Impact of Prefiltration
Prefiltration of the feed solution can have a dramatic impact on filter performance. Prefiltration is targeted to remove various
impurities or contaminants, such as protein aggregates, DNA, and other trace materials. Although larger impurities can be
removed by prefiltering using 0.2 μm or 0.1 μm microfilters, smaller impurities, such as protein aggregates that may be only
marginally larger than the protein product, are not easily removed using size-based removal methods. Prefiltration through
adsorptive depth filtration has been observed to provide significant protection for certain virus removal filters.7 The impact of prefiltration can be dramatic, with up to a 10-fold reduction in required filter area.
Impact of Hold Times and Freeze-Thaw Cycles
Some proteins exhibit time-dependent aggregate formation or will form low concentrations of aggregates when subjected to a
freeze–thaw cycle. If a hold step or a freeze-thaw cycle is expected to be a part of the process, it is important to evaluate
the effects of these during filter optimization. Furthermore, although the actual purification process may not have a freeze–thaw
step, feed samples required for virus retention testing are often conveniently submitted in a frozen form due to material
Parvovirus filters can be broadly classified into two groups: those with high protein flux but low-to-moderate volumetric
capacities, and those with high protein capacities but low-to-moderate protein fluxes. Both of these filters have advantages
and disadvantages. For example, for a process that must be completed in a short amount of time (2–4 h), high-flux filters
require less filtration area than high-capacity filters do (Figure 3). On the other hand, when processing times are extended
to 6 hours or more, high-capacity filters may be more economical. (This example assumes that all filters provide necessary
Figure 3. The graph shows the filtration area in m2 needed to filter 1,000 L of protein as a function of process time through different parvovirus filters. For short processing
times (2–4 h), high flux filters generally require less filter area than high capacity filters do.
Designing Virus Retention Qualification Studies
Spiking studies should be designed to reflect the virus clearance capability of the process-scale unit operation.2 Therefore, the level of purification of the scaled-down version should represent the production process as closely as possible,
by reproducing the key operating parameters that have an effect on purification and on virus clearance. The critical operating
parameters will vary depending on clearance technologies (due to the different mechanisms of action). For filtration, scale-down
will focus on such parameters as:
- Identity between process and retention study filter media
- Flow rate or pressure
- Ratio of volume processed to filter-surface area (V/A) or the extent of flow decay
- Equivalence of process and retention study feedstocks
- Product yield and quality.
Typically, the study sponsor may rely on the filter manufacturer to supply data required to support the claim of scale-down
Although not specified in regulatory guidance, some manufacturers choose to use worst-case process-variable settings in the
design of the spiking study.
Appropriate guidance on how to plan and carry out virus retention qualification studies is available from regulatory agencies.3 Documents such as the Parenteral Drug Association Technical Report 41 provide guidance on how to determine the quantity
of virus spike needed to achieve necessary log reduction values (LRV).10