, and epoxy floors can be produced in either the conductive or the static-dissipative ohms range. According to the ESD Association, a conductive floor measures below one million ohms (1.0 X 10 E6) when using test method ANSI/ESD S7.1-2005. Using the same test, a floor measuring between one million ohms and one billion ohms (1.0 X 10 E9) is defined as static dissipative. As a general rule, most experts believe that floors measuring below 10 million ohms (1.0 X 10 E7) offer the best static-control performance for electronic manufacturing and handling. Floors measuring above 10 million ohms drain static more slowly than floors measuring in the conductive or lower end of the static-dissipative range (< 1.0 X 10 E7).
Also, ESD floors that are too conductive may not be considered safe. Most safety engineers refer to NFPA 99 to define the minimum resistance of conductive floors. According to the 2005 version of NFPA 99, a floor should not measure below 25,000 ohms (2.5 X 10 E4). There is a caveat when referencing NFPA 99, however: This test requires measuring a floor’s resistance using an ohmmeter with a 500 volt output, and most of the meters used for testing conductive floors operate at 10 volts. Unfortunately, this creates a potential safety dilemma for specifiers because a floor measuring 25,000 ohms at 10 volts will measure far below the NFPA minimum of 25,000 ohms when tested at 500 volts. For this reason, we recommend setting the resistance minimum above 50,000 ohms to address the discrepancy caused by the two different test methods.
Upshot: Recommended floor range: greater than 50,000 ohms and less than 10,000,000 ohms (5.0 X 10 E4 – 1.0 x 10 E7).

FOOTWEAR
ESD floors should never be evaluated solely on electrical resistance parameters, since that is only part of the story. ESD standards like ANSI/ESD S20.20-2007 require testing of both the resistive properties (ohms) and the charge-generation properties (volts) of the floor. The ESD Association requires that the floor’s performance be evaluated in combination with static-control footwear. The first requirement in S20.20 evaluates a property called “system resistance,” which is determined using the ANSI/ESD S97.1 test method. In this test, the ohms resistance is measured from a person’s hand to the ground—through the body, the footwear, the floor itself, and the ground. As of November 2011, an acceptable reading requires a system resistance below 35 million ohms (3.5 X 10 E7). (Note that before writing this article, we interviewed several members of the ANSI/ESD S20.20 committee about possible changes in the system resistance requirement. We were informed that the requirement might be raised to a maximum of 1 billion ohms (1.0 X 10 E9). However, if this system resistance is increased, requiring body voltage testing at the same time will likely offset it.)
Body voltage generation is determined by measuring static charges using test method ANSI/ESD S97.2. With this method, subjects wearing special static-control footwear walk on ESD flooring while connected to an instrument that measures the amount of static charge the subject generates from the interaction of the footwear and grounded floor. To meet ANSI/ESD S20.20, a person wearing approved, grounded footwear cannot generate over 100 volts. Achieving this parameter may be difficult in cleanrooms, however, depending on the contamination-control footwear requirements. Standard cleanroom shoe covers generate static voltages in excess of 1,000 volts. Several suppliers offer disposable and permanent static-control shoe covers with conductive or static-dissipative material on the bottom side.
Regardless of the shoe cover specifications, they should always be tested with the grounded floor. Testing has shown that many conductive and static-dissipative vinyl and epoxy floors will generate well over the 100 volt maximum on test subjects wearing these types of booties and shoe covers. The same testing has proven that conductive rubber flooring will generate well under 100 volts combined with most static-control cleanroom footwear. This is because conductive rubber flooring generates significantly less static than vinyl or epoxy flooring, regardless of footwear.
Upshot: Current system resistance requirement: < 35 million ohms (3.5 X 10 E7). Proposed requirements: system resistance < 25 million ohms and body voltage < 100 volts.
CLEANLINESS
When determining the compatibility between construction materials like flooring and cleanroom processes, there are numerous considerations. Here, we will touch on the main factors: out-gassing and particle transfer. According to ESD and contamination-control consultant Carl Newburg, president of Microstat Laboratories and River’s Edge Technical Services in Rochester, Minnesota, “Out-gassing is a measurement of the quantity of volatile chemicals released from a material while it is heated. Condensable volatile residue (CVR), Static Headspace, and Dynamic Headspace are typical tests used to measure out-gassed materials. Test results offer an indication of the material’s tendency to contaminate surfaces in a controlled environment with airborne molecular compounds.”
Most vinyl flooring materials fail stringent, elevated temperature outgassing testing due to the inclusion of plasticizers in the flooring material. Plasticizers are problematic because they can migrate out of the flooring material and create significant contamination problems in cleanroom applications like optics and MR head manufacturing. We have all experienced plasticizer migration through what we refer to as “new car smell.” This smell is the result of airborne plasticizer out-gassing from all of the various plastics used in an automobile’s interior. Without thorough testing, this plasticizer migration would be difficult to identity and quantify. Many flooring manufacturers will state that their flooring will meet all out-gassing requirements at ambient temperature, but most contamination-control experts do not believe that ambient testing is adequate.
Before specifying any flooring for installation, we recommend discussing the application with an expert in contamination control and ESD. As a rule of thumb, conductive rubber flooring and conductive epoxy flooring will perform the best in elevated temperature out-gassing tests. Unlike vinyl, rubber and epoxy are made without plasticizers.
Upshot: Floors that pass elevated temperature outgassing tests: conductive rubber and conductive epoxy.

MECHANICAL PROPERTIES
Most cleanroom floors are installed using methods that create a seamless floor, which can be achieved with epoxy coatings because the material is coated onto the floor in liquid form and allowed to flow across the surface. The downside to seamless epoxy coatings involves the difficulty and time required to make repairs in the event of damage from scratches or cracking. A typical cure time for an ESD epoxy floor is between 24 and 72 hours, depending on the number of layers. If a repair is performed in an operational cleanroom, the epoxy could create contamination or odor problems during the time it takes to harden from its liquid state. Additionally, epoxy repairs usually require some form of abrasive floor preparation to make the surface fit for recoating. Abrasive floor preparation could generate particles that contaminate fixtures and HEPA filters.
Rubber and vinyl can also be installed without seams using a technique called seam welding, which fills in and fuses gaps resulting from the interactions of sheet floor flooring or adjacent tiles. Both tiles and sheet floors can be seam welded (similar to caulking), but most specifiers prefer sheet flooring since there are fewer seams to weld. The welds in rubber sheet flooring are less visible compared with vinyl welds because, unlike vinyl, rubber does not shrink. Rubber and vinyl floors can be repaired more easily than epoxy using simple techniques that don’t require abrasive floor preparation techniques. Conductive rubber sheet flooring can be installed with fast-drying, pressure- sensitive adhesives that can usually be applied in an operational cleanroom. Pressure-sensitive adhesives allow for foot traffic within an hour of the repair.
ERGONOMICS
Of the three most common cleanroom flooring options, rubber offers the most slip-resistant walking surface, wet or dry. Rubber is also softer underfoot, and it absorbs ambient noise better than hard epoxy and vinyl surfaces. Even though it is much harder than generic rubber used in forms like anti-fatigue matting, rubber can become damaged from rolling heavy loads over it. Compared with epoxy, it is also more difficult to roll heavy racks weighing thousands of pounds over rubber. In some cases, epoxy may be the only practical flooring option due to its toughness and ability to handle rolling loads and chemical spills.
Upshot: Rubber offers the most ergonomically friendly solution.
SIMPLIFYING THE SELECTION PROCESS
Indeed, there is a vast amount of technical information to consider when selecting static-free flooring in controlled environments—and it would be short-sighted to look for shortcuts in the process. In the final analysis, the key concepts are prevention and protection.
To prevent ESD problems, select the flooring option that best meets current and anticipated industry specifications. While there are different variables, here is what industry sources recommend for cleanrooms and electronic manufacturing facilities:

Conductive Rubber the only ESD flooring certified as Class-0 Qualified is rated “ideal;” it also has low body voltage generation.
Conductive Vinyl Tile and Conductive Epoxy may also be suitable.
Other flooring options are not recommended.

As far as protection, if you plan wisely from the get-go, you can avoid costly liability issues later on. We encourage installation floor audits to determine if you are ground safe.

Failure Modes and Effects Analysis (FMEA)

2012-01-20T22:20:00.000-08:00

By Scott Mackler

A tool to balance cost and schedule while maintaining facilities readiness.
In the October issue of Controlled Environments, Richard Bilodeau, “Ask the Facilities Guy” wrote about establishing an “Equipment Reliability” program. While clearly an important issue, it is one that often facilities departments have a hard time getting their arms around—as Richard points out. One tool that we have found quite useful in supporting high facility onstream time and process yield factors, as well as sustainability, is the equipment or hardware FMEA (Failure Modes and Effect Analysis). The FMEA exercise will provide the facilities team with a prioritized “risk burn-down” plan for ensuring readiness and can serve as a convenient basis for capital and operating expense budget creation and execution.
We have recently performed FMEA exercises for aerospace assembly, integration, and test facilities, aseptic filling laminar flow units and accompanying HVAC systems, thermal vacuum test chambers, powder metallurgy processing lines, precision cleaning equipment, and continuous web processing machinery. In many cases not only were predictive and preventive maintenance issues uncovered and addressed with corrective action plans developed as a consensus among customers, users, service providers, and subject matter experts, but in a few cases, serious life safety and product safety issues were brought to light and effectively dealt with before a catastrophe— likely one without warning—could occur.
An FMEA identifies the severity, occurrence, and detection of failure effects and then establishes priority- ranked corrective action plans. A cross-functional team including the customer or process owner, subject matter experts, facilities and maintenance specialists, quality assurance, and design engineering participate in a brain-storming exercise that identifies each potential failure and ranks the possible effects of each failure and develops a resulting RPN or “Risk Priority Number.” The RPN is the arithmetic product of the severity multiplied by the (probability of) occurrence multiplied by the (ability of) detection.
The objectives of the FMEA are to:

Ensure that potential failure modes and their effects are identified and ranked as to severity, occurrence, and detection.
Provide assessment as to risk ranking based on RPN (Risk Priority Number) and generate action register to burn down risk—thereby reducing life cycle costs, improving reliability and durability of systems.
Prioritize the engineering efforts and resources based on the assessment of potential failure impacts to the product and eliminate or minimize the impact of potential failures to the product.
Provide information for development of an efficient and effective preventive maintenance plan.
Establish closer links between production, quality, facilities engineering, and maintenance.

Examples of suggested scales for severity, detection, and occurrence might be:

Typically the deliverables of the FMEA include a Pareto Chart illustrating the number of failure items and risk effects that were identified and subsequently ranked by RPN during the brainstorming and analysis sections of the exercise, and then either a projected or an achieved burndown of the RPNs after development and execution of the Corrective Action Plan.

Point of View: An Apple To Apple Comparison on Cleanroom Proposals

2012-01-20T22:18:00.000-08:00

By Kelly Barton

That’s a tough one, but for simple, relatively small cleanroom projects there are a few things that you can do to help ensure everyone bidding the job is “singing from the same song sheet.” No matter how simple, it’s very important to generate a specification sheet and conceptual drawing or sketch and make sure all the prospective bidders get it and reference it in their proposal. The sketch must include the ceiling height in the cleanroom and room sizes. Ask them to separately itemize any items that they feel are needed but are not included in the spec sheet. If you do not have anyone in your organization qualified to generate this document, hire an outside consultant with the understanding that all you need is a very basic scope statement. The time you spend trying to figure out the differences between bids will cost you more time, money, and frustration than the consultant will charge. Plan on at least two bid phases, initial and final bids. As you evaluate the bids you will get a better understanding of what is needed and can make better decisions regarding what to specify in the final bid phase. The evaluation phase of the bids can almost be an education on cleanroom design for your staff. Don’t be shy about asking contractors why one proposal is different than another. Make sure you are comfortable with their explanation.
Here are some very important specifications to include in your document that are critical for a contractor to determine cost. If everyone gets a spec sheet with these variables defined, your final bids will be more accurate and should be more consistent.

Room Classification, either Fed Std or Iso spec should work here.
Temperature specification and tolerance. Example: 68 +/- 5 Deg.
Humidity specification and tolerance. Example: 50 +/- 10% RH. Let the bidder know if the temperature and humidity specifications are process critical or operator comfort. If humidity control does not affect your product, leave it out. It is the most costly specification to control for the HVAC system.
Amount of process exhaust in CFM. This is relevant since both outside air to generate room positive pressure and air removed from the cleanroom by the process has to be accounted for.
Process heat load in kilowatts (KW). Are there ovens, large process machines, or just operators in the cleanroom? If you are not sure about this item, list the connected electrical voltage and current draw (amps) to start with. This will ensure some A/C tonnage is dedicated to this specification.
Number of operators. This affects temperature and to some degree, humidity.
A brief definition of the manufacturing process or product in the cleanroom may also help.
A description of the host building is helpful since this may affect where the HVAC equipment can be placed.

Cleaning validation in the pharmaceuticals industry:

2012-01-09T03:09:00.000-08:00

Good pharmaceuticals manufacturing practice requires from pharmaceuticals companies that rooms and apparatus such as centrifuges and other devices must be cleaned according to written methods (“Good Manufacturing Practice” or GMP).
The most suitable method must be validated by the respective pharmaceuticals company on the basis of regulatory requirements [1] and their own expertise and technological advances in apparatus engineering. This takes place as part of cleaning validation; and this is precisely where the innovations of Ferrum in the area of vertical scraper centrifuges offer further alleviations.
Not without “my” risk assessment
The word cleaning validation represents a real challenge to the pharmaceutical, apparatus and plant engineering industries. This does not just simply involve complying with regulatory standards. The safety of pharmaceuticals, feasibility and efficiency are main aspects.

At the start of every cleaning validation is the validation plan, which can be divided into three phases, see also [2], [3]. The providers of centrifuge technology solutions can make an essential contribution in all three phases towards realisation and efficiency. This can only be achieved by working together and harnessing all available relevant knowledge available.
The three phases can be briefly described as follows:
1)Internal status inspection of planned production line
This concerns the question of which active and inactive ingredients are to be produced or used? The product change frequency has a considerable influence on the efficiency. One must therefore know which cleaning agent and method should or can be used.

This is where the latest innovative VBC centrifuge technology comes in; based on the expertise of the machine supplier in apparatus engineering and construction in line with the latest advances in mechatronics as well as design aspects and process sequences of solids-liquid separation and cleaning. The machine supplier is not responsible for the active ingredients however.

2)Risk assessment of products and facilities
The internal pharmaceuticals status inspection must be followed by risk assessment for all products, the aim being to identify substances that are a particular hazard. Responsible is the pharmaceuticals company, see graphic “worst-case” analysis according to Borchert [6].
Centrifuge manufacturers can make a valuable contribution with their expertise and years of experience in the assessment of design-related cross-contamination (e.g. difficult to access or absorbent surfaces, dead ends in pipes and extraction points, etc.).
3)Determination of extent of validation
On completion of the internal status inspection and joint risk assessment (machine supplier and pharmaceuticals company), the extent of validation can (must) be determined by the pharmaceuticals company, see also [4].

In this phase, Ferrum is able to offer the possibility of validating design-related critical points in its own assembly halls following assembly and so reduce by this verification item, time-consuming validation within the pharmaceuticals company. By means of a so-called riboflavin test, for example, the effectiveness and wettability within the centrifuge can be verified or also the effectiveness of CIP cleaning of inert material at “critical points”.
It is therefore in the interest of the pharmaceuticals company to complete validation quickly and if possible in the phase prior to commissioning. This is only possible in cases where existing facilities are duplicated. As maximum flexibility in the manufacture of pharmaceuticals is of the essence today, apparatus such as centrifuges must be appropriately flexible in design.

Precisely this step was taken at Ferrum with the latest VBC vertical pharmaceutical centrifuge. The processes of rinsing, washing, spraying, measuring, analysing, scraping, blowing out and even flooding have been greatly improved in the new generation of centrifuges over that which was state of the art a few years ago. The special advantage of Ferrum centrifuge technology solutions is that many of the different function modules can be integrated flexibly both in the initial design of the machine as well as retrofitting. The cleaning process can be optimally adapted to the respective production sequence in a highly flexible manner.
It is therefore extremely important that the experienced and innovative centrifuge supplier is included in process selection already in the planning phase.

Cleaning procedure validation and selection
In order to locate fouling on machine parts, specific samples are taken before and after the cleaning procedure. In the PIC document PI 006, sampling procedures using the wipe or swab test and flush or rinse test are considered suitable. [5]

One advantage of the swab test is that it provides information on where the fouling is located, e.g. in bends or branches of pipe systems.

Useful is the inclusion of global analytical methods. An example is TOC determination for organic loading, conductivity measurement for ionic residues and ph measurement for cleaning agent residue detection. These analytical methods can be included as online measurements or installed in the centrifuge. Such analytical methods can be used for multifunction systems to provide maximum flexibility during commissioning, as all possible active and inactive ingredients are often not known. Offered is a retrofitting option; this is usually possible in the majority of cases without redelivery to the manufacturer’s workshop due to the modular design of the VBC.

Modularity in use

Passive contribution towards cleanliness in scraper centrifuges
It may sound simple, but good access to the centrifuge is a precondition for its cleaning and analysis, even when fully automatic CIP systems are usually installed. The modular design of the VBC vertical pharmaceutical centrifuge takes this into account. The design of the cover opening, the position of the outlet and the basket drive can be selected in a wide range of variants and combinations. This enables the machine to be optimally adapted to local space conditions at the site of installation without additional expenditure; this is something that will be appreciated by structural engineers and plant constructors as well as those responsible for maintenance.

Until now, only so-called horizontal centrifuges where considered suitable for installation in a clean room. With the introduction of the VBC, a vertical scraper centrifuge now meets all requirements for installation in a clean room concept, as the complete drive can be arranged below the vibrating plate. This allows the technical area to be separated from the clean room area by means of a membrane in the floor/ceiling.
This method of installation complies with the wishes of many pharmaceuticals companies as the entire production flow takes place gravimetric vertical. The VBC vertical scraper centrifuge thus requires less space than a conventional horizontal centrifuge, as this additional clean room area is required for opening the horizontal housing and positioning the pipes with respect to the vertical product flow into the horizontal machine.

A further important item in the design of the VBC is the one-piece concept of the housing and base plate, avoiding numerous edges and transitions with the advantage of less fouling.
Active contribution towards cleanliness in scraper centrifuges
The function modules that contribute towards active cleanliness include CIP nozzles. The principle applies: As much as necessary, as little as possible. Especially in the case of multifunction systems, the use of an additional CIP nozzle may be necessary. In the modular concept of the VBC centrifuge, this does not present a problem as the CIP nozzles are flanged and easily retrofitted (during production) without welding. It goes without saying that these flanges are all provided with GMP compliant seals.

Those responsible in pharmaceuticals companies can face a far greater problem if the cleaning process must subsequently be changed from CIP cleaning to flood cleaning with a change of product, see “worst-case” analysis. This is another problem that can easily be solved with the design concept of the VBC, as the complete bearing and sealing system is in a modular design. The so-called bearing cartridge can be prefitted as a floodable version and exchanged for the fitted cartridge (during production); and all this without removing the complete centrifuge and sending it to the manufacturer’s works. After the conversion, the complete centrifuge can be flooded up to the cover.

Bibliography
[1] EG-GMP Guide, Appendix 15
[2] Jörg Koppenhöfer; Efficient and cost-saving cleaning validation in the area of active ingredients and substances in multifunction systems, gempex, GmbH, Mannheim, Source: http://www.gempex.com/
[3] Dr. Bernd Köhler und Dr. Carsten Richling; Planing, implementation and documentation of cleaning validation in the pharmaceuticals industry, SWISS PHARMA 25 (2003) No. 9.
[4] FDA Guide for inspection of the validation of cleaning processes; http://www.fda.gov/ICECI/Inspections/default.htm
[5] PIC/S PI 006; Recommendations for validation master plan; GMP Consultant, GMP-Verlag, Schopfheim (2003)
[6] D. Borchert; Cleaning validation (Bd. 1, Chapter 8.B-8.K), GMP Consultant, GMP-Verlag, Schopfheim (2003

BRET - a new method for assaying protein-protein interactions in living cells

2012-01-09T03:00:00.000-08:00

BRET - a new method for assaying protein-protein interactions in living cells

Category: BRET

This method, called bioluminescence resonance energy transfer (BRET), takes advantage of a naturally occurring phenomenon, namely, the Förster resonance energy transfer between a luminescent donor and a fluorescent acceptor. BRET can be observed in the sea pansy Renilla reniformis. This organism expresses a luciferase, which emits blue light when it is purified. If the luciferase is excited in intact cells, green light occurs, because in vivo the luciferase is associated with the green fluorescent protein (GFP), which accepts the energy from the luciferase and emits green light.
The transfer efficiency depends on the degree of the spectral overlap, the relative orientation, and the distance between the donor and acceptor. BRET typically occurs in the 1-10 nm regions, which is comparable with the dimensions of biological macromolecules and makes BRET an ideal system for the study of protein-protein interaction in living cells.
BRET – the assay method
BRET is an advanced, non-destructive, cell-based assay technology that is perfectly suited for proteomics applications, including receptor research and the mapping of signal transduction pathways. The assay is based on non-radiative energy transfer between fusion proteins containing a bioluminescent luciferase and a GFP mutant.
In most applications the fused donor is Renillaluciferase (Rluc) rather than aequorin, to avoid any intrinsic affinity for Aequorea-derived GFP mutant; the acceptor is the Yellow Fluorescent Protein (YFP), to increase the spectral distinction between the two emissions. When the donor and acceptor are in close proximity, the energy resulting from catalytic degradation of the coelenterazine derivative substrate is transferred from the luciferase to the YFP, which will then emit fluorescence at its characteristic wavelength.
To demonstrate the clear discrimination between positive and negative control of the BRET assay technology, the luminescence and fluorescence signals of the BRET^2™demo kit (Perkin Elmer Life Sciences) were quantified on the microplate reader POLARstar OPTIMA (BMG LABTECH, Fig.1), allowing the monitoring of the kinetic curves and the calculation of the BRET ratio. The POLARstar OPTIMA´s internal reagent injectors for 384-well plate format combined with high-end simultaneous dual emission detection offer a unique advantage for fast kinetic assays where simultaneous emission detection at two wavelengths is required.
The BRET²™demo kit applies the cell-permeable and non-toxic coelenterazine derivative substrate DeepBlueC™ (DBC) and a mutant of the Green Fluorescent Protein (GFP²) as acceptor. These compounds show improved spectral resolution and sensitivity over earlier variants.
Fig 1: The POLARstar OPTIMA is perfectly suited for monitoring BRET assays due to its simultaneous dual emission detection system, which allows collecting 50 kinetic data per second, and its internal reagent injectors for 384-well plate format.
The BRET² kit was performed as described in the kit instructions. The reaction was measured in a white 384-well plate at two channels in simultaneous dual emission detection mode with the highest possible resolution of 0.02 s for every data point. Four sets of samples were run in triplicate, a blank (non-transfected cells), a positive control (Rluc-GFP²), a negative control (Rluc + GFP²), and a buffer control (BRET² assay buffer). Readings were started immediately after the automated injection of the luciferase substrate DBC.

The kinetic curves of the negative control are shown in Fig.2 for both channels. The low values of the 515 nm channel indicate that no resonance energy transfer occurred. Whereas the positive control shows reduced values at the 410 nm and elevated values at the 515 nm channel due to the BRET effect.
Fig 2: Resonance energy transfer is obvious for the positive control. No BRET occurs for the negative control.
The calculated BRET ratio indicates the occurrence of protein-protein interaction in vivo. This type of detection eliminates data variability caused by fluctuations in light output which can be found with variations e.g. in assay volume, cell types, number of cells per well and/or signal decay across the plate. In Fig.3 the blank corrected BRET² ratios for both, negative and positive control, are shown and were determined as:

The signal for negative and positive control here reveals a value of around 0.06 and 3.3 respectively, which leads to a factor of around 50 and a clear discrimination between these controls.
Fig 3: Ratio of negative and positive control.
The high factor between these controls is caused by the artificial fusion construct of the positive control (Rluc-GFP²) resulting in an extremely high BRET. Real assay samples will presumably result in lower ratios. Nevertheless, the large spectral resolution between donor and emission peaks in BRET² (115 nm) greatly improves the signal to background ratio over traditionally used BRET and FRET technologies that typically have only a ~50 nm spectral resolution.³
Advantages of BRET over FRET
The BRET technique is related to an existing method for monitoring biomolecular interactions and conformational changes, fluorescence resonance energy transfer (FRET). In FRET, the luminescent donor is replaced by a second fluorophore, which emission spectrum overlaps with the excitation spectrum of the acceptor fluorophore. By using two spectral mutants of GFP, it is possible to genetically attach donor and acceptor fluorophores to proteins, which allows the study of protein interactions in native organisms under physiological conditions.
The main disadvantages of FRET, as opposed to BRET, are the consequences of the required excitation of the donor with an external light source. BRET assays show no photo bleaching or photoisomerization of the donor protein, no photodamage to cells, and no light scattering or autofluorescence from cells or microplates, which can be caused by incident excitation light. In addition one main advantage of BRET over FRET is the lack of emission arising from direct excitation of the acceptor.
This reduction in background should permit detection of interacting proteins at much lower concentrations than it is possible for FRET. However, BRET requires the addition of a cofactor and for some applications, e.g. determining the compartmentalization and functional organization of living cells, the GFP-based FRET method is superior to BRET due to the much higher light output.
BRET applications
The BRET technology was first described in 1999 from Xu and colleagues¹ and has been used successfully for a wide range assay types including protein-protein interactions (e.g. interaction of cardian clock proteins¹), GPCR functional assays⁴ (incl. orphan receptors), receptor oligomerization², and protease activity assays in living cells². BRET has been further used for Ca^{2 +} detection. By fusing GFP directly to the luminescent jellyfish luciferase aequorin, which metabolizes coelenterazine in response to binding free calcium ions, a sensor was produced, that reports calcium ion flux by increases in GFP fluorescence.⁵
Conclusion
BRET is a new energy transfer based technique that offers the ability to directly study complex protein-protein interactions in living cells. There is no need for an excitation light source. Therefore photosensitive tissue can be used for BRET, and problems associated with FRET-based assays such as photobleaching, autofluorescence and direct excitation of the acceptor are eliminated. This powerful technology has been applied in a range of interesting applications in academia and drug discovery. Its homogeneous nature and the development of sensitive plate readers, which offers injection features, have made high-throughput screening using BRET in live cells possible.
References

Xu Y, Piston DW, Johnson CH. A bioluminescence resonance energy transfer (BRET) system: application to interacting circadian clock proteins. Proc Natl Acad Sci USA 1999;96:151-6.
Angers S, Salahpour A, Joly E, Hilairet S, Chelsky D, Dennis M, Bouvier M. Detection of b₂-adrenergic receptors dimerization in living cells using bioluminescence resonance energy transfer (BRET). Proc Natl Acad Sci USA 2000; 97:3684-9.
Mahajan NP, Linder K, Berry G, Gordon GW, Heim R, Herman B. Bcl-2 and Bax interactions in mitochondria probed with green fluorescent protein and fluorescence resonance energy transfer. Nat Biotechnol 1998; 16:547-52.
Ayoub MA, Couturier C, Lucas-Meunier E, Angers S, Fossier P, Bouvier M., Jockers R. Monitoring of ligand-independent dimerization and ligand-induced conformational changes of melatonin receptors in living cells by bioluminescence resonance energy transfer. J Biol Chem 2002; 277:21522-8.
Baubet V, Le Mouellic H, Campbell AK, Lucas-Meunier E, Fossier P, Brûlet P. Chimeric green fluorescent protein-aequorin as bioluminescent Ca^{2 +} reporters at the single-cell level. Proc Natl Acad Sci USA 2000; 97:7260-5

Fig 3: Ratio of negative and positive control.
The high factor between these controls is caused by the artificial fusion construct of the positive control (Rluc-GFP²) resulting in an extremely high BRET. Real assay samples will presumably result in lower ratios. Nevertheless, the large spectral resolution between donor and emission peaks in BRET² (115 nm) greatly improves the signal to background ratio over traditionally used BRET and FRET technologies that typically have only a ~50 nm spectral resolution.³
Advantages of BRET over FRET
The BRET technique is related to an existing method for monitoring biomolecular interactions and conformational changes, fluorescence resonance energy transfer (FRET). In FRET, the luminescent donor is replaced by a second fluorophore, which emission spectrum overlaps with the excitation spectrum of the acceptor fluorophore. By using two spectral mutants of GFP, it is possible to genetically attach donor and acceptor fluorophores to proteins, which allows the study of protein interactions in native organisms under physiological conditions.
The main disadvantages of FRET, as opposed to BRET, are the consequences of the required excitation of the donor with an external light source. BRET assays show no photo bleaching or photoisomerization of the donor protein, no photodamage to cells, and no light scattering or autofluorescence from cells or microplates, which can be caused by incident excitation light. In addition one main advantage of BRET over FRET is the lack of emission arising from direct excitation of the acceptor.
This reduction in background should permit detection of interacting proteins at much lower concentrations than it is possible for FRET. However, BRET requires the addition of a cofactor and for some applications, e.g. determining the compartmentalization and functional organization of living cells, the GFP-based FRET method is superior to BRET due to the much higher light output.
BRET applications
The BRET technology was first described in 1999 from Xu and colleagues¹ and has been used successfully for a wide range assay types including protein-protein interactions (e.g. interaction of cardian clock proteins¹), GPCR functional assays⁴ (incl. orphan receptors), receptor oligomerization², and protease activity assays in living cells². BRET has been further used for Ca^{2 +} detection. By fusing GFP directly to the luminescent jellyfish luciferase aequorin, which metabolizes coelenterazine in response to binding free calcium ions, a sensor was produced, that reports calcium ion flux by increases in GFP fluorescence.⁵
Conclusion
BRET is a new energy transfer based technique that offers the ability to directly study complex protein-protein interactions in living cells. There is no need for an excitation light source. Therefore photosensitive tissue can be used for BRET, and problems associated with FRET-based assays such as photobleaching, autofluorescence and direct excitation of the acceptor are eliminated. This powerful technology has been applied in a range of interesting applications in academia and drug discovery. Its homogeneous nature and the development of sensitive plate readers, which offers injection features, have made high-throughput screening using BRET in live cells possible.
References

Xu Y, Piston DW, Johnson CH. A bioluminescence resonance energy transfer (BRET) system: application to interacting circadian clock proteins. Proc Natl Acad Sci USA 1999;96:151-6.
Angers S, Salahpour A, Joly E, Hilairet S, Chelsky D, Dennis M, Bouvier M. Detection of b₂-adrenergic receptors dimerization in living cells using bioluminescence resonance energy transfer (BRET). Proc Natl Acad Sci USA 2000; 97:3684-9.
Mahajan NP, Linder K, Berry G, Gordon GW, Heim R, Herman B. Bcl-2 and Bax interactions in mitochondria probed with green fluorescent protein and fluorescence resonance energy transfer. Nat Biotechnol 1998; 16:547-52.
Ayoub MA, Couturier C, Lucas-Meunier E, Angers S, Fossier P, Bouvier M., Jockers R. Monitoring of ligand-independent dimerization and ligand-induced conformational changes of melatonin receptors in living cells by bioluminescence resonance energy transfer. J Biol Chem 2002; 277:21522-8.
Baubet V, Le Mouellic H, Campbell AK, Lucas-Meunier E, Fossier P, Brûlet P. Chimeric green fluorescent protein-aequorin as bioluminescent Ca^{2 +} reporters at the single-cell level. Proc Natl Acad Sci USA 2000; 97:7260-5

Sustenance of Clean Room Conditions

2012-01-09T02:52:00.000-08:00

By Rashmi Nagabhushan
Director, Thermadyne Pvt. Ltd., Faridabad
Rashmi Nagabhushan is an M.Tech from IIT Delhi and has over 20 years experience in various capacities in group companies of Continental Device India Ltd. She has been associated with Thermadyne Pvt. Ltd., the complete clean room company, for the last 13 years.

Lakhs of Rupees spent in installing a clean room can go down the drain if cleanliness levels are not maintained on a continuous basis. Personnel training and discipline are the key factors. Sustenance guide lines should be followed both in letter and spirit. Clean room discipline can make a success or a failure of a clean facility. People must be motivated and trained to achieve cleanwork habits and follow timely maintenance practices.

Indoor air quality is of paramount importance for human comfort and health. Similarly, indoor air quality of manufacturing facilities plays a vital role in the yield and quality of many products.
There has been a great deal of advancement in building clean rooms suitable for different applications. Many individual national standards and now international ISO standards are available which define cleanliness classes and measuring procedures for their compliance. Guide lines are also available on the cleanliness levels required for various manufacturing facilities. Expertise now exists to achieve even Class 10 and Class 1. But little is said as to how to sustain the built cleanliness. In fact, by and large, the contract between the customer and contractor is on satisfying the ‘as built’ or ‘at rest’ requirements. And in most cases it is found that cleanliness levels in operational conditions are considerably poorer and some times may lead to conflicts between the two parties. First and foremost, many factors are required to be taken into consideration before designing and installing clean rooms to ensure achievement and sustenance of clean room conditions. Some of the important ones are listed below

Factors to be Considered at the Design Stage

Type of industry (to determine class of cleanliness).
Contamination generation by process, abrasion or by constructional materials.
Occupancy and physical activity level.
Contamination level in the surrounding areas.
Presence of heated surfaces.
Equipment lay-out in the room.
Process exhausts.
Future expansion plan.
Statutory requirements

Having given due consideration to the above factors and achieving the desired conditions at start-up why do most facilities fail to sustain the desired cleanliness levels on a long term basis?
The reasons sound very trivial, but their impact is unbelievable. The unhappy situation can be largely attributed to lack of personnel discipline which results in many short comings and ultimately in the failure of the clean room. If however the following practices are followed we can save the situation.

Steps to be Taken after Clean Room Installation

Personnel training.
Proper maintenance of air conditioning systems and other clean air systems to ensure that required air quantity is supplied on a continuous basis.
Timely cleaning / changing of filters, again to ensure that designed clean air quantity is maintained.
Proper and continued use of change room entry equipment.
Proper gowning.
Clean room discipline.
Scheduled cleaning of clean rooms.
Strict observance of equipment maintenance practices for clean rooms.
Regular monitoring of clean room conditions.

Some guide lines are available on the above which have been highlighted at the end however due to lack of appreciation of cause and effect, people tend to take them lightly thus causing degradation of facilities.
Being a clean-room company, installing and validating clean rooms, we have first hand experiences on the main culprits which lead to problems of sustenance of air cleanliness. To give you an insight into some of the problems, a few case studies are listed below.

CASE STUDY - 1

A class 10,000 facility was successfully installed. On revalidation after 6 months, particle counts were found to be in the range of 2 to 3 lakhs. It was observed that clean-room gowning procedures were not being followed properly, specially in terms of shoes. The surrounding area of the building also had rubble and people were bringing in a lot of dirt. The situation was worsened by the constant movement of people inside the clean area.
A thorough cleaning-up of the area, proper gowning and proper changing of clean room slippers brought back the clean conditions. See Table 1.

Table 1 – See case study 1
Designed Class - 10,000 0.5μ and above particle count (Average)			Number of Hepa filters - 16
	Production Area	Change Room	Observations
At Startup	3220	3100	Within specified limits
After 6 months	210,000	350,000	Dirty shoes inside change room. Gowning improper. Air shower not used properly. Sleepers washed once in a month. Water tap inside change room.
After one year	170,000	294,000	Shoes outside change room. Rest same as above.
After 1.5 years	81,000	140,000	Dresses in garment storage cabinet, new dresses. Three times floor cleaning (wet). Worker discipline better.
After 2 years	14,000	34,000	Air curtain at change room entrance. Proper discipline.
After 2.5 years After 3 years	6600 4930	32000 14300	Filters not changed.

CASE STUDY - 2

In another case filters choked unevenly due to dampers not being provided independently for each filter. The choked filters i.e. four out of twelve were replaced. This resulted in most of the air coming through the new filters resulting in pockets of unclean area and also, generally a higher count due to turbulence caused by high velocity through clean filters. See Figure 1.

CASE STUDY - 3

One small company asked us to do their clean-room validation. We found the particle counts to be high. On investigating we found that out of four terminal Hepa filters two were replaced by fine filters after choking. Since the air discharge from the filters was through perforated grilles it took us some time to figure out the problem.

CASE STUDY - 4

Filters were choked and sufficient air quantity was not reaching the area, resulting in negative pressure inside the area. Each time the entry door was opened, dirty air was coming in. The problem was accentuated as an additional exhaust was fixed for a new process machine which was not taken into consideration in the initial design.

CASE STUDY - 5

An otherwise satisfactory facility failed miserably on start-up. It was found that machines were crowding the clean room very badly and not allowing proper air flow.
Besides, unclean compressed air was being used freely, resulting in a total collapse of the clean area.

CASE STUDY - 6

Window air conditioners were added subsequently to supplement cooling of an existing clean facility. Installation was done very poorly leaving gaps between the air conditioner and the window cut out. Also some unfiltered fresh air was getting added through the window air conditioner.

CASE STUDY - 7

In another case a good clean room (Class 10,000) deteriorated badly due to influx of additional people and machinery to cope with the sudden increase in production capacity.

CASE STUDY - 8

One laboratory which was near a railway line developed cracks in the ceiling due to vibration and resulted in high particle count. A patch up did help but ultimately the laboratory had to be shifted to another location.

CASE STUDY - 9

A satisfactory clean facility detiorated within six months. A thorough investigation revealed that the fan used in the air handling unit did not have the desired static pressure capability. Inadequate clean air supply resulted in negative pressure and ruining of the facility.

Other Observations

In some cases cracking and detaching of sealing compound in Hepa filters with time was found to be a major reason of failure.
In a number of cases, damaged Hepa filters were found to be in use in spite of the concerned people being aware that this would cause problems.
While choked filters are replaced, their installation does not get adequate attention due to inadequate training of maintenance staff. Thus we get either dirty clean rooms or quickly choked Hepa filters.
Validation is not done at regular intervals. If you don’t monitor the clean room, you can sit pretty without any warning till you collect losses due to rejection of products.
In many cases even at the first validation, initial results are poor. But a through cleaning of the area invariably results in the designed class of cleanliness.

Recommendations for Maintaining Existing Clean Room Conditions in the Clean Room Area

Only authorised personnel should enter the clean rooms. • No body should be allowed into the clean room without wearing clean room garments including cap and clean room shoes/chappals properly.
Always stay in the “air shower” for a specified time before entering clean rooms.
After the use of garments and shoes, these should be kept at a proper place. Never go into “nonclean” areas from change room with garments or shoes.
Do not walk into a clean room unless necessary.
Smoking in the clean room and change room is prohibited.
Do not take contamination producing material like tobacco, food, match boxes, purses, cosmetics, card boards and unnecessary papers inside the clean areas. Also do not apply cosmetics in the clean area.
Do not sharpen pencils in the clean room and use a ball point pen only. • Wear gloves and finger cots whenever required.
Do not touch contaminated articles or surfaces after wearing finger cots/ gloves.
Do not scratch your head or rub your nails inside the clean room or change room and keep finger nails clean.
Do not take personal items into clean room, keep them in lockers provided.
Keep your work table clean.
Clean / change filters in the air conditioning system as and when required.
Never sweep the clean room floor, vacuum them or wet mop them as per frequency specified.
Clean walls, ceilings and furniture as per frequency specified with wet mop.
Garments should be washed as per frequency specified.
Clean all furniture, equipment and raw material packages etc. properly before taking into clean room.
Do minimum maintenance of equipment inside the clean room. Take the equipment outside the clean room for maintenance.
Unpacking of the machinery required for the clean areas should be done outside the clean room.

Clean room discipline which covers all the points listed earlier and above can make a success or a failure of a clean facility. People must be motivated and trained to achieve clean work habits and follow timely maintenance practices

Pharmaceutical Quality assurance

2011-12-29T22:58:00.000-08:00

Pharmaceutical Quality assurance is an essential element of drug advancement in the undersized pharmaceutical world. It is a branch which is accountable to warrant that all suitable methods have been abide by and recorded so that scientific development can be achieved. Modern solutions in documentation and supervising tools for use concerning essential storage, individual packaging and labeling, research laboratory environments, and development management are considered to enhance total quality assurance.
Pharmaceutical Quality Assurance is a very vast range of concept that encompasses all resources that individually or jointly influence the quality of a product. In relation to drugging industry, quality assurance can be classified into almost 4 key divisions:supervision of the process, the production process, deliverance and evaluation. The development of traditional norms and regulations for the boost of assurance of quality is an important factor of the rule book of World Health Organization.
Significant vital factors are assurance of quality management manuals in the fields of manufacturing, assessment, and dissemination of medicines. These comprise regulation on: high-quality production procedures, quality assurance in endorsement control, pre-criterion of qualitative medicines, research laboratories, and source organizations; exemplary certifications for quality assurance-linked efforts; value management analysis; latest requirements for enclosure in the Basic Tests progression.
All these fundamental features are projected for application by the national board officials, manufacturers and other concerned organizations. The need to raise access to low-cost quality medicines for communicable disease in the less developed countries has raised numerous tests within pharmaceuticals realm.
These jobs happen on unsurpassed fact that among national regulatory authorities there is a variable quantity to justify and apply current practices and accepted advices and ideals on instructions, quality management, labeling and classification of pharmaceuticals. World Health Organization will work to build up and promote complete standards, norms and guidelines in setting up its significance, worth and benefits of medicine.
The enhancement of ethics, morals and beliefs in promoting quality assurance and quality control is an essential factor of the rule book of World Health Organization and a remarkable involvement. It has been encouraged and sponsored via numerous World Health statements, and with Revised Drug Strategy. World Health Organization carries out its purpose in the areas of medicines and essential drugs at global markets, isolated or rural areas. At WHO headquarters, activities were developed and put to use by the Department of Essential Medicines and Pharmaceutical Policies (EMP).
Traditional medicine is the whole information, expertise, and the habits found on hypothesis, principles, and practices of ethnic to diverse cultures, whether understandable or not, implication in the reservation of health as well as in the preclusion, analysis, recovery or cure of physiological or psychological diseases.
Traditional use of herbal medicines employs to the lengthy treatment of these medicines. Their treatment is extensively acknowledge and widely accepted to be helpful, beneficial and highly effective and therefore, approved by the national regulatory authorities.
Active ingredients connected to elements of herbal medicines with healing properties are the top attributes to promote cure. In herbal medicines where beneficial component has been categorized, the foundation of such drugs should be dealt to comprise a defined quantity of the active factor, when enough systematic process are reachable. As it was, where it is not possible to be able to find out the active components, the entire herbal medicine may be considered as one effective cure

Beyond Size Exclusion: There Is No Universal Model Organism

2011-12-28T23:28:00.001-08:00

Brevundimonas diminuta is typically used as the model for organisms that are expected to be found in pharmaceutical manufacturing environments. It was selected, based on its presence in pharmaceutical operations, and within native bioburden [4].
The organism has been found particularly suitable for validating sterilizing grade filters due to its size and ease of cultivation. However, it should only be used to model situations where its dimensions closely match the organisms of interest in a given application, relative to the filter pore size and shape.
Furthermore, in some cases, sieve retention may not be the mode of organism removal. For instance, in some cases, it may be adsorption, as the organism forms hydrogen bonds to the filter’s polymeric surface. This could account for the observation, 26 years ago, that Pseudomonas aeruginosa organisms are more strongly retained by polyamide membranes than by cellulose triacetate filters [5]. It also explains the removal of latex particles from aqueous suspensions by polyamide membranes in the presence of surfactant, but not in its absence (Table 1) [6].
Table 1: Retention (%) of 0.198-µm spheres by various 0.2-µm-rated membranes

Filter Type	In Water (% )	In 0.05% Triton X-100 (%)
Polycarbonate	100.0	100.0
Asymmetric polysulfone	100.0	100.0
Polyvinylidene fluoride	74.8	19.2
Nylon 66	82.1	1.0
Cellulose esters	89.4	25.1

From Tolliver and Schroeder (1983) courtesy of Microcontamination
B. diminuta should not be viewed as a universal model organism, as some native bioburden may be a better alternative as challenge organism, being close to the actual process settings. Unfortunately, rare penetrations of sterilizing grade filters have caused an exaggerated doubt in the reliability of filtration. Appropriate process validation though should render such doubts and be trusted by even the most critical reviewer of sterile filtration..

Sources of Variability: Size and Shape

B. diminuta varies in size and shape, depending on how it is cultivated. Back in 1967, Bowman and colleagues described the B. diminuta size as 0.3 × 1.0 μm [7]. However, in 1978, Leahy and Sullivan found [8] that the organism grown at 30 °C and incubated for 24 hours in saline lactose broth, a minimally nutritional medium for that microbe, yielded cocci-like cells approximately 0.3 × 1.0 μm (Figure 1). Similar considerations have to be accounted for when challenge tests are performed with native bioburden forms.
B. diminuta are typically cultivated to develop as spherical a form as possible, since spheres are least amenable to retention. Thus, Leahy and Sullivan proposed, back in 1978, that it be used as the model organism for 0.2/0.22-μm-rated membranes, partly because of its size relative to the 0.2-μm dimension [8].
Subsequently, the FDA designated it for that very purpose [9], defining a sterilizing filter as one that retains a minimum of 1 x 107 cfu of Brevundimonas diminuta ATCC 19146 / per cm2 of effective filtration area (EFA).
Although it isn’t the smallest organism known, B. diminuta was considered diminutive enough to represent whatever smaller organisms were likely to be present in pharmaceutical preparations. The smaller the test organism, goes the logic, the more likely that its removal by a filter would assure the sieve retention of larger organisms.
However, ease and safety of cultivation and handling are also important considerations. In 2001, Sundaram and colleagues found an increasing number of cases where filtration in 0.2/0.22-µm-rated membranes failed to yield sterile effluent [10]. Experimental studies showed that penetrating organisms had shrunken because they had been cultivated in broths that were nutritionally inadequate. In such cases, the physicochemistry of the suspending fluid may serve to alter the size of the suspended organisms as expressed by the Donnan equilibrium consequent to ionic strengths.

Organism Shrinkage During Processing

Sundaram’s team [11] also found that organisms underwent size changes after exposure to certain drugs. In cases with 0.2-µm-rated membranes, the researchers found, the larger pore size could only provide sterile effluent and/or a high titer reduction with regard to certain organisms for various lengths of time, before penetration occurred.
Penetration times varied from 24 to 96 hours, and the cumulative challenge at which penetration was first observed ranged from 1.2 x 107 to 1.1 x 108 cfu/cm2. Two 0.2-µm rated Nylon-66 filters in series were unable to fully retain Ralstonia pickettii (now Burkholderia pickettii) with penetration observed at 72 hours, corresponding to a cumulative challenge of 2.4 x 107 cfu/cm2. The more extensive penetration of the Nylon-66 membranes, compared with the PVDFs, is in keeping with their greater degree of openness, as Krygier and colleagues showed in 1986 [12].
As a result, it has been suggested that 0.1-µm-rated membranes be substituted for their 0.2-µm-rated counterparts.
Sundaram’s team evaluated five 0.1-µm-rated membranes and found that they yielded sterile effluent over the entire duration of the test (120-196 hours), up to challenge levels of 5.7 x 107 to 2.0 x 108 cfu/cm2. Similar results were obtained with the PVDF filters tested; no B. pickettii were detected at challenge levels of 5.9 x 107-6.0 x 108 cfu/cm2.
In addition, all 0.1-µm-rated filters tested provided consistent and complete retention of B. pickettii for the entire duration of the test (120-192 hours), suggesting that the smaller pores would ensure sterile product at conditions where penetration could occur through conventional 0.2- and 0.22-µm-rated sterilizing grade filters. Proponents argue that using 0.1-μm-rated membranes would permit longer term formulation and filtration operations. In fact, 0.1 µm-rated filters may be the best choice for long-term filtrations.
However, penetration has also been found in 0.1 µm-rated filters. In 1999, Sundaram’s team found that B. pickettii, when its size was so affected, could be retained by certain 0.1-µm-rated filters. But, in a similar situation, they found that only four of seven commercially available 0.1-μm-rated membranes could remove a particular organism. Just because one type of membrane so classified may provide proper retention, does not mean that any other 0.1-µm-rated membrane can also be depended upon for a like result.
It is important to remember that today, there are no industry standards by which 0.1 filters can be judged. In addition, more research clearly needs to be done into the kinetics behind the organism’s size changes, evaluating different organisms in different fluids.
Based on available data, long term filtrations may best be handled by 0.1-µm-rated filters, subject to validations being performed. However, in other cases, substituting 0.1-µm-rated for 0.2-µm-rated membranes may be unnecessary, and could result in significant penalties, including:

 Slower flow and processing rates, resulting in longer term operations.
 Higher costs for larger EFAs
 More leaching and extractables
 Higher product losses, due to adsorptive bonding to the ultimately greater filter area used.
A responsible choice requires that both the 0.1 µm-rated membranes and the 0.2 µm-rated membranes be validated.
If both types of filter prove appropriate, the higher pore size rating should be used to avoid the penalties of reduced flows. If, however, the validation data do not permit a clear resolution, the 0.1 µm-rated membranes should be used, since retention is more critical than flow rate or flux.
Below, we address some of the common sterile filtration concerns, requirements or practices that appear to be motivated by fear and can best be resolved by careful process validation.
“0.2-µm filters are penetrated by organisms. The industry is, therefore, required to switch to 0.1-µm-rated filters.”
In certain specific processes, 0.2-µm–rated filter can be penetrated by organisms, or by organisms which would normally be retained by such filters. In such cases, the flltrative removal of the organisms may well require the use of 0.1 µm–rated filters. Such instances are not new. Their occurrences have been considered by regulators for years, at least since the PDA and FDA held a special forum on this topic in 1995 [13].

Certain organisms, such as Burkholderia pickettii, Burkholderia cepacia, and Pseudomonas aerugenosa. shrink as a result of their immersion in fluid media that are only minimally nutritious for them [14]. Their reduction in size renders as invalid validations that use B. diminuta as a model. Brevundimonas diminuta can undergo shape alterations in minimally nutritious media but is not listed as undergoing size alterations occasioned by contacts with process fluids.
The fact that some microbes require 0.1-µm–rated filters to arrest them does not signify that all organisms are so disposed. The necessitated switch from 0.2-µm-rated to 0.1-µm–rated happens in only roughly 0.005% – 0.01 % of sterilizing grade filtration applications.
A mandated switch is therefore scientifically and statistically unfounded. Its promulgation may be shunned and process validation activities and data used as performance verification. Sole reliance on pore size ratings have been found obsolete anyway.
“Increasingly there are detectable but non-culturable organisms or L-forms or nano-bacteria in our processes.”
Conclusions cannot be made regarding the sterile filtration of microorganisms unless the methods of quantifying them by culturing and counting are available. Organisms such as the L-forms, nanobacteria, and “viable but non-culturable” entities may not be amenable to such analyses. Concerns about their presence may be justified, but without the means to cultivate and count them, it is impossible to attest to their complete absence.
It follows that a sterilizing filter can be judged only by its performance in the removal of identifiable and culturable organisms known to be present in the drug preparation [15]. The complex of influences governing the outcome of an intended sterilizing filtration necessitates a careful validation of the process, including that of the filter [4]. The very drug preparation of interest, the exact membrane type, the precise filtration conditions, and the specific organism type(s) of concern must be employed in the necessary validation.
“Redundant 0.2-µm filtration is necessary and should be used.”
Not necessarily. Again, proper process validation will disclose whether a single filter will do the job or not. However, there are some specific applications which traditionally, for whatever reason, utilize a second (redundant) filter as an “insurance filter,” i.e. if the first filter fails, the second may compensate. This holds, however, only when each filter has been validated to show specified retentivity.
Even so, the wisdom of the exercise deserves careful evaluation, as it assumes added costs for membrane EFA, increased leachables and extractables. The loss of drug product may needlessly be incurred by the filter’s heightened product hold-up, and unspecified adsorption.

“The maximum bioburden in front of a sterilizing filter should be 10 cfu per 100 mL of fluid.”
This is true if one wishes to accord with EMA regulations, and especially if one wishes to export product to Europe. The FDA makes no such stipulation, but bases its approval on process validation.
Seemingly in conflict, the two views arise from the same premise. The EMA regulation tries to establish the same sterility assurance level (SAL) for filtration as for thermal sterilization. EMA recognizes that, the greater the number of challenges, the more likely that at least one will succeed.
The FDA seems to agree, in that if the filter can sustain the removal of organism burdens far above those liable to be encountered in real life situations, it can assuredly withstand lesser insults. If, as the authors see it, the FDA’s massive challenge fails to breach the filter’s pores, it is needless to compel bioburden assessment in front of the filter. Filter validation would gainfully serve the intended purpose. Process validation, effectively conducted, would reliably demonstrate the filter action.
“I need an absolute 0.1- or 0.2-µm-rated filter.”

As a former FDA authority, since retired, once observed, “The word ‘absolute’ should be used only in conjunction with vodka.” Absoluteness implies a complete independence from conditions, an inherent ability to retain particles larger that than the filter’s pore size rating, regardless of any other considerations. Without a complete knowledge of the properties of the particles and filter pores at our disposal, the statement is devoid of technical significance or guidance. It may, perhaps, be used in ignorance (although cynics may suspect that its utility derives from marketing efforts, a practice not unknown in the competitive world of sales.)

Control vs. Fear

As Sandman elucidated, human beings like to be in control, and, if this status cannot be achieved, may move rapidly to fear. Unfortunately, when sterile filtration is concerned, fear can result in the installation of wasteful, unnecessary safety nets that can create more problems than they solve.
Being in control is the desired state, and such control can only come from process validation studies. Their authority is at least as old as Lord Kelvin’s basic scientific principle, “When you can measure what you are speaking about, and can express it in numbers, you know something about it.”
It speaks to validation. In sterile filtration, as in most areas of pharmaceutical manufacturing, science-based validation is the best cure for fear.

References
1. Hessler, A., Sandman, P.M. Squeaky Clean? Not Even Close. http://www.nytimes.com/2004/01/28/dining/squeaky-clean-not-even-close.html?sec=health?pagewanted=1
2. FDA. Guideline on General Principles of Process Validation, FDA CDER, 1987.
3. Agalloco, J.P. “Compliance Risk Management Using a Top-Down Validation Approach,” Pharmaceutical Technology, July 2008.
4. PDA Technical Report 26 (2008), Sterilizing Filtration of Liquids, Parenteral Drug Association, Bethesda, MD.
5. Ridgway, H.F., Rigby, M.G., and Argo, D.G. “Adhesion of a Mycobacterium to Cellulose Diacetate Membranes Used in Reverse Osmosis.” Applied and Environmental Microbiology 47, 1984, pp. 61-67.
6. Tolliver, D.L. and Schroeder, H.G. “Particle Control in Semiconductor Process Streams.” Microcontamination (l), 1983, pp. 34-43 and 78.
7. Bowman, F.W, Calhoun, M.P. and White, M. “Microbiological Methods for Quality Control of Membrane Filters.” J. Pharm. Sci., 56/2, 1967, pp. 453-459.
8. Leahy, T.J., Sullivan, M.J. “Validation of Bacterial Retention Capabilities of Membrane Filters.” Pharmaceutical Technology 2(11), 1978, pp. 64-75.
9. FDA. Guideline on Sterile Drug Products Produced by Aseptic Processing, FDA CDER, 1987.
10. Sundaram, S., Eisenhuth, J., Howard Jr., G.H., and Brandwein, H. “Part 1: Bacterial Challenge Tests on 0.2 and 0.22 Micron Rated Filters.” PDA Journal of Pharmaceutical Science and Technology, 55 (2), 1984, pp. 65-86.
11. Sundaram, S., Auriemma, M., Howard Jr., G.H., Brandwein, H., and Leo, F. “An Application of Membrane Filtration for Removal of Diminutive Bioburden Organisms in Pharmaceutical Products and Processes,” PDA Jour. Pharm. Sci. and Technol. 53 (4), 1999, pp. 186-201.
12. Krygier, V. Rating of Fine Membrane Filters Used in the Semiconductor Industry, Transcripts of Fifth Annual Semiconductor Pure Water Conference, (1986), pp. 232-251, San Francisco, CA
13. PDA/FDA Special Scientific Forum, Bethesda, MD; Validation of Microbial Retention of Sterilizing Filters, July 12-13, 1995.
14. Mittleman, M.W., Jornitz, M.W., Meltzer, T.H., “Bacterial Cell Size and Surface Charge Characteristics Relevant to Filter Validation Studies,” PDA Jour. of Pharm. Sci. and Technol. 52 (1), 1998, pp. 37-42.
15. Agalloco, J., Letter to the Editor—re: “It just doesn’t matter, It just doesn’t matter, It just doesn’t matter.” PDA Journal of Science and Technology. Vol 52, No. 3, pp. 149-150

How to set-up your own Aseptic Laboratory?

2011-12-28T23:25:00.000-08:00

The Aseptic Room
All openings except the openings for entry need perfect sealing and horizontal surfaces including windows should be eliminated. These surfaces may allow retention of dust and may create problems.
The structure should be tight enough to prevent infiltration of uncontrolled air. All exposed surfaces should be smooth and impervious, easily cleanable and in no way prone to settling of dust upon them.
The surfacing material should not be susceptible to hold dust, flaking or chalking under normal operative procedures. Uncoated smooth surfaces e.g. stainless steel, aluminium and chromium plating, plastic laminates and plastic films make satisfactory surfaces. Cement concrete is a very undesirable surface.
Air-conditioned Atmosphere
In view of the sealed structure, preventing any source of entry of air, air conditioning is essential. Since no special requirement of temperature and humidity are prescribed, conventional air conditioning may be suitable enough. It is however preferable to have the humidity on the lower side to avoid contamination by the perspiration of the workers.
Air cleaning part of the air conditioning system is a critical factor as the nature of the atmospheric air may differ considerably from time to time. Cooling and heating coils, humidity control apparatus, reheat coils; blowers etc. are the equipment that should be of standard specifications.
However, the fans selected should be such that can provide high pressure. The room has to be constantly maintained under positive pressure to prevent inlet of air from entry point whenever it is opened. Dust, temperature and humidity control are interdependent functions.
Dust control is impossible without confining the area. Confined space is unlivable without air-conditioning. If temperature and humidity are controlled by air conditioning, dust control measures are automatically taken care of.
Cleaning of Air
Cleaning of air is the key factor of the aseptic processing. The usual approach is a combination of the conventional cleaners e.g. regular filters or electronic air cleaners located within the system and some kind of super-interception or an ultra cleaner located down stream from all coils, blowers etc.
Bactericidal equipment is incorporated in the assembly for providing sterile air in addition to be devices stated above. The air in the aseptic area should be free from fibers, dust and microbes. This can be conveniently achieved by the use of High Efficiency Particulate Air (HEPA) filters which can remove particles up to 0.3 µm with an efficiency of 99.7% or more.
HEPA filters made use of in Laminar Air Flow in which air moves with uniform velocity along parallel lines with minimum of eddies. The air flow can be either horizontal or vertical and 100 ± 10 ft/min. is considered to be the minimum effective air velocity. Such laminar flow stations and work benches are commercially available and should find immense use in compounding and dispensing practice.
Air Distribution
Materials of construction for the air distribution system should be made of non-rusting and non- flaking materials (e.g. ducts, air outlets etc.). Duct insulation, if needed, should be applied on the outer side and only on the ducts that are located out of the clean area. Joints and other fittings, if any, should be sealed to prevent leakage and contamination. Air distribution is also employed for 'washing' the workers off dust who enter the sterile area. These are called air showers which are strictly blasts or air directed on the person to remove dust.
Controls
Conventional controls are used in the system for regulating humidity and temperature. Sometimes greater pressure may have to be maintained in critical areas and for this purpose additional controls may have to be installed. Interlocks may be required between the sterile area and the entrance or passing doors. In a highly elaborate system, alarm circuits are introduced to warn against the malfunctioning or inadvertent misuse of the air locks.
Instruments
Highly sophisticated instrumentation has to be installed which constantly or intermittently monitors and samples the clean atmosphere for analyzing its cleanliness. These devices immediately indicate the contamination, if any.
Furniture
Seating, work tables, racks etc. are essential requirements of furniture inside he clean area and have to be specially designed and built to meet certain rigid specifications. Adjusting mechanisms on the chairs should either not exist or ought to be sealed. These may be potential dust setting surfaces.
Conventional upholstering of the chairs is completely out of question. Dust catching surfaces should be minimum and all parts should be readily cleanable and resistant to the action of the cleaning agents. Worker comfort is a very important factor in a confined area. The requirements of the job in a sterile room keep the workers chained to the work over long periods of time continuously without leisure and with minimum exits from the room and thus warrants high degree of comfort for carrying out the critical operations.
Special jigs, fixtures and tools are developed for specific purposes but many operations need dust-free hoods-miniature aseptic rooms located on the working bench. Even when the aseptic hoods or chambers are located in dust free rooms, they may have to be provided with supply of pressurized air in each one of them. The air may be either super cleaned or an inert gas. Further these hoods have to be independently illuminated.
There are many several important factors to consider when called upon compounding a sterile ophthalmic preparation. Eyes are very sensitive to heat, light, drugs and chemicals. In many cases, the drugs involved have a narrow therapeutic range and even small errors when introduced have the potential to cause irreversible damage to the eye or loss of the eye sight. The following considerations are recommended whenever preparing such a product.
1. Ensure concentration is within the acceptable range or not before dispensing of the product.
2. Sterility of the final product is a must, strictly handled in aseptic area.
3. The pH of the final product must be within an acceptable range.
4. Stability of the final product must be known, as well as the recommended storage requirements.
5. Suitable knowledge of potential diluents or vehicles is required in order to ensure proper tonicity, viscosity, or dissolution of the final product.
6. Proper documentation of each step is an important consideration to reduce error.
7. If the preparation of a product requires the breaking of an ampoule or the reconstitution of a powder, it is recommended that the final product be made in sterile water for injection and free from particulate matter.
8. The preparation of intra-occular products requires the use of preservative-free ingredients. Many preservatives have been found to be toxic to the inner ocular tissues.
9. Finally, before dispensing the finished product, always indicate the storage requirements, concentrations of ingredients, and the expected expiration date.

2011-12-28T23:17:00.000-08:00

While sterility testing can be cumbersome and time consuming, there are FDA-approved options regarding container and closure system integrity testing. In fact, the Food and Drug Administration (FDA) recently released a guidance document on container and closure system integrity testing in lieu of sterility testing as a component of the Stability Protocol for Sterile Products. The document offers an alternative approach, if the approach satisfies the requirements of the applicable statutes and regulations.

Aimed at manufacturers, the guidance offers alternative testing methods other than sterility testing to confirm container and closure system integrity as a part of the stability protocol for sterile biological products, human and animal drugs, and medical devices.

The purpose of stability testing is to provide evidence on how the quality of a substance or product varies with time under the influence of a variety of environmental factors such as temperature, humidity and light. Products labeled as sterile are expected to be free from viable microbial contamination throughout the product's entire shelf life or dating period. This enables manufacturers to establish or modify recommended storage conditions, retest periods and shelf life or dating period, as the case may be.

Currently, manufacturers of drugs and biologics purporting to be sterile are required to test each batch or lot, to ensure that the product in question conforms to sterility requirements. They must also maintain a written testing program designed to assess stability characteristics and meet stability testing requirements.

Manufacturers of medical devices are required to validate processes, including sterilization, for a device purporting to be sterile, although stability testing should be part of the design validation of such devices. Also, in vitro diagnostic products for human use are required to be labeled with stability information.

The minimum sterility testing generally performed as a component of the stability protocol for sterile products is at the initial time point (release) and final testing interval (i.e., expiration). Additional testing is often performed at appropriate intervals within this time period (e.g., annually).

Alternatives to sterility testing as part of the stability protocol, such as replacing the sterility test with container and closure system integrity testing, might include any properly validated physical or chemical container and closure system integrity test (e.g., bubble tests, pressure/vacuum decay, trace gas permeation/leak tests, dye penetration tests, seal force or electrical conductivity and capacitance tests, etc.), or microbiological container and closure system integrity tests (e.g., microbial challenge or immersion tests).

Such tests may be more useful than sterility testing in demonstrating the potential for product contamination over the product's shelf life or dating period. The advantages of using such container and closure system integrity tests in lieu of sterility tests in the stability protocol for sterile products include: detecting a breach of the container and/or closure system prior to product contamination; conserving samples that may be used for other stability tests; requiring less time than sterility test methods which require at least seven days incubation; and reducing false positive results with some alternative test methods when compared to sterility tests.

To implement container and closure system integrity testing as an alternative to sterility testing, the FDA recommends manufacturers consider the following: a container and closure system integrity test may replace sterility testing in a stability program at time points other than the product sterility test prior to release; container and closure system integrity tests do not replace sterility testing methods for product sterility testing prior to release; any validated container and closure system integrity test method should be acceptable provided the method uses analytical detection techniques appropriate to the method and is compatible with the specific product being tested.

TOOLS OF THE TRADE - Analytical Instrumentation | The Power of FCS

2011-12-16T22:48:00.001-08:00

Samara Kuehne

ISS makes the Alba-FCS dual-channel spectrometer, which combines a confocal scanning microscope with FCS.

Newer fluorescence correlation spectroscopy instruments focus on portability, affordability, and ease of use

Fluorescence correlation spectro-scopy (FCS), originally developed in the early 1970s for use in physics and physical chemistry, has recently been applied to the fields of drug discovery and development, with powerful results.
Early FCS equipment used in physics labs was large, with bulky lasers that were not necessarily user friendly. Since 2005, instrumentation with specific application to the drug discovery field has entered the marketplace, and products released since have increasingly focused on reducing the size of a unit’s footprint to make it easier to use in the lab.

FCS Technology

FCS is an extremely effective tool for ultrasensitive measurements. The technique measures and correlates fluctuations in fluorescence intensity within a very small volume element, allowing for single-molecule inspection. A sharply focused laser illuminates this small element, and single molecules diffusing through the illuminated confocal volume produce bursts of fluorescent light. Each burst is recorded by a single-photon detector and analyzed via autocorrelation.
This autocorrelation provides data on concentration, the diffusion time of the individual molecules, and each molecule’s brightness, which subsequently allows differentiation of slower- and faster-diffusing particles. Ultimately, the binding and catalytic activity is calculated from the diffusion times and the ratio of faster and slower molecules.^1,2

Pharmaceutical Uses

Sensor Technologies’ QuantumXpert FCS Spectrometer includes optical and electronic components.

In drug delivery and discovery, FCS is used to measure the quantity and distribution of a drug in the nanoparticles used to deliver it to its target and, ultimately, to determine how fast the binding is and how low a concentration of the drug is necessary to achieve an effective binding. Study results published in 2010 concluded that FCS, used in combination with a confocal microscope, could in fact determine diffusion constants and concentrations of fluorescent molecules.³
FCS can be used to study molecular and cellular interactions in homogeneous assays. In drug discovery and production, FCS can be used to develop assay targets for protein-protein interaction, protein-nucleic acid interaction, kinase activation by complexing, and host-cell contamination.
The technology can be used to detect both direct binding and competitive inhibition of binding, a newer method of measurement that makes FCS useful in drug research and drug delivery analysis.

Instrumentation

There are several devices currently available on the FCS market. Carl Zeiss (Jena, Germany) offers the ConfoCor 3, which is designed to be paired with one of the company’s laser scanning microscopes. The unit’s software controls the detection module and can analyze single or multiple measurements and allow auto- and cross-correlation to be calculated at the same time as the current measurement. Software upgrades to the unit include options for photon-counting histograms and for defining start values and boundaries.

The ConfoCor 3 by Carl Zeiss is designed to be paired with one of the company’s laser scanning microscopes.

ISS (Champaign, Ill.) manufactures the Alba FCS dual-channel spectrometer, which combines a confocal scanning microscope with FCS. Its light sources are either single-photon or multi-photon lasers, and the device can be interfaced with Leica, Nikon, Olympus, or Zeiss epi-fluorescence microscopes. The Alba can acquire data either in time mode, which counts photons acquired in time intervals, or in photon mode, which measures the time delay between photons and builds a histogram.
Sensor Technologies’ (Shrewsbury, Mass.) QuantumXpert FCS Spectrometer includes both optical and electronic components. The unit’s confocal microscope optics are built in to the system, so it doesn’t require them as external add-ons. The result is a much smaller and more portable system that does not require daily realignment exercises, one that can cost less than other traditional FCS systems. The device also features a data analysis software system designed to simplify the task of analyzing FCS measurement data, offering single and batch correction algorithms, along with curve-fitting methods for both fluorescence correlation data and photon-counting histograms. It is available with either a manual or an automated sample changer.
Corrvus (Spokane, Wash.) is also developing a portable FCS system with a lower price tag than more traditional models. This unit, available in the next 12 months and designed for ease of use in the lab, will not need a dark room to develop the imaging.

The Future of FCS

Research over the past 10 years has shown FCS to be a powerful tool in drug discovery and delivery. It allows for single-molecule observation and can specifically measure how one molecule interacts with another. Parijat Sengupta, PhD, research assistant professor at Washington State University in Spokane and consultant to Corrvus, thinks “FCS might play a very important role in personalized medicine,” a model that emphasizes therapeutic care and treatment plans tailored to specific individuals. With the technology’s specific application to pharmaceuticals still in its relative infancy, its versatility and allowance for extremely sensitive measurements could pave the way for some exciting developments in drug formulation.

References

Seethala R. Homogeneous assays for high-throughput and ultrahigh-throughput screening. In: Seethala R, Fernandes PB, eds. Handbook of Drug Screening. New York: CRC Press; 2001:94-95.
Schwille P, Haustein E. Fluorescence correlation spectroscopy: an introduction to its concepts and applications. Biophysics Textbook Online. 2003. Available at: www.dpi.physik.uni-goettingen.de/Praktika/ Biophysik/Versuche/2006w/Fluoreszenzkorrelationsspektroskopie-Literatur-Schwille_ Haustein.pdf. Accessed Oct. 16, 2011.
Jung CC, Polier S, Schoeffel M, Drechsler M, Jerome V, Freitag R. Fluorescence correlation spectroscopy as a quantitative tool applied to drug delivery model systems. Nature Precedings. 2010. Available at: http://hdl.handle.net/10101/npre.2010.4140.1. Accessed Oct. 16, 2011

QUALITY CONTROL - Method Validation | Means to a Method

2011-12-16T22:47:00.000-08:00

Cliff Nilsen

A step-by-step guide for proper validation

Part 4 of 4: This final installment completes our series on method validation, continuing the discussion of the mechanics of the process.

Read the Complete Series

Part 1, PFQ February-March 2010: Described high-performance liquid chromatography (HPLC) procedures for demonstrating that a method is stability indicating through the use of forced degradation studies and evaluation of peak purity using a photo diode array UV detector.
Part 2, PFQ June-July 2010: Defined each of the other method validation components—selectivity; linearity and range; accuracy and recovery; assay precision; intermediate precision; limit of detection; limit of quantitation; ruggedness, robustness, and comparative studies—with the primary focus on assay methods.
Part 3, PFQ October-November 2010: Began the description of how to perform method validation for each validation component.

A placebo is spiked with different levels of active ingredient standard, bracketing the normal working concentration of the method. The amount of standard recovered at each level is compared with that added.
As with linearity, prepare a series of five standards that span a range of 50% to 150% of the analyte working range except that each standard will contain placebo in an amount proportional to the placebo/active ratio of the drug product for which the method is being validated. For example, if a tablet weighing 500 mg contains 50 mg of active pharmaceutical ingredient (API), 450 mg is placebo.

click for larger view

Table 1. API Linearity Standards

To perform the accuracy portion of the validation, assuming a working concentration of 0.1 mg/mL, make 200 mL of a solution of active in a suitable solvent, having a concentration of 1.0 mg/mL (10 times the working concentration). This is the active stock solution. Next, obtain a small quantity of placebo (drug product minus active). Prepare the five working standards according to Table 1.
Using the method being validated (assume it is an HPLC method), inject each spiked standard three times. For each spiked standard—50%, 75%, 100%, 125%, 150% of the working standard, respectively—each containing a proportional amount of placebo, calculate the area unit’s percent relative standard deviation (%RSD) to determine injection precision at each level. Use the mean values for active concentration at each of the five levels, computing the percent recovery from the placebo at each level.
In most cases, it is desirable to have an injection precision, in terms of area unit RSD, of less than 2% for each standard level, i.e., 50% to 150% of the working concentration. Recovery (accuracy) limits vary with method requirements, but are usually considered acceptable if the recovery of spiked active from the placebo at each level is between 98% and 102%.

Assay Precision

Multiple (six) sample preparations are made from a single homogeneous sample, and the six separate preparations are assayed, using the method under validation, versus a freshly prepared standard. Precision between individual assay results is calculated and expressed as %RSD. An assay precision of not more than 2% is generally considered acceptable for assays. Some applications, such as residual solvent determinations or trace analysis, will have different acceptance criteria.

Limit of Detection

The limit of detection (LOD) of the analytical method is determined by comparing the test results obtained from samples with known concentrations of analyte against those of blank samples and establishing the minimum level of analyte that can be detected. There are a number of ways to express LOD, including a multiple of noise level, a minimum area count %RSD, or a fixed percentage of the lower limit of the linearity curve (50% lowest level for example). LOD is of little importance for assay determinations but is of great importance to applications such as impurity analyses or determination of trace solvent levels.
Experimentally, LOD can be determined by serial dilution of a working standard until the sample peak is indistinguishable from baseline noise.

Limit of Quantitation

The limit of quantitation (LOQ) of the analytical method is the lowest level at which analyte can be reliably measured. Some common definitions of LOQ: three times the LOD and a level at which the %RSD of injection precision is less than 5%. As with LOD, LOQ can be determined by serial dilutions of a standard. LOQ is important in determination of trace components such as impurities and residual solvents.

Ruggedness and Intermediate Precision

The ruggedness of an analytical method is determined by analyzing multiple samples from homogeneous lots. Samples from the same lot are assayed in hextuplicate (six times) using six sample preparations (six assays). Separate sets of six assays from the same homogeneous sample are performed by different chemists on different days, using different columns, different instruments (if possible), and different standard preparations. The %RSD of each set of assay results for each chemist should be no greater than 2.0, and the pooled %RSD for all 12 assays should be no greater than 2.0.
As a point of information, ruggedness (inter-lab precision) refers to performing each of the two assay sets in different labs, whereas intermediate precision (intra-lab precision) refers to performing each of the two assay sets in the same lab.

Robustness

Robustness is determined by observing how a method responds to slight variations in normal operating parameters. In HPLC methods, for instance, this could be a change in flow rate, column length, column temperature, or mobile phase concentration. A simple way of doing this is by performing assay precision under various conditions that vary slightly from the method parameters.
For example, if a method calls for a 30 cm column, a 1.0 mL/minute flow rate, a column temperature of 30 degrees C and a mobile phase consisting of 80 parts water and 20 parts methanol, one could perform an assay precision (six replicate assays) at 0.8 mL/minute, 1.0 mL/minute and 1.2 mL/min, at column temperatures of 28 degrees C, 30 degrees C, and 32 degrees C, at method conditions on a 25 cm column and at mobile phase ratios of 78/22, 80/20 and 82/18 water/methanol. One set of sample preparations is used for all these experiments (six weighings and a ton of injections). The %RSD of the assay results at slightly varied conditions should be no greater than the maximum allowed under normal method operating conditions.

Acceptance Criteria

The validation protocol must include acceptance criteria for each validation parameter. The criteria are the performance requirements of the method—a yardstick against which the method’s validity is measured and which will vary depending on the intended application. The typical acceptance criteria for a drug product assay method are cited in each of the validation parts described above. Refer to part three in this series for information on stability indication, selectivity, and linearity and range.
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Cliff Nilsen’s latest book, “Generic Drugs: A Consumer’s Self Defense Guide,” has been published by iUniverse (http://177060.myauthorsite.com). Here is an excerpt: Imagine the horror as you rush your infant child to the hospital emergency room. “Doctor, what’s wrong?” “We can’t be sure miss, but it looks like some kind of poisoning.” After a short time, the doctor comes back out to tell the hysterical mother, “I’m sorry, but your baby died.” What happened? Upon investigation, it was determined that the mother had given the baby a generic brand of infant ear drops for an earache. It turns out that the ear drops contained glycerin as a main ingredient — glycerin from China that was contaminated with anti-freeze. How could that possibly happen?
Well, this story is fictional, but glycerin from China contaminated with anti-freeze is real. How can we as consumers avoid buying contaminated or substandard drug products? Do you know if the products sitting in your medicine chest or kitchen cabinets are safe to use? How can you be sure? This book uncovers and documents questionable manufacturing practices used by drug companies, mostly generic companies, that could have serious health consequences for American consumers who purchase the drugs produced by those companies.
No one wants to use drug products that are contaminated with dangerous chemicals, foreign matter such as metal and glass, or harmful bacteria, or wants to purchase and use drugs that are mislabeled or have the wrong strength, or that have been made using shoddy manufacturing procedures in dirty equipment by untrained workers. Yet, these things are commonplace, particularly with over-the-counter drugs.

The Validation Report

Once the validation has been executed, a validation report is prepared and submitted for approval. The elements described below should be included in the validation report.

Summary: The summary is a simple statement about the results of the validation study. For example, “The method for the assay of product XYZ by HPLC was found to be accurate, precise, selective, linear, and stability indicating, and thus suitable for its intended use.”
Analytical Validation Data: Analytical data should be presented in tabular and graphical form for ease of evaluation. The data presentation should show analytical results for each validation parameter, plus residuals and all calculations used to derive results from laboratory data. Be sure to include all graphs, curves, and copies of raw data (chromatograms and notebook pages).
Discussion: Describe the outcome of the validation in detail. The discussion/ conclusions should deal with any problems that were encountered and should include a rationale for accepting or rejecting the validation. Any experiments or failing results that were repeated and then accepted need to be explained and justified.

Any deviations from acceptance criteria must be explained, and the conditions under which the method may be used (method limitations) should be clearly defined, such as “only linear from 75% to 125% of the working concentration” or “meets all acceptance criteria and can be used throughout the ranges tested in the validation.” The validation protocol must be approved prior to beginning a validation study, and the validation report must be approved prior to using the method under validation.

INGREDIENTS - Excipients | Embrace Excipients

2011-12-16T22:45:00.001-08:00

James Netterwald, PhD

Critical drug components are necessary to enhance solubility and delivery

Other than the active pharmaceutical ingredient (API), one or more excipients make up the rest of the weight in a final drug product, whether it is a tablet, an injectible, a dermal, or an inhaler. Even water is an excipient in an injectable drug, because it dissolves the API and facilitates drug delivery.
“Excipients are needed to bind the tablet together so that it will not break into powder,” said Dale Carter, chairman of International Pharmaceutical Excipients Council of the Americas (IPEC-Americas). Excipients can also be used to protect the active ingredient in a tablet so it does not get destroyed by stomach acid and can reach the small intestine, where the drug can be absorbed. Excipients are also used to sugar coat an antibiotic to mask its normally bitter taste.

The current thinking on excipients is that not all of them are effective for drug delivery. For a variety of reasons, many are difficult to use in formulations.

Types and Uses

Anthony Hickey, PhD, president and chief executive officer of Cirrus Pharmaceuticals Inc., in Durham, N.C., is a particle scientist, specializing in aerosol and inhalation formulations. The major excipient Dr. Hickey uses in drug formulation is dry powder lactose. He uses lactose, he said, because there are limited options for additives to use in the formulation of asthma inhalants. Dr. Hickey explained that only a handful of excipients appear in approved inhaled products.
“If you use a propellant-based, metered dose inhaler, you will find phosphotidylcholine and sorbitan trioleate, for example,” Dr. Hickey said. “These excipients were approved in the old chlorofluorocarbon (CFC) products, but they don’t work in hydrofluroalkane (HFA) products.”
Oleic acid, also found in old chlorofluorocarbon products, has a slightly different role, but it is included in some newer products. HFA inhalants are environmentally safer than the older inhalers, which released dangerous CFCs into the environment. Other additives and excipients in nebulizer formulations include solvents, buffers, and salts, but although all these products are approved for use, the range of excipients found in other dosage forms such as tablets does not appear in inhaled products.
Lactose is the excipient of choice for Dr. Hickey’s work because of its inherent ability to assist in the aerosolization of a drug. Micronized drug particles used in aerosol products exist as small particles that tend to stick together due to their physicochemical properties. Lactose acts as a carrier, separating the drug particles in the powder and assisting in the formation of an aerosol as the powder is drawn into the inspiratory airflow.
“In essence, the small drug particles are stuck on the surface of lactose,” Hickey said. “When you inhale, those small particles are more easily stripped from the surface of the lactose.” Lactose is then separated from the API. Lactose does not enter the lungs. Instead, it is deposited in the back of the throat and swallowed.
Lactose, present in almost all inhaled, dried, powdered products, especially those indicated for asthma, is also a cause of food intolerance. So, what happens for asthmatics who are also lactose intolerant? The solution is inhaled products without lactose, including those used in metered-dose inhalers and nebulizer solutions.
“There is a litany of excipients that have been added to oral products and injectables,” Dr. Hickey explained. “The dominant dosage form is in tablet form, where you have the most excipients, some of which are added to help or allow the very small dosage of drug to be measured to make the dosage form. Like lactose, some of those excipients are voided in various ways once the drug is released, not affecting the drug biology at all.”
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CASE STUDY: Apricus Bio Validates an Excipient

Apricus is not only an excipient manufacturer. The firm also develops drugs that use its excipient in new drug formulations. “For our erectile dysfunction drug Vitaros, we performed a dose response of our excipient DDAIP,” said Bassam Damaj, PhD, president and CEO of the San Diego, Calif., firm. “Our goal there was to find a minimum percentage of excipient necessary to adequately deliver the API to the penile arteries to induce the clinical effectiveness in patients without inducing irritation.” He said the challenge took several years to complete, but finally, the company found that 2.5% of DDAIP with API in its formulation produced the best systemic levels of the API to induce significant clinical effectiveness. These results were partially attributed to the approval of this drug.—JN

Basic Regulatory Rules

Excipients are generally inert components in a drug formulation and, therefore, do not usually pose a safety risk. However, “excipients that are still being consumed need to meet all of the same regulations as an active pharmaceutical ingredient,” said William Kopesky, vice president of analytical services at Particle Technology Labs. “(The) FDA [U.S. Food and Drug Administration] does not set specifications for excipients, but they expect a client, a submitter, or a manufacturer to have specifications and a controlled process or control over their materials,” he said.
Clearly, there is not a huge safety risk with the presence of excipients in formulations. And although drug formulators do not always wish to add excipients, in some instances they are necessary in order to make an effective formulation.
Excipients serve a purpose in the dosage form and do not pose a safety risk for individuals taking the pharmaceutical. Many are sugars, lipids, or polymers that are of biological origin or are biologically compatible. “The reasons excipients are added often have more to do with making the dosage form and making drug delivery easier,” said Dr. Hickey. “I think the tendency these days is that if you don’t have to use excipients then you don’t. And then, of course, to make it clear, the final dosage form is the product that has to be evaluated as part of the approval process, which includes excipients. Excipients are part of the drug safety evaluation.”

Role in Drug Delivery

The current thinking on excipients is that not all of them are effective for drug delivery. In the past, many companies have tried to develop excipients, as well as permeation enhancers such as azone, polyethylene glycol, and dimethyl sulfoxide (DMSO). The problem these companies have faced is that, for a variety of reasons, many excipients are difficult to use in formulations.
Bassam Damaj, PhD, president and CEO of Apricus Biosciences in San Diego, Calif., described the characteristics of his company’s main excipient and permeation enhancer—dodecyl-2-(N,N-dimethylamino) propionate (DDAIP)—to illustrate the features necessary for any excipient.
Dr. Damaj said DDAIP’s mode of action is to loosen the tight junctions between cells to allow a drug to enter the cell. Like DDAIP, excipients should not be toxic and should not induce irritation. Also, the excipient should have a very short half-life once it enters the circulation. An excipient should also be water soluble. Many excipients and enhancers are not water soluble, limiting their use in the pharmaceutical formulation.
To handle the issue of excipient solubility, Apricus has developed two forms of its excipients: a base form that works well with hydrophobic formulations and an acid form that works best with hyphophilic formulations. The excipient you choose depends on the drug and the active pharmaceutical formulation to be delivered.

Analysis in Formulation

There are various technologies available for excipient analysis. For particle-size determination, methods such as laser diffraction and sieving light obscuration can be used. For particle surface area determination, nitrogen gas absorption or krypton gas absorption are used.
Particle Technology Labs in Downers Grove, Ill., is a service laboratory that performs analysis of particles—which include excipients—for its mainly pharmaceutical clients, who submit samples for physical characterization such as analysis of particle size and surface area. Those measured properties can affect the materials’ behavior in formulations or in processing conditions. “In other words, the physicochemical properties of excipients in a drug formulation can affect the drug’s behavior (in terms of) its dissolution, solubility, and processability,” Kopesky said. He encounters many excipients in his work, including microcrystalline cellulose, magnesium stearate, crosprovidone, and lactose.
Excipients are added and analyzed prior to addition to the formulation. “If the excipient is within a certain particle size range, it may behave differently than a larger particle-sized excipient,” Kopesky said. He added that it is important to know those physical properties in order to either predict or elicit a certain processing characteristic in the final formulation.
Excipients must also be tested for their relative chemical inertness to ensure that they do not chemically interact with the API. Pharmaceutical formulation scientists desire excipients that exhibit chemical inertness. However, their inertness must be tested prior to their use as an additive in drug formulation. “So I think that anybody that could formulate with limited excipients would do so,” Dr. Hickey said. “That’s not to say that they don’t see the value in excipients, because so many drugs need to be formulated with excipients.”

Hot or Not?

As with any component in the formulation process, some excipients are in favor, while others have fallen by the wayside. Among those still in favor are polyethylene glycol and lactose as well as DDAIP-HCl. The latter is preferred because of its enhanced activity in formulations, its purity, and the ease with which it is manufactured.
Excipients that have fallen out of favor include DMSO, oleic acid, and paraffin. These have lost ground primarily because of significant skin irritation, reduced stability in formulation, or difficulty of manufacturing. Excipients that have lost their luster have been associated with a wide range of adverse events, incl

FORMULATION - Parenteral Advances | Get Local with Targeted Delivery

2011-12-16T22:44:00.000-08:00

Maybelle Cowan-Lincoln

A new range of polymer implants and programmable microchips is paving the way to more personalized therapies

There are three major parenteral drug delivery routes: intravenous, intramuscular, and subcutaneous, as well as several more rarely used routes such as intra-arterial.¹ Recently, however, interest has piqued in new parenteral technologies that facilitate targeted local drug delivery.
Parenteral delivery can be the route of choice under several circumstances:

When the plasma levels of a drug must be carefully controlled;
When it is necessary to avoid the “first pass” metabolism through the liver or minimize the risk of harmful side effects resulting from systemic delivery;
When the patient is not conscious or not capable of taking the drug orally; and
When administering drugs with a short half-life.²

The parenteral route provides the best solution for overcoming the challenges of delivering proteins and peptides. These agents are easily degraded by enzymes found in the gastrointestinal tract, and the large size of the molecules makes transdermal delivery difficult. In addition, proteins and peptides have very short half-lives in vivo, so they must be injected multiple times over the course of a day.
Novel controlled release parenteral technologies can reduce injection frequency and the accompanying pain, thereby potentially improving patient compliance.³ But this solution comes with its own set of problems. In addition to the pain of multiple injections, drugs taken by injection follow the pattern of first-order kinetics—high levels in the blood after administration followed by a sharp fall in concentration. At peak levels, toxicity can be an issue, and efficacy can decrease as levels fall. An ideal implantable system would include an electronic feedback device to control drug release.

Zero-Order Release

Controlled drug release from implantable parenteral devices may also be able to achieve the elusive goal of sustained zero-order release. This means that the rate of drug release remains constant, minimizing the risk of toxicity and the inconvenience of frequent dosing. This is particularly important when the drug concentration must fall into a narrow window between the minimum effective concentration and the maximum safe concentration.⁴
Strategies to come close to zero-order release have included multiple injections and implantable pumps. But these methods fall short of ideal. Frequent injections are inconvenient and painful, and implantable pumps require surgery to implant, refill, or remove. In addition, these pumps can only be used with drugs that are stable at physiological temperature. Research continues to look for a more efficient technology to achieve zero-order release.
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CASE STUDY: The Electronic Implant Revolution

EMS and NEMS devices are the basis for technologies that will effect a sea change in the treatment of many diseases. Implantable devices complete with micro- or nanochip technology that allows them to respond to physiological changes can virtually automate the management of certain chronic diseases and provide lightning fast interventions for emergency situations. Two systems on the leading edge of this research are the “artificial pancreas” and the “personal paramedic.”¹

www.artificialpancreasproject.com

According to the American Diabetes Association, approximately 25.8 million Americans suffer from diabetes, with 7 million of these undiagnosed. And, each year, 1.9 million new cases are discovered in people aged 20 and older.² The most important thing diabetes patients can do to maintain their health is to strictly control their blood glucose levels. But for a variety of reasons—pain and inconvenience among them—many do not. This often results in debilitating pathologies later in life, including blindness, kidney failure, and amputations.³
To promote better disease management, the Juvenile Diabetes Research Foundation (JDRF) launched the Artificial Pancreas Project six years ago. The JDRF has formed a consortium of government and academic researchers, along with private corporations from the United States and Europe, to work collaboratively toward the development of the first fully functional unit.⁴
The artificial pancreas would be a miniature, closed-loop device composed of a glucose monitor, a miniature pump powered by a MEMS chip to deliver insulin, and a power source. The artificial pancreas would continuously monitor blood sugar levels and automatically release the precise amount of insulin needed, practically automating diabetes management.
Another breakthrough MEMS-powered device is the “personal paramedic” or Implantable Rapid Drug Delivery Device (IRD3). An implantable delivery system designed for ambulatory emergency care, the IRD3 allows for rapid delivery of cardiac resuscitation drugs such as vasopressin.
The IRD3 is made up of three layers: the reservoir where the drug is stored, the membrane that seals the reservoir to prevent the drug from leaking out or foreign substances from penetrating, and the actuation layer. In the actuation layer, micro-resistors heat fluid to form bubbles once certain cardiac symptoms are detected by the MEMS microchip. The increased pressure caused by the bubbles ruptures the membrane, allowing the medicine to be released from the reservoir at a rate of approximately 20 µl in 45 seconds.⁵
The treatment of diabetes and certain types of heart disease can be transformed by these drug delivery systems. Their success can also stimulate a creative and vibrant commercial environment, fueling the discovery of more ways MEMS devices can improve the management of many conditions.

References

Staples M. Microchips and controlled-release drug reservoirs. Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2010;2(4):400–417.
American Diabetes Association. Diabetes statistics. Available at: www.diabetes.org/diabetes-basics/diabetes-statistics. Accessed Oct. 3, 2011.
Schetky LM, Jardine P, Moussy F. A closed-loop implantable artificial pancreas using thin film nitinol MEMS pumps. Paper presented at: International Conference on Shape Memory and Superelastic Technologies; 2003; Pacific Grove, Calif.
MacRae M. The artificial pancreas project. American Society of Mechanical Engineers. August 2011. Available at: www.asme.org/kb/ news—articles/articles/bioengineering/the-artificial-pancreas-project. Accessed Oct. 3, 2011.
Elman NM, Ho Duc HL, Cima MJ. An implantable MEMS drug delivery device for rapid delivery in ambulatory emergency care. Biomed Microdevices. 2009;11(3):625-631.

Polymer Implants

click for larger view

Polymer scaffolds made from chitosan can serve as temporary biodegradable drug depots

A popular controlled delivery device is the polymeric implant. These systems fall into one of two categories: nondegradable and biodegradable. An example of the former technology is the Norplant five-year contraceptive device. Hollow polymer tubes are filled with a drug suspension that dissolves into the polymer, then diffuses through the tubing walls.
Biodegradable polymer implants are usually made of microspheres containing a drug. Once injected, the polymer dissolves, releasing the drug into the system. A new technology under development uses biodegradable polymer rods implanted in the marrow of infected bones to deliver fluconazole to treat fungal osteomyelitis.⁵
Some exciting developments are being pursued in creating polymer scaffolds to serve as temporary biodegradable drug depots. These structures can be made from natural polymers such as collagen or chitosan, or from synthetic polymers that do not promote inflammation and are bio-degradable, biocompatible, and nontoxic.⁶
One of the most successful drug scaffold models is the injectable polymer depot. A liquid liposomal solution or suspension is injected subcutaneously or intratumorally, where it forms a semi-solid scaffold that releases the drug right at the target site. This method offers several benefits, including local drug retention and sustained release.

In addition to delivering pharmaceuticals, polymeric scaffolds have been designed to continously release growth factor cells to promote tissue regeneration.

Prefabricated polymeric scaffolds have also gained attention as delivery systems for small-molecule drugs and various bioactive molecules. These systems are manufactured outside the body and must be implanted surgically. Biodegradable and nondegradable materials are being tested as possible scaffold materials; the drawback of nondegradable materials is, of course, the necessity of surgical removal at the end of therapy.
In addition to delivering pharmaceuticals, polymeric scaffolds have been designed to continuously release growth factor cells to promote tissue regeneration. For example, vascular endothelial growth factor—a signal protein manufactured in cells that promotes the growth of new blood cells, a process known as angiogenesis—has been incorporated into a scaffold. In a recent study, increased blood vessel density was noted at the implant site. These results demonstrate an increase in angiogenic potential. Tissue regeneration typically uses prefabricated scaffolds requiring surgical implantation and removal, but research on injectible hydrogel scaffolds is ongoing.⁷

Getting a Charge

Exciting advances are being made in the development of implantable drug delivery devices using micro- and nanoelectromechanical systems. Called MEMS and NEMS, respectively, this technology employs microchips that contain micro- and nano-scale programmable electronic circuits.⁸
MEMS and NEMS devices offer complex functionalities that could potentially overcome many of the shortcomings and inconveniences of conventional drug therapy, including complicated dosing regimens and fragile or easily degradable active ingredients. Drugs can be released from a reservoir by electrical signals programmed into the micro- or nanochip. With MEMS technology, timing and dose amount can be precisely controlled, and drugs can be delivered to precise locations. Myriad dosing options can then be available, including delivery on demand, programmable dosing cycles, and automated dosing of multiple drugs.
Another delivery system that utilizes MEMS chips is the micropump. Unlike mechanical micropumps, actuated by one of various mechanisms, including electrostatic, electromagnetic, and piezoelectric energy (utilizing the electric charge that naturally collects in crystals, bone tissue, DNA, and other material in response to mechanical stress), some micropumps utilize MEMS microchips. These pumps must minimize chip and device size and must be made of biocompatible materials. They must be able to operate for weeks to years without presenting much risk to patients, must deliver a relatively steady flow rate, and must either use minimal power or be remotely rechargeable.

Medtronic’s ACT Insulin pump.

In even more exciting research, MEMS and NEMS technology is contributing to the emergence of personalized medicine, the science of optimizing treatment based on individual genetics and physiology. Delivery devices containing standard drugs could include micro- or nanochips and possibly a biosensor to provide physiological feedback. This complex device could maximize therapeutic flexibility and efficiency by tailoring drug delivery to the patient’s needs as revealed by the biosensor.
This type of system could be used to create an “artificial pancreas” for diabetes patients. It would comprise an insulin reservoir and a biosensor to monitor blood glucose levels. The sensor would communicate with a MEMS/NEMS-based drug delivery device to regulate insulin release and also transmit data to an external device for use by the patient or physician. Data for dosing flexibility and control could also potentially be received by the device. This technology has not yet been perfected, but once successfully implemented, it could revolutionize the treatment of a large patient population.
In addition to the benefits for long-term drugs, MEMS- and NEMS-based systems can also provide emergency care. Currently, a “personal paramedic” is being developed to work in tandem with current cardiac devices such as a pacemaker. This device, called the IRD3 (Implantable Rapid Drug Delivery Device), could release drugs used in cardiac resuscitation, such as vasopressin, when needed. The IRD3 could also be used to treat angina patients by releasing vasodilators on demand.
Microreservoirs that employ MEMS devices are a combination of drug reservoir systems and polymer matrices. Microreservoirs are implanted drug delivery systems used for proteins, hormones, pain medications, and other drugs. Each tiny reservoir, covered with a gold membrane, contains a single dose. The dose is released when one microreservoir is exposed to anodic voltage from the MEMS chip, causing the membrane to rupture.

Smooth as Silk

An artificial pancreas would be a miniature, closed-loop device composed of a glucose monitor and a miniature pump to deliver insulin powered by a MEMS chip.

Silk fibroin is another material being studied as a polymer vehicle for sustained local drug delivery. A recent study conducted at Tufts University evaluated the efficacy of silk fibroin to encapsulate the anticonvulsant adenosine in a biocompatible and biodegradable drug reservoir.
Adenosine is a promising treatment for drug-resistant epilepsy. When given systemically, however, it causes severe side effects, including suppressed cardiac function. Local delivery using a reservoir placed in the brain may provide the answer. However, polymers commonly used to coat reservoir devices have significant drawbacks. Some are nondegrading or require organic solvents that can damage encapsulated drugs. Others release drugs too quickly.
Silk-based implantable systems, on the other hand, offer numerous advantages. Silk fibroin is biodegradable, biocompatible, and strong; it is used as suture material for the brain and nervous tissue. But the study demonstrated that silk fibroin can successfully achieve the parenteral drug superobjective. Adenosine reservoirs coated with eight layers of 8% silk fibroin material exhibited zero-order release. Reservoirs with four layers of 8% silk fibroin exhibited near zero-order release.

References

Gad SC, Cavagnaro JA, Nassar AF, et al. Formulations, routes, and dosage design. In: Gad SC, ed. Pharmaceutical Sciences Encyclopedia: Drug Discovery, Development, and Manufacturing. New York: John Wiley and Sons; 2010:10-15.
Paolino D, Sinha P, Fresta M, Ferrari M. Drug delivery systems. In: Webster JG, ed. Encyclopedia of Medical Devices and Instrumentation. 2nd Ed. New York: John Wiley and Sons; 2006:437-486.
Gad SC, Tamilvanan S. Progress in the design of biodegradable polymer-based microspheres for parenteral controlled delivery of therapeutic peptide/protein. In: Gad SC, ed. Pharmaceutical Manufacturing Handbook: Production and Processes. New York: John Wiley and Sons; 2008:393-427.
Pritchard EM, Szybala C, Boison D, Kaplan DL. Silk fibroin encapsulated powder reservoirs for sustained release of adenosine. J Control Release. 2010;144(2):159-167.
Soriano I, Martín AY, Évora C, Sánchez E. Biodegradable implantable fluconazole delivery rods designed for the treatment of fungal osteomyelitis: Influence of gamma sterilization. J Biomed Mater Res A. 2006;77(3):632–638.
Mufamadi MS, Pillay V, Choonara YE, et al. A review on composite liposomal technologies for specialized drug delivery. J Drug Deliv. 2011;2011:1-19.
Chung HJ, Park TG. Surface engineered and drug releasing pre-fabricated scaffolds for tissue engineering. Adv Drug Deliv Rev. 2007;59(4-5):249-262.
Staples M. Microchips and controlled-release drug reservoirs. Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2010;2(4):400–417.

DELIVERY - siRNA/RNAi | RNAi No Longer Blue Sky

2011-12-16T22:42:00.000-08:00

Neil Canavan

Despite significant delivery hurdles, researchers make progress toward genetic therapies

The use of double-stranded RNA to deliberately “interfere” with gene expression was first described in the journal Nature in 1998.¹ Though the work was performed in Caenorhabditis elegans, the potential was immediately clear: RNAi made for an excellent research tool and likely represented a new class of therapeutic agents.
In either application, a recent advance may revolutionize the optimal selection of RNA inhibitors.
“The potential for RNAi demands that you have to have the right trigger,” said Christof Fellmann, a graduate student at Cold Spring Harbor Laboratory, N.Y. Algorithms to successfully predict an optimal RNAi sequence, in this case for short hairpin RNAs (shRNAs), remain elusive, and current methods involve extensive time-consuming screens. To overcome this barrier, a “sensor assay” was developed that rapidly identifies optimized shRNAs at large scale.
For this method, a library of 20,000 expression vectors was created, each consisting of (a) a unique shRNA sequence, (b) a known target sequence (the sensor), and (c) a fluorescent reporter gene. Once expressed in transfected cells, the components were free to interact (or not) with resulting levels of fluorescence inversely relating to the degree of target-gene suppression. This innovative screen was able to identify shRNAs not only of exquisite potency, but also of unprecedented specificity, thereby greatly reducing off-target effects.²
The sensor assay is already bearing fruit at the bench, as demonstrated by Cold Spring Lab’s recent identification of a potential therapeutic target for acute myeloid leukemia, and the technology will soon be commercialized by Mirimus Inc., a Cold Spring Harbor spin-off.³

Enhanced In Vivo Survival

Working to optimize their proprietary siRNA platform for in vivo use, investigators at Ambion, a Life Technologies company in Austin, Texas, have been working on chemical modifications to synthetic oligos. “About three years back, we came out with a technology for high-throughput screening for siRNAs called Silencer Select,” said Nitin Puri, PhD, senior manager of R&D, Ambion. “With chemical and/or sugar modifications to the oligo we have now managed to both enhance specificity, while decreasing the immunogenicity of the siRNA.”
Along with other proprietary chemistries, the use of RNA analogues (so-called locked nucleic acids) in the Ambion process lends rigidity to the RNA structure that confers enhanced hybridization-to-target behavior, while at the same time blocking destructive ribonuclease activity. This last point is critical, because this dynamic has a marked effect on serum stability—a major issue for in vivo applications. “We have increased serum stability more than 150-fold in rat and mice serum,” said Dr. Puri. Unprotected, siRNA half-life is roughly five minutes; with the Ambion modifications, the half-life is about 24 hours.
Life Technologies has made other recent strides toward enhancing RNAi selection, quantification, and delivery: a method described by Cheng and colleagues, based on stem-loop real-time polymerase chain reaction technology, allows for easier quantification of siRNAs in vivo.⁴ A new vehicle reagent for RNAi duplex delivery, Invivofectamine 2.0, which enables high transfection rates in the liver, is also now available.
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CASE STUDY: RNAi May Replace Standard Stent Coatings

Angioplasty revolutionized the treatment of coronary artery disease; however, there are still improvements to be made. First-generation bare metal stents, scaffolds set in situ to maintain the balloon-opened artery, were vulnerable to rapid restenosis via the buildup of scar tissue; secondgeneration appliances, the so-called drug-eluting stents, prevent scar tissue proliferation but at the same time delay endothelial healing, which can lead to eventual thrombosis.¹

Extended recovery times require the prolonged use of expensive antithrombotic agents and are clearly suboptimal. “We had the idea to use siRNA because we think that we can have a faster re-endothelialization while stopping the inflammation,” explained Andrea Nolte, PhD, research scientist at the Children’s University Hospital in Tuebingen, Germany.
In her study, Dr. Nolte and colleagues conducted an in vitro assay using endothelial cells (ECs) exposed to a complex of polyethylenimine (PEI) and siRNA targeted to E-selectin, a molecule that plays an important role in the inflammatory response. The PEI/siRNA complex was mixed with a gelatin solution and then fixed to the bottom wells on culture plates. ECs and culture media were added, and after cells reached confluence, an inflammatory response was induced with the addition of TNFα. Results showed a 70% knockdown of expression of expected inflammatory factors.^2,3
Along with validation of the approach, there was a happy surprise, and a challenge to be considered. “Normally, when you put a PEI/RNAi complex on ECs, not a whole lot happens,” said Dr. Nolte, but the construct fixed in gelatin sustained therapeutic efficacy even in the presence of serum. “We were very happy to see that.”
On the downside, the release of siRNA from the gelatin coating was too rapid; a different matrix will have to be found for the approach to be viable for a coated stent. “We’re looking for a release profile of about four weeks,” said Dr. Nolte, and added that they are exploring the use of poly(lactic acid-co-glycol acid) films. A sustained release over time should prevent initial stent-induced scarring while posing little hindrance to vascular healing.
Dr. Nolte’s use of siRNA-coated surfaces has caught the attention of numerous players in the industry. “They’re interested because there are other applications that would be useful with a coating,” she said. Beads coated with siRNA could be inserted into tumors, and topical formulations could be applied to chronic inflammatory conditions of the skin. “There are numerous settings that require the immobilization of the siRNA.”
Dr. Nolte’s work has, in fact, been done in partnership with industry. The stent application is being developed with Qualimed, of Hamburg, Germany.

References

Zhao FH, Chen YD, Jin ZN, Lu SZ. Are impaired endothelial progenitor cells involved in the processes of late in-stent thrombosis and re-endothelialization of drug-eluting stents? Med Hypotheses. 2008;70(3):512-514.
Nolte A, Walker T, Schneider M, Deniz O, Avci-Adali M, Ziemer G, et al. siRNA eluting surfaces as a novel concept for intravascular local gene silencing [published online ahead of print July 22, 2011].
Walker T, Saup E, Nolte A, Simon P, Kornberger A, Steger V, et al. Transfection of short-interfering RNA silences adhesion molecule expression on cardiac microvascular cells [published online ahead of print June 20, 2011]. Thorac Cardiovasc Surg.

RNAi Delivery

Delivering enough therapeutically active molecules to the intended target remains the most vexing suite of obstacles for RNAi researchers. Challenges include achieving persistence in systemic circulation and effective reuptake by target cells.

Delivering enough therapeutically active molecules to the intended target remains the most vexing suite of obstacles for RNAi researchers. In brief, the challenges are achieving persistence in systemic circulation (no rapid clearance), localization of active moiety to target-tissue cell surface, efficient uptake by target cells, and rapid release of the internalized RNAi cargo from the transporting endosome into the cytosol.
It would take an entire textbook to delineate these issues. However, one author of a recent review of the field, Xudong Yuan, PhD, assistant professor in the division of pharmaceutical sciences at Long Island University in Brooklyn, N.Y., touches on some of the vehicular approaches that have recently caught his eye.⁵
“The good thing about (PLGA [D, L-lactide-co-glycolide]) is that it’s biodegradable, biocompatible, and, most importantly, it’s already approved by FDA,” Dr. Yuan said. Release of cargo from PLGA alone is too rapid, however. Dr. Yuan and colleagues are experimenting with PLGA combined with polyethyleneimines (PEIs). “This gives you better RNAi loading and also facilitates endosomal release through the proton sponge effect,” whereby cationic PLGA/PEI nanoparticles induce osmotic swelling, rupturing the endosome.⁶ With the same goal and mechanism in mind, Dr. Yuan is also working with chitosin, a biodegradable, linear polysaccharide, an approach that should have the added advantage of minimized toxicity.⁷
Currently not on his bench top but certainly on his radar are a few more applications for which Dr. Yuan also sees great promise:

RNAi-loaded nanoparticles composed of cyclodextrin, specifically, CALAA-01, the first targeted siRNA nanoparticle administered to humans, which is currently in clinical trials;⁸ and
“Lipidoids,” a combinatorial approach to constructing RNAi-bearing particles being developed by the Massachusetts Institute of Technology in collaboration with the RNAi therapeutics company Alnylam.⁹

References

Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998;391(6669):806-811.
Fellmann C, Zuber J, McJunkin K, Chang K, Malone CD, Dickins RA, et al. Functional identification of optimized RNAi triggers using a massively parallel sensor assay. Mol Cell. 2011;41(6):733-746.
Zuber J, Shi J, Wang E, Rappaport AR, Herrmann H, Sison EA, et al. RNAi screen identifies Brd4 as a therapeutic target in acute myeloid leukaemia [published online ahead of print August 3, 2011]. Nature.
Cheng A, Vlassov AV, Magdaleno S. Quantification of siRNAs in vitro and in vivo. Methods Mol Biol. 2011;764:183-197.
Yuan X, Naguib S, Wu Z. Recent advances of siRNA delivery by nanoparticles. Expert Opin Drug Deliv. 2011;8(4):521-536.
Nel AE, Mädler L, Velegol D, Xia T, Hoek EM, Somasundaran P, et al. Understanding biophysicochemical interactions at the nano-bio interface. Nat Mater. 2009;8(7):543-557.
Yuan X, Shah BA, Kotadia NK, Li J, Gu H, Wu Z. The development and mechanism studies of cationic chitosan-modified biodegradable PLGA nanoparticles for efficient siRNA drug delivery. Pharm Res. 2010;27(7):1285-1295.
Eifler AC, Thaxton CS. Nanoparticle therapeutics: FDA approval, clinical trials, regulatory pathways, and case study. Methods Mol Biol. 2011;726:325-338.
Siegwart DJ, Whitehead KA, Nuhn L, Sahay G, Cheng H, Jiang S, et al. Combinatorial synthesis of chemically diverse core-shell nanoparticles for intracellular delivery. Proc Natl Acad Sci U S A. 2011;108(32): 12996-13001.

CONTAMINATION - Risk Management | Manage Contamination Risk with a Lean Approach

2011-12-16T22:40:00.000-08:00

Judy Madden

How existing products can benefit from examining the true cost of quality

In an ideal manufacturing world, we would always purchase pure ingredients, consistently formulate products within the parameters of aseptic technique, and reliably ship sterile goods to market. Quality by Design (QbD) and process analytical technology (PAT) initiative adherents alike support an approach that makes certain that product quality is part of the production process from the start.
In the real world, even with the best intentions and plans, our products and processes are at risk of contamination. As a result, and with the exception of parametric release, products and processes are tested to ensure their quality prior to releasing product to market. This is a necessary step, but—for companies using traditional microbial testing methods—it is extremely time consuming and costly.
A lean quality approach using rapid testing methods will reduce the cost and impact of contamination events and accelerate your production cycle. Best of all, it can be applied readily to an existing manufacturing process.

The True and Total Cost of Quality

To find the right balance between risk and safety, it can be helpful to compare the costs of not having a quality control process with the costs of a good quality system (see Figure 1).

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Figure 1. The True Cost of Quality

Without quality control processes, products are manufactured and shipped into distribution quickly. While this is not an option for pharmaceutical products, the risk is, of course, that pallets of finished products will be contaminated. In the plant, that means expensive rework, scrap, and overtime. For goods that have left the company bound for pharmacies or retail stores or, worse yet, have already been sold to patients or consumers, the liability and brand impact are significant and potentially catastrophic.
To minimize this risk, companies will often test their products at several stages: when raw materials arrive; potentially at the bulk or prepackaging stage; and always as packaged products ready to leave the facility (see Figure 2).
At each step in situations where traditional test methods are used, materials and products sit idle for multiple days awaiting results. In addition to the cost of the testing itself, warehouse space and invested capital are tied up. Despite these disadvantages, traditional testing is widely accepted as a cost of having good quality. Yet there is an alternative.

Lean Quality Advantage

In lean manufacturing terms, even minutes during which value is not added to the product are considered waste. Imagine the field day your lean team would have if they learned you could free up several days of waiting time by using a rapid method.

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Figure 2. Traditional Quality Testing

A lean quality approach does just that by allowing you to remove many days of “waste” or waiting time from the manufacturing process and still release safe products to market (see Figure 3).
In terms of risk management, the lean quality approach has a significant advantage. A faster production cycle means faster problem detection. Corrective action can be initiated sooner and, therefore, more effectively.
After all, it is far easier to isolate and identify events that may have led to a contamination event yesterday than to try and troubleshoot those same events four, five, or more days later. I can easily remember what I had for breakfast yesterday, but recalling the precise details of a meal I ate last week is a lot trickier.

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Figure 3. Lean Quality Testing

The same is true of contamination events. Additionally, by the time a problem is detected with traditional methods, the company has five or more days of additional, potentially contaminated inventory to deal with. Beyond the cost of the goods themselves, this can have a significant impact on your ability to meet customer demands.

Rapid Methods Manage Risk

The benefits of a rapid detection method are made clear in this simplified contamination event timeline (see Figure 4).
Assume it takes this company one to two days to formulate and package a product and then, using traditional microbiological methods, an additional three to seven days to test finished product for microbiological quality. In this example, contamination is identified after five days of micro-hold and the seventh day of overall production. An investigation and corrective action are initiated.

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Figure 4. Contamination Recovery Timeline

Several days later, replacement product needs to be produced to replace the original contaminated batch. That product is also subject to microbiological testing. The 17-day point in our production timeline arrives before the product is available for distribution—a full 10 days beyond the planned seven-day production schedule.
On the second timeline, we see that using a rapid detection assay reduces the microbiological testing time to 18 to 24 hours. Detection of the problem and initiation of corrective action now happen within 24 hours of production.
The benefits of rapid detection also extend to the release of replacement product. In the example above, replacement product is released at the nine-day point. This is a full eight days faster than in the scenario in which the manufacturer uses traditional methods and is still in crisis mode at day nine.

Costs Are One Thing, Savings Are Another

Many discussions of rapid methods begin and end with the cost per test. “It’s too high,” say the lab managers who appreciate the lab efficiencies generated by rapid methods but feel constrained by their budgets and pressure to keep expenses low.
The biggest obstacle to the adoption of rapid methods is the fact that 100% of the cost of the method is charged to the labs, while 90% of the financial benefits are in manufacturing.
For successful adoption, Operations and Finance need to get involved to help everyone see that the overall benefit to the company is well worth a modest increase in the lab’s testing budget.
Rapid detection may be the best-kept secret outside the microbiological laboratory. Yet some of the largest and most successful companies in the pharmaceutical and consumer product industries benefit from the efficiencies offered by rapid testing systems. The company discussed below implemented their first Celsis rapid detection system in 1996 and now uses the technology at its facilities worldwide.
The company, a manufacturer of pharmaceutical products, was experiencing issues with inventory and periodic in-house contamination events. It was following traditional microbial testing methods for screening raw materials and finished goods with a five-day hold at each step. The value of the company’s daily finished goods production at the time was roughly $75,000.
Celsis worked with the company to complete a financial impact assessment to determine the value of implementing a rapid system.
Readily available data, including the value of daily finished goods, the reduction in micro-hold days, the frequency of contamination events, and instrument and reagent costs, were incorporated into the impact assessment. The model calculated a five-year net present value (NPV) of over $677,000, payback of less than nine months, and savings from faster contamination containment annualized at $64,000 per year.
Projecting these savings over the company’s five facilities with an 18-month rollout program increased the five-year NPV to almost $2.5 million, with a payback of just 15 months. The later rollouts were financed through the working capital efficiencies generated from the earlier placements. Annualized contamination savings alone rose to almost half a million dollars.
Further, with results available in 24 hours, the company was able to introduce some in-process testing at a critical point in production to detect contamination even earlier and reduce the potential impact of a contamination event even further. Total micro-hold was reduced from 10 days to only three days and was redistributed to better manage risk as it occurs in the operation.

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Figure 5. Impact Report

The impact report (see Figure 5) shows a typical output graph with the projected savings for adopting rapid methods at a single plant. The report identifies the economic impact by six-month periods, along with the cumulative discounted cash flow.
The average company’s investment is shown in red. The initial outflow represents the initial system investment followed by an implementation period and the ongoing cost of reagents.
The blue bars represent the positive impact of the reduction in working capital requirements driven by a reduction in inventory held in quarantine and safety stock. This includes the initial release of inventory upon validation and the ongoing value of redeploying that capital into productive investments.
The green bars are the estimated savings from the reduced impact of contamination events. In many cases, these savings alone pay for the program: The green bars are larger than the red bars in each period.
Celsis also offers an environmental impact report documenting sustainability improvements resulting from implementation—from reducing water and energy consumption to minimizing the amounts of liquid, solid, and hazardous waste requiring disposal..

Superfluous or Essential: Part 2

2011-12-16T02:30:00.000-08:00

By Barbara Kanegsberg, Ed Kanegsberg

In the first installment, we discussed how to avoid superfluous materials and activities in cleanrooms. To accompany our inspiration of a man dressed for success in cholera prevention, we present his fashion-plate companion (Figure1).¹* We now know much more not only about how to protect people from contaminants; we are also cognizant of steps needed to protect critical product from contamination by employees. People are a major source of contamination in the cleanroom. Those of us who are involved in manufacturing high value product can recount horrific and/or amusing stories about cleanroom “discipline” or the lack thereof. That is a topic for another column (or perhaps even a book).
“My background is in manufacturing, so I am very conscious that we utilize people to their best advantage. Making them spend their day doing unnecessary tasks may not be the best use of their time,” says Kelly Barton, Senior Sales Engineer at Clean Rooms West, Inc. in Tustin, California.

CLEANROOM DISCIPLINE?
Gowning procedures are exacting, complex, and are often required for product protection.
As Barton explains, even where air showers are considered prudent, workflow and personnel practices should be considered. “I have observed operators gowning with full coverage bunny suits that were processed in a Class 10 cleanroom laundry. They then step into an air shower to blow them off. Blow off what? After the air shower, they go into a Class 10,000 or higher class environment. This is a waste of money and of valuable production time.”
As an example, says Barton, “Consider a facility assembling product in a Class 100,000 cleanroom to minimize visible contamination. They require employees to use full bunny suits as well as to go through a 15 minute gowning procedure and a 15 minute exit procedure. Once you enter the cleanroom, you see broken drywall and bare concrete. The environment is causing the problem, not the people.”
Scott Mackler, Founder and Principal at Cleanroom Consulting LLC in Pittsford, New York, suggests that setting the gowning protocol according to the cleanroom classification rather than the workflow is naïve. Mackler advises that manufacturers establish the rationale for cleanroom activities. “Why clean but not verify? Why wear a bunny suit when a smock will do?”
We have observed that most cleanroom employees are fairly intelligent. We strongly suspect that, in at least some instances, the lack of cleanroom discipline may be symptomatic not of laziness, not a desire to destroy product, and not a desire to vex the supervisor. At least part of the lack of cleanroom discipline may be due to suboptimal cleanroom design and/or to incorrect process flow. If employees are asked to jump through arbitrary, unnecessary hoops in the name of “discipline” while the facilities or process contribute to contamination, they may be more likely to take shortcuts.
CLEANROOM PSYCHOLOGY
On the other hand, in the previous column, Kraft commented on the psychological effect of air showers. Keith Weber, Vice President of Engineering at Clean Rooms International Inc. in Grand Rapids, Michigan, recounts the rationale used by a manufacturing plant to justify the protocol at a rather large unclassified cleanroom. While it was not built to a specific classification, it was much cleaner than a typical office or manufacturing environment. Lab coats, hairnets, and the use of shoe scrubbers and tacky mats were required. “After a walk through,” recalls Weber, “I talked to the facility manager to question what appeared to be a waste of company assets. Her response was valid and insightful. She understood the room did not require this level of cleanroom protocol. Her goal was to make it visibly clear that the room was designated for special activities, that the room was off-limits to the general workforce. She felt that it was much easier to enforce well-defined rules than to have a system where the employees ‘should not’ enter the cleanroom with food or drink or ‘avoid’ tracking in mud.”
An initial capital investment that minimizes operator error may be prudent. Barton views a central vacuum system as one such example. “I’ve seen it many times, where a person assigned to clean the cleanroom only accomplishes moving the dirt from one point to another. Detailed cleaning takes concentration, dedication, and consistency. A central vacuum improves the operator’s chance of removing contamination every time he cleans. It also helps to provide a more consistent cleaning system for inconsistent cleaning people.”
SPEND WISELY
“Who wants to hear from an expert that you need to spend more money? Maybe you need an expert to help you see how little to spend and where to spend it to get the most bang for your buck!” suggests Mackler. As might be suspected, the search for the non-superfluous cleanroom is ongoing; and the correct approach is process-specific and site specific.
Are cleanrooms a “do it yourself project?” Not completely. We suggest you work with appropriate experts in the design, operation, and ongoing monitoring² of controlled environments and that you find experts who are also dispassionate advisers. We also suggest that you have to be involved, and that you take all recommendations with a grain of salt. Controlled environments are best thought of as part of a process, not as safeguards or window dressing. As the manufacturer, you are the one best suited to make the final decision.
References

From “Dirt,” an exhibit at the Wellcome Collection, London, UK (2011); we thank Anselm Kuhn, from Finishing Publications, for suggesting this exhibit to us.
R. Kraft, “Point of View–The Importance of Ongoing Facility Monitoring,” Controlled Environments Magazine, June 2011.

Ask the Facilities Guy: How do I develop a Disaster Recovery Plan?

2011-12-16T02:28:00.000-08:00

By Richard Bilodeau, PE

Question: How do I develop a Disaster Recovery Plan?
Answer: “Plans are nothing. Planning is everything.”
When General Dwight Eisenhower reflected on the monumental task of planning the invasion of Normandy in World War II, he summed up my core advice regarding the development of a disaster recovery plan in six words. Entire books have been written on how to develop effective disaster recovery plans, but any facilities engineer who forgets General Eisenhower’s simple truth will have wasted an inordinate amount of time and money.
An effective Disaster Recovery Plan (DRP) is a tool, not an end game, and certainly not a file-filler or a bookcase trophy. It’s an organic document that must be constantly tested, trained to, adjusted, and amended to address not only your company’s changing business conditions but a constantly changing world.
But first, back to the beginning—following is a high level overview to help you get started on developing a Disaster Recovery Plan tailored to your company’s needs. Today’s facilities engineer must make plans for disasters that are as varied as fire and flood, chemical spills and releases, ice storms, earthquakes, utility disruptions, and—unfortunately in today’s post-911 world— acts of terrorism here or abroad that disrupt your supply chain. While the threats may vary greatly, their impact is the same: all have the potential to interrupt production and other operations, causing untold damage to your bottom line. Even worse is the potential to injure or kill your employees or members of the public.
Step One: Laying the Groundwork
Developing an effective disaster recovery plan is an exercise in planning. Your first task is to build support and buy-in from management. You must build a solid business case addressing the concerns of management, employees and, in the case of a public company, investor scrutiny. To borrow a phrase from the game Monopoly, do not pass “Go” until management is squarely behind both funding the plan development and supporting the required ongoing training, testing, and amending. Management support must be both budgetary and highly visible—in fact the most successful plans include top management as active members of the DRP team. Don’t expect your C-suite members to attend team meetings but do require them to visibly support the program, schedule regular briefings, know their roles cold, and participate visibly and actively should a disaster strike.
Your second step is to identify and recruit key members of the DRP team. Effective and swift recovery from any disaster requires the talents of many individuals within an organization—this is not just a manufacturing challenge. To succeed in recruiting the best and the brightest across your organization (and those that don’t wither under pressure-cooker scenarios), make sure your recruiting efforts carry the gravitas of senior management. An e-mail from the CEO outlining the importance of developing a DRP works wonders. Your team members must have the seniority, experience, temperament, and authority to be able to analyze a wide variety of situations and make decisions quickly. When disaster strikes, the DRP team doesn’t have time to wait for purchase order approvals.
Bring representatives, with authority, on board from the following disciplines:

Senior management: buy-in/plan approval/public face
Finance and Purchasing: funding and supply procurement/ expenditure management/vendor relations
IT: systems recovery and/or management
Logistics: planning/staging recovery efforts
Operations: your lifeline to getting manufacturing up and running
Security and Emergency Response: their department
Sales: customer relations/communications conduit/ income stream champions
HR: employee and family issues
Legal: Make sure your legal rep is capable of enabling the process, not manufacturing roadblocks. You need a problem solver, who can address red flags, not surrender to them.
Government relations: Don’t underestimate the value of this function. Strong government relations allowed one manufacturer to bring their operations online during a massive regional power failure days before power was fully restored.
Communications: Your lifeblood to emergency responders, your employees, the press, your Board of Directors, your investors, your customers, and vendors— anyone with the power to impact the health of your company both immediately and for the long term.

Companies with multiple locations require both a core DRP team and localized teams capable of responding immediately. Lines of authority, coordination, and communications must be clearly delineated. Don’t treat your regional locations as second tier team members— invest in their plan development, training, and tie-in to the corporate effort.
Next Step: Risk Assessment
In order to plan, you need to understand what you’re planning for. Develop a thorough risk assessment, identifying potential risks for each location. You need to be sure your playbook contains a comprehensive list of scenarios that could impact your operations. It’s helpful to categorize these risks into natural disasters, technology disasters, and those caused by human error, accident, or intentional acts.
For each potential threat or hazard, identify your facility’s vulnerabilities and clearly articulate any potential impacts on your company. Make sure your impact analysis goes beyond operations—you need to plan for effects on people, property, the environment and both your company’s financial health and reputation.
The risk assessment phase will likely uncover opportunities to eliminate, minimize, or mitigate some of your company’s vulnerabilities. Set up risk specific mitigation teams to address these opportunities on a parallel track with your DRP efforts, and be sure the work of both groups is closely coordinated.
Your final risk assessment step is to prioritize the risks, based on likelihood of occurrence and its potential impacts to your company, its people, property, or the environment. Spend some time developing your rationale and guidelines for prioritization—the time spent up front will make managing any scenario easier.
Plan Development: Where the rubber meets the road
Rolling up your sleeves and developing a solid, actionable disaster recovery plan requires diligence.
Each risk requires a scenario review and analysis, an action plan, a delineation of core team responders and roles, a discussion of appropriate response levels and escalation triggers and complete resource information. Additionally, specific functions—such as communications— require the development of a separate crisis plan.
Structure your plan so that it’s reader friendly and usable. Keep your language concise, use charts, as well as bolds and bullets in your text. Make sure technical information needed by the disaster recovery team is readily available. Tab sections in hard copies for easy reference (and hard copies are a must in today’s power dependent world of online information).
In my next column, I’ll delve into the specifics of creating a disaster recovery plan tailored to the requirements of your business, including resources you can access for more information. In my third column in this series we’ll look at managing an incident, as well as training and ongoing testing of your plan to ensure required course corrections keep you on top of changing risks.
Fair to say that General Eisenhower had it right. Until next time.
Richard Bilodeau, PE, is director of engineering at SMRT, architects and engineers (www.smrtinc.com). His 30 year career includes plant engineering positions in clean manufacturing. Richard has designed, operated, and supervised the construction of advanced technology facilities, numerous industrial projects, healthcare facilities, and corporate offices. He’s engineered clean manufacturing facilities for lithium-ion batteries, medical devices, electronics, and pharmaceutical clients. Richard can be reached at: TheFacilitiesGuy@smrtinc.com.

Thermal Decontamination: The Key to Effective Culture

2011-12-16T02:26:00.000-08:00

By Verena Ruckstuhl

When culturing various organisms it is vital that they do not become cross-contaminated compromising their viability.
Contamination is a major concern for any microbiologist. When culturing various organisms (bacteria, yeast, or fungi) for use in a multitude of downstream applications, it is vital that potentially contaminating microorganisms are removed from the incubation environment. If cultures do become infected with microorganisms, or cross contaminated with foreign cells, the viability of the culture is significantly compromised. In order to prevent this from having a negative impact on subsequent experimental steps and resulting data, contaminated cultures are commonly destroyed. As such, time and money are wasted as the entire preparation and culture must begin again, using fresh organisms and reagents. However, since sources of contamination are ubiquitous and difficult to identify, they are often especially hard to completely eliminate. As cell culture research is taking an increasingly prominent position in the development of therapeutics and vaccines, for example, it is vital that laboratories across the pharmaceutical, medical, food, research, and clinical sectors are employing incubation techniques with proven, trustworthy decontamination methodologies.
THE MICROBIOLOGICAL INCUBATOR
Designed for laboratory use, microbiological incubators maintain optimal conditions for the effective culture of prokaryotic and/or eukaryotic cells. They provide advanced control over temperature to ensure that organisms proliferate and grow in a fast and efficient manner. As incubators are commonly shared between multiple users, it is often the case that if the internal chamber is not properly cleaned, one user’s culture can have a negative effect on the next user’s culture, through the cross contamination of cell types. Furthermore, micro - organisms can be introduced through regular door openings, contact with skin/hair/ clothes, or poor cleaning routines. Bacteria and fungi (including yeast and molds) are the easiest contaminants to detect. They are also ubiquitous to the environment and can colonize extremely fast. Other contaminating microorganisms, such as mycoplasma and viruses, are more challenging to identify, yet can cause significant damage to the culture and, on occasion, the user. Therefore, the ideal microbiological incubator would incorporate an effective decontamination method, to ensure that even trace amounts of potentially detrimental contaminants are removed from the incubation environment. This ensures that precious cultures are protected and integrity is maintained.
Here, we discuss a third party test (performed by IBFE Institut fϋr Biotechnische Forschung und Entwicklung, Germany),¹ in which the 140ºC dry heat decontamination cycle of an incubator is tested for its effectiveness in eliminating a spectrum of contaminating microorganisms.
METHODS
A microbiological incubator was tested to assess the efficiency of its built-in 140°C thermal decontamination cycle. Cell suspension was added directly to Phosphate Buffered Saline (PBS), diluted and plated on Tryptic Soy Agar (TSA), to determine the total colony forming units of the applied suspension. This allowed determination of the efficacy of the recovery of the cells from the surface after the experiment.
In order to test the effect of the surfaces on cell inactivation, two surfaces of the incubator interior (the bottom stainless steel panel and the inner surface of the glass door) were contaminated, as detailed in Table 1 above.

Cell suspensions were dried on the surfaces for six hours at 35ºC.
The organisms were recovered from the surfaces using sterile cotton plugs.
Following vortexing and ultrasonication, appropriate dilutions were spread on TSA agar and colony forming units (CFU) were consequently determined after 24 and 48 hours of incubation.

To test the efficacy of the decontamination routine on cell inactivation, 55 areas of the interior of the unit (right, bottom, top, left, back, interior of glass door, exterior of glass door, steel door, shelf 1, shelf 2, shelf 3) were contaminated with each of the microbial suspensions listed in the table.
The location of the contamination is represented in Figure 1.

The decontamination program was run until completion (140ºC, 6 hours).
The microorganisms were recovered using microbiological detection plates—replicate organism detection and counting (RODAC), with TSA agar as a growth medium. This method is more sensitive than the cotton plugs for detection of very low CFU levels on surfaces.
After an incubation period of 24 and 48 hours, the number of CFU were determined.
The entire protocol was performed twice, to ensure reliability of the resulting data.

(Click For A Larger Image)

RESULTS
After running one cycle of the thermal decontamination (140ºC for 6 hours), the study found that none of the microorganisms were recoverable from any of the artificially contaminated surfaces. This demonstrates that an inactivation of more than 99.99999% (>7 log) occurred. Results from the second decontamination cycle used in the study presented identical inactivation rates. No viable microorganism cells were recoverable from any part of the surface.
The comparative drying process of 6 hours at 35ºC used in the study did not have much of an impact on the spores (B. atrophaeus) and conidiospores (A. brasiliensis). The CFU levels of P. aeruginosa and S. aureus were influenced the most, reflecting their need for humid environmental conditions. However the inactivation was below 1 log and negligible, compared to the above demonstrated effectiveness of the 140ºC decontamination cycle.
CONCLUSION
Decontamination is a pivotal part of any culturing protocol to eliminate contamination from various micro - organisms. By incorporating an effective decontamination cycle into a microbiological incubator, such as the 140ºC decontamination cycle of the incubators, users can be confident that potentially contaminating micro - organisms present are effectively inactivated. Certified by an accredited microbiological institute (IBFE Institut fϋr Biotechnische Forschung und Entwicklung, Germany), this routine eliminates the need for separate autoclaving of interior fittings, freeing up valuable time for other experimental protocols. In addition, this level of confidence in the integrity of the cultured organisms ensures their viability for downstream use. Users can therefore be confident in the knowledge that their resulting data is reliable and reproducible, while being of the highest possible quality.
References

Report # 1420710-2a: Determination of the Efficiency of the Thermal Decontamination in a Microbiological Incubator (Heratherm IMH180- S/41112744). Dr. Heiko Ewen, IBFE Institut fϋr Biotechnische Forschung und Entwicklung.

Principles of Cleanroom Validation

2011-12-16T02:24:00.000-08:00

By David Muchemu

A cleanroom must be validated and certified to a particular class before operation.
A cleanroom is a modular environment in which the following environmental factors are kept under control; temperature, airborne particulates, microbes, relative humidity, differential pressure, and air flow.
Cleanroom Validation is performed for a variety of reasons. To ensure that the design of the facility is fit for its intended purpose; to ensure that the facility, equipment, and environment meets User Requirement Specifications (URS); to ensure that the facility, equipment, and environment meet defined regulatory requirements; to ensure that the facility, equipment, and its environment function together as a system to meet defined standards.
Cleanrooms are validated and then certified to a chosen class of ISO 14644-1. Each class of ISO14544-1 has its unique requirements that must be made for a facility to be classified in the specified classification.
CLEANROOM VALIDATION LIFE CYCLE
Validation of a new cleanroom follows a specified lifecycle. The life cycle comprises five phases each of which accomplishes particular tasks to control variation in the modular environment.
Cleanroom validation work is accomplished through five phases. It starts off with the design control phase and ends with monitor and control. Changes to equipment and control factors after the cleanroom has been validated are grounds for cleanroom re-validation.

PHASE ONE: DESIGN QUALIFICATION
Cleanroom validation starts with Design Qualification (DQ). The purpose of this phase is to prove through objective evidence that the design is fit for its intended purpose. Design Qualification is a verification exercise against requirements defined in the acceptance criteria of your DQ protocol.
The protocol should address the following:

User Requirement Specifications(URS)
Vendor documents and specifications
Facility layout
Purchase orders
Design documentation
Factory Acceptance Tests(FATs)
As build drawings
Data sheets

The output of the Design Qualification phase is a phase report and an Standard Documentation List (SDL) file that documents the following:

Design requirements
Bidding requirements
Purchasing and order documentation
Vendor supplied documents list
As build drawings
Component lists
Inspection lists
Factory Acceptance Tests

The approval of the Design Qualification, DQ phase is a pre-requisite for the initiation of the Installation Qualification, IQ phase.
PHASE TWO: INSTALLATION QUALIFICATION
The purpose of this Installation Qualification (IQ) phase is to confirm through verification that equipment— as installed—confirms to user requirements and design requirements. Verification is focused on the following items that should be called for in your IQ protocol:

HVAC calibration
P&ID loop verification
HEPA filter integrity test data review
Critical equipment calibration status
Site Acceptance Tests(SATs)
Installation Qualification tests
Piping and welding documentation
Utility verification
System standard operating procedures and work instructions

The output of this phase should be an IQ report addressing all the above elements, and an SDL file that documents the following elements:

Project changes
IQ tests performed
Calibration
Supplier supplied documents
Equipment certificate
Installation deviations
Site Acceptance Tests (SAT)
Consumable list
Spare part list
Environmental review report
List of Operational and Instructional documents

IQ approval is a pre-requisite for the start of the Operational Qualification (OQ) phase.
PHASE THREE: OPERATION QUALIFICATION
The objective for this Operational Qualification (OQ) phase is to show through objective evidence that the cleanroom operates in conformance with design requirements and user defined requirements, and that it consistently operates within a defined range of conditions.
The OQ protocol should address the following:

Testing HVAC (Heating-Ventilation-Air Conditioner) system operation against specified functional requirements
Critical Alarms
Interlock Alarms
Critical operating parameters defined on the room data sheet
Filter integrity tests
Standard operation for the cleanroom
Air speed and air flow
Air flow patterns
Pressure differential

The OQ phase should also address worst case scenarios. To design the worst case scenario for the operation of the cleanroom, critical operating parameters are identified from the cleanroom data sheet. Operation ranges, and extreme ranges, are set for each critical parameter and a worst case designed and documented. It should include the following:

Maximum and minimum temperatures
Maximum and minimum humidity
Maintenance schedules
Personnel contamination

The worst case scenario is usually carried out at the specified High and specified Low parameters.
The output of this phase is an OQ report addressing alarms and functional requirements of the cleanroom specified in the user requirement specifications.
PHASE FOUR: PERFORMANCE QUALIFICATION
The purpose of Performance Qualification (PQ) of the cleanroom is to demonstrate with objective evidence that the cleanroom consistently operates within defined parameters to produce the defined, desired environmental outcome. Cleanroom performance qualification involves testing and monitoring of the following:

Airborne particulate levels
Surface particulate levels
Viable microbial particulates
Relative humidity
Differential pressure
Temperature

The output of the PQ phase is a PQ report that analyzes the performance of the cleanroom using specified equipment parameters. PQ is a pre-requisite for certification.

CLEANROOM CERTIFICATION
Validated cleanrooms are validated to a required class of cleanliness. The level of cleanliness chosen is driven by user requirements. Cleanroom classes are defined in ISO1464-1:
Methods for evaluation and measurements for Certification are specified in ISO14644-3. It calls out for the following ten tests.

Airborne particle count test
Airflow test
Air pressure differencial test
Filter leakage test
Flow vizualization test
Airflow direction test
Temperature test
Humidity test
Recovery test
Containment leak test

Once certified to a particular class the cleanroom factors are monitored to ensure that parameters have not drifted, or changed, and that the environment is under control.
MONITOR AND CONTROL
A constant monitoring program is required after certification. Requirements for compliance are found in ISO 14644-2.
Statistical analysis for cleanroom parameters is encouraged as a tool for monitoring the cleanroom after certification to ensure compliance. The tool of choice is statistical process control, SPC.

On the Quality of Cleanroom Wipers

2011-12-16T02:17:00.000-08:00

By Sandeep Kalelkar, Ph.D., Jay Postlewaite, Ph.D.

In assessing the quality of cleanroom wipers, it is vital to consider the consistency of their cleanliness as an integral property that allows the expected performance measures to be achieved.
CLEANROOM WIPERS AND THEIR CLEANLINESS
Wipers are used in cleanroom environments to control contamination and minimize its effect on critical manufacturing processes. Whether one is manufacturing the next generation of semiconductor chips or the newest engineered vaccine, one must select a cleanroom wiper that is the best fit for the specific critical cleaning application. Various performance attributes are typically considered in selecting a cleanroom wiper. These may include sorption capacity/rate in addition to a series of measures of cleanliness of the wipers. Wiper cleanliness is a critical attribute because wipers carry the risk of leaving behind contamination through the burden that they might intrinsically carry into the cleanroom. A variety of testing protocols exist in order to assess the quality of cleanroom wipers. Specifically, one refers to the IEST Recommended Practices¹ for evaluating the particulate, ionic, and non-volatile residue (NVR) burden carried by the wipers. However, these test results are typically reported as single data points, sometimes accompanied by an average value and/or a standard deviation. This is a statistically inadequate method to compare the cleanliness of wipers. We discuss here an alternative approach to assess the true cleanliness of cleanroom wipers.
HOW TO REPRESENT LARGE AMOUNTS OF TESTING DATA
Statistical Process Control (SPC) is typically used to control the physical, chemical, and contamination characteristics of wipers for each lot manufactured. Usually, SPC data are plotted by sample number as shown in Figure 1.
If one must compare the test results for several wipers, then Figure 2 is a typical result, which can be confusing and not very informative.
There arises a need to find a better way to represent all the available test results that are accumulated over a period of time on a given wiper.

SHOULD YOUR CLEANROOM WIPER BE “AVERAGE”?
Another method of comparing such large data sets is to convert them into a table of averages and standard deviations as shown in Table 1.
Summarizing the data in this manner assumes that the data exhibits a statistical normal distribution, which is often not the case in real world data sets. Since the data is summarized using just these two measures, much information is lost. Using average and standard deviation constitutes an incomplete representation of large data sets and an inadequate means to compare cleanroom wipers. How is the cleanliness of the wiper currently in hand represented by the average value? What if it were to be a lot dirtier than “average”? How would it compromise the integrity of the manufacturing environment? Once again, one recognizes the need to better represent large sets of wiper test data in a manner that is statistically relevant and unbiased.

CONSISTENCY CHARTS
Singular “typical” values or average values and standard deviations inadequately represent the true quality of a cleanroom wiper. A more statistically unbiased method to evaluate many large sets of wiper testing data is through the use of Consistency Charts. These may also be referred to as “Box and Whisker Charts.”
The components that make up a Consistency Chart are:

Median – represents the median or middle value of a ranked data set. (Extreme values do not affect the median value as much as an average could be affected.)
Box – represents the range of values in which 50% of the data lie. If the median line is nearer to one end of the box, the data are skewed toward that end. A smaller box indicates that the data points are more similar to each other.
Whisker – the line at each end of the box, expresses a range of values in which 25% of the data set lies. A shorter whisker indicates that values within the whisker range are more similar to each other.
Outlier – indicates data points that are significantly different compared to the rest of the dataset. A five-point Box and Whisker chart shown in Figure 3 is a pictorial representation of large data sets that allows for straightforward evaluation and comparison of wiper test results with minimal loss of information. It is non parametric, in that no statistical distribution type is assumed.

A detailed statistical description of the construction of consistency charts from large data sets can be found elsewhere^2,3 and is beyond the scope of this article.
A minimum of three data points are necessary to construct a Consistency Chart. However, when used to evaluate product quality such as test data on cleanroom wipers, it is advisable to have more than 15 data points to ensure statistically valid representation and comparisons.
Inspecting the chart below, the following can be observed:

Wiper 1 has the smallest box and the shortest whiskers.
Wiper 2 and Wiper 4 have similar medians.
Wiper 3 has the lowest median.
Wiper 3 has an outlier value shown by the asterisk above the whisker along with the longest whisker.
The median for Wiper 3 is nearer to the lower end of the box indicating many values that are similar and low, that is, 50% of the data values are between 2 and 12; however, the other 50% of the values range between 12 and 58.
Wiper 4 has the largest box and largest range in the data.

Summarizing these observations, Wiper 1 is most likely to be the cleanest among the four. In comparing Wipers 1 and 3, the following observation can be made:

The data set for Wiper 1 or the entire box and whisker chart lies within the box for Wiper 3.
Of the test results for Wiper 3, 25% are lower than those for Wiper 1. More than 25% of the rest of the results for Wiper 3 are higher than those for Wiper 1.

This presents us with an improved method to truly assess the cleanliness of cleanroom wipers than comparing “typical” values or the average values and/or the standard deviation which are by their very nature incomplete representations of the data set. Consistency Charts provide a true picture of the cleanliness of each wiper since they represent all of the data that is available. The true measure of the quality of a cleanroom wiper should be measured by the consistency of its cleanliness test over a period of time.
What truly matters in a critical cleaning operation is that each wiper in a bag, each bag in a lot, and each lot of a given wiper product is delivered to the cleanroom with the highest assurance of the expected cleanliness level. Consistency Charts offer a statistically unbiased approach to evaluate the variability of wipers from within a bag or lot, over time.
CONCLUSION
Selecting the best cleanroom wiper for a particular application requires an unbiased scientific assessment of the available data for any given wiper. Consistency Charts allow for such a statistically valid assessment of cleanroom wiper quality. The quality of a cleanroom wiper should be evaluated not through a typical or average value, but through the use of Consistency Charts that serve to indicate how frequently that typical value is achieved in practice over a period of time. The critical manufacturing processes in a cleanroom must not be susceptible to the variation in the cleanliness of the wiper that may be evidenced in the test data.
References

“Evaluating Wiping Materials Used in Cleanroom and Other Controlled Environments,” IEST-RP-CC004.3, Institute for Environmental Sciences and Technology, Rolling Meadows, IL, 2004; www.iest.org.
John W. Tukey. “Exploratory Data Analysis.” Addison- Wesley, Reading, MA. 1977.
Robert McGill, John W. Tukey, Wayne A. Larsen (February 1978). “Variations of Box Plots.” The American Statistician 32 (1): 12–16. http://www.jstor.org/pss/2683468.

Point of View: Isolation Technology Looking Back and Into the Future in Healthcare Applications

2011-12-14T01:42:00.001-08:00

By Hank Rahe

PHARMACEUTICAL
Twenty five years ago, the concept of using an isolator in pharmaceutical and hospital applications was only a far out thought. The original “BUGS” (Barrier Users Group) was in its formative stages as an idea to improve sterility assurance of aseptically filled parenteral products. Containment of hazardous drugs was accomplished by putting the operator in a “space suit.”
By the early 1990s the pharmaceutical industry, working with the FDA, were evaluating the isolator concept as a means of not only increasing sterility assurance but reducing facility cost. Companies were testing isolation systems by running over a million vials without a single unit being contaminated. The isolators supported by effective decontamination technology through the use of vapor phase hydrogen peroxide allowed for larger batches and extended run times. The 2004 FDA guidance document “Sterile Drug Products Produced by Aseptic Processing—Current Good Manufacturing Practice” delivered to manufacturers a message that the agency not only recognized the technology, but encouraged it as a means of producing products with a higher sterility assurance level.
Societies embraced the education of both users and regulators by conducting seminars, workshops, and Web casts. ISPE (International Society of Pharmaceutical Engineering) held the 20th Annual Barrier Conference in June 2011. Jack Lysfjord has chaired the conference since the initial meeting in 1991. Jack has been the historian for the aseptic use of isolators publishing each year the number and type of aseptic filling lines using the technology.
The other focus of isolators in pharmaceutical and biotechnology is their use for containment of the highly potent drugs. Isolators as a barrier around the product, rather than individuals wearing PPE (personal protective equipment) while handling potent drugs, have become almost a necessity as the drug exposure levels of new compounds has become lower and lower. Targeted therapy of the new classes of drugs has caused the amount of drug causing an adverse effect to drop into the nanogram range. Such small quantities are beyond the safe handling capabilities of conventional downflow booths or Class II biological safety cabinets.
What does the future hold? The future lies in the support technologies in terms of the ability to measure both from an aseptic and containment application of the technology. As measurement improves, incremental improvements in terms of how individuals interact with the process within the isolator will occur. Robotics and on-line feedback systems will be two areas of advancement.
PHARMACY
On the pharmacy front, laminator flow workbenches for preparing IVs and biological safety cabinets were the main line of engineering controls used to protect compounded preparations twenty five years ago.
Pharmacy isolators were introduced in England in the early 1990s. The first isolators in the United States began appearing in the mid to late 1990s. The use of isolators for compounding was driven by regulations created by state pharmacy boards until 2004. In 2004 USP (United States Pharmacopeia) produced a standard (General Chapter <797>) requiring pharmacies compounding sterile preparation to provide facilities to improve the sterility assurance level of these preparations. Patient deaths in several states were the driver behind the need for a national standard. Although the standard, which was reissued in 2008, is technically enforceable by FDA, the enforcement is state by state through pharmacy boards. Some states have actually refused to adopt the standard.
USP <797> has adopted the terms CAI (compounding aseptic isolator) and CACI (containment aseptic compounding isolator) to differentiate between isolators used for compounding non-hazardous and hazardous preparations. The primary difference between the two is pressurization with the non-hazardous being positive pressure and the hazardous being negative pressure. Pressurization has been a means of secondary containment. Studies (“Exposure to Antineoplastic Drugs in Two UK Hospital Pharmacy Units”) have shown no difference in exposure levels between hazardous drugs prepared in positive and negative isolators. The same study did show that the use of isolators significantly reduces exposures compared to Class II biological safety cabinets used to compound antineoplastic drugs.
Currently USP <797> allows for either ISO Class 7 cleanroom or the use of an isolator not located in a cleanroom. Hazardous drugs can be compounded in either a Class II biological safety cabinet or an isolator.
What does the future hold? The future focus will be on improving sterility assurance levels to improve patient safety. Unlike aseptic pharmaceutical applications, the decontamination of materials being placed into the isolator is manual and therefore not consistent or repeatable. Companies in both Europe and the United States have introduced isolators with vapor phase hydrogen peroxide capabilities to reduce surface micro contamination in the last few years. This is a critical step in reducing secondary infections. The next major advancement yet to be introduced is the use of robotics to facilitate the transfer of drugs from vials to the final patient delivery package

TOOLS OF THE TRADE - X-ray Diffraction | Uses of X-Ray Powder Diffraction in the Pharmaceutical Industry

2011-12-13T01:48:00.000-08:00

Among the many experimental techniques available for the identification of solid forms, including polymorphs, solvates, salts, co-crystals, and amorphous forms, X-ray powder diffraction (XRPD) stands out as a generally accepted “gold standard.”
While this does not mean that XRPD should be used to the exclusion of other experimental techniques when studying solid forms, X-ray diffraction (XRD) has applications throughout the drug development and manufacturing process, ranging from discovery studies to lot release. The utility of XRD becomes evident when one considers the direct relationship between the measured XRD pattern and the structural order and/or disorder of the solid.
XRPD provides information about the structure of the underlying material, whether it exhibits long-range order as in crystalline materials or short-range order as in glassy or amorphous materials. This information is unique to each structure—whether crystalline or amorphous—and is encoded in the uniqueness of the XRPD pattern collected on a well-prepared sample of the material being analyzed.
One must draw a distinction between crystalline materials, which give rise to XRPD patterns with numerous well-defined sharp diffraction peaks, and glassy or amorphous materials, whose XRPD patterns contain typically three or fewer broad maxima (X-ray amorphous halos). In practice, when using XRPD, one can usually measure a sequence of crystalline materials that are progressively more disordered, ultimately resulting in glass. A classification system has been proposed by Wunderlich (Table 1) to describe the type of structural order and molecular packing present in molecular organic solid forms using three order parameter classes: translation, orientation, and conformation.¹
XRPD can be used to identify and characterize solid forms of a given molecule exhibiting long-range crystalline order (e.g., polymorphs, solvates, co-crystals, and salts) by their unique combination of order parameters. Amorphous solid forms do not exhibit any long-range order but are identifiable and characterized by their unique local molecular order, apparent in the X-ray amorphous diffraction pattern.²
Given XRPD’s sensitivity to structural order, some of its typical applications in the analysis of solid-state properties of a drug substance or product include:

Identification of existing forms of the API;
Characterization of the type of order present in the API (crystalline and/or amorphous);
Determination of physical and chemical stability;
Identification of the solid form of the API in the drug product;
Identification of excipients present in a drug product;
Monitoring for solid-form conversion upon manufacturing;
Detection of impurities in a drug product; and
Quantitative analysis of a drug product.

Where appropriate data are available, XRPD analysis can determine the solid-form structure and crystal-packing relationship among individual molecules in the solid. This information is essential to the understanding of solid-state chemistry of drugs and important from the regulatory perspective.

Applications in Drug Development

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TABLE 1. Types of Solid Forms Described by the Wunderlich Classification System

XRD has a broad range of applications in various stages of drug development and manufacturing. This section will address many of the common XRPD uses from a practical standpoint. In the broadest terms, these applications can be divided between API characterization and identification. While there is some overlap in both categories, the former is more commonly applied during drug development (before the drug is on the market), while the latter is directed more toward manufacturing, regulatory aspects, and intellectual property.

API Characterization

Guidelines from regulatory authorities regarding the need for characterization of a drug substance under development have been clearly stated. Below is an example relating to the issue of polymorphism: “Polymorphic forms of a drug substance can have different chemical and physical properties, including melting point, chemical reactivity, apparent solubility, dissolution rate, optical and mechanical properties, vapor pressure, and density. These properties can have a direct effect on the ability to process and/or manufacture the drug substance and the drug product, as well as on drug product stability, dissolution, and bioavailability. Thus, polymorphism can affect the quality, safety, and efficacy of the drug product.”³
While there are a number of methods to characterize polymorphs of a drug substance, the two broadly accepted methods for providing unequivocal proof of polymorphism that are recognized by the U.S. Food and Drug Administration are single-crystal XRD and XRPD.⁴ Other techniques like thermal or spectroscopic methods can be helpful in further characterizing drug products, but only X-ray provides the necessary structural information to uniquely identify different polymorphs. Therefore, in early drug development, XRPD is often used as a primary experimental technique and a means of differentiating among experimentally generated materials. Fully characterizing any material requires the use of complementary techniques (thermal or spectroscopic) but X-ray is typically done first because it is fast, is nondestructive, requires little material, and provides the necessary structural information.

Databases of known XRPD patterns for various pharmaceutical materials are published annually by the International Centre for Diffraction Data and the Cambridge Crystallographic Data Centre, which publishes the Cambridge Structural Database.

Synchrotron XRD has frequently been used to characterize pharmaceutical materials in applications that require additional sensitivity not provided by laboratory X-ray diffractometers (e.g., crystallization monitoring).^5-6 The tradeoff is the greater expense and time investment typically associated with such measurements. Because such applications tend to be specialized, this section will focus primarily on laboratory XRPD methods.

Qualitative Analysis of Materials (Phase Identification)

Because every structurally different crystalline material exhibits a unique XRPD pattern upon analysis, the use of XRPD for phase identification was recognized early and remains the most common application of XRPD to pharmaceuticals.⁷ This so-called qualitative analysis typically refers either to the initial characterization of material not previously analyzed by XRPD or to the identification of a phase or phases in a sample of material by comparison to reference patterns. Reference patterns are previously collected XRPD patterns of the same material.
Where available, XRPD patterns calculated from, for example, single-crystal structures can be substituted, but one should remember that the temperature at which the pattern is calculated can have a significant effect on the calculated XRPD profile. When dealing with mixtures of phases, qualitative analysis can provide an estimate of the relative proportions of different phases in the sample, usually based on the comparison of peak intensities for characteristic peaks of the different phases.
Due to sample artifacts such as preferred orientation and poor particle statistics, this type of analysis should never be confused with quantitative analysis of mixtures. Databases of known XRPD patterns for various pharmaceutical materials are published annually by the International Centre for Diffraction Data and the Cambridge Crystallographic Data Centre, which publishes the Cambridge Structural Database.

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FIGURE 1. Two polymorphs of sulfamerazine. Patterns offset for clarity.

XRPD patterns are usually compared by overlaying and aligning the data from different samples. This procedure is typically done electronically, either using the software provided by the XRPD instrument vendor or using custom-developed software. The primary assessment might include determining whether each sample is X-ray amorphous or crystalline based on the absence or presence of crystalline peaks.
When comparing XRPD data of crystalline samples, one notes any differences in peak positions (to within a certain precision, e.g., 0.1 º2Ø) which correspond to structural differences between the samples.⁸ Intensities are generally not relied upon for qualitative analysis due to the previously mentioned instrument and sample artifacts, although they have to be used to some degree to allocate the peak positions (based on local maxima).
It is not uncommon for two patterns to share some but not all of the peak positions. This can be a coincidence, or it can happen because one of the samples is a mixture of multiple phases, including the phase in the other sample. Experience and data from complementary experimental techniques are needed to resolve such ambiguous cases. It should also be noted that, at higher 2Ø values, peaks of most organic materials become considerably overlapped, which makes determining their exact positions difficult. Therefore, freestanding peaks at low angles are the primary means of differentiating structures, and XRPD data above approximately 30 º2Ø are rarely useful for qualitative analysis.
Figure 1 shows XRPD patterns of two crystalline polymorphs of sulfamerazine. The patterns in Figure 1 were collected from material crystallized in glass capillaries during a polymorphism screen. A polymorphism screen is typically run early in the drug development process to identify and (partially) characterize the different polymorphs of a drug substance. Assuming XRPD was the first analytical technique used on the samples, the data in Figure 1 could be used to make a qualitative assessment regarding the probable nature of the material generated during crystallization experiments. Therefore, one could designate the first of the patterns crystalline “Pattern A” and the other crystalline “Pattern B,” noting the sharp peaks and lack of diffuse halos as a sign of crystallinity and the structural differences, as evidenced by the different peak positions in each pattern.
There is insufficient information at this stage to designate either pattern as a polymorph of the material; they could be, for example, a solvate, a hydrate, or a mixture of two or more polymorphs. It is clear, however, that both materials are crystalline and structurally different. Further characterization using thermal methods (TGA, DSC), for example, would confirm that these materials are not solvates or mixtures but actual polymorphs and would aid in determining the thermodynamically stable polymorph. XRPD provides information about the structure of materials, not thermodynamics, although variable-temperature XRPD has been used to study changes in structure at different temperatures.

PANalytical’s Empyrean X-ray diffractometer is a 2011 winner of an R&D 100 award in the ‘winning technology’ category.

One can envision a large number of different crystallization experiments (using different solvents or conditions) performed on the API, some possibly in automated fashion, with the resulting material characterized initially by XRPD. This is in fact a common approach to polymorphism, salt, and co-crystal screening and is perhaps the most common application of XRPD in the drug development process. The latter two screens are usually performed when the polymorphs of the drug candidate itself are not sufficiently bioavailable, in an effort to produce a formulation that addresses the bioavailability problem. An XRPD pattern is taken—of the API, the guest material (e.g., acid), and the mixture of the two. If a salt or co-crystal forms, the XRPD pattern of the mixture should be more than just a sum of the reference patterns of the API and the guest.
Therefore, the first application for XRPD during drug development is typically to identify the materials generated using different experimental methodologies, often in automated, high throughput screening environments.^9-11 To simplify this pattern recognition problem, which often involves hundreds or thousands of experimental data sets per screen, people have developed various computational approaches to recognize, sort, and classify unknown XRPD patterns, either through comparison to a known database of materials or simply within the experimental set of unknown patterns.^12-15 The latter often uses an approach called hierarchical clustering.^16-17
XRPD data are often cataloged in databases using the so-called Hanawalt system.^18-19 In this system, the data are stored as d versus I/Imax pairs. The use of d-space eliminates the need to specify the radiation source wavelength and allows comparison between laboratories using different instrumentation.
A similar system is often used for intellectual property filings. However, there is considerable structural information available in a typical XRPD pattern that can be used to characterize the material. Making use of this information usually requires high quality laboratory data and the use of advanced computational methods.

References

Wunderlich B. A classification of molecules, phases, and transitions as recognized by thermal analysis. Thermochim Acta. 1999;340/341:37-52.
Yu L. Amorphous pharmaceutical solids: preparation, characterization and stabilization. Adv Drug Deliv Rev. 2001;48(1):27-42.
U.S. Food and Drug Administration. Center for Drug Evaluation and Research. Guidance for Industry: ANDAs: Pharmaceutical Solid Polymorphism Chemistry, Manufacturing, and Controls Information. FDA. Available at: www.fda.gov/OHRMS/DOCKETS/98fr/2004d-0524-gdl0001.doc. Accessed Aug. 3, 2011.
Brittain HG. Polymorphism in Pharmaceutical Solids. New York: Marcel Dekker, Inc; 1999.
Varshney DB, Kumar S, Shalaev EY, et al. Solute crystallization in frozen systems–use of synchrotron radiation to improve sensitivity. Pharm Res. 2006;23(10):2368-2374.
Blagden N, Davey R, Song M, et al. A novel batch cooling crystallizer for in situ monitoring of solution crystallization using energy dispersive X-ray diffraction. Cryst Growth Des. 2002;3(2):197-201.
Jenkins R, Snyder RL. Introduction to X-ray powder diffractometry. In: Winefordner JD, editor. Chemical analysis. Vol. 138. New York: John Wiley & Sons; 1996.
United States Pharmacopeial Convention. General chapter 941: X-ray diffraction. In: USP 31-NF 26. Rockville, Md.: United States Pharmacopeial Convention; 2008:374.
Hertzberg RP, Pope AJ. High-throughput screening: new technology for the 21st century. Curr Opin Chem Biol. 2000;4(4): 445-451.
Barberis A. Cell-based high-throughput screens for drug discovery. Eur Biopharm Rev website. Winter 2002. Available at: www.samedanltd.com/magazine/12/issue/43/article/1231. Accessed August 3, 2011.
Johnston PA, Johnston PA. Cellular platforms for HTS: three case studies. Drug Discov Today. 2002;7(6):353-363.
Ivanisevic I, Bugay DE, Bates S. On pattern matching of X-ray powder diffraction data. J Phys Chem B. 2005;109(16):7781-7787.
Marquart RG, Katsnelson I, Milne GWA, et al. A search-match system for X-ray powder diffraction data. J Appl Cryst. 1979;12(6): 629-634.
Gurley K, Kijewski T, Kareem A. First- and higher-order correlation detection using wavelet transforms. J Eng Mech. 2003; 129(2):188-201.
Gilmore CJ, Barr G, Paisley J. High-throughput powder diffraction. I. A new approach to qualitative and quantitative powder diffraction pattern analysis using full pattern profiles. J Appl Cryst. 2010;37:231-242.
Johnson SC. Hierarchical clustering schemes. Psychometrika. 1967;32(3):241-254.
Borgatti SP. How to explain hierarchical clustering. Connections. 1994;17(2):78-80.
Hanawalt JD, Rinn HW, Frevel LK. Chemical analysis by X-ray diffraction. Ind Eng Chem Anal Ed. 1938;10(9):457-512.
Byrn SR, Pfeiffer RR, Stowell JG.

IN THE LAB - Outsourcing | Perfect Partners

2011-12-13T01:43:00.000-08:00

Maurizio Sollazzo, Paul Luchino, and Ted Gresik

A 70-person PerkinElmer OneSource on-site team takes complete responsibility for maintaining and qualifying more than 50,000 Merck Research Laboratories assets in six facilities.

Merck Research Laboratories reduces equipment maintenance costs and improves productivity with PerkinElmer OneSource team

Maintaining laboratory instruments is critical to the productivity of pharmaceutical researchers at Merck Research Laboratories (MRL). In the past, individual MRL departments were responsible for arranging their own instrument maintenance using original equipment manufacturers (OEMs). With the broad array of instrumentation in their labs, this meant administration of 120 maintenance contracts, often by multiple people at multiple sites within the organization.
To curb inefficiencies, MRL initiated a program that centralized responsibility for the maintenance of more than 35,000 assets in three facilities with a single service provider—PerkinElmer OneSource. Using a combination of asset management models (providing the ideal level of insurance and service for each instrument), on-site service engineers, and third-party parts, the consolidated approach delivers substantial cost savings while enhancing the quality and timeliness of service.
Based on the success of the program, MRL is implementing the model across the organization, shifting all maintenance responsibilities and contracts to PerkinElmer. A 70-person PerkinElmer OneSource on-site team maintains and qualifies more than 50,000 assets in six facilities. Assets in the “maintain” category are serviced by PerkinElmer directly, while those in the “manage” category are maintained by service partners under the management of PerkinElmer OneSource. With a single point of contact, MRL only has to make one call to manage the entire program.
The bottom line? Response times have typically been reduced from a day or two to an hour or two, on-time preventive maintenance exceeds 90%, and the cost of asset management has been reduced by 20% since the program’s inception.

Consolidated Approach

When MRL was dealing with myriad OEMs, each department spent a considerable amount of time negotiating and administering multiple contracts and found itself in a weak negotiating position because of the relatively small number of instruments under its control. There was no systematic way to determine whether or not the service level agreements (SLAs) were being met, and it was difficult to determine how many pieces of equipment each vendor managed.
With a single contract and single point of contact, those inefficiencies disappear. At the same time, the approach improves instrument qualification and validation by shifting the focus from the capabilities of the instrument to a more focused, customized model geared toward the end user’s specific applications and environment. Consistent protocols (IQ/OQ/PQ) are utilized across different makes, models, and types of instruments so that they are easier to review and audit, reducing compliance risk. In MRL’s single-provider partnership, an ISO 17025-certified platform is being used to harmonize procedures among many sites, resulting in a 21 CFR part 11-compliant digital record. Preventive maintenance is performed at the same time as qualification to reduce instrument downtime, and automated qualification techniques save time and provide more rigorous testing, more concise electronic reports, and more secure data backup.

The Choice

PerkinElmer OneSource personnel service equipment from most top manufacturers.

MRL is partnering with PerkinElmer OneSource for several reasons. PerkinElmer OneSource supports more than 400,000 laboratory assets globally, giving it the experience and capability to handle MRL’s high volume of instruments. PerkinElmer OneSource personnel are experienced in servicing equipment from virtually any manufacturer, including Agilent, Waters, Millipore, Thermo Fisher Scientific, AB Sciex, Dionex, and, of course, PerkinElmer.
Furthermore, PerkinElmer OneSource is the only major service provider capable of qualifying hyphenated systems such as liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry. This capability eliminates the need for multiple service calls by multiple vendors and facilitates a swift return to scientific throughput in the lab. PerkinElmer OneSource also provides the ability to schedule maintenance and compliance services together, usually by the same scientifically trained personnel, dramatically reducing instrument downtime.
When the process started, MRL was searching for a vendor who could provide insights into what equipment was being used, why, and how often, and could significantly improve return on assets and return on invested capital. PerkinElmer OneSource is proving to be that vendor.
Upon selection, a team of 22 PerkinElmer OneSource certified service personnel was assigned to provide on-site support at the three MRL facilities at the start of the program. The program was administered by a PerkinElmer OneSource management team that provided a single point of contact for all of the services. The services were defined by an SLA that was developed jointly by MRL and PerkinElmer and included metrics on all maintenance and qualification details, including response time, instrument downtime, and completion rate.
All assets among the three facilities were managed from installation and warranty to disposition by PerkinElmer OneSource. Asset management software tracks each piece of equipment and each critical event in the life of these assets. Standardized operational performance data is delivered through a comprehensive asset management program.

The Benefits

Recently, MRL needed to move approximately 800 pieces of equipment among its locations around the world. PerkinElmer OneSource experts handled the move from start to finish.

The improved reporting provided by PerkinElmer OneSource helps MRL’s managers maintain better control over the assets in their facilities. Managers now have access to reports that show what equipment they have, where it is, its service history, response time, downtime, total cost of ownership, and other key data. Utilization information for each instrument, regardless of technology or manufacturer, coupled with operational service and financial metrics, improves decision-making capabilities. With this type of information, the lab manager can justify capital requests using quantifiable data about instrument utilization. The lab manager can pinpoint the time when assets become too costly to maintain and need to be decommissioned or auctioned. He or she has information to justify redeploying an underutilized asset to another branch or laboratory to distribute workload.
Recently, MRL decided to rationalize its clinical areas globally. This change necessitated moving approximately 800 pieces of equipment among MRL locations around the world. PerkinElmer OneSource experts handled the move from start to finish, relocating and recommissioning all instrumentation to minimize downtime and ensure continued regulatory compliance.
As part of the agreement, PerkinElmer OneSource demonstrated that every piece of equipment was working in its new location. Whenever equipment did not perform exactly as expected, PerkinElmer OneSource personnel responded and repaired the items. PerkinElmer OneSource also provided reporting to track the location and status of every asset throughout the move.
By streamlining the entire vendor management process, significantly reducing the daily administration burden on scientists and allowing them to focus on research instead of managing multiple vendors, the consolidated maintenance program delivers significant cost savings. At the same time, improvements in record keeping and service tracking are enabling significant improvements in purchasing decisions.
In light of these results, MRL is expanding the partnership, assigning to PerkinElmer OneSource the vendor management of all its OEM service contracts as well as responsibility for the procurement and storage of parts. This approach provides endless possibilities, all based on the fundamental approach of letting specialized experts do what they do best so that the laboratory can focus on being a laboratory and generating science.

FORMULATION - Nanoparticles | Strides for Small Cancer Fighters

2011-12-13T01:15:00.000-08:00

James Netterwald, PhD

Nanoparticles used to formulate and deliver drugs to cells and tumors show increasing promise

Nanometer-sized particles, typically made of iron oxide, are beginning to transform the world of medicine. In particular, nanomedicine’s impact has been defined by the potential use of nanoparticles in the formulation and delivery of cancer drugs.
When a nanoparticle-based drug is developed, some thought must be put into how biodistribution, targeting, and post-delivery mechanism of action will be incorporated into its design.
“The research we are doing is really based on the premise that altering the temperature of the tumor can dramatically change its response to things like radiation therapy or chemotherapy,” said Theodore DeWeese, MD, professor and chairman of radiation oncology and molecular radiation sciences at The Johns Hopkins University in Baltimore, Md. Doing that in a reproducible way has been a challenge.
That was until about four to five years ago when cancer biologists decided to employ nanoparticles to do the heating “using so-called iron oxide particles, which, in the right configuration and when placed in an alternating magnetic field, will actually heat to a very high temperature and lead to the sensitization of cancer cells to chemotherapy and radiation therapy,” said Dr. DeWeese. Just heating the tumor by three degrees nearly doubles its sensitivity to therapy.
One of the major challenges in the path to building a nanoparticle delivery system for cancer therapy has been targeting, the process by which the nanoparticles are coated with either antibodies, RNA molecules, or small proteins so that they are targeted to a certain cancer cell type. Dr. DeWeese and his colleagues coat their particles with dextran as well as polyethylene glycol, which aid in biodistribution of the drug when it is administered either intratumorally or intravenously.
However, these iron particles, which range from 80 to 100 nanometers in diameter, do not specifically carry a drug. In fact, the iron itself is what is delivered to the tumor cell. “When the iron reaches the cell and when that cell is placed in an alternating magnetic field, substantial heating of the targeted cell results,” said Dr. DeWeese. “Even in the untargeted state, these particles are taken up by pinocytosis. Cancer cells like to take up these particles, but non-cancer cells take up the particles as well. So, nonspecific targeting is also possible.”

Targeting a Challenge

Howard Soule, PhD

Although heating the nanoparticle to destroy a target cancer cell is one possible mechanism of action, it is somewhat nonspecific.
“The holy grail is to take a highly toxic substance and target it within a nanoparticle to a specific tissue—and in the case of prostate cancer, that would be the metastatic tumor,” said Howard Soule, PhD, executive vice president and chief science officer of the Prostate Cancer Foundation in Santa Monica, Calif.
The toxic substance referred to is a chemotherapeutic agent for cancer. Just as they are being developed for solid tumors, nanoparticles are also being crafted to target prostate cancer cells. The targeting is made possible by labeling the particles with ligands that selectively bind to prostate-specific membrane antigen (PSMA), a clinical biomarker that is highly expressed on the surface of metastatic prostate cancer cells and many solid-tumor blood vessels.
“So you have a particle filled with the chemotherapy medication decorated with a targeting entity on the outside of the nanoparticles,” said Dr. Soule. “When you introduce these things systemically to the patient, the theory is that these particles will go into circulation and, based on their specificity, they will find the tumor.” He explained that the tumor would take up the particle, degrade it, and then release the drug inside the tumor cell, thereby sparing bystander (normal) cells.
“Targeting is really a complex issue,” he explained. “These are virus-sized particles that distribute in ways that chemicals don’t. However, there are still questions and challenges about the potential of nanotherapies for cancer.” For instance, how specific will a given particle be for a tumor, and how much of the tumor-targeting specificity is due to the vascular leakiness of the tumor, which is a property of meta-static malignancies?
“We just don’t know the answers to these questions yet,” he said.

Nanoparticles to Nanomedicine

Omid Farokhzad, MD, an associate professor at Harvard Medical School and director of the Laboratory of Nanomedicine and Biomaterials at Brigham and Women’s Hospital in Boston, Mass., has made some seminal discoveries in the world of nano-medicine. His academic pursuits have led to the development of a platform that enables one to target nanoparticles for a number of therapeutic applications.
That success ended the academic challenges and opened a new set of issues: commercial scale-up and development. The solution was start a company to license the technology from the university so that it could be further developed and eventually marketed. The company, BIND Biosciences, was co-founded in 2007 by Dr. Farokhzad and Robert Langer, ScD, David H. Koch Institute Professor at the Massachusetts Institute of Technology (MIT).
“The company eventually made a modification to the formulation to make the particles much more stable and much more appropriate from a drug development standpoint … BIND started human trials of BIND-014, a targeted nanoparticle therapeutic for treatment of solid tumors, in January 2011,” explained Dr. Farokhzad. “The technology is composed of very long circulating, controlled release, polymeric nanoparticles that are targeted to specific receptors on the surface of disease cells for targeted and controlled release of drugs.”
BIND’s platform enables the company to engineer nanoparticles with the appropriate sizes and surface properties, targeting the ligand density, circulation times, and drug release profiles that would be required for optimizing a drug’s performance for various therapies.
“Based on the research of MIT nano-particle guru Dr. Robert Langer, BIND’s nanoparticles provide the unique opportunity to control the drug load and release profile while actively targeting diseased cells with ligand-directed receptor-mediated binding,” said Jeff Hrkach, PhD, senior vice president of pharmaceutical sciences for BIND. The particle’s surface is coated with polyethylene glycol, which enables it to reach its drug target by evading recognition by the immune system. Ligands can also be attached to the surface of the particles, allowing them to bind directly to the desired cells or tissues to be treated. BIND’s nanoparticles were developed in collaboration with Drs. Langer and Farokhzad.
“BIND has spent the last four years translating that academic bench work into more robust processes for development and clinical translation,” said Dr. Hrkach.
BIND’s lead program is a targeted nanoparticle loaded with docetaxel, the active ingredient in Taxotere—a well-known and successful Sanofi-Aventis cancer drug that has recently gone off patent. The product, BIND-014, which is in Phase 1 clinical trials for a number of solid-tumor indications, targets PSMA.
“We are working with partners who have existing approved drugs or candidates in their pipeline and are looking for opportunities to improve them or expand their existing indications,” said Dr. Hrkach. “Some of these products are currently in clinical development and show signs of promise but have limitations related to their therapeutic index. Our technology can increase a drug’s efficacy and reduce its toxicity by keeping the drug sequestered in our long-circulating nanoparticles until they reach and actively bind to their specific target cells for maximal concentration at the site of action and minimal systemic exposure.”
The Prostate Cancer Foundation funds both the work done by Dr. Langer at MIT and that of Dr. Farokhzad at Harvard

GREEN CHEMISTRY - Waste Reduction | A Green Sweep

2011-12-13T01:10:00.000-08:00

Neil Canavan

Big pharma is drafted by ethical and fiscal responsibilities to collaborate on waste reduction efforts

The EPA defines green chemistry as “the design of chemical products and processes that reduce or eliminate the use or generation of hazardous substances.” This definition is taken to include the entire life cycle of the product from bench to bedside.
While such endpoints are easily expressed, even the experts find it a bit much to comprehend in practical terms. Thus the formation of the American Chemical Society’s Green Chemistry Institute (ACS GCI) and within that—and more to the medicinal point—the collaborative working group known as the ACS GCI Pharmaceutical Roundtable (GCIPR).

Green Team

It could almost be said that that big pharma’s awareness of green chemistry and the roundtable’s creation in 2005 were prompted by a growing embarrassment. “A paper came out by Roger Sheldon that looked at a metric called e-factor,” explained Julie Manley, senior industrial coordinator with the ACS GCI and the GCIPR. “E-factor generally referred to the amount of waste generated per kilo of product produced, and he showed that, on that basis, pharma generated the most waste” as compared with other chemical-based industries.¹
Because the waste comprises chemicals, it is rarely benign. Corporate reputations are at risk, liabilities accrue, and waste in this or any context can be measured in terms of resources squandered. “The roundtable looks to combine the ethical and fiscal objectives,” Manley explained. “This not only helps the bottom line of the company, but improves its environmental health and safety standards as well.”
But why and how do pharmaceutical companies collaborate on what should be a competitive issue? “There are, in fact, common challenges across the industry,” said Manley, “and while each company has a focus on their unique molecule, in general, much of the chemistry is very similar. Everyone uses certain solvents, certain reactants… .”
One company looking for green alternatives on its own cannot match the creativity of 16 companies—the current number of roundtable members—working together. “The key is to interact in a noncompetitive way, and that is how the roundtable is set up,” Manley said. Shared data sets are blinded to retain corporate privacy, and co-company authored papers are legally vetted by all contributors.
Further, the roundtable is able to encourage, and hopefully share in, green chemistry innovation beyond its membership by using a portion of membership dues, ranging from $10,000 to $25,000 a year, to fund research grants that have totaled more than $950,000.
“Right now the program is limited to academics,” said Manley. Part of the reason for this restriction is to focus on spreading the word, to influence academic curricula. “If people are doing the research, then green chemistry is being communicated internally within that institution.”
Available to non-GCIPR members are analytic tools that can be accessed through the ACS green chemistry website.² For example, there is a tool for calculating the so-called process mass intensity (PMI), defined as the kilos of mass of all materials that go into producing an active pharmaceutical ingredient (API), normalized by the mass of the end product; this is taken as a measure of the “greenness” of a given process.³ “The benchmark of PMI has been a very useful tool so that companies can compare apples to apples,” of particular use when considering the greenness of third-party manufacturers that may be a part of your supply chain.
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CASE STUDY: Green Means

The 15th annual Green Chemistry and Engineering Conference—the premier green chemistry event—saw BioAmber Inc., win the Presidential Green Chemistry Challenge Award, bestowed by the Environmental Protection Agency and the American Chemical Society in Washington, D.C., in June.
BioAmber, a renewable chemistry company, received the honor for their innovation in the biosynthesis of succinic acid, which is normally produced with petrochemicals. BioAmber’s proprietary platform uses microbes that have been optimized for succinic acid production.
Last year’s co-winners, Merck and Codexis, were honored for their green chemistry approach in retooling the synthesis steps for making the diabetes drug sitagliptin. Along with numerous green optimizations, the critical alteration was finding an alternative to the catalytic use of rhodium, a rare metal that became prohibitively expensive during the scale-up of manufacture for sitagliptin; for this, scientists were able to substitute a transaminase enzyme for a rhodium-based hydrogenation catalyst.¹
Another example of biocatalysis in green pharmaceutical chemistry is seen in the production of the neuroactive agent pregabalin. In this case, the initially developed API synthesis was highly wasteful, producing 86 kg of waste per one kilogram of product. In addressing this issue, the manufacturer, Pfizer, performed an enzymatic screen for a problematic cyanodiester. The resulting hit was a lipase derived from Thermomyces lanuginosus, resulting in a marked reduction of useless byproduct.²
A final example of green pharmaco-chemistry comes from the familiar class of drugs known as statins—specifically, rouvastatin. In this instance, an initially wasteful chemical reaction was replaced with an enzymatic step that uses deoxyribose phosphate aldolase (DERA) an innovation pioneered in an academic lab. Once the efficacy of this approach was established, a nagging problem remained involving the irreversible deactivation of the enzyme by a chloroacetaldehyde. This was solved with DERA 2.0, if you will, which was created using the biotech method of directed mutagenic evolution.³
For a review of these and other green chemistry options see: Dunn PJ. The importance of green chemistry in process research and development [published online ahead of print May 12, 2011]. Chem Soc Rev

References

Grate J, Huisman G. A greener biocatalytic manufacturing route to sitagliptin. Paper presented at: 13th Annual Green Chemistry and Engineering Conference; June 23, 2009; College Park, Md.
Martinez CA, Hu S, Dumond Y, et al. Development of a chemoenzymatic manufacturing process for pregabalin. Org Process Res Dev. March 18, 2008. Available at: http://pubs.acs.org/doi/abs/10.1021/ op7002248. Accessed August 1, 2011.
Jennewein S, Schürmann M, Wolberg M, et al. Directed evolution of an industrial biocatalyst: 2-deoxy-D-ribose 5-phosphate aldolase. Biotechnol J. 2006;1(5):537-548.

This process mass intensity calculator is one of several analytic tools available at the ACS Green Chemistry Institute website: http://bit.ly/qb5buA

Green Team Player

click for larger view

This process mass intensity calculator is one of several analytic tools available at the ACS Green Chemistry Institute website: http://bit.ly/qb5buA

“I hope we’re reaching a tipping point of awareness for green chemistry,” said Concepción Jiménez-González, PhD, director and team leader of operational sustainability at GlaxoSmithKline (GSK), a GCIPR member. “That’s part of what we wanted to do with the roundtable.”
An engineer by training who has published on the subject, Dr. Jiménez-González is concerned with the production issues beyond the flask: “There are very common techniques outside of pharma that are not really as practiced within pharma, like life cycle assessment, process identification, or the use of continuous processes. We need to move away from emulating what happens in the lab when considering scale up.”⁴
For example, GSK has recently finalized a carbon footprint analysis for its global operations. “We wanted to identify the main contributors to the footprint—what we call ‘hotspots,’ ” Dr. Jiménez-González said. The chief suspect of un-greenness she identified overall is GSK’s use of solvents. “We did some case studies going from cradle to gate in manufacture, from the moment you extract raw materials to the moment you finish the API, and we found out that the impact of solvents is, on average, around 70% to 75% of all the overall environmental impact of the process.”⁵
So what to do? Recycling is one possibility, and it can be done is such a way that it does not affect good manufacturing practices. For instance, you can use recycled solvent to serve the same step in a synthesis. “The other option, when you are looking at the process from the life cycle standpoint, is [that] it really doesn’t matter if you recycle through the same process or you down-cycle, say, to a paint manufacturer,” Dr. Jiménez-González noted.
Or, you could simply use a more benign solvent. Though chemists may be loath to make changes to a set process, there are now references available to guide them in selecting alternative solvents; resources include advice from GSK, Pfizer, and the GCIPR.^6-8
“In general, it makes life easier for us if we include those types of changes prior to filing the IND [investigational new drug application],” Dr. Jiménez-González said. Beyond that, a retooling of the process could cost you valuable patent expiration time.

Green Think

Retooling, or even thinking de novo, can often be a challenge for creatures of habit. If you’re stuck in a circle of self-referencing ideas, you may want to bring someone in from outside—someone like John Warner, PhD, president and chief technology officer of the Warner Babcock Institute for Green Chemistry in Wilmington, Mass.

“The most amazing, most shocking thing is that a chemist can go through six years of higher education and never have a single course in toxicology. Never have a course in environmental mechanisms, never a course in anything at all to prepare them for understanding the regulatory consequences of chemistry.”

—John Warner, PhD, president and chief technology officer of the Warner Babcock Institute for Green Chemistry

“It happens all the time: a company has enormous resources working on a problem, they’re poring over the literature, the textbooks, people are scouring this material, pushing to get that incremental change to do something new, and they come up against a brick wall,” Dr. Warner explained. The problem is the starting point of having an outdated chemical methods perspective.
To start fresh in green chemistry, you might want to first check out the bible of the field, Dr. Warner’s Green Chemistry: Theory and Practice, cowritten with Paul Anastos, PhD, of the Environmental Protection Agency.⁹ In it you will find the 12 guiding principles of practicing green chemistry, which are, though initially intended for use by the chemical industry, easily applied to medicinal chemistry. Not so surprising, given the fact that Dr. Warner’s career started by contributing to the synthesis of the anticancer agent Alimpta.
In Dr. Warner’s opinion, the impediments to green chemistry adoption are not merely intellectual but institutional as well. “There is a love-hate relationship between discovery and process,” he asserted. “The people in discovery are always very grumpy that the people in process don’t take their pearls of wisdom and bring them to amazing fruition, and the people downstream look at the discovery people and say, Why do you keep sending us stuff that can’t be scaled up? Why these solvents, and these toxic reagents? Green chemistry is the language they should both be speaking. If you think about it, the least changes that are made in a process from the bench to the bottle, the more profitable the company will be.”
Dr. Warner acknowledged that progress is being made. Great strides, for instance, have been made in biocatalysis (see case study). And he sees the possibility of one day attaining the holy grail of pharma manufacture: continuous-flow reactions, which would make for a much smaller footprint at greater cost savings. But he remains concerned about the generation of toxic byproducts.
Of particular note is the book’s green principle No. 4: Chemical products should be designed to preserve efficacy of function while reducing toxicity. “The most amazing, most shocking thing is that a chemist can go through six years of higher education and never have a single course in toxicology,” said Dr. Warner. “Never have a course in environmental mechanisms, never a course in anything at all to prepare them for understanding the regulatory consequences of chemistry.”
He is also concerned about competition: “India is mandating that all chemists in training take a yearlong course in green chemistry. China has opened up 15 national research centers dedicated to green chemistry.” These developing economies are going to become far more competitive and innovative because they are putting green chemistry into the front end of innovation and creativity, “and we are still scratching our heads about whether we should do it.”

References

Sheldon RA. Catalysis: the key to waste minimization. J Chem Technol Biotechnol. 1997;68(4):381-388.
American Chemistry Society. ACS GCI Pharmaceutical Roundtable. American Chemistry Society website. Available at: http://portal.acs.org/portal/acs/corg/content?_nfpb=true&_pageLabel=PP_TRANSITIONMAIN&node_id=1407&use_sec=false&sec_url_var=region1&__uuid=a628a938-683c-4c0d-8cac-2aba9f3f06ab. Accessed August 1, 2011.
American Chemistry Society. PMI worksheet. Available at: http://portal.acs.org:80/portal/PublicWebSite/greenchemistry/industriainnovation/roundtable/CNBP_026644. Accessed August 1, 2011.
Jiménez-González C, Poechlauer P, Broxterman QB, et al. Key green engineering research areas for sustainable manufacturing: a perspective from pharmaceutical and fine chemicals manufacturers. Org Process Res Dev. February 22, 2011. Available at: http://pubs.acs.org/doi/abs/10.1021/op100327d. Accessed August 1, 2011.
Constable DJC, Jiménez-González C, Henderson RK. Perspective on solvent use in the pharmaceutical industry. Org Process Res Dev. December 14, 2006. Available at: http://pubs.acs.org/doi/abs/10.1021/op060170h. Accessed August 1, 2011.
Jiménez-González C, Curzons AD, Constable DJC, et al. Expanding GSK’s Solvent Selection Guide—application of life cycle assessment to enhance solvent selections. Clean Technol Environ Policy. April 8, 2004. Available at: www.springerlink.com/content/bk59v8me1l6pv85q/. Accessed August 1, 2011.
Alfonsi K, Colberg J, Dunn PJ, et al. Green chemistry tools to influence a medicinal chemistry and research chemistry based organisation. Green Chem. November 16, 2007. Available at: http://pubs.rsc.org/en/content/articlelanding/2008/gc/b711717e. Accessed August 1, 2011.
Hargreaves CR. Collaboration to deliver a solvent selection guide for the pharmaceutical industry. Paper presented at: American Institute of Chemical Engineers Annual Meeting; November 17, 2008; Philadelphia.
Anastas PT, Warner JC. Green Chemistry: Theory and Practice. New York: Oxford University Press; 1998.

DELIVERY - Cyclosporine | TBI’s Miracle Drug

2011-12-13T01:07:00.001-08:00

Steve Campbell

First discovered by Sandoz (now Novartis) scientists in Norway in 1969, cyclosporine is isolated from the fungus Tolypocladium inflatum.

An accidental discovery about 20 years ago has led to a cyclosporine pharmaceutical on the threshold of approval

Often called the silent epidemic, traumatic brain injury (TBI) afflicts approximately 1.7 million Americans annually. More than 52,000 are killed, and 275,000 are hospitalized.¹ Most are left in various states of disability—from almost full recovery to mild symptoms but able to function with some or moderate disability to severe disability requiring around-the-clock intensive care and support. The annual costs of TBI, both direct and indirect, including such factors as lost work time or reduced productivity, have been estimated at more than $60 billion, and there may be more than six million TBI survivors in society.
Over the past decade, TBI has come to the fore as tens of thousands of wounded soldiers return home from the Middle East suffering both hidden and visible TBIs and trauma caused by blast injuries from improvised roadside explosions.²
What is called post-traumatic stress disorder may actually be the long-term effects of TBI.
Due to the economic and social costs of TBI, a significant ongoing effort is being made to develop and apply emerging new clinical and pre-clinical pharmaceuticals with the potential to mitigate the cascading additional brain damage that occurs during the critical secondary phase in TBI. Among these is an interesting pharmaceutical compound called cyclosporine (also known as cyclosporin-A, or CsA), which has been found to have significant neuroprotective capabilities and the ability to moderate the resulting damage and long-term disability in TBI.^3-6
Pre-clinical mouse model studies show an 80% reduction in neural damage after the application of this pharmaceutical.^7-8 More than 17 years in development for neuroprotection, CsA is working its way toward approval as a treatment that can greatly ameliorate the effects of TBI in humans.
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Cyclosporine Mitigates Heart Attacks

Mitochondria are present and produce effective energy in almost all cells in the body. It turns out that mitochondrial collapse may be associated with a variety of acute injuries, such as myocardial infarctions and chronic diseases like amyotrophic lateral sclerosis, multiple sclerosis, and other neurological disorders. In myocardial infarctions, reperfusion of the blocked artery can cause reperfusion injury and extra damage and disability to the heart muscle, as well as increased mortality. Mitochondrial protection in heart muscle tissue is one answer to moderating the long-term impact of heart attacks on health and lifestyle.
Every year, an estimated 500,000 people in the United States suffer a myocardial infarction. Infarct size is a major determinant of mortality. During myocardial reperfusion, the abruptness of the reperfusion can cause additional damage—a phenomenon called myocardial reperfusion injury. Studies indicate that this form of injury can account for up to 50% of the final size of the infarct.¹ Focusing on reducing the additional infarct resulting from reperfusion would protect heart muscle and allow the patient to live longer and in better health after the initial attack.
Interestingly, a number of proposed interventions, such as ischemic postconditioning, have been claimed to mediate cardioprotective actions by acting on the opening of the mitochondrial permeability transition pore (MPT), which is directly inhibited by cyclosporine. CsA has been studied for its cardioprotective capabilities and found to be a potentially significant pharmaceutical for ameliorating long-term damage from heart attacks.
A small proof-of-concept clinical study by Christophe Piot, MD, PhD, and his colleagues, published in The New England Journal of Medicine in 2008, found that the administration of CsA with the aim of inhibiting the induction of the MPT was associated with a 40% reduction in infarct size.² An editorial in the journal called for large, multi-center studies to determine if this new treatment option can positively influence clinical outcomes. In addition, targeting the MPT “may also offer protection in other clinical contexts, such as stroke, cardiac surgery, and organ transplantation.”
Following that lead, in April, a European investigator-initiated multi-center phase III study of NeuroVive’s cyclosporine-based cardioprotection pharmaceutical CicloMulsion in myocardial infarctions enrolled the first of 1,000 patients.³ —SC

References

Hausenloy DJ, Yellon DM. Time to take myocardial perfusion injury seriously. N Engl J Med. 2008;359(5):518-520.
Piot C, Croisille P, Staat P, et al. Effect of cyclosporine on reperfusion injury in acute myocardial infarction. N Engl J Med. 2008; 359(5):473-481.
AktieTorget. NeuroVive: first heart attack patient treated in European cardioprotection phase III trial with NeuroVive’s Ciclomulsion. AktieTorget website. Available at: www.aktietorget.se/NewsItem.aspx?ID=58252. Accessed Aug. 12, 2011.

Two Stages

Cyclosporine is a cyclic peptide of 11 amino acids and contains a single D-amino acid, rarely encountered in nature. Cyclosporine protects mitochondria in TBI, myocardial infarction and other acute injury applications.

TBI has two stages. The first stage occurs at the time of injury, whether it is caused by a gunshot, blast, fall, or hit. This initial stage could be either a closed-head or open wound, and medical emergency personnel focus on treating the wound or injury and stabilizing the patient’s vital signs.
The secondary stage of damage to the brain takes place after the initial insult, as the injury continues to ripen and worsen in the hours and days after the initial trauma. This is when the doctor says, “Now we just wait and see,” because there’s nothing more that medicine can do. In this secondary stage, the trauma to the brain triggers a series of cascading intra-cellular biochemical reactions that cause severe demise of brain cells, brain damage, and expanded disability. If this secondary stage can be mitigated, the potential damage and disability can be reduced significantly, enabling the victim to get closer to full recovery.
Some of the secondary-stage mechanisms believed by researchers to be involved in brain-cell death after TBI include uncontrolled release of signalling molecules (neurotransmitters), cellular calcium overload, inflammation, energy failure, oxidative damage, and the overactivation of enzymes such as calpains and caspases.⁹
All of these are believed to create the intra- and extra-cellular conditions that lead to the destruction of millions of additional brain cells, along with the damage and disability that result. Many of these are being targeted by a variety of pharmaceutical compounds and medical treatments that are in various stages of clinical development—including forcing oxygen into the brain through the use of hyperbaric chambers. Because it targets the protection of mitochondria inside brain cells, cyclosporine is perhaps the most promising of these.
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Pharmaceutical Approaches to TBI

There are a number of TBI pharmaceuticals in a variety of stages of development. The most promising of these approaches are “multipotential,” targeting at least two secondary-stage injury mechanisms, including excitotoxicity, apoptosis, inflammation, edema, blood– brain barrier disruption, oxidative stress, mitochondrial disruption, calpain activation, and cathepsin activation.¹
The value of multipotential agents is their potential to modulate one or more of these multiple secondary injury factors, greatly increasing the chance of achieving clinical value. Previously, more than 30 phase III clinical studies for single-factor targeted TBI pharmaceuticals failed to find significance. Multipotential agents may have a better chance of delivering a successful therapeutic result for TBI patients and, ultimately, recouping the costs of development and trials.
Promising pharmacological multipotential agents fall into two categories: those that have been studied clinically and those that constitute emerging pre-clinical strategies.
Clinically studied pharmaceuticals include the statins (targeting excitotoxicity, apoptosis, inflammation, edema), progesterone (excitotoxicity, apoptosis, inflammation, edema, oxidative stress), and cyclosporine (mitochondrial disruption, calpain activation, apoptosis, oxidative stress).
Emerging multipotential neuroprotective agents showing promise in pre-clinical studies include diketopiperazines (apoptosis, calpain activation, cathepsin activation, inflammation), substance P antagonists (inflammation, blood–brain barrier, edema), SUR1-regulated NC channel inhibitors (apoptosis, edema, secondary hemorrhage, inflammation), cell cycle inhibitors (apoptosis, inflammation), and PARP inhibitors (apoptosis, inflammation). —SC

Reference

Loane DJ, Faden AI. Neuroprotection for traumatic brain injury: translational challenges and emerging therapeutic strategies. Trends Pharmacol Sci. 2010;31(12):596–604.

Role of Mitochondria

Stick model of cyclosporine, as found in the P2₁2₁2₁ crystalline form, demonstrates the complexity of this peptide.

Research confirms that mitochondria, the cellular energy (adenosine triphosphate, or ATP) producers inside the brain cells, play a pivotal role in neuronal cell death or survival, and that mitochondrial dysfunction in brain injuries is considered an early event that causes neuronal cell death. The uncontrolled release of signalling molecules with resulting overstimulation/stress of brain cells and accumulation of high levels of intracellular calcium may be the initial mechanism that leads to neuronal cell death.¹⁰
How does this affect brain cells? Increases in calcium lead to its rapid uptake into the mitochondria, which act as cellular sinks for calcium. However, the excessive transport and uptake of calcium negatively impacts mitochondrial energy production, because the driving force for both ATP production and calcium transport relies on the “proton motive force” (the proton gradient created over the mitochondrial inner membrane by the respiratory chain). Further, excessive calcium uptake by mitochondria, in combination with energy failure, leads to the formation of protein channels (pores) in the inner membrane—the induction of the so-called mitochondrial permeability transition (MPT).
The increased permeability of the inner membrane caused by the MPT pores immediately collapses mitochondrial function and structure, because when the pores are opened, the osmotically active inner compartment (matrix) of the mitochondria attracts water, and the mitochondria swell and pop like balloons. In addition to causing the cessation of energy production, upon induction of the MPT, the stored calcium and harmful proteins are then released from mitochondria, resulting in an avalanche of further mitochondrial collapse, cellular energy depletion, and subsequent cell death. When brain cell death is repeated millions of times during the cascading biochemical imbalances that characterize the secondary phase, the extent of brain damage and eventual disability are greatly increased.
Protecting the mitochondria by targeting the MPT is a viable neuroprotective approach that has emerged in the last decade. Published research has found that the protein cyclophilin D is an essential component to opening the MPT pores and that cyclosporine binds to cyclophilin D and inhibits the induction of MPT.^11,12 The result is that mitochondria can absorb much more calcium without collapsing, allowing them to survive. As mitochondria survive to produce energy for brain cells, fewer brain cells die during the secondary stage. This is the core battleground in the war against TBI.
continues below...

What is TBI?

CRUSHED: After the initial brain injury, excessive calcium imbalances during the all-important secondary damage phase cause brain cell mitochondria to swell and burst, releasing calcium that creates a cascading avalanche of further mitochondrial collapse, cellular energy depletion, and subsequent brain cell death. By protecting mitochondria, cyclosporine limits overall brain damage and eventual disability.

BRUISED: Limiting the secondary stage brain damage that occurs after the initial injury is a key strategy in treating TBIs. Cyclosporine does this by protecting the brain cell mitochondria from collapse during the secondary stage, enabling non-injured brain cells to continue energy production and operation while recovery from the initial injury occurs.

A traumatic brain injury is defined as a blow or jolt to the head or a penetrating head injury that disrupts the function of the brain. Not all blows to the head result in a TBI. The severity of a TBI may range from “mild,” involving a brief change in consciousness, to “severe,” featuring an extended period of amnesia or unconsciousness. A TBI can result in problems with independent function, either short- or long-term.
Millions of Americans have a long-term need for help in performing their daily activities as a result of suffering a TBI. By one estimate, there are up to 6 million survivors of TBI. Statistics on the full extent of TBI are not known, however, because the number of people with TBI who were not seen in an emergency department and/or who have received no formal care cannot be determined.
The leading causes of TBI include falls, car crashes, hitting or being hit in sports, and physical assault. In war zones, blasts from roadside improvised explosive devices (IEDs) and other explosions are a leading cause of TBI for soldiers. Males are 1.5 times as likely as females to suffer a TBI, and the two age groups at highest risk are children aged 0–4 years and teenagers aged 15–19. African Americans have the highest death rates from TBI, and it is the fourth-leading cause of death for males under age 45.¹
More recently, the Iraq and Afghanistan wars have brought the issue to the attention of the public and Congress, as advances in combat protection and helmets have allowed soldiers to survive blasts that would previously have killed them.
Post injury, there is little that can be done for soldiers returning home with TBI. It’s been estimated that some 200,000 returning soldiers have varying degrees of TBI, ranging from mild to severe. Symptoms include depression, an inability to concentrate, moodiness, and frustration as the TBI sufferer struggles to complete formerly routine tasks. Moreover, much anti-social behavior exhibited in society may be related to diagnosed and undiagnosed traumatic brain injuries sustained in battle, on sports fields, on the streets, or around the home. —SC

Reference

U.S. Centers for Disease Control and Prevention (CDC). National Center for Injury Prevention and Control. Injury prevention and control: traumatic brain injury. CDC website. Available at: www.cdc.gov/traumaticbraininjury/statistics.html. Accessed Aug. 12, 2011.

Cyclosporine Protects

Cyclosporine protects brain cells by preventing the cascading biochemical imbalances of the TBI from causing the mitochondria to collapse and stop powering the brain cells, exacerbating brain damage and leading to disability.

Cyclosporine was discovered in 1969 when it was first isolated from the fungus Tolypcladium inflatum in Norway by researchers working for Sandoz (now Novartis). Its impressive immunosuppressive properties led to its use as a pharmaceutical to prevent tissue rejection in organ transplant recipients. It has been in use for immunosuppressive applications since the early 1980s as a commercially successful Novartis product called Sandimmune.¹³
CsA’s ability to protect the mitochondria in the brain by binding to cyclophilin D and preventing the induction of the MPT was discovered in 1993–1994, a period during which medical researcher Eskil Elmér, MD, PhD, and his Japanese colleague Hiroyuki Uchino, MD, PhD, were conducting experiments in cell transplantation. An unintended finding was that CsA was strongly neuroprotective when it crossed the blood–brain barrier.¹⁴ This startling discovery became the starting point for basic research and patent applications in a promising new avenue of neuroprotection.
Basic research mapping out CsA’s extensive neuroprotective capabilities has been running continuously since 1993, and many international and independent research teams have since conducted and published numerous studies confirming that CsA is a powerful nerve-cell protector in TBI, stroke, and brain damage associated with cardiac arrest. Advanced studies also show that CsA is useful in protecting mitochondria in heart tissue facing reperfusion injury during heart attacks (see sidebar).¹⁵
Together with U.S. neurosurgeon Marcus Keep, MD, Dr. Elmér and his colleagues formed a company with the aim of commercializing and patenting their work of developing cyclosporine-based products for acute conditions and diseases affecting the brain. In 1999, the U.S. patent was approved and, in 2000, their CsA product name, NeuroSTAT, was registered. Later, the patent portfolio around CsA’s impact on the central nervous system and other areas was expanded greatly under their company, NeuroVive Pharmaceutical AB (Sweden).

Cyclosporine acts to protect the brain cell’s mitochondria from the cascading biochemical imbalances that cause these cellular power sources to collapse and stop powering millions of brain cells. This reduces the additional brain damage and disability that occurs during the secondary damage phase of TBI.

Today, NeuroVive’s NeuroSTAT version of cyclosporine is a fully developed product. An important advancement in NeuroSTAT is that its formulation is made using a patented non-allergenic lipid emulsion to keep CsA a lipophilic drug in solution.

Next Steps

It’s been almost two decades since Dr. Elmér and his colleagues discovered cyclosporine’s neuroprotective capabilities, and there is still some way to go. However, CsA’s promise as a TBI pharmaceutical continues to make progress. Full commercialization is in sight.
In 2010, NeuroSTAT received orphan drug status from both the U.S. Food and Drug Administration and its European counterpart for the treatment of moderate and severe TBI. In March, the company announced it would be working with the European Brain Injury Consortium to conduct a phase II/III adaptive study on NeuroSTAT.¹⁶ These clinical trials should provide the basis for the registration of NeuroSTAT in Europe, and possibly in the U.S. and other countries. U.S.-based clinical trials are also being planned, and NeuroVive is seeking partnering organizations in China for similar trials. Of course, the challenges in such development, where many drugs have failed in the past, involve translating the research results into clinical benefits in patients and recruiting a sufficient number of patients within a reasonable time.

Cyclosporine is isolated from the fungus Tolypocladium inflatum. In the early 1990s, NeuroVive’s chief scientific officer Eskil Elmér and his Japanese colleague Hiroyuki Uchino discovered cyclosporine was strongly neuroprotective when it crossed the blood–brain barrier.

Assuming all goes according to plan, cyclosporine’s early promise from its serendipitous discovery as a neuroprotectant in the 1990s could be fulfilled within the next two to five years. And neurologists and neurosurgeons worldwide will finally be able to trumpet an exciting new weapon in the war against the silent epidemic of traumatic brain injuries.

References

U.S. Centers for Disease Control and Prevention (CDC). National Center for Injury Prevention and Control. Injury prevention and control: traumatic brain injury. CDC website. Available at: www.cdc.gov/traumaticbraininjury/statistics.html. Accessed Aug. 12, 2011.
Hoge CW, McGurk D, Thomas JL, et al. Mild traumatic brain injury in U.S. soldiers returning from Iraq. New Engl J Med. 2008;358(5):453-463.
Sullivan PG, Sebastian AH, Hall ED. Therapeutic window analysis of the neuroprotective effects of cyclosporine A after traumatic brain injury. J Neurotrauma. 2011;28(2):311-318.

Fluoropolymers High Purity Acid Handling

2011-08-15T03:02:00.000-07:00

Fluoropolymers survive acidic environments, but extractables must be examined.
For several years many companies that process or utilize high purity acids in the semiconductor industry have settled upon fluoropolymers as a material of construction to ensure that limited amounts of impurities could be leached into the acids.¹
Fluoropolymers offer an advantage over other polymers since they are often manufactured in such a manner that no foreign additives that later could become extractables are needed for processing stability.² Fluoropolymers have advantages over metal in that they are not subject to chemical change (oxidation, rouging, etc.) and hence, will not have greater particle generation over time. In other words, a fluoropolymer’s greatest period of leaching is the initial week of installation whereas a metal will become a greater source of leachate over time.^3,4
A large number of case histories (ten of thousands) exist in the general chemical industry for the use of polyvinylidene fluoride (PVDF, PVF2), polytetrafluoroethylene (PTFE), and perfluoroalkoxy (PFA) resins. These materials of construction have many applications in strong acids even at high temperatures, and as long as they are used within the manufacturer’s recommended temperature ranges for the named chemical, these resins have proven success for over 25 years.^5,6,7,8,9
With all of this history, answering the question as to whether the polymer will stand up to a chemical and temperature environment is easy. The less obvious part of the equation is whether the fluoropolymer could be adding extractables to high purity versions of semiconductor-grade acids? For example, it is well proven in deionized water testing that a polymer simply maintaining its physical properties in exposure to water does not necessarily qualify it as a fluid handling component for high purity application due to extractables that can be emitted as high as part per million (ppm) levels.¹⁰
High Purity Acid Testing:
HNO₃, HF, H2SO₄, HCl
Data generated on extractables in this study involved high purity water (ionic extractions ), HNO₃ (70%), HF (49%), H₂SO₄ (96% and 20%), and HCl (37% and 30%). Other extractable data in acid has been generated on fluoropolymers and published in earlier works.¹¹ Due to the permeation effect of polarity of the polymer and chemical being tested for extraction, the aggressiveness of a particular acid for certain elements, and the molecular size of an acid molecule plus additional water in its composition, it was thought to test four different acids to best develop a cross section of data.
The fluoropolymers selected were emulsion type PVDF (hereafter referred to as E-PVDF) and a designated specific high purity (HP) version of PFA (hereafter referred to as HP-PFA). Both of these polymers are commonly used by professionals in the above acids in general chemical containment as well as in high purity applications.

Test Methods: IC, ICP-MS, TOC
The scope of this data generation was to compare resins in their raw form to negate the effects of processing. Since processing could be largely dependent on the quality and cleanliness of a manufacturer, pellets were used in the corresponding method of extraction. The authors thought it could be unfair to represent sticks of tubing from one PVDF processor and one PFA processor when there are several established processors of each resin type commercially promoting the product lines. Designers should be cautioned that the same resin molded or extruded by different processors could have widely different results depending on handling techniques utilized by the processor.¹²
The test methods used to provide data were:
* Ion Chromatography (IC): leachable ions and leachable anions in high purity water
* Inductively Coupled Plasma/Mass Spectroscopy (ICP-MS): leachable elements in high purity acids.
* Total Oxidizable Carbon (TOC): TOC measured from acid exposure.
Extraction Results and Discussion
* Water Testing. More than 20 years of data exists on PVDF and PFA in water.^{13, 14}The water industry has settled, that for rigid piping and components, PVDF is more than suitable to handle 18 megohm/cm water and is typically chosen over PFA due to strength considerations and overall costs.^{15, 16, 17} Tables 1 and 2 list IC results for leachable anions and leachable cations from 100 ml of ultrapure water at ambient conditions in exposure of 1 gram of pre-cleaned HP-PFA and E-PVDF for 7 days. The results are given in ppb (ug/L) and in neither case were extractables even near 1 ppb for any ion tested.
* Acid Testing. TOC extractions are a consideration for high purity acid in that while bacteria typically will not survive in strong acidic environments, TOCs themselves added to the acid from fluid handling components act as system contaminants. TOC measurements were generated after 7 days exposure to 49% HF, 96% H₂S0₄ and 20% H₂S0₄ at ambient temperature. The results are listed in Table 3. Since the control blank measurement in HF was above the detection limit, it is appropriate to consider actual extractions from the tested resin to be less than the measured value. PFA and PVDF TOC extractable results were in the part per billion range.
ICP-MS was used to determine the extractable contributions of HP-PFA and the emulsion PVDF resin in contact with strong acids for 50 elements for 7 days at room temperature. The data was generated by using 1 gram of polymer to 150 grams of chemical. Extraction results are presented in Tables 4, 5, 6, 7. No data is listed for the control blanks for each chemical because the laboratory reported only a few measured leachables attributed to the blanks (0.1 ppb Al in the 37% HCl blank; 1.0 ppb Al and 0.2 Na 96% H₂SO₄ blank).
Discussion of Results
The testing in acids by IC, ICP-MS, and TOC confirm, in a test with a high ratio of contact surface to liquid volume, that both the PFA grade tested and the PVDF grade tested yield extractables below the part per million level in total cumulative leachate. TOC was the highest contributor of measured extractions for the three acids tested.
PFA does not change color in long term acid exposure and has gained wide use based on the above performance and the aesthetic nature of the color stability attributed to this resin. The E-PVDF (ASTM D3222, Type I, Class 1 and 2) used in this test does not substantially change color over time when exposed to this set of acids. Some commercial PVDF resins do change color substantially when exposed to concentrated hydrochloric acid, concentrated sulfuric acid and even water over time and this has created industry caution in specifying the less expensive PVDF fluoropolymer for use in some high purity facilities. The scope of this study did not include extraction tests on such color sensitive suspension type PVDF (ASTM D3222 Type II) resins.

Throughout the testing, the elements detected from each resin were consistent. In no case were the following elements detected in this test: Sb, As, Be, Bi, B, Li, Hg, Nb, Pd, Pt, Rh, Rb, Ru, Sc, Se, Si, Ag, Ta, Tl, Th, U, V, and Zr. In most cases, calcium was the largest leachate contributor from HP-PFA, followed by sodium and potassium. In all cases, sodium was the largest leachate contributor from E-PVDF and usually calcium was the second highest contributor.

Nitric acid seemed to be the most effective extractive media for both resins, but none of the acids were able to extract above 150 ppb elemental leachate from either fluoropolymer in any test. In analyzing this data at such a minute level, the reviewer must understand that these extractions are typically non-exact. For example, at this level of sensitivity, 0.3 ppb and 0.1 ppb are essentially the same number and within limits of error. One would not say that one product is three times better than the other, but with enough data, trends can be determined as to the frequency expectations of finding various elements in any tested polymer. In other words, the data developed in this study does not appear to suggest that either resin is largely superior to the other in any of the test acids, but perhaps the data will help in understanding what can be looked for when attributing low level extractables to these two fluoropolymers.
Historical testing and conventional wisdom support that continued testing on the same samples would lead to lower extractions each time a new rinse is performed. This is a great advantage of the use of plastics for high purity fluid handling. The plastic materials only contain a finite amount of entrapped extractables and because the materials are not subjected to a corrosion rate as would be typical for a metallic product, the extractions do not increase or have unpredictable changes over time.¹⁸
References
1 Parker, Kevin. “Containers Prevent Product Contamination, Allow Safe Handling of Hazardous Materials,” Chemical Processing, pp. 43-48 (May 1991).
2 Holton, James; Seiler, David; Fulford, Kenneth; Cargo, James T. “Extractable Analysis of Modified PVDF Polymers Utilized in DI Water Applications,” Ultrapure Water, pp. 47-52 (May/June 1993).
3 Banes, Patrick H. “Fundamentals of Passivation in Water Systems,” Ultrapure Water, pp. 60-69 (April 1998).
4 Burkhart, M.; Klaiber; F. Wermelinger, J. “Is Polyvinylidene Fluoride Piping Safe for Hot Ultrapure-Water Applications?,” Microcontamination, pp. 27-31 (February 1995).
5 McCallion, J. “PVDF Exchangers Thrive in Pickling Acids,” Chemical Processing, pp. 59-61 (September 1995).
6 Fusco, Joseph C. “Plastics Developments Advance Pipe Performance and Safety,” Chemical Processing, pp. 48-52 (December 1990).
7 Sixsmith, Tom. “Selecting the Right Plastic Piping System,” Plant Services, pp. 16-18 (September 1991).
8 Dennis, Gary. “Picking the Best Thermoplastic Lining,” Chemical Engineering, pp. 122-124 (October 1998).
9 Glein, Gary A. “Dual-Laminate Tanks and Piping,” Chemical Processing, pp. 82-85 (February 1996).
10 Hanselka, R. Williams, R,; Bukay, M. “Materials of Construction of Water Systems - Part 1: Physical and Chemical Properties of Plastics,” Ultrapure Water, 4 (5), pp. 46-50 (July/August 1987).
11 Mikkelsen, Kirk, J. Alberg, Michele J.; Prestidge, Janice K. "Comparing the Purity of Commercially Available Fluoropolymers,” MICRO, pp. 37-48 (June 1998).
12 Patrick, Frank N. “High Purity Product Qualification Using Electron Spectroscopy for Chemical Analysis (ESCA) Methodology,” Ultrapure Water, pp. 54-58 (October 2000).
13 Goodman, J.; Andrews, S. “Fluoride Contamination from Fluoropolymers in Semiconductor Manufacture,” Solid State Technology, pp. 65-68 (July 1990).
14 Balazs, Majorie K. “A Five Year Study Using PVDF Pipes in an Ultrapure Water System,” ISPEAK Newsletter (July 1997).
15 Henley, M. "PVDF Remains Favorite Piping in Semiconductor Plants,” Ultrapure Water 14 (10), pp. 16-22 (December 1997).
16 Governal, Robert A. “Ultrapure Water: A Battle Every Step of the Way,” Semiconductor International, pp. 176-180 (July 1994).
17 Wulf, Brian "Pristine Processing - Designing Sanitary Systems,” Chemical Engineering, pp. 76-79 (November 1996).
18 Meltzer, Theodore H. “Extractables from PVDF Piping Systems Conveying High Purity Waters,” Pharmaceutical Technology (March 1997)

BENEFICIAL CONTAMINATION: PART 2

2011-08-15T03:01:00.000-07:00

BENEFICIAL CONTAMINATION: PART 2
Last month we discussed how, under certain circumstances, a little contamination can be good for you. Commercially-produced heart valves of an alloy primarily of cobalt, chromium, molybdenum, and tungsten, Stellite 21, were nonthrombogenic; they did not induce potentially deadly clots because an easy-release hydrocarbon coating had been introduced as a manufacturing artifact. Two techniques, internal reflection infrared spectroscopy (Multiple Attenuated Internal Reflection, or MAIR) and critical surface tension, were used to study and monitor essential surface characteristics and, in effect, detect beneficial contamination.
MAIR allows analysts to obtain a fingerprint of films as thin as 10 A. Internal reflection spectroscopy involves placing the surface of interest in contact with an internal reflection plate. A beam of light is directed toward the plate in such a manner that it repeatedly reflects inside the plate where it contacts the sample surface; an augmented IR scan is obtained. Some clinicians prefer IR measurements over high vacuum techniques such as Electron Spectroscopy for Chemical Analysis (ESCA), which may provide more definitive identification of molecular species at a specific location on the sample than does IR spectroscopy; but because ESCA involves placing the sample in a vacuum chamber, there is the nagging concern that certain materials on the surface may “turn away” from the surface toward the bulk or even vaporize. While MAIR does not provide complete molecular identification, the infrared scans indicate functional groups, like methyl groups. In the case of the metal heart valves, identifying part of a molecule was sufficient. Methyl groups were found on the surface of vigorously polished, non-thrombogenic metal implant material; and, while vigorous aqueous cleaning had little effect, heavy mechanical scrubbing eroded the surface.
The second technique, critical surface tension (CST), is related to contact angle measurement, which is a refinement of the water-drop test. Contact angle measurement provides an indication of non-specific organic contamination. The contact angle between a liquid droplet and the surface is determined by the nature of the gas/liquid/surface interface. Looking beyond water, the contact angle is also influenced by the quality of the liquid with solutions being less accurate than pure compounds and actual material solutions being worst of all because they attack the surface. Surface quality of a variety of materials from paper to metal have been grossly characterized by marking the surface with dyne pens, which look at bit like magic markers and contain various solvents.
In the study of surface quality of heart valves, researchers determined the critical surface tension (CST) of a solid as measured in dynes/cm. CST, the highest surface tension any liquid (actual or theoretical) can have and still completely wet the surface of the solid, involves measuring contact angles for as many as 16 liquids of known surface tension. The surface tension of each liquid in dynes/cm is plotted on the x axis and the cosine of the contact angle on the y-axis (a Zisman plot). The CST of the solid is surface tension where the cosine of the contact angle equals one.
CST characterized the desirable surface quality of the heart valve material. The CST of the properly contaminated alloy, the alloy polished with organic-based compounds, was 20 to 25 dynes/cm. Alloy polished with organic-free abrasives had a CST of over 35 dynes/cm. Alloy polished with organics but then detergent-scrubbed also showed an increased CST of 27 dynes/cm. Contact angle measurements predicted performance of the implanted device. Lower contact angles, for example, were obtained if the coating was applied with rigorous polishing but with incomplete coverage or if the coating was applied without rigorous polishing and then water-washed. In both instances, devices were thrombogenic.
What can we extrapolate from these studies? First, the mere presence of material on a surface does not necessarily mean that it is a contaminant; it may be essential to proper performance. Further, surface characterization techniques should be selected because they provide the most useful information, not necessarily complete identification. While MAIR-IR does not provide complete molecular identification, it gives enough information about the immediate surfaces of implantable clinical devices, without the concern of altering the surface during sample preparation. Similarly, the overall indication of contamination using CST was predictive of surface performance. Finally, analytical and surface testing were used not dogmatically but rather pragmatically in conjunction with actual performance studies, in this case, in living systems.

Making Informed Choices in Wet Bench Fire Safety

2011-08-15T03:00:00.000-07:00

Making Informed Choices in Wet Bench Fire Safety
As the costs and consequences of failure grow, so does the list of fire-safe, approved materials.
In the precision manufacturing and process industries, the term “wet bench” generally refers to cleanroom process equipment that contains, dispenses, rinses, or in some manner processes or utilizes corrosive chemicals. The sheet plastics traditionally used in the construction of wet benches, polypropylene or polyvinyl chloride (PVC), provided good construction properties and corrosion resistance along with relative ease of fabrication and welding. These early materials, however, did not offer a high level of fire or flame resistance. The introduction of fire retardant polypropylene in the mid-1990s improved the flame resistance of wet bench materials.

Changing Standards
Until recently, the industry relied on UL94-V0 and V5a and ASTM E-84 standards to evaluate the performance of plastics when exposed to flame. UL94 measures the flammability of plastics used as components in appliances and other devices. E-84, also known as the Steiner Tunnel Test, measures the tendency of building materials to spread flame and produce smoke. None of these standards addressed specific concerns about plastics used in cleanrooms.
Risk underwriters and insurers, facing financial losses from major fires in wafer fabrication plants, demanded a reduction in fire risk from their insureds. Wet bench plastics were identified as culprits in accelerating flame spread and worsening damage to work in process and equipment from excessive smoke. In 1997 Factory Mutual Research Corporation (FMRC) released a new standard to measure the performance of plastics in these areas. This standard, titled Cleanroom Material Flammability Test Protocol 4910, references a fire propagation index (FPI) to measure a plastic’s fire propagation potential and cites a smoke damage index (SDI). Together, these indices are used to evaluate a plastic’s suitability for cleanroom use.
Using the FM4910 standard, plastic sheet manufacturers began to develop and modify sheet formulations to meet it. Submitted products were tested by FMRC. Successful candidates were given official listings as compliant with FM4910. In 2000 Underwriters Laboratories (UL) released |L2360, Test Methods for Determining the Combustibility Characteristics of Plastics Used in Semi-Conductor Tool Construction. This protocol also measures the fire propagation and smoke generation qualities of wet bench plastics. In addition, it provides a class rating of 1, 2, or 3, depending on the material’s FPI and SDI levels. Manufacturers have submitted materials to UL for testing and several sheets and resins have been UL2360 listed.

Spreading the News
With more options for wet bench construction, material selection has become problematic. For those buyers/specifiers whose insurers favored a protocol (FM or UL), their choices are limited to the listed fire-safe materials. In other cases, the end user has mandated fire-safe plastic use. In some municipalities the authority having jurisdiction (AHJ) has stipulated fire-safe plastic use or compliance with a protocol or industry standard. Yet there are also those situations in which no clear-cut directives are in place for the buyer/specifier that would limit their choices. Wet benches made from polypropylene and FRPP are still being built and installed in retrofitted and newly constructed fabs.
Dissemination of information about fire-safe plastics has been a slow process. Industry associations such as SEMI, Semiconductor Industry Association (SIA), Semiconductor Safety Association, National Fire Protection Association and others have reviewed the FM4910 and UL2360 protocols. In some cases they have endorsed them or included them in their own standards.
Adoption of FM4910 and UL2360 into building codes is slowly under way. Fire and building approval professionals learn about new materials through official documentation, their membership in relevant organizations, their industry contacts, the reading of published data and sales materials, and through their personal research.
The promotion by FM and UL of their own standards has become a primary source of information. The publishing and announcement of their protocols are important methods for tooling buyers, specifiers, builders, AHJs and interested third parties.

Choosing the Right Material
At best this objective approach provides the designer/builder with merely a list of approved materials. Still to be resolved is how to determine which listed materials are best suited for the wet bench in question. Three significant factors must be considered to help narrow down the choices:
* Functionality. The types of process chemistries which will come in contact with the wet bench must be examined to ensure that the appropriate fire-safe plastics are used. In some cases, tooling models offered by manufacturers may be used with a variety of chemistries. Thorough research will ensure that the material selected is compatible and rugged enough for all intended applications. The average workload of the installed bench may also affect the decision of which sheet material to choose. The conditions in which the bench will be located are a key factor. A hostile manufacturing environment may require a more durable, corrosion-resistant cabinet than if the only process chemistries contact were in the wetted areas. A trend toward hybrid tools has developed among equipment designers. In such units the shell is constructed from a fire-safe but less expensive cabinet-grade material.
* Budget. Opinions vary about the relative cost of wet bench plastics. In the overall cost of a tool the plastic sheet content may represent only 15-30% of the total expense. This is not to say that the comparative increase from one fire-safe material to another is not relevant or important, however. When multiple benches are constructed, significant savings may be realized when materials are selected according to performance requirements. The hybrid bench built from cabinet-grade materials (outer shell) and process chemical-grade materials (wetted areas) can be both cost-effective and well suited to the cleanroom environment.
* Builder Selection. The importance of which tool builder is selected cannot be minimized. Experience, technical expertise, reputation, and quality are important considerations. Judgment based on hands-on knowledge of fire-safe materials is invaluable. A savvy fabricator can save money, time and labor and build in tool longevity by helping select the best plastics for the job.

Communication Channels
There are many ways specifiers, designers, builders, AHJs, or third parties with approval responsibilities, can find up-to-date fire-safe plastics information in trade journals, at trade shows, and on vendor websites. If one were to ask wet bench fabricators, specifiers, buyers, and especially plastic sheet manufacturers, which information source has the greatest impact on fire-safe product selection, it would be the FM and UL websites. Proof that a resin, compound or sheet is indeed fire-safe must be documented by a listing on the respective websites. Until the product is posted on the FM or UL site, even with the written test and listing results in the manufacturer’s hands, the industry is reluctant to accept it.
The FM Global website, www.fmglobal.com, encompasses more than 1000 pages with information about its standards, insurance services, and its resource center. While its main function is to help its clients prevent and control property loss, it also provides information for customers, including manufacturers of listed products. A downloadable listing of FM4910 materials (40, as of this writing) is included the Sidebar.
The UL website, www.ul.com, unlike the FM Global site, does not offer a unified posting of all UL2360-listed products, but instead offers search capability by company name and product number.
It is clear that the web has become a viable, vital research, sales, and teaching tool. An internet presence is now a necessity; since 1995, when many companies were either starting their websites or merely thinking about the idea, the number of sites has exploded exponentially. The ways we use the web now to make decisions about fire-safe plastics, for example, have changed how we do business and on which experts and data sources users rely.

For Further Reading
FM4910: Cleanroom Materials Flammability Test Protocol, September 1997, Factory Mutual Research, 1151 Boston Providence Turnpike, Norwood MA.
NFPA318, Standard for Protection of Cleanrooms, 2000 Editions, National Fire Protection Association, 1 Batterymarch Park, Quincy MA.
“Process Compatibility Parameters for Wet Bench Plastic Materials,” International Sematech, 2706 Montopolis Drive, Austin, TX.
SEMI S2-0200, Environmental, Health & Safety Guideline for Semiconductor Manufacturing Equipment, Semiconductor Equipment and Materials International, 805 E. Middlefield Road, Mountain View, CA.
UL2360: Test Methods for Determining the Combustibility of Plastics Used in Semi-Conductor Tool Construction, Underwriters Laboratories, Inc., 333 Pfingsten Road, Northbrook IL.
“Wet Bench Fire Safety: Update,” A2C2, February 2000, pp 17-19.
“Wet Bench Fire Safety: One Issue Fizzles, New Ones Ignite,” A2C2 February 2001, pp 19-26.

Fundamentals of Cleaning: Drying, Part 2

2011-08-15T02:59:00.000-07:00

Evaporation rates are slow, we learned, because the rate of heat transfer from air to parts is quite low because gas-solid heat transfer coefficients are so poor. In last month’s column, a figure showed how these coefficients have little dependence on temperature, but de-pend greatly on air velocity.
Is heat transfer coefficient the whole story? No, indeed. It is the rate at which heat can be transferred from the moving air stream to the water which controls drying rate. What’s the difference be-tween coefficient and rate? The defining equation is:
heat transfer rate = (heat transfer coefficient) x (part surface area) x (temperature difference)
The units are:
BTU/hr = (BTU/hr - ft2 - °F) x (ft2) x (°F)
Temperature difference is that between the free stream air and the water on the parts.
A key point is that heat transfer coefficient has little dependence upon air temperature, but heat transfer rate is highly dependent upon air temperature, and that rate limits drying rate. Let’s examine some practical situations. We’ll use 10 ft2 for surface area and ambient temperature (75°F) for water temperature on parts. Remember, that the heat of evaporation of water is about 1000 BTU/lb. Figure 1 of this column will control.
The same format is shown above for heat transfer rate. Suppose we need to evaporate ~10 lb/hr of water. The heat transfer rate is ~10,000 BTU/hr.

Which should we prefer?
From the standpoint of utility costs, the one with the highest free air velocity will be significantly cheaper. It costs much more to heat air than to make it flow. How about part integrity? Some parts can’t stand temperatures of up to 350°F. Plastic components may warp. Metal components may be damaged by unwanted chemical reactions. From the standpoint of particle contamination, the least air flow is the better choice.
There are two aspects to this point. First, suppose this operation is done in a cleanroom. Then particle loading is probably light, and it is of sub-micron particles. The velocities required for drying of parts are at least 10X higher than would be observed at the face of a filter. A higher air velocity, or volumetric flow, will contact the presumably cleaned part surface with more sub-micron particles. Thus the good cleaning work may be negated.
Second, suppose the operation is not done in a cleanroom. These air velocities, 25 to 50 ft/sec, are large and will convey large, small, and sub-micron particles onto the cleaned part surface. The velocity in the center column of Table 1 is equal to the calculated terminal velocity for a particle whose size is given in the right column of that table. Here, the cleaned part surfaces become infected with particles of all sizes.
There are valid reasons to chose both high and low air velocities, depending upon what will happen to your parts after processing.

Vacuum drying, another method of evaporating water, can resolve the dilemma. Vapor pressure and vaporization temperature are correspondingly reduced. Hence evaporation rate is enhanced without having to use high air velocities.

Drying can be done by converting the liquid water to a solid, liquid, or a gas. One saves time and money by using impact-based methods to remove liquid water before using evaporation. The latter is expensive and can make clean parts dirty.

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