Cleaning Validation in Active pharmaceutical Ingredient manufacturing

2. Objective
The intention of this document has been to define a comprehensive approach to the
Validation of Cleaning procedures in Active Pharmaceutical Ingredient manufacturing
facilities.
Cleaning Validation in the context of Active Pharmaceutical Ingredient manufacture
may be defined as:
The process of providing documented evidence that the cleaning methods
employed within a facility consistently controls potential carryover of product
(including intermediates and impurities), cleaning agents and extraneous
material into subsequent product to a level which is below predetermined levels.
It is necessary to Validate Cleaning procedures for the following reasons:
a. It is a customer requirement - it ensures the safety and purity of the product.
b. It is a regulatory requirement in Active Pharmaceutical Ingredient product
manufacture.
c. It also assures from an internal control and compliance point of view the quality
of the process.
Scope
This Document will serve to:
1. Define the basic concepts and terms associated with Cleaning Validation in the
Active Pharmaceutical Ingredient industry.
2. Serve as a guide from which Masterplans, Protocols and Reports may be
compiled.
Note: General validation principles and a glossary of terms also relevant to cleaning
validation are detailed in the CEFIC / EFPIA Guide entitled ‘Good Manufacturing
Practices for Active Pharmaceutical Ingredient Manufacturers’.
It applies to sterile API’s only up to the point where the API is rendered sterile.
4. Potential residues
The Active Pharmaceutical Ingredient Industry involves (in general) the manufacture of
Active Pharmaceutical Ingredients by both chemical and physical means through a
series of multiple step processes. Plants or individual pieces of equipment, including
ancillary equipment, may be used in multi-product manufacture or dedicated to
individual products.
The result of inadequate cleaning procedures is that any of a number of contaminants
may be present in the next batch manufactured on the equipment such as:
1. Precursors to the Active Pharmaceutical Ingredient
2. By-products and/or degradation products of the Active Pharmaceutical
Ingredient
3. The previous product
4. Solvents and other materials employed during the manufacturing process.
5. Micro-organisms
This is particularly the case where microbial growth may be sustained by the
product.
6. Cleaning agents themselves and lubricants
Current regulatory guidance
Refer to the reference section of this document for details of current Regulatory
Guidance.
Cleaning validation policy
The main focus of this document will be to describe equipment and ancillary equipment
/ process Cleaning Validation in an Active Pharmaceutical Ingredient manufacturing
plant. However, it is appropriate to start by giving a brief introduction as to how the
concept of Cleaning Validation should be approached in a facility.
It is advisable for Active Pharmaceutical Ingredient manufacturing facilities to hold an
official Cleaning Validation Policy. Specific department responsibilities should be
outlined in this and it should be approved by senior management. This policy should
serve to provide a general guideline and direction for company personnel, regulatory
authorities and customers as to how the company deals with areas associated with
Cleaning Validation.
The policy should incorporate the following types of statements:
· Definition of terms employed during validation i.e. rinse vs. flush vs. wash etc.
· A statement specifying what company policy is on validation of cleaning
procedures related to equipment (including ancillary) and processes.
· Company policy re dedication of equipment in certain cases (if products are
deemed too dangerous and / or highly active to manufacture on multi-product
equipment).
· Analytical validation policy.
· The policy should also state the rational for the methods by which acceptance
criteria is determined.
· Revalidation policy.
Levels of cleaning
The degree or level of cleaning and validation required for processes in Active
Pharmaceutical Ingredient manufacturing depends largely on:
· The equipment usage (i.e. dedicated equipment or not)
· The stage of manufacture (early, intermediate or final step)
· The nature of the potential contaminants (toxicity, solubility etc.)
Each of the above three bullets must be evaluated based on the next product, not
only toxicology etc. The rational for this statement is given below:
In general, the higher the potential for finished Active Pharmaceutical Ingredient
contamination the greater the requirement to validate cleaning methods to ensure
product safety.
Active Pharmaceutical Ingredient manufacturers may have different levels of cleaning
requirements in facilities based on the stage of the process being cleaned and the
subsequent product to be manufactured.
Table 1 on page 7 illustrates an example of how a company may decide on the level of
cleaning between lots.
It is the responsibility of the manufacturer to demonstrate that the level of cleaning and
validation performed is adequate based on each individual situation and on a justifiable
scientific rational.
Cleaning should be carried out as soon as practical after the end of processing and
should leave the plant in a suitable condition for next use.
Table 1: levels of cleaning
LEVEL USED WHEN CLEANING
VALIDATION REQUIRED
LEVEL 2 i.e.
· Product changeover of
equipment used in final
step
· Intermediates of one
batch to final step of
another
yes – essential
LEVEL 1 i.e.
· Intermediates or final
Step of one product to
intermediate of another
· Early Step to
intermediates in a
product sequence
progression between level
0 and 2 depending on
process and nature of
contaminant based on
scientific rational
LEVEL 0 i.e. in-campaign, batch to
batch changeover
no validation required
NB: ALL PROCESSES MUST BE EVALUATED INDIVIDUALLY
Elements of cleaning validation
A brief outline of the various elements of a basic cleaning validation study is given
below (see also Figure 1 on page 11).
This is followed by a more detailed view of the individual elements in this section.
I. Establishment of acceptance criteria
II. Cleaning procedure
· Identification of the equipment
· characterization of the products (Previous: activity/toxicity,
solubility, subsequent: dosage, lot size)
· determination and characterization of the cleaning agents
III. Analytical method and its validation
IV. Sampling Procedure and necessary validation of same
V. Validation protocol
VI. Validation report

Putting Paper in the Past

An environmental sampling process that employs technology, ensures cleanroom efficiency and security.

Today’s industry standard for collecting and recording the environmental monitoring (EM) process is paper. This paper-based method, which, for the most part, has not varied much since its inception in the late 1970s, can be time consuming for everyone involved, from the technician to the supervisor, and additionally has the potential for multiple errors introduced inadvertently at any step. Consider the popular grade school game “telephone.” One person starts a story that is continually twisted as it transfers from person-to-person until the last individual receives it and shares a totally different story from what was originally told. This misunderstanding and twisting of facts is not exclusive to verbal transmission of stories, as this can also be initiated by people’s handwriting and is often seen in the healthcare as well as the microbiology industries. What a technician writes on the media container or test worksheet in the lab may be misinterpreted by the next person to handle the document or sample. This misinterpretation that occurs during EM data collection, especially when it is a paperbased process, leads to significant recording errors that require time-consuming, labor-intensive corrective action procedures subsequently increasing overtime and lowering productivity.

Realizing the advantages afforded by wireless technology, the microbiology industry is spurring a shift to put paper in the past. A significant investment is being made in information technology solutions to move from paper-based systems to automated-data collection, management, and reporting. Regulatory agencies are encouraging the use of computer- based systems. According to the Parenteral Drug Association (PDA), the recommended procedure to collect data is to utilize a computer-based system for the following reasons:

• “Based on the large number of samples tested by a given facility, a computer-based data tracking system is recommended.”¹

• “A manual data entry or image scanner system with advantages of speed and accuracy can be used to populate tables.”¹

With new regulations emerging and the significant costs associated with paper-based data collection and recordkeeping, there is increasing demand to improve the efficiency and accuracy of quality control (QC) operations. In reaction to this demand, manufacturers supporting EM testing are beginning to integrate new intuitive wireless technologies into their product portfolios, such as barcode labeling, wireless communications, and remote control operations, for easy integration with EM data-management systems. The following examines how these new applications are addressing recently introduced regulations and helping the industry take full advantage of its information technology investments by automating the EM process from start to finish. CURRENT

PAPER-BASED EM SAMPLING
Today, the majority of EM processes require the technician to document every step in the process on paper, including sample location, date, time, lot numbers (in the case of plated media or strips), and other infor mation directly on the container. Other factors that need to be noted by the technician include “collection site, date and time of collection, person collecting the sample, current activity at the time of sampling, the media type if appropriate, any deviations of the plan.”² Given the amount of information required, this becomes a very time-consuming process especially when recorded manually according to ISO14698-1.².

Once samples have been taken and data recorded, supervisors must review the paper records for accuracy and approve the documents. Then the information may be entered into a database system which again must be reviewed and approved by management. This doubles the amount of time devoted to handling, recording, and approving EM information.

In the case of surface, employee, and active air sampling, the media used must be linked to the corresponding paperwork to ensure accurate result interpretation and record keeping by the microbiology lab personnel. Barcodes are often generated and placed on the media to assist with proper identification, but this process still leaves the potential for error open for the following reasons:

• the technician deviates from the established workflow plan
• the barcode interferes with result interpretation.

The analysis step in the QC process also requires that critical information be included on the test report according to ISO14698-1.2:

Type of sample, method(s) used, the number and title of the standard, collection device used, sampling site, type of activity underway at the time of sampling including occupancy state, number of persons within the sampling ar ea, sampling date and time of sampling, sampling duration, time of examination of samples, conditions and duration of incubation, test results from the examination of the collected samples after initial and final reading, when quantitative tests have been perfor med the results expr ess using appropriate SI units, description of the isolate, name of the organization responsible for the test report and date of completion of the test, name and signature of the individual( s) responsible for performing the test.³

With all the information required, it is easy to see how one could write down or copy the wrong information throughout this extensive and cumbersome paper-based process.

EM SAMPLING GOES AUTOMATED
Instrumentation manufacturers have taken the first step in improving the paper-based process by incorporating new technology into the instrumentation such as data storage to eliminate the need for tedious paper records. Also, instrumentation is now being equipped with wireless data transfer so data can be sent and received either to a PC or scanner. Instrumentation is also being equipped with safety features to prevent the user from changing any settings, which will help prevent any deviations from the sample process. If any changes need to be made to the instrumentation, it would need to be facilitated by the supervisor and all changes would be tracked with a software program and easily recalled later for review, if and when needed. Similarly, media manufacturers have also started barcoding its media, which will help prevent mix-ups with labeling or using the wrong media during a sample. One media manufacturer has even taken barcoding one step further by using two-dimensional data matrix codes (DMC) on their media. The DMC has features such as error checking so if part of the label has been covered up or destroyed it can still be read. The DMC codes also allow the labels to be smaller than a barcode and even contain more information.

In using the new EM process, paper will be eliminated and automated information technology embraced. There will still be a set of steps the user must take to ensure the accuracy of the sample being taken, however, the chance of errors will be greatly reduced because of having barcoding already in place.

1. Scan the media being used in the sample. The media-ID information recorded in the scan may include the media being used, lot number, expiration date, and sample ID.
2. The technician will then scan his or her user-ID, which may include name and employee number.
3. The next scan will be the location-ID, which may include the room and sample point in the room. Along with this information, the facility can also scan in user defined fields, what is being manufactured, if the room is at rest or in use at the time of the sample, or any other pertinent details according to the standard operating procedure (SOP).
4. Once all of the information is recorded in the scanner, it may then be transferred wirelessly to the viable air sampler which already contains more required information. The data in the sampler may include the calibration expiration date, serial number, and volume of air to be sampled.
5. Now that all of the required data has been transferred to the viable air sampler, the sample can be taken.

The steps listed above are repeated for all of the sample locations in the room. The last step in the sampling process is to wirelessly transfer all of the sample data to the PC which is saved in a secure database.

The media can now be incubated according to the facility SOP. Once this is complete, the microbiologist can easily recall all of the information stored in the database. To access the data, the microbiologist will scan the barcode on the media and the record will automatically appear according to the specific identifier of the media. The results can be entered into the record with comments and then saved. This step is repeated for all the media samples taken. Once all of the results are entered, the supervisor can verify and release the record so it may not be altered. Also, if more detailed analysis is needed, the facility can easily integrate this new software with an existing Laboratory Information Management System (LIMS) system.

The same can be also said for media being used without an air sampling instrument. Media that has barcodes from the manufacturer can also be used independently with a scanner and software.

So, in cases where media is used on its own, this new technology can also be leveraged. According to a leading manufacturer of plated media:

• “…the user no longer needs to carry out additional procedures such as labeling or inscribing plates. This leads to greater GMP compliance of the documentation of the used nutrient media as well as to increased process safety as far as secondary contamination is concerned”⁴
• “There is no such risk when it comes to nutrient media with plastic labels, which means that the danger of data loss or mix-ups is considerable reduced.”⁴

INSTRUMENTATION, MEDIA, AND SOFTWARE COMPANIES MAKE A TEAM EFFORT
Specific companies have their own specialties and are utilizing one another’s strengths to make the EM sampling plan state-of-the-art. For the most part, the EM sampling plan has been divided into instrumentation, media, and software — primarily LIMS. Today, there is a team effort to combine all or use a combination of these components together. As stated before, the instrumentation can be equipped with data storage and wireless technology to send and receive data to eliminate the need for recording information on paper. Along the same lines, media is now being barcoded from the manufacturer to eliminate another step in the process. Now, if a complete cleanroom computer system is integrated with the instrumentation and media to include sampling plans, instructions, and trending, all steps can be computerized and for the most part completed during the sampling process. This new process will surely be to the benefit of the manufacturer for its enormous time savings. Not to mention, receiving the results of the sampling much faster and catching any problems before product is shipped saves the manufacturer from potential product recalls and possible lawsuits.

CONCLUSION
Instrumentation, media, and software companies are now redefining the EM sampling process with “Instrumentation Systems” instead of having separate pieces to complete the EM sampling puzzle. The new parts of the puzzle include instrumentation with data storage and wireless technology, media which have been barcoded during the manufacturing process, barcode scanners to read and transmit data, and computer software that tie it all together. This new partnership between companies will create a seamless transition to make paper-based EM a process of the past.

References

1. J. Moldenhauer. Fundamentals of Environmental Monitoring Program – Technical Report No.13 (Revised) Volume 55 No.5 page 9
2. Cleanrooms and associated controlled environments, Biocontamination control – ISO/DIS 14698-1.2 section 5.3.7
3. Cleanrooms and associated controlled environments, Biocontamination control – ISO/DIS 14698-1.2 section 10
4. M. Stein, heipha Dr. Muller GmbH, “Increasing process safety and GMP compliance through the use of barcode ready-to-use nutrient media,” Reinraumtechnik.

Process Gas Chromatograph PGC2000 Edition 2



ABB Process Analytics raises the process GC technical standards with a series of new features added to the PGC 2000. The PGC 2000 Edition 2 Electronic Pressure Control, which improves chromatography resolution and repeatability, is now standard on all PGC 2000 E2s. Two new detectors, dielectric barrier discharge ionization (DBDID) and photo-ionization (PID) add new industry applications for ppm and ppb

Cleaning Tools for Production, Packaging Processes



The FTS Flexi-Cool Immersion Cooler from SP Industries Inc. eliminates the inconvenience of handing dry ice or liquid nitrogen while saving both time and money. Using remote refrigerated probes, the FTS Flexi-Cool provides reliable, rapid cooling capability down to -100ºC, just where you need it. Benefiting from a durable, heavy-duty refrigeration system, the FTS Flexi-Cool is designed to provide years of continuous trouble-free operation.

Caron Introduces New Incubators

Caron has introduced a new line of refrigerated (models 6012 and 6022) and diurnal (models 6011 and 6021) incubators. The product line includes the new 25 cubic foot and the redesigned 10 cubic foot refrigerated and diurnal incubators. These new incubators are designed with the end user in mind, including more customer-friendly and earth-friendly features and innovations.

Automatic Bottle Handling Systems



Faster production and reduced production costs can be achieved with integration of Watson-Marlow’s FF20 and FF30 automatic bottle handling systems. Watson-Marlow announces the availability of its Flexicon FF20 and FF30 automatic bottle handling systems for integration into systems already using the 520Di peristaltic or other filling units from Watson-Marlow. Additionally, with the benchtop automated bottle handling system, there is virtually no risk of bottles falling over during the filling. The FF20 and FF30 work nicely with existing systems, requiring minimal investment since existing dispensers or filling units can be employed.

3M Expands Inhalation Portfolio

3M Drug Delivery Systems introduces two new tools for inhalation drug delivery with pressurized

metered dose inhalers (pMDIs), 3M Plasma Coating Technology and the 3M Face Seal Valve. Both technologies help pMDI systems achieve significant improvement in product performance, expanding the capabilities and robustness of these devices.

Real Time, IR Spectroscopy System

Syrris, a chemistry automation product company, has introduced the new Atlas FT-IR System for in situ reaction monitoring. With integrated mid-IR capabilities, this fully-automated system combines the technology of the Atlas automated reactor with Bruker Optics’ Matrix-MF, a process-ready fourier transform infra red (FT-IR) spectrometer. Available in a wide range of vessel sizes, from 50 ml to 5 L, this compact system fits easily into fume cupboards, making it ideal for any laboratory performing real-time analysis of chemical reactions and processes.

The Vaccine Conundrum

Three years ago, when I worked at a publication that served researchers in drug discovery and development, I wrote an article on efforts to develop vaccines and update production techniques for them.

The story, and the efforts that inspired it, arose from intense public fear about the H5N1 strain of influenza, the so-called "bird flu." At that time, researchers around the world were working tirelessly to isolate strains like H5N1 and H9N2, clone them, and develop vaccines that could be used for inoculation.

But there was one catch: Even after the vaccines were developed, there was still the problem of production to contend with. The sad truth is that the vast majority of flu vaccines are produced today the same way they were 50 years ago. Although this process is proven, it is painstaking and can take several months.

To address this problem, government agencies like the National Institutes of Health (NIH) and pharmaceutical companies investigated the use of cell culture techniques and novel methods such as DNA approaches.

"We definitely would like to see the cell-based vaccine technology come up to speed, not only for the avian flu, but also for the regular circulating strains," David Cho, PhD, the influenza program officer at NIH’s National Institute on Allergy and Infectious Diseases, said in an interview with me at the time. "Having that technology up to speed would really enhance vaccine development."

I’ve been thinking of this story, and the interviews I did for it, quite a bit over the last several weeks in the wake of the recent H1N1 swine flu outbreak.

The day I wrote this letter, Reuters reported that cases of the new H1N1 flu virus have been found in all 50 states, and tests have confirmed the virus in more than 10,000 Americans. There have been 19 fatalities nationwide.

The World Health Organization said that 17,564 people in 64 countries have contracted the swine flu and 115 have died. There were fears early on that this outbreak could be very deadly, so those relatively low numbers are reassuring. But scientists fear that a mutation or two could change the situation quickly.

It’s important to remember how deadly a severe outbreak could be. The infamous 1918 outbreak of Spanish flu (H1N1) was the most devastating flu pandemic in recent history. It killed more than 500,000 in the United States and 20 to 50 million people worldwide. The 1957-58 outbreak of Asian flu (H2N2), although much less severe, caused roughly 70,000 deaths in the United States.

Although some progress has been made in modernizing vaccine production since my article three years ago, not nearly enough has been done, and the infrastructure that is needed to produce millions and millions of doses is nowhere near realization. If a full-scale pandemic were to emerge, we would be in very serious trouble indeed.

The main problem is that vaccines are not moneymakers, so they don’t draw the attention of the vast majority of pharma and biotech companies. And organizations like NIH and the Centers for Disease Control and Prevention are at the mercy of governments that tend to think about money for vaccine research only when the threat of a pandemic emerges, when it is clearly too late.

Here’s hoping that this latest outbreak has gotten the attention of legislators and will move them to put real money and resources behind this vital public health issue

Vibrational spectroscopy can verify the identity of raw materials

ALL IMAGES COURTESY OF ahura scientific inc.

A handheld Raman scanner being used to check the quality of a pharmaceutical raw material.

Manufacturers of pharmaceuticals and other products serving consumer health are faced with an increasingly fragmented, global supply chain of raw materials. At the same time, in the aftermath of the highly publicized tainted pet food and heparin scares of the last years, consumers are growing more concerned about the safety of the products they purchase and consume. As a result, the demand for cost-effective, accurate ways for manufacturers to verify the identity of incoming raw materials is growing.

Along with the resurgent Raman technology, mid-infrared (M-IR) and near-infrared (NIR) spectroscopic techniques form a trident of vibrational spectroscopy tools that are well suited for raw material identification and verification. In vibrational spectroscopy, vibrations at the molecular level produce a unique spectral fingerprint for each compound and enable accurate and conclusive differentiation.

M-IR Theory

M-IR spectroscopy, configured as Fourier transform infrared (FTIR) spectroscopy, has for the last 60 years been used extensively for material identification and authentication.1 When infrared light is passed through a compound, some wavelengths of the light may be absorbed, while others merely pass through the sample unaffected. The frequencies of the light that are absorbed correspond to the vibrational frequencies of the chemical bonds within the sample’s molecules. The absorption frequencies in FTIR (expressed as wave numbers [cm-1]) range from 400 cm-1 to 4,000 cm-1.

Because absorptions correspond to the vibrational frequencies of different bonds within the molecule, they can be used to identify a particular functional group such as C-O, C=O, O-H, or N-H, because the vibrational frequencies of these bonds in the mid-infrared range are well known. Because each bond may have several vibrational frequencies and a molecule may incorporate many different bonds, the infrared spectrum of a material may be complex. Fortunately, it is not necessary to identify every absorption frequency, because it is possible to match an infrared spectrum against others, providing they were all obtained under the same conditions. The fingerprint region (900 cm-1 to 2,000 cm-1) is especially interesting because of its greatest number of absorptions.

M-IR Sampling

The Fourier transform infrared spectrum of alpha-lactose monohydrate.

FTIR absorptions are strong, a characteristic that offers benefits but also poses challenges. Because glass absorbs FTIR wavelengths strongly, it cannot be used to contain materials during FTIR measurements. Materials must be measured directly, and the use of transmission sampling makes it necessary to dilute the sample by preparing a KBr pellet or Nujol (oil) mull. More recently, attenuated total reflectance (ATR) sampling techniques have been improved. They are now popular for FTIR because they remove the need for tedious sample preparation. Despite improvements, current sampling techniques introduce uncertainties into the analysis.

For ATR (the most common sampling method today), the sample of interest must be in direct contact with the ATR crystal, and only a few microns of sample are actually interrogated. This procedure raises significant issues for analyzing heterogeneous materials. ATR accessories can be difficult to use and are breakable and somewhat operator dependent. They also introduce characteristic distortions to the FTIR spectrum, making it necessary to use a software algorithm before searching against libraries generally created from transmission measurements. This correction procedure introduces additional uncertainties into the interpretation.

Traditional FTIR instruments are large, have moving parts, and generally require operation via a computer, making them impractical for use outside the laboratory. While fiber optics for the mid-infrared spectral region exist, they are delicate, have extremely poor transmission (one meter maximum), and are very expensive and are therefore not routinely used. Despite the practical difficulties, FTIR is still extremely popular for all chemical identification needs.

Because FTIR has exquisite molecular selectivity, it is simple to interpret, making it useful in sample identification. Compared to NIR and Raman, there are many more reference texts, correlation charts, and electronic libraries containing spectra of compounds. Automatic search methodologies can be employed to compare the spectrum of an unknown sample against different spectra in a library, including simple peak matching and discrimination analysis (Euclidean distance). Analysts must use caution when interpreting the results, however. Searching techniques can easily generate spurious results, because the closest known match may actually be significantly different from the unknown material; the search simply reports the best-known match from the spectral library, even though the unknown may be from a very different class of materials.

NIR Theory

NIR characterizes the material based on its absorption in the approximately 4,000 to 12,500 cm-1 wavelength region, corresponding to vibrational overtone (harmonic) and combination modes that are much weaker than the fundamental modes measured in the mid-infrared. Each fundamental absorption in the mid-infrared region has several corresponding overtone and combination bands, many of which overlap and are broadened. Because of this, NIR spectra are characterized by much broader features than FTIR spectra, and it is often impossible to make direct bond assignments to particular frequencies.

The bands observed in NIR arise predominantly from stretching of O-H, C-H, and N-H bonds. Because these substructures are common across organic molecules, the differences between NIR spectra of different compounds are often subtle, resulting in a much lower inherent molecular selectivity than FTIR. The sampling method is generally diffuse reflectance, making the physical nature of the sample extremely important in NIR analyses; NIR spectra contain both chemical information like peak shape and position and physical state information such as baseline slope.

The weakness of NIR bands can often be used as an advantage because there is no requirement to specially prepare a sample as there is in FTIR. Additionally, samples can be analyzed through some translucent packaging. This characteristic has led to interest in the use of NIR to identify raw materials in pharmaceutical quality control. The most commonly used sampling mode for solids with NIR spectroscopy is diffuse reflectance. Trigger-operated fiber probes—usually a multi-fiber bundle for solids—and integrating spheres made of polytetrafluoroethylene or coated with gold, are both popular diffuse-reflectance sampling configurations.

Probes and spheres pose challenges for solids interrogation, however. Probes can produce operator-dependent results because the pressure on a powder sample causes baseline movement in the NIR spectrum. The baseline of the spectrum also rises and falls if there is any movement in the fibers during sample collection. Integrating spheres necessitate collecting and placing a sample directly onto the window of the accessory—or putting it into a suitable container first, albeit an inexpensive one, negating one of NIR’s benefits and exposing the operator to the material.

NIR Interpretation

NIR bands are generally broad and ill-resolved, lacking the specificity of FTIR, which is so prized for identification of chemical spectra. As such, peak picking algorithms are not used to identify a material. Chemometric techniques such as multivariate discriminant analysis are required for sample qualification with NIR data. There are many different methodologies available, but they can be thought of as methods to reduce the dimensionality of the data and model the inter- and intra-class dispersion of the data, thereby classifying new samples that are in accordance with historical training/calibration data.

Extensive and comprehensive library construction is critical to accurate interpretation of NIR spectra. Generally, an NIR library can be produced by training the system to recognize what is representative of a sample. Because NIR spectra are affected by moisture content, as well as particle size and density, however, compiling a library for any method requires great care to ensure that representative materials are used and a full validation procedure takes place. Otherwise, the system may fail quite soon after commissioning, as an acceptable sample may display some subtle physical change from the samples used to prepare the library. The new sample may then have to be included as a representative sample and the library updated. The cost of preparing and maintaining an NIR library is often recognized as one of the most significant costs of operation.

Raman Theory

The near infrared spectrum of alpha-lactose monohydrate.

Unlike FTIR and NIR, which are absorption techniques, Raman is a scattering technique. The sample is illuminated with an intense single wavelength light source. Most of the light scatters from the sample without any change in wavelength; this is the elastic scatter or Rayleigh-scattered light. A very small proportion of the light is inelastically (Raman) scattered. The frequency of the Raman-scattered light has shifted from the original wavelength, with the difference in frequency corresponding to the vibration frequency of bonds within the molecules of the sample. Typically, one photon in 106 to 108 is Raman scattered; the rest are Rayleigh scattered or absorbed.

The mechanism causing Raman scatter is different from that found in FTIR or NIR absorption. FTIR and NIR require a change in dipole movement in the vibrating bond for absorption to occur; a change in polarizability in the vibrating bond is needed for Raman scatter to occur. A molecule showing the change in polarizability required to make it Raman active may or may not show the FTIR/NIR activity (i.e., a change in the dipole).

Many absorptions that are weak in FTIR are strong in Raman; mid-IR and Raman are therefore said to be complementary. Symmetric vibrations give rise to intense Raman lines; nonsymmetric ones are often weak and sometimes unobservable. In Raman, as in FTIR, fundamental vibrational modes are interrogated, resulting in outstanding molecular selectivity with little dependence on physical properties such as particle size.

One of the greatest strengths of Raman over other technologies is the ease of sampling it affords. Glass, plastic film, and water are very weak Raman scatterers, enabling sampling through containers and packaging that would not be possible in FTIR; for example, Raman technology can sample through an ultraviolet cuvette, NMR tube, capillary tube, vial, plastic bag, or bottle. Raman sampling is non-contact, non-destructive, and can be made through the double-bagged internal containment in drums. Raman technology can be used to measure aqueous solutions, interrogating the dissolved analytes while analytically ignoring the water. The sensitivity of contemporary Raman instrumentation is such that acquisition times of seconds, comparable to FTIR and NIR, are now the norm.

Raman Interpretation

The Raman spectrum of alpha-lactose monohydrate.

A major benefit of Raman over NIR is its insensitivity to the physical form of the sample. This advantage results in a much simpler approach to data interpretation. Peak picking routines can be used with Raman libraries because the peaks are distinct and sharp and do not move unless the chemical moiety is affected. Raman measurements may be limited by the phenomenon of fluorescence; fortunately, this effect is not frequently observed with today’s 785 nm or longer Raman excitation lasers.

All three vibrational spectroscopy techniques have their place in the pharmaceutical industry. FTIR is still the most widely implemented for identity testing. Its sharp peaks make it ideal for qualitative analysis, and its output is most often used with a peak picking routine or full-spectrum search to confirm the identity of incoming raw materials.

The sampling and current size requirements of FTIR limit it primarily to lab use, however. In addition, water’s extremely strong and broad absorbance in FTIR can pose a problem. Significant amounts of water in a solid sample will likely prevent the measurement of some useful information. The costs associated with lab-based FTIR analysis, including sample collection, health and safety issues, quarantine requirements, and training of highly skilled lab personnel, have encouraged the investigation of both NIR and Raman as alternative and complementary techniques.

Having gained popularity over the past 20 years mostly due to its sampling convenience through some container materials, NIR is present in a variety of applications such as blend uniformity, raw material verification, moisture determination, and particle sizing.2 For raw material verification, the sensitivity of NIR to physical and chemical attributes makes it superior to FTIR because FTIR sample preparation destroys the original particle size distribution, and sensitivity to water prevents measurement of high moisture content samples.

This combination of physical and chemical information also renders simple and direct interpretation of the spectrum an arduous task at best, however, leaving NIR at a disadvantage when compared to Raman.3 In a raw material identification application, a quality control group faces many suppliers and products with vastly different physical characteristics, a situation that leads to lengthy and continual calibration set maintenance, making validation difficult and expensive.4 Subtle differences in particle size and other sample characteristics have to be captured, leading to the requirement of multiple calibration reference samples.

Raman Advantages

Raman instrumentation, on the other hand, is sample-container independent with easily interpreted results and small calibration sets. Raman combines the selectivity of FTIR and ease of sampling of NIR to make an ideal raw material inspection tool that will likely see widespread implementation in the pharmaceutical industry. Because a Raman spectrum is largely unaffected by particle size and moisture, continual library maintenance is not required.

In the past, Raman was not as popular as FTIR and was relegated to the research and development lab. Laser sources were large, unreliable, expensive, and difficult to maintain, and sensitivity was poor, requiring many hours for each measurement. The development of FT-Raman in the 1980s and 1990s improved matters significantly, and the development of high-performance optical blocking filters and charge-coupled device detectors in the 1990s was another significant improvement.

But the instruments that emerged from these advances were still large, costly, and suited only to controlled laboratory environments. Recently, technological advances have enabled the development of compact, rugged, self-contained Raman instruments that can be reliably used in harsh environments. Miniaturization and improved speed of analysis, for instance, have allowed use in warehouse and loading dock locations.

One instrument is the TruScan from Ahura Scientific. Handheld Raman instruments such as the TruScan feature rugged hardware designed to withstand field use, an easy-to-use interface, and fast, cost-effective validation and analysis. n

Bradley is the director of business development and Prulliere is TruScan product manager at Ahura Scientific Inc. Reach Bradley at (978) 642-2563 or jbradley@ahurascientific.com; reach Prulliere at (978) 342-2536 or fprulliere @ahurascientific.com.

References

1. Ryan JA, Compton SV, Brooks MA, et al. Rapid verification of identity and content of drug

formulations using midinfrared spectroscopy.

J Pharm Biomed Anal. 1991;9(4):303-310.

2. Blanco M, Coello J, Iturriaga H, et al. Near-infrared spectroscopy in the pharmaceutical industry. Analyst. 1998;123(8):135R-150R.

3. Ulmschneider M, Wunenburger A, Pénigault E. Using near-infrared spectroscopy for the noninvasive identification of five pharmaceutical active substances in sealed vials. Analusis. 1999;27(10):854-856.

4. Gemperline PJ, Webber LD, Cox FO. Raw materials testing using soft independent modeling of class analogy analysis of near-infrared reflectance spectra. Anal Chem. 1989;61 (2):138-144.

Hyperspectral imaging technology can help control your

A hyperspectral scan image of four generic tablets (left). The corresponding "blur" scan (above) represents a magnified slice of the field of view as the tablets move across the process line.

Scaling pre-clinical drug discovery and development to meet high-volume manufacturing requirements is a challenge for pharmaceutical manufacturers. Spectral imaging and, in particular, hyperspectral imaging represent an opportunity to gain greater analytical understanding and improved process control capability with the deployment of "in-line" or "at-line" hyperspectral instruments.

Spectral imaging encompasses two techniques. One, Raman imaging, measures the molecular vibrational shift associated with exciting a sample compound with a specific laser frequency. The other technique that is emerging, hyperspectral imaging, is a highly specific analytical approach for pharmaceutical manufacturers that some say has advantages over Raman imaging, including:

• It has a large field of view for wide area sampling versus single tablet analysis;

• It has high-throughput screening for real-time analysis and control;

• When utilized in the near infrared (NIR) or short wave infrared (SWIR), hyperspectral instruments offer much greater spectral specificity than mid-wave infrared (MWIR) techniques;

• No sample preparation or sample handling are required; and

• Sampling is in line and non-destructive.

Consistent with the goals of the Food and Drug Administration’s (FDA) process analytical technology (PAT) initiative, hyperspectral imaging instruments greatly enhance the knowledge and understanding of the pharmaceutical process by capturing all of the spectral and spatial attributes of material samples within the sensor’s field of view (FOV).

When combined with spectral libraries established during the drug discovery phase and multivariate analytical models, hyperspectral sensors such as Headwall Photonics’ Hyperspec NIR (900 to 1700 nm) or the Hyperspec SWIR (1000 to 2500 nm) can make accept or reject decisions when deployed in line or at line. As a result, hyperspectral imaging allows pharmaceutical manufacturers to establish critical control points (CCPs) from the post-discovery phase through pre-production and high-volume manufacturing.

Applying Hyperspectral Imaging

A hyperspectral scan showing spectral signatures of generic tablets.

Utilizing high-efficiency diffractive optics, hyperspectral sensors can be configured to offer peak optical efficiency in wavelengths across broad spectral regions. With the underlying advantage being deployment for wide area spectral analysis over a conveyor processing line versus an off line, tablet-by-tablet approach, hyperspectral sensors enable a new set of application capabilities as pharmaceutical manufacturers ramp to volume with precise control over key steps in the production process.

Pharmaceutical manufacturers can use hyperspectral analysis to increase production yields in several areas, including active pharmaceutical ingredient (API), content uniformity, polymorph analysis, quality control over spray dry dispersion, and anti-counterfeit verification and authentication.

In a post-discovery production application, capturing precise spectral information from pharmaceutical manufacturing control points has traditionally involved either simple machine vision or single-point NIR spectral instruments deployed off line. The limitation of these systems is that they are only capable of sampling a very small area of the overall product flow and do not lend themselves to high-speed production processes. In addition, these options are costly due to redundant equipment, poor sampling rates, and time required for analysis.

Defining Hyperspectral Analysis

Pharmaceutical manufacturers can use hyperspectral analysis to increase production yields in several areas, including active pharmaceutical ingredient, content uniformity, polymorph analysis, quality control over spray dry dispersion, and anti-counterfeit verification and authentication.

Hyperspectral imaging technology is an established chemical sensing and imaging technology that allows for the spectroscopic analysis of any particular sample or point within a scene of interest. After its introduction during the mid-1980s as a means to conduct remote sensing experiments and its success as a military and defense sensor technology, hyperspectral imaging is poised for rapid adoption in pharmaceutical applications involving the complex manufacturing of chemical materials and products.

Within the post-discovery manufacturing environment through the ramp to high-volume manufacturing, hyperspectral imagers capture and build a wavelength intensity map of a scene with high spatial resolution. The combination of spectral data and spatial detail enables the high-speed analysis of chemical content, uniformity, quality, and a host of other spectral characteristics and attributes. Traditionally, because these hyperspectral imaging systems were designed to perform under ambient lighting conditions such as available sunlight, they required innovative instrument designs to optimize environmental parameters such as signal-to-noise, optical efficiency, and dynamic range.

Hyperspectral imaging yields the following results within a pharmaceutical manufacturing operation:

• A rendered view of the scene of interest based on known chemical spectra or established spectra libraries;

• For in-line or at-line deployment, spectral wavelengths of interest can be interrogated based on defined intensity thresholds as material and samples pass by the hyperspectral imaging sensor; and

• For any point or pixel within the FOV, the chemical spectra or spectral signature of any particular point can be determined while maintaining the integrity of spatial information obtained.

All hyperspectral imaging instruments consist of the following key elements: a high-performance, aberration-corrected imaging spectrometer; a fore-optics lens selected for the appropriate field of view and distance from the production line; stable illumination; a reference tile such as Spectralon (a reflectance material); a processing unit or computer; and application software for acquiring the images, creating the hyperspectral data cube, and rendering a control decision. Often, depending on the harsh nature of the production environment, the instrument enclosures may be rated for industrial use. For pharmaceutical manufacturing operations, transmissive optics or prisms are not used within the spectrometer, because high imaging performance is required and these components lead to excessive stray light within the system.

Hyperspectral imagers are deployed as a scanning "push-broom" spectral imager. For each moment in time or frame capture by the sensor, the scene observed by the instrument fore-optic lens is imaged onto a tall slit aperture of the hyperspectral instrument. The scene that fills the slit aperture of the sensor is re-imaged through the spectrometer with the wavelengths dispersed by a diffractive grating onto a two-dimensional focal plane array (FPA) such as a charge coupled device.

One axis of the focal plane array (pixel-rows) corresponds to the imaged spatial positions within the FOV all along the slit height. The second axis (spectral for pixel-columns) corresponds to the spectral wavelength that is linearly dispersed and calibrated. Each two-dimensional image or frame capture is digitized by the FPA to build a dataset that comprises all of the spectral and spatial information within the scene or FOV of the sensor.

While scanning a wide conveyor of moving tablets or pills, multiple two-dimensional image frame captures are rapidly taken as tablets pass by the hyperspectral imager; these individual frames are taken at very high speed and are stacked like a deck of cards to produce a data file commonly called a hyperspectral data cube. The value of each pixel within this hyperspectral data cube represents the wavelength-calibrated spectral intensity of that pixel’s small FOV on the scene.

Imaging performance attributes that are critical to the successful deployment of hyperspectral sensing for pre-clinical development in pharmaceutical manufacturing operations are the achievement of high spectral and spatial resolution and exceptional photometric accuracy.

Tangible Results

A hyperspectral rendered view of tablets based on spectral signature.

To demonstrate the value of hyperspectral imaging technology, Headwall application engineers scanned four tablets of similar appearance and size, composed of the following generic drug compounds: aspirin, acetaminophen, vitamin C, and vitamin D. These four compounds were scanned simultaneously with NIR hyperspectral imaging sensors utilizing a moving linear stage to simulate a conveyor line manufacturing process. The samples were scanned in both powder and tablet form and the corresponding image slices were "stitched" together.

One obvious advantage of hyperspectral imaging is the ability to scan multiple tablets simultaneously as they move across a process line. The number of tablets that can be scanned is based on required FOV and the spectral and spatial resolution required (instantaneous field of view). These parameters are application specific and can be modeled to identify the specific sensor components needed to achieve the required performance. Once key spectral features are identified, the hyperspectral imager can be set to bin areas of pixels or regions of interest in order to achieve high volume throughput.

The diagram in Figure 1 (see p. 36) represents a single image slice of the four generic tablets, taken during a process line scan experiment. The corresponding "blur" scan in the photograph represents a magnified slice of the FOV as the tablets move across the process line; the data content comprises all of the spectral and spatial information that will used to build the hyperspectral data cube from which spectral imaging data will be rendered and analyzed.

Derivatives of spectra from aspirin tablets.

Hyperspectral sensor technology analytical information can be displayed in real time while the entire analytical data set (spectral and spatial) is captured and stored for further analysis at a later time. Applications involving NIR chemical imaging such as spray dry dispersion, content uniformity, or polymorph analysis are well suited to hyperspectral imaging techniques. Standard applications like bulk quality control, blending, and packaging also benefit from hyperspectral imaging.

For any pixel or spatial position within the FOV, the corresponding chemical spectrum is obtained and can be graphed as required. Figure 2 (see p. 37), for example, shows the spectral imaging results generated as a result of the hyperspectral scan of the generic tablet compounds. For the purposes of this illustration, all of the corresponding spectra for each of the pharmaceutical tablets are shown.

Can Be Customized for CCP

Given the specific pharmaceutical application or analytical need at that CCP, specific or relevant spectral data can be rendered in the required level of detail or wavelength resolution based on the speed of the production environment. For example, not all hyperspectral data need to be displayed; in some cases, it may be appropriate only to display spectral bands of interest to the researcher or manufacturing engineer.

Compounds and chemical formulations comprised of different spectral signatures can be color coded based on user-defined parameters and established spectral libraries developed during the drug discovery phase. For example, with spray dry dispersion techniques, the presence of multiple compounds or lack of spectral homogeneity in a sample or pill of interest can be identified as in the hyperspectral image in Figure 3 (above, left).

The power of the hyperspectral NIR imaging technique becomes apparent when standard techniques of spectroscopy are applied to the spectral signatures of each tablet. The results of applying hyperspectral spectroscopy to pharmaceutical tablets in the post-discovery phase show that high-speed screening extracts sufficient information from the spectral imaging data to allow discrimination of each tablet from the next with complete certainty.

In demonstrating the spectral features derived from hyperspectral scanning, Figure 4 (see p. 38) displays a series of derivatives taken of the spectra for a series of five random spatial points of each tablet. By applying the standard methods of spectral chemometric analysis and creating a special material index to define each tablet as an independent material variable, the chemometric model can recreate with a high degree of statistical certainty the material index of that particular table or of a similar tablet.

These techniques can be applied to the movement of tablets in real time, and early work suggests that tablet discrimination can be achieved at a very high throughput rate. In many cases, the technique can also be applied to the real-time quantification of the material makeup of tablet samples within the same material population (see Figure 4).

While hyperspectral imaging has been established as a proven, hardened technology for the harsh environments of military, defense, and remote sensing deployments, the technique’s use in pharmaceutical manufacturing operations has demonstrated considerable value in the past few years. Understandably, a key driver in the adoption of hyperspectral imaging instruments is the FDA’s PAT initiative to gain increased control and process understanding at various points in the production process.

With the introduction of commercially available hyperspectral instruments, these spectral imaging sensors can be deployed to increase production yields in a cost-effective manner at many points along the production process, with attractive return on investment and very short payback periods

Local Delivery of Modified Paclitaxel-Loaded Nanoparticles

Images of paclitaxel-loaded DMAB-modified PCL/F68 nanoparticles.

The purpose of this research was to test the possibility of localized intravascular infusion of didodecyldimethylammonium bromide (DMAB)-modified paclitaxel-loaded poly(∑-caprolactone)/Pluronic F68 (PCL/F68) nanoparticles to achieve long-term inhibition of hyperplasia in a balloon-injured rabbit carotid artery model.
Paclitaxel-loaded nanoparticles were prepared by the modified solvent displacement method using commercial poly(lactide-co-glycolide) (PLGA) and self-synthesized PCL/F68, respectively. DMAB was adsorbed on the nanoparticle surface by electrostatic attraction between positive and negative charges to enhance arterial retention. Nanoparticles were found to be of spherical shape with a mean size of around 300 nm and polydispersity of less than 0.150.
The surface charge was changed to positive values after the DMAB modification. The in vitro drug release profile of all nanoparticle formulations showed a biphasic release pattern. Drug release from DMAB-modified PCL/F68 nanoparticles (DPNP) was significantly slower than DMAB-modified PLGA nanoparticles (PGNP). After 90 days, the DPNP group showed very significant inhibition of neointimal proliferation (P <>

Mei L, Sun H, Song C. Local delivery of modified paclitaxel-loaded poly(∑-caprolactone)/pluronic F68 nanoparticles for long-term inhibition of hyperplasia. J Pharm Sci. 2009;98:2040-2050. Correspondence to Hongfan Sun, The Tianjin Key Laboratory of Biomaterial Research, Institute of Biomedical Engineering, Peking Union Medical College & Chinese Academy of Medical Sciences at sunhf@bme.org.cn or +86-22-87892052.

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Surface Stabilization During Drug Nanocrystal Production

Success rate (%) in nanosuspension production for the high-viscosity stabilizers (black bars = 10 wt%, dark gray bars = 2.5 wt%, light gray bars = 1 wt%, white bars = accumulated results for all concentrations).

In order to establish a knowledge base for nanosuspension production, a screening was performed on 13 different stabilizers at three concentrations for nine structurally different drug compounds. Concerning the stabilizers tested, the group of semi-synthetic polymers was the least performant (stable nanosuspensions were obtained in only one out of 10 cases).
For the linear synthetic polymers, better results were obtained with povidones; however, poly(vinyl alcohol) did not result in adequate stabilization. The synthetic copolymers showed even higher success rates, resulting in nanosuspensions in two out of three cases when applied at a 100 wt% concentration (relative to the drug weight). Finally, the surfactants gave the best results, with TPGS being successful at concentrations of 25 or 100 wt% of the drug weight for all compounds tested.
From the point of view of drug compound, large differences could be observed upon evaluation of the relative number of formulations of that compound resulting in nanosuspensions. It was found that the hydrophobicity of the surfaces, as estimated by the adsorbed amount of TPGS per unit of surface area of nanosuspensions stabilized with 25 wt% TPGS was decisive for the agglomeration tendency of the particles and hence the ease of nanosuspensions stabilization.

Van Eerdenbrugh B, Vermant J, Martens JA, et al. A screening study of surface stabilization during the production of drug nanocrystals. J Pharm Sci. 2009;98:2091-2103. Correspondence to Guy Van den Mooter, Laboratory for Pharmacotechnology and Biopharmacy, K.U. Leuven at guy.vandenmooter@pharm.kuleuven.be or +32-16330304.

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Minitablets as Gastroretentive Floating Drug Delivery Systems

Design of floating multiple-unit dosage forms.

A gastroretentive floating drug delivery system with multiple-unit minitablets based on a gas formation technique was developed for furosemide. The system consists of core units (solid dispersion of furosemide: povidone and other excipients), prepared by direct compression process, which are coated with two successive layers, one of which is an effervescent (sodium bicarbonate) layer and another that is an outer polymeric layer of polymethacrylates.
The formulations were evaluated for pharmacopoeial quality control tests and all the physical parameters evaluated were within the acceptable limits. Only the system using Eudragit RL30D and a combination of them as polymeric layer could float within an acceptable time. The time to float decreased as the amount of the effervescent agent increased and when the coating level of the polymeric layer decreased. The drug release was controlled and linear with the square root of time. By increasing the coating level of the polymeric layer, drug release was decreased.
The rapid floating and the controlled release properties were achieved in this study. The stability samples showed no significant change in dissolution profiles (f2 = 81). Radiograms were used to examine the in vivo gastric residence time and it was observed that the units remained in the stomach for about six hours.

Surface analysis techniques can identify counterfeit drugs

ALL IMAGES COURTESY OF Ceram surface and materials analysis

This time-of-flight secondary ion mass spectrometry image shows distribution of active pharmaceutical ingredient (blue) and lubricant (green) across a cross-section of a tablet. This allows the distribution of ingredients to be monitored; if the lubricant distribution differs from that expected from the genuine manufacture route, a counterfeit can be identified.

The pharmaceutical industry has spent billions of dollars trying to tackle the issue of fake medicines, from working with global regulatory authorities and educating healthcare professionals and patients to using technology such as radio frequency identification (RFID) tags and 3D barcodes. Yet, despite these efforts, criminals are still able to move huge quantities of counterfeit drugs through the supply chain and into the hands of millions of unsuspecting patients.

Experts at Ceram Surface and Materials Analysis (Stoke-on-Trent, UK) are responding to this issue by using surface analysis techniques not only to successfully detect fake medicines, but also to determine whether the drugs in question were manufactured using licensed or unlicensed manufacturing processes. Pharmaceutical companies can effectively use many of these analytical technologies in their battle against counterfeiters.

A Global Issue

The problems associated with counterfeit medicines are global and well documented; their costs to patients, drug manufacturers, governments, and society have been in the media spotlight for many years. The World Health Organization estimates that sales of fraudulent drugs will hit $75 billion by 2010, and the pharmaceutical industry estimates that counterfeit medicines account for 10% of the global supply chain—a figure that rises to 70% in some developing countries. Currently, counterfeit drugs can be split into the following categories:

• products without active pharmaceutical ingredients (APIs);

• products with incorrect quantities of APIs;

• products with the wrong ingredients;

• products with the correct quantities of APIs but with fake packaging;

• products with high levels of impurities and contaminants; and

• copies of the original product made using unlicensed manufacturing pro- cesses.

The contents of counterfeits are always unreliable because their sources are unknown or vague. Those with the wrong chemical composition or contaminants can be investigated using a range of techniques, including nuclear magnetic resonance spectroscopy, infrared spectroscopy, thin layer chromatography, desorption electrostatic ionization, Raman spectroscopy, X-ray diffraction, and liquid chromatography-mass spectrometry.

More difficult to identify are counterfeit products that contain the correct ingredients in the correct amounts. These drugs, which are copies of originals, can actually be just as deadly as those with the incorrect ingredients. Other tools that can detect fake drugs and unlicensed manufacturing methods include X-ray photoelectron spectroscopy (XPS), secondary ion mass spectroscopy (SIMS), depth profiling, and time-of-flight secondary ion mass spectrometry (ToF-SIMS).

Reveal More Than Face Value

Images generated by time-of-flight secondary ion mass spectrometry show molecular color-coded images of a tablet cross-section. The images show the three main constituents—core, drug, and coating—as well as the overlay. This allows determination of the molecular distribution.

Surface analysis techniques have proven invaluable to the pharmaceutical industry in the rapid identification of counterfeit drugs, in the trace contamination of pharmaceuticals, and in the assessment of manufacturing processes.

XPS, for example, is a quantitative spectroscopic technique that is able to measure the elemental composition, empirical formula, and chemical state of the elements at the surface of a material and can also detect the elements that contaminate a surface. The technique uses X-rays to dislodge photoelectrons from the surface of the sample; the kinetic energies of the electrons that escape from the top 1–10 nm of the material are then analyzed. This non-destructive quantitative testing method can detect elements down to 0.1 At% concentration (atomic percent, or the ratio of atoms of a particular element to all atoms in a given volume). Every element except hydrogen and helium can be detected with a typical sampling depth of 5–8 nm.

SIMS can examine the surfaces and sub-surfaces of a drug using a depth profiling technique involving continuous bombardment of the surface with a primary beam of ions, resulting in the emission of secondary ions from the sample. The secondary ions are then analyzed, and the associated mass spectra and chemical maps reveal the chemical composition (elemental, isotopic, or molecular) that lies within several microns of the drug surface. SIMS is an incredibly sensitive surface analysis technique, enabling elemental detection in the parts per billion range.

In one form of SIMS, ToF-SIMS, a primary focused ion beam is also used, but the beam is pulsed to provide packets of primary ions that generate packets of secondary ions when they make contact with the sample surface. These secondary ions are then passed through a mass analyzer. Given that the "time of flight" of the ions to the detector is related to their mass, the mass analyzer reveals an extremely detailed breakdown of the molecular composition of the pharmaceutical surface.

Most importantly, the low primary ion doses and sampling depth of 1-2 nm used in this technique means that the sample is effectively undamaged by the analysis. The ion beam can be rastered across the surface under examination to produce a color-coded chemical map. A full spectrum can then be obtained for every pixel point within the image. Although the technique is not quantitative like XPS, it does provide more detailed structural information, particularly for organic and polymeric components.

Used individually or together, these three techniques can provide a very clear picture of the composition of a pharmaceutical product.

Identify Manufacturing Routes

Identifying whether a medicine was manufactured using unlicensed processes is critical to patients. The manufacturing process determines certain characteristics of the drug, and any deviations from the approved practice can threaten the lives of patients who take it.

For example, Ceram Surface and Materials Analysis has used surface analysis techniques to identify differences in the distribution of magnesium stearate, a common pharmaceutical lubricant readily detectable by XPS and ToF-SIMS. This technique pinpoints differences in the manufacturing processes of tablets that have the same chemical composition but are prepared using direct compression and wet granulation.

The analysis also involves careful sectioning of different test tablets in a controlled atmosphere to expose an inner surface. Conventional microscopy and ToF-SIMS (see Figure 1, left) can be used to determine variations in the surface composition and distribution of the lubricant between the two pharmaceutical tablet samples.

It is vital that the pharmaceutical industry have the techniques and expertise to detect counterfeits in order to protect its own reputation and commercial interests and to ensure that patients receive the correct medication. Many pharmaceutical companies now regularly turn to surface analysis techniques, not only for the detection of fakes, but also for routine detailed analysis as part of a long-term quality assurance process. n

Dr. Bentley is a technical sales consultant at Ceram Surface and Materials Analysis. Reach her at justine.bentley @ceram.com.

Resources

1. World Health Organization. Counterfeit medicines. WHO Web site. November 14, 2006. Available at: http://www.who.int/medicines/services/counterfeit/impact/ImpactF_S/en/. Accessed May 7, 2009.

2. Center for Medicine in the Public Interest. Moderator’s guide: 21st century health care terrorism: the perils of international drug counterfeiting. September 20, 2005. Available at: www.cmpi.org/uploads/File/21st-Century-Terrorism.Report.pdf. Accessed May 7, 2009.

3. Bentley J. The counterfeit detective. Pharm Technol Eur. 2008;20(9):20-23.

Novel technologies improve oral drug delivery performance

ALL IMAGES COURTESY OF ELAN DRUG TECHNOLOGIES

An illustration of the Spheroidal Oral Drug Absorption System technology, a controlled-release system.

Oral delivery is the most popular route of administration due to its versatility, ease, and, probably most importantly, high level of patient compliance. A recent PricewaterhouseCoopers report stated that compliance was one of the major issues for the healthcare industry and the leading cause of those recurring health problems that result in a significant rise in healthcare costs. The cost of noncompliance in the United States alone is an estimated $77 to $300 billion a year.1 Providing patients with simplified, convenient oral medications that improve compliance has been a major driver of innovation in the oral drug delivery market.

Within this market, controlled-release tablets and capsules will continue to create the largest demand, with adaptations such as chewable, orally disintegrating, nanoparticle, and combined technology formulations expected to broaden applications and revenues. The total market for oral medication adapted to delivery systems is forecast to reach $56.7 billion in 2012, up 7.1% percent annually from 2007 for the United States alone.2 Oral products represent about 70% of the value of pharmaceutical sales and 60% of the drug delivery systems market.3 The introduction of widely prescribed proprietary medicines in new controlled-release forms will be the driver of market gains.

Generally, controlled-release medicines can be categorized into two groups based on actions. Extended-release formulations deliver a portion of the total dose shortly after ingestion and the remainder over an extended time frame. One example of this formulation is Avinza, a once daily rapid onset extended-release morphine product. Delayed-release systems provide steady dosing after passage through the stomach. Typically, oral drug delivery systems are developed as matrix or reservoir systems.

Two of the most widely commercialized controlled-release technologies are the OROS delivery system developed by Johnson & Johnson’s Alza and Elan Drug Technologies’ Spheroidal Oral Drug Absorption System (SODAS) technology. Both of these technologies were initially developed for calcium channel blockers to improve the drug compliance of hypertensive patients. Other successfully commercialized technologies include Skye- Pharma’s Geomatrix, Eurand’s Diffucaps, and Flamel’s Micropump.

Evolution Continues

The majority of FDA drug approvals from 2002 to 2007 were for reformulations or combinations of previously approved products.

These and other technologies have evolved to address specific therapeutic needs such as pain and blood pressure. A number of companies are engaged in the development of pulsatile release systems, in which the drug is released in pulses separated by defined time intervals. Ritalin LA and Focalin XR, both used to treat attention deficit hyperactivity disorder, use pulsatile release to mimic the twice-daily dosing of a conventional immediate release tablet. These once-daily pulsed profiles offer the patient efficacy throughout the day, negating the need for a second dose during school hours. Ritalin LA and Focalin XR both utilize Elan’s SODAS technology.

Further manipulation of delivery systems has led to the development of chronotherapeutic systems, which take advantage of the natural biorhythms of the human body. Examples include Biovail’s Cardizem XL and UCB’s Verelan PM.

Orally disintegrating tablets (ODTs) are evolving into an important delivery system for drugs that treat medical conditions vulnerable to a sudden onset of symptoms. Such conditions include allergies, nausea, migraine headaches, and schizophrenia. Among the available ODT technologies are Catalent Pharma Solutions’ Zydis, CIMA Labs’ (Cephalon) Durasolv and Orasolv, and SPI Pharma’s Pharmafreeze systems. Catalent’s Zydis technology has been the most commercially successful, with numerous products launched through licensees.

Eli Lilly’s Zyprexa is among the widely prescribed drugs that have been adapted to the ODT delivery system. GlaxoSmithKline’s Lamictal ODT is the most recent product approved by the Food and Drug Administration (FDA). In May, GlaxoSmithKline announced that this product, which uses Eurand’s technology, is the first antiepileptic treatment available in an orally disintegrating formulation.

While there are a number of other delivery systems being developed, including chewables and transmucosals, advances in nanotechnology have recently provided one of the most significant opportunities for growth by addressing the estimated 40% of drugs with poor water solubility issues.4 Elan Drug Technologies’ NanoCrystal technology is one of the players in this area.5 Compared to conventional forms, oral formulations using NanoCrystal technology can enhance bioavailability, thereby reducing dose and size of dosage form; provide for rapid adsorption and onset; extend the range of dose proportionality, allowing for more drug to be delivered to the body; and reduce fed/ fasted variability, enhancing safety and efficacy (see Figure 1, p. 32).

Several of these benefits are embodied in marketed solid oral products, including Abbott’s TriCor 145 mg, Merck’s Emend, and Wyeth’s Rapamune. In-market sales for these three products were over $1.8 billion in 2008. Other technologies designed to overcome problems associated with poor water solubility include Skye-Pharma’s IDD solubilization technology, which has been used to launch Triglide for Sciele and Life Cycle Pharma’s MeltDose technology. Over the coming years, many more poorly water-soluble products should be launched with the aid of these and similar technologies.

Future Opportunities

Many new technologies are being developed to address the problem of oral controlled-release drugs that are poorly soluble in water.

While oral drug delivery is now considered an important drug optimization feature, many more advances are underway.3 For example, new drugs made up of a number of mini-tablets, each one formulated individually and designed to release drug at different sites, allow higher dose loading within the gastrointestinal tract and provide very flexible oral dosage options.

Incorporating mini-tablets of different sizes makes high drug loading possible. The TriLipix fenofibrate product launched by Abbott in January is composed of a number of mini-tablets. The PRODAS (programmable oral drug absorption system) delivery system from Elan Drug Technologies uses a similar approach.

Another area of opportunity is abuse-resistant delivery systems. At present, there are a number of initiatives working to minimize the risk associated with abuse of drugs, strong pain medications in particular. A record 36 million Americans have abused prescription drugs at least once in their lifetime, a U.S. government study found.6

Pain Therapeutics is considered the front-runner with its abuse-resistant formulation of oxycodone, which was made using Durect’s sustained-release gel-cap Oradur technology. In December 2008, Pain Therapeutics received a complete response letter from the FDA for its new drug application submitted for Remoxy in June 2008. To date, Remoxy has not been approved for marketing; the FDA believes additional non-clinical data will be required to support its approval.7

Other drug delivery companies at late-stage development include Alpharma with its morphine-based anti-abuse opioid Embeda, Elite with EL-216, and Acura with Acurox.8 It is estimated that the anti-abuse market, driven by oxycodone and morphine anti-abuse formulations, will create a $1.2 billion market by 2017.9

Another challenge—and opportunity—for the controlled-release market is alcohol-induced dose dumping. In 2005, Palladone capsules were withdrawn from the market in the United States and Canada due to dose dumping when co-ingested with alcohol. This problem is being addressed by a significant number of companies, including Flamel, which has developed Trigger Lock technology based on its Micropump platform. The Trigger Lock formulation of an opioid analgesic is being studied in two clinical trials.

Egalet’s key technology is an oral drug delivery system of capsules comprising a coat and a drug release matrix. The drug is distributed throughout the drug release matrix and is released over time as the coat and matrix are eroded within the gastrointestinal tract. Egalet says that because its technology is neither crushable nor injectable, it is difficult to extract, so it is not subject to alcohol-induced dumping. Other technologies designed to avoid or reduce alcohol dose dumping include Durect’s Saber technology, Soliqs’ Meltrex, and Banner’s Versatrol, a controlled-release softgel technology.

Combination Approaches

Advances in the oral controlled-release (OCR) market have seen companies looking to combine products and/or technologies to achieve better therapeutic effects. One newer initiative in this category, NitroMed’s BiDil product, involves combining hydralazine hydrochloride and isosorbide dinitrate. Drug combinations designed to help improve patient compliance have been a significant driver in the pharmaceutical industry for many years.

Combining approaches to overcome delivery problems of certain drug candidates will become more prevalent as companies push the limits of their technologies. Combining the NanoCrystal technology with their OCR platform, Elan Drug Technologies seeks to overcome problems associated with poorly water-soluble candidates while applying one of their OCR technology platforms to offer the additional benefits of modified or controlled-release properties and allow the drug to be processed into a solid oral dosage form.

Other approaches with significant potential include the targeting of drug directly to the colon and the stomach.10-11 Colonic drug delivery has attracted interest primarily for local delivery in diseases of the colon such as Crohn’s diseases, ulcerative colitis, and colorectal cancer. The colon has also been proposed as a better site than the small intestine to promote oral macromolecule uptake and is typically a site of drug absorption from extended-release preparations.

A variety of approaches are being investigated, including Alizyme’s Colal delivery system, which is in Phase 3, and Cosmo’s MMX technology, in Phase 2. Researchers continue to study gastro-retentive delivery—dosage forms are retained in the stomach to achieve a prolonged and predictable drug delivery profile in the gastrointestinal tract. One example of this type of delivery is Depomed’s AcuForm, a multi-hour, gastric retentive, controlled-release drug delivery system that allows for targeted, controlled delivery of pharmaceuticals to the upper gastrointestinal tract.

A review of the number of FDA approvals over the past years shows that new chemical entities (NCEs) have accounted for only 25% of all products approved; the majority of approvals have been reformulations or combinations of previously approved products (see Table 1, p. 34).12 With a new formulation costing approximately $40 million and taking four to five years to develop compared to the average cost of a next-generation product—in the region of $330 million—the potential for reformulation using oral controlled-release technologies has never been greater.13-14 Moreover, development of an NCE has been estimated to cost between $1.3 and 1.7 billion.15

In the face of financial pressures, it is no surprise that many pharmaceutical companies are turning to drug delivery companies to optimize their marketed products. An extra five years of patent life could generate 50% to 100% more revenue.16 Numerous drug delivery companies now offer a range of OCR technologies, many of which have been validated by product launches. Ongoing developments like the ones described here ensure that the OCR market will continue to grow in order to satisfy the demands of pharmaceutical companies and patients alike. n

Dr. Singh is senior director of oral controlled release, product development, and early stage development at Elan Drug Technologies. Reach him at gsingh.rekhi@elan.com or (770) 538-6321.

References

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2. The Freedonia Group. Drug Delivery Systems to 2012—Demand and Sales Forecasts, Market Share, Market Size, Market Leaders. Study # 2294. Cleveland, Ohio: The Freedonia Group, Inc.; 2008.

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8. IMS Health Inc. IMS LifeCycle R&D Focus: Overview. IMS Web site. Available at: www1.imshealth.com/web/product/0,3155,64576068_63872702_71263548_71480111,00.html. Accessed May 21, 2009.

9. Wheeler H. Short acting and anti-abuse technologies set to fragment and grow the market. Datamonitor Group. March 26, 2008.

10. Touitou E, Barry BW, eds. Enhancement in Drug Delivery. Boca Raton, Fla.: CRC Press; 2006:77-80.

11. Dubin CH. Discussing gastro retentive drug delivery systems. Drug Deliv Technol. 2007;7 (6):26-29.

12. United States Food and Drug Administration. FDA Web site. Available at: www.fda.gov/. Accessed May 21, 2009.

13. Business Insights. Lifecycle management strategies: maximizing ROI through indication expansion, reformulation and Rx-to-OTC switching. Business Insights report. February 2006.

14. Grudzinskas C, Balster RL, Gorodetzky CW, et al. Impact of formulation on the abuse liability, safety and regulation of medications: the expert panel report. Drug Alcohol Depend. 2006;83 (Supp 1):S77-S82.

15. Collier R. Drug development cost estimates hard to swallow. CMAJ. 2009;180(3):279–280.

16. PricewaterhouseCoopers. Pharma 2020: The Vision. June 2007. Available at: www.pwc.com/extweb/pwcpublications.nsf/ docid/91BF330647FFA402852572F2005ECC22. Accessed May 21, 2009.

Characterization of predominant bacteria isolates from clean rooms in a pharmaceutical production unit

	INTRODUCTION Clean rooms are essential in aseptic pharmaceutical or food production. Monitoring microbial distribution and identifying the predominant isolates is part of good manufacturing practices (Akers, 1997). The commonly used protocol for monitoring involves the use of media such as soybean casein digest agar (SCDA) and incubation at 30 °C for 4 d (Akers, 1994). According to the European Union’s good manufacturing practice directive, the permissible number of colony forming unit (CFU) on surface contact plates for grades A and B is 1 and 5 respectively (Schicht, 1998). The number permitted by USP (US Pharmacopeia) for classes 100 and 10 000 is 3 and 5 respectively (USGSA, 1992). To meet such requirements, all the surfaces within the clean room, air, floor and personnel hands, are disinfected routinely using a variety of disinfectants. Ultraviolet (UV) irradiation, Lysol (phenol) solution swabbing and Maskin (chlorhexidine gluconate, CHG) solution immersion are three widely used ways for disinfecting clean room air, furniture surface and personnel skins in many pharmaceutical factories. Maintaining the integrity of a clean room is a constant battle. To decide which method, or combination of methods, to be employed in disinfecting aseptic workshop, there is a need to understand the kind of bacteria that are the prime sources of contamination (Nagarkar et al., 2001). Therefore, knowledge of the microbial diversity of clean rooms, as well as any extreme characteristics these microbes might possess, is essential to the development of disinfection technologies. The aim of this study was to isolate and identify the predominant bacteria strains distributed in clean rooms of a pharmaceutical workshop. In on-going investigations to determine and document possible microbial contamination in clean rooms, bacteria strains were isolated and their colony and cell morphology characteristics were compared. To analyze the phylogenetic relationships of the predominant isolates, the 16S rDNA genes were amplified and their partial sequences were determined. To determine the extreme characteristics of these isolates, their resistance levels to 3 disinfectants (UV, phenol and CHG) were tested.
Top Abstract INTRODUCTION " style="border-style: none;">MATERIALS AND METHODS RESULTS DISCUSSION References	MATERIALS AND METHODS Bacteria isolation Bacteria strains were isolated in the routine aseptic monitoring courses from the clean rooms of a pharmaceutical factory, using settle plate counts for air flora, swabs from a variety of surfaces in the working area, and finger dabs of the working personnel after routine disinfection procedures (MHPRC, 2002). For air sampling, SCDA (casein hydrolysate, 1.5 g; soybean digest, 0.5 g; sodium chloride, 0.5 g; agar, 1.5 g; and distilled water to make 100 ml, pH 7.3) plates were exposed in the clean room after UV irradiation. For surface sampling, a 25-cm2 area of floor was swabbed by tampon after disinfection with 5% (w/v) phenol solution. The hands of workers were sampled after disinfection with 1% (w/v) CHG solution. Hands were washed using the axenic water to collect the bacteria. All the plates were cultivated at 30 °C for 96 h, and bacteria were purified further using streaking method. Morphological and biochemical characterization of predominant bacteria Representative bacteria strains were selected based on colony morphology. On divergent colonies, Gram staining, as well as oxydase and catalase tests was performed. The method of these tests followed John et al.(1994). With this process, and based on the distribution frequencies of these isolates, 5 predominant strains named F01~F05 were selected. Their cell morphologies were further observed under JEM-1200EX transmission electron microscopy (JEOL, Japan) with uranyl acetate staining, and physiological/biochemical characteristics were tested according to John et al.(1994). Molecular characterization of the predominant bacteria DNA was extracted from 5 isolates with freezing and thawing method (Wu and Zhou, 2005). Polymerase chain reaction (PCR) amplification was conducted in Eppendorf Mastercycler using 341F and 907R as primers (Teske et al., 1996). The PCR amplification mixture contained 0.2 mmol/L (each) dNTP, 400 nmol/L (each) primer, 5 mmol/L MgCl2, and 1 U Taqplus (Bioasia, China) in a final volume of 50 μl. After a hot start at 94 °C for 3 min, 30 cycles of PCR reaction were run as follows: denaturation at 94 °C for 1 min, annealing at 55 °C for 45 s, and extension at 72 °C for 1 min. In addition, a final extension at 72 °C for 10 min was added. The resulting products were analyzed by electrophoresis in 1.0% agarose gel and purified with QIAquick PCR purification kit column (Qiagen, Germany). Sequences were determined in an ABI PRISM 3730 DNA automated sequencer using 341F as a sequencing primer, and their closest matches were found by blasting against the short and nearly exact matches from NCBI (National Center for Biotechnology Information) databases (http://www.ncbi.nlm.nih.gov). Sequences were aligned and the phylogenetic tree was generated using DNAMAN package (Lynnon Biosoft, Canada) with evolutionary distances method (bootstrapping 1000 trials). The determined partial 16S rDNA sequences described in this study were deposited in European Molecular Biology Laboratory (EMBL) nucleotide sequence database under accession Nos. AM268420 to AM268424. Preparation of bacteria cell suspension The isolated bacteria were inoculated into nutrient liquid (beef extract 0.5%, peptone 1%, NaCl 0.5%, pH 7.2) and cultivated in a rotating shaker (200 r/min) at 30 °C for 12 h, 5 ml fermentation liquid was centrifuged at 10 000×g for 5 min, then the precipitated cells were washed twice and diluted to 106 cells/ml with sterilized distilled water. Effect of UV irradiation on survival rate of bacteria isolates A 15 W UV light [irradiation intensity is 30 μJ/(cm2·s)] was used to determine the UV resistance abilities of the isolated strains. After turning on the UV light for 30 min to stabilize the irradiation intensity, 5 ml cell suspension was placed into a sterilized disk, exposed to UV light (20 cm) for 0~5 min per half minute, with slow stirring using a magnetic stirring apparatus, using a serial dilution method and then the surviving cells were counted on nutrient agar plates (Park and Noguera, 2007). All procedures were performed under red light. Effect of phenol and CHG solution on survival rate of bacteria isolates Two methods were used to determine the disinfection effects of phenol or CHG solution on the isolated bacteria. The first one, named short term treatment test, was to inoculate 107 bacteria cells into 5 ml at various concentration of the disinfectants and was kept at 30 °C for 30 min. The cells were then collected by centrifugation and washed twice with sterilized distilled water to remove adherent phenol or CHG. Surviving bacteria cells were counted on nutrient agar using colony forming unit method. The second method, named long term co-culture test, was to cultivate bacteria cells in liquid nutrient with varying contents of disinfectants for 48 h. Bacteria growth was determined using colorimetry based on an increase in absorbance at 650 nm wave length. Statistical analysis All the disinfectant resistance tests were performed in triplicate. Since there was no interaction between the isolates and disinfectants, two-ways analysis of variance (ANOVA) were performed to determine the treatment differences between the isolates and Staphylococcus aureus (Sa) using statistical software in Microsoft Excel.
Top Abstract INTRODUCTION MATERIALS AND METHODS " style="border-style: none;">RESULTS DISCUSSION References	RESULTS Bacteria isolation and characterization Bacteria were isolated in the routine microbiological monitoring processes for clean rooms of a pharmaceutical factory. Based on the colony and cell morphology, as well as distribution frequency and sampling origin, 5 predominant bacteria strains named F01~F05 were selected. Isolates F01, F02 and F04 are cocci, about 0.5 µm in diameter, while isolates F03 and F05 are rods. In addition, isolate F05 has an endospore and several flagella (Fig.1 ). Further investigations showed that all the 5 isolates are positive in Gram staining reaction, and have no mobility except isolate F05. Fig. 1 Transmission electron micrograph of 5 isolates. Bacteria cells were directly stained with uranyl acetate and observed under JEM-1200EX electron microscopy The biochemical and physiological tests also showed that isolates F01, F02 and F04 are similar in most of the properties (Table 1), and 16S rDNA sequence analyses further demonstrated that these 3 isolates share more than 99% similarities. Blasting analyses of partial 16S rDNA sequences confirmed that these 3 isolates belong to the genus Staphylococcus. However, molecular characterizations were at the limits of resolution for the differentiation of species in this genus. Combined with the results of biochemical and physiological tests (Table 1), it can be preliminarily concluded that F01 and F04 closely match S. saprophyticus and S. cohnii subsp urealyticus, respectively, and F02 may be S. warneri or S. pasteuri. With the same methods, F05 was identified as a Firmicutes Bacillus fusiformis, while F03 was an Actinobacteria Microbacterium oleivorans (Fig.2 ). Table 1 Biochemical and physiological characterization of the predominant bacteria isolates Fig. 2 Phylogenetic affiliation of the bacteria isolated from clean rooms as revealed by comparative analysis of partial 16S rDNA sequences and those stored in public nucleotide databases. F01 to F05 are sequences determined in this study. Accession numbers (more ...) Effect of UV irradiation on the growth of the isolates UV irradiation is a widely used method for disinfection of clean room air in pharmaceutical factories. Isolate F03 had much higher UV resistance than the other strains. When exposed to UV rays for 3 min, 7% of suspension cells were still alive, while only about 4% of the F05 cells survived after being irradiated for 1 min (Fig.3 ). However, it cannot be simply deduced that isolate F05 is more sensitive to UV rays than the other 4 isolates because the irradiated cells in this study were in the state of log phase, when endospores have not yet been formed. In 3 isolates belonging to Staphylococcus, F01 and F04 exhibited weaker UV resistance than the control S. aureus (Sa), while the contrary was true for the isolate F02. However, differences among these strains are not notable (Table 2), demonstrating that the 3 isolates are the general strains distributed in natural environments and not the UV-resistant mutants. Fig. 3 Effect of UV irradiation on the survival of the isolates Table 2 P-value in F-test on the survival rates between isolated bacteria and Staphylococcus aureus Effect of phenol solution on the survival of isolates From the median lethal dose (LD50) of the phenol solution on the 5 isolates (Fig.4 ), phenol resistance abilities were as follows: F02>F01>F03>Sa>F04>F05 in the short term treatment tests and F04>F02>F01>Sa>F05>F03 in the long term co-culture tests. Similar results were obtained when evaluated with 90% lethal dose (LD90), that is: F02>F01>F04>F05>Sa>F03 in the short term treatment tests and F04>F01>F02>F05>Sa>F03 in the long term co-culture tests. This leads to the conclusion that isolates F02 or F01 are less sensitive, whereas isolate F03 is sensitive to phenol treatment. Isolate F05 is special in that more than 60% of cells were killed at 0.1% phenol treatment solution for 30 min. However, 3.1% and 0.25% of cells were still alive under treatments with 1% and 2% phenol at the same conditions. It seems that endospores had been formed in a small proportion of test cells. Fig. 4 Effect of phenol solution on the survival of the isolates in a short term treatment test (a) and a long term co-culture test (b) It is odd that at low phenol concentration (for example, 0.1%), survival rates of the 3 isolates belonging to Staphylococcus (F01, F02 and F04) are higher in the long term co-culture tests than in the shot term treatment tests. Different culture methods in the two kinds of tests may be one of the reasons. It seems that some injured bacteria cells that could not recover on nutrient agar could still grow in liquid media, making the optical density of the culture to increase slowly. Also the efficacy of the phenol may be reduced by high organic load in the co-culture test. Effect of CHG solution on the survival of 5 isolates The LD50 of the 5 isolates follows the order of F04>F05>F01>F03>Sa>F02 in short term treatment tests and F04>F01>Sa>F03=F05>F02 in long term co-culture tests. Similar results were obtained when evaluated with LD90, that is: F04=F05>F01=F02>F03=Sa in short term treatment tests and F04=F01>Sa>F05>F02>F03 in long term co-culture tests (Fig.5 ). Isolates F04 and F01 had high resistance abilities while isolates F02 and F03 were sensitive to this disinfectant in both tests. Like on the effect of phenol solution, isolate F05 showed strong resistance to 30 min treatment with CHG solution, but was easily inhibited in co-culture tests. It seems that the endospore of F05 can endure 30 min treatment of CHG solution and therefore could recover on nutrient agar after the disinfectant was removed, whereas in long term co-culture test, low dosage of disinfectant was active during the entire culture period and the endospore was therefore not able to germinate. Fig. 5 Effect of chlorhexidine gluconate (CHG) on the survival of the isolates in a short term treatment test (a) and a long term co-culture test (b) Osmotic resistance tests (Table 1) show that 4 of the 5 isolates can grow on nutrient agar with 10% of NaCl and that the isolate F05 can even grow on 15% NaCl, suggesting that the 5 isolates have the abilities to resist severe environment pressures. Significant test of disinfectants resisting abilities between 5 isolates and Staphylococcus aureus Although the 5 isolates have different resisting abilities to 3 disinfectants, two-ways ANOVA showed no significant differences between these 5 isolates and S. aureus (Table 2), suggesting that these isolates are just the normal bacteria in the environment, and that the resisting abilities are the inherent characteristics of these strains, rather than plasmid hold or DNA changed mutants.
Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS " style="border-style: none;">DISCUSSION References	DISCUSSION Maintaining the integrity of a clean room is a constant battle (Nagarkar et al., 2001). There are 3 prime sources of contamination. The first is from human errors. To control this source of contamination, human hands must be washed with disinfectant. CHG, as a cationic surfactant, is the widely used skin disinfectant because of its mild nature. Contamination may also result from the room surface areas. To avoid such contamination, floors, walls and ceilings must be swept with phenol or other disinfectants. The third contamination source is from the room air. UV irradiation is the most convenient way to sterilize room air although it is not very penetrating and needs direct exposure. To ensure a clean room conforming to the designated classification, constant monitoring of contaminant sources and identification of the predominant contaminant bacteria is usually necessary (Rosch et al., 2005). This study found that the predominant contaminant bacteria were a group of Gram positive bacteria: either spore-forming Bacillus, or non-sporulating Staphylococcus and Microbacterium. Further experiments showed that these bacteria could endure UV irradiation and phenol or CHG treatment. These results are in agreement with the findings of other workers (Onaolapo, 1995). Shaban et al.(1997) and Newcombe et al.(2005) found that Staphylococcus and spore-forming Bacillus were more resistant to UV than the other vegetative bacteria. Ajaz et al.(2004) screened 4 phenol resistant bacteria strains, two of which belong to genera Staphylococcus and Bacillus. Ogunniyi et al.(2000) also found that Bacillus and Staphylococcus were resistant to CHG treatment. The mechanisms associated with resistance have received uneven attention. For most disinfectants, the studies are largely on phenomenological descriptions of the occurrence. Much less is known about the frequency with which resistance develops and the impact of environmental factors on resistance development (Chapman, 2003). This study found that all the 5 identified isolates were Gram positive bacteria, either spore-forming Bacillus, which is known to confer resistance to extreme environmental conditions, or non-sporulating Staphylococcus and Microbacterium, which have a thick cell wall. The thick wall of a cell or spore is a reasonable explanation for resistance to UV irradiation because this kind of non-ionizing radiation penetrates weakly. However, to phenol and CHG solutions, the cell wall could not be a reasonable explanation for retarding disinfectant entrance. It seems that physiological or genetic changes, such as phenotypic adaptation, genetic alteration, or genetic acquisition, must have been developed (Cloete, 2003). It has been reported that some genes special resistant phenotypes are located together on mobile genetic elements such as a plasmid, transposon, or integron (Chapman, 2003), and therefore, the development of resistance to one antibacterial agent is always accompanied by the appearance of resistance to another agent (Dukan and Touati, 1996). However, two-ways ANOVA showed no significant differences between the isolates and the control (Table 2), suggesting that these isolates are just phenotype adapted wild strains, rather than mutants resulting from gene acquisition.
	Acknowledgments We thank engineer H. Weng and engineer Z.L. Zhou from a pharmaceutical factory in Hangzhou for supplying bacteria isolates.