The value of upfront virus filtration validation studies

Well-defined viral filtration validation studies lead to successful commercial scale processes that save time and money while optimizing quality.

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The need for pre-filtration

In addition to understanding the relationship between process and validation targets and taking into consideration the target reduction level, spike purity, spike ratio, sample volume, impact of freezing and thawing, and the use of highly sensitive assays, upfront studies can be highly beneficial for the development of effective virus filtration validation studies.

This is particularly true if pre-filtration of the process solution is necessary prior to performance of the validation study to remove aggregate impurities that may foul the virus filter. Prefilters, while having the potential to dramatically improve throughput, are not validated for virus removal. Removal claims can therefore only be associated with the validated virus filter. The level of virus removal achieved by the virus filter alone must be determined.

To decouple or not?

If a prefiltration step is needed prior to the virus filtration validation study, there are two approaches that can be used: coupled or decoupled. When coupled, the process solution with the virus spike is passed through both the prefilter and virus filter. When decoupled, the process solution is passed through the prefilter and then the virus spike is added before the solution passes through the virus filter.

Decoupling is preferred if it does not impact throughput of the virus filter, because it is easier to determine validated virus removal. The use of prefilters can add complexity, however, and their impact must be evaluated to determine whether a decoupled strategy is possible.

For instance, adsorptive prefilters may bind and remove viruses, resulting in less virus being delivered to the actual virus filter. It is also possible that protein aggregates may reform after the prefiltration step. Aeration during prefiltration may also affect the process solution. Consequently, determination of the impact of the prefilter on virus filter throughput performance must be determined via a decoupling study before committing to a coupled or decoupled approach.

The decision tree shown in our application note ‘Validating Pegasus™ Prime Virus Membrane Filters – How do I Incorporate a Prefilter in my Virus Clearance Study’ which provides a systematic method for choosing the most appropriate test approach to ensure maximum virus retention and optimum throughput based on the results of the decoupling study. Please contact us for a copy.

If decoupling does not impact virus filter performance, then the validation study should use a decoupled prefilter. The test solution is prefiltered and the filtrate is pooled. The pooled, prefiltered solution is then spiked with the virus before challenging the test virus filter. The flow rate of the prefilter should match the flow rate of the virus filter as closely as possible to maintain the correct prefilter residence time.

If a coupled approach is necessary, the design of a virus filtration validation study will depend on whether the spike contains smaller or larger viruses. This difference dictates when the virus spike is added during the study.

Coupled validation for small viruses

For coupled validation studies involving spikes containing smaller, non-enveloped viruses, it is necessary to determine the virus retention of the prefilter alone. The typical log reduction value (LRV) of minute virus of mice (MVM) under moderate pH and conductivity is typically ≤0.5 for Pegasus Protect. The virus retention of the prefilter is then subtracted from the virus retention of the coupled filters to determine the LRV for the virus filter by itself.

To obtain the two required LRVs, two filtrations are performed in parallel, one with the prefilter coupled to the virus filter and one with the prefilter alone (to see a diagram of this please refer to the previously mentioned application note). The spiked sample solution should be passed through both setups at the same flow rate to ensure equal residence time through the two prefilters and give confidence that the LRV results for the prefilters are comparable.

Coupled validation for large viruses

For some studies, the spike contains a larger virus that will be removed by the prefilter to a significant degree, but decoupling is not possible due to issues with fouling of the virus filter. In these cases, it is necessary to perform the virus validation study using inline spiking.¹

The test solution is challenged to the prefilter and filter in series, but in between the filters, a virus spike is pumped in at a small percentage of the test solution flow and mixed before being challenged to the virus filter.

Providing options even for challenging systems

Each of these strategies for virus filter validation studies incorporating prefilters has its own considerations. Decoupling the prefilter and virus filter is the simplest approach and provides the most accurate LRV determination but does not provide the best scale-down model. While comparing the LRV of the coupled prefilter and virus filter to that of the prefilter alone is the closest representation of virus filtration at the process scale, the LRV determined for the virus filter in this manner cannot be directly measured. Inline spiking is even more complex to operate and verify, but this approach allows for validation of virus filtration processes for even challenging proteins and viruses.

Virus filtration and validation at Cytiva

Aligning process development and validation teams internally with a comprehensive supplier that offers guidance, support, and expertise can offer another layer of value when determining a suitable virus filter validation strategy.

Careful consideration of virus and product stability and regulatory feedback are understood and built into the technical support and guidance we offer. This experience is combined with next-generation virus filters to deliver robust performance, even in unique design spaces. The result is virus filtration processes that are robust and reliable.