Wet Behind the Ears

By Barbara Kanegsberg, Ed Kanegsberg

Most cleaning processes involve a fluid. The purpose of the fluid is to get the cleaning agent to the areas of surfaces that are contaminated and then carry the contamination or soil away from the surface. One parameter that determines the efficacy of a cleaning fluid is how readily the cleaning agent contacts the surface. Does it really get in there to do the job of soil removal? This is especially important when the cleaning agent needs to fit into small holes or spaces to reach the soils. The contact of the fluid to the surface is the process of “wetting.”
WETTING INDEX
The wetting of a surface depends on characteristics of both the fluid and the surface. The Wetting Index (WI) was developed by Bill Kenyon in the 1980s (perhaps even earlier) as a means of comparing the ability of various cleaning agents to wet a surface and to access tightly-spaced components.
WI = 1000 x Density / (Viscosity x Surface Tension)
WI is based on the physical characteristics of a solvent or cleaning agent. It was originally designed as a teaching tool to help electronics assemblers choose an effective cleaning fluid. Over the years, based on empirical observations, it has proven to have much broader utility.
UNDERSTANDING WI
The WI is largely intuitive. For a fluid to wet a surface effectively, it must flow easily and get into any tight spaces. The lower the viscosity of a fluid, the easier it flows. If the surface tension is high, it is hard for the fluid to penetrate a small space. Therefore both these parameters appear in the denominator of the formula. Explaining the idea that wettability is directly proportional to the density is a bit more difficult. Because we were having trouble expressing the rationale in one phrase, we contacted a colleague, a very experienced formulator¹ who explained that density appears in the numerator because denser fluids exert a higher pressure on the surface, making it easier to drive a liquid into tight spaces. In a sense, density is acting as a molecular version of macro methods of increasing fluid pressure, such as agitation and spray.
Table 1² contains a number of common cleaning fluids with the parameters that contribute to the Wetting Index. Molecules with large polar and/or hydrogen bonding forces,³ such as water, tend to have higher viscosity and surface tension, thus resulting in a low WI. Surfactants are frequently added to water based cleaners to reduce the surface tension and thus increase the wettability. However, water with surfactant is not as effective at wetting as many organic solvents, which have little or no polar bonding. There is also the issue of rinsing, of removing the cleaning agent additives that may themselves become contaminants. Pure water has a lower wetting index and therefore will not be as effective as the aqueous cleaning agent in accessing blind holes.

For a given fluid, one parameter that can be adjusted to improve wettability is temperature. The viscosity of liquids decreases with increased temperature. For example, water is 3.6 times less viscous at 100°C as at 20°C.
Wetting Index eludicates part of the story but not the whole story, because it describes only the properties of the wetting fluid. The WI is a measure of the upper limit of wettability. The nature of the surface being wetted is not part of the WI equation; the molecular forces between the surface and the wetting fluid play a strong role in how effective the wetting of the surface will be. The most familiar examples of surface effects in wetting are illustrated by the Water Drop test and Contact Angle measurement to describe how well water wets a surface. If the surface forces are primarily non-polar, as in the case of an oil coating, the surface does not attract the polar water molecules. Water will bead into drops and exhibit a high contact angle, rather than flow or wet the surface effectively.
While successful residue removal requires a cleaning agent with appropriate wettability, it is not sufficient. Designing the cleaning process requires understanding of multiple factors as well as experience. This means that assigning development of critical cleaning processes to someone who is, in fact, “wet behind the ears,” may not be the wisest move.
Getting the fluid to the destination is crucial, but the real question of importance is, does it clean? This is where such issues as agitation, impact force or pressure, temperature, and solubility enter the picture. For example, while many hydrofluoroethers (HFEs) and hydrofluorocarbons (HFCs) have a very high wetting index, the solvency characteristics are not favorable for removing most industrial soils of interest. To fully characterize cleaning is a complex mixture of the properties of the cleaning agent, the method of application of the cleaning agent, the soils and the substrate surface. Parameters such as Wetting Index can help in narrowing the choices, but there can be no substitute for actually testing the efficacy of cleaning.
References:

1. K. Dishart, personal communication.
2. B. Kanegsberg and E. Kanegsberg, “Defluxing for New Assembly Requirements,” On-Board Technology, Nov 2011.
3. J. Burke, “Chapter 4: Solvents and Solubility,” Handbook for Critical Cleaning: Cleaning Agents and Systems, Second Edition,” CRC Press, B. Kanegsberg and E. Kanegsberg, Editors (2011).

A systematic approach to educating pharmaceutical employees

Achieve the Holy Grail of Training Effectivene

A systematic approach to educating pharmaceutical employees

As a pharmaceutical industry training professional, I am regularly asked, “How do we make training more effective?” It’s an important question, but it doesn’t have a simple answer.
Training touches most of what we do as an industry. Our operations are based on two systems—training and documentation—and everything we do hinges on our people appropriately executing our procedures. Training is what ties them together. It affects everyone in our businesses, how well our businesses run, and what results we’re able to obtain. And we know it’s not working when we see large numbers of deviations and increasing manufacturing costs.
Training effectiveness results from an effective training process—probably the least understood process in our industry. Our industry values meaningful numeric data, and we use it to define, describe, and understand our processes, but it’s difficult to identify and provide meaningful numeric data that defines and describes a training process—especially if the process is suboptimal. Worse, many of the outcomes of training hinge on an individual’s motivation and ability to learn, characteristics for which numeric data doesn’t exist. It’s not impossible to generate data around the training process, but it tends to be difficult. Anecdotal data from around the industry on evaluation practices shows that we typically focus on happiness with the training and/or whether the trainees possess knowledge, rather than on the success and effectiveness of our process.
The regulations don’t help us much, either. The FDA provides sparse guidance on training in the regulations, and what the agency does provide, while helpful, are only criteria to be met, like:

• “Each manufacturer shall have sufficient personnel with the necessary education, background, training, and experience to assure that all activities...are correctly performed” 21CFR820.25(a)¹;
• “Each manufacturer shall establish procedures for identifying training needs and ensure that all personnel are trained to adequately perform their assigned responsibilities” 21CFR820.25(b);
• “Training shall be in the…operations the employee performs…including the cGMP regulations and written procedures…as they relate to the employee’s functions” 21CFR211.25(a)²;
• “Training...shall be conducted by qualified individuals on a continuing basis” 21CFR211.25(a); and
• “Training shall be documented” 21CFR820.25(b).

Even if we meet these requirements, there’s no guarantee that our training is effective. And regulators don’t often look deeply into our training effectiveness, as long as our process runs without many deviations and product safety is assured. Training even receives a somewhat cursory review in inspections unless a particular weak spot is identified.
In reality, “training effectiveness” results from a combination of two things: a training system that includes all of the elements of an effective training process and processes that support the training system. Both must be present and working together—training effectiveness suffers if either of these elements is missing.

Elements of a Training System

To develop role-based curricula and training plans, we need to know who is responsible for performing which roles and what baseline knowledge, experience, and skills are required.

To ensure its effectiveness, we must first know what training is needed—our curricula. In simplest terms, curricula define groups of requirements to be met. We should define curricula for all employees, including requirements for onboarding (corporate, site, and departmental), ongoing GMP and safety training, and task training. Curricula should also define professional development requirements appropriate for specific positions or levels, such as leadership development training for management or specific certifications required for IT, engineering, and project management positions. Requirements for specific tasks and roles should be relatively standard; we should not define requirements based on a specific person. Finally, we should express these as role-based curricula for all levels of employees, including senior management and contract employees.
Next, we need to determine when and how to provide training and capture this information in training plans. Our departmental training plan should document the progression of tasks in the development of an employee in that role and department, and it should be structured such that training builds on previous skills acquired earlier in the development process. Then, in alignment with our defined curricula and training plans, but based on the individual’s specific experience and needs, we define an individual training plan, tailored to meet these needs. (In many cases, tailoring requirements to an individual happens through performance management and/or a personal development plan.) The combination of departmental and individual training plans allows for flexibility while individuals learn the skills to perform the department’s tasks. What each individual needs to be able to meet the requirements may vary, but the requirements and expectations should be the same for anyone performing the same task.
In order to develop role-based curricula and training plans, we need to know who is responsible for performing which roles and what baseline knowledge, experience, and skills are required. Combine that with the information used to build curricula and training plans, and we’ve identified the need for job descriptions—truly the starting point of our training system.
A job description should exist for each position, defining the role and tasks performed, as well as describing the level of performance required. Defining these expectations enables all the other elements of the training system. Job descriptions also allow us to assess an individual’s knowledge, skills, and experience against defined criteria, which helps to avoid putting individuals into positions for which they are unprepared. Organizational charts visually structure job descriptions to show reporting relationships, for the purpose of determining accountability for functions and operations. Because neither of these are currently GMP requirements in the U.S., we often consider them a non-GMP HR function; however, the EU GMPs require job descriptions and organizational charts—and a company marketing products in the EU is expected to have them.³
Because our curricula identifies who to train in what areas, and because job descriptions describe what employees need to be able to do when trained, we can now provide appropriate training activities. Training effectiveness discussions typically focus on training activities because they’re the most visible piece of the process, and we usually base our decisions on the delivery of the training activity and whether or not it met expectations. But basing judgment of our training system on this element alone overlooks the many other factors critical to our ability to provide effective training! The quality of the training activities we design, develop, and deliver directly affects training effectiveness, but the quality of training results from the combined outputs of three different process elements: instructional design, accurate content, and qualified trainers.

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FIGURE 1. Components of an effective training process.

Instructional design allows us to define and build appropriate training. It is a specialized skill set that enables an instructional designer to break a task into its multiple components and determine how to effectively teach and assess each one. It begins with a needs analysis, which identifies what the training must include and accomplish—and whether training is actually needed at all. Then, the instructional designer, usually in conjunction with a subject matter expert, builds the content and practice activities and determines the most appropriate type of assessment based on the intended use of the content.
Effective training must provide accurate content. During the development process, a subject matter expert, and quality assurance where appropriate, should review training materials for accuracy. This should be part of a training content approval process, which should also include a system to control the approved content and any changes that have been made to this content. Where approved content is used on a continual basis, it should undergo periodic review and should also be reviewed when system or process changes are made, to ensure continued accuracy and appropriateness.
Next, qualified trainers must deliver our training. Qualified trainers should be subject matter experts who are able to properly perform the task, answer questions about it, and assess trainee performance. They also need interpersonal and training delivery skills. Where they deliver training will define the skills sets they need; providing one-on-one task training in an operational environment is very different than providing group training in a classroom. Trainers without training skills are just as detrimental to training effectiveness as those who have no knowledge of the content or process for which they’re providing training.
Once we’ve delivered the training, we need to ensure that it accomplishes our desired outcomes. To do this, we assess and evaluate both the trainee and the training program itself. When we assess and evaluate, we want trainees to demonstrate that they have the desired knowledge, have developed and can use the skills required to perform tasks, and, where necessary, can react appropriately to different situations. The most common assessment is a test that demonstrates knowledge, but those don’t allow trainees to demonstrate their ability to perform appropriately. The training program is typically evaluated using Donald Kirkpatrick’s Kirkpatrick Model, which helps us determine whether or not we obtained the desired results, and if not, what areas to investigate to understand why we didn’t and what to do about it.4
Last, we need to be able to provide records for our training system. We commonly think of training records as proof of training completion, but we should also include content approvals and change control, assessment results, curricula, training plans, and more. These records help demonstrate that the system is effective and training is happening appropriately, as well as providing proof of qualification to auditors.

What Processes Support the Training System?

If there is any doubt regarding management’s role in and effect on training, the FDA’s “Guidance for Industry: Quality Systems Approach to Pharmaceutical CGMP Regulations” clearly assigns responsibility for all aspects of training to management.

The most basic support to any GxP system is a standardized process documented in our operating procedures, because an effective system requires consistency of application and standards for operating. For training, our procedure should include a standard approach for how we perform and document training, how we qualify trainers, how we process and maintain our training documentation, and what we define as passing scores on knowledge assessments, along with a standardized on-boarding process through which all GMP employees are processed. Where appropriate, we should also include standardized time frames for completion in our training procedure.
Appropriate operational procedures are critical to an effective training process, because we derive much of our training content from them. Procedures should clearly define processes, responsibilities, and tasks to an appropriate level of detail, providing standard performance requirements for each process. Ambiguity of performance requirements or responsibilities, or the lack of documented processes, causes training effectiveness to suffer—it’s difficult to teach, learn, or assess a process that everyone does differently. However, if procedures provide on-the-job reinforcement of items learned in training—even wording as simple as “at this point in the investigation, perform a root cause analysis to identify the root cause of the problem”—they automatically improve training effectiveness, because they tie directly to what was learned and define when to use it.
Systems and repositories—preferably electronic—ensure that we can manage the process and the associated materials and records, and access the information we need. While this can be done in a well-managed paper system, electronic learning management systems and learning content management systems make this process much easier to manage. For example, the ability to easily access records enables us to quickly verify an operator’s qualification on a specific task. Having a location where people can access controlled, approved training materials and information provides the same benefit as a controlled documentation system: access to current, consistent information.

The Most Critical Factor

Of all the system elements, management support has the largest impact on training effectiveness. So what does “management support” mean? In a word: expectations. Our management teams should expect the following:

• Training occurs, is done well, and is completed on time;
• Results are measured, reported, and acted on appropriately;
• Training is meaningful, and skills learned in training are appropriately applied to operations;
• All parts of the training system and support processes are in place and functioning appropriately, and people are accountable for their parts of the process;
• Training helps meet the goals of the business; and
• Training is part of the job and part of ongoing employee performance management processes.

If there is any doubt regarding management’s role in and effect on training, the FDA’s “Guidance for Industry: Quality Systems Approach to Pharmaceutical CGMP Regulations” clearly assigns responsibility for all aspects of training to management:
“Under a quality system, managers are expected to establish training programs that include the following:

• Evaluation of training needs
• Provision of training to satisfy these needs
• Evaluation of effectiveness of training
• Documentation of training and/or re-training.

When operating in a robust quality system environment, it is important that managers verify that skills gained from training are implemented in day-to-day performance.”⁵
The guidance also requires senior leadership and management to define and provide policies, procedures, and desired work culture, and prescribes these topics for inclusion in our training courses and content. So, the FDA expects management to have a vested interest in the content, the quality, and the process of training.
Finally, consider this: The attitude the organization’s leadership takes toward training—particularly their own training requirements—sets a powerful example, either good or bad, for the rest of the organization. Training takes time away from operations and is often viewed as a necessary evil. But, when employees see leadership attending GMP refreshers or completing applicable procedural training, they understand that training is a business activity that has value and is worth the time. However, if people see their leadership regularly excused from or habitually late in completing training, they think, “Training isn’t important enough for management to spend their time on.” Training, in their eyes, is now a punishment for those at lower levels, one that can be bypassed by management in favor of other activities.

Achieving the Holy Grail

So this is why improving training effectiveness doesn’t have a simple answer. We’re dealing with a fundamental business process that touches many parts of the organization, along with other processes built upon or around it.
At heart, improving training effectiveness is about problem solving and continuous improvement, and it will likely lead to changes in systems, processes, and procedures, as well as training practices. But finally achieving effective training “nirvana” provides measurable benefits to the business: Imagine having competent and motivated employees making fewer errors and better decisions, along with fewer wasted resources, increased efficiency, and, ultimately, more compliant and more productive operations. Who’d have thought a necessary evil could be so positive?

References

1. U.S. Government Printing Office. United States Code of Federal Regulations, 21 CFR 820.25: Quality System Regulation: Personnel. Updated April 1, 2011. Available at: www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?fr=820.25. Accessed June 7, 2012.
2. U.S. Government Printing Office. United States Code of Federal Regulations, 21 FR 211.25: Current Good Manufacturing Practice for Finished Pharmaceuticals: Personnel Qualification. Updated April 1, 2011. Available at: www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?fr=211.25. Accessed June 7, 2012.
3. European Commission Health and Consumers Directorate-General. EudraLex Volume 4: EU Guidelines to Good Manufacturing Practice Medicinal Products for Human and Veterinary Use, Part 1, Chapter 2. Personnel, Section 2.2. Dec. 2010. Available at: http://ec.europa.eu/health/files/eudralex/vol-4/pdfs-en/cap2en200408_en.pdf. Accessed June 7, 2012.
4. Kirkpatrick Donald L. The Kirkpatrick Philosophy. Kirkpatrick Partners website. Available at: www.kirkpatrickpartners.com/OurPhilosophy/tabid/66/Default.aspx. Accessed June 7, 2012.
5. U.S. Food and Drug Administration. Guidance for Industry: Quality systems approach to pharmaceutical CGMP regulations. September 2006. Available at: www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM070337.pdf. Accessed June 7, 2012.

FORMULATION - TARGETED THERAPIES | Liposomes an Important Force in New Cancer Drug Directions

Tim Donald

Nanoparticulate siRNA systems and therapeutic complexes directed by targeting moieties are under intense study

Liposomes are becoming increasingly valuable tools in the development of cancer therapeutics. These lipid bilayer vesicles can be loaded with a variety of therapeutic payloads, ferrying their cargoes to tumor sites while circulating in the bloodstream.
The first cancer drugs to use liposome technologies were established chemotherapeutic agents, packaged in long-circulating lipid-based delivery systems to increase their efficacy and reduce side effects. More recently, liposomes containing short interfering RNA, designed to specifically inhibit the production of disease-causing proteins, are showing promise in early clinical trials. Some of these systems are equipped with targeting components that direct them specifically to tumor cells.

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FIGURE 1. Design of the liposome nanoparticle, with gene payload inside a liposome and the targeting moiety on the outside.

“The initial work we and others did in the late 1980s and early ’90s that led to clinical products was loading anticancer drugs, such as doxorubicin, into liposomal systems,” said Pieter R. Cullis, PhD, FRSC, director of the NanoMedicine Research Group and a professor in the department of biochemistry and molecular biology at the University of British Columbia in Vancouver.
Dr. Cullis, who has been working with liposomes for drug delivery for about 30 years, said early research included devising the methodology for producing liposomes in the desired dimensions, approximately 100 nm or less, and loading them efficiently with drug.
Delivery of these chemotherapy-loaded liposomes depended on the phenomenon of enhanced penetration and retention, or EPR. In areas in and around tumors, the vasculature is leaky, and liposomes accumulate preferentially in those places, but not in healthy tissue vessels, Dr. Cullis said, thereby concentrating drug accumulation at the site of disease.
“With anticancer drugs, for example, you can get up to 50 times more drug at a tumor site than you would get by injecting the same amount of free, non-encapsulated drug, by putting it inside these nanoparticles,” he said.
The first liposome-based cancer drug to enter the market was Doxil (Johnson & Johnson), a liposomal formulation of doxorubicin. Doxil is approved in Europe and the United States for treatment of ovarian cancer.
Dr. Cullis and coworkers developed an alternative formulation of doxorubicin (Myocet, Enzon Pharmaceuticals) that has been approved in Canada and Europe for treatment of metastatic breast cancer. They also developed a liposomal version of vincristine sulfate (Marquibo, Hana Biosciences) that has been granted orphan drug status by the FDA and is being evaluated for the treatment of adult lymphoblastic leukemia. In phase 1 and 2 clinical trials, patients tolerated doses of liposomal vincristine that were approximately 100% greater than conventional vincristine doses.
“Reduced toxicity is probably the major benefit from those systems, allowing us to achieve higher doses and see enhanced efficacy,” Dr. Cullis said.
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CASE STUDY: Tumor-Specific Delivery to Metastatic Lesions

Researchers at Georgetown University Medical Center, along with collaborators, recently reported tumor-specific delivery of a systemically administered therapy to metastatic lesions for the first time in patients.¹
A liposome complex containing a plasmid that encodes for the tumor suppressor gene p53, decorated with a targeting moiety, was administered to 11 patients with solid tumors who had exhausted all standard therapies. In this phase 1 dose-escalation trial, primarily grade 1 and 2 easily managed adverse events were observed. A single grade 3 (fatigue associated with massive tumor necrosis) and no grade 4 adverse events were seen. The patients were treated by John Nemunaitis, MD, and Neil Senzer, MD, at the Mary Crowley Cancer Research Centers in Dallas.
Although this was a safety trial, there were signs of efficacy, the researchers reported. Eight of the 11 patients showed stable disease, and two showed more than disease stability: One patient’s adenoid cystic carcinoma was reclassified from inoperable to operable, and in another patient with leiomyosarcoma with metastases in liver and lung, computed tomography showed necrosis in all metastases after the treatment.
This liposome therapeutic is designed to work in combination with standard cancer therapies, said Kathleen F. Pirollo, PhD, a research professor at Georgetown University Medical Center. “The idea is to make standard therapy, either radiation or chemotherapy, more effective in these cancers,” she said. The p53 tumor suppressor gene activates the cell death pathway in the cancer cells, “so when we expose the tumors to conventional chemo and radiation, they can now respond and die.”
Esther H. Chang, PhD, a professor at Georgetown, noted that this is a truly targeted therapy.
“It was important for us to see whether in patients the p53 gene actually ends up in the tumor only and not in normal tissue,” she said. “We extracted DNA from the metastatic tumors, looking for the specific payload, the therapeutic gene. We found that even in patients treated with the lowest dose, we were able to see the specific unique presence of the gene we put in. There was also a dose response, with strong presence of the transgene at the highest dose administered. Also, when we look at the normal skin, it’s clean. We’re very proud of that—truly tumor-targeted delivery.”—TD

References

1. Senzer N, Nemunaitis J, Nemunaitis D, et al. Results of a Phase I trial of SGT-53: a systemically administered, tumor-targeting immunoliposome nanocomplex incorporating a plasmid encoding wtp53. Paper presented at: American Society of Gene and Cell Therapy Annual Meeting; May 15-19, 2012; Philadelphia.

siRNA Delivery

In their ongoing work, begun in the previous decade, Dr. Cullis and coworkers are principally focused on the use of liposomal nanoparticulate systems to deliver siRNA for therapeutic applications in the liver.
“siRNA could be a major therapeutic, as long as we can get it to the inside of target cells,” he said. “The issue is to protect it from degradation in circulation and get the material to the target tissue and then inside the target cells. These delivery systems have to be more sophisticated, with more components, than those that use the EPR effect.”
The Vancouver researchers take advantage of a naturally occurring phenomenon in hepatocytes (liver cells). Their liposomes accumulate the serum protein apolipoprotein-E, which is then taken up by so-called scavenging receptors on hepatocytes. Once the particles reach the endosome of the hepatocytes, they must then be taken into the cytosol to do their job, and this is accomplished by another component of the system, cationic lipids.
“Much of our work has been focused on getting very potent cationic lipids for delivery from the endosome to the inside of the cell. These systems are now very viable therapeutics with low toxicity,” Dr. Cullis said.
Three such therapeutics, in development by Alnylam Pharmaceuticals of Cambridge, Mass., are being investigated in humans, including one directed at liver cancer. A phase 1 clinical trial of that compound, ALN-VSP, showed that the drug was well tolerated and demonstrated evidence of antitumor activity in patients with advanced malignancies. Dr. Cullis said Alnylam is looking for partners to move the compound forward.

Targeting Moiety

Researchers at Georgetown University have taken a different tack for targeting therapeutics to cancer cells. They have designed therapeutic complexes composed of cationic liposomes that can encapsulate multiple types of payloads and can be cancer directed by attaching a targeting moiety to the outside of the liposome

Researchers at Georgetown University in Washington, D.C., have taken a different tack for targeting therapeutics to cancer cells. They have designed therapeutic complexes composed of cationic liposomes that can encapsulate multiple types of payloads and can be cancer directed by attaching a targeting moiety to the outside of the liposome. The prototype of these targeted complexes has successfully completed a phase 1 clinical trial (see case study) to deliver the therapy to a range of solid tumors and is now in a phase 1b trial.
The therapeutic complex is a platform technology, said Kathleen F. Pirollo, PhD, a research professor in experimental therapeutics in the department of oncology of the Lombardi Comprehensive Cancer Center at Georgetown University Medical Center.
“We can mix and match,” she said. “We can switch out the targeting moiety, switch out the payload. It’s applicable for a number of molecular medicines and contrast agents. We have shown in preclinical studies that we can successfully deliver a number of payloads, including plasmid DNA, siRNAs, miRNAs, antisense oligonucleotides; we can even encapsulate chemotherapeutic agents to increase their efficacy and reduce their side effects because of the targeting nature of this platform technology.”
The targeting moiety that the group has explored most extensively is a single-chain antibody fragment that is designed to target cancer cells by binding to the transferrin receptor, she said.
“The transferrin receptor is a good target because most if not all cancer cells have elevated levels of expression of this receptor. Cancer cells grow so rapidly they need to bring iron in, so we take advantage of that to bind to these receptors and transport the complex into the cell,” Dr. Pirollo said.
In addition to the gene therapy work, the Georgetown researchers have also investigated the use of the liposome technology for delivery of chemotherapy agents in animal models, said Esther H. Chang, PhD, a professor of oncology and otolaryngology at Georgetown University Medical Center.
“We found that after encapsulation, the safety profiles look better than the original form, and we can edit or change the capacity or function of the chemotherapy drugs,” she said. “It turns out that when you encapsulate a conventional chemotherapeutic agent, it broadens its use and significantly increases its efficacy.”
In collaboration with SynerGene Therapeutics, of Potomac, Md., Dr. Chang and colleagues are developing a number of liposomal therapeutic entities that are in late translational stages.

FORMULATION - BIOMIMETIC LIPOPHILICITY | Liposome Water Partitioning

Alex Avdeef EDITOR’S NOTE: This chapter is from the book Absorption and Drug Development: Solubility, Permeability, and Charge State, 2nd Edition by Alex Avdeef, published in May by John Wiley & Sons (http://www.wiley.com/WileyCDA/WileyTitle/productCd-1118057457.html). For further information on the book, visit the author’s website: www.in-adme.com/book.html.
This chapter considers “biomimetic” lipophilicity, where partition coefficients of drugs are determined in the liposomes–water system. Unilamellar vesicles formed with phosphatidylcholine provide a lipophilicity scale, expressed as log P_MEM, which is different from that of octanol. The log P_MEM can be used as a component in property or biological activity prediction models. For ionizable molecules, the coefficients depend on pH and are called distribution or apparent partition coefficients, log D_MEM.
Given a wide range of pH, monoprotic molecules show a sigmoidal curve when log D_MEM is expressed as a function of pH. At the asymptotic top of such curves, log D_MEM is equal to the log P_MEM constant describing the liposome–water partition of the neutral species. At the asymptotic bottom of the sigmoidal curve, log D_MEM is equal to log P_^SIPMEM, the constant describing surface ion-pairing, charged drug paired with surface charge components in the bilayer. In the liposome system, charged species partitioning—association with the surface—is about 100 times greater than ion-pair partitioning in octanol. Consequently, the “diff 3–4” approximation in the octanol system becomes the “diff 1–2” approximation in the liposome system. The phospholipid-drug interaction discussed here serves as the foundation for the PAMPA model in Chapter 7. A database of log P_MEM and log P_^SIPMEMfor 114 molecules is listed at the end of the chapter.
The legacy octanol–water partition model has some shortcomings. Notably, it is not very “biological.” Given that liposomes—vesicles with internal aqueous compartments separated from the bulk solution by a phospholipid bilayer—are made of the main ingredients found in all biological membranes, a substantial effort has been made to characterize drug partitioning in the more biomimetic liposome–water system.^1-68

Tetrad of Equilibria and Surface Ion Pairing

Figure 5.1 shows a tetrad of equilibrium reactions related to the partitioning of a drug between an aqueous environment and that of the bilayer formed from phospholipids. (Only half of the bilayer is shown in Figure 5.1.) The subscript “MEM” designates the partitioning medium as that of a membrane vesicle formed from a phospholipid bilayer. Equations (4.1)–(4.4) apply.
The pK_a^MEM in Figure 5 .1 refers to the “membrane” pK_a. Its meaning is similar to that of pK_a^OCT: When the concentrations of the uncharged and the charged species in the membrane phase are equal, the aqueous pH at that point defines pK_a^MEM, which is described for a weak base as:
BH⁺_MEM B_MEM + H⁺ K_a^MEM = [B]_MEM[H⁺]/[BH⁺]_MEM

---

The salt dependency of constants discussed in Sections 4.2 and 4.3 also applies to the pK_a^MEM and log P^SIP_MEM constants in Figure 5.1. Although the surface ion-pair and membrane-pK_a are conditional constants, the dependence on solution counterion concentration differs from that of octanol.^{57, 66}

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FIGURE 5.1. Phospholipid membrane–water tetrad equilibria. Only half of a bilayer is shown.

It is thought that when a charged drug migrates into the lipid environment of a liposome, the counterion that at first accompanies it may be exchanged with the zwitterionic phosphatidylcholine head groups, as suggested in Figure 5.1, while still maintaining local charge neutrality. As the nature of the ion pair may be different with liposome partitioning, the term “surface ion-pair” is used to denote it. The term diff log P_MEM will be used to designate the difference between the neutral species partitioning and the surface ion-pair partitioning [cf. Eq. (4.6)].

Data Sources

There are no convenient databases for liposome log P values. Most measured quantities need to be ferreted from original publications.^{1,2,5-11,67,69} The handbook edited by Cevc is a comprehensive collection of properties of phospholipids, including extensive compilations of structural data from X-ray crystallographic studies.⁴ Constituent-lipid distributions in various biological membranes have been reported.^4,12,57

Location of Drugs Partitioned into Bilayers

Based on the nuclear Overhauser effect in a ^31P{¹H} NMR study of egg phosphatidylcholine (eggPC) bilayers, Yeagle and colleagues concluded that the N-methyl hydrogen atoms were in proximity to phosphate oxygen atoms in neighboring phospholipids, suggesting that the surface of the bilayer was a “shell” of interlocking (intermolecular) electrostatic associations.²³ Added cholesterol bound below the polar head groups and did not interact with them directly. However, its presence indirectly broke up some of the surface structure, making the surface more polar and open to hydration.
Boulanger and colleagues studied the interactions of the local anesthetics procaine and tetracaine with egg PC multilamellar vesicles (MLV,52–650 mM), as a function of pH, using deuterium NMR as a structural probe.^44,45 They proposed a three-site model, similar to that in Figure 5.1, except that the membrane-bound species, both charged and uncharged, had two different locations, one a weakly bound surface site (predominantly occupied at pH 5.5) and the other a strongly bound deeper site (predominantly occupied at pH 9.5).

The partition of lipophilic drugs into lipid phases is often thought to be entropy-driven, a “hydrophobic” effect. Bäuerle and Seelig studied the thermodynamics of amlodipine and nimodipine binding.

Membrane partition coefficients (D_MEM) were estimated for both sites. Westman and colleagues further elaborated the model by applying the Gouy–Chapman theory.46 When a positively charged drug partitions into the bilayer, a Cl− is likely bound to the surface, to maintain local charge neutrality. They found unexpected low values of diff log P_MEM of 0.77 for tetracaine and 1.64 for procaine (cf. Section 4.6), much smaller than the value expected in octanol–water partitioning. Kelusky and Smith, also using deuterium NMR, proposed that there was an electrostatic bond formed at pH 5.5 between the protonated drug and the phosphate groups, )=P–O…+H₃N–(, and a hydrogen bond formed between the aminobenzene proton and the acyl carbonyl oxygen.⁴⁷ At pH 9.5, the electrostatic bond breaks as the secondary amine moves deeper into the interior of the bilayer; however, the aminobenzene H-bond, )=CO…H₂N–(, continues to be an anchoring point.
Bäuerle and Seelig studied the structural aspects of amlodipine (weak base, primary amine pK_a 9.24 [2]) and nimodipine (nonionizable) binding to phospholipid bilayers, using NMR, microcalorimetry, and zeta–potential measurements.¹⁹ They were able to see evidence of interactions of amlodipine with the cis double bond in the acyl chains. They saw no clear evidence for )=P–O−…+H₃N–( electrostatic interactions.
Herbette and co-workers studied the structures of drugs bound to liposomes using a low-angle X-ray diffraction technique.^49-52,70 Although the structural details were coarse, it was apparent that different drugs position in different locations of the bilayer. For example, amlodipine is charged when it partitions into a bilayer at physiological pH: The aromatic dihydropyridine ring is buried in the vicinity of the carbonyl groups of the acyl chains, while the –NH⁺₃ endpoints toward the aqueous phase, with the positive charge located near the phosphate negatively charged oxygen atoms.^50-52 A much more lipophilic molecule, amiodarone (weak base with pK_a 10.24; Table 3.14), positioned itself closer to the center of the hydrocarbon interior.⁴⁹

Thermodynamics of Partitioning: Entropy or Enthalpy Driven?

TABLE 5.1. Energy of Transfer (kJ·mol−1) into Lipid Phase for 4-Methylphenol

Davis and colleagues studied the thermodynamics of the partitioning process of substituted phenols and anisoles in octanol, cyclohexane, and dimyristoylphosphatidylcholine at 22° C (below the gel-liquid transition temperature of DMPC).¹⁸ Table 5.1 shows the results for 4-methylphenol.
The phenol partitioned into the lipid phases in the order DMPC > octanol > cyclohexane, as indicated by ΔG_tr. That is, the free energy of transfer into DMPC was greater than into octanol or cyclohexane. Partitioning was generally entropy-driven, but the enthalpy and entropy parts of the free energy of transfer differed greatly among the three lipid systems (Table 5.1).
Octanol was the only lipid to have an exothermic heat of transfer (negative enthalpy), due to H-bond stabilization of the transferred solute, not found in cyclohexane. Although ΔH_tr in the DMPC system is a high positive number (endothermic), not favoring partitioning into the lipid phase, the entropy increase (+114.1 J·mol−1) was even greater, more than enough to offset the enthalpy destabilization, to end up an entropy-driven process. The large ΔH_tr and TΔS_tr terms in the DMPC system are due to the disruption of the ordered gel structure, found below the transition temperature.
The partition of lipophilic drugs into lipid phases is often thought to be entropy-driven, a “hydrophobic” effect. Bäuerle and Seelig studied the thermodynamics of amlodipine and nimodipine binding to phospholipid bilayers (above the transition temperature) using highly sensitive microcalorimetry.¹⁹ The partitioning of the drugs into the lipid bilayer was enthalpy-driven, with ΔH_tr − 38.5 kJ·mol−1 bound amlodipine. The entropy of transfer is negative, contrary to the usual interpretation of the “hydrophobic” effect. Thomas and Seelig also found the partitioning of the calcium antagonist, flunarizine (a weak base), to be predominantly enthalpy- driven, with ΔH_tr− 22.1 kJ·mol−1, again at odds with the established ideas of entropy-driven partitioning of drugs.²¹ The same surprise was found for the partitioning of paclitaxel.²² So, these observations appear to suggest that these drugs partition into membrane phases because they are lipophilic and not because they are hydrophobic.

Novel Stability Testing Methods Save Time and Money

TOOLS OF THE TRADE - TESTING METHODS |

Neil Canavan

Advances with names like ASAP and RobX are taking evaluations to the next level

Time is money; in the business world, there is little argument with this premise. In the pharmaceutical business, the time between the identification of a lead compound and its regulatory approval for sale represents a great deal of money indeed.
In the interest of saving time, a small army of researchers is dedicated to shortening the time it takes for drug development, as well as extending the marketable shelf life of a product once approved, and the potential to achieve both aims can be enhanced through knowledgeable, innovative stability testing.
One such innovation is ASAP, the accelerated stability assessment program. “Normally, when companies are determining their product’s shelf life, that process takes six to 12 months,” said Ken Waterman, PhD, CEO and founder of FreeThink Technologies in Storrs, Conn. Multiple formulations must be tested, as well as a variety of packaging types. “Obviously, that’s a lot of time, and it may delay the introduction of a product,” he said, “but I’ve figured out how to do it in just two weeks.”
ASAP, a software package recently launched by FreeThink, is a method whereby, with a limited dataset of stability tests under specific conditions, longer time durations under different conditions may be extrapolated.
The theoretical foundation of ASAP is the isoconversional approach, a method that relies on performing a series of experiments at different temperature programs that reveal the values of effective activation energy as a function of molecular conversion.¹

ASAP, a software package by FreeThink, is a method whereby, with a limited dataset of stability tests under specific conditions, longer time durations under different conditions may be extrapolated.

“Before I did this, people would use different temperatures, certainly, but they didn’t adjust the time so that the amount of conversion to degradation products stayed the same,” explained Dr. Waterman. The results do not usefully inform as to the kinetics of the reaction. Using isoconversion as a basis for extrapolation, Dr. Waterman is able to greatly shorten the time needed for a stability test.
“The other thing I did with ASAP was to consider the importance of water in reactions, and how relative humidity determines reaction rates in the solvents.” This understanding is important, said Dr. Waterman, because even after packaging, the humidity inside the package will change over time.² “Rates go up exponentially with humidity, so a little change in humidity can result in a large change in the reaction rate.” Reliable estimates for temperature and relative humidity effects are accounted for by using a humidity-corrected Arrhenius equation.
According to Dr. Waterman, the ASAP calculation provides an inside-the-package shelf-life determination that is not only quicker than standard stability testing methods, but is also more accurate—and accounts for humidity. “We can determine the course of the drug degradation, and we know it out to the specification limit. In essence, we know when the drug is going to fail at room temperature because we know when it will fail at higher temperatures.” This knowledge allows for more time to get the formulation just right.
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CASE STUDY: State of the Solid-State Stability

The solid-state chemistry of APIs is of growing importance in the pharmaceutical industry, yet many companies lack the expertise to answer the important questions. “Many are not even looking at this because they don’t realize the impact of the solid-state,” said Robert M. Wenslow Jr., PhD, vice president of business development at Crystal Pharmatech in Suzhou, China.
To illustrate the importance of solid-state awareness, Dr. Wenslow offered a case in point: the HIV drug ritonavir. “This was developed as a liquid formulation, a gelatin, because the compound had very low solubility.”
Initially, all went well. Over 200 batches were manufactured with no stability issues reported. “Then, all of a sudden, they noticed their dissolution profiles slowing down,” said Dr. Wenslow, “and when they broke open the capsules, they saw solid particulates—a new solid-state crystal form—and this form had a much lower bioavailability than the original.”
And the result of this belated observation? The drug was pulled off the market for reformulation. “They lost millions because of solid-state stability issues.”^1-2
This loss could have been avoided if more attention had been paid to the solid-state early on in development, Dr. Wenslow said. “Even if you are dealing with a liquid formulation, you need to understand the thermodynamic and kinetic landscape of your solid-state forms,” and if you can’t do it in-house, you can always outsource to the experts.
Chrystal Pharmatech does all types of solid-state research, be it a crystal, a salt, a free-base, free-acid… “We’ll get a complete picture of not just what’s happening to the chemical state, but what’s happening to the physical state as well,” said Dr. Wenslow. “And once you understand the impact of solid-state on your formulation, then you can actually increase shelf life if you chose the appropriate excipients based on solid-state properties.”
For a review of one solid-state detection method, see:
Lin SY, Wang SL. Advances in simultaneous DSC-FTIR microspectroscopy for rapid solid-state chemical stability studies: some dipeptide drugs as examples. Adv Drug Deliv Rev. 2012;64(5):461-478.—NC

References

1. James JS. Ritonavir capsule manufacturing problems will require switch to liquid formulation. AIDS Treat News. 1998;(No 300):1,5.
2. Morissette SL, Soukasene S, Levinson D, Cima MJ, Almarsson O. Elucidation of crystal form diversity of the HIV protease inhibitor ritonavir by high-throughput crystallization. Proc Natl Acad Sci U S A. 2003;100(5):2180-2184.

For Biologics, Try RobX

To be clear, the utility of ASAP does not extend to proteins. For stability information in that context, you may want to consider the robustness index, or RobX, a new measure being proposed to easily and quickly gauge how to design robustness into biologic formulations across different temperatures over time.³ RobX=B in the equation K_T2/K_T1 = e^Bx(T2−T1).
Driving the need for RobX is the intrinsic complexity of macromolecules, a quality that reveals Arrhenius calculations as too simplistic (though some researchers have recently proposed just such an approach).⁴
“When you have something that is strictly a function of purity, it’s not such a problem,” said Anthony Lonardo, associate vice president of statistics and quantitative sciences for Imclone Systems in Branchburg, N.J. “But when you’re looking at a quality measure like charge heterogeneity, as opposed to simply purity, what does Arrhenius mean in that case?” Lonardo wants a way to look at the multiple critical quality attributes of amino acid-based tertiary structures in a given formulation.
“I’m really interested in being able to define (formulation) robustness as a constant that defines rates of change at different temperatures,” and the objective is to minimize that number to achieve an optimally stable formulation. Put another way, and as simply as only someone who really loves math can do: “For RobX, if you subtract the log of the rates, it turns out really nice—it turns out the RobX index defines a ratio of the rates of change for two temperatures.”
The beauty of this method, as Lonardo described it, is that the calculated RobX is a unitless constant and can therefore be used across a number of quality characteristics. It’s also very easy to understand the meaning of the value once derived: The number represents a ratio, graphically illustrated as a line with an inclination, a slope, which flattens as you approach the value of 1—that being the ratio of no change at all.

One expert is seeing stability protocols with built-in extended time points out to 48, or even 60, months.

“Another insight was that I can put all of this in a table,” said Lonardo. “I know what I want—I want the number to near 1 when considering two different temperatures, say 25-48°C, for all the quality characteristics, and then I identify which of those (based on the RobX number) is going to give the most trouble.” For example, if RobX indicates that the acidic peak group has the greatest potential for change, the drug formulators can focus on that issue.
“From our standpoint, we’re relentless in terms of wanting to produce robust and stable formulations,” Lonardo said. “And what RobX does is allow for a single way of thinking about the robustness of a molecule.”
It’s not just theoretical. The accuracy of the approach, as compared to known data points, has already been demonstrated (see Lonardo and colleagues). Lonardo is adamant about the utility of RobX: “Take the data up and see if you can reproduce my results.” (Lonardo is quick to note the vital contributions of his coauthors in the conception of RobX.)

That’s a Wrap

While innovation is wonderful, the CROs doing the large-scale testing will likely wait for the validation. “There’s always been the idea that you can do some extrapolation based on short periods at much higher temperatures and humidities,” said Jeannine Schreiber, manager of stability services at Lancaster Laboratories of Lancaster, Pa. “But so far that hasn’t really flowed through to the regulators.”
With nearly 30 years of tenure at Lancaster, Schreiber does note some recent changes in the focus of regulators when it comes to the stability of drug packaging. This shift may be motivated by the recent publicity surrounding the use of bisphenol A in plastic compounds ^.5 “Right now we’re doing a lot of studies that are looking at extractables/leachables, so they are using elevated temperatures and humidities to look at how their container closures may be impacting product.”
Schreiber mentioned one example, an IV bag. “Because it’s a plastic product, there are some extractables/leachables in that plastic that may come out of it.” The contaminant itself may pose a problem, or it may even react with the API in solution^.6
Schreiber has also witnessed a shift in stability protocols for drug formulations, with clients building in options for time extensions. “Let’s say your approved product currently has a 36-month shelf life,” she recounted. “In most cases, part of the requirements are that [that] company put an annual lot of product on stability, and traditionally that would end at 36 months.” What she is seeing now, though, are protocols with built-in extended time points out to 48, or even 60, months. “They’ve provided enough material so that if they want to push the button and gather the extra data to support an extended expiration period, they have the product and the ability to do that.”
That is if they need the time, and want to save some money.

References

1. Waterman KC. The application of the Accelerated Stability Assessment Program (ASAP) to quality by design (QbD) for drug product stability. AAPS PharmSciTech. 2011;12(3):932-937.
2. Waterman KC, MacDonald BC. Package selection for moisture protection for solid, oral drug products. J Pharm Sci. 2010;99(11):4437-4452.
3. Lonardo AJ, Srivastava A, Singh S, Goldstein J. Robustness index score: a new stability parameter for designing robustness into biologic formulations. J Pharm Sci. 2012;101(2):485-492.
4. Martin-Moe S, Lim FJ, Wong RL, Sreedhara A, Sundaram J, Sane SU. A new roadmap for biopharmaceutical drug product development: Integrating development, validation, and quality by design. J Pharm Sci. 2011;100(8):3031-3043.
5. Geens T, Goeyens L, Covaci A. Are potential sources for human exposure to bisphenol-A overlooked? Int J Hyg Environ Health. 2011;214(5):339-347.
6. Chang JY, Xiao NJ, Zhu M, et al. Leachables from saline-containing IV bags can alter therapeutic protein properties. Pharm Res. 2010;27(11):2402-2413.

INGREDIENTS - STABILITY | Tools for Evaluating the Stability of Human Recombinant Albumins Used in Human Therapeutics

Karl Nicholls, Neil Dodsworth, Phil Morton, Dr. Mark Pothecary, Dr. Oksana Leszczyszyn, and Dr. Hanna Jankevics

Dynamic light scattering and size exclusion chromatography with light-scattering detection

Aggregation and misfolding during long-term storage is a major challenge in the development of structurally altered proteins for therapeutic applications. While a liquid formulation is both easy to handle and economical during manufacture, many proteins are difficult to formulate as stable solutions. Storage stresses such as temperature changes, shear strain, surface absorption, and high protein concentration can cause conformational changes and precipitation.
Implicated in adverse immunogenic side effects, aggregation must be minimized during handling, shipping, and long-term storage. Formulations must be optimized to ensure the efficacious delivery of clinical applications and rigorously tested to provide a detailed understanding and confirmation of their stability under a variety of conditions.
Here we use dynamic light scattering in cuvette mode and size exclusion chromatography with light-scattering detection to demonstrate both the short- and long-term stability of novel recombinant human albumins (rAlbumins) that have been specifically developed and optimized to deliver a stable, safe, and regulatory-compliant product for the formulation markets.

Novel Recombinant Human Albumins

Excipients incorporated into the formulation process not only stabilize the drug product but also assist in the administration and release of the active pharmaceutical ingredient. However, strict regulatory limitations in the use of blood- and plasma-derived materials have arisen due to safety concerns regarding the potential risk of transmitting infectious agents such as HIV, hepatitis, variant Creutzfeld-Jacob disease, and West Nile virus. This challenge has given rise to the development of high-quality, animal-free, recombinant human albumins for use in a range of applications, including the formulation of pharmaceutical drugs and vaccines and medical device manufacturing.
Albucult and Recombumin (Novozymes Biopharma, UK) are examples of fully characterized, regulatory-compliant multipurpose excipients that are optimized to prevent or minimize physical, as well as chemical, degradation of drug substances. Providing a tertiary level of animal-free status and lowering the risk of potential contamination, recombinant supplements are suitable for any stage of the clinical pathway.
Both Albucult and Recombumin are derived from proprietary Saccharomyces cerevisiae yeast expression technology. Formulated at pH 7, Recombumin is structurally identical to human serum albumin. Recombumin is the world’s first commercially available rAlbumin approved for use in the manufacture of human therapeutics and has been shown to have a shelf life of greater than five years at 5°C.¹ Albucult was further developed to deliver process and performance consistency for applications such as drug and vaccine manufacture, device coating, IVF media, specialized cell culture, and cell therapy applications. The GMP formulation is stable for at least four years to date.²
Typical storage times for both protein and non-protein formulations depend on a number of factors. Storage times are generally between one and three years. However, shelf life for protein-based formulations is typically shorter than that of non-protein based formulations.

Testing Protein Stability with Light-Scattering Techniques

The effects of pH, temperature, and concentration on oligomerization and aggregation behaviors can be studied in order to demonstrate short-term stability and predict shelf life. Dynamic light scattering provides a rapid measurement—about two minutes per sample—that allows the comparison of a number of sample conditions within a short time and without the need to alter or dilute the sample. As the following case studies show, DLS can provide much information about the stability and relative composition of different protein formulations. Pre-screening by DLS can therefore minimize total analysis time, directing the experimenter to undertake more time-consuming measurements, such as size exclusion chromatography—typically 15-20 minutes per sample—on only the most promising formulations.

Case Study

Understanding Protein Thermal Stability with DLS

FIGURE 1. Recombumin: DLS-determined aggregation temperature plotted as a function of pH.

DLS was used to study the thermal stability of Recombumin over the range of pH conditions in which APIs are commonly formulated. In this technique, the mean size of the molecule was monitored as a function of temperature, and the stability of a given formulation was indicated by the delayed onset of unfolding and aggregation. For Recombumin, the knowledge of the aggregation temperature allows determination of the most appropriate pH conditions required for a stable liquid formulation.
The samples were measured with the Zetasizer APS (Malvern Instruments, UK), an automated plate sampler system that allows unattended dynamic light scattering measurements of multiple samples. An algorithm is applied to the DLS data, combining a number of factors to detect where unfolding and aggregation begins. The aggregation temperatures determined for Recombumin samples at various pH conditions are shown in Figure 1.
The lowest aggregation temperatures are observed for buffers at and below pH 4, which suggests that these conditions are less favorable for a stable formulation. In more basic formulations—those at and above pH 5—the aggregation temperatures are all greater, with the maximum aggregation temperature observed at pH 6. Therefore, under the conditions studied here and at least over the times taken for these measurements, citrate and phosphate buffers with a pH of 6 provide the most stable formulations.
To understand how these aggregation temperatures correlate with observations from long-term stability studies, DLS measurements were carried out on the same Recombumin formulations at six- and nine-month intervals after storage at 2-8ºC (Figures 2 and 2b, respectively). Shifts in the mean hydrodynamic size to larger values are indicative of aggregation, and on this premise the formulations at and below pH 4 are deemed the least stable. This observation is in agreement with the indications from aggregations temperature studies.
From these comparisons, it is clear that the determined aggregation point is related to the low temperature stability of the sample and can be used as an indicator of longer-term stability. However, at higher pHs, other factors, such as ionic strength, sample concentration, and the presence of additives, may become more significant players in contributing to longer-term formulation stability.

Case Study

Monitoring Protein Structural Stability

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FIGURE 2. Z-average size for Recombumin formulated at different pHs. A: 6 months and B: 9 months after storage at 2-8°C.

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FIGURE 3. Chromatogram of Albucult and ADH mixtures. The two protein peaks are only partially separated.

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TABLE 1. SEC-LS results for the range of ADH-spiked Albucult sample.

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FIGURE 4. DLS Z-average radius and derived count rates (in kilo counts per second) for the ADH-spiked Albucult samples.

DLS gives a clear indication of not only large aggregates but also small oligomers or aggregates present in the sample. In comparison, SEC can provide even more detailed insight and, when combined with light-scattering detection, is an excellent tool for characterizing the type of oligomers or aggregates. This example demonstrates the resolving and detection capabilities of size exclusion chromatography light scattering and dynamic light scattering.
To mimic the appearance of small oligomers, mixtures of two different proteins were used. An Albucult sample (66 kDa recombinant human albumin) was used for the monomer protein. This protein shows very good stability in its formulation buffer and is therefore a useful DLS reference sample. Alcohol dehydrogenase was used for the second sample. At about 150 kDa, ADH is similar in molecular weight to a dimer of Albucult.
Stock solutions of both proteins were prepared in phosphate buffered saline and filtered to ensure that no aggregates or dust particles were present in either sample. The Albucult sample was spiked with increasing amounts of ADH. The level of ADH is given in %ADH (in terms of moles of ADH compared to total moles of protein in the sample). The samples were first measured by DLS on a Zetasizer Nano ZS (Malvern Instruments, UK). Duplicate measurements of 100 µl of each sample were injected for analysis by SEC-LS on a Viscotek TDAmax (Malvern Instruments, UK).
SEC-LS measurements show partial separation of the Albucult and ADH peaks as shown in the refractive index chromatograms (Figure 3). In samples in which the concentration of the ADH was below 1.5%, neither the UV nor the RI detectors were sensitive enough to allow accurate determination of the molecular weight. However, at concentrations of ADH above 7.7%, the detection of both light-scattering and RI responses was sufficient to allow determination of the molecular weight. As seen in Table 1, the values determined for Albucult and ADH are close to the known molecular weight values. In this case, the measured %ADH value is slightly below the input value due to the incomplete resolution between the two peaks and could be improved by additional columns, although at the cost of a longer measurement time.
In contrast to SEC-LS measurements, DLS in batch mode is not able to resolve these two proteins, because the resolution in DLS requires a minimum threefold difference in population sizes. In the case of these proteins, this would be equivalent to a molecule of RH ~10.8 nm (where Albucult RH =3.6 nm), which corresponds to a globular protein complex with a molecular weight of around 880 kDa, a value far larger than the 150 kDa ADH.
Nonetheless, DLS is extremely sensitive to changes in the sample, and although it will not discern these two populations, the resulting distribution will change in its mean size and width, allowing comparisons between samples and identification of samples in which substantial oligomerization or aggregation is occurring. This comparison can be used to monitor the same sample over time, for example, or to determine the difference between samples, as in this case study.
In Figure 4, the change in scattering intensity (derived count rate) and the intensity weighted mean radius (Z average) are plotted as a function of the %ADH in the total sample. It is clear that, just as in the SEC-LS measurements, there is a substantial change detected at 7.7% ADH and above, but the mean size of the sample is already increasing at 1.5% of ADH, indicating the presence of oligomers.
Light scattering is capable of detecting the ADH at very low levels, both in SEC-LS and DLS measurements. The limiting factors determining the molecular weight in the SEC-LS measurements are the concentration signal and the resolving power of the SEC column used.
High-quality, animal-free recombinant human albumins provide pharmaceutical manufacturers with the fully characterized and regulatory-compliant multipurpose excipients needed to optimize process development timelines and product quality, and hasten regulatory acceptance of the final drug product.
SEC-LS and DLS are useful tools in the rigorous testing required to provide a detailed understanding and confirmation of protein stability under a variety of conditions. High-quality rAlbumins provide useful reference samples when evaluating the effects of such parameters as pH, temperature, and concentration on oligomerization and aggregation behaviors.
Whether applied to formulation optimization, quality monitoring, or testing for long-term stability, SEC-LS is an excellent tool for the detailed characterization of the type of oligomers or aggregates present in samples of therapeutic proteins. In comparison, DLS provides a rapid measurement that allows the comparison of a number of sample conditions within a short time and gives a clear indication of the presence of large aggregates and small oligomers or aggregates. In this case, these tools provide detailed stability data supporting the confident inclusion of Recombumin and Albucult in pharmaceutical formulations.

References

1. Novozymes Biopharma US Inc. Recombumin Product Specification sheet. Novozymes Biopharma website. Available at: www.biopharma.novozymes.com/en/information-centre/brochures-and-datasheets/Documents/Recombumin_prod_spec_FINAL.pdf. Accessed June 3, 2012.
2. Novozymes Biopharma US Inc. Albucult Product Specification sheet. Novozymes Biopharma website. Available at: www.biopharma.novozymes.com/en/information-centre/brochures-and-datasheets/Documents/Abucult_prod_spec_FINAL.pdf. Accessed June 3, 2012.

Improve Transdermal Insulin Delivery

Maybelle Cowan-Lincoln

Several techniques for penetrating the stratum corneum are being investigated

The first transdermally delivered medication, a nitroglycerin ointment introduced in 1954, was developed after it was observed that fewer angina attacks were reported in munitions workers who handled it than in the rest of the population. In the 1980s, a transdermal nitroglycerin patch was released, followed more recently by patches that deliver compounds like fentanyl, lidocaine, estradiol, and nicotine.¹
By the early 21st century, transdermal delivery products constituted a significant portion of the drug candidates under clinical evaluation. According to a 2008 estimate, more than 1 billion transdermal patches were manufactured around the world in that year.²
The transdermal drug delivery route is particularly desirable for the treatment of diabetes. More than 21 million Americans suffer from this condition, and the CDC forecasts an annual growth rate of 43%. Multiple studies have demonstrated that insulin-dependent patients would benefit from five to six insulin injections per day, but many are not willing to suffer that much discomfort. This results in a “compromise regimen” of two or three injections per day.
A recent study estimates that only 30% of patients with diabetes achieve good control of blood glucose levels (defined as HbA1c lower than 7%). Poorly controlled diabetes can result in short-term symptoms that include low energy and difficulty concentrating and long-term complications such as neuropathy, blindness, and amputation.³
Although transdermal insulin can provide more comfortable therapy, potentially improving patient compliance and therefore diabetes control, this delivery system is best suited to low molecular weight compounds: 100–500 Da. Insulin is a macromolecular compound: 5800 Da. Traditional transdermal methods like patches are not an option. To meet the challenge of moving insulin across the stratum corneum, several penetration enhancement techniques are being investigated.
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CASE STUDY: The Thinking Person’s Diabetes Management

Ultrasonically enhanced transdermal delivery has been demonstrated in preclinical studies to reduce blood glucose levels as well as subcutaneous injections.¹ Using this technology, a “smart” diabetes management system—a possible step toward the elusive goal of an artificial pancreas—is being developed by Penn State University.
Sponsored by the Army, this project endeavors to create a small, portable device to control blood glucose monitoring and deliver insulin transdermally. The system will work like the body—continually sensing the body’s condition and reacting with a sufficient drug dose to maintain a healthy blood glucose level.²
This novel device is composed of three parts:

• Blood glucose sensor;
• Closed-loop feedback controller to vary the insulin dose according to blood glucose levels; and
• Transdermal drug delivery mechanism.²

The insulin delivery device employs ultrasound generated by a 3 x 3 rectangular array of cymbal transducers, facilitating transport across the stratum corneum. The rectangular array has been proven more effective than earlier devices that employed a circular pattern.³ —MCL

References

1. Park EJ, Dodds J, Smith NB. Dose comparison of ultrasonic transdermal insulin delivery to subcutaneous insulin injection. Int J Nanomedicine. 2008;3(3):335-341.
2. Smith N, Pishko M, Gabbay R, Werner J. Closed-loop noninvasive ultrasound glucose sensing and insulin delivery. September 2007. Award Number W81XWH-05-1-0617. Available at: www.dtic.mil/dtic/tr/fulltext/u2/a477332.pdf. Accessed June 3, 2012.
3. Luis J, Park EJ, Meyer RJ, Smith NB. Rectangular cymbal arrays for improved ultrasonic transdermal insulin delivery. J Acoust Soc Am. 2007;122(4):2022-2230.

Microneedles Enhance Delivery

One option is microneedles. These projections, typically ranging in length from 25 µm to 2000 µm, physically breach the stratum corneum to facilitate drug penetration. Solid microneedles are being studied to evaluate how successfully they deliver pharmaceuticals using one of the following two methods:

• “Poke and patch”: An array of microneedles is inserted into the skin and then removed after a short time. The area is then coated with a drug-loaded formulation. However, because this is a two-step process, there are concerns about patient compliance. There are also safety issues regarding non-biodegradable microneedles, particularly those made of silicon, breaking off in the skin during the process.
• “Coat and poke”: Microneedles are coated with a drug formulation that is released into the body, where the coating dissolves after insertion.

A 2003 study at the Georgia Institute of Technology using the poke and patch method demonstrated that solid microneedles can significantly increase transdermal insulin delivery. In the trial, an array of 105 microneedles was inserted into the skin of three groups of live hairless rats for 10 seconds, 10 minutes, and four hours, respectively. A flanged glass chamber of Humulin insulin was adhered to the skin around the array and left on for four hours in all three groups. Blood samples were collected and tested for blood glucose levels.⁴

In addition to microneedles and ultrasound, formulation development is focusing on excipient research into penetration enhancers.

All groups demonstrated a significant decline in blood glucose levels compared with pre-treament, but the largest decrease was seen in the group whose microneedles were left in for 10 seconds. This supports the use of a technique involving brief pre-treatment with microneedles followed by a long drug treatment on the area.
Hollow microneedles have also been shown to be effective in the administration of insulin across the skin. Successful attempts at blood glucose reduction have been achieved in rats using both passive diffusion—an array of microneedles attached to a drug reservoir—and a mechanically driven device employing an electronically controlled drug dispenser.
More importantly, hollow microneedle delivery of insulin has been demonstrated in Type I diabetic humans. Microneedles were inserted to three depths—1, 3.5, and 5 mm. The needles were attached to a 3 mL syringe containing insulin and connected to a syringe pump. The microneedles inserted 1 mm into the skin demonstrated rapid insulin absorption and blood glucose reduction. The efficacy of this depth may be attributable to the layer of capillaries found at that level.
In response to safety concerns about fracturing microneedles, there is an increasing push to manufacture these systems using biodegradable materials, including maltose, galactose, and water-soluble polymers. Any fragments made of these materials would be broken down by skin enzymes. These technologies are currently being evaluated for the delivery of proteins and peptides such as insulin.
Another means to increase drug penetration is electroporation, a technique in which an electric pulse is applied to the skin in order to create transient aqueous paths that increase the permeability of the stratum corneum approximately fourfold. Recently, it has been observed that anionic lipids driven into the stratum corneum extend the finite life of these channels, increasing drug penetration. This theory was tested at Roswell Park Cancer Institute in Buffalo, N.Y., evaluating the transport of insulin across porcine epidermis. When electroporation was enhanced with 1,2-dimyristoylphophatidylserine, an anionic lipid, transport was increased by nearly eighteenfold.⁵
Sonophoresis uses low-frequency ultrasound (20–150 kHz) to increase the transport of insulin. The drug is either incorporated into the hydrogel coupler or applied to the skin in an aqueous solution. The ultrasound enlarges pores, which remain open in the skin for several hours, along with low-pressure air bubbles on the skin’s surface. When the bubbles collapse, they create microjets, propelling the insulin through the stratum corneum.⁶
Although sonophoresis shows promise for successful transdermal insulin delivery, traditional ultrasound devices are large and relatively immobile. A small portable device is needed before this delivery system is feasible.
In response to this need, a portable apparatus that uses hard lead zironate-titanate disks is being tested. These circular caps are 0.25 mm thick with a 12.7 mm diameter and a cavity depth of 0.32 mm. Nine of these “cymbals” were wired into 3 x 3 arrays, driven by a radio frequency waveform generator, digital oscilloscope, RF amplifier, and matching circuit. Through multiple rabbit experiments, it has been determined that this small device can likely deliver the same amount of insulin as a conventional ultrasound machine, resulting in a similar reduction in blood glucose levels.^7,8
In addition to technologies such as microneedles and ultrasound, formulation development is focusing on excipient research into penetration enhancers. An Australian pharmaceutical company, Phosphagenics, claims to have developed a novel transdermal insulin that has achieved positive results in preclinical trials. These innovations may change the lives of more than 200 million diabetes patients worldwide.⁹

References

1. Donnelly RF, Singh TRR, Morrow DIJ, Woolfson AD. Transdermal delivery applications. In: Microneedle-Mediated Transdermal and Intradermal Drug Delivery. Chichester, U.K.: John Wiley & Sons, Ltd; 2012:79-112.
2. Grice JE, Prow TW, Kendall MAF, Roberts MS. Electrical and physical methods of skin penetration enhancement. In: Benson HAE, Watkinson AC. Topical and Transdermal Drug Delivery: Principles and Practice. Hoboken, N.J.: John Wiley & Sons, Inc.; 2012:43-66.
3. Sadrzadeh N, Glembourtt MJ, Stevenson CL. Peptide drug delivery strategies for the treatment of diabetes. J Pharm Sci. 2007;96(8):1925-1954.
4. Martanto W, Davis SP, Holiday NR, Wang J, Gill HS, Prausnitz MR. Transdermal delivery of insulin using microneedles in vivo. Pharm Res. 2004;21(6):947-952.
5. Murthy SN, Zhao YL, Marlan K, Hui SW, Kazim AL, Sen A. Lipid and electroosmosis enhanced transdermal delivery of insulin by electroporation. J Pharm Sci. 2006;95(9):2041-2050.
6. Owens DR, Zinman B, Bolli, G. Alternative routes of insulin delivery. Diabet Med. 2003;20(11):886-898.
7. Park EJ, Dodds J, Smith NB. Dolse comparison of ultrasonic transdermal insulin delivery to subcutaneous insulin injection. Int J Nanomedicine. 2008;3(3):335-341.
8. Snyder B, Lee S, Smith NB, Newnham RE. Ferroelectric transducer arrays for transdermal insulin delivery. J Mater Sci. 2006;41(1):211-216.
9. Barnes K. World’s first transdermal insulin shows promise. In-Pharma Technologist.com. June 19, 2006. Available at: www.in-pharmatechnol