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A cleaning process should remove materials such as media, buffers, storage solutions, cell culture fluids, cell debris, non-active pharmaceutical ingredient, and formulations and concentrations of active pharmaceutical ingredients. Manufacturing and cleaning equipment must be designed for effective and consistent cleaning to avoid cross-contamination and the cleaning processes must be verified as effective. Part 1 of this article provided background on cleaning validation and the associated regulations, cleaning methods, validation strategy, and new product introduction. Part 2 covers Genentech's grouping strategy, 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 that a 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 cycle development, cleaning validation, and maintenance. The program begins with equipment design evaluation such as sanitary equipment, sprayball, rinse water, and compatibility of construction materials with product and cleaning solutions, followed by cleaning process development and cleanability studies. Cleaning validation must be performed using a pre-approved protocol. Selection of appropriate sampling to demonstrate 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 visual inspection can reduce the number of samples required for cleaning validation. Ongoing monitoring, change control, and revalidation constitute the maintenance program. This article covers Genentech's grouping strategy, validation samples, acceptance criteria, clean hold time, training, change control, and revalidation.
To simplify cleaning validation, similar equipment may be grouped into equipment families. These families are based on equipment design, construction material, geometry, complexity, functionality, or cleaning procedure. Such families involve only the equipment for manufacturing similar products that are cleaned by the same or similar cleaning processes. Grouping may apply to equipment that is cleaned by an automated clean-in-place (CIP) process, a semi-automated CIP process, a manual cleaning process, or an automated parts 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 smallest portable tanks in a group), or it may involve testing only the worst case in a group (for example, the most difficult to clean transfer line). The following are some examples of equipment families: bracketed equipment, identical equipment, small equipment or parts cleaned manually, and small equipment or parts cleaned 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. Identical equipment such as lyophilizers, 400-liter fermentors, transfer pumps, and 120-liter freeze-thaw tanks, are of the same scale.
Equipment that is validated individually and not as a family, because of significant differences in design and cleaning processes, is called unique equipment or unique systems. Examples of unique equipment include large-scale harvest cell culture systems, centrifuge systems, and chromatography systems.
Cleaning following the manufacture of products may be validated for individual products or may be validated by product group. Grouping by products includes considerations of potency, toxicity, and cleaning difficulty. Products in groups must be manufactured using the same equipment categories and cleaned by the same or similar cleaning processes. Chemical and physical properties of ingredients (including excipients) 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 may be validated individually or by product group. Acceptable validation of extreme or worst case constitutes acceptable validation for all group members. Grouping must be justified and documented.
Sampling depends on the characteristics of soiling and cleaning agents. At Genentech, swabbing and visual inspection are used to inspect product-contact surfaces directly for assessment of surface cleanliness. Visual inspection and surface Fourier transform infrared spectroscopy (FTIR) are sometimes called "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 methods should 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 for injection) is not acceptable; however, a risk-based method can be applied to determine appropriate sampling type.1 Both rinse and swab samplings are considered acceptable methods of sampling in cleaning validation guidance documents.2,3
In the early 1990s, there was an inclination toward swab sampling for cleaning validation. This was possibly because at that time, equipment was designed to be disassembled for effective cleaning, and this made swab sampling very convenient. More recently, as more large equipment has been designed for CIP cleaning and therefore designed not to be disassembled, it makes less sense to require tank entry to perform swabbing. Opening up the system for entry—as opposed to depending on rinse sampling alone for cleaning validation purposes—leads to concerns about operator safety, as well as concerns about equipment cleanliness and resulting product quality. The use of a remote camera to inspect the interior of a large 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 aqueous alkaline cleaning solutions. This means that the specific analytical method for measuring the native protein may not be an appropriate method for measuring residues following cleaning. Thus, TOC is a good measure of the overall cleanliness of equipment following a cleaning process. A TOC assay will detect organic carbon in product residues including degraded protein, cell culture or fermentation media, buffers with organic carbon, and other organic materials, including organic components of formulated cleaning agents. Rinse sampling is less technique-dependent, but swab sampling is like manual cleaning in that it is highly operator-dependent. Collecting a swab sample requires that the equipment surface be exposed to the 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. Equipment surfaces 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 visual residue limit provides a nonselective cleaning assessment and visual detection limits should be established for 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 the analytical 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 cleaning process, no acceptance limit is established for a cleaning agent. If the cleaning agent is solely a chemical species that is subsequently used in manufacturing as a process chemical (e.g., sodium hydroxide for pH adjustment), 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, and they may include swab sampling, rinse sampling, or both. Swab sampling sites are readily accessible and include 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 potential residues that may be on the product-contact surfaces of equipment. A laboratory sampling recovery for protein residues must be performed for each combination of chemical residue, sampling method, and sampled surface material of construction specified in a validation protocol. Recovery studies are not appropriate for bioburden and endotoxin measurements in cleaning validation. Validated analytical methods 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 effectively detect residual carbonaceous materials and that rinse recovery is greater or equal to swab recovery, swab sampling is not required in cleaning validation studies. Justifications for not performing swab sampling such as swab results from cleanability and visual inspections of hard-to-clean (worst-case) locations, are documented. Note that swab sampling can nevertheless be used in cases where incomplete cleaning solution coverage is suspected and for investigation purpose when visual inspection fails. Genentech biopharmaceutical products are water-soluble proteins in aqueous-based solutions, and rinse water sampling has been shown to be an effective method for collecting cleanliness data. It has been found at Genentech that swab sampling routinely exhibits lower recovery levels than rinse sampling (the typical rinse recovery is greater than or equal to 80%).
Residue acceptance criteria are established for the active pharmaceutical ingredient (API), the cleaning agent, the bioburden, and the endotoxin after cleaning. These acceptance criteria are based on process capabilities 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 earlier manufacturing steps are removed by subsequent purification steps, then those earlier cleaning processes may not require cleaning validation. At Genentech, process characterization and validation demonstrate removal of process- and product-related residues in the purification steps. However, for process efficiency reasons, validation is required for fermentation and purification equipment.
Maximum allowable carryover (MAC) must be evaluated, and the rationale for choosing that MAC should be justified and documented. MAC calculation is typically performed on worst-case equipment in formulation and filling areas. For a finished drug product, the acceptance limit for an API residue on cleaned equipment surfaces should be no more than 0.001 of the normal therapeutic dose of an API that appears in a maximum dose of a subsequently manufactured product. Normal therapeutic dose means the recommended 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 multiplying the limit for the protein by the fraction of carbon in the protein. If the API has unusual health effects such as being cytotoxic, or being a reproductive hazard, the safety concerns should be evaluated in setting residue limits for the protocol. This evaluation may result in the need for limits at the boundary of detection of the protein, 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 toxic chemicals, the acceptance limit for the cleaning agent on cleaned equipment surfaces is determined using calculations in Parenteral Drug Association Technical Report 29.4 Control of the bioburden through adequate cleaning and storage of equipment is important to ensure that subsequent sterilization or sanitization procedures achieve the necessary assurance of sterility. There should be documented evidence 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 acceptance criteria are based on the equipment process step, final rinse water quality, and the capability of the cleaning 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 for any rinse sample in final purification equipment and fill or finish equipment is derived from rinse water quality.
Validation sampling and testing methods are used for determining equipment cleanliness, but they may not measure the actual carryover of cleaning process residues from one use of the equipment train to the next use. Because of very low concentrations of active ingredients in biopharmaceuticals, a MAC calculation using the surface areas of the entire equipment train may not be feasible. Water-fill carryover studies can be conducted at the end of cleaning or in conjunction with postcleaning hold time validation studies, and can 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 all biochemical processes use water-soluble solutions. These studies are very useful in documenting actual process carryover, and they are typically performed in fill or finish areas at Genentech.
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 are specified 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 equipment or 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 equipment undergoes autoclave (or SIP) or production usage after CIP within a manufacturing shift (or for approximately eight hours), then a clean hold time need not be established, provided the circumstances are justified and documented. Clean hold time validation is not required for indirect product-contacting equipment 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 potential accumulation 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 and endotoxin levels in the equipment after a predetermined storage time. One protocol study, comprising three individual runs, for validation of an expiry period for a given equipment family may apply to all pieces of 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 that may occur subsequent to cleaning and storage condition. Therefore, clean hold time bioburden criteria may exceed the end-of-cleaning (time-zero) bioburden criteria. The justification for the bioburden acceptance criteria should be documented. If the validated clean hold time elapses, the equipment must be cleaned again before use.
Changes to a validated cleaning process, and changes to a manufacturing process or equipment that may affect a validated cleaning process, must be made in accordance with approved change control procedures. Such a change may result in additional testing to demonstrate that cleaning processes remain in a state of control. Revalidation of cleaning processes is performed following any significant change to a cleaning process that may affect its validated state. A change evaluation process determines the extent of validation required.
Periodic review and revalidation are methods by which the performance of a validated cleaning process is evaluated to ensure that a state of control is maintained. At Genentech, a risk-based approach is used for setting periodic review and revalidation frequencies. The rationale for the revalidation frequencies should be 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 equipment family) according to an established frequency. Such a review includes an assessment of cleaning-process documentation, including changes implemented under the change control program since prior revalidation to 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 each unique equipment or system on an established frequency, provided that the equipment has been used to manufacture a product that year. Such a validation run may include any product manufactured using the unique system. For each equipment family cleaned by the same or similar cleaning procedures, one successful cleaning validation run is also conducted on an established frequency using one equipment item from the applicable equipment family. This validation run may include any product manufactured using the equipment family. Each major equipment item in each family is included in cleaning validation runs in a predetermined period. Major equipment refers to product-contacting equipment that serves a significant purpose in the manufacture of product (i.e., the main equipment used in process unit operation). Examples include fermentors, hold tanks, purification equipment, and lyophilizers.
Training regarding approved processes or protocols used for cleaning validation studies such as cleaning processes and sampling processes, is performed and documented by approved training policies or procedures. Personnel who collect samples for cleaning validation and who perform visual inspections must be trained before these collections and inspections. Operators are routinely retrained for validated manual cleaning procedures, particularly when there has been a change in any validated cleaning procedure.
Cleaning validation is driven by regulatory requirements to ensure that residues from one product will not carry 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 by approved procedures, training, change control, monitoring, and revalidation. A cleaning validation strategy that is based on the evaluation of potential risks increases success rate and reduces execution time.
The author is thankful to Lara Collier for management support and to Jenna Carlson and Ahmed Bassyouni for reviewing the manuscript and providing comments.
A. Hamid Mollah, PhD, is a senior technical manager for corporate quality and validation at Genentech, Inc., South San Francisco, CA, 650.467.1095, email@example.com
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. Validation master 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 to consider for cleaning validation. Bethesda, MD; 1998 Aug.