It is also important to note that, as with any study, some additional runs should be performed in the region of greatest interest
to verify the indications from the study. In this example, the final test before proceeding to conformance runs could be a
robustness test, centered around the indicated set point (red dot) with narrow variation ranges that are still practical in
manufacturing (the blue lines).
A combination of small-scale and manufacturing-scale runs typically are used to validate a downstream process. In chromatography,
the scale-down runs are used for prospective resin lifetime studies and clearance studies, where appropriate. For viral clearance
studies, it is essential to validate the scale down prior to performing virus spiking studies. Column scale down is best designed
and validated at the manufacturer's site, where all the analytical methods are available for demonstrating comparable purity
and impurity profiles at both small and production scales.
The selection of analytical methods and their validation is critical for validating a downstream process. For clearance of
impurities other than viruses or other hazardous materials, the need to validate a small-scale model will depend on the availability
of sensitive analytical methods. For example, Q-PCR sensitivity enables the detection of DNA removal at pilot or even manufacturing
A risk assessment can be conducted to determine whether to perform a clearance study (and the scale of that study) or to include
an assay as an in-process test or lot release test. The emphasis today is on applying meaningful, in-process tests with corrective
actions enabled by automated systems, in other words process analytical technologies (PAT). The issue of replacing validation
with PAT is frequently discussed. At this time, it does not appear that better in-process control will negate the need for
formalized process validation (i.e., conformance batches), but it should certainly aid in attaining the goal of process validation
(i.e., control of variability).8
Not-to-be-Forgotten Downstream Processing Validation Issues
Column packing, storage, cleaning of packed columns and multiuse equipment, and resin lifespan studies should all be included
in the validation plan.
Column packing procedures at large scale should be validated. Repacking at large scale is expensive. Labor, buffer, and water
costs, as well as resin attrition, are all reasons to maintain a well-packed column that delivers the expected process intermediate
quality. The packing quality at different scales can vary and measurements to determine how well a column needs to be packed
to achieve requisite performance should be established so that the specifications are realistic and achievable. Several companies
have recently implemented transitional analyses to determine column packing efficiency. This technique allows routine measurements
of column efficiency by measuring a step change that is part of the manufacturing protocol (e.g., a change in conductivity
in an ion exchange step).9 Time needs to be allotted to validate column packing at large scale. Up to three months or more may be required for this
Storage of Columns and Process Intermediates
Regulatory agencies direct attention to the storage of process intermediates and packed columns. Stability studies for process
intermediates should demonstrate control over bioburden, proteolytic degradation, aggregation, and other potential product
modifications. The stability of column storage solutions and the removal of storage agents prior to column reuse should be
validated. Validated rapid microbiology methods may now enable faster turnaround times and reduce the amount of processing
that is carried out at risk while waiting for quality control bioburden results.
Cleaning packed columns should be addressed early in development. Designing a robust cleaning protocol is essential to prevent
carryover of impurities and residual target molecules, some of which may be degraded. New developments include the use of
in-line total organic carbon, but product-specific or protein assays may also be needed for validation. As with other validation
activities, a risk assessment can dictate which assays are most appropriate. Factors that should be evaluated include feedstream
impurities; chromatography mechanism (i.e., binding or flow-through); and location of step in purification train (i.e., early
capture or final polishing step).