(199b) Understanding Prefiltration and Fouling of Virus Filters

Virus clearance is an essential component of the downstream purification of biopharmaceutical products. Virus filtration is routinely conducted towards the end of the purification train to ensure effective removal of both endogenous and adventitious virus during the manufacture of protein therapeutics. Membrane fouling and the performance of virus filters (filtration flux and throughput) could be strongly affected by product properties (charge, hydrophobicity and conformational stability), solution conditions, and the properties of the virus filter. Fouling is typically dominated by product related foulants such as soluble aggregates (dimers, trimers) insoluble aggregates, and product variants (such as denatured or mis-folded product and product(s), with markedly different post translational modifications), although other impurities such as host cell proteins (HCPs), DNA and spiked virus particles can also contribute to the overall flux decline. Use of a prefilter is often critical when developing a practical virus filtration step. The specific mechanisms for foulant removal during prefiltration include size exclusion, ion-exchange and hydrophobic interaction. Here the effects of prefilter type, with a single mechanism of action, on the filtration performance of one commercial virus filter was investigated. In addition, analytical tools have been used to identify specific foulant(s) that plug the virus filter by analysis of material eluted from the filter using mass spectroscopic tools including LC-MS, MALDI-MS, and capillary electrophoresis. Results show that size-based prefilters are largely ineffective, demonstrating that the foulants are not large aggregates. Hydrophobic interaction and cationic exchange prefilters provided significant improvements in the performance of the virus filter, suggesting that the foulants may be denatured product or product variants differing in charge due to post-translational modifications or differences in glycosylation. This was confirmed by the presence of high concentrations of product in the eluted samples and that the eluted samples have different molecular weight and charge profiles to those from the filtrate. These results provide significant insights on the nature of foulants that affect the performance of the virus filter and suggest important strategies for improving virus filtration processes.