Cleaning Validation Principles

Type: Classroom Training Course
Level: Intermediate
Level: Advanced

Date	Location	Country	Instructor(s)
9-10 November 2010	Annual Meeting Orlando, FL	USA	Becky Brewer
21-22 February 2011	Tampa Conference Tampa, FL	USA	Becky Brewer
9-10 March 2011	San Francisco, California	USA	Becky Brewer

Description

As cleaning technology and detection methodology advance, so do the challenges associated with establishing, managing, and maintaining a scientifically sound cleaning validation program. FDA's risk-based regulatory initiatives focus new attention on the risks of cross-contamination. The solution is to understand life cycle management techniques for an effective cleaning validation program.
This course will cover elements of a cleaning validation program from start to finish, exploring such concepts as the determination of residues to be targeted, selection of analytical and sampling methods, determination of appropriate limits in various pharmaceutical and biotechnology processes, and establishment of scientific rationales acceptable to regulatory inspectors. For mature cleaning validation programs, concepts such as understanding process control, capability, learning to effectively self-audit a cleaning validation program, and documentation will be essential takeaways.
This course contains knowledge related to the CPIP™ technical knowledge competency element Facilities and Equipment. For complete information concerning the knowledge elements or the CPIP Credential, please visit www.ispe-pcc.org.

Course Modules

• Developing, Deploying and Maintaining
• Regulatory Requirements
• Fundamentals of Cleaning Validation
• Master Plans
• Equipment Characterization
• SOP Development
• Selecting Residues, Developing and Maintaining Limits
• Methods Validation and Recovery Studies
• Engineering Studies and Cycle Development
• Creating Cleaning Validation Protocols
• Collecting and Testing Validation Samples
• Validation Reports

Take Back to Your Job

• Identify and characterize potential residues including product, processing aids, cleaning agents, and adventitious agents
• Apply appropriate analytical methodology for selected residues
• Determine suitable sampling techniques and the selection of sampling locations that present a challenge for the cleaning process
• Calculate residue limits that meet all necessary regulatory requirements
• Create scientifically sound rationales, validation protocols, and reports
• Manage the challenges of multi-product facilities in the establishment of limits, determination of validation strategies, and maintaining the validated state
• Understand campaign-based production strategies for effective and scientifically sound validation
• Differentiate the requirements for cleaning validation when using manual, semi-automatic, and automatic cleaning technologies
• Determine scientific grouping or bracketing approaches
• Comprehend the pitfalls inherent in cleaning after the production of biopharmaceutical and pharmaceutical products
• Accomplish analytical method validation and recovery study requirements in cost-effective studies that provide the necessary assurance of an analytical system
• Evaluate cleaning practices, limit calculations, scientific rationales, and validation documents through internal self-audits to ensure compliance with ever-changing regulatory needs
• Practice hands-on exercises designed to reinforce core competencies and job-focused skills

Attendance Suggested For

• Professionals responsible for all aspects of cleaning validation programs, including development, deployment, and maintenance
• Quality assurance specialists, quality control technicians, regulatory affairs professionals, pharmacologists and toxicologists, validation scientists, and validation service personnel
• Manufacturing supervisors, technical support personnel, and engineers responsible for evaluating cleaning systems, reviewing equipment, and supporting the cleaning validation program on the plant floor
• All levels of management who need to understand the science of cleaning and cleaning validation including the aspects of residue selection, sampling method and analytical detection method validation, limits determination, and strategies for managing multi-product facilities

Continuing Education Units 

ISPE will provide continuing education units (ISPE CEUs) for all North American seminars and courses, including educational programming at the ISPE Annual Meeting. CEUs are nationally recognized units of achievement designed for those individuals continuing their education in their chosen field or profession.
Delegates attending European seminars and courses will receive a Certificate of Attendance, as ISPE CEUs are currently not offered at European events. Verification of CEUs is based on attendance as well as satisfactory completion of all evaluation materials. Statements of credit will be sent via email within four weeks of evaluation materials. One hour of education programming equals 0.1 ISPE CEU credits.

THE APPLICABILITY OF CLEANING VALIDATION

THE APPLICABILITY OF
CLEANING VALIDATION

Cleaning validation is primarily applicable to the cleaning of process manufacturing equipment in the pharmaceutical industry. Cleaning validation focuses on those cleaned equipment surfaces that, if inadequately cleaned, potentially could contaminate the product subsequently manufactured using that same equipment. This validation primarily covers product contact surfaces in the cleaned equipment (PIC/S, 2004), for these surfaces directly contact the next product. For clarification, the regulatory and scientific requirements for the cleaning of process equipment surfaces are different from those requirements for the cleaning of environmental cleanroom surfaces. While both involve aspects of cleaning, the focus of this book’s cleaning validation is product contact surfaces of process equipment.
This includes the interior surfaces of vessels, agitators, piping, hoses, pumps, and other items that directly contact the manufactured product and thus can transfer residues directly to the next product. There are some applications where indirect residue transfer may occur. I like to call these surfaces “indirect product contact” surfaces. Some examples of indirect transfer are clear. The reflux condenser in the organic synthesis of an active ingredient may not directly contact the next manufactured product; however, the

4 Cleaning Validation: Practial Compliance Solutions . . .
refluxing solvent does contact the condenser surfaces and potentially could carry residues from the condenser surfaces to the solvent containing the active ingredient.
A more controversial example of indirect transfer involves the interior surfaces of a lyophilizer (or freeze dryer). Although debate about transfer from lyophilizer surfaces may be controversial, there is a fairly clear regulatory expectation that cleaning validation will be performed for lyophilizers (FDA, 1993). It is possible that residues left behind (on shelves, for example) potentially could transfer by an airborne route to the manufactured product. Such a transfer has not been demonstrated in real life cases. Such a transfer is more likely to occur with lyophilizing of bulk product on trays than it is with lyophilizing of product in vials. One possible rationale for cleaning validation for lyophilizers is that most companies that lyophilize parenterals will sterilize the lyophilizer surfaces prior to use. The ostensible reason is that bioburden from the lyophilizer surfaces somehow could be transferred to the lyophilized product. If it is possible that bioburden from the surfaces could be transferred, though, is it also not likely that product could be transferred? One interesting feature of residues in lyophilizers is that adherent residues (which are generally most difficult to clean) are least likely to transfer via an airborne route, whereas loosely adherent residues (which are more likely to be removed in cleaning) are more likely to transfer via an airborne route. Whatever the situation, because the products manufactured in a lyophilizer are usually parenteral products, some companies have been asked by regulatory authorities to validate the cleaning of lyophilizers, while others have chosen to pursue cleaning validation for other reasons.
Other types of cleaning cannot be validated because of the frequency of performing the identical cleaning procedure (SOP). For example, for clinical trial materials or for drugs made infrequently (every year or two, for example), it is doubtful that the exact same cleaning SOP would be used three successive times in order to obtain three PQ (performance qualification) runs. While three runs no longer are the regulatory expectation for process validation purposes (FDA, 2004a; FDA, 2004b), in most cases companies still require a minimum of three PQ runs unless a different number is justified. In such cases, the cleaning process cannot be validated; however, it is

The Applicability of Cleaning Validation 5
still necessary to determine that the equipment is suitably cleaned for the manufacture of the next product. This calls for cleaning verification and involves performing tests similar to those done for the three PQ runs in cleaning validation, except that the tests are performed for each and every cleaning event. Although cleaning verification can be contrasted with cleaning validation, cleaning verification ordinarily should be defined in a cleaning validation policy or cleaning validation master plan.
Still other types of cleaning require neither validation nor verification. For example, cleaning of the outsides of tanks and the cleaning of walls and floors is required under GMPs. There should be SOPs defining those cleaning processes. However, those processes are not critical because the possibility of transfer to the product is remote, making validation unnecessary. When a significant possibility of transfer of residues from such surfaces to the next products exists, that situation should be rectified not by cleaning validation but rather by manufacturing controls. Furthermore, for extremely hazardous drug actives present on non-contact surfaces, there may be more of a concern related to personnel safety as compared to potential cross-contamination of the next manufactured product.
The applicability of cleaning validation should be written into a facility’s Cleaning Validation Master Plan to define clear situations that require validation, but also to permit professional judgment in cases that may require considered reflection.
The above chapter is based on a Cleaning Memo originally published in October, 2000.
www.pda.org/bookstore
6 Cleaning Validation: Practial Compliance Solutions . . .
REFERENCES
Food and Drug Administration (FDA). 1993. Guide to inspections of lyophilization of parenterals. July 1993. Washington, D.C.: U.S. Government Printing Office. Accessed 26 May 2006 at http://www.fda.gov/ora/inspect_ref/igs/lyophi.html
Food and Drug Administration (FDA). 2004a. FDA takes first step in recognizing the role of emerging technologies in the area of process validation. Mar. 17, 2004. Washington, D.C.: U.S. Government Printing Office. Accessed 26 May 2006 at http://www.fda.gov/cder/gmp/processvalidation.htm
Food and Drug Administration (FDA). 2004b. Sec. 490.100 process validation requirements for drug products and active pharmaceutical ingredients subject to pre-market approval (CPG 7132c.08). Revised Mar. 12, 2004. Washington, D.C.: U.S. Government Printing Office. Accessed 26 May 2006 at http://www.fda.gov/ora/compliance_ref/cpg/cpgdrg/cpg490-100.html
Pharmaceutical Inspection Convention Pharmaceutical Inspection Co-Operation Scheme (PIC/S). 2004. Recommendations on cleaning validation. Document PI 006-2. July 1, 2004. Geneva, Switzerland: PIC/S.

WHAT’S A CONTAMINANT?
Once at a training session at the FDA’s Basic Drug School, I was asked the question whether allowing a specified level of residue in a manufactured product (such as 0.001 of a therapeutic dose of a previous active) was, in fact, allowing manufacturers to produce and release drug products which were “contaminated.” My answer at the time was related to the fact that with newer analytical methods, we were able to measure residues at even lower levels, so that it was not feasible to specify that “no residues” of previous products appear in any other product. In giving this matter more thought, my answer would be slightly different. Rather than concede that any measured residue is a “contaminant,” I would now answer that question by stating that a contaminant is defined by both the presence of a “foreign” substance as well as the level of that substance in the drug.
This definition is related to the argument that “the dose makes the poison.” For example, selenium is considered a poison (causing selenosis) at doses of 500 micrograms, but is necessary in human diets at levels of about 50 micrograms. In fact, selenium is included in some vitamin and dietary supplement formulations at around 100 micrograms (NIH, 2004). This analogy doesn’t apply directly to residues in drugs because for the most part we are not considering substances that may have beneficial effects at extremely low levels.
However, consider situations where it is only certain levels of a given substance in a drug that render the substance to be classified

8 Cleaning Validation: Practial Compliance Solutions . . .
an “objectionable” contaminant. For example, bioburden in a non-sterile oral drug product can be present at certain levels, such as 75 CFU per gram, and not be considered objectionable. However, if those 75 CFU were E. coli, then one readily would conclude that the material was objectionably contaminated. It is both the nature of the substance and the level that are important.
This suggests that perhaps we should be more precise in our language as we discuss acceptable levels of residues. Thus, we should avoid phrases like “acceptable level of a given contaminant,” because in some cases it is the level that makes the “residue” a “contaminant.” Therefore, to say that a drug contains a residue of a previous drug at a given level is not to state that it is necessarily contaminated (or even adulterated). One way to look at this is to say that “a contaminant is an objectionable residue” (realizing, of course, that there may other types of contaminants).
This should not be used as an excuse to be sloppy in our cleaning efforts and say that any residue is okay as along as it is below the acceptance threshold. We should be conscientiously applying good manufacturing practices in our cleaning procedures so that any potentially contaminating residue is kept as low as practical. In a Human Drug CGMP Note, the FDA states that although equipment does not have to be absolutely clean as measured by the best available analytical technique, the equipment surfaces should be as clean as “reasonably achieved” by good cleaning procedures (FDA, 2001).
This is also not to be interpreted as saying that for any substance, some measurable amount may be acceptable. Consistent with the PIC/S cleaning validation guide (PIC/S, 2004), for certain allergens and cytotoxic substances, any residue should be below the limit of detection by the best available analytical technology. In such a case, one must still concede that a possibility exists that the drug will have a small, but not measurable (with current technology) residue of the allergen or cytotoxic material. If that unmeasured level of residue could still be objectionable, then in such a case it makes sense to use dedicated equipment.

Nor is it to ignore the fact that, with new information, levels that we regard acceptable today may become objectionable in the future. However, this is one of the tradeoffs we live with in trying to advance medical care.
The above chapter is based on a Cleaning Memo originally published in March, 2002.
What’s a Contaminant? 9

10 Cleaning Validation: Practial Compliance Solutions . . .

REFERENCES
Food and Drug Administration (FDA). 2001. Human drug CGMP notes. Second Quarter 2001. Washington, D.C.: U.S. Government Printing Office.
National Institutes of Health (NIH). 2004. Dietary supplement fact sheet: Selenium. Aug. 1, 2004. Bethesda, MD: Office of Dietary Supplements, NIH. Accessed 26 May 2006 at http://dietary-supplements.info.nih.gov/factsheets/selenium.asp
Pharmaceutical Inspection Convention Pharmaceutical Inspection Co-Operation Scheme (PIC/S). 2004. Recommendations on cleaning validation. Document PI 006-2. July 1, 2004. Geneva, Switzerland: PIC/S.

ADEQUATE
“DOCUMENTED EVIDENCE”
FOR CLEANING VALIDATION

A short definition of cleaning validation, consistent with a definition of process validation (FDA, 1987), is “documented evidence with a high degree of assurance that a cleaning process will consistently produce equipment and products meeting predetermined quality specifications.” In an internal audit or regulatory investigation, a key is reviewing that “documented evidence.” What could be included as part of that documented evidence?
One usually first thinks of the cleaning validation summary report. This is an important item. However, there is much more to the documented evidence than just this summary report. The consistency of the cleaning process is not demonstrated solely by the three (or whatever number is required) validation runs. It may be useful to think of the three validation runs as “confirming” the consistency of the cleaning process. The three validation runs are not experiments to determine if residues are acceptable. The experiments should be done earlier in the design of the cleaning SOP. At the point of cleaning validation, one should have a reasonable assurance that acceptable results will be the outcome. Therefore, other documents that could be considered part of the relevant “documented evidence” include the following:

12 Cleaning Validation: Practial Compliance Solutions . . .
• Cleaning validation master plan or high level policy
• Cleaning validation procedure
• Cleaning procedure or cleaning instruction
• Cleaning process development report or technology transfer report
• Cleaning validation protocol
• Analytical procedure
• Sampling procedure
• Report on rationale for selection of sampling locations
• Report on rationale for challenges (e.g., worst cases) to the cleaning process
• Analytical method validation
• Analytical/sampling method recovery
• Limits calculation report
• Deviation investigation report
• Training records
• Change control documents
• Monitoring records and/or trend reports
• Revalidation report and/or annual cleaning review report
Certainly under the new FDA investigation program (FDA, 2002), a key point to consider is the cleaning validation master plan or high level cleaning validation policy. This higher-level document—a practical necessity though not absolutely necessary—ties together most of the items listed above. One would question, for example, whether a cleaning process can still be considered validated (beyond the initial validation protocol) if it is not covered under a change control policy/procedure. Supporting the higher-level policy may be a more specific cleaning validation procedure document.
Another important document is the cleaning process procedure or instruction (or whatever the detailed cleaning process followed by the cleaning operator is called in a facility). One may have excellent validation data (for example, with all the swab and rinse

samples meeting the properly calculated acceptance criteria), but the cleaning process may be inadequately detailed and controlled such that there is no reasonable assurance that the cleaning process will produce the same data if carried out in the future. Appropriate design of the cleaning procedure is equally as important as appropriate design of the cleaning validation protocol.
The rationale for a cleaning process development report (which sometimes is called a technology transfer report) is twofold. One function is to provide future scientists in your company with a good rationale on how the cleaning process and the various parameters (cleaning agent, cleaning agent concentration, times, temperature, hold times, etc.) were selected. Furthermore, although such reports may not be critical to the validation investigation by regulatory authorities, those regulatory authorities may ask for this information. It is part of the emphasis of the FDA in asking pharmaceutical manufacturers to understand their manufacturing processes (FDA, 2004). A second reason is to provide assurance that the cleaning validation will be successful once the protocol is executed. As mentioned previously, the execution of the cleaning validation protocol should not be viewed as an experiment to test whether the cleaning process is effective; rather the “experiments” should be performed before the execution of the protocol in order to have a high degree of assurance that the cleaning process will be successfully validated when the cleaning validation protocol is carried out.
Other protocol related documents, such as the cleaning validation protocol itself, analytical procedure(s), sampling procedure(s), a report on the rationale for selection of sampling locations, a report on rationale for challenges (e.g., worst cases) to the cleaning process, analytical method validation, sampling/sampling method recovery, limits calculation report, training records, and any protocol deviation investigation report may exist as “stand alone” documents (e.g., analytical method validation), but some may also just be incorporated into the cleaning validation protocol (e.g., justification for sampling locations).
Other documents related to demonstrating consistency after initial validation is complete include the training records (particularly for any retraining on process clarifications and for operators
Adequate “Documented Evidence” for . . . 13

14 Cleaning Validation: Practial Compliance Solutions . . .

in manual cleaning processes), monitoring/trending after protocol execution, process-related deviations/investigations, change control, and revalidation.
The purpose of this chapter is not to proscribe certain ways to document cleaning validation but rather to consider all the evidence that can be part of the assurance of consistency of a cleaning process. This chapter should serve as a reminder that any of these documents may be requested as part of an audit or investigation of cleaning validation for a process or a facility.
The above chapter is based on a Cleaning Memo originally published in July, 2003.

REFERENCES

Food and Drug Administration (FDA). 1987. Guideline on general principles of process validation. May 1987. Accessed 26 May 2006 at http://www.fda.gov/cder/guidance/pv.htm
Food and Drug Administration (FDA). 2002. Compliance program guidance manual 7356.002, drug manufacturing inspections. Feb. 1, 2002. Washington, D.C.: U.S. Government Printing Office. Accessed 26 May 2006 at www.fda.gov/ora/cpgm/7356_002/7356-002FINAL.doc
Food and Drug Administration (FDA). 2004. Guideline for industry: PAT–a framework for innovative pharmaceutical development, manufacturing, and Quality Assurance. Sept. 2004. Washington, D.C.: U.S. Government Printing Office. Accessed 26 May 2006 at www.fda.gov/cder/guidance/6419fnl.pdf
Adequate “Documented Evidence” . . . 15

WATER QUALITY FOR

VALIDATED CLEANING PROCESSES

The quality of the water used for aqueous cleaning is critical for performance. This includes the water quality for any pre-rinse, for the washing step itself, and for any rinses. Quality includes chemical properties—pH, conductivity, hardness, Total Organic Carbon (TOC), etc.—and biological properties, including bioburden and endotoxin. Unfortunately, there are few regulatory guidelines that deal with this subject. (One guidance will be covered later.) However, there are a number of good scientific principles to apply. We’ll start with water quality for washing, then cover rinsing, and finally cover pre-rinsing.
Water for Washing
For the washing process, perhaps the most critical element to control is the water hardness (calcium and magnesium ions). Hardness ions are well known to affect the efficacy of cleaning of aqueous surfactant solutions (LeBlanc, 2000). If tap (potable) water is used for cleaning, hardness can be accounted for by using chelants in the cleaning formulation. It can also be a problem if the hardness in the water varies, either seasonally or by source. For example, some municipalities obtain their water both from surface waters and from deep wells. The deep-well water is more likely to have

18 Cleaning Validation: Practial Compliance Solutions . . .

an elevated degree of hardness. If a cleaning process were designed using the surface water, that cleaning process might not be effective if the deep-well water (with higher hardness) were used. A second concern with hardness ions in any tap water source is that, if alkaline cleaning agents are used (for example, those with potassium or sodium hydroxide), hardness ions may precipitate as calcium carbonate at high pHs. Depending on the conditions of precipitation, the result may cause a white residue on surfaces. That white residue may cause a surface to fail a “visually clean” criterion. This can be minimized by utilizing a cleaning agent with chelants, or by using an acidic post-rinse. The later approach is a common one and a carryover from cleaning processes in the dairy industry where the precipitation of “milk stone” (from the calcium in the milk) in the alkaline cleaning step is a routine feature.
Water for Rinsing
A general principle involving the manufacture of finished drugs utilizing water in the formulation is that the quality of the water for the final rinse should be at least as good as the quality of the water added in the next manufacturing process. For example, if a parenteral drug product is formulated with Water for Injection (WFI), then the final rinse of the previous cleaning process should be with WFI. If an oral drug product is formulated with Purified Water (PW), then the final rinse of the previous cleaning process should be with PW. The rationale for this is that any residues left behind from the final rinse are residues that would be added in the next product anyway. In this way, concerns about residues from the final rinse water itself are minimized. If water is used for rinsing, and the subsequently manufactured product does not have water in the manufacturing process, then additional information is required. For example, if the product is a non-sterile oral, solid dose product, PW clearly would be acceptable for a final rinse. If the aqueous cleaning process involves cleaning an active pharmaceutical ingredient (API) made by an organic synthesis route (in which no water is used in the synthesis), the most common approach is to use deionized water as the final rinse (such facilities rarely have a validated

Purified Water system). Of course, in that specific situation, some solvent rinse would be used after the water rinse to remove any water from the equipment, being that water would interfere with the organic synthesis.
One additional concern about the final rinse quality is that one also should be aware that using a lower quality water for the final rinse may leave behind mineral deposits, which in and of itself would not be a problem; however, those mineral deposits may be visible when the rinse water dries, and therefore would cause the equipment to fail any “visually clean” criterion.
Water for Pre-rinsing
In most cases, the quality of the water for pre-rinsing is the least critical of the three cases. After all, why be scrupulous about the quality of the water for this step when the greater issue is removing all the previous product? Water for pre-rinsing is solely used to flush residue out of the system prior to the washing step itself. Some companies will choose to recycle their water, and use for the pre-rinse the water from the previous final rinse (not from the initial post-rinses, for these will be highly contaminated with residues and cleaning agent). Choices of water quality to use for the pre-rinse sometimes are based on practical issues, such as using the same water as was used for the washing step.
Regulatory Guidance
One useful regulatory guidance on water quality for cleaning processes is the EMEA’s “Note for Guidance on Quality of Water for Pharmaceutical Use.” (EMEA, 2002). The relevant comments regarding water for cleaning are given in Table 5 of that document. That table is summarized as follows—
Water Quality for Validated Cleaning Processes
20 Cleaning Validation: Practial Compliance Solutions . . .
Note that this guidance does not address the quality of water used for the washing step.
Although not regulatory documents per se, FDA 483’s have been sent to some companies if they have used potable (tap) water for cleaning. However, the main issue was not the use of potable water but rather the lack of a monitoring program to measure and control the quality of the water. Such a monitoring program generally includes both chemical and microbiological quality of the water. Although records from testing at the water source by the municipality may help, it is best to have an onsite monitoring program in place in the individual pharmaceutical facility. This is a better measure of the water quality as it is used by the facility.


REFERENCES
European Agency for the Evaluation of Medicinal Products (EMEA). Note for guidance on quality of water for pharmaceutical use. May 2002. London, England: EMEA. Accessed 26 May 2006 at http://www.emea.eu.int/pdfs/human/qwp/015801en.pdf
LeBlanc, D. A. 2000. Validated cleaning technologies for pharmaceutical manufacturing. Englewood, CO: Interpharm Press.

CLEANING VALIDATION

PRINCIPLE
1.1
Pharmaceutical products and active pharmaceutical ingredients (APIs) can be contaminated by other pharmaceutical products or APIs, by cleaning agents, by micro-organisms or by other material (e.g. air-borne particles, dust, lubricants, raw materials, intermediates, auxiliaries). In many cases, the same equipment may be used for processing different products. To avoid contamination of the following pharmaceutical product, adequate cleaning procedures are essential.
1.2
Cleaning procedures must strictly follow carefully established and validated methods of execution. This applies equally to the manufacture of pharmaceutical products and active pharmaceutical ingredients (APIs). In any case, manufacturing processes have to be designed and carried out in a way that contamination is reduced to an acceptable level.
1.3
Cleaning Validation is documented evidence that an approved cleaning procedure will provide equipment that is suitable for processing of pharmaceutical products or active pharmaceutical ingredients (APIs).
1.4
The objective of the Cleaning Validation is the confirmation of a reliable cleaning procedure so that the analytical monitoring may be omitted or reduced to a minimum in the routine phase.
2.
PURPOSE AND SCOPE
2.1
These guidance notes describe the validation of cleaning procedures for the removal of contaminants associated with the previous products, residues of cleaning agents as well as the control of potential microbial contaminants.
2.2
These guidance notes apply to the manufacture of pharmaceutical products (final dosage forms) and of active pharmaceutical ingredients (APIs).
3.
GENERAL
3.1
Normally only cleaning procedures for product contact surfaces of the equipment need to be validated. Consideration should be given to non-contact parts into which product may migrate. For example, seals, flanges, mixing shaft, fans of ovens, heating elements etc.
3.2
Cleaning procedures for product changeover in the case of marketed products should be fully validated.
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3.3
Generally in case of batch-to-batch production it is not necessary to clean after each batch. However, cleaning intervals and methods should be determined.
3.4
Several questions should be addressed when evaluating the cleaning process. For example:
-
At what point does a piece of equipment or system become clean?
-
What does ‘visually clean’ mean?
-
Does the equipment need to be scrubbed by hand?
-
What is accomplished by hand scrubbing rather than just a solvent wash?
-
How variable are manual cleaning processes from batch to batch and product to product?
-
What is the most appropriate solvent or detergent?
-
Are different cleaning processes required for different products in contact with a piece of equipment?
-
How many times need a cleaning process be applied to ensure adequate cleaning of each piece of equipment?
3.5
Cleaning procedures for products and processes, which are very similar, do not need to be individually validated. It is considered acceptable to select a representative range of similar products and processes concerned and to justify a validation program, which addresses the critical issues relating to the selected products and processes. A single validation study under consideration of the “worst case” can then be carried out which takes account of the relevant criteria. This practice is termed "Bracketing".
3.6
At least three consecutive applications of the cleaning procedure should be performed and shown to be successful in order to prove that the method is validated.
3.7
Raw materials sourced from different suppliers may have different physical properties and impurity profiles. Such differences should be considered when designing cleaning procedures, as the materials may behave differently.
3.8
Control of change to validated cleaning procedures is required. Re-validation should be considered under the following circumstances:
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3.8.1
Re-validation in cases of changes to equipment, products or processes; and
3.8.2
Periodic re-validation at defined intervals.
3.9
Manual methods should be reassessed at more frequent intervals than clean-in-place (CIP) systems.
3.10
It is usually not considered acceptable to "test until clean". This concept involves cleaning, sampling and testing, with repetition of this sequence until an acceptable residue limit is attained. For the system or equipment with a validated cleaning process, this practice of "test until clean" should not be required. The practice of "test until clean" is not considered to replace the need to validate cleaning procedures.
3.11
Products that simulate the physicochemical properties of the substance to be removed may be used instead of the substances themselves, where such substances are either toxic or hazardous.
4.
DOCUMENTATION
4.1
A Cleaning Validation Protocol is required laying down the procedure on how the cleaning process will be validated. It should include the following:
-
The objective of the validation process;
-
Responsibilities for performing and approving the validation study;
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Description of the equipment to be used;
-
The interval between the end of production and the beginning of the cleaning procedures;
-
Cleaning procedures to be used for each product, each manufacturing system or each piece of equipment;
-
The number of cleaning cycles to be performed consecutively;
-
Any routine monitoring equipment;
-
Sampling procedures, including the rationale for why a certain sampling method is used;
-
Clearly defined sampling locations;
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Data on recovery studies where appropriate;
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Analytical methods including the limit of detection and the limit of quantitation of those methods;
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The acceptance criteria, including the rationale for setting the specific limits;
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Other products, processes, and equipment for which the planned validation is valid according to the “bracketing” concept; and
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When Re-validation will be required.
4.2
The Cleaning Validation Protocol should be formally approved by the Plant Management, to ensure that aspects relating to the work defined in the protocol, for example personnel resources, are known and accepted by the management. Quality Assurance should be involved in the approval of protocols and reports.
4.3
A Final Validation Report should be prepared. The conclusions of this report should state if the cleaning process has been validated successfully. Limitations that apply to the use of the validated method should be defined (for example, the analytical limit at which cleanliness can be determined). The report should be approved by the Plant Management.
4.4
The cleaning process should be documented in an SOP.
4.5
Records should be kept of cleaning performed in such a way that the following information is readily available:
-
The area or piece of equipment cleaned;
-
The person who carried out the cleaning;
-
When the cleaning was carried out;
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The SOP defining the cleaning process; and
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The product, which was previously processed on the equipment being cleaned.
4.6
The cleaning record should be signed by the operator who performed the cleaning and by the person responsible for Production and should be reviewed by Quality Assurance.
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5.
PERSONNEL
5.1
Operators who perform cleaning routinely should be trained in the application of validated cleaning procedures. Training records should be available for all training carried out.
5.2
It is difficult to validate a manual, i.e. an inherently variable/cleaning procedure. Therefore, operators carrying out manual cleaning procedures should be supervised at regular intervals.
6.
EQUIPMENT
6.1
The design of the equipment should be carefully examined. Critical areas (those hardest to clean) should be identified, particularly in large systems that employ semi-automatic or fully automatic clean-in-place (CIP) systems.
6.2
Dedicated equipment should be used for products, which are difficult to remove (e.g. tarry or gummy residues in the bulk manufacturing), for equipment which is difficult to clean (e.g. bags for fluid bed dryers), or for products with a high safety risk (e.g. biologicals or products of high potency which may be difficult to detect below an acceptable limit).
7.
MICROBIOLOGICAL ASPECTS
7.1
The existence of conditions favorable to reproduction of micro-organisms (e.g. moisture, temperature, crevices and rough surfaces) and the time of storage should be considered. The aim should be to prevent excessive microbial contamination.
7.2
The period and when appropriate, conditions of storage of equipment before cleaning and the time between cleaning and equipment reuse, should form part of the validation of cleaning procedures. This is to provide confidence that routine cleaning and storage of equipment does not allow microbial proliferation.
7.3
In general, equipment should be stored dry, and under no circumstances should stagnant water be allowed to remain in equipment subsequent to cleaning operations.
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8.
SAMPLING
8.1
Samples should be drawn according to the Cleaning Validation Protocol.
8.2
There are two methods of sampling that are considered to be acceptable, direct surface sampling (swab method) and indirect sampling (use of rinse solutions). A combination of the two methods is generally the most desirable, particularly in circumstances where accessibility of equipment parts can mitigate against direct surface sampling.
A.
Direct Surface Sampling
(i) The suitability of the material to be used for sampling and of the sampling medium should be determined. The ability to recover samples accurately may be affected by the choice of sampling material. It is important to ensure that the sampling medium and solvent are satisfactory and can be readily used.
B.
Rinse Samples
(i)
Rinse samples allow sampling of a large surface area. In addition, inaccessible areas of equipment that cannot be routinely disassembled can be evaluated. However, consideration should be given to the solubility of the contaminant.
(ii)
A direct measurement of the product residue or contaminant in the relevant solvent should be made when rinse samples are used to validate the cleaning process.
9.
DETERGENTS
9.1
The efficiency of cleaning procedures for the removal of detergent residues should be evaluated. Acceptable limits should be defined for levels of detergent after cleaning. Ideally, there should be no residues detected. The possibility of detergent breakdown should be considered when validating cleaning procedures.
9.2
The composition of detergents should be known to the manufacturer. If such information is not available, alternative detergents should be selected whose composition can be defined. As a guide, food regulations may be consulted. The manufacturer should ensure that he is notified by the detergent supplier of any critical changes in the formulation of the detergent.
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10.
ANALYTICAL METHODS
10.1
The analytical methods should be validated before the Cleaning Validation Study is carried out.
10.2
The analytical methods used to detect residuals or contaminants should be specific for the substance to be assayed and provide a sensitivity that reflects the level of cleanliness determined to be acceptable by the company.
10.3
The analytical methods should be challenged in combination with the sampling methods used, to show that the contaminants can be recovered from the equipment surface and to show the level of recovery as well as the consistency of recovery. This is necessary before any conclusions can be made based on the sample results. A negative result may also be the result of poor sampling techniques.
11.
ESTABLISHMENT OF LIMITS
11.1
The pharmaceutical company's rationale for selecting limits for product residues should be logically based on a consideration of the materials involved and their therapeutic dose. The limits should be practical, achievable and verifiable.
11.2
The approach for setting limits can be:
-
product specific Cleaning Validation for all products,
-
grouping into product families and choosing a ‘worst-case’ product,
-
grouping into groups of risk (e.g. very soluble products, similar potency, highly toxic products, difficult to detect).
11.3
Carry-over of product residues should meet defined criteria. For example, the most stringent of the following three criteria:
(a)
No more than 0.1%of the normal therapeutic dose of any product will appear in the maximum daily dose of the following product,
(b)
No more than 10ppm of any product will appear in another product,
(c)
No quantity of residue should be visible on the equipment after cleaning procedures are performed. Spiking studies should determine the concentration at which most active ingredients are visible,
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(d)
For certain allergenic ingredients, penicillins, cephalosporins or potent steroids and cytotoxics, the limit should be below the limit of detection by best available analytical methods. In practice this may mean that dedicated plants are used for these products.
11.4
One cannot ensure that the contaminate will be uniformly distributed throughout the system. It is also an invalid conclusion to make the assumption that a residual contaminant would be worn off the equipment surface uniformly or that the contamination might only occur at the beginning of the batch.
11.5
In establishing residual limits, it may not be adequate to focus only on the principal reactant since chemical variations (active decomposition materials) may be more difficult to remove.
12.
REFERENCE
12.1 PIC/S document, PI 006-3: Recommendations on Validation Master Plan, Installation and Operational Qualification, Non-Sterile Process Validation, Cleaning Validation.

Cleaning Validation in Active pharmaceutical Ingredient manufacturing plants

Table of contents
1. Foreword....................................................................................................…..... 2
2. Objective....................................................................................................…..... 3
3. Scope..……................................................................................................…..... 4
4. Potential residues …………………………….................................................. 5
5. Current regulatory guidance……………….................................................... 6
6. Cleaning validation policy........................................................................…..... 7
7. Levels of cleaning.………….....................................................................…..... 8
8. Elements of cleaning validation...............................................................…..... 10
8.1 Establishment of acceptance criteria….................................................... 12
8.1.1 chemical determination ........................................................................ 12
8.1.2 physical determination ......................................................................... 13
8.1.3 microbiological determination.............................................................. 13
8.2 Cleaning procedures ............................................................................... 13
8.3 Sampling ................................................................................................ 15
8.4 Analytical methods…. ............................................................................ 16
8.5 Validation protocols ............................................................................... 17
8.6 Validation reports................................................................................... 18
9. Minimum requirements...........…..............................................................…..... 20
10. Change control…………..........…..............................................................…..... 21
11. Summary………………...........…..............................................................…..... 22
12. References…………..…...........…..............................................................…..... 23
Guide to Cleaning Validation in API plants
2
1. Foreword
This Guideline has been produced by the Active Pharmaceutical Ingredients Committee
(APIC) Working group.
Different organizations will be influenced by their companies and the markets that they
serve in the approaches that they take and the policies that they have with respect to
the subject.
It is also valuable to bear in mind that this is an area that is changing rapidly and what
was considered as being acceptable 2-5 years ago is now not adequate. Therefore,
companies should be aware of the need to continuously update themselves on current
regulatory requirements.
Guide to Cleaning Validation in API plants
3
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.
Guide to Cleaning Validation in API plants
4
3. 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.
Guide to Cleaning Validation in API plants
5
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
Guide to Cleaning Validation in API plants
6
5. Current regulatory guidance
Refer to the reference section of this document for details of current Regulatory
Guidance.
Guide to Cleaning Validation in API plants
7
6. 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.
Guide to Cleaning Validation in API plants
8
7. 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.
Guide to Cleaning Validation in API plants
9
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
Guide to Cleaning Validation in API plants
10
8. 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
Guide to Cleaning Validation in API plants
11
EVALUATE EQUIPMENT SURFACES
AND DETERMINE
1. Worst case locations to sample (swab
sampling)
1. Volume and type of rinse solvent to be
employed (rinse sampling)
1. Equipment surface area (necessary to
calculate carryover into subsequent
batches)
DETERMINE THE MOST APPROPRIATE CLEANING
PROCEDURE FOR THE EQUIPMENT -
1. Generate acceptance criteria data for the contaminant.
2. The cleaning method will be determined by the process,
the equipment the cleaning agents and the cleaning
techniques available.
3. All aspects of the cleaning procedure should be clearly
defined in SOPs be they manual / CIP or COP
DEVELOP AND VALIDATE THE
SAMPLING AND CHOSEN
ANALYTICAL METHODS
FOR THE COMPOUND(S) BEING
CLEANED
1. Swab
2. Rinse
(determine % recovery, limit of detection,
limit of quantitation, accuracy of method,
reproducibility, stability over time ...etc.)
INTERIM REPORT:
GENERATE INTERIM CLEANING
VALIDATION REPORTS ON A CLEAN BY
CLEAN BASIS DETAILING THE
ACCEPTABILITY OF THE CLEANING
PROCEDURE FOR THE EQUIPMENT AND
THE PRODUCT.
This is only required where there is a long
period of time between manufacture of the
validation runs (see stage 4 for reporting
requirements).
Figure 1: Cleaning Validation Process
STAGE 1:
STAGE 2: STAGE 3 STAGE 4:
DEVELOP A CLEANING VALIDATION PROTOCOL
FOR THE PRODUCT AND THE EQUIPMENT BEING
CLEANED
That should encompass for example:
1. Introduction
2. Scope
3. Equipment
4. Cleaning procedure
5. Sampling procedures
6. Analytical testing procedure
7. Acceptance/Cleaning limits.
8. Acceptance criteria for the validation.
GENERATE A CLEANING VALIDATION
REPORT DETAILING THE ACCEPTABILITY
OF THE CLEANING PROCEDURE FOR THE
EQUIPMENT AND THE PRODUCT
The report should give a full detailed background and
introduction to the cleaning Validation study and
should evaluate all data generated with respect to the
acceptance criteria employed for the study. The report
should also indicate the requirement if any for
revalidation (period of time /change control etc.)
Guide to Cleaning Validation in API plants
12
8.1 Establishment of acceptance criteria
The Cleaning Validation should demonstrate that the procedure consistently
removes residues of the substance previously manufactured down to levels that
are acceptable and that the cleaning procedure itself does not contribute
unacceptable levels of residual materials to the equipment. The limits set
should be practical, achievable and justifiable.
In Active Pharmaceutical Ingredient manufacture there may be partial reactants
and unwanted by-products which may not have been chemically identified.
Therefore, it may be necessary to focus on by-products as well as the principle
reactant. Companies should decide on which residue(s) to quantify based on
sound scientific rational.
8.1.1 Chemical determination
It is generally the residual Active Pharmaceutical Ingredient or intermediate,
which is of greatest concern rather than reaction side products or residual
impurities.
There are a number of options available when determining acceptance criteria.
Where either toxicological or therapeutic data if available then calculation A or
B is preferable. If data is not available for either of these calculations or if the
result is more stringent calculation C should be used.
A. Limiting the level based on toxicity data.
An Acceptable Daily Intake (ADI) is calculated with suitable safety
factors applied and this is converted to the maximum allowable
carryover to the API.
B. Pharmacological Dose Method:
The philosophy is to reduce the levels of residual product in each piece
of equipment, such that no greater than 1/1000 of the normal
therapeutic dose will be present per typical dose of the next product to
be run in the equipment. The validation protocol should include a
calculation, which ties this philosophy to the acceptance criteria for the
samples to be tested.
C. Limiting the level of product which could appear in the following
products.
Limits from 10ppm up to 0.1% (based on the ICH impurity document
which indicates that up to 0.1% of an individual unknown or 0.5% total
unknowns material may be present in the product being tested )
Note FDA Statement on 0.1% impurities
Guide to Cleaning Validation in API plants
13
FDA statement: P. Alcock, in Human Drug cGMP Notes, P. Motise,
June 98: „...we have found that some firms have incorrectly applied as
their acceptance limit the 0.1% impurity identification threshold as
discussed in both the ICH impurity guideline and the U.S.P. General
Notices. This application of the 0.1% impurity threshold is
inappropriate because the limit is intended for qualifying impurities that
are associated with the manufacturing process of related compound and
not extraneous impurities caused by cross contamination. ...“ ) may be
used depending on the stage of the process.
It is also necessary to evaluate the ability of the cleaning procedure to
remove any cleaning agents introduced. The acceptance criteria for the
residual-cleaning agents should reflect the absence of these materials,
within the range of the capabilities of the assay and sampling methods.
The individual company must decide on the Acceptance Criteria which
are justifiable for their particular situation.
8.1.2 Physical determination
There should be provision during routine cleaning for a visual examination of
the equipment, verifying that it is free of visible residues. The validation
protocol should include this requirement as an acceptance criteria. During
validation, special attention should be given to areas that are ‘hard to clean’
(e.g. agitator shafts, thermowells, discharge valves etc.) and areas that would
be difficult to verify on a routine basis.
8.1.3 Microbiological determination
Appropriate studies should be performed (e.g. swabs and/or rinse sampling)
where the possibility of microbial contamination of subsequent product is
deemed possible and presents a product quality risk.
8.2 Cleaning procedures
Written cleaning procedures for each piece of equipment and process1 must be
prepared. It is vital that the equipment design is evaluated in detail in
conjunction with the product residues to be removed, the available cleaning
agents and cleaning techniques when determining the optimum cleaning
procedure for the equipment.
1 If one cleaning procedure has been shown to be adequate for a number of products, then it is only
necessary to have one cleaning SOP for those products for each piece of equipment.
Guide to Cleaning Validation in API plants
14
Cleaning procedures should be sufficiently detailed to remove the possibility of
any inconsistencies during the cleaning process.
A. Equipment parameters to be evaluated
· Identification of the equipment to be cleaned
· Difficult to clean areas
· Property of materials
· Ease of disassembly
· Fixed or not
· Etc.
B. Residues to be cleaned
· Cleaning limits
· Solubility's of the residues
· Length of campaigns
· Etc.
C. Cleaning agent parameters to be evaluated
· Preferably materials that are normally used in the process
· Detergents available (as a general guide, minimize use of detergents unless
absolutely required)
· Solubility properties
· Environmental considerations.
· Health and safety considerations
· Etc.
D. Cleaning techniques to be evaluated
· Manual cleaning
· CIP (Clean-in place)
· COP (clean-out-of-place)
· Semi automatic
· Automatic
· Time considerations
· Number of cleaning cycles
· Etc.
E. Other requirements
Guide to Cleaning Validation in API plants
15
Procedures must be determined to be operator independent i.e. rugged and
reproducible, during the validation studies.
The Cleaning documentation should include the following items in order to ensure that
it can be followed reproducibly and maintained subsequent to Validation.
· Detailed definition of levels of cleaning to be performed.
· Detailed description of cleaning methods.
· The necessity to inspect and verify equipment cleanliness prior to manufacture of
next batch should be stated in the SOP and recorded on the batch record.
· The SOP should detail where verification of cycle parameters (if automated) and
checklists (for complex manual procedures) is necessary.
· Where microbial contamination may be an issue, consideration should be given to
the integrity of the vessel prior to manufacture.
Written cleaning procedures may also include additional items not specified above,
these would include, as an example, the steps needed to protect the equipment from
contamination after cleaning.
8.3 Sampling
In developing the sampling plan for a validation study, it makes scientific sense
to
incorporate an understanding of the acceptance criteria and the limitations of the
sampling method relative to the surface to be sampled.
The two methods of sampling generally employed are swab and / or rinse
sampling. (If neither or these methods is shown be a scientifically sound method
for testing in a specific instance then an alternative is to consider testing the next
product.)
The selection of either of these techniques must be consistent with sound
scientific judgment and must support the objective of the study, which is to
demonstrate that the amount of residual material in the equipment has been
reduced to acceptable levels.
Each method is described in brief below.
1. SWAB:
· Swab sampling does not cover the entire equipment surface area therefore
sites must be chosen with care. It is important that, as a minimum, the
Guide to Cleaning Validation in API plants
16
swab sites represent worst case locations on the equipment and that the
result is then extrapolated to account for the total product contact surface
area. This calculation makes it possible to make a worst case determination
of potential carryover into subsequent product.
· Due to the nature of this method which employs physical forces as well as
chemical forces it may be necessary to perform sampling technique
evaluation.
· Swabbing efficiency (% recovery) for the swabbing method must be
determined.
· It is necessary to ensure that extractables of the swab do not interfere with
the sampling method.
· Using this technique it is possible to sample insoluble residues due to the
physical action associated it.
2. RINSE:
· The solvent rinse occurs after cleaning has been completed
· This method is not as direct as swabbing but will cover the entire surface
area (and parts inaccessible to swabs)
· It is important to ensure chosen solvent has appropriate recovery for
residues being quantified
· This method allows much greater ease of sampling than swabbing
· A reduced no of samples are required to generate a carryover figure.
(Other sampling methods which may be employed in addition to swab / rinse
sampling during a validation may include: placebo sampling, testing subsequent
batches for residues, use of coupons (test pieces), etc. )
8.4 Analytical methods
In order for the analytical testing of the cleaning validation samples (swabs or
rinses) to yield meaningful results, the analytical methods used should be
validated. This should be documented.
The basic requirements are:
· The ability to detect the target substance(s) at levels consistent with the
acceptance criteria
· The ability to detect the target substance(s) in the presence of other
materials that may also be present in the sample (selectivity)
(Companies might want to consider the following:
Guide to Cleaning Validation in API plants
17
Where more than one impurity is suspected (which is probably the normal
case in API manufacturing) a method could be proposed that is not
necessarily specific for each of the impurities but detects them all
together. Then additionally the assumption must be made, that the worst
case (e.g. most active) impurity represents the whole residue. This is
secure approach for the patients and could be accepted by the authorities.
It is also an practicable approach for the industry because such methods
are for example dry residue determination for non volatile impurities or
TOC determination for water rinses, which are very simple methods. )
· The analytical method should include a calculation to convert the amount
of residue detected in the sample to 100% if the recovery data generated
indicates a recovery outside of an allowed range.
· Stability of samples over time if the time interval between removal and
testing of samples potentially effects sample integrity.
8.5 Validation protocols
A Validation Protocol is necessary to define the specific items and activities
that will constitute a cleaning validation study. It is advisable for companies to
have drawn up a Master Validation plan indicating the overall Cleaning
Validation strategy for either the product range / equipment type / entire site.
The protocol must be prepared prior to the initiation of the study and must
either include or reference the documentation required to provide the following
information:
· The objective of the study:
What cleaning process is to be validated (indicating the product to be
removed and the equipment from which it is to be removed)?
If this study is to be employed to demonstrate the acceptability of the
cleaning procedure for a group of products the rational for doing so
should also be detailed here.
The cleaning procedure(s) to be validated should be identified i.e.
cleaning agents, soakage times, equipment parameters, number of
cleaning cycles etc.
Guide to Cleaning Validation in API plants
18
· Scope of the study:
The company must evaluate the process and determine which residues are
to be tested for and which are not to be based on sound scientific
rational.
What residues (including cleaning agents) are to be tested for, why those
residues (if more residues may be present than are being tested for all
residues should be under control see comments at 8.4). How many times
should the study be run before a report is compiled and recommendations
made.
· Listing of the process parameters to be verified
This is particularly necessary when automated or semi-automated
cleaning techniques are to be employed.
· Sampling and inspection procedure to be used.
The types of sampling methods to be used, where the samples are to be
removed from and how many samples are to be taken. Any particular
requirements should also be stated i.e. for sterile sampling / sampling
light sensitive products.
An equipment sampling diagram should be referenced.
· Personnel responsibilities during the study
· Test methods to be used (should be referenced): See Section 8.4.
· Acceptance criteria
Physical: see section 8.1.2
Chemical: see section 8.1.1
(The rational for this criterion should be given along with a calculation
step.)
· Change control: See section 10.
· Approval of protocol before the study.
Guide to Cleaning Validation in API plants
19
8.6 Validation reports
A validation report is necessary to present the results and conclusions and
secure approval of the study. The report should include the following:
· Summary of or reference to the procedures used to clean, sample and test
· Physical and analytical test results or references for same, as well as any
pertinent observations
· Conclusions regarding the acceptability of the results, and the status of
the procedure(s) being validated
· Any recommendations based on the results or relevant information
obtained during the study including revalidation practices if applicable.
· Approval of conclusions
· Review any deviations for the protocol that occurred.
· In cases where it is unlikely that further batches of the product will be
manufactured for a period of time it is advisable to generate interim
reports on a batch by batch basis until such time as the cleaning validation
study has been completed. (Typically, in Active Pharmaceutical
Ingredient Pharmaceutical manufacture, verification is deemed
appropriate during development of the cleaning methods. Where
products are manufactured infrequently, verification may be applied over
a period of time until all measuring data has been collected for the
Validation Report.)
· The report should conclude an appropriate level of verification
subsequent to validation.
Guide to Cleaning Validation in API plants
20
9. Minimum requirements
If company policy is not to validate all equipment cleaning procedures for all products
then as a minimum requirement the validation policy should encompass conditions
which represent the most appropriate challenges (worst case) to the procedure.
These would include, as an example, such things as:
· Removal of products which contain the products with the greatest biological
activity.
· Removal of products containing the products/intermediates/byproducts with the
least solubility.
These represent studies that are minimally required as part of a validation, the results
from which could be used to support lesser challenges to the procedure. It is often
termed product grouping.
· The maximum idle time before cleaning.
A validation program generally encompasses three consecutive successful replicates to
establish that the procedure is reproducibly effective although companies should
evaluate each situation individually.
Where equipment of similar size, design and construction is cleaned by the same
procedure, studies need not be conducted on each unit, as long as a total of three
successful replicates are done on similar pieces of equipment (equipment grouping).
Concurrent Validation may be appropriate when product is manufactured infrequently.
Guide to Cleaning Validation in API plants
21
10. Change control
Validated cleaning procedures should be included in the change control program. This
will ensure that any proposed changes are evaluated fully for their impact on the
validated state of the procedure. Where deemed necessary, the proposed revised
procedure may need to be validated prior to routine implementation.
Cf. Change control chapter in the CEFIC / EFPIA Guide entitled ‘Good
Manufacturing Practices for Active Ingredient Manufacturers’
In the absence of an intentional change to a procedure, it is reasonable to assume that
properly trained operators or a properly qualified automated system will be able to
execute the procedure reproducibly and obtain the desired outcome - reduction of
residue to acceptable levels. There may exist special circumstances that would suggest
that this assumption be verified via testing. This may be addressed by periodic reviews
or re-evaluations.
Guide to Cleaning Validation in API plants
22
11. Summary
A validation policy should be written for a plant including cleaning validation.
An cleaning validation program should contain the following elements:
1. Assess equipment and products (previous, following)
2. Assess impact of this process on routine processes. If covered under bracketing
then no further validation is required.
3. Determine an appropriate cleaning agent and method
4. Determine acceptance criteria for the residue(s) (including cleaning agents).
5. Determine degree of evaluation required to validate the procedure.
6. Decide what residue(s) (including cleaning agents), are to be tested for based on
solubilities, toxicities etc. and document rational behind decision.
7. Develop sampling and analytical methods for recovery and detection of residues
(swab/rinse, HPLC/dry residue etc.)
8. Acceptance Criteria for the Validation
9. Compile and approve Validation protocol
10. Perform Validation Studies in accordance with protocol
11. Compile and approve a Validation report documenting studies, conclusions and
recommendations.
12. Revalidation policy
Guide to Cleaning Validation in API plants
23
12. References
ICH:
· ICH Good Manufacturing Practice Guideline for Active Pharmaceutical
Ingredients. (July 23 1999)
PIC :
· Principles of Qualification and Validation in Pharmaceutical Manufacture -
Recommendations on Cleaning Validation. (ref. Document PR 1/ 99 March
1999)
FDA:
· Guide to inspections of validation of cleaning processes (July 1993)
· Biotechnology inspection guide (1991)
· Foreign inspection guide (1992)
· Guide to inspection of bulk pharmaceutical chemicals
· Guide to inspections of topical drug products
· Manufacture, processing or holding of active pharmaceutical ingredients, draft
document, FDA, March 1998.
CEFIC / EFPIA:
· Good Manufacturing Practices for Active Ingredient Manufacturers - August
1996.
PHRMA :
· Draft PhRMA BPC Cleaning Validation Guideline. (November 1996 Edition)
OTHER PUBLICATIONS
· S.W. Harder, ‘The validation of cleaning processes’, pharmaceutical technology.
(1984)
· James Agalloco, ‘Points to consider in the validation of equipment cleaning
procedures’, Journal of parenteral science and technology. (October 1992)
Guide to Cleaning Validation in API plants
24
· Fourman Mullen, ‘Determining cleaning validation acceptance limits for
pharmaceutical manufacturing operations’, pharmaceutical technology. (April
1993)
· Mc Cormick, Cullen, ‘Cleaning validation’, pharmaceutical process validation,
second edition. (1992)
· Mc Arthur, Vasilevsky, ‘Cleaning validation for biological products: case study’,
pharmaceutical engineering. (November / December 1995)
· Zeller, ’Cleaning Validation and residue limits: a contribution to current
discussions’, pharmaceutical technology Europe. (November 1993)

Cleaning validation, stability SOP trip up API manufacturer

Industriale Chimica, Sacronno, Italy, received a two-item 483 for incomplete cleaning validation in the manufacture of active pharmaceutical ingredients (APIs).
The EIR stated that investigator Vlada Matusovsky from the Center for Drugs conducted the inspection, which covered quality, laboratory control, production, facilities and equipment systems.
Cleaning validation performed in 2003 on the manufacturing equipment used in production of several APIs was incomplete because it did not include the evaluation of effectiveness of the manufacturing equipment cleaning procedures to remove an undisclosed residue, according to the 483.
Also, the report noted that the validation did not include the determination of the existence of an undisclosed substance used by the firm to store intermediates and APIs during processing, FDA added.
According the report, management conceded, "the effectiveness of these cleaning procedures was not established with respect to their ability to remove [undisclosed substance]."
The firm further explained that the acceptance criteria for the cleaning validation was determined based on the pharmacological activity of the most and least potent products.
Then, the products were grouped into different categories depending on their solubility in the solvent used for cleaning and one product from each category was then selected as the worst-case scenario. "These products were used to evaluate the effectiveness of the cleaning procedures," the report added.
Next, the EIR stated that the SOP for long-term stability was not followed in that samples were collected outside of the specified time frame for various stability stations. It stated that stability samples were to be analyzed during the week due or in subsequent weeks.
For example, one lot initially was tested Jan. 23, 2004, while the three-month station for normal stability testing was analyzed March 4, 2004, which was more than one month early. Also, another lot initially was tested May 17, 2004, while the three-month station for normal stability testing was analyzed Oct. 4, 2004, which was more than one month later.
Industriale Chimica, Sacronno, Italy, 10/25-28/04, Doc. 109918M, $15 plus retrieval.

Introduction to cleaning validation

Introduction to cleaning validation

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Cleaning Validation Wiper

Cleaning Validation Wiper selection to clean critical surfaces in sterile clean rooms has always presented a tradeoff between a wiper that offers efficient pickup of stubbornly adhered residues and fiber generation that results from the wiping action itself. MiraWIPE® through a unique micro fiber construction with a sealed edge perimeter bridges this gap by enabling excellent pick up and entrapment of the most stubbornly adhered residues while minimizing abrasion related fiber generation. Cleaning Validation SOP's can now be made much more robust and expensive re-cleaning incidents can be greatly reduced.
	Sealed Edge
Cleaning Validation Wiper
MiraWIPE® is constructed from a continuous filament, super soft polyester/nylon micro fiber cloth resulting in enhanced absorbency and contamination pickup versus traditional polyester wipers. Woven from an abrasion resistant fabric to minimize In-Use Particles from the face of the wiper. Sealed edge construction and proprietary washing process ensure compatibility with ISO Class 1 clean rooms.
	«« Click on Picture For Comparison Pictures
Improved Cleaning Validations With Efficient Wiping
Micro Fiber construction offers excellent cleaning efficiency allowing MiraWIPE® to remove trace disinfectant residues from Medical Device and Pharmaceutical cleanrooms. It is often said that without proper cleaning, disinfection is hard to achieve. MiraWIPE offers unsurpassed cleaning allowing effective disinfection.
MiraWIPE®'s non-symmetrical micro fiber structure allows it to scrub and remove API, microbial bio-films and sporicidal residues making it ideal for disinfection procedures.
Nylon/Polyester engineered micro denier fibers and super strong sealed edge offer robust abrasion and tear qualities to prevent fiber generation when cleaning critical surfaces in isolators and filling machines.
MiraWIPE® is a micro fiber cloth engineered to resist abrasion damage resulting in cleaner process tools.

MiraWIPE® is a micro fiber cloth which is abrasion resistant to eliminate fiber contamination			Traditional Class 10 Wipers generate abrasion related fibers as a matter of routine cleaning.

The Race Is On

By Walter Armstrong

GETTY IMAGES / PHOTOLINK

"I really, really, really want to die and have had enough of being so sick and in so much pain every second of every day and, basically, one serious health crisis after another," wrote Lynn Gilderdale in a 2006 Web post during one of many discussions the 31-year-old British woman had with parents and friends on whether to hasten her own death. In July 2009, Gilderdale decided to act, injecting herself with what she believed to be a lethal quantity of morphine. An hour later, she was unconscious but still alive, so her mother, Kay, took over the duty of assisting her daughter's suicide. She crushed antidepressants and sedatives and inserted the powder into her daughter's nasogastric tube. When that remedy failed, Kay gave Lynn several more injections of morphine, and later, increasingly desperate, several injections of air. Finally, toward dawn, Lynn's spirit made good her longed-for escape from a body ravaged for 17 years by severe chronic fatigue syndrome (CFS).

Judith Mikovits, Whittemore Peterson Institute

The Gilderdales' personal tragedy became a public story following Kay Gilderdale's arrest for attempted murder. With the British government inching toward legalizing assisted suicide, Lynn's CFS-related loss of almost every physical function, coupled with her mother's steadfast devotion, rendered the Gilderdales the most sympathetic in a series of highly publicized right-to-die cases. In January, a British jury unanimously found Kay Gilderdale not guilty of attempted murder. Her exoneration marked a triumph for advocates of the legalization of assisted suicide. But lost in that debate was what patients with CFS view as a more urgent story: The disease that took Lynn Gilderdale's life remains as untreatable in 2010 as it was when the first known outbreak occurred in Lake Tahoe in 1984.

Cort Johnson, CFS Patient, Activist, and Blogger

"CFS simply gets no respect. It has been underfunded, understudied, underdiagnosed, and the healthcare system would like nothing better than to sweep it under the rug," says Donnica Moore, a women's health expert and CFS advocate. "But we're not going to allow that." Medicine's "Problem Child"
From almost any angle, CFS presents a vexing picture. No cause—not even a single biomarker—has been identified. Symptoms are as diverse as they are unpredictable, including debilitating fatigue, post-exertion malaise, and an enduring flu-like state ranging from aches and pains to severe headaches, cognitive disturbances, paralysis, and myriad complications. "CFS defies the established structure of medical disease," says Kimberly McCleary, who has headed the CFIDS Association of America for 20 years. "Many doctors still don't 'believe' in it. They treat a single symptom without seeing the whole. Or, worse, they dismiss it as a psychological problem." In turn, a fierce mistrust of not only the medical profession but the federal research establishment is endemic in the CFS community. Conspiracy theories abound.

Suzanne Vernon, CFIDS Association of America

Some 200,000 Americans have been diagnosed with CFS, while anywhere from 1 million to 4 million may suffer from it, according to the CDC. Average life expectancy is about 55, with suicide the third most frequent cause of death. Depression is rampant. "CFS is not a death sentence—it's a life sentence," is a CFS community truism. Meanwhile, skeptics persist in dismissing it as "yuppie flu" and "shirker syndrome." Yet recent studies show that most CFS patients did not experience clinical depression prior to getting sick. And increasing diagnoses of pediatric and adolescent cases reveal that kids who fall victim to the disease include many high achievers, whose parents can trace the onset of the illness to a routine infection of unusual severity or duration. Still, the CDC's sole treatment recommendation is cognitive-behavioral therapy. The agency's longtime CFS program head was finally axed in February, following years of public criticism by doctors for favoring a research focus on early sexual abuse rather than the search for pathogens.
The tenacity of its "disputed diagnosis" status has earned CFS the dubious distinction as the only orphan disease with literally millions of "silent sufferers." Pharma's longstanding disinterest in CFS is predictable, given the disease's unforgiving uncertainties. "I don't blame the drug industry—CFS is medicine's 'problem child,'" says virologist Suzanne Vernon, the CFIDS Association's scientific director. "If so many doctors do not recognize CFS, how can a drugmaker sell a treatment?"
CFS presents a kind of Gordian Knot to any pharma wishing to brave clinical trials: the lack of a biomarker confounds diagnosis; the lack of quantitative measurements of fatigue—the telltale symptom—confounds evaluation of a drug's efficacy; the presence of such diverse symptoms confounds validation of data.
"The drug industry works best on a 'bug and drug' model, and CFS has been slow to deliver a target," says McCleary. Early on, hopes were high that basic science would uncover a single virus behind CFS's devastating immune-system collapse—as took place in HIV. Academic research into the human retrovirus HTLV-II yielded especially promising preliminary results in 1991, raising patients' hopes, but replication studies foundered and funding was cut.
Until now, pharma's contribution to CFS treatment has been largely limited to the off-label use of a panoply of drugs, such as stimulants, sedatives, antidepressants, and anti-migraine medications to treat symptoms. However, with the success of Lyrica and Cymbalta for fibromyalgia (another "disputed diagnosis") drugmakers may find themselves inching into the CFS market.
Pharma may in fact stand to gain considerably by investing in CFS R&D. Expert consensus is that CFS is actually a suite of diseases, with some overlapping symptoms but many differences—and multiple causes. Advanced research is identifying biological trends, including chronic low-grade immune activation, latent activation of infections, and specific abnormalities in cognition, metabolism, and blood pressure. Deeper forays into CFS pathogenesis could yield finds that apply to many other conditions.
"CFS is a huge opportunity for pharma," says Moore. "The market is big, the bar is low, and they don't need a home run. Even incremental improvements to quality of life would be fantastic."
Unfortunately, the first CFS drug to face FDA review bombed in December: Hemispherx's sloppy NDA for Ampligen, an antiviral and immune booster in experimental use since the late '80s, contained 15-year-old data that "did not provide credible evidence of efficacy." The drug, which requires twice-weekly IVs and costs thousands of dollars a month, appears to work well in about 15 percent of patients. "This is the right drug in the wrong hands," says McCleary. "They cut too many corners."
In XAND Land
Into this bleak landscape last October blazed an unpredictable claim by an obscure researcher from a little-known institute that the cause of CFS may have been discovered: a human retrovirus called xenotropic murine leukemia virus–related virus (XMRV). Biochemist Judith Mikovits at the Whittemore Peterson Institute (WPI) in Reno, NV, along with colleagues at the National Cancer Institute and the Cleveland Clinic, reported in the journal Science that DNA from the mouse-derived retrovirus were found in 67 out of 101 blood samples of CFS patients. Testing of 300 additional samples was said to hit 98 percent. What's more, 3.7 percent of the 218 control samples also contained XMRV.
The media predictably amplified the remarkable, if preliminary, findings into a "cause-of-CFS" story, and WPI was only too happy to oblige. "This is the breakthrough that we have been hoping for. Now we have scientific proof that this infectious agent is a significant factor in CFS," Annette Whittemore, WPI founder and president, proclaimed in the initial press release, which also announced that WPI had renamed CFS as XMRV-associated neuro-immune disorder (XAND).
WPI did not discover the XMRV virus, however. That distinction goes to scientists at the Cleveland Clinic and the University of California San Francisco, who in 2005 detected this fourth human retrovirus in the cancerous prostate tissue of 40 percent of men with a particular defective gene. WPI's Mikovits made the opportune leap from prostate cancer to CFS when she learned of the high incidence of lymphoma among the original Lake Tahoe cohort. XMRV seemed a possible culprit because it decimates natural killer blood cells, the immune defense against cells infected by HTLV-I. In addition, some CFS patients carry the same genetic mutation as men with prostate cancer who tested positive for XMRV. The working hypothesis at WPI is that XMRV indirectly causes CFS by inflicting so potent an assault on the immune system that it reactivates other viral infections and a chronic inflammatory response. "XMRV is the sort of agent that could create that effect on the immune system," Daniel Peterson, WPI's medical director and the co-discoverer of the original Lake Tahoe outbreak, told The New York Times in a piece headlined "A Big Splash by an Upstart Medical Center."
WPI was founded in 2006 by Whittemore and her husband, Harvey, a prominent Nevada couple whose daughter, Andrea, 31, has lived with a severe case of CFS for 20 years. Frustrated by Andrea's marginalization by doctors and by the lack of leadership, funding, and research at CDC, Annette Whittemore invested $5 million to launch her own research institute at the University of Nevada Medical School in Reno.
A flurry of activity followed on the heels of the discovery. Other researchers raced to confirm the WPI study. Patients flocked to the Internet for more information: Was XMRV fatal? How was it transmitted? Could they get tested for it? The answer to the last question was yes. A diagnostic test for the virus was already being marketed at $650 a shot by VIP Dx, which just happens to be owned by Annette and Harvey Whittemore. "Leaving aside the issue of who's right and who's wrong, the original paper did not establish the virus [causes CFS] and didn't establish it as a viable marker," Tufts University retrovirologist John Coffin, who wrote the editorial accompanying the original Science study, told the journal. Nevertheless, VIP Dx reported a six-to-eight-week backlog for results.
In general, patients' emotions bordered on the euphoric. Cort Johnson, whose Phoenix Rising Web site is one of the most trusted sources of information in the CFS community, says, "Patients are starved for good news. A discovery like this excites researchers, brings in funding, and gives patients hope—something they haven't had for many years." Meanwhile, the nation's handful of CFS specialists tried to temper patients' expectations with YouTube educational lectures on XMRV and its potential treatment implications.
For public health officials, the most alarming data point was XMRV's 3.7 percent prevalence rate in the control group. Extrapolating a worst-case scenario led to the prospect that as many as 10 million Americans could be carrying an infectious retrovirus already linked to two serious diseases. In January, a federal task force was convened to safeguard the nation's blood supply, an operation that could take a year or more, according to member Suzanne Vernon. Then again, a little public panic has its upside. "As we saw in the early years of HIV, fear among the general population at least gets the money flowing," says Moore.
A Pharma Screening
XMRV is exactly the kind of bug that hooks Big Pharma. "Two of the world's biggest drug companies contacted us the day our Science paper appeared," says Judith Mikovits. "By showing that XMRV is an infectious agent, we think we've convinced them to become interested in this target." Although Mikovits refused to disclose the identity of the two companies—"for fear that patients might seek out the treatments before studies"—she said that both were already screening HIV antiretroviral compounds in WPI cell lines for a hit.
Given the similarities among human retroviruses, an HIV drugmaker may already possess an effective anti-XMRV agent—if not a drug already on the market, then one of the thousands of marginally variant molecules made in the painstaking process of discovery—and currently gathering dust. Two classes of HIV drugs are in the running.
Both HIV and XMRV replicate by virtue of reverse transcriptase, the enzyme that links their viral RNA to the host cell's DNA. Reverse-transcriptase blockers were the first victory Big Pharma scored against HIV. Ironically, in the February Virology, Mayo Clinic researchers reported that after testing 10 HIV drugs against XMRV in vitro, the virus was susceptible only to AZT, a nucleoside reverse-transcriptase inhibitor (NRTI) notorious for its toxicity. "No CFS patient wants to go near AZT," says Mikovits.
Other RTs (or experimental versions) that may show promise include Bristol-Myers Squibb ddI and d4T, GlaxoSmithKline's Ziagen, and Gilead's Emtriva and Viread. Merck's first-in-class integrase inhibitor, Isentress, may work "because of its broad-spectrum activity," according to Coffin. In the best case, an already-approved antiretroviral will reveal XMRV-busting prowess, allowing the drugmaker to bypass safety and other early tests and advance straight into humans. "If one of the drugmakers currently screening candidates gets lucky, we could start a clinical trial in a month," says Mikovits.
Veteran advocates like Kimberly McCleary do a double-take at the news that two global pharmas are on the trail of CFS. "Now what we need is a race between them to see which can be first to market," she says.
WPI and Full Disclosure
When XMRV was first discovered in 2005, pharma held back because it was reported that the virus appeared to be inactive in prostate cancer cells. But Abbott Diagnostics jumped at the challenge of developing assays to detect XMRV. Last month, Abbott HIV Global Surveillance Program's John Hackett reported early progress on several fronts. But the main takeaway was that detecting XMRV in human blood samples is proving far more difficult than the WPI study had led anyone to expect. Using their new assay that can detect three different antibody proteins, the Abbott team found XMRV in only three of 2,851 random human samples. That's good news for the general population—a .01 percent extrapolated prevalence rate—but bad news for CFS patients.
Nor is Abbott alone in judging XMRV hard to find. Since January, three confirmation studies—two British, one Dutch—have reported results, and none found the retrovirus in either their CFS blood samples or their controls. As doubt is increasingly cast on WPI's theory that XMRV causes CFS, arguments have raged across the Atlantic. Accusations of sloppiness, bias, and even fraud have been hurled, mostly by Judith Mikovits and WPI's defenders. Old suspicions of patients have reappeared.
When asked for a more considered opinion, others choose their words carefully. "Validation and confirmation are not coming as fast as one might like, that's for sure," says John Coffin. "If you can't establish a disease association, then there is less interest in developing a drug, obviously." Coffin also notes that uncertainty remains about whether or not the virus is replicating. "If it does so, like HIV, then an antiretroviral would be very effective. But if not, as it appears in prostate cancer, a drug would not make any difference."
Writing on the CFIDS Association of America's Web site, Suzanne Vernon made a valiant effort to keep hope in the causal hypothesis flickering by emphasizing that none of the three studies is a "proper and robust replication study." And she concluded by throwing down the gauntlet: "Until methods are standardized and the scientific community is provided information about the specific characteristics of the CFS subjects who tested positive in the Science paper, be prepared to read more negative studies. Hopefully the Science investigators will make this information available before interest in XMRV being associated with CFS fades."
Given the great diversity in CFS symptoms, disclosure of the medical histories and clinical conditions of the high number of WPI's XMRV-infected CFS patients is critical. "Of course, this would generate more questions, but a cleaner association is needed," Vernon says. "I don't know why WPI won't provide this."
So far, Mikovits has refused to budge. "No additional medical histories or anything about the patient population would shed any light on XMRV," she says.
Sleuthing on her own, Vernon was able to uncover some suggestive information about the 32 CFS patient samples about which WPI originally reported assay results. Only 12 tested positive on more than one assay (WPI ran four assays); of those 12, four had been diagnosed with cancer. Another 13 of the total 67 XMRV-positive CFS samples also had cancer.
Whether XMRV is a cause or a passenger or merely a geographical coincidence of a particular CFS outbreak remains to be learned. But one thing is clear: With its big discovery, the upstart medical center has made more than a big splash. WPI has placed CFS—and itself—at the center of the perfect storm. "I knew how serious a retrovirus is," Annette Whittemore told the Times. "I was very concerned, knowing the implications. My second thought was, 'Of course, it was going to be something serious like that. Look at my daughter and how ill she is.'"