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Cleaning Validation for Biopharmaceutical Manufacturingat Genentech, Inc. Part 2By A. Hamid Mollah, PhD

ABSTRACT

A clean ing process should remove mater ials such as media, buf fers, s torage solu tions, cell culturefluids, cell debris, non-active pharmaceutical ingredient, and formulations and concentrations ofactive pharmaceutical ingredients. Manufacturing and cleaning equipment must be designed foreffective and cons istent cleaning to avoid cross-contamination and the cleaning processes mustbe verified as effective. Part 1 of this article provided background on cleaning validation and theassociated regulations, cleaning methods, validation strategy, and new product introduction. Part 2covers Genentech's grouping st rategy, validation samples, acceptance criteria, clean hold time,training, change control, and revalidation.

Cleaning validation refers to establishing documented evidence providing a high degree of assurance thata specific specific cleaning process will produce consistent and reproducible cleaning results that meet a

predetermined level. A cleaning program can be divided into three phases: cleaning process and cycledevelopment, cleaning validation, and maintenance. The program begins with equipment design evaluationsuch as sanitary equipment, sprayball, rinse water, and compatibility of construction materials with productand cleaning solutions, followed by cleaning process development and cleanability studies. Cleaningvalidation must be performed using a pre-approved protocol. Selection of appropriate sampling todemonstrate that residues are removed to an acceptable level is vital for the success of cleaning validation.In addition, use of sampling techniques such as recovery study for swab and rinse and thorough visualinspection can reduce the number of samples required for cleaning validation. Ongoing monitoring, changecontrol, and revalidation constitute the maintenance program. This article covers Genentech's groupingstrategy, validation samples, acceptance criteria, clean hold time, training, change control, and revalidation.

GROUPING OF EQUIPMENT

To simplify cleaning validation, similar equipment may be grouped into equipment families. These familiesare based on equipment design, construction material, geometry, complexity, functionality, or cleaningprocedure. Such families involve only the equipment for manufacturing similar products that are cleaned bythe same or similar cleaning processes. Grouping may apply to equipment that is cleaned by an automatedclean-in-place (CIP) process, a semi-automated CIP process, a manual cleaning process, or an automatedparts washer. Grouping into equipment families must be justified and documented.

Grouping may involve separately validating the extremes of a group (for example, the largest and smallestportable tanks in a group), or it may involve testing only the worst case in a group (for example, the mostdifficult to clean transfer line). The following are some examples of equipment families: bracketedequipment, identical equipment, small equipment or parts cleaned manually, and small equipment or partscleaned in an automated parts washer. Bracketed equipment such as portable stainless steel tanks,fermentors, glass vessels, transfer lines, filler parts, and glass carboys may vary in scale. Identicalequipment such as lyophilizers, 400-liter fermentors, transfer pumps, and 120-liter freeze-thaw tanks, areof the same scale.

Equipment that is validated individually and not as a family, because of significant differences in designand cleaning processes, is called unique equipment or unique systems. Examples of unique equipmentinclude large-scale harvest cell culture systems, centrifuge systems, and chromatography systems.

GROUPING OF PRODUCTS

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Cleaning following the manufacture of products may be validated for individual products or may bevalidated by product group. Grouping by products includes considerations of potency, toxicity, and cleaningdifficulty. Products in groups must be manufactured using the same equipment categories and cleaned bythe same or similar cleaning processes. Chemical and physical properties of ingredients (includingexcipients) must be considered when grouping by products; excipients may be more difficult to clean.

Cleaning following the manufacture of buffers, media, and similar indirect product-contacting surfaces maybe validated individually or by product group. Acceptable validation of extreme or worst case constitutesacceptable validation for all group members. Grouping must be justified and documented.

VALIDATION SAMPLING

Sampling depends on the characteristics of soiling and cleaning agents. At Genentech, swabbing andvisual inspection are used to inspect product-contact surfaces directly for assessment of surfacecleanliness. Visual inspection and surface Fourier transform infrared spectroscopy (FTIR) are sometimescalled "real" direct surface sampling. Rinsate sample testing for pH, conductivity, total organic carbon(TOC), bioburden, and endotoxin are indirect testing methods. Both direct and indirect sampling methodsshould be used to measure residues in cleaning validation. Establishing limits for final rinse water based

solely on compendial water specification (such as final rinse meeting conductivity specification for water forinjection) is not acceptable; however, a risk-based method can be applied to determine appropriatesampling type. Both rinse and swab samplings are considered acceptable methods of sampling incleaning validation guidance documents.

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In the early 1990s, there was an inclination toward swab sampling for cleaning validation. This waspossibly because at that time, equipment was designed to be disassembled for effective cleaning, and thismade swab sampling very convenient. More recently, as more large equipment has been designed for CIPcleaning and therefore designed not to be disassembled, it makes less sense to require tank entry toperform swabbing. Opening up the system for entryas opposed to depending on rinse sampling alone forcleaning validation purposesleads to concerns about operator safety, as well as concerns aboutequipment cleanliness and resulting product quality. The use of a remote camera to inspect the interior of alarge tank (e.g., a 20,000-liter bioreactor) is being introduced at Genentech.

In biotech manufacturing, protein actives are degraded during the cleaning process using hot aqueousalkaline cleaning solutions. This means that the specific analytical method for measuring the native proteinmay not be an appropriate method for measuring residues following cleaning. Thus, TOC is a goodmeasure of the overall cleanliness of equipment following a cleaning process. A TOC assay will detectorganic carbon in product residues including degraded protein, cell culture or fermentation media, bufferswith organic carbon, and other organic materials, including organic components of formulated cleaningagents. Rinse sampling is less technique-dependent, but swab sampling is like manual cleaning in that it ishighly operator-dependent. Collecting a swab sample requires that the equipment surface be exposed tothe environment, often by equipment disassembly, and be exposed to the sample-collection technician.These situations can cause false positive TOC results that require further investigation. Equipmentsurfaces that can be examined must be visually clean after cleaning procedures are performed. Visually

clean means that the surfaces have no visible residues when viewed under appropriate lighting. A visualresidue limit provides a nonselective cleaning assessment and visual detection limits should be establishedfor residues.

Removal of the cleaning agent is demonstrated through selection of a freely rinsable cleaning agent,establishment of a correlation between analytical method and concentration, and sensitivity of theanalytical method. Indicator species are measured to detect residual cleaning agent levels. For example,residual CIP200 is indicated by phosphorus or rinsate conductivity. If water alone is used for the cleaningprocess, no acceptance limit is established for a cleaning agent. If the cleaning agent is solely a chemical



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species that is subsequently used in manufacturing as a process chemical (e.g., sodium hydroxide for pHadjustment), then complete removal of the species as part of a cleaning validation may not be applicable.

Sampling techniques must be appropriate for the equipment surfaces and for the nature of the study, andthey may include swab sampling, rinse sampling, or both. Swab sampling sites are readily accessible andinclude any worst-case locations, as well as locations representative of different functional parts and

different construction materials. Rinse samples are collected in a manner representative of potentialresidues that may be on the product-contact surfaces of equipment. A laboratory sampling recovery forprotein residues must be performed for each combination of chemical residue, sampling method, andsampled surface material of construction specified in a validation protocol. Recovery studies are notappropriate for bioburden and endotoxin measurements in cleaning validation. Validated analyticalmethods are used at Genentech to measure each residue for which an acceptance limit is established.Compendial methods do not require validation.

When it is established during a cleanability study that rinsate TOC and visual inspection can effectivelydetect residual carbonaceous materials and that rinse recovery is greater or equal to swab recovery, swabsampling is not required in cleaning validation studies. Justifications for not performing swab samplingsuch as swab results from cleanability and visual inspections of hard-to-clean (worst-case) locations, aredocumented. Note that swab sampling can nevertheless be used in cases where incomplete cleaningsolution coverage is suspected and for investigation purpose when visual inspection fails. Genentechbiopharmaceutical products are water-soluble proteins in aqueous-based solutions, and rinse watersampling has been shown to be an effective method for collecting cleanliness data. It has been found atGenentech that swab sampling routinely exhibits lower recovery levels than rinse sampling (the typicalrinse recovery is greater than or equal to 80%).

ACCEPTANCE CRITERIA

Residue acceptance criteria are established for the active pharmaceutical ingredient (API), the cleaningagent, the bioburden, and the endotoxin after cleaning. These acceptance criteria are based on processcapabilities or industry practices. Because of the purification that occurs during the processing steps of the

API, upstream process acceptance criteria may be less stringent than for downstream processes. In

accordance with the ICH Q7 principle for API manufacturing, if residues from cleaning for earliermanufacturing steps are removed by subsequent purification steps, then those earlier cleaning processesmay not require cleaning validation. At Genentech, process characterization and validation demonstrateremoval of process- and product-related residues in the purification steps. However, for process efficiencyreasons, validation is required for fermentation and purification equipment.

Maximum allowable carryover (MAC) must be evaluated, and the rationale for choosing that MAC shouldbe justified and documented. MAC calculation is typically performed on worst-case equipment informulation and filling areas. For a finished drug product, the acceptance limit for an API residue oncleaned equipment surfaces should be no more than 0.001 of the normal therapeutic dose of an API thatappears in a maximum dose of a subsequently manufactured product. Normal therapeutic dose means therecommended minimum daily dose of the active drug substance for an average-size patient. Measured

TOC in analytical samples is treated as if it were all from the API, which represents a worst-case condition.If the protein is to be measured by TOC, the limit for the protein may be converted to TOC by multiplyingthe limit for the protein by the fraction of carbon in the protein. If the API has unusual health effects such asbeing cytotoxic, or being a reproductive hazard, the safety concerns should be evaluated in setting residuelimits for the protocol. This evaluation may result in the need for limits at the boundary of detection of theprotein, or in the need to manufacture the product using dedicated equipment.

The final rinse step is aimed at complete removal of cleaning agents. For a cleaning agent containing toxicchemicals, the acceptance limit for the cleaning agent on cleaned equipment surfaces is determined usingcalculations in Parenteral Drug Association Technical Report 29. Control of the bioburden through4



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adequate cleaning and storage of equipment is important to ensure that subsequent sterilization orsanitization procedures achieve the necessary assurance of sterility. There should be documentedevidence that routine cleaning and storage of equipment do not allow microbial proliferation. Hence,bioburden is monitored during cleaning validation and clean hold time studies. Bioburden acceptancecriteria are based on the equipment process step, final rinse water quality, and the capability of thecleaning and sampling processes. Depending on the system and sampling technique, it may not be

feasible to achieve final rinse water bioburden quality in a grab sample. The endotoxin acceptance limit forany rinse sample in final purification equipment and fill or finish equipment is derived from rinse waterquality.

Water-Fill Carryover Studies

Validation sampling and testing methods are used for determining equipment cleanliness, but they may notmeasure the actual carryover of cleaning process residues from one use of the equipment train to the nextuse. Because of very low concentrations of active ingredients in biopharmaceuticals, a MAC calculationusing the surface areas of the entire equipment train may not be feasible. Water-fill carryover studies canbe conducted at the end of cleaning or in conjunction with postcleaning hold time validation studies, andcan express their results in terms of worst-case carryover into a minimum volume subsequent batches.Water is used to mimic the next batch (i.e., filling product vials using fill or finish equipment), as allbiochemical processes use water-soluble solutions. These studies are very useful in documenting actualprocess carryover, and they are typically performed in fill or finish areas at Genentech.

CLEANED EQUIPMENT STORAGE AND HOLD TIMES

The duration between the end of equipment cleaning and the start of subsequent operations (e.g.,autoclave, sterilization-in-place [SIP], or production usage) is called clean hold time. Following cleaning,equipment to be reused should be stored to protect it from contamination. Storage instructions arespecified in a document such as that delineating the cleaning procedure or the storage procedure.

Equipment should be stored dry following cleaning. There should be no visible water pool in the equipmentor line after draining (including air blowing) when viewed under appropriate lighting conditions. An

exception to the dry storage recommendation occurs when equipment is stored in solution. If equipmentundergoes autoclave (or SIP) or production usage after CIP within a manufacturing shift (or forapproximately eight hours), then a clean hold time need not be established, provided the circumstancesare justified and documented. Clean hold time validation is not required for indirect product-contactingequipment that undergoes autoclave or SIP, when operational controls are in place to minimize bioburden,and when a documented risk assessment demonstrates no risk to product quality from potentialaccumulation of bioburden and endotoxin as a result of prolonged storage.

A validation study is used to establish an expiry period. The study includes measuring bioburden andendotoxin levels in the equipment after a predetermined storage time. One protocol study, comprisingthree individual runs, for validation of an expiry period for a given equipment family may apply to all piecesof equipment in the family, to all products using that equipment, and to all cleaning processes for that

equipment, provided the final state of the cleaned equipment and the storage conditions are consistent.The bioburden criteria for clean hold times takes into account the relevant microbial control procedures thatmay occur subsequent to cleaning and storage condition. Therefore, clean hold time bioburden criteriamay exceed the end-of-cleaning (time-zero) bioburden criteria. The justification for the bioburdenacceptance criteria should be documented. If the validated clean hold time elapses, the equipment must becleaned again before use.

CHANGE CONTROL AND REVALIDATION



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Changes to a validated cleaning process, and changes to a manufacturing process or equipment that mayaffect a validated cleaning process, must be made in accordance with approved change controlprocedures. Such a change may result in additional testing to demonstrate that cleaning processes remainin a state of control. Revalidation of cleaning processes is performed following any significant change to acleaning process that may affect its validated state. A change evaluation process determines the extent ofvalidation required.

Periodic review and revalidation are methods by which the performance of a validated cleaning process isevaluated to ensure that a state of control is maintained. At Genentech, a risk-based approach is used forsetting periodic review and revalidation frequencies. The rationale for the revalidation frequencies shouldbe appropriately documented and justified. The revalidation frequency for product-contact surfaces (e.g.,bioreactor and pool tanks) and manual cleaning should be higher than for indirect product-contact surfaces(e.g., media and buffer tanks).

A periodic review is performed on each combination of cleaning process and equipment (or equipmentfamily) according to an established frequency. Such a review includes an assessment of cleaning-processdocumentation, including changes implemented under the change control program since prior revalidationto determine if processes are still in a state of control. The periodic review should be documented.

In addition to a periodic document review, one successful cleaning validation run is conducted for eachunique equipment or system on an established frequency, provided that the equipment has been used tomanufacture a product that year. Such a validation run may include any product manufactured using theunique system. For each equipment family cleaned by the same or similar cleaning procedures, onesuccessful cleaning validation run is also conducted on an established frequency using one equipmentitem from the applicable equipment family. This validation run may include any product manufactured usingthe equipment family. Each major equipment item in each family is included in cleaning validation runs in apredetermined period. Major equipment refers to product-contacting equipment that serves a significantpurpose in the manufacture of product (i.e., the main equipment used in process unit operation). Examplesinclude fermentors, hold tanks, purification equipment, and lyophilizers.

TRAINING

Training regarding approved processes or protocols used for cleaning validation studies such as cleaningprocesses and sampling processes, is performed and documented by approved training policies orprocedures. Personnel who collect samples for cleaning validation and who perform visual inspectionsmust be trained before these collections and inspections. Operators are routinely retrained for validatedmanual cleaning procedures, particularly when there has been a change in any validated cleaningprocedure.

CONCLUSION

Cleaning validation is driven by regulatory requirements to ensure that residues from one product will notcarry over and cross contaminate the next product. The cleaning program consists of equipment design

and qualification, a cleanability study, sampling evaluation, and meeting predetermined acceptance criteria.Dirty and clean hold times are established during cleaning validation. Cleaning validation is supported byapproved procedures, training, change control, monitoring, and revalidation. A cleaning validation strategythat is based on the evaluation of potential risks increases success rate and reduces execution time.

ACKNOWLEDGMENTS

The author is thankful to Lara Collier for management support and to Jenna Carlson and AhmedBassyouni for reviewing the manuscript and providing comments.



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A. Hamid Mol lah, PhD, is a senior technical manager for corporate quality and validation at Genentech,Inc., South San Francisco, CA, 650.467.1095, [email protected][http://mailto:[email protected]]

REFERENCES

1. Mollah AH, White EK. A risk based cleaning validation in biopharmaceutical API manufacturing.BioPharm International 2005;18(11):54-67.

2. US Food and Drug Administration. Guide to inspections of validation of cleaning processes. Rockville,MD; 1993 Jul.

3. Pharmaceutical Inspection Convention and Pharmaceutical Inspection Co-operation Scheme. Validationmaster plan installation and operational qualification: Non-sterile process validation. Cleaning validation.Geneva, Switzerland; 2004 Jul.

9. Parenteral Drug Association Office of Science and Technology. Technical report no. 29: Points toconsider for cleaning validation. Bethesda, MD; 1998 Aug.

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