Cleaning Validation Volume III

Institute of Validation Technology4

E D I T O R I A L A D V I S O R Y B O A R D

J O U R N A L M I S S I O NThe Journal of Validation Technology is a peer-reviewed publication that provides an objective forum for the dis-semination of information to professionals in FDA-regulated in dustries. The Journal’s Editorial Advisory Board reviews all submissions to ensure that they have been researched thoroughly, reflect current industry standards, and are not promotional in nature. The Journal will not publish articles which have not been approved by the Board.

Gamal amer, Ph.D.Validation and Process

Associates, Inc.

louis a. anGelucci, iii Foster Wheeler Corporation

GeorGe n. Brower Analex Corporation

Kenneth G. chaPman Drumbeat Dimensions, Inc.

Dennis christensen Consultant

roBert c. colemanUS Food & Drug Administration

shahiD Dara Independent Consultant

DaviD r. Dills Medtronic Xomed

michael Ferrante Catalytica Pharmaceuticals

Patricia stewart Flaherty

Bayer Corporation

roBerta D. GooDe Consultant

cynthia Green Northwest Regulatory Support

Daniel harPaz, Ph.D.

PCI, Pharmachem International

william e. hall, Ph.D.Hall & Associates

elDon henson Boehringer Ingelheim

Animal Health

Jay h. KinG LifeScan, a Johnson & Johnson Company

John G. lanese, Ph.D. The Lanese Group, Inc.

BarBara mullenDore AstraZeneca

roBert a. nash, Ph.D. St. John’s University

charlie neal, Jr.BE&K

toD e. ransDell Bio-Rad Laboratories

melvin r. smith Independent Consultant

roBert w. stotz, Ph.D. Validation Technologies, Corporation

eric D. veit Johnson & Johnson

DaviD w. vincentValidation Technologies, Inc.

sPecial eDition n cleaninG valiDation iii

Editor and Publisher Glenn Melvin

Vice President Terri Kulesa

Production Director Edward Eick

Associate PublisherBrandon Melvin

Disclaimer:

Any reproduction of the contents of this publication in whole or part is strictly prohibited without permission. Views and conclusions expressed in articles herein are those of the authors. The publisher accepts no responsibility for the accu-racy of information supplied herein or for any opinion expressed. No liability can be accepted in anyway. The informa-tion provided does not constitute legal advice.

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PO B ox 6004Duluth, MN 55806Telephone: 218-723-4977 U.S. only: 888-524-9922 Fax: 218-723-9308 or E-Mail: [email protected] site: www.ivthome.comISSN 1079-6630

equiPment cleaninG valiDation: microBial control issues . . . . . . . . . . . . . . . . . . . . . . 6 by Destin A. LeBlanc, M.A.

cleaninG valiDation: maximum allowaBle resiDue: question anD answer. . . . . . . 13 by William E. Hall, Ph.D.

DeveloPment oF total orGanic carBon (toc) analysis For DeterGent resiDue veriFication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 by James G. Jin and Cheryl Woodward

total orGanic carBon analysis For cleaninG valiDation in Pharmaceutical manuFacturinG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 by Karen A. Clark

DeterGent selection – a First critical steP in DeveloPinG a valiDateD cleaninG ProGram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 by Mark Altier

analysis cleaninG valiDation samPles: what methoD? . . . . . . . . . . . . . . . . . . . . . . . . . 35 by Herbert J. Kaiser, Ph.D., Maria Minowitz, M.L.S.

control anD monitorinG oF BioBurDen in Biotech/Pharmaceutical cleanrooms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 by Raj Jaisinghani, Greg Smith and Gerald Macedo

a cleaninG valiDation ProGram For the eliFa system. . . . . . . . . . . . . . . . . . . . . . . . . . . 56 by LeeAnne Macaulay, Jeff Morier, Patti Hosler and Danuta Kierek-Jaszczuk, Ph.D.

a cleaninG valiDation master Plan For oral soliD Dose Pharmaceutical manuFacturinG equiPment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 by Julie A. Thomas

ProPoseD valiDation stanDarD — vs-3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

BONUS

BONUS

Special Edition: Cleaning Validation III 5

C O N T E N T ST A B L E O F

sPecial eDition n cleaninG valiDation iii

The PDA spring conference was held in Las Vegas, Nevada in March 20, 2001.

The conference showcased cleaning validation, residue limits, bioburden, micro bial limits, and sanitization. This paper is based on a pre sentation at that conference.

The initial focus of regulatory documents relating to cleaning validation for process equipment in pharmaceutical manufacturing in volved measuring residues of the drug active and the cleaning agent. For example, the introduction to the Food and Drug Ad mini stra tion (FDA) guidance document on clean ing validation1 states: “This guide is intended to cover equipment cleaning for chemical residues only.” While admitting that microbial re si dues are beyond the scope of the guideline, that guidance document further states, “microbiological aspects of equipment cleaning should be considered,” particularly with reference to preventive measures so that microbial proliferation does not occur during storage. The European PIC/S document,2 that was issued several years later, does explicitly mention microbial re sidues. In Section 6.2.1, contaminants to be re moved in clude “the previous products, residues of cleaning agents as well as the control of potential microbial con

taminants.” How ever, Section 6.7 of this document that covers “Micro biological As pects” focuses exclusively on the same issue discussed in the FDA guidance document, namely the issue of preventing microbial proliferation during storage.

As a practical matter, microbial residues on equipment surfaces are part of the contaminants that should be reduced to an acceptable level; that acceptable level being what is safe for the manufacture of the subsequently manufactured pro duct. Unfortunately, very little has been written on what is a safe level for microorganisms following cleaning and/or sanitation.3,4 Part of the reason for this is that microbial resi dues are significantly different from chemical re sidues. Chemical resi dues are “in ert” in the sense that it is easy to cal culate (especially using scenarios

of uniform contamination in the subsequently manufactured product) the potential levels and effects of those chemical residues in the subsequently manufactured pro duct should they be transferred to that subsequently manufactured pro duct. With microbial residues left after the cleaning process, the situation is somewhat different. Because microorganisms are living organisms, those left as residues on equipment may change in number after the cleaning process, but


Equipment Cleaning Validation:

Microbial Control Issues By Destin A. LeBlanc, M.A.

Cleaning Validation Technologies

v

}…it is becoming more

common for regulatory authorities

to cite manufacturers for deficiencies

related to microbial control in cleaning

validation programs.~

Special Edition: Cleaning Validation III

Destin A. LeBlanc, M.A.

before the manufacture of the subsequently manufactured pro duct. Those microbes transferred to the subsequently manufactured product may also change in number after they are incorporated into the subsequently manufactured product in the manufacturing step. This change may be a significant reduction in bioburden, either due to drying of the equipment or due to a preservative in the finished drug product, for example. This change may also involve rapid proliferation, either due to suitable growth conditions in wet equipment during storage, or due to suitable growth conditions in the finished drug product. Or, they may result in no significant change in microbial level, because the bioburden was due to bacterial spores (that will survive readily in dried equipment), or because the subsequently manufactured product was a dry product (with low water activity). There fore, knowing the levels of microorganisms left on the equipment following cleaning does not necessarily give one the full story of the po ten tial hazards of those microbial residues. Addi tional in formation is required to assess those potential hazards.

Why has microbial evaluation during cleaning of process equipment been a little discussed topic? Part of the reason is that it is not a significant problem in process manufacturing. Yes, it could conceivably be a problem if cleaning and storage were inadequate. How ever, for the most part, cleaning and storage of pro cess equipment, in so far as it applies to microbial residues, probably is done relatively well in most pharmaceutical manufacturing facilities. On the other hand, it is becoming more common for regulatory authorities to cite manufacturers for deficiencies related to microbial control in cleaning validation programs. One reason for this seeming anom aly is that while firms are adequately controlling microbial contamination of process equipment, there may be little documentation to support this. This lack of documentation includes any measurement of microbial residues during the cleaning validation and/or during routine monitoring. Some companies will measure the change in microbial levels on equipment surfaces during storage of

the cleaned equipment. However, many times this does not include any assessment as to the effect of that unchanged bioburden level on the subsequently manufactured product.

This paper will address issues covering ap proaches to control of microorganisms in process equipment, setting of acceptance limits, sampling techniques, and approaches to providing acceptable documentation.

Microbial Control Measures

Control measures to reduce the bioburden on cleaned process equipment include control of bioburden of raw materials, the cleaning process itself,

a separate sanitizing step, and drying of the equipment following cleaning. Bioburden of raw materials in cludes the active, excipients, water, and any processing aids. In many cases, the manufacturer may have little control over the bioburden of raw materials other than to accept a specification by the raw material supplier. The most critical raw materials probably will be natural products, in which there may be considerable variation in the levels and types of microorganisms. A solid monitoring program to control in coming bioburden of raw material is necessary. If there could be significant variation in bioburden, then that should be addressed in the cleaning validation Performance Qualification (PQ) trials. At least one PQ trial should utilize the worstcase incoming bioburden of raw materials to demonstrate adequate cleaning and microbial control under those conditions.

7

}Some companies will measure the change in microbial levels on

equipment surfaces during storage of the cleaned equipment. However,

many times this does not include any assessment as to the effect

of that unchanged bioburden level on the subsequently manufactured product.~

Institute of Validation Technology


A second means of microbial control is the cleaning process itself. The conditions of aqueous cleaning are often hostile to microbial survival. These con ditions include high temperature (commonly 6080ºC), pH extremes (>11 and <4), and the presence of oxidizers (such as sodium hypochlorite in biotechnology manufacture). In addition, the presence of surfactants in the cleaning solution can assist in providing good physical removal of microbes (without necessarily killing them). Good cleaning is also beneficial to microbial control in that chemical residues left behind can provide a physical “microbial trap” to allow microorganisms to survive even in the presence of chemical sanitizers. Those chemical residues left behind might also serve as a nutrient source that allows microbes to proliferate during improper storage. Based on the author’s experience, in most cases, effective control of microorganisms in pharmaceutical process equipment can be achieved with the use of an effective cleaning process, without the need for a separate chemical sanitizing step.

In some cases, a separate sanitizing step may be necessary. This may include sanitation by steam or by chemical sanitizers. Suitable chemical sanitizers for process equipment include sodium hypochlorite (chlorine bleach), quaternary ammonium compounds, alcohol (ethyl or isopropyl), hydrogen peroxide, and peracetic acid. It should be noted that, with the exception of alcohol and hydrogen peroxide, additional rinses would be necessary to remove any chemical residues of the sanitizer from the equipment. Those chemical residues may also have to be evaluated as residues to be measured in the cleaning validation protocol. For such chemical treatments, it is not an expectation that the equipment be sterile. Unless the final rinse is with sterile water, microorganisms will be reintroduced into the equipment from the use of WaterforInjection (WFI) or purified water as the final rinse.

Some companies will use an alternative to sanitizing immediately after cleaning. This usually involves sanitizing after storage and immediately before use. This may be used in situations where it is difficult to control microbial recontamination or proliferation during storage. It should be noted that control of storage conditions, if possible, is preferable. The practice of relying solely on a separate sanitizing step immediately before manufacture should be discouraged. If this is practiced, then the sanitization step should be shown to be effective in reducing bioburden under the worstcase storage

conditions (“initial” bioburden, time, temperature, and humidity). Needless to say, if the chemical sanitizing step is performed im mediately prior to manufacture of the subsequently manufactured product, then removal of the sanitizer chemical residues to an acceptable level should also be demonstrated.

A fourth consideration for control of microorganisms is drying the process equipment surfaces following the final rinse. Drying the surfaces will further reduce the levels of vegetative organisms on the surface. In addition, drying will assist in preventing microbial proliferation during storage. Drying can be achieved by heated air, heated nitrogen, or by rinsing with alcohol. In all cases, the process can be assisted by application of a vacuum (to speed the evaporation of the water or, in the case of an alcohol rinse, of the alcohol itself).

Limits for Microbes

As mentioned earlier, it is possible to reasonably predict levels of chemical residues in subsequently manufactured products based on the levels present on equipment surfaces.5,6 With microorganisms, it is possible to measure levels on equipment surfaces; however, the effect of those residues will depend on what happens to those microorganisms once they come in contact with the subsequently manufactured product. Areas that may have to be evaluated include the species (including the socalled “objectionable” organisms), type of organism (vegetative bacteria versus bacterial spore, for ex ample), the presence of preservatives in that subsequently manufactured product, the water activity of the subsequently manufactured product, as well as any subsequent sterilization process performed on that product. As a general rule, if the water activity is less than 0.6, then it can be expected that microorganisms will not proliferate (although they may continue to survive without reproducing).7 Water activity is a physicalchemical measurement that ex presses the water vapor pressure above the test sample as a fraction of the water vapor pressure of pure water at the same temperature as the test sample. For aqueous products with a neutral pH, microbial proliferation can generally be expected unless there is a preservative in the product. If there is a possibility of microbial proliferation because the product is unpreserved and neutral, then that should be addressed in setting limits.

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Three methods to set microbial limits will be ad dressed. The first (Case I) involve limits where the sub sequent product does not allow microbial proliferation and is not subject to any further sterilization process. The second (Case II) involves subsequently manufactured products that are terminally sterilized. The third (Case III) involves subsequently manufactured products that are processed aseptically.

Case I Limits If the subsequently manufactured product does not

allow microbial proliferation, then the determination of acceptable microbial limits in the cleaned equipment can be calculated using the same principles used for chemical residues with one important exception. This process involves first determining the acceptance limit in the subsequently manufactured product. This limit is typically given in Colony Forming Units (CFU) per gram of product. Once this is determined, then the limit per surface area of equipment (assuming uniform contamination) can be calculated based on the batch size of the subsequently manufactured product and the equipment surface area.

How is the limit in the subsequently manufactured product determined? For chemical residues, it is based on dosing information for actives or toxicity in for mation for cleaning agents. Such concepts cannot be directly applied to microbes. Fortunately, there are two good sources of information relating to levels of microorganisms in products. One is the manufacturer’s own Quality Control (QC) specifications for the product, that may include a limit for bioburden in the product. A second source is information given in the proposed United States Pharmacopeia (USP) <1111> relating to “Microbial Attributes of Non sterile Pharma copeial Articles.”8 Examples of those limits are given below:

Solid oral: ≤1000 CFU/gLiquid oral; ≤100 CFU/gTopicals: ≤100 CFU/g

Note: Although these limits were discussed and proposed in the Pharmacopeial Forum, these specific recommendations were not adopted officially as part of the 24th edition of the USP.

Unfortunately, this is where the one exception to the conventional treatment arises. When one looks at the bioburden in a finished drug product, the equip

ment surfaces are not the only source of bioburden. One must also consider the raw materials themselves, as well as the primary packaging, as potential sources of microorganisms. The best way to deal with this issue is to develop information on the bio burden of the raw materials and the primary packaging, and factor these into the limits calculation. For example, if one were dealing with an oral liquid, one might calculate the contribution from the raw materials (assuming the upper limit bioburden for each raw material) as a maximum of 27 CFU/g. At the same time the contribution from the primary packaging is determined to be 3 CFU/g. Therefore, the amount allowed from equipment surfaces would be 70 CFU/g (100 minus 27 minus 3). An additional safety factor should be used to account for the significant variability in microbiological enumeration. An appropriate factor may be on the order of 5. There fore, in this case, the limit (in CFU/g) that would be allowed solely due to the cleaned equipment surfaces would be 14 CFU/g (obtained by dividing 70 by 5). Higher safety factors also could be considered. These numbers are given for illustration purposes only. It should be realized that the contribution percentage allowed from cleaned equipment would vary depending on the contributions from the raw materials and the primary packaging.

Once the limit in the subsequently manufactured product allowed from the cleaned equipment surfaces is determined, the next step is to determine the limit per surface area (CFU/cm2). This is calculated exactly as it would be for chemical residues:

Limit per surface area = LSP x MBS SA

whereLSP = Limit in the subsequent product MBS = Minimum batch size SA = Product contact surface area

In the example above, if the batch size is 200 kg and the product contact surface area is 260,000 cm2, then the microbial surface limit of the cleaned equipment is:

Limit per surface area = (70 CFU/g)(200,000g) = 54 CFU/ cm2 (260,000 cm2)

9



If sampling were done with a typical contact plate of 25 cm2, this would correspond to a limit of over 1300 CFU per contact plate. Since it is reasonable to count a maximum of only 250 CFU on a typical contact plate, this would clearly be in the TNTC (too numerous to count) category. Needless to say, this will vary with the limit in the subsequently manufactured product, the portion allowed from cleaned surfaces, the safety factor used, batch size, and the shared surface area. However, under most reasonable scenarios, the calculated limit due to microorganisms on the cleaned equipment surfaces will be significantly above what should be (and can be) achieved by proper cleaning. As a general rule, a good cleaning process should produce surfaces that contain no more than 25 CFU per contact plate (<1 CFU/cm2). When failures occur, generally they will be gross failures, with counts generally above 100 CFU perplate.

Case II LimitsThis involves setting limits for cleaned equipment

when the product subsequently manufactured in that equipment is to be sterilized. In this case, the microbial limit in the subsequently manufactured product can be established based on the assumed bioburden of that product at the time of sterilization. In other words, any validated sterilization process depends on an assumed bioburden of the item being sterilized. That assumed bioburden then becomes the limit in the subsequently manufactured product. Once that limit in the subsequently manufactured product is established, then the calculations are the same as for Case I – a certain portion of that total limit is allowed from cleaned equipment surfaces, a safety factor is applied, and then the limit per surface area is calculated using the minimum subsequent product batch size and the product contact surface area. It is significant that this issue is actually addressed in the FDA’s cleaning validation guidance document; that states:

“…it is important to note that control of bioburden through adequate cleaning and storage of equipment is important to ensure that subsequent sterilization or sanitization procedures achieve the necessary assurance of sterility.”9

Case III LimitsThis third case involves setting limits on equip

ment surfaces where the subsequently manufactured product is aseptically produced. This case is slightly different from Case II in that it is the equipment itself, and not the product, which is subsequently sterilized. This case is relatively straightforward, because the microbial limits on the surfaces of cleaned equipment are established based on the assumed bioburden of the equipment surfaces for sterilization validation of that equipment. No information on batch sizes or surface areas is necessary. The assumed bioburden for the sterilization validation can be used directly for limit purposes. The only adjustment may be the incorporation of a safety factor (to accommodate normal variation in microbiological enumeration).

Measurement Techniques

Conventional tools used for microbial enumeration from surfaces can be used. These include rinse water sampling (usually with membrane filtration), swabbing (with desorption of the swab into a sterile solution and then a pour plate count), and use of a con tact plate. The choice of recovery medium and incubation conditions is usually dictated by the expected organisms. As a general rule, the initial focus is on aerobic bacteria. However, if anaerobic bac teria or molds/yeasts are suspected problems, these should be also evaluated.

One issue that does not translate directly from chemical residue measurements is the idea of determining percent recovery using the sampling method. In the measurement of chemical residues, the target residue is spiked onto a model surface and the quantitative percent recovery is determined. The amount re covered as a percent of the amount spiked is considered the sampling method percent recovery. Per cent recoveries in chemical sampling measurement are generally above 50 percent. This percent recovery is then used to convert an analyzed sample value; for example, if a chemical residue measured by a swabbing technique gives 0.6 µg of residue, then with a 50 percent recovery, this actually represents the possibility of 1.2 µg being on that surface. This concept cannot be applied directly to microbiological sampling. The reason for this is partly the inherent variability in microbiological testing. If one measured 10 CFU in one test and 5 CFU in a duplicate test (a 50 percent difference), one would be hard pressed to say that

10



those numbers are significantly different. In addition, how would one actually measure the percent recovery in a microbiological test? If a model surface is spiked with a specific number of a certain bacterium, and then that surface is allowed to dry and is sampled, just the process of drying might cause a low recovery of bacteria (due to the dying of vegetative bacteria by drying). In addition, what species of bacteria would be used for the recovery study?

It is recognized that microbiological sampling methods may understate the number of microbes on a surface (indeed the concept of a CFU, that may

contain any number of bacteria, also clouds the issue). There are two ways to view such an issue. One is to make it clear that whatever variation exists in measuring micro organisms on surfaces is probably equally an issue when one sets limits based on product limits or sterilization bioburden limits. Therefore, the variability issue becomes a “wash.” The other perspective is to ac count for such variation by choosing extremely high safety factors. In the calculation example for Case I, a factor of 5 was used as a safety factor. Even if that safety factor were increased to 10 or 20, the calculated acceptance limits would have still been ex tremely high, and still beyond what one should achieve with a welldesigned cleaning program.

Documentation Strategies

How these issues will be addressed will depend on the stage of the cleaning process development. For a new process being designed, the best strategy is to prepare a calculation of microbial limits, and then design the cleaning process to meet those acceptance criteria. Included in that evaluation should be any change in bioburden (in particular, any increase or proliferation) on storage of the equipment. The micro bial acceptance

limits should be included in the validation protocol, and measured as part of the three PQ trials. One should also include the absence of “ob jectionable” organisms as part of the acceptance criteria.

To deal with processes for which cleaning validation has already been completed, but for which no microbial evaluation has been done, there are two strategies available. The objective of each is to develop documentation that the cleaning process consistently provides equipment surfaces with acceptable bioburden. One option is to perform a cleaning validation PQ, measuring only bioburden on sur

faces for comparison to calculated acceptance limits. The other option is to initiate a routine microbiological mon itoring program as part of the monitoring of cleaning. This may involve something as simple as monitoring the bioburden in the final rinse water to demonstrate consistency. This data, combined with product QC data on bioburden, may satisfy the need for adequate docu

mentation. One should also consider one’s motivation for

wanting to obtain assur ance that the bioburden is ac ceptably low after cleaning. If the im petus for action is due to lack of data, one should resist the impulse to immediately add a sanitizer into the cleaning program. The focus should be on developing data to demonstrate the sufficiency of the current cleaning process. Adding a separate sanitizing step only complicates matters by adding additional residue concerns. If the impetus for action is due to observed high microbial counts on equipment surfaces or (more likely) in manufactured product, then it is important to determine by careful investigation whether that unacceptable contamination is due to issues with the cleaning process, with storage, or to both. In such a case, a separate sanitizing step should only be added if the data fully support it.

Conclusion

Bioburden on cleaned equipment is an important concern in the cleaning process. Fortunately, most aqueous cleaning processes, properly designed, should provide low and acceptable bioburden levels on equipment surfaces following the cleaning pro cess.

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}One issue that does not translate directly from chemical residue measurements is the idea of

determining percent recovery using the sampling method.~



Proper drying and storage should provide assurance that microbial proliferation does not occur be fore the manufacture of the subsequently manufactured product in that equipment. Any scientifically justified determination of acceptable bioburden levels, particularly for nonsterile products, is generally far higher than what should be achieved in conventional practice. This is becoming more of a regulatory and compliance issue, not because microbial contamination is a widespread pro blem, but rather because pharmaceutical manufacturers may lack appropriate documentation to support their practices. This can easily be remedied by a separate validation protocol to address microbial issues, or by routine monitoring to demonstrate consistency. o

About the AuthorDestin A. LeBlanc, M.A., is with Cleaning Validation Technologies, providing consulting in the area of pharmaceutical cleaning validation. He has 25 years experience with cleaning and microbial con-trol technologies. He is a graduate of the University of Michigan and the University of Iowa. He can be reached by phone at 210-481-7865, and by e-mail at [email protected].

References 1. FDA. “Guide to Inspections of Validation of Cleaning Pro

cesses.” 1993. 2. Pharmaceutical Inspection Cooperation Scheme. Recom men

da tions on Cleaning Validation. Document PR 1/992. Geneva, Switzerland. April 1, 2000.

3. A.M. Cundell. Microbial Monitoring. Presented at the 4th IIR Cleaning Validation Conference, October 2022, 1997. (http://microbiol.org/files/PMFList/clean.ppt, accessed May 29, 2001).

4. S.E. Docherty. “Establishing Microbial Cleaning Limits for Nonsterile Manufacturing Equipment.” Pharmaceutical En gineering. Vol. 19 No. 3. May/June 1999. Pp. 3640.

5. G.L. Fourmen and M.V. Mullen. “Determining Cleaning Validation Acceptance Limits for Pharmaceutical Manufact uring Operations.” Pharmaceutical Technology. Vol. 17 No. 4. 1993. Pp. 5460.

6. D.A. LeBlanc. “Establishing Scientifically Justified Ac ceptance Criteria of Finished Drug Products.” Pharma ceutical Technology. Vol. 19 No. 5. October 1998. Pp. 136148.

7. R.R. Friedel. “The Application of Water Activity Measurements to Microbiological Attributes Testing of Raw Materials Used in the Manufacture of Nonsterile Pharma ceutical Products.” Pharmacopoeial Forum. Vol. 25 No. 5. SeptemberOctober 1999. pp. 89748981.

8. <1111> Microbial Attributes of Nonsterile Pharmacopoeial Articles (proposed). Pharmacopoeial Forum. Vol. 25 No. 2. MarchApril 1999. Pp. 77857791.

9. FDA. “Guide to Inspections of Validation of Cleaning Processes.” 1993.

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CFU: Colony Forming UnitsFDA: Food and Drug Ad mini stra tionPQ: Performance QualificationQC: Quality ControlUSP: United States PharmacopeiaWFI: WaterForInjection

Article Acronym Listing

W e are involved in the production of soft gel atin capsules and tablets in

our newly built facility. Our products consist of at least 17 minerals and multivitamins in a single product, while other products consist of the same ingredients having some quantity (in MG) varying with the previous one. In some products, some vitamins are not present. I want to know how to conduct a cleaning validation study of each product. Again, I want to know which ingredients I have to check after cleaning of the equipment to determine the residues?

• What will the limit be for the micro bial contamination for the cleaning validation studies, and what will be the rationale for the same?

• If I’m using some cleaning agent, then what rationale is used for keeping the limit the same?

A: Thank you for your question. It is a very good one because it represents cleaning from the

point of view of a manufacturer of vitamins and minerals, which in some countries, are considered drugs, and in other countries, are considered as “nutraceuticals,” an important and emerging part of our business.

The first specific question you asked related to how to conduct a cleaning validation for each product, and how to select which ingredient to check after cleaning to verify that the cleaning is adequate.

The choice of which ingredient in a multiingredient product should serve as the focus of the cleaning validation is often a difficult one for vitamin and mineral products. For classical pharmaceutical products, the choice is usually based on choosing the most potent ingredient, or the least water soluble ingredient, or a combination of these two factors. For vitamins and minerals the choice may be more difficult because of the many ingredients present in the formulation and the relatively small amounts present. Coup led with these difficulties is often the difficulty in assaying the very small amounts of active re sidues that might be present after cleaning. My suggestion would be to identify an ingredient for which there is a good sensitive assay available. For example, if one of the in gredients hap pens to show good de tectable levels of fluorescence

(e.g., riboflavin, folic acid, and certain B vitamins show good fluorescence) in water, then this material could be selected as the “marker” material, and could serve as the ingredient to focus on during the analysis of the rinse samples. In the case of vitamins and minerals, it may be necessary, and even highly desirable, to take this ap proach because of the extremely low levels of residues present after cleaning. It may also be possible to examine equipment in a dark room with the use of an ultraviolet light to identify areas of equipment that are not cleaned sufficiently (an enhanced visual examination), again utilizing the known fluo


Cleaning Validation: Maximum Allowable Residue

Question and Answer}…sometimes

the many possible

combinations of products and

equipment would result in so many studies that the company would never be able to complete them

during a reasonable

period of time.~


William E. Hall, Ph.D.

rescent behavior of certain vitamins. A brief study will need to be carried out to determine if this approach is appropriate and adequate for your particular situation. I would suggest that you not try to con duct cleaning validation for every product. The reason I say that is be cause sometimes the many possible combinations of products and equipment would result in so many studies that the company would never be able to complete them during a reasonable period of time. If, for example, you have 50 products, and each could be run on ten (10) different pieces of equipment, then you would need 500 studies to cover all the possible combinations and permutations. That is simply too much of a re source and cost issue for the average company to face. It would be much better to divide your products into groups or families, and choose one or two representatives from each group to conduct full cleaning validation. The assumption is that you can pick some “worstcase,” most difficult to clean, potent products from each group. The first step is to divide the products into groups. I don’t know the names and ingredients of the products your company manufacturers; however, you did mention that some products are vitamin products and others are mineral products. So I think there would be two major groups – vitamins and minerals. Then each of these groups might be further divided, if necessary. For example, in the vitamin category you may have some products that contain water sol uble vitamins, and some that contain fat soluble vitamins. So now we have three (3) major groups (water soluble vitamins, fat soluble vitamins, and mineral pro ducts). So you begin to see our approach. It might be that if you have vastly different types of mineral products you might want to also further divide that group into smaller groups. In any event, you want to have pro bably four (4) to ten (10) products in each group, and then pick a worstcase representative from each group. So by choosing this “grouping approach,” you have re duced the work from a very large resource requirement to a doable or achievable project.

The choice of the worstcase representative should be based on a combination of aqueous solubility and po tency. The potency can be determined for some pro ducts by determining the amount present in the product from the label or package insert. Sometimes this may be a little confusing for vitamin products because the amounts are listed in units instead of quantitative amounts, such as milligrams. In these

cases, I would sug gest that you refer to the Internet, and conduct a search on the toxicity or potency of these materials. You may be surprised to find that a vitamin, such as folic acid, is quite potent in terms of its medical effect and dosage.

The limits for these products can be calculated by allowing a certain small fraction of vitamins or minerals to carry over to each dose of the following product. Again, you will need basic information, such as the medical dosage of the initial product, the batch size and dosage of the next or subsequently manufactured product. In terms of the safety factor, i.e., the factor that is used to reduce the allowable dosage, I suggest that you use a factor of 1/100th for vitamin and mineral products. A factor of 1/1000th is often used for pharmaceuticals, but I feel a more generous factor of 1/100th is appropriate for vitamin and mineral products. You could refer to some of the articles published in the Journal of Validation Technology for the details of how to calculate specific limits.

Your last question related to what rationale should be used for the cleaning agent itself. The basic re quirement is that you be able to provide data that de monstrates that the cleaning agent itself is re moved during the cleaning process, usually by the final rinse. You will need to go through the same rationale for the product residue limits, i.e., establish a scientific basis or justification that shows that the most potent ingredient in the cleaning agent is reduced to a medically insignificant level. It is beyond the scope of this answer to go into the mathematical details of how to calculate this data, but again the details can be found in the various articles published in the Journal of Validation Technology. You will need to know about the ingredients in your cleaning agent, as they are typically multiingredient formulations, just like our pharmaceutical products, and you will need to get that information from your supplier of cleaning agents. The good news is that if you use the same cleaning agent and cleaning procedure for many products, then you only have to do a single cleaning validation study (three runs) for the cleaning agent. o

This answer was provided by an Editorial Advisory Board Member, William E. Hall, Ph.D. Dr. Hall be reached by phone at 910-458-5068, or by fax at 910-458-1087, and by e-mail at [email protected].

14

The 1993 FDA Guideline for cleaning validation states that the removal of deter

gent residues should be evaluated and there should be no or very low detergent levels left after cleaning.1 Currently, the pharmaceutical in dustry employs varieties of detergents for cleaning and different clean ing validation programs. Many companies have not included detergent residue evaluation as part of their cleaning validation programs mainly due to unavailability of ef fective methodologies or lack of aware ness of the requirement by man agement. In the late 1970s, To tal Organic Carbon (TOC) analysis had been used for monitoring water quality in pharmaceuticals and en viron mental controls. More re cent ly, the biotechnology and pharmaceutical industry has be come in creasingly interested in the use of TOC as an analytical tool in cleaning validation programs. TOC analysis has been used as an analytical tool for cleaning validation in the biotechnology industry for years.2,3 Westman and Karlson recently conducted a comparison study for different analytical methods – visual detection of foam, pH, conductivity measurements, and TOC for detergent residue evaluation. They

concluded that the visual detection of foam was the best method for the detergents they tested.4 The method of visual detection of foam is only effective for foaming detergents, but is invalid for low foaming detergents. From a user’s point of view, this paper documents that TOC is an effective and quantitative method for detergent residue verification.

Total Organic Carbon Methodology

TOC is a nonspecific method for the compound analyzed. How ever, TOC analysis is sensitive to very low levels of 0.0020.8 ppm carbon, depending on whether the sample is a water sample or a swab sample. Cur rent ly, two major oxidation technologies dominate the TOC market: combustion and Ultra Violet (UV)/ persulfate. There has been debate

about which technique is better suited for TOC testing since the late 1980s. The major differences for each technique5 are described in Figure 1, and give the user appropriate information to make an informed decision as to which technique better serves their needs.

The best TOC oxidation technology is the one that meets the application and analytical needs of the


Development of Total Organic Carbon (TOC) Analysis for Detergent

Residue Verification By James G. Jin

and Cheryl Woodward Boehringer Ingelheim Pharmaceuticals, Inc.

v

}…the biotechnology and

pharmaceuti-cal industry has

become increasingly interested in

the use of TOC [Total

Organic Carbon] as an analytical tool in cleaning

validation programs.~


James G. Jin

user’s situation. The UV/Persulfate method meets precision and accuracy requirements for lowlevel cal ibration check standards such as 0.5 ppm carbon in detergent residue evaluation. However, if capturing the particulate organic matter in the TOC value is important, then combustion would be the better oxidation technology. The instrument we chose is a TekmarDohrmann Phoenix 8000 with the UV/Persul fate oxidation technique.

Chemistry of Oxidation and Total Organic Carbon Analysis of UV/Persulfate

Wet chemistry oxidation of carbon compounds utilizes two chemical reactions to complete the analysis. A 21 percent solution of phosphoric acid is utilized in converting inorganic carbon species. Acid ification of the sample allows for attack on inorganic species such as carbonates and bicarbonates to convert them to carbon dioxide. This, along with any dissolved carbon dioxide in the sample is then sparged out, and either exhausted to vent or routed to the Non Dispersive Infrared detection (NDIR) for quantification when analyzing for Inorganic Carbon (IC) or TOC by difference (TCIC).

H+ + CO32 → H2O + CO2

Persulfate is used to do the rest of the oxidation chemistry that is required for analysis. Sodium persulfate, at a concentration of 10 percent, and phosphoric acid, five percent are added to the UV chamber for analysis. The persulfate species in the presence of UV light breaks down at a weak oxygenoxygen bond yielding two radicals per molecule. These radicals start chain reactions that ultimately lead to the

degradation of all carbon species to carbon dioxide, water, and other oxides of heteroelements. The UV light alone induces breakdown of many carbon species with the persulfate providing additional help to attack compounds difficult to oxidize. The radical reactions are aggressive and indiscriminate in their attack.

S2O82 → SO41 + R → H2O + CO2

The NDIR is constructed in such a way as to be sensitive and selective for carbon dioxide present in the gas flow. An infrared beam from the source is passed through a chopper and down the sample chamber to a dual chamber detector. Each chamber is filled with carbon dioxide and is separated by a thin membrane. Varying intensity of the light hitting the cell causes fluctuation in temperature and thus the pressure of the gas inside the detector. This causes the membrane to deflect, which is ultimately read as a millivolt output signal from the detector.

Detergent Evaluation

Three detergents (CIP100, CIP200, and Sparquat 256) were tested both inhouse using the Tekmar Dohrmann Phoenix 8000 TOC Analyzer and at a contract lab, Quantitative Technologies Inc. (QTI), to ver ify the total amount of organic carbon in each de tergent at its original concentration. The method and instrument used at QTI was a PerkinElmer CHN Analyzer 2400. This experiment was performed to make a comparison between our instrument and the instrument in a qualified contract laboratory for information purposes only. One detergent (ChlorMate) was tested inhouse and compared with the available

16

Figure 1types of total organic carbon techniques

oxidation Detection technique analytical range (toc) official methodsCombustion Thermal Conductivity Detector (TCD) 0.5 – 100% AOAC 955.07Combustion Coulometric 1 – 100% ASTM D4129UV/Persulfate Non-Dispersive Infrared Detector (NDIR) 0.002 – 10,000 mg/L USP 643Heated Persulfate NDIR 0.002 to 1,000 mg/L USP 643Combustion NDIR 0.004 – 25,000 mg/L USP 643UV/Persulfate Membrane/Conductivity 0.0005 – 50 mg/L USP 643UV Conductivity or NDIR 0.0005 – 0.5 mg/L USP 643


James G. Jin

vendor’s specification. The TOC results for all the detergents are shown in Figure 2.

The differences between the inhouse and QTI results with respect to the TOC assay for CIP100 and CIP200 are 5.0 percent and 9.6 percent, respectively. These differences are relatively low compared to the 20 percent recovery criteria during recovery studies. The difference between the inhouse and QTI results with respect to the TOC assay for Sparquat 256 is 28.4 percent. The inhouse result was reviewed and no error was noted in the performance of the testing procedure. The major differences may be due to in strument and testing method variations. The result for ChlorMate is within the vendor’s specification.

Swab Selection

It has been known for years that polyester is a suitable material for TOC swabbing analysis. Over 20 different kinds of polyester swab samples were received from The Texwipe Company LLC. Five of them were chosen for TOC evaluation based on sample design and the convenience for use. The purpose of this experiment was to select a type of swab that has little TOC background interference and with consistent TOC results over time. Ultra purified water with 0.05 to 0.08 ppm carbon was used for swab analysis. The TOC results obtained from our TOC analyzer are shown in Figure 3.

Swabs TX761 and TX741A showed increasing TOC results from 0.0813 to 0.9692 ppm carbon and from 0.1724 to 1.1246 ppm carbon over five days, re spectively. Swab TX700 showed an unacceptably high TOC result of 46.1991 ppm carbon at the beginning of the experiment, and was therefore not tested further. None of these swabs are suitable for our TOC analysis.

Both polyester wipers AlphaSorb® HC TX2412

and TX2418 show acceptable results with respect to result consistency. The average of the seven TOC results from TX2412 and TX2418 found in Figure 3 is 0.8327 ± 0.1860 ppm carbon. The variation is acceptable compared to the acceptance criterion of three ppm carbon. These two swabs with the same material were selected to be our TOC swabs (cut to 5x5 cm2) for detergent residue verification.

The TX3340 TOC cleaning validation kit including Eagle EP Picher 0346440mL clear vials, Tex wipe® TX714Llarge SnapSwabsTM, and blank vial labels may be chosen since it is specially de signed for TOC swabbing purposes.

Detergent Recovery Evaluation from Stainless Steel Surface

Ten stainless steel templates were spiked with detergent solution and swabbed using the polyester wipers AlphaSorb® HC TX2418 (5x5 cm2) for the detergent recovery study. The spiking and swabbing procedures were the same as those used for drug substance recovery studies. Forty mL of ultra purified water was added to each test tube as the extraction solution, vortexed about one minute, and then sonicated for five minutes for testing. The results are shown in Figure 4.

The recoveries for CIP100, CIP200, and ChlorMate are over 80 percent and no correction factor is necessary.

For Sparquat 256, a correction factor of 0.61 will be used. For example, if a result of 0.5 ppm carbon is obtained from the TOC analyzer, the final reported result would be 0.82 (0.5 ÷ 0.61) ppm carbon.

Detergent Recovery Evaluation from Non-Stain-less Steel Surfaces

The aforementioned study was repeated using nonstainless steel templates. Two or three nonstain

17

Figure 2total organic carbon results for Detergent evaluation

Detergent manufacturer/lot total organic carbon result toc results identification From BiPi* From qti/vendorCIP-100 Vestal Convac lot 211097 4.0208 ± 0.0139% 4.22%CIP-200 Convac lot 213915 2.4986 ± 0.0114% 2.26%Sparquat 256 ISSA (lot: n/a) 14.0232 ± 0.9336% 18.0%Chlor-Mate WestAgro® lot J8G0489AR 1.29% ± 0.0086% 1 – 1.5%

*Boehringer Ingelheim Pharmaceuticals, Inc.


James G. Jin

less steel templates were spiked with each detergent solution and swabbed using the polyester wipers AlphaSorb® HC TX2418 (5x5 cm2). The results are shown in Figure 5.

For CIP100 and CIP200, the recoveries from each nonmetal surface are over 80 percent. Therefore, no correction factor is needed with respect to the TOC recovery. For Sparquat 256, the recoveries vary with different surfaces. The correction factors are as follows:

For Delrin surface: correction factor = 0.74For Glass surface: correction factor = 0.75For Nylon surface: correction factor = 0.43For Lexan surface: correction factor = 1.0

Evaluation of Detergent Residue After RinsingThe purpose of this experiment was to evaluate:

∂ The suitability of the Acceptance Criterion (AC) of three ppm carbon

∑ The effect of detergent concentration on detergent residue after rinsing

∏ Recovery of detergent from different surfaces with and without rinsing

π Rinsing efficiency and rinse time

Four detergents (CIP100, CIP200, Sparquat 256, and ChlorMate) were used in both a concentrated form and at a working concentration of 0.5 oz/gal. Approximately one mL of detergent solution

was pipetted and spiked onto the templates with different materials of construction and dried with ventilation under a hood in the research and devel

18

Figure 3total organic carbon results (ppm c) for swab selection

swab toc/two hours toc/Four hours toc/one Day toc/two Days toc/Five Days Description in h2o in h2o in h2o in h2o in h2oPolyester Alpha 0.0813 0.3221 0.3926 0.9410 0.9692 swab TX761 ± 0.0041 ± 0.0853 ± 0.0166 ± 0.0288 ± 0.0299Polyester Alpha 0.1724 0.2509 0.5330 0.8091 1.1246 swab TX741 A ± 0.0144 ± 0.0068 ± 0.0250 ± 0.0200 ± 0.0394Polyester wipers 1.1665 0.6091 0.8602 0.7535 0.9723 AlphaSorb® ± 0.0406 ± 0.0490 ± 0.0264 ± 0.0328 ± 0.0668HC TX2412Polyester wipers 0.7406 0.7269 N/A(1) N/A(1) N/A(1)

AlphaSorb® ± 0.0056 ± 0.0297HC TX2418Polyester Alpha 46.1991 N/A N/A N/A N/A swab TX700 ± 8.07611. Polyester wipers AlphaSorb® HC TX2412 and polyester wipers AlphaSorb® HC. TX2418 is same material cut to different sizes.

Figure 4total organic carbon recovery

results from a stainless steel surface

Detergent Percent number Percent recovery of relative samples standard Deviation

CIP-100 111.7 30 5.92CIP-200 92.4 10 4.10Sparquat 256 61.0 20 8.47Chlor-Mate 99.1 10 2.76Note: Results were automatically corrected for the

instrument blank effect.

Figure 5total organic carbon recovery

results from a non-stainless steel surface

Detergent lexan Delrin Glass nylon surface Percent Percent Percent Percent recovery recovery recovery recovery

CIP-100 106.9 113.8 107.6 127.0CIP-200 90.3 92.3 97.4 93.2Sparquat 83.3 74.0 75.1 42.5 256


James G. Jin

opment manufacturing area for a minimum of four hours. The templates were swabbed per standard swabbing procedure either before or after rinsing, using the polyester wipers AlphaSorb® HC TX2412 cut to 5x5 cm2. The rinse was first conducted using tap water and then purified water United States Pharmacopoeia (USP), both at room temperature and with a slow flow rate of approximately 2.7 L/min. Two different rinse times (30 seconds and 60 seconds) were evaluated for different detergents on different templates to simulate the final rinse step in our manual cleaning process. The recovery results are reported in Figure 6.

The Tekmar Dohrmann Phoenix 8000 TOC analyzer was easily able to detect the nonrinse samples with the results of 3.911 ppm carbon, 2.0928 ppm carbon, and 10.0868 ppm carbon for CIP100, CIP200, and Sparquat 256, respectively. The results indicate that the AC of three ppm carbon is still high for detergents CIP100, CIP200, and Sparquat 256. The AC of one ppm carbon is acceptable. There were no differences in detectable residue for all four detergents (both concentrated and at 0.5 oz/gal) on stainless steel after a 30second tap water rinse followed by a 30second purified water, USP rinse. Delrin was chosen for a typical material of construc

19

Figure 6total organic carbon results on Detergent residue by rinsing

sample concentration templates rinse time area toc results identification swabbed (ppm c)d

CIP-100 0.5 oz/gal SS a No rinse 100 cm2 3.9111CIP-100 0.5 oz/gal SS a 30”/30” b 100 cm2 Less than blankCIP-100 Concentrated SS a 30”/30” b 100 cm2 Less than blankCIP-100 0.5 oz/gal Delrin 30”/30” b 100 cm2 Less than blankCIP-100 0.5 oz/gal Delrin 60”/60” b 100 cm2 Less than blankCIP-100 0.5 oz/gal Nylon 30”/30” b 100 cm2 0.6682CIP-100 0.5 oz/gal Glass 30”/30” b 100 cm2 0.0001CIP-100 0.5 oz/gal Lexan 30”/30” b 100 cm2 Less than blank

CIP-200 0.5 oz/gal SS a No rinse 100 cm2 2.0928CIP-200 0.5 oz/gal SS a 30”/30” b 100 cm2 Less than blankCIP-200 Concentrated SS a 30”/30” b 100 cm2 Less than blankCIP-200 0.5 oz/gal Delrin 30”/30” b 100 cm2 Less than blankCIP-200 0.5 oz/gal Delrin 60”/60” b 100 cm2 Less than blankCIP-200 0.5 oz/gal Nylon 30”/30” b 100 cm2 0.7720CIP-200 0.5 oz/gal Glass 30”/30” b 100 cm2 0.0133CIP-200 0.5 oz/gal Lexan 30”/30” b 100 cm2 Less than blank

Sparquat 256 0.5 oz/gal SS a No rinse 100 cm2 10.0868 c

Sparquat 256 0.5 oz/gal SS a 30”/30” b 100 cm2 0.2693 c

Sparquat 256 Concentrated SS a 30”/30” b 100 cm2 Less than blankSparquat 256 0.5 oz/gal Delrin 30”/30” b 100 cm2 Less than blankSparquat 256 0.5 oz/gal Delrin 60”/60” b 100 cm2 Less than blankSparquat 256 0.5 oz/gal Nylon 30”/30” b 100 cm2 0.3866 c

Sparquat 256 0.5 oz/gal Glass 30”/30” b 100 cm2 Less than blankSparquat 256 0.5 oz/gal Lexan 30”/30” b 100 cm2 Less than blank

Chlor-Mate 0.5 oz/gal SS a 30”/30” b 100 cm2 Less than blankChlor-Mate Concentrated SS a 30”/30” b 100 cm2 Less than blankNotes: a. Stainless steel. b. 30”/30” or 60”/60” – rinse time in seconds, tap water/purified water United States Pharmacopoeia (USP). c. Result without correction factor.


James G. Jin

tion and 30/60 seconds were chosen for evaluation of the rinse time. There was no difference in detectable residue for CIP100, CIP200, and Sparquat 256 on the Delrin surface after 30second and 60second rinse times. The results also show that it is more difficult to remove residues of CIP100, CIP200, and Sparquat 256 from a Nylon surface than from other materials.

Acceptance Criterion for Detergent ResidueThere is no universal AC for detergent residue

allowed to be left on GMP equipment surfaces. In our detergent residue verification program, the AC for each detergent residue left on equipment surfaces depends on the sensitivity of the instrument used for analysis. This means we must set a low AC that is still quantifiable and applicable. Toxicity of the detergent is not a concern at these trace amounts de tergent level. Effects on human health from re sidue left on equipment surfaces should be insignificant at a low concentration such as 0.5 oz/gal and with a routine rinse procedure. Our objective in this program is to demonstrate that we are able to verify whether or not the detergent residues are removed to an acceptable lowlevel we can achieve.

Therefore, the AC should be established as close to the instrument’s level of detection as possible. We tighten the initial limit of three ppm carbon to AC = 1.0 ppm carbon (net reading automatically corrected with blank by the instrument in a 40 mL solution), which is less than two times the blank baseline. The AC can also be expressed as AC ≤ 10 ppb carbon/cm2. This AC is practical and verifiable.

The significance of the 1.0 ppm carbon AC for each detergent can be explained in Figure 7.

We can see from the above calculations that AC = 1.0 ppm carbon means, for all detergents at 0.5 oz/gal, that we allow the maximum of 1 ÷ 3.92 = 0.26 mL of CIP100, 1 ÷ 2.44 = 0.41 mL of CIP200, 1 ÷ 13.68 = 0.07 mL of Sparquat 256, and 1 ÷ 1.26

= 0.79 mL of ChlorMate to be left on 100 cm2 of equipment surface after cleaning, respectively.

Detergent Residue Verification ProgramOur detergent verification program is designed

to be a onetime verification for each detergent used. This was based on the rinse experiment and the assumption that our routine rinsing procedures performed by well trained operators are sufficient to remove detergent residues to the level of less than the AC. This assumption has been verified from the results shown in Figure 6 that all the residues are easily removed by a 30second tap water rinse followed by a 30second purified water, USP rinse with very low spray rate. Verification rather than validation is currently required by the 1993 FDA, Guide to In spec tions of Validation of Cleaning Procedures due to the fact that detergent residue is less significant than drug substance residue left after cleaning.

Summary

The detergent residue verification program has been successfully established using the Tekmar Dohrmann Phoenix 8000 TOC analyzer. This paper has shown the program development, and presents critical data to support the detergent verification reports for each detergent used.

The instrument Installation Qualification (IQ), Operational Qualification (OQ), system calibration, and the TOC analysis method development were performed but not discussed in this paper. The polyester wipers AlphaSorb® HC TX2412 and TX2418 cut to 5x5 cm2 have been selected as the swabs for sampling detergent residue from equipment surface for TOC analysis. The AC for the detergents CIP100, CIP200, Sparquat 256, and ChlorMate with respect to TOC has been established as AC ≤ 10 ppb car bon/cm2. Two different rinse times, 30 seconds and 60 seconds, were evaluated. The results show

20

Figure 7significance of total organic carbon results for Detergent at 0.5 oz/gal

ciP-100 ciP-200 sparquat 256 chlor-mate1 mL at 0.5 oz/gal 3.92 ppm 2.44 ppm 13.68 ppm 1.26 ppm diluted to 40 mL1.0 ppm C per 100 cm2 0.26 mL 0.41 mL 0.07 mL 0.79 mLcorresponding to


James G. Jin

that 30second/30second rinse time (30second rinse with tap water and then 30second rinse with purified water, USP) is sufficient to remove the detergent re sidues from different material templates including stainless steel, Delrin, Glass, Nylon, and Lexan to a level below the AC. The correction factors were de termined based on the results of the recovery studies and will be used by analytical sciences to report the final TOC results for the detergent residue verification. o

About the AuthorsJames G. Jin is Chairman of the Cleaning Validation Committee for Boehringer Ingelheim Pharma ceuti-cals, Inc., which is responsible for clean ing valida-tion program development and implementation. He has more than ten years experience in pharmaceuti-cal science and business arenas. He can be reach-ed by phone at 203-798-5309.

Cheryl Woodward is Associate Director of Research and Development (R&D) Manufacturing, for Boeh-ringer Ingelheim Pharmaceuticals, Inc. She is re sponsible for all aspects of GMP manufacturing for clinical supplies and has over 18 years experi-ence in the pharmaceutical and related industries. She can be reached by phone at 203-798-5367.

References 1. FDA. Guide to Inspections of Validation of Cleaning Pro ce

dures. July, 1993. 2. Jenkins K.M., Vanderwielen A.J, Armstrong J.A, Leonard L.M,

Murphy G.P, Piros N.A. 1996. “Application of Total Organic Carbon Analysis to Cleaning Validation.” PDA. Journal of Pharma ceutical Science and Technology. 50. Pp 615.

3. Guazzaroni M., Yiin B., Yu J., 1998. “Application of Total Or ganic Carbon Analysis for Cleaning Validation in Pharma ceutical Manufacturing.” American Biotechnology Laboratory. September. Pp. 6667.

4. Westman L., Karlsson G., 2000. “Methods for Detecting Re sidues of Cleaning Agents During Cleaning Validation.” Re search Article, Vol. 54, No. 5. September/October.

5. Furlong J., Booth B., Wallace B. 1999. “Selection of a TOC Analyzer: Analytical Considerations.” TekmarDohrmann Ap pli cation Note. Vol. 9.20.

21

In the pharmaceutical industry, Good Manufacturing Practice (GMP) requires that the clean

ing of drug manufacturing equipment be validated.1 Many different validation techniques can demonstrate that the manufacturing equipment is cleaned and essentially free from residual active drug substances and all cleaning agents.

Common analytical techniques in the validation process include High Performance Liquid Chromatography (HPLC), spectrophotometry Ultraviolet/Visible (UV/Vis) and Total Organic Carbon (TOC). HPLC and UV/Vis are classified as specific methods that identify and measure appropriate active substances. TOC is classified as a nonspecific method and is ideal for detecting all carboncontaining compounds, including active species, excipients, and cleaning agent(s).2,3,4,5

The disadvantage of specific methods, particularly HPLC, is that a new procedure must be developed for every manufactured active drug substance. This development process can be very time consuming and tedious, plus important sampling issues must also be considered. In addition, HPLC analyses must be performed in a relatively short time period after sampling to avoid any chemical deterioration of the active substance. Finally, the sensitivity of HPLC methods can be limited by the presence of degradation products. Of course the disadvantage to non

specific methods like TOC is that they cannot identify exactly what the residue material is. Depending on the chosen cleaning process and established acceptance limits, a non specific method may be all that is needed to validate the process.

TOC analysis can be adapted to any drug compound or cleaning agent that contains carbon and is “adequately” soluble in water. Studies have been conducted to demonstrate that TOC methods can also be applied to carbon containing compounds that have limited water solubility, and recovery results are equal to those achieved by HPLC.6

TOC methods are sensitive to the parts per billion (ppb) range and are less time consuming than HPLC or UV/Vis. United States Pharmacopoeia (USP) TOC methods are standard for WaterforInjection and Purified Water,7 and simple modifications of these methods can be used for cleaning validation.

Methodology

TOC analysis involves the oxidation of carbon and the detection of the resulting carbon dioxide. A number of different oxidation techniques exist, including photocatalytic oxidation, chemical oxidation, and hightemperature combustion. In this study, an Anatel A2000 WideRange TOC Analyzer, equipped with an autosampler, was used. The Anatel A2000 Wide


Total Organic Carbon Analysis for Cleaning Validation in

Pharmaceutical ManufacturingBy Karen A. Clark Anatel Corporation

v

}TOC analysis can be adapted

to any drug compound or

cleaning agent that contains carbon and is ‘adequately’

soluble in water.~


Karen A. Clark

Range Analyzer measures TOC in accordance with American Society for Testing and Materials (ASTM) methods D 477988 and D 483988. It measures TOC directly by adding phosphoric acid to the water sample to reduce the pH from approximately two to three. At this low pH any inorganic carbon that is present is liberated as CO2 into a nitrogen carrier gas and is directly measured by a nondispersive infrared (NDIR) detector. Any remaining carbon in the sample is assumed to be TOC. A sodium persulfate oxidant is then added to the sample, and in the presence of UV radiation, the remaining carbon is oxidized to CO2. The amount of CO2 generated is then measured by the NDIR to determine the amount of TOC originally present in the water.

For equipment cleaning validation there are two types of TOC sampling techniques. One is the direct surface sampling of the equipment using a swab. The second consists of a final rinse of the equipment with highpurity water (typically <500 ppb TOC) and collecting a sample of the rinse for analysis. In general, direct surface sampling indicates how clean the actual surface is. This study demonstrates how to develop and validate a TOC method to measure a variety of different organic residues on stainless steel surfaces. Performance parameters tested include linearity, method detection limit (MDL), limit of quantitation (LOQ), accuracy, precision, and swab recovery.

Linearity

TOC analysis should provide a linear relationship between the measured compound concentration and the TOC response of the analyzer. We evaluated four different types of cleaning agents for linearity:

∂ CIP100 ® (alkaline)∑ CIP200 ® (acidic)∏ Alconox® (emulsifier)π TritonX 100 (wetting agent)

Results are shown in Figures 14. Correlation coefficients ranged from 0.9787 to 0.9998. Alconox and TritonX 100 have a tendency to foam, depending on the concentrations that are analyzed and this foaming phenomena can have a negative effect on the accuracy of the TOC result (reduced R2). Three

23

Figure 1linearity of ciP-100

900080007000600050004000300020001000

0

Mea

sure

d T

OC

(p

pb

)

CIP 100 Concentration (ppm) 0 50 100 150 200 250

y=39.254x + 1.462R2=0.9997

Figure 2linearity of ciP- 200

900080007000600050004000300020001000

0

Mea

sure

d T

OC

(p

pb

)

CIP 200 Concentration (ppm) 0 100 200 300 400 500

y=19.132x + 51.042R2=0.9998

Figure 3linearity of alconox

454035302520151050

Mea

sure

d T

OC

(p

pm

)

Alconox Concentration (ppm) 0 200 400 600 800 1000

y=0.0355x + 1.1983R2=0.9787


Karen A. Clark

representative examples of active substances were also tested for linearity: an excipient (sucrose), an antibiotic (vancomycin), and endotoxin. Results are shown in Figures 57. All three compounds demonstrated excellent linearity with correlation coefficients (R2) ranging from 0.9996 to 0.9998.

Method Detection Limit and Limit of Quantitation

We determined the Method Detection Limit (MDL) by measuring the TOC response of the method blank.

A method blank consists of the sampling vial, swab, and recovery solution. In this study, the recovery solution was low TOC (< 25 ppb) water. Ten precleaned vials were filled with the low TOC water. One swab was placed in each vial (Texwipe Alpha Swab TX761; tips cut off). Solutions were vortexed and allowed to stand for one hour prior to analysis. Four replicates from each vial were analyzed. The four replicates from each of the ten blank vials were averaged. These ten values were averaged again and a standard deviation was calculated. The standard deviation was multiplied by the Student t number for n1 degrees of freedom (3.25 for n=10), at 99% confidence levels to determine the method detection limit. The MDL was calculated to be 50 ppb. The Limit of Quantitation (LOQ) was calculated by multiplying the MDL by three. A value of 150 ppb was obtained (see Figure 8).

Precision and Accuracy

24

Figure 4linearity of triton-x 100

12500

10000

7500

5000

2500

0

Mea

sure

d T

OC

(p

pb

)

Triton-X 100 Concentration (ppm) 0 5 10 15 20 25

y=415.76x + 16.997R2=0.9982

Figure 6linearity of vancomycin

8000

6000

4000

2000

0

Mea

sure

d T

OC

(p

pb

)

Vancomycin Concentration (ppb) 0 2000 4000 6000 8000

y=0.8758x + 62.133R2=0.9998

Figure 5linearity of sucrose

12000

10000

8000

6000

4000

2000

0

Mea

sure

d T

OC

(p

pb

)

Sucrose Concentration (ppb) 0 2000 4000 6000 8000 10000 12000

y=1.003x + 45.185R2=0.9996

Figure 7linearity of endotoxin

80007000600050004000300020001000

0

Mea

sure

d T

OC

(p

pb

)

Endotoxin Concentration (ppb) 0 2000 4000 6000 8000

y=0.9287x + 30.8R2=0.9998


Karen A. Clark

To demonstrate the precision and accuracy for this TOC method, a representative solution of CIP100 as 1000 ppb, or one ppm as carbon, was analyzed sequentially ten times. This carbon concentration was chosen to evaluate these method parameters because, in general, TOC residual limits are typically around one ppm. Results are listed in Figure 9. At this TOC level, the precision was ± 1% and the accuracy was ± 5%.

Swab Recovery

Stainless steel plates were used in the swab recovery test to simulate manufacturing equipment. One side of each plate was spiked with a solution of active substance or cleaning agent. The plates were allowed to completely dry overnight at room temperature. A Texwipe alpha swab TX761 was moistened with low TOC (< 25 ppb) water and the spiked plate surface was swabbed both vertically and horizontally. The swab end was cut off, placed into a vial to which we added 40mL of low TOC water. The vial was capped tight, vortexed, and allowed to stand for one hour prior to analysis. The same volume of each solution that was spiked onto the plates was separately spiked directly into 40mL of low TOC water and analyzed. The percent recoveries of the different substances are listed in Figure 10. Reported values are the average of three individual swab samples for each substance. The swab recoveries varied between 79.3% to 95.9%

Conclusion

This study demonstrates that TOC analysis is suitable for measuring organic residues on stainless steel surfaces, and that it is a reliable method for cleaning validation as demonstrated by surface residue recoveries of 79%96%. This methodology

25

Figure 8calculated toc averages

from 10 Blank vials vial number average toc (ppb) 1 58 2 72 3 75 4 93 5 79 6 102 7 60 8 83 9 67 10 54Average 74.3Standard Deviation 15.5MDL (Student t, n=10) 50 ppbLOQ 151 ppb

Figure 9calculated accuracy and Precision from 10 replicates of a 1ppm ciP-

100 solution as carbon vial number measured toc (ppb) 1 1041 1 1025 1 1039 1 1057 1 1054 2 1034 2 1042 2 1048 2 1054 2 1055Average 1045Standard Deviation 10.5% CV (precision) 1.0%% Recovery based on 105%1 ppm C (accuracy)

Figure 10representative examples of swab recoveries from cleaning agents

and active substances substance ppm c of spike ppm c of spiked % recovery % rsD standard solution Plate CIP-100 1810 1710 94.5 1.8 Sucrose 2663 2112 79.3 4.9 Vancomycin 661 634 95.9 3.0 Endotoxin 902 736 80.0 2.8


Karen A. Clark

shows that low limits of detection, excellent linearity, precision, and accuracy can be obtained. All of these TOC results, with the exception of Alconox and TritonX 100, were generated using the same TOC method, making TOC analysis a low cost and less time consuming alternative for cleaning validation. o

About the AuthorKaren A. Clark is a Product Manager at Anatel Corporation. She has over 15 years experience in the pharmaceutical/biotechnology industry focus-ing on drug formulations, analytical methods devel-opment and validation, and GLP/GMP laboratory management. Clark holds a B.S. in Biochemistry from Millersville University and an M.S. in Chemical Engineering from the University of Colorado. She can be reached by e-mail at [email protected] or at Anatel Corporation, 2200 Central Avenue, Boulder, CO 80301.

References 1. FDA. Current Good Manufacturing Practice Regulations, 21

CFR 211.220. 2. Baffi, R. et al. 1991. “A Total Organic Carbon Analysis Method

for Validating Cleaning Between Products in Bio pharmaceutical Manufacturing.” Journal of Parenteral Science and Technology 45, no. 1: 139.

3. Jenkins, K. M. et al. 1996. “Application of Total Organic Carbon Analysis to Cleaning Validation.” PDA Journal of Pharmaceutical Science and Technology 50, no. 1: 615.

4. Strege, M. A. et al. 1996. “Total Organic Carbon Analysis of Swab Samples for the Cleaning Validation of Bioprocess Fer men tation Equipment.” BioPharm (April).

5. Guazzaroni, M. et al. 1998. “Application of Total Organic Carbon Analysis for Cleaning Validation in Pharmaceutical Manufacturing.” American Biotechnology Laboratory 16, no. 10 (September).

6. Walsh, A. 1999. “Using TOC Analysis for Cleaning Val idation.” Presented at The Validation Council’s Conference on Cleaning Validation, 27 October, Princeton, New Jersey.

7. USP 23, Fifth Supplement, 15 November 1996.

26


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The FDA recognizes the im portance of effective cleaning and sanitizing pro

tocols as a proactive measure in preventing crosscontamination in the pharma ceutical and cosmetic in dus tries:

21CFR 211.67: “Equip ment and utensils shall be cleaned, maintained, and sanitized at appropriate intervals to prevent malfunctions or contamination that would alter the safety, identity, strength, quality, or purity of the drug product beyond the official or other established requirements.”

In order to comply with this regulatory requirement, sound cleaning and sanitizing protocols must be developed and followed. One of the most critical components of any cleaning program is detergent se lection. Different pro cesses and po tential contaminants may require different de tergents that are appropriate for the application. In certain cleaning ap plications, a neutral foaming de tergent might be appropriate, where as in others, a nonfoam ing alkaline detergent is de sirable. The choice of

detergent for a given ap plication should be based on sound, scientific reasoning.

A sound rationale for detergent selection begins at the man ufacturing site, where the process and clean ing program will take place. A full evaluation of the pro cess, cleaning strategies, potential contaminant levels, and available utilities is a good first step. Follow ing this step, laboratory testing is re quired to de termine the exact nature of the po tential contaminant. Next, identifi ca tion and testing of various cleaning chem istries against the potential contaminant is performed to determine which de tergent type is best suit ed for con taminant re moval. The next step is to return to the manufacturing site, test the cleaning chemistry, and optimize the program. This ap proach provides a sound, scientific rationale for the detergent selection and lays a firm foundation to the formal cleaning protocol, once de veloped.

This article will discuss the key factors that must be ad dres sed when selecting a detergent. Each factor will be discussed in detail and examples are given when appropriate. The roles


Detergent Selection – A First Critical Step in Developing a Validated Cleaning Program

By Mark Altier Ecolab, Inc.

v

}This article will discuss the key

factors that must be

ad dres sed when selecting a

detergent. Each factor will be discussed in detail and

examples are given when appropriate. The roles of

laboratory testing and plant

optimization are also addressed.~


Mark Altier

of laboratory testing and plant optimization are also addressed.

The Five Factors for Determining Detergent Suitability

There are five key factors that must be ad dres sed when determining which detergent is most suitable for a cleaning application. These are:

∂ Nature of the residue (or potential contaminant)

∑ Surface to be cleaned∏ Method of applicationπ Role of water∫ Environmental factors

All five of these factors must be addressed when developing a cleaning program. Failure to address any of these issues in sufficient detail can result in a less than desirable cleaning program and could place the successful completion of the cleaning validation at serious risk.

The Nature of the ResidueA residue can be defined as any unwanted matter

or potential contaminant on the surface of the ob ject or equipment being cleaned. Oftentimes, what is re ferred to as a “residue,” is in fact a finished product, drug active, or other component that is produced us ing the process equipment that is being cleaned. The terms “residue,” “contaminant,” and “potential con taminant” will be used interchangeably throughout this article.

Determination of the nature of a residue is a fundamental component in the development of any clean ing program. In some cases, the exact nature and composition of a residue is known. For example, if the residue is a finished product, the exact composition and physical properties are almost always known. However, the identity and nature of the re sidue may be completely unknown if the re sidue is composed of an intermediate, byproduct, or result of thermal, chemical, or other degradation of a previously known substance.

The nature of the potential contaminant plays a central role in determining what type of detergent is most appropriate for the application. Individual re si dues require different detergent chemistries. All

residue types will fall into one of the following three categories: organic, inorganic, or a combination of these. Most potential contaminants are a combina

tion of organic and inorganic components. Com mon residue types in the pharmaceutical industry are given in Figure 1.

A number of powerful analytical instruments are available that can provide tremendous insight into the nature and composition of almost any unknown potential contaminant type. Some of the more useful tools include:

• Fourier Transform Infrared Spectroscopy (FTIR)

• Energy Dispersive XRay Spectroscopy (EDS)• Scanning Electron Microscopy (SEM)• Compound microscopic imaging• Nuclear Magnetic Resonance imaging (NMR)• Inductively Coupled Plasma detector (ICP)• Atomic Absorption Analyzer (AA)

Often, a combination of two or more of these tools is required to provide a full picture of a potential contaminant in question. For example, Fig ure 2 and Fig ure 3 are typical images generated to help characterize unknown potential contaminant samples. This type of analysis is invaluable in determining the ex act residue type and breakdown of the organic and inorganic portions of a residue.

Figure 2 is an FTIR image of an unknown re sidue. This characterizes and gives a general breakdown of

29

Figure 1common residue types in the

Pharmaceutical industryorganic residues inorganic residues

Eudragit Titanium Dioxide

Acetaminophen Zinc Oxide

Carbopols Iron Oxide

Albuterol Sulfate Calcium Carbonate

Neomycin Sulfate Inorganic Salts

Water/Oil – Oil/Water Silicon Dioxide Emulsions

Glyburide


Mark Altier

30

Figure 2Ftir scan of unknown sample. this analysis indicates the Presence of alkyl and amide Protein components and Possible inorganic content

3933

.71

2958

.86

2926

.38

2854

.86

2287

.55

2257

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2211

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2165

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2155

.88

2033

.97

2013

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1631

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1545

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.04

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.26

1077

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1044

.64

980.

456

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204

694.

924

3500 3000 2500 2000 1500 1000Wave Number (cm-1)

.35

.30

.25

.20

.15

.10

.05

0

Abso

rban

ce

Figure 3eDs scan of unknown sample

this analysis confirms the Presence of inorganic components such as silicon, aluminum, and iron, in addition to organic compounds

0.000 1.000 2.000 3.000 4.000 5.000 6.000 7.000 8.000Key

600580560540520500480460440420400380360340320300280260240220200180160140120100806040200

Cou

nts

C

O

Fe Mg

Al

Si

Fe

Fe


Mark Altier

the organic portion of the residue. FTIR imaging gives valuable insight into the functional groups that may be present in the organic component of a re si due. Figure 3 confirms the presence of inorganic ma terial and identifies the specific inorganic components present in an unknown sample. This information is useful when determining which chelant or surfactant family is most suitable for re moving or tying up the free metal ions and other inorganic material.

Combined, FTIR and EDS imaging can give a com plete picture of most unknown residues. These analyses provide the information needed to select a group of detergent chemistries that are formulated and known to be effective against the residue type.

Surfaces To Be CleanedDifferent substrates (i.e., product contact surfaces,

such as stainless steel, glass, or plastic) will interact differently with the contaminant and the de tergent system. Some materials, such as glass, and alumi num, are not tolerant to high pH systems. Other substrates

may tolerate high pH, but may not tolerate chlorine or chlorides. It is important to have a clear understanding of how the substrate being cleaned will interact with the detergent system, otherwise serious damage to equipment surfaces can result. A SEM image, shown in Figure 4, is a stainless steel surface that has been pitted by using an in compatible detergent. The prospective customer in this case felt that the residue was becoming more tenacious with time and was using higher detergent concentrations to remove the residue.

A close look at the surface revealed that the surface was actually being pitted by the detergent, providing microscopic crevices where the residue was able to harbor during the cleaning cycle. This problem was aggravated by the fact that the customer continued to increase the detergent concentration, which accelerated the rate and degree of corrosion, and provided the residue with even more locations to harbor during the cleaning cycle.

These images clearly demonstrate the problems

31

Figure 4microscopic corrosion of a stainless steel surface caused by improper

Detergent selection. inset shows Boxed region at 1000 times magnification


Mark Altier

that can be caused by improper detergent selection. In this case, the customer was advised to discontinue the use of the incompatible detergent, and a compatible detergent chemistry was identified and tested. The customer was also required to re place or re pair damaged equipment.

When developing a cleaning protocol, it is necessary to identify all components of the process that will be exposed to the cleaning chemical(s). This includes equipment surfaces, gasket materials, nozzles, piping, pumps, etc. It is also important to consider surfaces that will be exposed to the vapor phase of the cleaning solution, such as overhead spaces in enclosed vessels and pipes. A common mistake is to concentrate only on items that will have direct contact with the liquid solution, neglecting the vapor phase.

Method of ApplicationThere are several common methods of applying

a detergent to equipment surfaces. Some are more common than others in the pharmaceutical industry. Some of the more common methods of application in the pharmaceutical industry include:

• CleaninPlace (CIP)• Clean OutofPlace (COP)• Manual scrubbing/wiping• High and low pressure spray• Soaking/immersion

Each of these application methods dictate certain desirable or undesirable detergent properties. For example, a high pH detergent is ideal in a CIP application where little, or no direct contact is made be tween the detergent and the operator. In a manual application, however, a high pH detergent creates a significant safety risk to an operator handling the de tergent concentrate and use solutions. In a manual application, a neutral or mildly alkaline de tergent (pH 7.0 – 10.0) is much more desirable as it significantly reduces the risk for accidental chemical burn to the operator’s eyes, skin, and mucous membranes.

Other detergent characteristics, such as foam properties, are important considerations in light of the method by which the cleaning solution will be applied to a surface. A moderatetohigh foaming detergent is not desirable when used in an agitated immersion or

CIP application, as both create highshear and thus are prone to foam formation. The result of this is a detergent solution that foams outofprocess or CIP vessels, cavitates pumps, and pro vides inefficient surface coverage when sprayed on the inside of a vessel through a spray ball. Con versely, a high foaming detergent is desirable in a manual application, as this gives the operator a visual indication of where the detergent solution has been applied to the surface.

Some cleaning application technologies exist that are widely used in other industries, but have not taken hold in the pharmaceutical industry. These application methods include:

• Thin film cleaning• Stabilized foam generators• Built, solvated detergents (Generally Recog

nized As Safe [GRAS])

Discussion regarding these application methods are outside of this article and will not be addressed.

The Role of WaterIn general, 9599% of a cleaning solution is

com posed of water. It is important to know the purity level of the water being used for cleaning and sanitizing. In many pharmaceutical applications, the water being used for cleaning and sanitizing is high purity water. However, this is not the case in every application and in these cases, know ing and understanding how the purity level of the process water affects the cleaning process is critical. Some of the factors that can affect the cleaning process include water hardness, pH, metals, salts, and microbial contamination. Refer to Figure 5.

Of the factors listed above, water hardness has the most significant impact on cleaning and sanitizing solutions. Water hardness can be classified as temporary or permanent hardness. Temporary hard ness indicates the presence of bicarbonates of mag nesium or calcium. Both of these compounds are readily water soluble and can be present at high levels. When heated, these compounds react to form the carbonate salts, which are water insoluble. Permanent hardness refers to a condition where the chloride or sulfate salts of magnesium and/or calcium are present in the water. These compounds are also very water soluble, but are unaffected by temperature.

32


Mark Altier

Both temporary and permanent hardness cause problems in alkaline solutions, as they both precipitate in high pH solutions and cause scaling on equipment surfaces. Water hardness is responsible for scaling, film formation, excessive detergent consumption, and formation of precipitate. Water hardness can be addressed by installing a water softening system, or by using a detergent that is formulated to handle hard water.

Environmental FactorsMany pharmaceutical plants have some type of

effluent restrictions mandated by local municipalities, or by the plant’s internal effluent treatment facility. Common factors that must be considered are pH, phosphate levels, Biological Oxygen De mand (BOD) or Chemical Oxygen Demand (COD) loading, Total Organic Carbon (TOC) levels, and solids levels. In many cases, the correct choice of detergent can help reduce the impact on components of the effluent stream that are a concern. For example, if phos phates are a concern, a detergent that contains low levels of phosphate can be used. Another example is a situation where the pH of the effluent must not exceed 10 and

must not fall below four. If a strong acid or alkaline detergent is used, the pH restrictions could be violated. In this case, choosing a neutral, mildly alkaline or mildly acidic detergent may be the solution. However, in some cases, a strongly acidic or alkaline detergent might be re quired to effectively re move the potential contaminant from equipment surfaces. If a strong alkaline detergent is re quired, the cleaning cycle could be designed to include an acid rinse. The acid rinse will help reduce the amount of rinse water re quired to neutralize residual alkalinity in the system, will help remove any inorganic residues, and can be captured and mixed with the alkaline wash water to neutralize.

In general, detergents will have the greatest im pact on pH and phosphate levels. Relative to the residue load, detergents generally have little im pact on BOD, COD, TOC, or solids levels.

If effluent restrictions exist, these should be ad dressed in the early stages of the development of a cleaning program to avoid compounded problems later on when the cleaning protocol is implemented.

At this point, five key factors that should be considered when selecting a detergent to be used as a part of a validated cleaning program have been discussed. Once these factors are addressed and an appropriate detergent chemistry is identified, laboratory testing should be done to verify that the chemistry is effective against the potential contaminant. Other cleaning parameters such as cleaning time, temperature, and concentration can be evaluated in the laboratory as well.

Laboratory Testing

Cleaning studies conducted in the laboratory can be designed to closely mimic the actual application method, such as a CIP system, or they can be

33

Figure 5typical water impurities that can

impact a cleaning Processcomponent chemical Problem Formula caused

Barium Sulfate BaSO4 ScaleCarbon Dioxide CO2 CorrosionCalcium Bicarbonate Ca(HCO3)2 Scale and CorrosionCalcium Sulfate CaSO4 Scale and CorrosionIron Fe ScaleManganese Mn ScaleMagnesium Bicarbonate Mg(HCO3)2 ScaleMagnesium Chloride MgCl2 Scale and CorrosionMagnesium Sulfate MgSO4 Scale and CorrosionOxygen O2 CorrosionSodium Chloride NaCl CorrosionSilica Si ScaleSuspended Solids r Deposit and Corrosion

Figure 6water hardness

(reported as caco3) ratinghardness Grains Parts Per Per Gallon million (PPm)

Soft 0 – 3.5 0 – 60Moderately Hard 3.5 – 7.0 60 – 120Hard 7.0 – 10.5 120 – 180Very Hard >10.5 >180


Mark Altier

de signed to stress the system to differentiate between similar cleaning chemistries. An example of the latter is a designed study that removes all mechanical action from the system, forcing the chemistry type and concentration, thermal energy, and contact time to act on the residue. This ap proach is especially effective in differentiating between similar chemi stries that appear to be equally effective when ap plied using some type of mechanical action.

An important component of designed cleaning studies is the preparation of the residue being tested.

Typically, the residue is applied to a 304 or 316 stainless steel coupon and the treated coupon is then subjected to the cleaning solution. The application of the residue to the coupon is critical to obtain results that can be directly applied to the actual system in the plant. For example, a manufacturing process may involve a heating step that causes some of the finished product to “bake” onto a vessel side wall. To obtain re sults that are ap plicable to this situation, the residue should be applied to the coupon surface, heated, and then allowed to bake for an equivalent amount of time as is experienced in the actual process. If this is not done, the results of the study will have little relevance to the development of a cleaning program aimed at removing a baked on residue from equipment surfaces.

Prior to implementing any cleaning studies, a set of success criteria must be established. Once the cleaning studies have been completed, quantitative measurements against the success criteria should be made. Based on the results of this work, a final detergent chemistry recommendation is derived. Ideally, the detergent chemistry should meet or exceed all established success criteria.

The end result of the laboratory work will be a scientifically sound recommendation of detergent chemistry and other important cleaning parameters such as cleaning time, temperature, and concentration. This is the basis for the overall cleaning program that will be tested at the production facility. The importance of performing preliminary laboratory testing is that it provides a sound, scientific rationale of why the selected chemistry is appropriate for the cleaning application.

Plant Optimization

Once a cleaning chemistry has been identified and verified in the laboratory and other cleaning parameters such as cleaning time, temperature and concentration have been established, testing and optimization must be carried out at the production plant. Initial optimization and testing is usually done on a pilot scale, prior to scaling up. The re sults of the laboratory cleaning studies should be used as a guide or a starting point for the optimization process at the plant site.

Conclusion

Process cleaning is an integral component of any pharmaceutical process. The five key factors that must be addressed to help identify a detergent when developing a cleaning program have been defined and discussed. The interaction of these factors with each other and with the development of a cleaning program must be understood. Lab oratory testing is critical for documenting the ap propriateness of the detergent selection for the cleaning application. Plant optimization is a final critical step prior to starting the validation process at the production facility. When these steps are taken, a complete, scientifically sound approach to the development of a cleaning program can be documented. o

About the AuthorMark Altier is a Principal Chemical Engineer for Ecolab Inc., where he manages their pharmaceu-tical and cosmetic programs. Mark has worked for Ecolab for seven years and has held positions in quality assurance, process engineering, and re search and development. He can be reached at 651-306-5876, by fax at 651-552-4899, or by e-mail at [email protected].

34

Cleaning validations are very difficult to perform. They can be made easier if an

ap propriate method for analyzing the samples is used. The method used should be based on the previously established residue limits of the active and cleaning agents. There are many choices of an alyt i cal techniques that can potentially be used. This article will de scribe various analytical technologies avail able for use, particularly for cleaning agent residues. Ref er ences are provided to guide the reader to more indepth information.

Cleaning validation in the pharmaceutical industry is of critical im portance.1,2,3 There are many analytical techniques available that can be used in cleaning validations.4 The choice of the technique used in analyzing a particular sample is very important in cleaning validation. The technique must be appropriate for measuring the analyte at and below the acceptance residue limit. Today’s analytical chemist has a wide variety of techniques available for use. These choices in clude specific and nonspecific methods. Many methods are complementary to each other. The pros

and cons of each technique will be ex am ined. Validating the methods will be discussed, as well. The references included with this paper can be used to provide more indepth information to the reader and act as guides to the available literature.

Choosing the appropriate analytical tool depends on a variety of factors.5,6 The most important factor is determining what species or parameter is being measured.7 Is it an or ganic compound or inorganic compound? The next question is mea sure ment. How is this compound going to be measured? Is it going to be swabbed from a surface or determined from a rinse water sample? If it is going to be swabbed from a surface, where will this swabbing oc cur? Another im portant factor in choosing an analytical tool is establishing the limits of the residue. The limit should always be

established prior to selecting the analytical tool.8,9 The limits should not be established solely based on detection limits of a particular method. Yet, another important factor in choosing an analytical tool is whether or not the method can be validated. If the method can’t be validated, then another technique


Analyzing Cleaning Validation Samples:

What Method? By Herbert J. Kaiser, Ph.D.

and Maria Minowitz, M.L.S. STERIS Corporation

v

}This article will describe various

analytical technologies

available for use, particularly for

cleaning agent res-idues. References are provided to

guide the reader to more in-depth information.~


Herbert J. Kaiser, Ph.D. & Maria Minowitz, M.L.S.

needs to be chosen.Sampling Technique

The sampling technique plays a large role in determining which analytical technique to use. Some techniques are more applicable for swab samples, and other techniques are more applicable for rinse water sampling. The acceptable sampling techniques include direct surface sampling (swab) and rinse water samples.10 The rinse water sample is a direct measure of potential contaminants, but the analysis should not just be a compendial test for water. Rinse water analyses should be directed toward responses peculiar to the possible contaminants. A questionable form of sampling is placebo sampling. The placebo method sampling is when the product, not containing the active ingredient, is processed in the specific piece of equipment. This is analyzed for any active that may have been picked up from the equipment. A problem with placebos is the potential lack of uniformity. The contaminant may not be evenly distributed throughout the placebo. Another problem is the analytical power of the tools that are used to analyze the samples. The residue levels may be extremely low if in fact the contaminant is evenly distributed throughout the sample. The use of placebos is only acceptable if used with swab or rinse water data. Therefore, placebos are generally not used because of the additional work involved.

Another important factor to consider in choosing an analytical method is the type of residue being analyzed. Residues can be drug actives, formulation components, cleaning agents, organic, inorganic, water soluble, water insoluble, particulate, microbial, and/or endotoxins. If the residue being detected is a drug active, and the method used for detection is the same method that is used for quality control purposes of the final formula, it must be established that the active has not changed its chemical nature during the cleaning process. That is, it must be established that the active is still detectable and quantifiable using the analytical method. This can easily be established by performing forced degradation studies. Exposing the active to the cleaning compound at an elevated temperature and then analyzing that sample will help determine the compatibility of the cleaner with the active. If the active has indeed changed its chemical nature during the cleaning process, a new technique

will need to be established for its analysis.Limit of Detection and Quantitation

Before choosing a method, some definitions need to be established. The Limit of Detection (LOD) is the lowest amount of a compound that can be detected. The Limit of Quantitation (LOQ) is defined as the lowest amount of a compound that can be quantified. The LOD is usually lower than the LOQ, but is never higher. The LOD should never be used to establish residue acceptance limits. The residue ac cep tance limit should be well above the LOQ so that it can be accurately quantitated.

Specific and Nonspecific MethodsA specific method is a method that detects a

unique compound in the presence of potential contaminants. Some examples of specific methods are High Performance Liquid Chromatography (HPLC), ion chromatography, atomic absorption, inductively coupled plasma, capillary electrophoresis, and other chromatographic methods. It should be noted that HPLC is not inherently specific. What is meant is that the conditions in an HPLC measurement can usually be adjusted to separate out known potential contaminants.

Nonspecific methods are those methods that detect any compound that produces a certain response. Some examples of nonspecific methods are Total Organic Carbon (TOC), pH, titrations, and conductivity. A very interesting and sensitive nonspecific technique is dynamic contact angle.11 Titrations may be specific for acids or bases, but they are not specific for particular acids or bases. There are, however, specific titrations for classes of surfactants.12

InterferencesA good nonspecific strategy that could be fol

lowed is to first identify possible interferences. These interferences can be either positive or negative. The nonspecific property is then measured, and the residue is calculated as if all of the measured property is due to that residue. For example, if the cleaning agent was the analyte and TOC was the method used, all of the TOC would be assumed to have come from the cleaning agent and calculated as such. This would then provide a worstcase upperlimit value.

There are many possible sources of interferences. Cleaning agents and compounds can be a source of

36



interferences, for example. Active agents and their byproducts, water system components, maintenance materials, and the atmosphere can all be sources, as well as people, if samples are not handled properly. The materials used to perform the analytical method can also be a source of interference. For example, if a swab that has a high TOC value is used to sample, it could increase the level of TOC detected.

For specific methods, there should be no interference if the method is properly designed. Again, it should be stressed that the method must be able to follow the analyte after exposure to the cleaning en vironment. It is necessary to establish that the cleaning environment or the cleaning process does not change the analyte. For nonspecific methods (which measure a nonspecific property), any compound with the property that is introduced into the sample will interfere. For example, if the method being used is TOC, atmospheric carbon that may enter the sample could cause interference. With all nonspecific methods, there is a need to identify potential sources of interference.

n High Performance Liquid ChromatographyThe first technique that will be discussed is HPLC.

Almost every pharmaceutical company has an HPLC instrument. HPLCs utilize a variety of detectors. These include ultraviolet (UV), fluorescence, electrochemical, refractive index, conductivity, evaporative light scattering, and many others. The ultraviolet de tector is by far the most common. However, Evaporative Light Scattering Detection (ELSD) may be the most appropriate detector for cleaning agents. We will discuss the use of both UV and ELSD detectors in depth.

• Ultraviolet DetectorsThere are many advantages of using UV detec

tors. Many compounds have chromophores and therefore, they can be easily detected by UV. Many in struments are equipped with diode array spectral capabilities. This allows for easy detection of impurities or potential contaminants within peaks. Ultraviolet detection usually requires no additional reagents or post column or precolumn reactions. UV detectors are not harmful to the sample, if that is important. They are generally inexpensive and readily available. Also, molar absorptivities are generally not affected by temperature and therefore, there

is no need for heating or cooling the detector.While there are many advantages of UV detec

tors, there are also some significant disadvantages. UV detectors cannot detect all types of compounds and therefore are not considered to be universal. All compounds do not have chromophores. This is particularly true of surfactants that are used in the pharmaceutical industry. Dirty cells, air bubbles, and the use of gradients can affect baseline drift and detection capability. The limits of detection can be higher than other detector types due to background interferences.

• Evaporative Light Scattering DetectionIn ELSD, the compound is separated on an HPLC

column as usual, and then enters a nebulizer that is combined with a gas stream and passed through a heated column. The heated column evaporates the mobile phase leaving the solid analyte in the column. The solid analyte then passes through a detector that consists of a laser or light source. The laser or light source is scattered when it hits the solid analyte. The detector then picks up this scattering.

There are many advantages associated with evaporative light scattering detectors. ELSD is claimed to be universal. It is called universal because it can detect any type of compound. ELSDs are simple, versatile, and rugged in use. Since it is a mass detector, all compounds produce similar responses. Additionally, there is no baseline drift due to mobile phase effects.

There are two primary disadvantages of ELSD. First, there is a very limited choice of buffer salts that can be used. Recall that the mobile phase is evaporated or removed, leaving the analyte. Any buffers that will not evaporate will also produce solid particles that will then be detected and cause interferences. The second disadvantage is that the nebulizer and detector must produce consistent particle sizes. This requires careful cleaning and monitoring of the nebulizer.

Actives and DetergentThere are many types of residues that can be ana

lyzed using HPLC techniques. These include both actives and detergent residues. When dealing with detergent residues, it is important to identify what is being analyzed: surfactant, builder components,

37



chelating agents, etc. The separation and quantitation of surfactants at low levels is difficult, at best. Industry literature is full of references for surfactant analyses using HPLC. The vast majority of techniques described in the literature are for the determination of surfactants in concentrated products.13,14 There fore, the limits of quantitation and the limits of detection are rather high. There are also references for the analysis of surfactants related to the environment.15,16 In environmental analysis, the sample is preconcentrated so that the limits of quantitation are very low. The preconcentration can be up to one thousand fold.

Suggested ReadingAuthors Lin, et. al., compared

the analysis of anionic, cationic, and amphoteric surfactants containing ndodecyl groups using HPLC and capillary electrophoresis.17 They found that HPLC was best for all classes of surfactants, especially for formulated surfactants. Authors Carrer, et. al., utilized ELSD for amphoteric type surfactants.18 Amphoteric surfactants are a class of surfactants that display cationic behavior in an acidic solution and anionic behavior in an alkaline solution. The lowest calibration standard that they utilized was 50 ppm, but they probably could have gone much lower. Authors Guerro, et. al., obtained a limit of quantitation of 0.49 ppm for alkyl polyethylene glycol ethers using ELSD.19

n Capillary ElectrophoresisAn interesting method of analysis is Capillary

Electrophoresis (CE). There are many different types of CE. Capillary Zone Electrophoresis (CZE) is by far the most common. CE instrumentation is fairly simple, consisting of a high voltage source, a capillary, and a detector. The high voltage source is used to apply a potential across two solutions. One of the solutions contains the analyte, and the potential ap plied to the solutions causes the analyte to migrate through the capillary, through the detector, and into the other solution. The column or capillary is typically composed of fused silica with a polyimide coating. The diameter of the capillary is typically 2575µm in diameter. The capillary has a polyimide

coating simply to make it more rugged. All common detection techniques (UV, fluorescence, etc.) can be used in capillary electrophoresis detection. The capillary itself serves as the detector cell. A small portion of the polyimide coating is scraped off prior to use, and the bare portion of the capillary is placed in the light path. This detection is different from that seen in HPLC because the detection occurs while the separation is taking place, rather than after separation has been completed. Using a Zcell can increase the sensitivity of the technique. This is accomplished by

using a special accessory that bends the capillary, causing the source radiation to penetrate lengthwise through the capillary rather than a crosssectional sampling. This, in effect, increases the path length of the cell. The Zcell can be used in all types of CE where UV detection is used.

CE can be used for many different types of analyses. Surfactants can be determined quite readily using this technique.20,21 However, detection limits typically are higher than with HPLC. This can be overcome by preconcentrating the samples on the capillary itself. A voltage is applied to the capillary in a manner that allows the compounds to collect at one end of the capillary without flowing through to the detector. An advantage that capillary electrophoresis holds over HPLC is the ease with which indirect detection can take place. Indirect detection is where a highly UVabsorbing material is included in the mobile phase. As the analyte is eluted or travels along the capillary through the detector, a negative peak is seen for the analyte. This typically is done for compounds that display low UV absorption. In addition to being useful for the analysis of surfactants, capillary electrophoresis can be used to analyze organic acids, inorganics,22 and trace drug residues.23

Suggested Reading

38

}The TOC is then computed by subtracting the inorganic carbon

concentration from the total carbon concentration

of the sample.~



Vogt, et. al., provided a good overview of the separation of cationic, anionic, and nonionic surfactants using capillary electrophoresis.24 They indicated that one can easily adjust the parameters of the separation to coelute or separate oligomers. Coelution of the oligomers increased the sensitivity at the ex pense of increasing the potential for coeluting positive interferences. Direct UV detection could be used for UVabsorbing materials and indirect or non UV absorbing materials.

Heinig, et. al., utilized micellar electrokinetic capillary chromatography for the separation of nonionic alkylphenol polyoxyethylene type surfactants.25 However, the use of this method was limited because of insufficient peak resolution and relatively low detection sensitivity. Heinig, et. al., also compared HPLC and CE analyses of surfactants.26 The surfactant types they studied were linear alkylbenzenesulfonates, nonylphenolpolyethoxylates, cetylpyridinium chloride, and alkylsulfonates. For the CE analyses, they utilized UV detection either in the direct or indirect modes, depending on the nature of the surfactant. For the HPLC analyses, they utilized either direct UV detection or conductivity detection. An ionic surfactant samples were preconcentrated one thousand fold through the use of solid phase extraction. This allowed for detection limits in the parts per billion range to be obtained.

Kelly, et. al., utilized CE with indirect detection to determine sodium dodecylsulfate concentrations.27 They also indicated that it is important to look at the absorption of the surfactants onto filters if the samples are indeed filtered prior to analysis. This is most important in dilute solutions. Filtering large volumes of sample can minimize this. Again, appropriate studies need to be done to determine if this indeed is a problem.

Altria, et. al., examined the use of CE in the analysis of sodium dodecylbenzenesulphonate.28 They ob tained a limit of quantitation of 0.6 ppm and a 0.3 ppm limit of detection. They utilized direct UV detection. Shamsi, et. al., utilized CE with indirect de tection for the determination of cationic and anionic surfactants.29 The authors obtained limits of detection of 0.25 and 0.5 ppm, respectively. Heinig, et. al., also utilized CE in the analysis of cationic surfactants using indirect UV detection.30 They compared this with HPLC. They obtained a limit of quantitation for

CE of 4.0 ppm; and for HPLC, they obtained a limit of quantitation of 5.0 ppm.n Total Organic Carbon

TOC is used widely in the pharmaceutical industry.31,32,33 The TOC is determined by the oxidation of an organic compound into carbon dioxide. This oxidation can occur through a number of mechanisms depending on the instrument being used. Some typical methods are persulfate, persulfate/UV oxidation, and direct combustion. The carbon dioxide that is produced from these oxidations is either measured using conductivity or infrared techniques. In stru ments generally measure the inorganic carbon content of a sample. The inorganic carbon consists of carbon dioxide, bicarbonate, and carbonate. They then determine the total carbon content of the sample. The TOC is then computed by subtracting the inorganic carbon concentration from the total carbon concentration of the sample.

There are two primary advantages associated with TOC. The first is that it does not take long to develop a method. There are not a lot of variables in the actual analysis. The second advantage is that it is relatively quick. A third potential advantage (which can also be a disadvantage) is that it will detect and analyze any compound containing carbon.

As with most techniques, there are disadvantages in using TOC. A significant disadvantage is that the compound or the analyte must be water soluble. This does not mean that the compound must be soluble in the hundreds of parts per million range but soluble in the low parts per million range. Another disadvantage is that organic solvents cannot be used. If organic solvents were used, the TOC of the solvents would be measured instead of the residue. There are also many sources of contamination that can occur using TOC. These sources can include the atmosphere, the swab it self, personnel, and many other sources. Methods de veloped using TOC should be written to include controls and blanks to identify or account for possible contamination. For example, a common source of contamination is the technique used to cut the handles of the swabs so that they fit into the TOC vials. Many times, the scissors or utensils are not clean enough for TOC use. This introduces contamination into the sampling vial when the swab is cut.

Excipients

39



Some methods/techniques can be used in certain situations to complement each other. Examples in clude TOC and HPLC. Consider the case of a drug in the presence of excipients. The excipients are very soluble in water while the drug active has ex tremely low solubility in water. The excipients con tribute to the TOC values because they are very soluble in water; however, the drug active does not show up in the TOC analysis. An HPLC analysis is performed to monitor the loss of the drug. The ex ci pients are removed much faster from a surface during cleaning than the drug active is removed. In this case, TOC analysis is not a good standalone method. It is, however, a good complement for the HPLC assay. The TOC analysis enables the analyst to see what water soluble matter is left behind, if any.

Suggested ReadingGuazzaroni, et. al., examined the use of total

or ganic carbon for the analysis of detergents, endotoxins, biological media, and polyethylene glycol.34 For detergents, they were able to obtain a 0.7 ppm limit of quantitation. Endotoxins were found to have a 0.2 ppm limit of quantitation. The biological media produced a total organic carbon limit of quantitation of 20.3 ppm; and the polyethylene glycol produced a 0.5 ppm limit of quantitation. They examined swab and rinse water recoveries. They were able to obtain 78101 percent recoveries utilizing swabs, and 93 percent or better for rinse water recoveries.

There are many examples in the literature that utilize ion chromatography as the method for analysis of surfactants.35 The surfactants have to be charged in order to be analyzed using ion chromatography, that is, only anionic or cationic surfactants can be detected. Pan, et. al., recorded limits of quantitation down to 0.5 ppm for linear alkane sulfates and sulfonates.36 Takeda, et. al., recorded a limit of quantitation of 0.1 ppm for dodecyl alkyl sulfates.37 Nair, et. al., separated different sulfate, sulfonate, and cationic type surfactants using ion chromatography with suppressed conductivity detection.38 They reported detection limits at less than 1.0 ppm.

n Ion ChromatographyIn addition to its use for surfactants, ion chro

matography can be used for the analysis of inorganics and other organic compounds present in

cleaners.39,40,41 Most cleaners contain sodium and/or potassium. The ion chromatography detection technique of suppressed conductivity is more sensitive to potassium than it is to sodium. Very low levels of cleaning agent can be detected using this technique. This assumes that the rinse water used contains no potassium. Ionizable organic acids are also readily quantitated using ion chromatography. This includes chelating agents that are often found in cleaning compounds.

Suggested ReadingIn determining surfactants, an excellent review

concerning their analysis was done by Vogt, et. al..42 They compared the use of HPLC, CE, ion chromatography, Liquid ChromatographyMass Spectroscopy (LCMS) and Gas ChromatographyMass Spectro scopy (GCMS). They also discussed preconcentration of the samples. They compared the use of solid phase extraction, super critical fluid extraction, Soxhlet extraction, and steam distillation as means of preconcentrating samples. They found, by far, that the best method was solid phase extractions for the preconcentration of surfactants. They also examined the use of titrimetric methods of analysis for surfactants. For detecting anionics, substances like methylene blue, pyridinium azo, and triphenylmethane dye was used to complex the surfactants prior to photometric determination. Non ionics were determined indirectly by forming a cationic complex with barium. This complex was then precipitated by bismuth tetraiodide ion in acidic acid. The bismuth was then quantified by potentiometric titrations. Cationics were complexed with anionic dyes such as disulfine blue.

Theile, et. al., brought up an excellent point that surfactants tend to concentrate at interfaces.43 This can be a problem in extremely dilute solutions of surfactants. The surfactants can collect at the surface of the containers that they are stored in. This may cause errors in analysis. Proper controls in studies should be done to determine if this is a problem. The authors indicated that preconcentration was re quired to determine very low levels of surfactant. Solid phase extraction was the best method for this. They were also able to obtain detection limits for linear alkylbenzenesulfonates of 2.0 ppb with fluorescence detection and 10.0 ppb using HPLC with

40



UV detection after preconcentration.n ThinLayer Chromatography

There are many examples in the literature for the use of ThinLayer Chromatography (TLC) for the qualitative determination of surfactants.44,45 Henrich described the TLC of over 150 surfactants in six different TLC systems.46 This was excellent for identification of the surfactants, but the author did not attempt to quantify the surfactants. Buschmann and Kruse combined diffuse reflection infrared spectroscopy and TLC, along with SIMS and TLC for surfactant identification.47 Although these techniques are tedious and timeconsuming, there is no doubt that these methods could be developed into quantitative analyses. Novakovic has used high performance TLC for two generic drugs.48

Other TechniquesOther excellent techniques for inorganic con

taminants, and in some cases actives, are Atomic Absorption (AA)49 and Inductively Coupled Plasma (ICP) atomic emission. These techniques can detect inorganic contaminants down to extremely low levels. Inorganic contaminants in a system are often ignored. These can come from rouge that forms in Water for Injection (WFI) systems. They can also come from the detergent utilized in cleaning the equipment.

n FourierTransform Infrared SpectroscopyFourierTransform Infrared (FTIR) spectroscopy

is never used as a standalone method for analyzing residues on equipment. This is because of the lack of portability of FTIR equipment and the semiquantitative nature of the reflectance techniques used for these types of analyses. However, it is very useful in performing screening studies and in evaluating po tential cleaning agents. This is done by soiling standard coupons with the cleaning agent, allowing them to dry, and performing static rinsing studies. These types of studies can indicate whether or not the cleaning agent is readily removed from surfaces. The height or area of a particular peak is measured versus the concentration of the standard coupon.

n BioluminescenceBioluminescence is quite useful for biologicals.

This type of analysis usually uses Adenosine Tri

phosphate (ATP) bioluminescence.50 This is based on the reaction of ATP with Luciferin/Luciferase. This technique is often used in biopharmaceutical facilities. It has extremely high sensitivity and a very high reproducibility. In many cases, the instruments can be used at the equipment site. This technique utilizes swabs for surface analyses.

n Optically Stimulated Electron EmissionIn some cases, a company’s established limits of

residue are so low that they cannot be detected by conventional methods. A very sensitive method that may be applicable is Optically Stimulated Electron Emission (OSEE).51 The instrumentation for OSEE is fairly portable, and can be readily taken to tank side for analysis. The technique uses a probe that is placed against a surface, and a UV source illuminates and activates the surface. When some surfaces are exposed to UV light at certain wavelengths, electrons are emitted from the surface. The instrument measures the current that is produced. If even small amounts of residues are present on the surface, the current will be affected. The current can be affected either in a positive or negative way depending on the nature of the residue. This is an extremely sensitive technique. It can be used in either a qualitative or quantitative manner.

n Portable Mass SpectrometerFor those companies that require ultrasensitive

measurements and identification of the residues, a technique has been developed – Lawrence Livermore National Laboratories has developed a portable mass spectrometer.52 The unit consists of a gun portion of the instrument that is connected with cables to vacuum pumps. The gun portion is held against the surface to be analyzed. A seal is formed, and the surface is heated to volatilize any compounds that are present. This instrument is used not only to measure how much of something is present, but also what that something is. This piece of equipment has been utilized in the aerospace industry. One drawback of the portable mass spectrometer is that it requires relatively flat surfaces. However, they are currently working on adaptors to be used on nonflat surfaces.

Additional Techniques

41



In the biopharmaceutical industry, a wide variety of techniques are utilized.53 These include the EnzymeLinked Immunosorbent Assay (ELISA),54 the Limulus Amoebocyte Lysate (LAL), and a wide variety of protein determinations. These are all contaminant specific assays. For example, the LAL test measures the level of endotoxins present. There is also the anthrone assay that can be used to monitor the levels of carbohydrates on surfaces. These techniques are usually used in combination with TOC.

The nonspecific techniques of pH, conductivity, and titrations can be used throughout all areas of pharmaceutical manufacturing. Ob viously, these techniques are most often utilized in rinse water monitoring. The conductivity and pH of rinse water is typically monitored and compared to the conductivity and pH of the water prior to introduction to the equipment. If acidic or alkaline materials are being measured, titration is a very useful technique. In some cases, titration can be more sensitive than performing TOC analyses. The sample size can be adjusted, and/or the normality of the titrant can be adjusted to increase the sensitivity of the titration.

Method Validation

It is very important to scientifically establish the residue limit prior to choosing the method of analysis. This includes the limit in the analytical sample and the limit in the next product. This will ensure that the method chosen will be able to detect and quantitate the limit chosen. Once the technique for analysis has been chosen, it is very important to validate the method used.5560 The validation of a meth od is very different than the validation of recovery. A validated method is one that is rugged and robust enough to measure the residue limit established. The validation of a recovery is to determine the amount that can be recovered from a surface. Again, it should be stressed that these are two completely different validations.

“Twos” of Validation

A minimum validation requires two different analysts, instruments, columns (if chromatographic method), days, and prepared standards and samples.60 These are the “twos” of method validation. The point of any method validation is to show that the method can be utilized by different analysts and/or laboratories, along with the ability to produce the same results. If a validated method is given to a lab

oratory, that laboratory must revalidate the method for their laboratory. It is not sufficient or accurate to assume that another laboratory’s validation will apply in all laboratories. For example, if a surfactant is being quantitated, typically a low wavelength is used in a UV detector for HPLC. UV detectors vary in their noise levels at these low wavelengths. A detector used in one laboratory may have significantly less noise than a detector in another laboratory. The second laboratory may not be able to detect at the same low level as the first laboratory.

Coupons and SwabsCoupons can be prepared for recovery studies

through the use of aerosol bottles available through laboratory supply companies. A known weight percent of a solution containing the analyte can be sprayed fairly evenly over the surface of a coupon. The coupon can be swabbed using a standard technique. It does not matter how you swab the coupon, as long as the complete surface is covered and that the coupons are swabbed the same way – each and every time. The type of swabs used in recovery studies must be the same as those used in the validation protocol. If this is a simulated rinse procedure, then the coupons are rinsed and the rinse water is analyzed.

42

}For those companies that require ultra-sensitive measurements and

identification of the residues, a technique has been developed

– Lawrence Livermore National Laboratories has developed a portable mass spectrometer.~



For swabs, a desorption process is carried out. This can consist of simply shaking the sample vial or using an ultrasonic bath. The samples are then analyzed. Recovery studies are always done below ac ceptance limits in the test solution. This ensures that the limit will be (or can be) measured in the analysis. A recovery of greater than 80 percent is good. If the recovery is greater than 50 percent, it may be acceptable. However, if the recovery is less than 50 percent, questions arise and the source of the poor recovery should be investigated. A possible cause of a poor re covery can be that the residue is being too tightly held by the swab. This can be investigated by spiking a swab with a known amount of residue, allowing it to dry, trying to desorb the res idue, and following up with analysis. If the analyte is held too tightly by the swab, another type of swab material should be investigated. The recovery factor should be included in analytical calculations or in the acceptance limit calculation. It should not be included in both of the calculations.

ContainersThe choice of containers used in the analysis of

samples is very important. It has been shown that, in very dilute solutions, surfactants can adsorb onto the surfaces of sample vials. This will produce artificially low results in the analysis. This, however, typically only occurs in static systems. There is no need to worry about the adsorption of the surfactant on the walls of manufacturing equipment. This is because the agitation that is involved in cleaning removes the surfactants from the surfaces. This is another matter in sample vials. Appropriate spiking studies should be performed to ensure that this phenomenon is not occurring and will not interfere with the analytical method. This includes both HPLC or ion chromatography sample vials, as well as TOC sample vials. This phenomenon is not limited to surfactants. Proteins have been shown to adsorb readily onto glass surfaces. These proteins are much more difficult to remove from surfaces than surfactants.

Specific Versus Nonspecific

The choice of using a specific or nonspecific method can be difficult. If a drug active is highly toxic, a specific method is always recommended.61

Detergents can be quantitated either using specific or nonspecific methods; however, care must be taken in choosing which component is measured. For example, a detergent may contain five percent of a surfactant and 20 percent of another organic ingredient. Assuming equal sensitivities of the analytical methods, the limit of quantitation of the whole detergent system will be lowered by a factor of four if the ingredient present in the greater amount is determined.

If a nonspecific method (i.e., TOC) is used for the same system, a much lower limit of quantitation could be determined simply because there would be a tremendous amount of carbon present in the sample. In addition, if detergent systems are combined, such as in the case of adding a detergent additive to another product, the choice of a specific method would be made even more difficult. The question would be, “Which detergent do I determine?” A disadvantage of using a nonspecific method for the entire cleaning validation analysis is that, if there is a failure in the future, it would not be known where the failure originally occurred. The failure could be due to the active, excipients, detergent system, or even an unknown source.

Conclusion

There are many different analytical techniques available that can be used to detect residues. These range from simple titrations to more complex LCMS. The choice of technique should be based on what equipment is available, the type of residue, and the scientifically established residue limit. It is important for an analytical chemist to keep abreast of the literature and what techniques are available. There are techniques available that will analyze any residue at any level. At the end of the day, however, it is always wise to choose the simplest technique that can be used to reach the desired goal. o

About the AuthorsHerbert J. Kaiser, Ph.D. is Manager – Hard Surface Products at STERIS Corporation. He has 18 years of experience in cleaning and surface technologies, which includes developing products and methods for the cleaning and analyzing of a wide variety of

43



surfaces. Dr. Kaiser has developed a wide variety of products for the healthcare, industrial, and pharma-ceutical markets. He is the sole inventor listed in five United States Patents for various industrial treat-ment schemes. Dr. Kaiser received his B.A. degree from St. Mary’s University in San Antonio, Texas, his M.S.(R) from St. Louis University, and his Ph.D. from the Un iversity of Missouri. He is a member of the American Chemical Society and the Association for the Ad vancement of Medical Instrumentation. Dr. Kaiser can be reached by phone at 314-290-4725, by fax at 314-290-4650, or e-mail at [email protected].

Maria Minowitz, M.L.S., Information Associate at STERIS Corporation, has 10 years of experience in corporate research and development librarianship. She has been responsible for information manage-ment in the disciplines of chemistry, medicine, and engineering. Minowitz received her A.B. degree from St. Louis University and an M.L.S. from the University of Missouri-Columbia. She is a member of the Special Libraries Association, Midcontinental Chapter of the Medical Library Association, and the St. Louis Medical Librarians.

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41. Masters, M. B. “Use of Ion Chromatography in Surfactant Analysis.” Analytical Processing. London. Vol. 22(5). 1985. pp. 146147.

42. Vogt, C. and Heinig, K. “Trace Analysis of Surfactants Using Chromatographic and Electrophoretic Techniques.” Fresenius Journal of Analytical Chemistry. Vol. 363. 1999. pp. 612618.

43. Theile, B., Günther, K., Schwuger, M. “Trace Analysis of Surfactants in Environmental Matrices.” Tenside Surfactants and Detergents. Vol. 36(1). 1999. pp. 812, 1418.

44. Bosdorf, V., Krüßmann, H. “Analysis of Detergents and Cleaning Agents with ThinLayer Chromatography.” Fourth World Surfactants Congressional Asociacion Espanola de Productores de Sustancias para Aplicaciones Tensioactivas. Barcelona, Spain. Vol. 4. 1996. pp. 9295.

45. Read, H. “Surfactant Analysis Using HPTLC and the Latroscan.” Proceedings of the International Symposium on In strumental High Performance ThinLayer Chromatography. Third Edition. Ed. Kaiser, R. Institute of Chromatography. Bad Duerkheim. Federal Republic of Germany. 1985. pp. 157171.

46. Henrich, L. H. “Separation and Identification of Surfactants in Commercial Cleaners.” Journal of Planar Chromatography — Mod. TLC. Vol. 5(2). 1992. pp. 103117.

47. Buschmann, N., Kruse, A. “InSitu TLCIR and TLCSIMS: Powerful Tools for the Analysis of Surfactants.” Comunicaciones Presentadias a las Jornadas del Comite Espanolde la Detergenteia. Vol. 24. 1993. pp. 457468.

48. Novakovic, J. “Validation of a High Performance ThinLayer Chromatographic Method for Trace Analysis for Some Generic Drugs Affecting Gastrointestinal Function.” Journal of AOAC International. Vol. 83(6). 2000. pp. 15071516.

49. Raghavan, R., Mulligan, J. A. “LowLevel (PPB) De ter min a tion of Cisplatin in Cleaning Validation (Rinse Water) Samples. I. An Atomic Absorption Spectrophotometric Technique.” Drug Device Industry Pharmaceutical. Vol. 26(4). 2000. pp. 423428.

50. Davidson, C. A., Griffith, C. J., Peters, A. C., Fielding, L. M. “Evaluation of Two Methods for Evaluating Surface Cleanliness – ATP Bioluminescence and Traditional Hygiene Swabbing.” Luminescence. Vol. 14. 1999. pp. 3338.

51. Chawla, M. K. “Is It Clean?” Precision Cleaning. Vol. 8(6). 2000. pp. 36,38.

52. Meltzer, M., Koester, C., Steffani, C. “Criteria Evaluation for Cleanliness Phase 0.” Lawrence Livermore National Lab oratory. UCRLCR133199, PPG99003. 1999.

53. Inampudi, P., Lombardo, S., Ruezinsky, G., et. al. “An

Integrated Approach for Validating Cleaning Procedures in Biopharmaceutical Manufacturing Facilities.” Annals of the New York Academy of Sciences. Vol. 782. 1996. pp. 363374.

54. Rowell, F. J., Miao, Z. F., Neeve, R. N. “Pharmaceutical Analysis Nearer the Sampling Point, Use of Simple, Rapid OnSite Immunoassays for Cleaning Validation, Health and Safety, and Environmental Release Applications.” Journal of Pharma cy and Pharmacology. Vol. 50. 1998. p. 47.

55. Seno, S., Ohtake, S., Kohno, H. “Analytical Validation in Prac tice at a Quality Control Laboratory in the Japanese Pharma ceuti cal Market.” Accreditation and Quality Assur ance. 1997, 2(3), 140145.

56. Kirsch, R. B. “Validation of Analytical Methods Used in Pharmaceutical Cleaning Assessment and Validation.” Pharmaceutical Technology. 1998 (Analytical Validation Supplement). pp. 4046.

57. Brittain, H. G. “Validation of Nonchromatographic Analytical Methodology.” Pharmaceutical Technology. Vol. 22(3). 1998. pp. 8290.

58. Ciurczak, E. “Validation of Spectroscopic Methods in Pharmaceutical Analyses.” Pharmaceutical Technology. Vol. 22(3). 1998. pp. 92102.

59. Swartz, M. E., Krull, I. S. “Validation of Chromatographic Methods.” Pharmaceutical Technology. Vol. 22(3). 1998. pp. 104120.

60. USP 23, United States Pharmacopoeia Convention. Rock ville, Maryland. 1995.

61. Segretario, J., Cook, S. C., Umbles, C. L., et. al. “Validation of Cleaning Procedures for Highly Potent Drugs. II. Bisnafide.” Pharmaceutical Device Technology. Vol. 3(4). 1998. pp. 471476.

45

This paper describes results of monitoring biotech clean rooms and a pharmaceutical

cleanroom equipped with an Electrically Enhanced Fil tration (EEF) system that significantly reduces airborne bioburden in cleanrooms. The EEF High Ef ficiency Particulate Air (HEPA) system traps and kills bacteria and also im proves the filtration performance of a filter me dia by two to three orders of magnitude. In laboratory tests the EEF tech nology has been shown to kill Staphyl o coccus epidermidis and Escherichia coli. These field test results support laboratory testing and show that basically there is no airborne bioburden in both a Class 10 room, with terminal HEPA in addition to the EEF, and in a Class 1000 room that utilizes only the EEF without any terminal HEPA filters. In the case of an old laboratory converted to a cleanroom, direct comparison of the EEF with respect to conventional

HEPA fan filter units (FFUs) was possible. The results showed that at the same flow rate the EEF resulted in significantly lower bioburden as compared to the FFUs.

Background

The fundamental purpose of cleanrooms in the pharmaceutical, medical device, biotechnology, and hospital applications is to control the amount of bioburden due to both internal operations and transport from the air. From a particulate point of view, cleanrooms in

these industries are classified and specified according to the same cleanroom standards (e.g., Federal Stand ard 209E) as in other industries. It is often assumed that the particle (total) concentrations will generally correlate to concentration of viable microorganisms. This may not always be valid. Hence, the concentration of viable organisms is also


Control and Monitoring of Bioburden in Biotech/

Pharmaceutical Cleanrooms

By Raj Jaisinghani Technovation

& Greg Smith Encelle, Inc.

& Gerald Macedo

Med-Pharmex, Inc.

v

}The FDA has specific

requirements and guidelines1 for bio-

burden for various

pharmaceutical operations and

processes.~


Raj Jaisinghani, Greg Smith, & Gerald Macedo

directly measured – both at the work surfaces (or at the process) and in the air.

Cleanrooms in these industries must meet separate standards for bioburden. The FDA has specific requirements and guidelines1 for bioburden for various pharmaceutical operations and processes. Similarly, the United States Pharmacopoeia (USP) and the European Union’s GMP2 guidelines give specific recommended limits for microbial contamination for each class of room. A cleanroom that meets the particle concentration requirements, but does not result in the desired level of bioburden, will clearly be inadequate.

One of the main obstacles in achieving the required bioburden levels is that the measurement of bioburden is time consuming. Typically, bioburden measurement involves sampling, incubation, and counting of colonies. This is a time consuming pro cess and thus “real” time monitoring is not possible. Thus, it is not always possible to relate higher incidents of bioburden to operating events. Recently, however, ultraviolet (UV) fluorescence (cf. Seaver and Eversole3, Pinnick et al.4) technology has made it possible to achieve real time monitoring of particles of biological origin. This technology will find increasing use in the real time monitoring of air in hospitals, cleanrooms, and military nuclear, biological and chemical (NBC) warfare protection systems – as a real time supplement to the standard methods of determining bioburden. As this happens, more attention will be focused on cleanroom contamination control systems – currently mainly mechanical filtration.

One problem with mechanical filters is that under certain conditions common bacteria caught on the filter can start growing on the filters, grow through the media, and start shedding into the room.5,6,7 The wellknown case of the Legionnaire’s outbreak at the veterans convention in Philadelphia has been attributed to this phenomena. In that case the filters were supposedly in a wet state. Generally, it is accepted that bacteria is difficult to grow on clean glass fiber filter media, used in HEPA filters, under normal humidity conditions. However, since the function of these filters is to capture all particulate contamination, filters eventually get dirty. The experiments conducted by Jaisinghani et al.8 show that very little contaminant is needed for growth of Staphylococcus epidermidis and Escherichia coli on HEPA glass filters. In their experiments Jaisinghani et al.8 found that very little of the

applied E. coli survived on the clean glass filter after four hours of airflow, keeping in mind that E. coli is not a hardy organism. Next about one gm of colloidal kaolin was added to the E. coli solution that was to be aerosolized. This time the recovery of E. coli was about 104 – 105 CFU/square inch of the filter media. Similar tests with S. epidermidis recovered a little more S. epidermidisthan with E. coli even without the colloidal kaolin, due to the hardier nature of S. epidermidis. With 1 gm of colloidal kaolin in the 25 ml S. epidermidis solution (in tryptic soy broth) the recovery of S. epidermidis was about 105 – 106 CFU/square inch of filter media. Tests with airflow continuing for seven hours (following the aerosol) did not result in any significant reduction in bacteria recovery. This result suggests that, even in normal environments, bacteria can survive or grow on the filters. As the trend towards using HEPA cleanroom filters for longer periods continues, the possibility of bacterial growth on the filter, and thus the rise in the airborne bioburden, also increases.

EEF Technology

Jaisinghani7 has played a significant role in the development and commercialization of EEF technology. The most recent version (see Figure 1) of this technology7 maintains the filter under an ionizing (as opposed to a simple electrostatic field)

47

Figure 1technovation’s ionizing

eeF technology

Flow

Control Electrode Ionizer

Electrode

Downstream Ground Electrode

H.V. Supply

Ionizing Wires

Filter



field. Another higher intensity ionizing field charges incoming particles, stabilizes the electrical fields, and increases the safety and reliability by ensuring that no spark over can occur towards the filter. This method provides two fundamental benefits:

1. Bacteria are killed as they pass through a first high intensity ionizing field and then killed as they are subjected to continuous ionizing radiation when they are trapped on the filter. This inhibits growth of bacteria on the filter.

2. The same ionizing fields enable penetration re duction by about two to three orders of magnitude.

Since the cost of the additional electrical compo

nents is partially offset by the increase in filtration performance (either higher flow at the same pressure drop and filtration efficiency or lower pressure drop at the same flow and efficiency, as compared to mechanical filtration of the same size) this is a highly cost effective method for the control of bioburden.

Laboratory Evaluation of the EEF

Jaisinghani et al.8 have demonstrated the bactericidal properties of the EEF under laboratory conditions. This study, conducted at Virginia Polytechnic Institute, is summarized in this section.

Experimental MethodsS. epidermidis was grown in Tryptic Soy Broth

(TSB) to a concentration of 3 x 109 colony forming units (CFU)/ml. The culture was lyophilized in Wheaton vials in 5 ml aliquots – 1.5 x 1010 CFU per vial. All vials were stored in a desiccator at 4 – 6ºC.

Pleated 15.24cm by 15.24cm by 5cm (6” x 6” x 2”) deep filters were first coated with colloidal kaolin and TSB using an Aztek airbrush. The airbrush cup was filled with 1g kaolin suspended in 25ml TSB and sprayed onto a filter inside a laminar flow hood and allowed to air dry before being used. The pleated filters were placed in a miniature version of the EEF. One vial of lyophilized S. epidermidis was resuspended with 1 ml of sterile distilled water. A small aliquot of this suspension was serially diluted tenfold to 108 and plated on Columbia Blood Agar (CBA) plates to

confirm the initial viable concentration of bacteria. The rehydrated culture was then sprayed onto the filter using a Meinhard nebulizer, which was placed eight inches from the center of the filter.

A control assay was performed to determine the amount of viable S. epidermidis on the filter, without application of high voltage. The bacteria were sprayed onto the filter as previously described, and the temperature and humidity were monitored every 15 minutes for four hours or seven hours during which the airflow was on. The relative humidity was held between 4555% using a Kaz steam vaporizer. At the end of each control run three pieces of the filter were extracted using a sterilized scalpel and forceps. The pieces of filter were approximately one square inch on the face of the filter, which when unfolded measured approximately 28 square inches of filter material. Filter pieces were removed from the center of the filter, directly above the center, and directly below the center.

The samples were cut into small pieces and placed into 10 ml of sterile phosphate buffered saline (PBS) pH 7.4 in a Nasco WhirlPak bag. The bags were then processed in a Tek Mar Stomacher LabBlender 80 for one minute. Each sample was then serially diluted tenfold to 102, 103, and 104, then spread on CBA plates to determine the number of viable bacteria per sample filter piece.

Similar tests were then conducted by applying high voltage to the EEF. In addition to monitoring the temperature and humidity, the current was also monitored at fifteen minute intervals during the four or seven hour period of airflow with the applied high voltage on.

Results and DiscussionThe results are summarized in Figure 2. In the

absence of any voltage applied to the EEF unit (i.e., control tests), viable bacteria were recovered from one square inch of filter in the range of 1 x 105 CFU to 2 x 106 CFU. Counts greater than about 3 x 106 CFU were too crowded to be accurately counted and were considered to be too numerous to count. When high voltage was applied for four hours, the majority of the bacteria were killed. The kill rate increased with increased voltage or with the first applied field strength (applied voltage divided by the distance of the ionizer wires from the control ground electrode – see Figure 1), V/d1. At a field strength (V/d1) of 4.2 kV/cm, there was no growth after 24 hours of incu

48



bation. After 48 hours, there was either no growth or small (in size and in number) colonies grown. These small colonies were identified as S. epidermidis, and were identical in biochemical profile as the isolate used in the tests. It was concluded that four hours at 4.2 kV/cm (V/d1) did not completely kill the S. epidermidis. If the bacteria were not all killed, some of them were damaged sufficiently so that no growth or very limited growth could occur after 24 hours incubation. When the ionizing time was increased to seven hours, over 99% of the bacteria (as compared to the control) were killed.

When the applied field strength, V/d1, was increased to 4.5 kV/cm or higher, no growth occurred on any of the filter pieces except for one experiment. This exception may have occurred because the starting dose of bacteria for this experiment was three times higher than for the control and up to 10 times higher than for any other experiment. Nonetheless, there were still three to four logs of killing using an applied field strength, V/d1 of 4.5 kV/cm or higher, as compared to the control experiments. It should be noted that, in practice, bacteria caught on the filter are held within the ionizing field for an almost infinite amount of time, thus receiving an almost infinite radiation dosage. Hence, in practice, the killing efficiency should be higher even at lower field strengths. Similar results were obtained using E. coli in a previ

ous study conducted with the EEF at the University of Wisconsin.

Field Results in Cleanrooms

Model 3001B or Model 1001B BIO PLUS® EEFs (Figure 3) manufactured by Technovation Systems, Inc., were used in the cleanroom discussed here.

49

Figure 2eeF Bactericidal test summary using S. epidermidis

Filter incubation eeF exposure eeF Field average comment time time strength colonies

control or hours hours (v/d1) kv/cm #/sq. inch eeF

Control 24.00 0.00 0.00 1.00E+06 No additional growth Control 24.00 0.00 0.00 1.02E+05 After 24 Hours EEF 24.00 4.00 4.64 00.0E+00 100% Killed EEF 24.00 4.00 3.99 3.44E+02 99.93% Killed EEF 24.00 4.00 4.24 0.00E+00 100% Killed EEF 24.00 4.00 4.50 0.00E+00 Some growth EEF 24.00 4.00 4.20 0.00E+00 After 48 Hours EEF 24.00 4.00 4.20 6.26E+03 98.75% Killed EEF 48.00 7.00 4.20 5.44E+02 99.9% Killed EEF 48.00 4.00 4.80 2.16E+02 99.95% Killed EEF 48.00 4.00 4.20 3.51E+03 99.3% Killed

Figure 3model 3001 eeF Filter



Both models have a flow rating of about 3000 scfm without attached ductwork.Comparison to FFU in a Converted Cleanroom

Jaisinghani et al.8 have conducted a field study comparing FFUs to an EEF in a small laboratory converted to a cleanroom. This will be referred to as the “older Encelle cleanroom.” (Encelle, Inc., Greenville, NC.) Encelle had four conventional HEPA fan filter units (FFUs) installed in this tissue culture laboratory, prior to replacing these with one Model 1001 EEF. One model 1001 provides HEPA filtered air at about the same total flow (approx. 4250 m3/h (2500 scfm) in this case) as the four FFUs. This allowed direct evaluation of the effect of EEF on the bioburden in the room, under field conditions. Airborne bioburden in the room was reduced by as much as 75% after switching to the EEF system. The airborne bioburden in the Class 10K room was 0.021 cfu/ft3 (no process) and 0.392 cfu/ft3 (in process) after installation of the EEF filter.

Figure 4 shows the Federal Standard 209E, USP, and European Union recommended airborne bioburden and particulate concentration for various class cleanrooms. Clearly, from a bioburden perspective Encelle’s older Class 10K room performs (at rest) at a level equivalent to a Class 100 room – without incurring the higher cost associated with building a Class 100 room. Most of this benefit should be attributed to the EEF filter system.

New Class 1000 Biotech Tissue Cul ture Cleanroom

Facility DescriptionA 1,300 square foot Class 1,000 cleanroom and

900 square feet of Class 10,000 surrounding space was constructed at Encelle, Inc., Greenville, NC facility. It utilized eight 3001B BIO PLUS® EEF filters. The total

flow rate utilized here (22 fpm average air velocity) was on the lower end of flow normally used in Class 1,000 cleanrooms. This room will simply be referred to, henceforth, as “the Encelle cleanroom.”

All processing and manufacturing conducted within the cleanroom areas are done aseptically. Workers are gowned in sterile coveralls, shoe covers, goggles, or face masks with shields, hair nets, and sterile gloves.

The Class 1K cleanroom and clean Class 10K surrounding zone are cleaned daily with a monthly rotation of sterile chemicals using cleanroom equipment and trained personnel. Disinfectants include Hypochlor®, Process LpH®, Process Vesphene®, and as needed, treatments with a sporekilling agent called SporKlenz®.

Sampling MethodsAn environmental monitoring program has been

designed to establish the standards and limits that are acceptable to the facility management and to regulatory agencies that will audit the manufacturing within the cleanroom environments.

Daily activities for monitoring include temperature and pressure readings. Relative humidity is also reported on sampling days.

Surface samples are collected on a routine basis using only sterile supplies. Surfaces monitored in clude floors, equipment, walls, and ceilings. A five percent sheep’s blood agar plate (three inch diameter) is swabbed with a sterile, moist, cotton swab after sampling various surface areas. Plates are labeled and incubated at 37ºC, with five percent carbon dioxide for 72 hours. Colony forming units that grow are counted and identified using standard microbial techniques.

Particle counting is conducted biweekly or as needed for monitoring during critical processes. A Biotest® APC Plus particle counter measures concentration at particle sizes of 0.3, 0.5, 1.0, and 5.0µm.

50

Figure 4recommendations for airborne Bioburden for various cleanroom classes

class Parameter 209e eu usP

10K 0.5 Um particles #/ft3 <10K <10K10K CFU/ft3 <2.83 <2.51K 0.5 Um particles #/ft3 <1K <1K1K CFU/ft3 NA NA100 0.5 Um particles #/ft3 <100 <100100 CFU/ft3 <0.028 at rest; <0.283 Process <0.1 Process



Data is collected in nearly 200 areas within the filteredair areas. These areas are categorized by process and particle counts are reported to the facilities manager for evaluation and disposition if warranted. Data is downloaded via an RS232 port for digital documentation of these counts.

Microbial air sampling is performed in parallel with particle counting to provide data on airborne viable particulate counts and comply with Federal Standard 209E Cleanliness classes for cleanrooms and clean zones. A Biotest® Centrifugal Air Sampler collects 500 liters of air in each location on sterile tryptic soy agar strips that are designed for this type of sampler. Strips are labeled and incubated similarly to the surface agar plates. Classification and identification are performed using the standards described in the current edition of the USP.

USP standards9 for microbial growth follow in Figure 5.

Results – Particle ConcentrationThe particle concentration measurements are

shown in Figure 6.From the perspective of particle counts alone, the

design Class 1,000 cleanroom is functioning very close

to a Class 100 cleanroom in operation. It should be noted that the cleanroom certified as Class 100 at rest (i.e., without personnel). Similarly, the design Class 10,000 area is functioning as a borderline Class 1,000 in operation. It should be noted again, that the airflow rate used in this Class 1,000 cleanroom (22 fpm) was on the low end for a normal Class 1,000 cleanroom. The high performance of this room can be attributed to the high degree of ceiling coverage (i.e., Airflow is highly distributed throughout the room) which is an inherent feature of the Technovation BIO PLUS® EEF system.

Results – Airborne MicroorganismsFigure 7 shows the results of airborne microor

ganism monitoring in the Class 1,000 cleanroom.The results clearly indicate that, from the perspec

tive of airborne bioburden, the Class 1,000 area is performing at a level that is to be expected for a Class 100 to Class 10 level. Note that (by comparing to the particulate data in Figure 8) the airborne bioburden does not correlate to the particulate concentration. During the months of December and January, the submicron particulate concentration actually went up while the airborne bioburden was reduced.

Figure 8 shows the results of airborne microorganism monitoring in the Class 10,000 cleanroom.

Note again that the bioburden does not relate to the particulate concentration. One probable reason for this is that the bacterium are larger than the size of the submicron particles being monitored. Also note that on the average the Class 10K area performs between a USP Class 100 and a USP Class 10K from a bioburden perspective.

Results – Surface Microorganisms Figure 9 shows the results of surface microorgan

ism monitoring in the Class 1,000 cleanroom.The surface concentrations of bacteria are almost

zero throughout the cleanroom suite. This can be attributed to:

n The cleaning protocols instituted at Encellen The use of the disinfecting compoundsn The low airborne bioburden

Observations from the Encelle Cleanroom Mon-itoring

1. The Encelle cleanroom operates significantly

51

Figure 5usP standard for Bioburden

in cleanrooms class 100 requirements

class 10,000 requirements

class 100,000 requirements

Air 0.1 cfu/ft3

Surface 3 cfu/plate* Gowns 5 cfu/plate

Air 0.5 cfu/ft3

Surface 5 cfu/plate (10 from floor) Gowns 10 cfu/plate

Air 2.5 cfu/ft3

Surface 20/plate Gowns 30/plate

* 2 in2 surface



better than the design classification although the flow rate (average room velocity = 22 fpm) used is at the low end of what is normally used in such a cleanroom. This is due to the higher distribution of flow rate – an inherent feature of the BIO PLUS® filter system.

2. The airborne bioburden in both the Class 1K and 10K areas is lower than what would be expected for such rooms based on USP recommendations. The Class 1K room has the bioburden of what would be expected (based on USP) for a Class 100 room. Coupling this observation to the laboratory studies on the bactericidal properties of the EEF technology and the direct comparison with respect to conventional FFU HEPAs, it may be inferred that

the low airborne bioburden is due to the BIO PLUS® EEF filters.

3. The surface bio contamination is almost non existent in the Class 1K cleanroom. This may be attributed to the good cleaning practices used at Encelle and due to the low airborne bioburden in the suite.

4. The airborne bioburden seems to be lower in the winter months, although the room temperature is held constant at 66ºF. This may be due to lower humidity in the winter months.

New Class 10 Pharmaceutical Cleanroom

Facility DescriptionA 12’x20’ Class 10 cleanroom (including a

52

Figure 6Particle concentration measurements in the encelle

tissue culture laboratory air Particle concentration (per ft3)

11/14/99 11/14/99 12/1/99 12/1/99 12/31/99 12/13/99 1/6/00 1/6/00 1/28/00 12/28/00 size – > 0.3 um 0.5 um 0.3 um 0.5 um 0.3 um 0.5 um 0.3 um 0.5 um 0.3 um 0.5 um class 10,000 Design areas approx. sq. Ft.

Mechanical Corridor 235 1959 506 786 602 1163 903 801 899 852 512

Side Corridor 555 2240 642 4559 1035 3598 762 4058 687 7989 1422

Water Filtration Area 171 3649 2598 4657 2429 11406 4312 8350 4368 6378 1295

Labware Processing 212 343 102 981 719 1233 1798 243 326 618 530

Gowning Room 139 236 87 853 1063 1506 913 2874 2808 957 235

Materials Pass Through 40 257 88 450 539 2461 2463 3887 6064 1451 595

total square Feet – > 1312

Actual Area Classification 482 671 683 1065 1187 1859 1123 2525 1014 765

class 1,000 Design areas

Specialized Cleanrooom 152 158 66 223 145 350 337 313 379 754 237

Formulations Mfg. Area 142 208 24 84 52 415 228 322 307 360 126

Product Testing Area 132 28 5 9 9 43 49 19 74 19 76

Product Finishing Area 127 264 26 129 49 474 139 400 179 472 184

Product Mfg. Area 145 293 83 250 87 534 180 639 698 829 731

Refrigerator/Freezer Storage 212 262 55 233 157 832 692 427 335 659 381

total square Feet – > 910

Actual Area Classification 67 43 52 83 147 271 118 329 172 289



4’x12’ Class 10K gowning room) was constructed at MedPharmex in Pomona, CA using two BIO PLUS® Model 3001B filters with eight terminal 2’x4’ HEPA filters. The Model 3001Bs were used for the 12’x16’ Class 10 inner room. The resultant average room ve locity in the Class 10 area was 24 fpm (4500 scfm). The design specification for the room was Class 100. This airflow was much lower than used in a Class 100 cleanroom – normally, with conventional single terminal HEPAs, at least 40 fpm average room velocity is used in a Class 100 room. However, due to the double HEPA filter system (each Model 3001B powered Airflow through four terminal HEPAs) the cleanroom easily classified as Class 10 as per Federal Standard 209E. This resulted in significant energy savings. The room was validated for bioburden initially and then has been shut down since the facility is now being moved to a new location. The facility was cleaned with 0.25%

hypochlorite solution.

Sampling MethodsAir sampling was done using Tryptic Soy Agar

(TSA) and Sabouraud Dextrose Agar (SDA). The TSA values reflect total bacterial counts while the SDA reflects molds and yeast, although it contains no bacterial inhibitors. In some cases Rose Bengal Agar (RBA) was used. This reflects a better value for molds/yeast since the RBA contains bacterial growth inhibitors. Surface monitoring was done using 2430 cm2 RODAC plates with TSA and SDA. The TSA plates were incubated for a minimum of 48 hours at 32.5 +/ 2.5ºC while the SDA plates were incubated for a minimum of 72 hours at 22.5 +/ 2.5ºC.

Results – Airborne MicroorganismsThe gowning room was sampled in two zones

53

Figure 7airborne Bioburden in the encelle class 1,000 cleanroom

new Facility’s microbial air sample results collected with Biotest Plus centrifugal air sampler

Design class 1,000 11/14/99 11/30/99 average cfu av. cfu/ft3 cfu/ft3/24hr* cfu/ft3/72hr* average cfu av. cfu/ft3 cfu/ft3/24hr* cfu/ft3/72hr*

Device Testing 0 0 0 0 0 0 0 0

Coating 0 0 0 0 1 0.028 0.028 0.028

Formulations 0 0 0 0 0 0 0 0

Device Manufacturing 3 0.085 0.085 0.085 2 0.057 0.057 0.057

Refrigeration 0 0 0 0 0 0 0 0

Isolation 3 0.085 0.085 0.085 15 0.425 0.425 0.425

Class 1,000 Average 1 0.028 0.028 0.028 3 0.085 0.085 0.085

12/13/99 1/28/00Design class 1,000 average cfu av. cfu/ft3 cfu/ft3/24hr* cfu/ft3/72hr* average cfu av. cfu/ft3 cfu/ft3/24hr* cfu/ft3/72hr*

Device Testing 0 0.000 0 0 0 0.000 0 0

Coating 0 0.000 0 0 0 0.000 0 0

Formulations 0 0.000 0 0 0 0.000 0 0

Device Manufacturing 0 0.000 0 0 0 0.000 0 0

Refrigeration 0 0.000 0 0 0 0.000 0 0

Isolation 0 0.000 0 0 0 0.000 0 0

Class 1,000 Average 0 0.000 0 0 0 0.000 0 0

The time period refers to the incubation time in hours.



while the Class 10 cleanroom was sampled in five zones. All plates (TSA and SDA) were negative (i.e., zero counts) in all the areas. The Class 10 area was also sampled using RBA and once again the results were negative – zero counts.Results – Surface Microorganisms

The surface measurements were made before and after cleaning the newly constructed cleanroom. The results are shown in Figure 10. The 0.25% Hypochlorite cleaning is obviously very effective in eliminating surface bacteria.

Observations From the Medpharmex Clean room Validation

1. The new Encelle Class 1000 and the Med Pharmex Class 10 room have about the same airflow average velocity. From the particulate point of view the MedPharmex room operates at Class 10 simply

because of the double HEPA filter system used. The MedPharmex cleanroom validates as a Class 10 cleanroom, although the airflow used was lower than what is normally used in a Class 100 room.

2. It should be noted that the MedPharmex room was simply validated and then shut down in order to move it to an adjacent facility, while the Encelle room is being continuously monitored and is operational. How ever, from the point of view of airborne bioburden, after the first month of operation the Encelle Class 1000 room operates at an equivalent level as the MedPharmex Class 10 room – with essentially zero airborne bacterial counts. The low bioburden benefit to Encelle (this Class 1000 room is operating at essentially zero airborne bioburden) may be attributed to the bactericidal properties of the EEF system. o

54

Figure 8airborne Bioburden in the encelle class 10K area

new Facility’s microbial air sample results collected with Biotest Plus centrifugal air sampler

Design class 10,000 11/14/99 11/30/99 average average cfu av. cfu/ft3 cfu/ft3/24hr* cfu/ft3/72hr* average cfu cfu/ft3 cfu/ft3/24hr* cfu/ft3/72hr*

Water Filtration Area 30 0.850 0.850 0.850 2 0.057 0.057 0.057

Side Corridor 3 0.085 0.085 0.850 3 0.058 0.085 0.085

Manufacturing Corridor 19 0.538 0.538 0.538 11 0.312 0.312 0.312

Labware Processing 5 0.142 0.142 0.142 4 0.113 0.113 0.113

Gowning Room 0 0 0 0 3 0.085 0.085 0.085

Materials Pass Through 3 0.085 0.085 0.085 11 0.312 0.312 0.312

Class 10,000 Average 10 0.283 0.283 0.283 5.67 0.160 0.160 0.172

12/13/99 1/28/00Design class 10,000 average cfu av. cfu/ft3 cfu/ft3/24hr* cfu/ft3/72hr* average cfu av. cfu/ft3 cfu/ft3/24hr* cfu/ft3/72hr*

Water Filtration Area 2 0.057 0.057 0.057 2 0.057 0.057 0.057

Side Corridor 2 0.057 0.057 0.057 0 0 0 0

Manufacturing Corridor 1 0.028 0.028 0.028 0 0 0 0

Labware Processing 0 0 0 0 0 0 0 0

Gowning Room 0 0 0 0 0 0 0 0

Materials Pass Through 2 0.057 0.057 0.057 0 0 0 0

Class 1,000 Average 1.17 0.033 0.033 0.033 0.333 0.009 0.009 0.009

The time period refers to the incubation time in hours.



About the AuthorsRajan (Raj) Jaisinghani is a chemical engineer with thirty years of research, product development, and business development experience. Jaisinghani holds a B.S. from Banaras Hindu University, India, and an M.S., with additional graduate work, from

the Un iver sity of Wisconsin. Jaisinghani has exten-sive re search experience in air and liquid filtration, colloid and aerosol science, fluid mechanics, heat transfer, and physical surface chemistry. He holds 10 patents and has many publications in technical journals and handbooks. He can be reached by phone at 804-744-0604, by fax at 804-744-0677, or by e-mail at [email protected].

Greg Smith is facilities manager at Encelle, Inc. He holds a B.A. in Psychology from West Virginia University and a B.S. in Chemistry from East Carolina University. Smith has assisted in the development of medical devices and has five years experience as a hospital pharmacy aseptic compounding techni-cian. He can be reached by phone at 252-355-4405 or by e-mail at [email protected].

Gerald Macedo has a B.S. degree in Pharm acy and an M.S. in Pharmaceutical Sciences. He has over 30 years experience in pharmaceutical manufactur-ing, with extensive experience in the manufacture of sterile injectables. He has ser ved as head of man ufactur ing, quality control, quality assurance, research and development, and regulatory affairs. Macedo currently heads Med-Pharmex, Inc., a pharmaceutical manufacturing company. He can be reached by phone at 909-593-7875 or by fax at 909-593-7862.

References 1. FDA. “Guideline on Sterile Drug Products by Aseptic Pro

cessing.” Rockville, MD. 2. EU. 1998. “The Rules Governing Medicinal Products in the

EU.” Good Manufacturing Processes 4. Luxembourg. 3. Seaver, M. and Eversole, J.D. 1996. “Monitoring Biological

Aerosols Using UV Fluorescence.” Proceedings 15th Annual Meeting AAAR. October. Orlando, FL: 270.

4. Pinnick, R.G., Chen, G., and Chang, R.K. 1996. “Aerosol Analyzer for Rapid Measurements of the Fluorescence Species of Airborne Bacteria Excited with a Conditionally Fired Pulsed 266 nm Laser.” Proceedings 15th Annual Meeting AAAR. October. Orlando, FL.

5. Rhodes, W.W., Rinaldi, M.G., and Gorman, G.W. 1995. “Reduction and Growth Inhibition of Microorganisms in Commercial and Institutional Environments.” Environmental Health 12 (October).

6. Tolliver, D.I. 1988. “Domestic and International Issues in Contamination Control Technologies.” Microcontamination 6, no. 2 (February).

7. Jaisinghani, R. A. U.S. Patent 543,383. 4 April 1995. 8. Jaisinghani, R.A., Inzana, T.J., and Glindemann, G. 1998.

“New Bactericidal Electrically Enhanced Filtration System for Cleanrooms.” Paper presented at the IEST 44th Annual Technical Meeting. April. Phoenix, AZ.

9. “Microbial Evaluation of Cleanrooms and Other Controlled Environments.” United States Pharmacopoeia, <1116>, p. 20992106.

55

Figure 9new Facility surface

contamination summaryDesign classification10,000 average number of cfu/plate grown in 72 hours Date – > 11/14/99 11/30/99 12/13/99 01/28/00Water Filtration Area 0 0 2 0Side Corridor 1 0 0 0Manufacturing Corridor 0 0 0 0Labware Processing 1 0 0 0Gowning Room 0 0 0 0Materials Pass Through 0 0 0 0Design classification1,000 average number of cfu/plate grown in 72 hours Date – > 11/14/99 11/30/99 12/13/99 01/28/00Device Testing 0 0 0 0Coating 0 0 0 0Formulations 0 0 0 0Device Manufacturing 0 0 0 0Refrigeration 0 0 0 0Isolation 0 0 0 0control 255 210 134 104

Figure 10surface Bioburden in the

class 10/10K suite(counts per 25 cm2 roDac Plates)

area Before cleaning after cleaning tsa sDa tsa sDa counts counts counts countsGowningTable-gowning 22 5 0 0Wall-gowning 3 0 0 0class 10Tank 0 1 0 0Fill 21 12 0 0Filter table 9 4 0 0Wall 1 0 0 0

The EnzymeLinked Immunofiltration Assay (ELIFA) provides high sensitivity of

de tection with rapid results. For this reason we developed a very sensitive, semiquantitative ELIFA to determine IgA in therapeutic Win Rho SDF™ im munoglobulin. In the course of the development we no ticed that nonuniform and unusually high background (blank) re sponses, that occurred infrequently, greatly interfered with the test results obtained. We hypothesized that such background responses resulted from inadequate cleaning of the ELIFA apparatus. Accordingly, a cleaning program for the apparatus has been devised and validated. In this paper the results supporting the hypothesis will be presented, and the rationale and core aspects of the developed program delineated.

Cleaning Validation Programs for Research

and Development?

The establishment of Cleaning Validation Pro grams (CVP) in the pharmaceutical industry is dictated by the regulatory requirements to develop and observe, in a fully documented way, effective cleaning procedures. Regulatory guidelines for validation of cleaning pro cesses1 are meant to sup

port individual CVPs and enforce compliance. The guidelines and programs may cover a plethora of different types of equipment but they usually refer to equipment used in the manufacture, processing, holding, filling, and packaging of raw materials, inter mediate/ final products, and associated components. The guidelines do not refer to equipment used in Research and Development (R&D), and to our understanding, there is no regulatory requirement for the development of CVPs for equipment used in these areas. The EasyTiter™ ELIFA system2 is a small, micro titer format compatible apparatus developed and manufactured by Pierce Chemical Company. As shown in Figure 1 (adapted from Product Instructions, Pierce Chemical Company) the apparatus utilizes a nitrocellulose mem brane (NC) sandwiched be tween the sample application plate and vacuum collection chamber. Similar to the widely used EnzymeLinked Immunosorbent Assay (ELISA), the ELIFA is an

immunoassay well suited for testing of multiple samples over a range of serial dilutions.3 In the ELIFA, the immunological reaction between NC immobilized ligand and ligandspecific analyte in the test sample followed by an enzymatic reaction with a chromo


A Cleaning Validation Program for the ELIFA SystemBy LeeAnne Macaulay, Jeff Morier, Patti Hosler,

& Danuta Kierek-Jaszczuk, Ph.D. Cangene Corporation

v

}The establishment

of Cleaning Validation

Programs (CVP) in the

pharmaceutical industry is

dictated by the regulatory

requirements to develop

and observe, in a fully

documented way, effective cleaning

procedures.~


LeeAnne Macaulay, Jeff Morier, Patti Hosler, & Danuta Kierek-Jaszczuk, Ph.D.

genic substrate gives rise to colored dots. The color intensity of the individual dots varies proportionally to the amount of the analyte in the samples and dots produced by the samples devoid of analyte (blanks or background) are very pale or even colorless. We used the ELIFA system to research and develop a screening assay for human IgA.4 The developed IgA ELIFA will be used for testing of the licensed WinRho SDF™ therapeutic, hence, its performance characteristics need to be established and validated. In prevalidation studies, however, we observed that the developed ELIFA lacked reproducibility. The color of the blank dots varied sometimes from experiment to experiment or, even within the same experiment, from well to well. We also observed that the color of dots produced by the replicated test samples occasionally varied. We postulated that the observed variability is a result of external contamination carried over from previous experiment(s). An inadequately cleaned ELIFA apparatus would then be the cause of obscured test results. We, therefore, decided to develop a CVP for the ELIFA apparatus before proceeding to assay validation.

ELIFA CVP – Approaches and Hallmarks

A body of experience at Cangene with validation5,6 or cleaning7 programs, as well as manufacturer’s cleaning instructions for the ELIFA system (Figure 2, adapted from Product Instructions, Pierce Chemical Company) was the foundation when developing the ELIFA CVP. Among others, the developed program addressed the following:

n Specific design of apparatus, its individual parts and accessories that require cleaning

n Disassembling and reassembling the unit before and after cleaning

57

Figure 1exploded view of the easy-titer™ eliFa system

MembraneSupport

Plate

Tubing

Collection Chamber

Transfer Cannula

Position Stops (Acrylic Balls)

Gaskets

Vacuum Relief Valve

Thumb Screws

Sample Application

Plate

Guide Pins

Pump Tubing

Port

Microtiter Plate

Clamp

Nitrocellulose

Figure 2cleaning of the easy-titer™

eliFa systemClean all of the pieces to the Easy-Titer™ ELIFA System unit in a two percent PCC-54 solution and then rinse with distilled water. The unit may also be soaked in the PCC-54 solution to remove stains from the unit caused by the substrate solution.



n Cleaning operationsn Cleaning procedures including cleaning ves

sels, agents and utensilsn Compatibility of cleaning agents with equip

ment and assayn Decontaminating abilities of cleaning agents n Sampling on cleaned equipmentn Analytical methods for monitoring of cleaning

processesn Storage of cleaned partsn Inspection of apparatus for cleanliness before usen Recording and documenting the cleaning pro

ceduresn Establishing acceptance criteria, andn Maintaining cleaning records

Strategy for Validation of Cleaning Procedures

Two cleaning procedures (procedure 1 and 2 in Figure 3) utilizing either enzyme or detergentbased cleaning agents were developed and tested in conjunc

tion with two ELIFA experiments; the standard IgA ELIFA and the mock ELIFA. Such a combination of analytical methods allowed for instantaneous monitoring of the effectiveness of the cleaning pro cess. Standard IgA ELIFA method4 involved testing 96 replicates of a sample at a high (worstcase condition) IgA concentration, which were applied into 96 wells of the ELIFA apparatus. As expected, these experiments invariably produced highly colored dots (Figure 4). A tested cleaning regimen (procedure 1 or 2 in Figure 3) followed by the second mock experiment was then executed. The mock ex periment involved the use of the diluting buffer in lieu of a sample with high IgA concentration that was also applied into 96 wells of the ELIFA apparatus. Providing that the cleaning regimen was effective, the mock experiment should not produce colored dots, as there was no specific analyte that could attach to the immobilized ligand to facilitate subsequent enzymatic and color reactions. The results ob tained show that whereas Procedure 1 did not re move the contaminants from preceding experiments well enough (Figure 5), procedure 2 was fully effec

58

Figure 3cleaning Procedures 1 and 2

Procedure 1 Procedure 2

Disassemble the unit by first removing the thumb Disassemble the unit by first removing the thumb screws screws on the top of the sample application plate, then located on the top of the sample application plate, thenremoving the application plate and top gasket, and removing the application plate and top gasket, and finally unclamping the membrane support plate from finally unclamping the membrane support plate from the collection chamber. the collection chamber.Rinse all parts for two minutes under running Reverse Rinse all parts for two minutes under running RO water. Osmosis (RO) water. Immerse them into a vessel with two percent TERG-A- Immerse them into a vessel with five percent RBS10 solutionZYME (Alconox Inc., New York, NY, U.S.A.) solution and at 50ºC and wash for five minutes by agitating the vessel.wash for five minutes by agitating the vessel.Rinse all parts for two minutes under RO water. Rinse all parts for two minutes under RO water.Clean all 96 wells of the sample application plate with Clean all 96 wells of the sample application plate with TOC swabs by dipping the swabs into the detergent TOC swabs by dipping the swabs into the detergent solution, inserting them into wells once from top and solution, inserting them into wells once from the top and once from the bottom, and swabbing the inner part of once from the bottom, and swabbing the inner part ofeach well by turning the swab first to the right and each well by turning the swab first to the right and then to the left. then to the left.Clean all 96 wells of the top gasket in a similar way. Clean all 96 wells of the top gasket in a similar way.Raise all 96 cannulas on the membrane support plate Raise all 96 cannulas on the membrane support plate andand soak the plate for five minutes in the detergent clean them with TOC swabs by dipping the swabs into thesolution. detergent solution and swabbing the surface of individual can-

nulas and also spaces between cannulas and bottom gaskets.Rinse each part and the spaces between the bottom Rinse each part and the spaces between the bottomgasket and the membrane support plate for two gasket and the membrane support plate for two seconds seconds under running RO water. under running RO water.



tive (Figure 6). Procedure 2 was then validated, in two independent experiments performed by two analysts. It was shown that it invariably leads to results similar to those presented in Figure 6.

Assessment of the Effectiveness of the Validated Procedure

The Total Organic Carbon (TOC) method is widely utilized in industrial CVPs as it measures low levels of carbon and is compatible with swab sampling techniques. The standard IgA ELIFA4 followed by the validated cleaning procedure and swab sampling of the surface of three randomly selected wells were, therefore, used to assess the cleanliness of the apparatus by standard TOC. A procedure used at Cangene8 was followed. The results obtained confirm that the validated cleaning procedure was fully effective as the carbon concentration determined in

59

Figure 7results From total organic carbon analysis (in ppb)

sample sample replica replica replica average sD Percent number name 1 2 3 cv

1 Well B11 277.8 271.7 268.9 272.8 4.55 1.67 2 Well D8 235.4 234.3 225.9 231.8 5.20 2.24 3 Well G2 228.4 219.1 255.3 234.3 4.73 8.03 4 Water 185.2 165.4 167.4 172.7 10.9 6.29

Figure 4iga eliFa results obtained for a test sample containing human

iga at a concentration of 2µg/ml

Figure 5iga eliFa results obtained for a replicated test sample Deprived

of human iga.

The experiment was performed in an apparatus cleaned with TERG-A-ZYME (Procedure 1).

Figure 6iga eliFa results obtained for a replicated test sample Deprived

of human iga.

The experiment was performed in an apparatus cleaned with RBS (Procedure 2).



water extracts of test samples was only slightly greater than that of water used for extraction (Figure 7).

Implementing of the ELIFA CVP

The validated ELIFA cleaning procedure will become part of a written Standard Operating Procedure (SOP). Although addressing R&D instrumentation, the SOP document will detail the activities that were conducted by adhering to industrial standards for cleaning validation.1,9 The document will also advise on safety precautions, cleaning schedule, and assignment of responsibility for cleaning and storage of the cleaned apparatus. The SOP document will be observed not only when validating the performance of the IgA ELIFA, but also during routine use of the ELIFA system. It will be the subject of a periodic evaluation and, if deemed necessary, be updated and/or revised.

Conclusions

n A comprehensive, credible CVP designed and developed at Cangene for the EasyTiter™ ELIFA system has been shown to effectively remove contaminants and residues entrapped in the apparatus after the conclusion of the ex periment(s) and/or subsequent cleaning.

n The CVP has been demonstrated to vastly re duce the analytical background of the IgA ELIFA, improve its signal to background ratio, increase the quality of the test results and may, therefore, be expected to notably support the upcoming assay validation.

n The CVP, by virtue of antiviral and antibacterial properties of the RBS10, allows for simultaneous decontamination and sanitization of the ELIFA unit, thus facilitating its safe use with infectious samples.

n The CVPs generated for R&D equipment that fulfill the standards of industrial cleaning validation not only improve the quality of the as says utilizing this equipment but may become vital components of assay validation. o

About the AuthorsLeeAnne Macaulay is a Technician at Cangene Corporation. She completed the first year towards

a B.Sc. degree at the University of Winnipeg and received a diploma in Chemical and Bioscience Technology from Red River College in Winnipeg. She has experience in QC/QA Laboratories in the areas of microbiology and biochemistry.

Jeff Morier is a Senior Assay Development Tech-nologist at Cangene Corporation. He received his B.Sc. degree in Microbiology from the University of Manitoba. He has seven years experience in the pharmaceutical industry in the areas of QC microbi-ology, QA biotechnology, and R&D experience in the validation of immunoassays of various formats.

Patti Hosler is a Technician at Cangene Corporation. She completed the first year of a B.Sc. degree pro-gram at Brandon University and received a diploma in Chemical and Bioscience Technology from Red River College. She has seven years experience as a QA/QC laboratory technician in the food production industry.

Danuta W. Kierek-Jaszczuk is a Senior Research Scientist/Assay Development Supervisor at Cangene Corporation. She obtained her M.S. degree in Biology from the Nicolaus Copernicus University, and a Ph.D. degree in Agricultural Sciences from the Polish Academy of Sciences Institute of Genetics and Animal Breeding. She can be reached by phone at 204-275-4263, by fax at 204-269-7003, and by e-mail at [email protected].

References 1. FDA. 1993. “Guideline to Inspection of Validation of Cleaning Pro

cesses.” Office of Regulatory Affairs, USFDA, Washington, D.C. 2. Pierce Chemical Company. Product Instructions, EasyTiter™

ELIFA System. Rockford, IL. 3. Paffard, S.M., Miles, R.J., Clark, C.R., and Price, R.G. 1996.

“A Rapid and Sensitive Enzyme Linked Immunofilter Assay (ELIFA) for Whole Bacterial Cells.” Journal of Immunological Methods 192, no. 1–2: 1336.

4. Morier, J., Macaulay, L., and KierekJaszczuk, D. “Screening for the Presence of Human IgA in a Hyper Immune Product Using An EnzymeLinked ImmunoFiltration Assay.” Poster Presentation at IBC Conference on Assay Development for Future HighThroughput Screening. 8 – 9 November 1999. Annapolis, MD.

5. Faurschou, A. 2000. General Procedure for Validation Program. SOP Document # 11.001.0001.RR. Cangene Corporation. Winnipeg, MB, Canada.

6. Alejo, M. and Faurschou, A. 1998. Process Validation Qualification. SOP Document # 11.001.0002.RR. Cangene Corp oration. Winnipeg, MB, Canada.

7. Heise, R. and Poschner, E. 1999. Manual Cleaning and Sanitizing Equipment. SOP Document # 2.010.0017.RR, Cangene Corporation. Winnipeg, MB, Canada.

8. Shinkarik, T. 1998. Surface Sampling for Total Organic Carbon (TOC). SOP Document # 500602.RR, Cangene Corporation. Winnipeg, MB, Canada.

9. Chudzik, G.M. 1998. “General Guide to Recovery Studies Using Swab Sampling Methods For Cleaning Validation.” Journal of Validation Technology 5, no. 1: 7781.

10. Pierce Chemical Company. Product Information, RBS. Rockford, IL.

60

With the benchmark constantly being raised, many companies find

that they are in perpetual validation mode. Often, companies have executed validations for equipment, cleaning, and processes, but the doc umentation no longer stands up to the latest in validation standards. Although these validations are generally complete and on file, there are many opportunities to improve both the supporting documentation and the execution. One way to en sure that your company’s policies and procedures regarding cleaning validation are stateoftheart is to assemble a multidisciplined team from the appropriate manufacturing sites that can review and revise all components associated with cleaning validation. What follows are ex cerpts from a Cleaning Validation Master Plan (the Plan) that was painstakingly composed and has now be come the standard for planning and executing cleaning validations at several manufacturing sites.

An outline of the Plan contains the following seven elements, the concepts of which are taken directly from the FDA publication, “Guide to Inspections of

Validations of Clean ing Processes – July 1993.” Each of these will be discussed in greater detail in the sections below.

n Objectiven Scopen Introductionn Responsibilitiesn Philosophyn Methodologyn Schedule

Objective

This section should state the purpose of your cleaning master validation plan and define whether you will be revalidating current procedures or prospectively validating new ones. Often, the plan will have provisions for both situations.

Scope

The scope needs to list exactly which aspects of validation will be covered in the document and to which types of products and/or processes the Plan applies. For example, “This document provides steps for planning, executing, and maintaining equipment cleaning valida


}Often, companies have

executed validations for

equipment, cleaning, and pro-cesses, but the doc-

umentation no longer stands up to the latest

validation standards.~

A Cleaning Validation Master Plan for Oral Solid

Dose Pharmaceutical Manufacturing Equipment

By Julie A. Thomas McNeil Consumer Healthcare

v


Julie A. Thomas

tions for oral solid dose products at Your Company’s manufacturing facility in Your City, State.”

Introduction

The introduction should let the reader know what elements will be addressed in the Master Validation Plan and why a formal plan is necessary. For in stance, “This plan is intended to be a roadmap clarifying the course the Company will take as it plans and executes the cleaning validations required by current Good Manufacturing Practices (cGMP). This program describes and defines the various categories of cleaning validation, provides the necessary protocol elements, and offers guidance for un ex pected results. Furthermore, it includes provisions for revalidation and monitoring and serves as a mechanism to organize and store critical information that supports the cleaning validation process.”

Responsibilities

There are many departments and disciplines involved in planning for and executing a cleaning validation. It is necessary to list each contributing area and the associated tasks for which it is responsible. This serves to clarify roles and to ensure that tasks are not overlooked. Typically, representatives from Validation, Manufacturing, Quality Control, En gineer ing, and Research and Development (R&D) will be needed. The following are some examples of departmental responsibilities:

Validation Specialist • Review cleaning procedures • Assist the cleaning validation team in iden

tifying equipment test sites for swab or rinse samples

• Write cleaning validation protocols • Coordinate execution of the cleaning pro

cess with the appropriate departments and laboratories

• Prepare the sampling schedule • Assemble the test data into final report

form for approvalManufacturing • Provide technical information for the devel

opment of protocols and reports • Review and approve protocols and reports

for accuracy and agreement with operating practices

• Create and/or revise related SOPs and cleaning checklists

• Perform cleaning processes per SOP as referenced in the validation protocol

• Provide documented training for all personnel responsible for cleaning the equipment

Quality Assurance • Review and approve protocols and reports

for conformance with cGMPs and internal procedures

• Provide analytical technical support • Provide documented training for all person

nel responsible for sample collection and testing

• Collect analytical samples as specified in the protocol

• Perform analytical testing using validated procedures

• Label, package, and send out those samples that need to be analyzed by an external laboratory

• Review and approve analytical results • Notify departments of test resultsEngineering • Inform the affected department in advance

of any anticipated change to the facility or equipment

• Include all utilities and cleaning equipment in the calibration and maintenance program

• Review and approve equipment drawings and surface area calculations

Research and Development • Provide swab and surface recovery data for

active ingredients and cleaning agents • Validate analytical test methods for chemi

cal and cleaning agent analyses • Transfer validated methods to the site QC

laboratories and/or contract laboratories • Provide recommended cleaning procedures

for new active ingredients and/or cleaning agents

Cleaning Validation Philosophy

This section discusses the considerations you

62


Julie A. Thomas

have made in developing a comprehensive cleaning validation program, such as how to define equipment holding time, equipment storage time, and campaign length. In general, the philosophy section presents the Company’s position on what is being achieved by the cleaning validation and how it will be demonstrated. For instance, “Cleaning validation is required for all manufacturing and packaging equipment that comes into contact with the product or product components during production. Prior to validation, acceptance criteria will be developed for active ingredient and cleaning agent residues. Verification of acceptable equipment holding time will be included as part of the validation. Holding time is defined as the time between the end of the last product manufactured and the start of the cleaning process. This will demonstrate that the cleaning procedure effectively removes residue after the equipment has remained idle for a specified period of time. Additionally, holding time will be evaluated to ensure storage conditions are adequate for a predetermined length of time. Storage time is defined as the time between cleaning completion and the next batch processed on the equipment. Campaign length will be determined jointly by Operations and R&D and validated with at least three iterations using the maximum number of batches or maximum length of time. This approach fully challenges the cleaning procedure by providing worstcase residues.”

Cleaning Validation Methodology

To ensure all of the elements are in place for a thorough and successful validation, a chronological methodology should be followed. One such design is illustrated through the following four phases: development, planning, execution, and maintenance. (See Figure 1) In this section of the Plan, it is appropriate to include the number of sampling/testing iterations required for each piece of equipment and/or each analyte. (See Figure 2.)

If you intend to reduce the number of tests re quired to validate cleaning after various products by using a grouping approach, it should be explained in this section.1

Development Phase

The initial phase of the cleaning validation plan is

preparatory and includes analytical methods validation, recovery studies, surface types, degradants, and methods transfer. There is a considerable amount of scientific activity that must be completed before the validation can begin. These steps are explored in the following sections.

1. Analytical Methods ValidationDescribe how the analytical methods will be

developed and validated for active ingredients, degradants (if applicable), and cleaning agent residue. Validation of the method should assess reproducibility, linearity, specificity, limit of detection (LOD), and swab and surface recovery. Other elements for consideration are the instrumentation, swabbing and dilution solvents, dilution volume, and sample handling and storage.2,3

2. Recovery StudiesRecovery studies evaluate quantitative recovery

of chemical residue from both the surface to be sampled and the swab material to be used for sampling. The results confirm the appropriateness of the sampling method and material used. You should determine the minimum recovery criteria for each surface type and state that percentage in this section. For instance, you may want recovery values of at least 70% of actual readily soluble residues, but may choose a much lower recovery value for relatively insoluble proteins.4 Most important, you must provide data to justify the chosen value.

3. Surface TypesSince different surface types have different affini

ties, you may want to choose a few surface materials to represent the many product contact surfaces used in manufacturing. For oral solid dose manufacturing, you may determine that stainless steel, silicone, and polypropylene are the most abundant surfaces and that they also provide varying degrees of porosity. A matrix of all surface types and the representative material that will be used in recovery studies is appropriate. (See Figure 3)

4. DegradantsMany degradant products are more soluble in the

cleaning solvent than are the active ingredients; there fore, you should determine the degree of degradant

63


Julie A. Thomas

64

Figure 1cleaning validation Flow Diagram

Development Phase

Planning Phase

execution Phase

maintenance Phase

Analytical Method Development

• Recovery • Surface types

Equipment

• Sample site selection

• Surface area calculation

• Schematic

Analyte Selection and Acceptance

Criteria

• Active ingredient• Cleaning agent

Cleaning SOP

• Write• Approve• Train

Analytical Method Validation

• Degradant identification

• Transfer

Protocol Development

• Write• Approve• Train

Validation Report

• Write• Approve

Protocol Execution

• Clean• Sample• Test

Incident InvestigationPass?

Monitoring

Revalidation

Change Control

No

Yes


Julie A. Thomas

testing required based on the toxicity and solubility of potential degradants. Likewise, active ingredients should be exposed to the selected cleaning agent under normal usage conditions to determine if degradants are formed as a result of the cleaning process.

5. Analytical Methods TransferIn this section, you can state how sampling and

analytical methods will be transferred from the R&D laboratories to the site QC laboratories and how the analysts conducting validation testing will be qualified. Reference appropriate SOPs and/or De velopment Transfer Report.

Planning Phase

The next phase of preparation is the planning phase. This is a broad category that focuses on equipment information, analyte selection, acceptance criteria, cleaning procedures, and protocol development. At this point, you are starting to think about what equipment will be included in the validation, which analytes will be chosen, and how you will determine acceptance criteria. This leads to an indepth review of the procedures and, finally, to protocol development.

1. Equipment InformationThis section should detail the methodology for

providing specific equipment information. One option is to prepare a binder containing detailed surface area calculations, swab sampling sites (with justification), photos, and schematic diagrams for each piece of equipment. This binder can be maintained separately and used as an attachment to the cleaning validation protocol as needed.

a) Sample Site SelectionExplain how you will select sampling sites to rep

resent the product contact surface area of the equipment. One of the best sources of information is the operator who routinely cleans the equipment. He or she can certainly point out the areas they find most difficult to clean. Make the operator part of a larger team of experts to include representatives from Validation, QA, and Operations, and let the team determine the product contact surface areas that are most difficult to clean and those that are most representative of the equipment. Sampling these sites will represent the entire equipment surface area using the assumption that residue will be evenly distributed over the equipment and that the most difficult to clean locations will represent the worst case for residue removal. Include the basis for selecting each

65

Figure 2cleaning iteration summary requirements

sample total number of iterations conditions

Active Residue 3 1 at maximum campaign length or maximum time period plus holding time.

2 at maximum campaign length or time period.

Cleaning Agent Residue 3 3 per cleaning procedure, per piece of equipment.

Figure 3surface recovery matrix

recovery surface: 316l stainless steel Polyethylene silicone

Material Used: 316L Coupon Plastic Bulk Container Hose

To Represent: 304 Stainless Teflon Rubber Aluminum Lexan EPDM Brass HDPE Neoprene


Julie A. Thomas

site. For example, sampling sites may be deemed to be the most difficult to clean, most difficult to dry, or of different material of construction. Swab sites

can be indicated with either digital photographs or suitable diagrams. (See Figure 4)b) Surface Area Calculation

An accurate surface area must be calculated for each piece or section of equipment. This can be done with manufacturer’s drawings, but should be confirmed by field measurements. If drawings are not available, the equipment must be measured to determine surface area (see Figure 5). Although not shown here, it is advisable to include the calcula

tions with the schematic diagram in the equipment information binder mentioned above.c) Schematic Diagram

To clearly illustrate each piece of equipment, pre

pare schematic diagrams labeled with the major sections of the equipment. (See Figure 6) The drawings do not have to be to scale, but should appropriately represent the equipment. If a schematic is not practical (i.e., a packaging line), a photograph is acceptable. The intent is to depict the product contact surfaces that are included in the calculations. This helps to ensure that the swab samples are taken from the intended location.

2. Analyte SelectionAnalyte selection is required for active, excipi

ent (possibly), and cleaning agent residues. Keep in mind that you are validating a cleaning procedure, not a manufacturing process. In the situation where the same cleaning procedure is used for many product formulas, there is an opportunity to select a representative analyte to cover multiple active ingredients and reduce the amount of testing.

a) ActivesIf several active ingredients are processed in a single

piece of equipment, a marker active, or guiding substance, can be selected based on the active ingredient solubility in water, potency, previous production experience, and R&D studies. This reduces the number of studies required to validate the cleaning procedure.5

b) Excipients

66

Figure 4swab site

Figure 5surface area

swab number area swabbed

1 Screen/ring interface gasket

2 Discharge port – inside of top circular area (top seam)

Total contact S.A. of Kason Separator (in2) 3,171.2

Total contact S.A. of Filter Socks (in2) 15.6

Figure 6Kason Separator


Julie A. Thomas

The removal of excipients can either be confirmed by visual inspection or through analytical testing. The approach should be stated here along with training requirements for individuals performing visual inspection.

c) Cleaning AgentsTesting for cleaning agent residue is essential but

is often an area in which current cleaning validations are deficient. For most cleaning agents, a marker compound can be selected for analysis based on the recommendation of the cleaning agent manufacturer. Removal of volatile cleaning agents that do not leave a residue, such as isopropyl alcohol, may not need to be validated.

3. Acceptance CriteriaThe equipment must pass visual and olfac

tory inspection, where appropriate, as defined in the cleaning validation protocol prior to initiation of swabbing.6 This is a critical step in the validation process that, if skipped, can lead to failed results.

a) Active IngredientAcceptance criteria for active ingredients should

be based on medical and pharmacological properties and scientific information. Calculations using the maximum allowable carryover (MAC) and/or 10ppm formulas can be used.7

To ensure that all active contact surfaces are considered in the carryover calculation, you may want to identify equipment trains. Acceptance criteria are calculated using the surface area from the entire equipment train; however, protocols are executed per each piece of equipment. Equipment trains could be designated as follows:

n Granulation – granulator system through the product container

n Compression through printing – compression, filmcoating, and printing phases

n Packaging – product contact surfaces for each type of packaging line

b) Cleaning AgentAcceptance criteria for the cleaning agent marker

should be based on toxicity, limit of detection of validated assay method, and/or data gathered during certification studies. Acceptance criteria can be

calculated using a formula such as the No Observed Effect Limits (NOEL).8

4. Cleaning ProceduresThis section should indicate that cleaning procedures

will be developed (or existing procedures reviewed) prior to the validation. It should also list the required elements for cleaning procedures, such as temperature, pressure, water quality, cleaning agent concentration, spray nozzle location, etc., or it should reference where these requirements can be found.9 Additionally, you should describe the process for training the operators who will be executing the validation studies.10

5. Protocol DevelopmentThe next step is to write a cleaning validation pro

tocol for each cleaning procedure that you intend to validate. The protocol should describe all documentation required to complete the cleaning validation. It should also present the rationale for using a marker active to cover validation for multiple products. For ease of review, include a matrix of the products and equipment that are covered by each validation, or reference where this information can be found. For example, if there are three active ingredients processed in Fluid Bed Granulator #1, indicate which active will be used to represent the other two. Likewise, indicate which pieces of equipment will be used to validate

the removal of active ingredient and cleaning agent residues. (See Figure 7)

Execution Phase

When all of the supporting documentation is complete, it is time to execute the plan. During the execution phase, you will complete the protocol, investigate any nonconformances that may have occurred, and write a report to summarize your findings.

1. Protocol Execution

67

Figure 7equipment cleaning matrix

active a active B active c cleaning agent a

Fluid Bed Gran 1 X – – XFluid Bed Gran 2 – – – –Starch Kettle 1 – – – X


Julie A. Thomas

Typically, three iterations of cleaning, sampling, and testing using the same procedure are required. Acceptance criteria for all cleaning iterations must be met for both the active ingredient and the cleaning agent. Be sure to reference the procedure where a detailed description of the chemical swab preparation and sampling methods can be found.

2. Incident InvestigationThis section explains how the Company will

handle test failures and nonconformances during execution of the validation. Once the root cause of the failure has been identified, options are to addend the protocol or start over with a new protocol. For any incident that occurs during validation, document the investigation along with corrective and preventive actions. The incident report may contain elements such as:

n Cleaning validation protocol numbern Incident report numbern Equipment model and locationn Initiator and daten Incident descriptionn Root cause analysisn Corrective actions recommended/takenn Assessment of effect on product

3. ReportsDescribe the report format and content that will

be used to summarize the validation. Reference appropriate SOPs for detailed report information. An explanation of all deviations should be included in the validation report.

Maintenance Phase

The final phase of the Plan should specify how you will maintain the conditions you have just validated. This includes periodic monitoring, using a control of change process, and potentially, revalidating.

1. MonitoringThis section details how you will ensure that the

conditions used during validation remain in control during routine production. This is especially important for manual cleaning procedures, where

repeatability is highly dependent on the quality and consistency of training. Monitoring should include, at a minimum, a review of changes made to the cleaning procedure or equipment, visual inspection of the equipment, and direct observation of employees executing the cleaning procedure. For some equipment, swab samples for active ingredients may be necessary in addition to the visual inspection and observation. Indicate the frequency that you intend to monitor the cleaning process. Reference the appropriate SOP for detailed requirements of the monitoring program.

2. Change ControlIndicate how changes will be managed to ensure

the validated state is maintained. Any change in the facility, process equipment, cleaning procedure, cleaning agent, product formulation, or addition of new products to the equipment train should be documented and approved via the Change Control System. The change should be reviewed by the Validation Group, Operations, and QA, who will decide if revalidation is necessary. Reference appropriate SOPs.11

3. RevalidationIndicate the criteria that will be used to determine

the need for revalidation. Based on the nature of the change, a determination will be made as to which, if any, phases of the validation must be repeated. Ref erence where documentation of the revalidation will be filed.12,13

Cleaning Validation Schedule

PrioritizationAs is usually the case, all cleaning validations

cannot commence at one time; therefore, it is necessary to set up a priority list. Some situations to consider are:

n Equipment shared between products containing different active ingredients

n Equipment in contact with raw material with high potential for contamination

n Unshared primary equipment currently in use with outdated validations

n Unshared auxiliary equipment used for pro

68


Julie A. Thomas

duction with limited potential for product contamination

Tactical ScheduleA proposed schedule, including equipment pri

oritization and target initiation dates, should be presented in this section. This gives an indication that you have contemplated the order of execution, and it also provides a tool that can be used to track your progress.

Summary

There are many aspects of cleaning validation that must be carefully planned to guarantee a successful validation program. If you begin with a philosophy, this will set the stage for you to develop a structured approach. By dividing the approach into sections, such as development, planning, execution, and maintenance, it breaks down the project into manageable segments. To complete the Plan, generate a tactical schedule and begin monitoring pro g ress towards your new and improved cleaning validation status. o

About the AuthorJulie Thomas is Validation Manager at McNeil Consumer Healthcare in Round Rock, Texas. She has 14 years of experience in various aspects of solid dose pharmaceutical manufacturing. Most recently, she chaired a company-wide commit-tee to enhance cleaning validation practices and procedures for all McNeil facilities. She can be reached by phone at 512-248-4470 or by e-mail at [email protected].

This article presents only one alternative for preparing a Master Validation Plan. The views ex pressed in this article are strictly those of the author and in no way represent the view of McNeil Con sumer Healthcare, Johnson & Johnson, or this publication.

References 1. Jenkins, K.M. and Vanderwielen, A.J. “Cleaning Validation: An

Overall Perspective,” Pharmaceutical Technology, April 1994, p. 62.

2. McCormick, P.Y. and Cullen, L.F., Pharmaceutical Process Validation, 2nd ed., edited by I.R. Berry and R.A. Nash, 1993, p. 334.

3. Kirsch, R.B., “Validation of Analytical Methods Used in Pharmaceutical Cleaning Assessment and Validation,” Pharmaceutical Technology, Analytical Validation, 1998.

4. Chudzik, G.M., “General Guide to Recovery Studies Using Swab Sampling Methods for Cleaning Validation,” Journal of Validation Technology, Vol. 5, No. 1, pp. 7781.

5. Hall, W.E., “Your Cleaning Program: Is It Ready for the PreApproval Inspection?” Journal of Validation Technology, Vol. 4, No. 4, August 1998, p. 302.

6. Alvey, A.P. and Carrie, T.R., “Not Seeing is Believing – A NonTraditional Approach for Cleaning Validation,” Journal of Validation Technology, Vol. 4, No. 3, pp. 189193.

7. Fourman, G.L. and Mullen, M.V., “Determining Cleaning Validation Acceptance Limits for Pharmaceutical Manufact ur ing Operations,” Pharmaceutical Technology, 17 (4), 1993, pp. 5460.

8. Hall, W.E., “Validation of Cleaning Processes for Bulk Pharmaceutical Chemical Processes,” Cleaning Validation An Exclusive Publication, p. 4.

9. Hall, W.E., “Proper Documentation and Written Procedures,” Journal of Validation Technology, Vol. 4, No. 3, pp. 199201.

10. Tunner, J., “Manual Cleaning Procedure Design and Validation,” Cleaning Validation An Exclusive Publication, p. 28.

11. PDA Technical Report No. 29, “Points to Consider for Cleaning Validation,” March 1998, p.43.

12. Coleman, R.C., “How Clean is Clean?” Journal of Validation Technology, Vol. 2, No. 4, August 1996, p. 278.

13. Jenkins, K.M. and Vanderwielen, A.J., “Cleaning Validation: An Overall Perspective,” Pharmaceutical Technology, April 1994, p. 70.

69

© Advanstar Communications Inc. All rights reserved.

PROPOSED VALIDATION STANDARD VS-3

Cleaning Validation

VALIDATION TECHNOLOGY

Journal of Validation Technology ~

Proposed Validation Standard VS-3

PROPOSED VALIDATION STANDARD VS-3 Cleaning Validation

Introduction

T his document is the third in a series of new proposed validation standards issued by the Institute of Validation Technology Standards Committee (IVT/SC). The initial proposed standard (Process Validation Standard VS-l: Nonaseptic Pharmaceutical Processes) was issued in February 2000, and

is intended to help practitioners worldwide who develop, implement, control, and validate processes that produce Active Pharmaceutical Ingredients (APIs) and drug products. Our second proposed validation standard VS-2: Computer-Related System Validation was issued in May 2001. The current document (Cleaning Proposed Validation Standard VS-3) is intended to offer more specific proposed standards for the cleaning processes for equipment used to manufacture APIs and drug products. These proposed standards, will be used by reviewers of manuscripts intended for publication in the lournal of Validation Technology (1VI).

Just as with the previous proposed standards, readers are encouraged to offer comments, questions, and recommendations. Such feedback will be useful to the IVT/SC and JVT editors in updating this document and in developing future proposed standards. Technologies are continually changing, sometimes in ways that can influence the way validation is best conducted. Therefore, the IVT/SC plans to periodically update each proposed validation standard, including its corresponding Preamble and reference list. In order to be dynamically responsive to changing industrial practices and regulatory requirements, and make it easier for readers to cut and paste the contents for their own use, all three proposed standards are available on the IVT web site at www.ivthome.com.

A fundamental need the IVT/SC intends to meet with its new proposed standards stems from the fact that most Good Manufacturing Practice (GMP) regulations today call for numerous written procedures; for example, more than 100 different kinds of written procedures are required to comply with current GMP regulations in the United States. Many firms find it helpful to issue written policies in order to coordinate and reduce the number and length of required Standard Operating Procedures (SOPs). Thus, the IVT proposed validation standards format includes statements and definitions that can be excised and used directly or with minor editing in a firm's policies and SOPs.

Contents of the Proposed Cleaning Validation Standard In order to be consistent with the prototype standard (Validation Standard VS-l) the Proposed Cleaning

Validation Standard VS-3 will be divided into the following five sections:

I. Policy statements - Proposed standards that indicate what is required II. Procedural Statements - Proposed standards that describe how to meet requirements

III. Acronyms - Meaning of each acronym/abbreviation used in the document IV. Glossary - Definition of key terms, which are highlighted and asterisked (*) when first used in the

proposed validation standard

~ Institute of Validation Technology


V. Regulatory Excerpts - Regulatory language (United States, Australia, Canada, World Health Organization [WHO], Japan, and European Union) related to each Standard

The following proposed standard is intended to reflect desirable contemporary practices, is not binding in any way, and can be modified to suit a firm's specific needs. This proposed standard incorporates imperative verbs (e.g., shall, will, must) to provide users with unambiguous quality assurance auditing tools,

and is prefaced by a Preamble that provides rationale for several of the more complex concepts. This document is also directed toward users located at a given plant site that mayor may not be a part of a larger corporation. Terms that are bold and asterisked (*) the first time they are used are defined in Section IV - Glossary.

I. POLICY STATEMENTS

POL 1.1 The critical cleaning processes associated with the manufacture of Active Pharmaceutical Ingredients (API)*, critical Intermediates*, Drug Products*, or In-Process Materials* shall be validated or verified.

POL 1.2 The critical cleaning processes associated with products in the development stage of the product lifecycle shall be verified. The administrative responsibility for such products will reside in either the appropriate development group or in the Site Validation Steering Committee (SVSC)*. If the company decides that responsibility for cleaning verification shall reside in the appropriate development group, then the documentation describing the verification procedure and the Cleaning Verification Protocols* must also be approved by the site Quality Authority*.

POL 1.3 During development of the new product, the manufacturing equipment, batch size, and formulation is constantly changing and the cleaning procedure must be appropriate and customized for each manufacturing event. The lifecycle for the development and validation of a new cleaning procedure consists of the following steps:

l.3.1 1.3.2

1.3.3 1.3.4 l.3.5 1.3.6 1.3.7 1.3.8

l.3.9

1.3.10 1.3.11

1.3.12

Determine what materials need to be cleaned from the equipment or surfaces. Determine what methods should be used to evaluate the anticipated residues (from Section 1.3.1). Determine the sensitivity and reproducibility of these methods. Define the Critical Product Cleaning Specifications*. Define the specific equipment to be used for each development batch. Define the specific formulation to be used for manufacturing each individual development batch. Identify the cleaning agents to be used, if appropriate. Determine what other products are manufactured in the same equipment. Calculate Cleaning Verification Limits* for the specific equipment taking into account the critical product cleaning specifications as well as the other products made in the same equipment. Draft a Cleaning Procedure* for the specific combination of product and manufacturing equipment. Identify Critical Cleaning Process Operating Parameters* and Cleaning Agents*. Prepare a cleaning verification protocol. Manufacture a single product batch, clean the equipment; then test the equipment, as specified in the cleaning verification protocol. Once development is complete, perform Cleaning Validation* on the first three (3) commercial batches.

Journal of Validation Technology


1.3.13 1.3.14

1.3.15 1.3.16

1.3.17

1.3.18 1.3.19 1.3.20

POL 1.4

Validate analytical methods to be used for cleaning validation samples. Determine recovery factors of expected residues from representative materials (stainless steel, glass, plastics). Prepare and obtain approval of a Cleaning Validation Protocol*. Train and qualify operational and supervisory laboratory and production personnel in product-specific cleaning procedures, sampling procedures, and analytical procedures. Ensure that interrelated systems (automated clean-in-place, utilities, Programmable Logic Controllers [PLCs]) are all validated. Conduct Cleaning Performance Qualification (CPQ)*. Assemble and document evidence that the cleaning process is acceptable and consistent. Provide for retention of archived cleaning validation files for required periods following the last commercial lot expiration date.

The cleaning processes associated with products in the marketed stage of the product lifecycle shall be validated for all products manufactured with a normal frequency of production. For rare instances where products are infrequently manufactured (e.g., one batch per year or less frequently), it may be difficult to achieve fully validated cleaning processes and the principle of cleaning verification should be utilized. The administrative responsibility for cleaning validation and cleaning verification of products will reside in the Site Validation Steering Committee (SVSC). The SVSC shall adjudicate cleaning validation issues and appoint project-specific validation teams as needed that include principal(s) having experience in the cleaning processes involved. Such SVSC responsibilities extend to cleaning processes used by contract vendors and suppliers of the firm's drug products and/or APIs, as well as to those cleaning processes employed on-site.

POL 1.5 A written Cleaning Verification Policy (CVP) shall be used to define and describe the strategies and approaches used to verify cleaning procedures associated with drug products, biotechnology products, medical devices, and APIs during the development stage of the lifecycle.

POL 1.6 A written Cleaning Validation Master Plan (CVMP)* shall be used to define and coordinate validation activities related to any cleaning process associated with the manufacture of a commercially marketed drug product, biotechnology product, medical device, and API.

POL 1.7 Cleaning Verification Protocols shall be used to define individual cleaning verification runs.

POL 1.8 Cleaning Validation Protocols shall be used to define individual cleaning validation runs.

POL 1.9 Cleaning Verification Reports* shall be used for documenting and summarizing results of cleaning verification studies. Definite statements must be used, especially in describing the scientific rationale for the limits chosen and whether the cleaning process was effective in meeting the limits.

POL 2.0 Cleaning Validation Reports* shall be used for documenting and summarizing results of cleaning validation studies. Definitive statements must be used, especially in describing the scientific rationale for the limits chosen and whether the cleaning process was effective in ensuring that these limits were met.



POL 2.1 All Cleaning Verification Policies, Cleaning Validation Master Plans, Cleaning Verification Protocols, Cleaning Validation Protocols, Cleaning Verification Reports and Cleaning Validation Reports must be approved and available to the SVSc. All such cleaning documents created on-site must be approved by the site Quality Authority and, when production is involved, also by the site Production Authority*.

POL 2.2 Relevant cleaning process verification and validation information from other divisions, departments (including Research and Development), production sites, and outside contract services is to be gathered, evaluated, utilized, and maintained by the SVSC.

POL 2.3 Certain cleaning processes are considered critical manufacturing steps and thus require validation (it should be noted that not all cleaning procedures are considered critical and thus require validation). Once the cleaning procedures are validated, they must not be altered without prior review, and any changes should be subjected to a formal Change Control* review process prior to making the change. The site Quality Authority must approve all changes to validated cleaning procedures.

II. PROCEDURAL STATEMENTS

PROC - 2.a [ref. POL 1.3.2] Critical product cleaning specifications are known factors that can influence the development of the cleaning process. These can be physical in nature such as solubility in a variety of solvents, polymorphic crystal form, and stability. These factors could also be chemical in nature such as reactivity with water or other solvents. They could also include medical information such as potency, toxicity, and allergenicity. They could also be safety factors such as toxicity when inhaled and could require personal protection attire to protect the operator. These factors, which are normally determined during pre-formulation, are vital information that must be known before meaningful cleaning procedures and limits can be developed.

PROC - 2.b [ref. POL 1.3.3] During development, various types of equipment may be used in an effort to develop an optimum process or effective product. This means that normally the specific equipment or the scale of the equipment may vary from batch-to-batch. Because of this variability in the equipment used, the cleaning procedures may also vary from batch-to-batch even for the same product. Therefore, the cleaning verification results apply only to the specific cleaning event (i.e., the specific combination of equipment, processes, and materials) used for the individual study. The cleaning verification report should contain the details of the specific equipment (size, model number), formulation, and processes used.

PROC - 2.c [ref. POL 1.3.4] During development, the formulation may vary from batch-to-batch in order to identify the combination of ingredients that presents the best product performance 'in vitro' and 'in vivo'. Excipients may be varied as well as the concentration of active ingredient. These combinations will present different degrees of cleaning challenges. A given cleaning procedure may be adequate for one formulation but inadequate for another formulation of the same active ingredient. This data will be useful for the selection of the ultimate cleaning procedure that will be used for commercial product. It will be necessary to include the formulation in the cleaning verification study, either by reproducing in total, or by reference to a formula number.



PROC - 2.d [ref. POL 1.3.5] Since it is other products made in the same equipment that will be contaminated due to inadequate cleaning, it is necessary to evaluate the other products made in the same equipment. Some of the factors pertaining to other products that will be needed are:

• Batch sizes • Normal daily doses • Route of administration

PROC - 2.e [ref. POL 1.3.6] In order to develop a scientific basis for cleaning verification limits, information will be needed for both the product being cleaned as well as other products made in the same equipment. The following information should be assembled:

• For product being cleaned - Solubility in various solvents - Potency - Toxicity - Stability (wet and dry) - Allergenicity - Route of administration - Daily dosage - Difficulty of cleaning - Physical and chemical interaction with cleaning agent

• For other products made in same equipment - Batch sizes - Daily doses - Stability - Chemical interaction with product being cleaned - Route of administration

The pharmacological relationships between the potential contaminating product and other products, which could be possibly cross contaminated, may also be significant and should be considered if known. The contaminating product has the potential to amplify the medical activity of other products resulting in a synergistic effect. The contaminant could also partially negate the medical effect of the other products by having an antagonistic effect.

PROC - 2.f [ref. POL 1.3.7] Just as there are critical parameters for the manufacturing process, there are critical parameters for the cleaning process. These factors will lead to either inadequate or inconsistent cleaning if not controlled. Critical parameters for the cleaning process must be determined and may vary from one cleaning process to another. Some potential critical cleaning parameters (list is not all inclusive) are:

• Temperature of wash solutions • Temperature of rinse solutions • Amount of mixing or agitation during cleaning • Mechanical wiping or brushing



• Flow rates • Concentration of cleaning agent • Time of washing • Time of rinsing • Length of time and environmental conditions (temperature, humidity) between manufacturing and

cleaning • Nature and amounts of excipients • Concentration or amount of residue left on equipment • Physical properties of residues • Chemical properties of residues • Cleaning solvent chosen • Soak times • Contact time with cleaning agent • Rinse volumes • Order of application of cleaning solvents (acid, alkaline, and organic solvents)

PROC - 2.g [ref. 1.3.8] Each cleaning verification protocol shall include, and is not limited to, the following:

o Statement of objective or purpose @ Justification for cleaning verification limits, if applicable ~ Descriptions of sampling procedure(s), and locations, types, and numbers of samples to be taken o Indications of most difficult-to-clean locations in equipment o Experimental plan to be executed, including number of samples, and how data will be calculated <D Descriptions of analytical methodology and sensitivity of analytical method as well as recovery factors o Descriptions of all testing instruments to be used and specific calibration plans for each o Complete description of acceptance criteria including visual examination (if possible) and quantitative

analytical data o Training records of operators and analytical personnel

PROC - 2.h [ref. 1.3.11] Prior to cleaning validation studies, analytical methods must be validated to demonstrate that they are suitably sensitive to detect residues at levels below the allowable limits. Analytical Method Validation* for cleaning validation shall include, and is not limited to, the following:

o Accuracy @ Precision ~ Linearity o Robustness o Sensitivity-Limit of Detection (LOD)*, Limit of Quantitation (LOQ)* <D Specificity

The specificity of the analytical method may not be as critical for cleaning validation as for process validation due to the fact that the levels of residue detected is very low, and often non-specific analytical methods are available that may be at least or more sensitive than specific methods. The assumption is often made that all of the residue detected is composed of the most potent ingredient (usually the active) present and, if this amount is still below the established limits, then the exact nature of the residue is irrelevant, i.e., the 'worst case' assumption was made and limits were met.



PROC - 2.i [ref. 1.3.12] Following validation of the analytical method, the analytical method should be challenged concurrently with the sampling procedure(s) to determine the percentage recovered from representative manufacturing surfaces. The determination of recovery is important and will differ according to the composition of the surface sampled (e.g., stainless steel, glass, plastics), the nature of the sampling technique, and the nature of the residues themselves. The recovery factor must be used to correct observed analytical results to account for portions of residue that remain on equipment even after swab and rinse sampling.

PROC - 2.j[ref. 1.3.13] Each cleaning validation protocol shall include, and is not limited to, the following:

o Statement of objective or purpose @ Justification for cleaning validation limits @) Descriptions of sampling procedure(s) and diagrams of locations o Indications of most difficult-to-clean locations in equipment o Experimental plan to be executed, including number of cleanings to be evaluated, number of samples

from each cleaning, and how data will be calculated <D Descriptions of analytical methodology and sensitivity of the analytical method as well as recovery factors fi Descriptions of all testing instruments to be used and specific calibration plans for each «l) Complete description of acceptance criteria including visual examination (if possible) and quantitative

analytical data CD Criteria for determining when the cleaning process may be considered validated, i.e., how many suc

cessful consecutive cleanings (normally at least three (3) are required) @ Training records of operators and analytical personnel

PROC - 2.k [ref.1.3.14] Prior to implementation of the cleaning validation protocol, it is important to verify the training of the production operators who actually conduct the cleaning, sampling personnel (production, analytical, validation) who sample the equipment, analytical personnel who analyze cleaning validation samples, as well as personnel who implement the protocol and process the documentation. If documentation does not already exist that demonstrates each of these types of training, then the training should be done before any actual validation runs are carried out.

PROC - 2.1 [ref. 1.3.15] Special equipment and critical utilities such as water and steam must be qualified prior to implementation of the cleaning validation protocol. In addition, any automated cleaning equipment such as Clean-inPlace (CIP)* systems and their associated automated controllers must also be validated or qualified prior to implementation of the cleaning validation protocol. In the case of CIP, Sprayball Pattern Analysis* should be carried out to verify that cleaning solutions reach all locations in closed systems. The qualification of equipment and utilities is normally accomplished by means of an Installation Qualification (IQ) * and an Operational Qualification (OQ) * (see next two sections).

PROC - 2.m [ref. 1.3.15] An Installation Qualification (IQ) must exist for all equipment that is critical to the cleaning process including specialized cleaning aids such as Spray Devices (Sprayballs)*, equipment that delivers cleaning solutions, high pressure wands, water heating devices, steam generators, and utilities. The IQ is to include at least the following:



o List of all equipment, the operation of which has potential bearing on the quality of the cleaning process @ As-built drawings of all specialized cleaning equipment such as pumps, high pressure delivery devices,

and hose cleaners @) Verification that all such equipment and the installation thereof meets original intent, including applic

able building, electrical, plumbing, and other such codes o Preventative maintenance plans and schedules for all such equipment

PROC - 2.n [ref. 1.3.15] An Operational Qualification (OQ) must exist for all equipment that is critical to the cleaning process and should include at least the following:

o A list identifying each step of the cleaning process that relates to the specific equipment @ Process operating parameters for each piece of equipment that is critical to the cleaning process @) An OQ protocol that is designed to demonstrate via appropriate tests that the equipment operates as in

tended throughout the cleaning process o Report that describes the successful execution of each OQ protocol for each piece of equipment criti

cal to the cleaning process

PROC - 2.0 [ref. 1.3.16] At least three consecutive, successful cleanings shall be completed on the equipment used to produce the commercial product. Normally, the cleanings follow the production of each of the batches used for the validation of the manufacturing process. A Cleaning Performance Qualification (CPQ) shall be performed when the following items are complete and commercial production has been authorized.

• The cleaning process is fully defined in writing, including identification of critical cleaning process operating parameters

• A justification for Cleaning Validation Limits* has been prepared that takes into account the potency and toxicity of the material, as well as the other products to be made in the same equipment

• IQ and OQ steps are complete for critical utilities and any specialized equipment used in cleaning such as pumps, sprayballs, high pressure wand cleaners, etc.

• Operating, sampling, and analytical personnel are trained and qualified and the training is documented • An appropriate change control procedure is in place

PROC - 2.p [ref. 1.3.17] Once the cleaning validation protocol has been implemented on three cleanings and the sampling and testing has been completed, the data must be assembled and evaluated for each cleaning event. A cleaning validation report should be prepared that consists of:

• The cleaning validation protocol • All data assembled in a logical format • An analysis of the data that addresses any deviations in the protocol, explains any failures, compares

the data to the acceptance criteria, and ultimately states whether the cleaning process mayor may not be considered validated

PROC - 2.q [ref. POL 1.5] The Cleaning Verification Policy (CVP) can be considered to be the master plan for cleaning for a product during the development phase of the lifecycle of the product. Since each cleaning is a unique event because of the variability in the manufacturing equipment, formulation, and batch size between batches of the same



product, it is not possible to validate the cleaning process during the development phase. Still it is possible to prepare a policy describing the testing of development equipment and what criteria will be used to determine if the equipment has been suitably cleaned. The strategy and approach to cleaning in the development areas must be in writing and clearly explain how equipment will be sampled and tested, and how limits will be determined, recognizing that only a single set of data will be available. Since only a single set of data is available, it would be erroneous to refer to this situation as "validation". Thus the term "cleaning verification" is a more appropriate description of this scenario.

PROC - 2.r [ref. POL 1.6] The Cleaning Validation Master Plan (CVMP) may take different forms in companies around the world. Some may have a separate independent document. Others may have a Standard Operating Procedure* that describes in general terms how the cleaning program will operate. Still others will devote a section of the Validation Master Plan* to cleaning. Regardless of the exact form taken, it is essential to have a written plan describing how the cleaning program will be organized and controlled. The essential elements of the CVMP are:

• A description of the approach and strategy to be used for controlling, verifying, and/or validating in the various departments such as Basic Research * , Research and Development * , Scale-Up! Pilot Plant*, Production*, Packaging*, Contract Manufacturing Facilities * , and Contract Packaging Facilities * .

• A mechanism for defining what is adequate cleaning, based on the potency, toxicity, potential allergenicity, potential teratogenicity, and potential carcinogenicity of the material, as well as other factors such as route of administration and properties of the other products made in the same equipment.

• Sampling methods to be used to evaluate cleaned equipment. Examples are Swab Sampling* and Rinse Sampling*, or a combination of these two methods depending on the nature of the equipment or product.

• Selection of sampling locations to include 'worst case' and/or most difficult-to-clean locations. • For equipment used for manufacturing multiple products, how the Worst Case Product* for cleaning pur

poses might be selected from a group of very similar products. Typically, a Product Matrix Approach* is used to compare the critical cleaning properties of the products in the group. Critical cleaning properties are potency/toxicity, solubility, and the inherent difficulty of cleaning.

• Provision for how documentation will be developed, reviewed, and approved. This would include a list of those responsible for preparing, reviewing, and approving Cleaning Verification Protocols, Cleaning Verification Reports, Cleaning Validation Protocols, Cleaning Validation Reports, Cleaning Procedures, Change Control Procedures, and Cleaning Monitoring Programs * .

• Criteria for Revalidation* of cleaning procedures. • Provision for creation of a Site Validation Steering Committee (SVSC), that would serve as the group

immediately responsible for all cleaning issues. This group would normally select project teams related to cleaning activities, e.g., for a new product.

• Training of development, pilot plant, sampling, and analytical testing personnel. • Definition of resources required and allocated. • Schedule of cleaning activities including cleaning validation and assignment of responsibilities.

III. ACRONYMS

API BPC

CGMPs CIP

CPQ

Active Pharmaceutical Ingredient Bulk Pharmaceutical Chemical Current Good Manufacturing Practice (U.S.) Clean-in-Place Cleaning Performance Qualification


CVMP CVP IPEC

IQ OQ PIC

SOP SVSC

Cleaning Validation Master Plan Cleaning Verification Policy International Pharmaceutical Excipients Council Installation Qualification

Operational Qualification Pharmaceutical Inspection Convention Standard Operating Procedure Site Validation Steering Committee


IV. GLOSSARY

Reference Standard Number

POL 1.1 Active Pharmaceutical Ingredients (API) - (synonymous with drug substance). A substance that is represented for use in a drug and, when used in the manufacturing, processing, or packaging of a drug, becomes an active ingredient of a finished drug product. Such substances are intended to furnish pharmacological activity or other direct effects in the diagnosis, cure, mitigation, treatment, or prevention of disease, or to affect the structure and function of the body of humans or other animals.

Bulk Pharmaceutical Chemical (BPC) - includes active pharmaceutical ingredients (APIs) as well as non-active excipients such as starch, lactose, rnicrocellulose, and other materials that have no direct therapeutic effect but may indirectly affect the performance of drug dosage forms.

PROC·2.h Analytical Method Validation - documented evidence that an analytical procedure will consistently detect and/or quantitate materials.

PROC-2.r Basic Research - the segment of the pharmaceutical industry that evaluates new chemical entities for potential application to treatment of disease. This includes, but is not limited to, basic disciplines such as biochemistry, molecular biology, toxicology, pharmacology, and pharmacokinetics.

POL 2.3 Change Control Procedure - A procedure for:

POL 1.3.9

PROC-2.r

(a.) Identifying all modifications or alterations that are potentially significant to a state of control, qualification, or validation.

(b.) Implementing corrective action, such as repair, readjustment, requalification, and/or revalidation.

(c.) Implementing interim measures to be taken until effective corrective actions are complete. (d.) Documenting all of the above.

Cleaning Agents - any chemical or solvent that facilitates the cleaning of equipment by dissolution, hydrolysis, or other chemical or physical action.

Cleaning Monitoring Program - a formal, written program describing how cleaning procedures can be monitored on a regular schedule to evaluate the effectiveness and consistency of the cleaning process.



POL 1.3.18 Cleaning Performance Qualification (CPQ) - documented evidence that a cleaning procedure is consistent in removing product residue and cleaning agent from equipment.

POL 1.3.9 Cleaning Procedure - a detailed written procedure (SOP) that describes how equipment will be disassembled, cleaned, examined, and reassembled.

POL 1.3.12 Cleaning Validation - documented evidence that a cleaning procedure is consistent in removing product residue and cleaning agents from equipment (sometimes also referred to as Cleaning Performance Qualification [CPQ]).

POL 1.6 Cleaning Validation Master Plan (CVMP) - a comprehensive, written plan that describes the company's strategy in ensuring that all cleaning procedures are effective and in a state of control to ensure that all products are free of contamination and of high qUality. The plan includes or references all appropriate cleaning procedures, and SOPs describes how protocols, cleaning validation reports, and other documentation will be assembled, provides for the testing and analysis of data, identifies resources to be allocated, provides for training of personnel, describes qualification of equipment, indicates the process for assigning responsibility for the various activities, provides a criteria for revalidation of cleaning procedures, and describes a mechanism for controlling changes to validated procedures and equipment.

PROC-2.o Cleaning Validation Limits - The maximum allowable amounts of material that can remain on equipment after cleaning without compromising the safety of the consumer or the quality of the product. These limits are applied during the cleaning validation study and depending on the manufacturing circumstances, limits may be for:

• Residues of active ingredients • Residues of excipients • Degradation materials • Intermediates • Cleaning agent or by-product residuals • Bioburden • Endotoxin • Other foreign materials

POL 1.3.15 Cleaning Validation Protocol - a product specific plan of sampling and testing of equipment after at least three consecutive cleanings to establish that equipment is appropriately cleaned after a specific product is manufactured in a development area by a specific, detailed written cleaning procedure.

POL 2.0 Cleaning Validation Reports - a written report that summarizes results and conclusions of the cleaning validation study and includes:

• Protocol • Test results • Analyses • Conclusions • Discussions of any deviations from procedures specified in the original protocol • Discussion of any failures


POL 1.3.8

POL 1.5

POL 1.2

POL 1.9

PROC-2.1

PROC-2.r

PROC-2.r


• Indication as to whether the testing met the acceptance criteria specified in the protocol

Cleaning Verification Limits - Maximum amount of residue that may remain on equipment during a cleaning verification study. These limits are derived in a similar fashion to those for a cleaning validation study, but are applied to a single cleaning event, as versus multiple cleaning runs (at least three) for cleaning validation studies. Just as for cleaning validation, the limits may be for any of the following:

• Residues of active ingredients • Residues of excipients • Degradation materials • Intermediates • Cleaning agents or by-product residuals • Bioburden • Endotoxin • Other foreign materials

Cleaning Verification Policy (CVP) - a written document describing how equipment will be verified as clean after a single manufacturing event in a development area. This is a general document that will pertain to all cleaning in development areas.

Cleaning Verification Protocol- a product specific plan of experimental sampling and testing to verify that equipment is appropriately cleaned after a specific product is manufactured in a development area.

Cleaning Verification Reports - a written report that summarizes results and conclusions of the cleaning verification study and includes:

• Protocol • Test results • Analyses • Conclusions • Discussions of any deviations in procedures from those specified in the original protocol • Discussion of any failures • Indication as to whether the testing met the acceptance criteria specified in the protocol

Clean-in-Place (CIP) - cleaning of equipment that is accomplished without disassembly of the equipment but rather through the application of cleaning solutions delivered internally by one or more internal spray devices (sprayballs) or recirculation of cleaning solution throughout the equipment. CIP may be entirely automated or the cycle parameters may be controlled by the operator. This type of cleaning is also known as closed system cleaning.

Contract Manufacturing Facilities - facilities or companies that manufacture products for customers on a contractual basis.

Contract Packaging Facilities - facilities or companies that package products for customers on a contractual basis.



POL 1.3.9 Critical Cleaning Process Operating Parameter - an operating variable that is assigned a required control range with acceptability limits, outside of which exists potential for product or process failure. A critical process operating parameter is determined by process development and/or investigational work.

POL 1.3.3 Critical Product Cleaning Specifications - physico-chemical properties as well as therapeutic or medical information that are used to determine cleaning procedures and set limits for cleaning processes. Examples are solubility, stability, hydrophobicity, therapeutic potency, and toxicity.

POL 1.1 Drug Products - a finished dosage form (e.g., tablet, capsule) that contains an API, generally in association with excipients. Synonymous with finished drug product.

POL 1.1 In-Process Materials - (as applied to drug product manufacture) - any material manufactured, blended, compacted, coated, granulated, encapsulated, tableted, or otherwise processed that is produced for and used in the preparation of a drug product. (Corresponding materials used in the preparation of APIs are referred to as intermediates.)

PROC-2.1 Installation Qualification (IQ) - documented verification that equipment, system, or a subsystem has been properly installed, adheres to applicable codes and approved design intentions, and supplier recommendations have been suitably addressed.

POL 1.1 Intermediate - a material produced during steps in the synthesis of an API that must undergo further molecular change or processing before it becomes an API. The degree to which a given intermediate should be rated "critical" with respect to cleaning must be determined by a firm's experts based on such criteria as:

• Potential toxicity or other physiological activity • Degree to which equipment used is dedicated to the process, as opposed to having multiple uses • Ease or difficulty of removing process residuals when cleaning equipment

PROC-2.h Limit of Detection (LOD) - the lowest amount or concentration of a material that can be detected by an analytical instrument or chemical test. Although detectable, the amount of material in the sample cannot be determined at this level.

PROC-2.h Limit of Quantitation (LOQ) - the lowest amount or concentration of a material that can be quantitatively determined by an analytical instrument or chemical test.

PROC-2.1 Operational Qualification (OQ) - documented verification that equipment, system, or process performs as specified throughout representative or anticipated operating ranges. (Note: Overlap between IQ and OQ often occurs and is considered allowable, but should be addressed in the VMP.)

PROC-2.r Packaging - The area or department that receives bulk product and incorporates the product in packaging that will either be sent to the customer or sent to another area for additional packaging and/or labeling.

PROC-2.r Production - The unit of the company responsible for the manufacture of bulk product. This mayor may not include the packaging function depending on the size and organization.



POL-2.1 Production Authority - counterpart of quality authority, sometimes referred to as production head or, in the case of FLP (Fill-Label-Pack) operations, packaging head.

PROC-2.r Product Matrix Approach - a chart that presents medical, toxicity, solubility, and other pertinent data so that a comparison can be made between products in the group in order that the most risky product can be selected for cleaning validation. This 'worst case' approach obviates the need to perform cleaning validation studies on every combination of product and equipment.

POL 1.2 Quality Authority - one or more persons who, collectively, have formal responsibilities for specified quality-related operations, such as approval of manufacturing materials, release of finished products, review and approval of documents, and adjudication of quality assurance investigations. Titles of quality authority principals vary throughout the world; for example, in the U.S., one term "the Quality Control (QC) unit," is all embracing; in the E.U. and Canada, the head of quality control has some of the responsibilities, while a qualified person has others; terms as responsible head (or person) and quality assurance (and/or control) department are also used in other areas.

PROC-2.r Research and Development - The division of a company that is responsible for developing the optimal manufacturing techniques and dosage form for a pharmaceutical product. It is also responsible for the development of preliminary cleaning procedures for new products.

PROC-2.r Revalidation - repeating the original validation or selected portions for the purpose of demonstrating that the process is still in a state of control and delivers acceptable product and processes. As applied to cleaning procedures, the purpose would be to demonstrate that the cleaning procedures are still effective in removing residues. Revalidation is a natural consequence of making significant changes to equipment, manufacturing procedures, components, cleaning procedures, and cleaning agents.

PROC-2.r Rinse Sampling - a type of sampling of cleaned equipment used in cleaning validation and cleaning verification studies to determine if product-contact manufacturing surfaces are clean. Controlled amounts of solvent are subjected to the equipment either under pressure or allowed to stand in the equipment to allow dissolution of the residues. Mixing, spraying, and recirculation may also be used to facilitate the detection of residues. Rinse solvents are usually selected on the basis of residue solubility in that solvent. The rinses may be either heated or at ambient temperature.

PROC-2.r Scale-UplPilot Plant - Functionally, this area of responsibility is between development and full-scale production. This group is charged with scaling a formulation up from small scale to large production scale and troubleshooting problems that arise as a result of the scale-up process. They are also responsible for further refinements of the cleaning procedures handed over by development.

POL 1.2 Site Validation Steering Committee (SVSC) - a standing committee with authority and responsibilities for validation policies, practices, and adjudication of issues. Must include quality authority and Production Authority representation, and often includes representatives of other involved disciplines. The name of the SVSC may vary from firm-to-firm.



PROC-2.m Spray Devices (Sprayballs) - a device that may be either permanently installed or inserted into closed systems such as tanks specifically to provide a thorough, even coverage of the equipment surfaces by the cleaning solutions. If fixed and spherical in shape, the device is usually referred to as a "sprayball." Sprayballs may have fixed heads or they may rotate in complex patterns.

PROC-2.1 Sprayball Pattern Analysis - a study that establishes that cleaning solution delivered by sprayballs will reach all equipment surfaces, especially difficult to access or shadowed areas. The study usually consists of coating equipment with easily detectable residue (dyes or fluorescent material), activating the sprayball mechanism for a normal cycle cleaning time, and then examining the equipment to see if any material remains on the equipment surfaces.

PROC-2.r Standard Operating Procedure (SOP) - A written document describing, in detail, a specific process or procedure. These written procedures are required by the current Good Manufacturing Practice regulations for all critical processes. These procedures must be current, detailed, controlled, and revised when necessary. All personnel must be trained in a new or revised SOP prior to its implementation. Some companies have function specific procedures, e.g., cleaning procedures, that take the place of SOPs.

PROC-2.r Swab Sampling - a type of sampling of cleaned equipment used in cleaning validation and cleaning verification studies to determine if product-contact manufacturing surfaces are clean. This type of sampling makes use of small pieces of fabric (usually polyester or other synthetic material) fused to the end of a plastic strip. The swab is typically wetted with solvent (although they can be used dry). Defined surface areas of equipment, including the most difficult-to-clean locations, are swabbed. The swab is then immersed in a vial of solvent. The residue on the swab is dissolved in the solvent, which is subsequently analyzed for product residues. Limits are calculated on the basis of the area swabbed.

PROC-2.r Validation Master Plan (VMP) - a comprehensive, project-oriented action plan that includes or references all protocols, key SOPs and policies, existing Validation Task Reports * , and other relevant materials on which the specific system or process validation effort will be based. The plan also identifies resources to be allocated, specific personnel training, and qualification requirements of relevant, organizational structure, and responsibilities of the validation team, and planned schedules. The VMP is subject to periodic revisions as defined in change control procedures.

PROC-2.r

Validation Task Report - a written report that summarizes results and conclusions following execution of all or any portion of a Validation Master Plan (VMP) (often referred to as a final report if summarizing all activities of the VMP).

Worst Case Product - the product selected from a group of similar products that presents the greatest risk of carryover contamination to other products made in the same equipment by virtue of its poor solubility, unstable chemical properties, potency, toxicity, or a combination of these factors.


v. SELECTED REGULATORY EXCERPTS

Regulatory Reference

FDA Proposed Amendments to current Good Manufacturing Practice Regulations Section Ill. C (May 3, 1996)


Under CGMP, a manufacturer will set contamination limits on a substance-by-substance basis, according to both the potency of the substance and the overall level of sensitivity to that substance.

Because other substances, such as cytotoxic agents, or other antibiotics, pose at least as great a risk of toxicity due to cross-contamination, FDA is proposing to expand the contamination control requirements to encompass other sources of contamination.

The Agency has refrained from establishing a list of drugs or drug products that present such an unacceptable risk, because such a list would quickly become obsolete.

FDA Guidance for Industry: Manufacturing,Processing, or Holding Active Pharmaceutical Ingredients Section IV.D (March, 1998)

Nondedicated equipment should be thoroughly cleaned between different products and, if necessary, after each use to prevent contamination and cross-contamination. If cleaning a specific type of equipment is difficult, the equipment may need to be dedicated to a particular API or intermediate.

The choice of cleaning methods, cleaning agents, and levels of cleaning should be established and justified.

FDA Guidance for Industry: Manufacturing, Processing, or Holding Active Pharmaceutical Ingredients Section IV.E (March, 1998)

Equipment cleaning methods should be validated, where appropriate. In early synthesis steps, it may be unnecessary to validate cleaning methods where residues are removed by subsequent purification steps.

If various API's or intermediates are manufactured in the same equipment and the equipment is cleaned by the same process, a worst-case API or intermediate can be selected for purposes of cleaning validation. The worst-case selection should be based on a combination of potency, toxicity, solubility, stability, and difficulty of cleaning.

The cleaning validation protocol should describe the equipment to be cleaned, methods, materials, extent of cleaning, parameters to be monitored and controlled, and analytical methods.

Sampling should include swabbing, rinsing, or alternative methods (e.g., direct extraction), as appropriate, to detect both insoluble and soluble residues. Swab sampling may be impractical when product contact surfaces are not easily accessible due to equipment design and/or process limitations (e.g., inner surfaces of hoses, transfer pipes, reactor tanks with small ports or handling toxic materials, and small intricate equipment such as micronizers and microfluidizers).

Validated analytical methods sensitive enough to detect residuals or contaminants should be in place.

Residue limits should be practical, achievable, verifiable, and based on the most deleterious residue.



FDA Guidance for Industry: Manufacturing, Processing, or Holding Active Pharmaceutical Ingredients Section IV.F (March, 1998)

When practical, equipment in CIP systems should be disassembled during cleaning validation to facilitate inspection and sampling of inner product surfaces for residues or contamination, even though the equipment is not normally disassembled during routine use.

FDA Part 211-Current Good Manufacturing Practice for Finished Pharmaceuticals Subpart D, Section 211.67 (1990)

Equipment and utensils shall be cleaned, maintained, and sanitized at appropriate intervals to prevent contamination that would alter the safety, identity, strength, quality, or purity of the drug product beyond the official or other established requirements.

Written procedures shall be established and following for cleaning and maintenance of equipment, including utensils, used in the manufacturing, processing, packing, or holding of a drug product.

FDA Guide to Inspections of Validation of Cleaning Processes, (July, 1993)

FDA expects firms to prepare specific written validation protocols in advance for the studies to be performed on each manufacturing system or piece of equipment, which should address such issues as sampling procedures, and analytical methods to be used, including the sensitivity of those methods.

FDA expects firms to conduct the validation studies in accordance with the protocols and to document the results of studies.

FDA expects a final validation report which is approved by management and which states whether or not the cleaning process is valid. The data should support a conclusion that residues have been reduced to an "acceptable level."

Examine the design of equipment, particularly in those large systems that may employ semi-automatic or fully automatic clean-in-place (CIP) systems since they represent significant concern. For example, sanitary type piping without ball valves should be used. When such nonsanitary ball valves are used, as is common in the bulk drug industry, the cleaning process is more difficult.

Examine the detail and specificity of the procedure for the cleaning process being validated, and the amount of documentation required.

When more complex cleaning procedures are required, it is important to document the critical cleaning steps (for example certain bulk drug synthesis processes).

Determine the specificity and sensitivity of the analytical method used to detect residuals or contaminants.

The firm's rationale for the residue limits established should be logical based on the manufacturer's knowledge of the materials involved and be practical, achievable, and verifiable.

Check the manner in which limits are established.

If a detergent or soap is used for cleaning, determine and consider the difficulty that may arise when attempting to test for residues.

FDA Guide to Inspections of Bulk Pharmaceutical Chemicals (May, 1994)

Cross contamination is not permitted under any circumstances.



The cleaning program should take into consideration the need for different procedures depending on what product or intermediate was produced.

Where mUltipurpose equipment is in use, it is important to be able to determine previous usage as an aid in investigating cross-contamination or the possibility thereof.

Cleaning of multiple use equipment is an area where validation must be carried out.

Validation data should verify that the cleaning process will remove residues to an acceptable level.

There should be a written equipment cleaning procedure that provides details of what should be done and materials to be utilized.

We expect the manufacturer to establish an appropriate impurity profile for each BPC based on adequate consideration of the process and test results.

PIC Document PR 1/99-2 "Cleaning Validation" Section 1.0 (April, 2000)

Cleaning procedures must strictly follow carefully established and validated methods of execution. This applies equally to the manufacture of pharmaceutical products and bulk active ingredients.


Normally only cleaning procedures for product contact surfaces need to be validated.


Cleaning procedures for product changeover should be fully validated.


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.


Control of change to validated cleaning procedures is required. Revalidation should be considered under the following circumstances:

(a) Revalidation in cases of changes to equipment, products or processes. (b) Periodic revalidation at defined intervals.


A validation protocol is required laying down the general procedures on how cleaning processes will be validated. It should include the following:

• The objective of the validation process • Responsibilities for performing and approving the validation study • 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. • Any routine monitoring requirement • Sampling procedures, including the rationale for why a certain sampling method is used • Data on recovery studies where appropriate



• Analytical methods including the limit of detection and the limit of quantitation of those methods • The acceptance criteria, including the rationale for setting specific limits • When revalidation will be required


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 management.


The cleaning process should be documented in an SOP. 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 • The SOP defining the cleaning process • The product which was previously processed on the equipment being cleaned


The cleaning record should be signed by the operator who performed the cleaning and by the person responsible for the Production and should be reviewed by Quality Assurance.


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.


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.


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).


The existence of conditions favorable to reproduction of microorganisms (e.g., moisture, sub-strength, crevices, and rough surfaces) and the time of storage should be considered. The aim should be to prevent excessive microbial contamination.


Samples should be drawn according to a written sampling plan.




There are two methods of sampling that are considered to be acceptable: direct surface sampling (swab method) and the 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.


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.


The analytical methods should be validated before the cleaning validation study is carried out.


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.


The pharmaceutical company's rationale for selecting limits for product residues should be logically based on a consideration of the materials involved and their dosage regimes. The limits should be practical, achievable, and verifiable.


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)


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 10 ppm of any product will appear in another product. (c) No quantity of residue will be visible on the equipment after cleaning procedures are performed. Spiking

studies should determine the concentration at which most active ingredients are visible (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. 0

About the Author William E. Hall, PhD., is the President of Hall & Associates, where he provides consulting on cleaning validation, process validation, and compliance issues for the pharmaceutical industry. Dr. Hall is internationally recognized as an authority on the subject of cleaning validation. Dr. Hall serves on the Editorial AdviSOry Board of the Journal of Validation Technology, and is a member of the Institute of Validation Technology Hall of Fame. Dr. Hall received his PhD. from the University of Wisconsin, and is a former professor at the University of North Carolina. Dr. Hall can be reached by phone at 910-458-5068, by fax at 910-458-5068, or bye-mail at [email protected].


Documents

Cleaning Validation Volume III