VALIDATION SAMPLING

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.

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%).

ACCEPTANCE CRITERIA

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.

Water-Fill Carryover Studies

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.