Filter preconditioning prior to virus spiking: A complementary tool in the validation of virus filters

Virus filtration is a critical dedicated viral clearance step in mammalian cell derived recombinant protein manufacturing processes. Regulatory agencies require demonstration of virus filter performance using model viruses in validation studies. However, scaled down virus filter validation studies usually involve introduction of virus stocks containing impurities to highly purified recombinant protein feeds and these nonrepresentative virus stock impurities can result in sub-optimal filter performance. Filter preconditioning followed by virus spiking has previously been proposed as an alternative validation methodology to minimize the impact of virus stock impurities during virus filter validation. However, previous work has not evaluated the effect of different variables on operating limits or the effect of preconditioning on the filter itself. The objectives of this dissertation research were to characterize filter preconditioning as an alternative virus filter validation methodology for use with ViresolveRTM NFP filters and to investigate factors affecting the operational limits.;Virus retention comparability and volumetric throughput improvement of filter preconditioning relative to conventional spiking was determined under a range of conditions. The impact of preconditioning endpoints, total virus loading, virus stock impurities, filter lots, virus stock types, buffer conditions and protein concentration were investigated. Operating limits of the filter preconditioning method were identified where virus retention was elevated relative to conventional spiking.;The distribution of viruses in the filter's component membrane layers was found to be unaffected by preconditioning followed by spiking compared to conventional spiking. Preconditioning was shown to affect the sieving properties of individual virus titter membrane layers using dextran rejection testing. A strong inverse relationship between dextran R90 values and filter preconditioning flow decay was demonstrated.;The eventual passage of viruses retained earlier in the filtrations was shown. Factors influencing the passage of previously retained viruses were investigated and presence of protein in the feed was shown to strongly influence this virus passage. Finally, elevation in virus retention of filter preconditioning relative to conventional spiking was linked to the passage of previously retained viruses during conventional spiking.