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
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.