A key consideration in bioprocessing is that the active ingredient is usually degraded by cleaning processes that involve
hot, aqueous, alkaline cleaning solutions. Although this degradation is a key mechanism of the cleaning process, and in many
cases is required to remove denatured proteins from surfaces, it affects various elements of cleaning validation. For example,
after a cleaning process, the active ingredient itself should not be present on cleaning surfaces; if residues are present,
they may exist as degradation products of the active ingredient. Also, a specific analytical method for the active ingredient
is not usually an appropriate analytical technique to determine whether the cleaning process is effective. These effects are
discussed in more detail in Technical Report No. 49.
Because of the degradation of the active ingredients in the cleaning processes for biotech manufacture, the most common practice
is to use TOC as the analytical procedure to indicate the removal of the active. TOC also measures other sources of organic
carbon, including media, cellular materials, detergents, and organic process materials. If total protein is used as a nonspecific
analytical method for the active, that method may measure various protein species. Other methods, such as conductivity, may
be used for the cleaning agent.
The appropriate analytical methods must be validated. Typically, validation involves principles from the International Conference
on Harmonization's Q2 (R1) (4). Although degraded fragments of the active ingredient are measured in a cleaning-validation
protocol, analytical method validation is typically performed with the bulk active itself because this typically reflects
the worst case.
ACCEPTANCE LIMITS FOR ACTIVES
Personnel demonstrate the effectiveness and consistency of a cleaning procedure by showing that the cleaning process can reduce
the amounts of potentially adverse residues to acceptable levels. For nonbiotech applications, limits for the active ingredient
are typically established using a carryover calculation, which is based on the safety or toxicity of the active ingredient.
For the manufacture of biotech products, however, that approach only works for fill–finish manufacture, where it is assumed
that all measured organic carbon comes from the active ingredient (a worst-case assumption).
Table IV provides a list of the methods that can help set limits for the various cleaning applications (1). Limits for cleaning
validation generally contain a measure related to the active protein or other major component of interest, a measure related
to the cleaning agent, a measure related to bioburden levels, a measure related to endotoxin levels, and a requirement that
the equipment be visually clean. In addition, if the active protein or other process components raise specific toxicity concerns
(e.g., cytotoxicity, allergenicity, or reproductive hazards), the manufacturer's toxicology or pharmacology groups may determine
whether limits should be modified or whether dedicated equipment is needed.
That approach does not work for limits for the active ingredient in bulk active manufacture. If the carryover limits are calculated
using the entire equipment train's surface area, the limits are extremely low. If the active ingredient were undegraded after
the cleaning process, it might be possible to measure the active ingredient using a specific analytical technique, such as
enzyme-linked immunosorbent assay (ELISA). When the carryover limit for the active is converted to a TOC value, it typically
is below a quantifiable TOC value for a swab or rinse value. That quantifiable value is close to 100 or 200 ppb carbon because
of the background subtraction (i.e., the correction for the blank values).
A further complicating factor is that in the manufacture of the bulk active, residues left after earlier cleaning steps (e.g.,
until the first purification-process step) may be removed by subsequent purification processes, such as chromatographic purification.
Therefore, consistent with ICH Q7, the cleaning of these earlier steps may not be critical for the carryover of residues to
the final bulk active (5). Only a few literature references document the degradation of specific drug active proteins during
the cleaning process. However, the literature contains ample evidence that proteins generally will degrade in hot, alkaline
cleaning solutions. Although not well documented, this effect further mitigates the concern about carryover of residues in
bulk active manufacture.
For these reasons, limits for the manufacture of bulk actives in biotech are generally established based on industry standard
practice of about 5–10 ppm TOC for upstream processes and 1–2 ppm for downstream processes for any analytical sample, whether
a swab sample or a rinse sample. The industry needs to provide more scientific rationales and data to support that practice,
and such improvements in support documentation have started to occur.