Regulatory authorities place stringent guidelines on the removal of contaminants during the manufacture of biopharmaceutical products. Monoclonal antibodies, Fc-fusion proteins, and other mammalian cell-derived biotherapeutics are heterogeneous molecules that are validated based on the production process and not on molecular homogeneity. Validation of clearance of potential contamination by viruses is a major challenge during the downstream purification of these therapeutics. Virus filtration is a single-use, size-based separation process in which the contaminating virus particles are retained while the therapeutic molecules pass through the membrane pores. Virus filtration is routinely used as part of the overall virus clearance strategy. Compromised performance of virus filters due to membrane fouling, low throughput and reduced viral clearance, is of considerable industrial significance and is frequently a major challenge. This review shows how components generated during cell culture, contaminants, and product variants can affect virus filtration of mammalian cell-derived biologics. Cell culture-derived foulants include host cell proteins, proteases, and endotoxins. We also provide mitigation measures for each potential foulant.

Monoclonal antibodies (mAbs) are an essential class of biotherapeutics. A platform process is used for mAb development to ensure clinically safe and stable molecules. Regulatory authorities ensure that mAb production processes include sufficient viral clearance steps to achieve less than one virus particle per million doses of product. Virus filtration is used for size‐based removal of enveloped and nonenveloped viruses during downstream processing of mAbs. Process development in mAb purification relies on empirical approaches and often includes adsorptive prefiltration to mitigate virus filter fouling. Opportunities for molecular‐level prediction of mAb filterability are needed to plug the existing knowledge gap in downstream processing. A molecular‐level approach to understanding the factors influencing mAb filterability may reduce process development time, material loss, and processing costs due to oversized virus filters. In this work, pH step gradient fractionation was applied on polished bulk mAb feed to obtain concentrated pools of fractionated mAb variants. Biophysical properties and quality attributes of fractionated pools, including oligomeric state (size), isoelectric point profile, diffusion interaction parameters, and glycoform profile, were determined using bioanalytical methods. Filterability (loading and throughput) of fractionated pools were evaluated. Statistical methods were used to obtain correlations between quality attributes of mAb fractions and filterability on the Viresolve Pro virus filter.

Virus filtration is used to ensure the high level of virus clearance required in the manufacture of biopharmaceutical products such as monoclonal antibodies. Flux decline during virus filtration can occur due to the formation of reversible aggregates consisting of self-assembled monomeric monoclonal antibody molecules, particularly at high antibody concentrations. While size exclusion chromatography is generally unable to detect these reversible aggregates, dynamic light scattering may be used to determine their presence. Flux decline during virus filtration may be minimized by pretreating the feed using a membrane adsorber in order to disrupt the reversible aggregates that are present. The formation of reversible aggregates is highly dependent on the monoclonal antibody and the feed conditions. For the pH values investigated here, pretreatment of the feed using a hydrophobic interaction membrane adsorber was the most effective in minimizing flux decline during virus filtration. Ion exchange membranes may also be effective if the monoclonal antibody and membrane are oppositely charged. Consequently, the effectiveness of ion exchange membrane adsorbers is much more dependent on solution pH when compared to hydrophobic interaction membrane adsorbers. Size based prefiltration was found to be ineffective at disrupting these reversible aggregates. These results can help guide the development of more effective virus filtration processes for monoclonal antibody production.

A rapid assessment of manufacturability for drug candidates is crucial for advancing a prospective biotherapeutic from a candidate to a bulk drug substance. A lot‐to‐lot approach to manufacturability is adopted where each biologic batch is assessed for manufacturability as a bulk, unfractionated pool. Manufacturers may explore a more granular approach, independently enriching and evaluating the filterability of antibody variants within each lot, especially within the confines of relative hydrophobicity and surface charge. This study examined the use of bind‐and‐elute chromatography to alter the proportions of monoclonal antibody (mAb) proteoforms in eluate sub‐pools from a mixed‐mode chromatography resin‐packed column. Filterability of each sub‐pool through a virus‐retaining filter was subsequently examined. Circular dichroism and Fourier transform infrared spectroscopy were performed for each sub‐pool to probe for higher‐order structure differences between mAb variants enriched therein. Bioanalytical techniques were also used to assess colloidal stability, surface hydrophobicity, surface charge, and size differences. Results showed that basic charge variants, high‐mannose glycovariants, high relative hydrophobicity proteoforms, and high‐molecular‐weight species were enriched in the last‐eluting (terminal) sub‐pools. The first sub‐pool and the final sub‐pool showed the most fouling propensity on VPro virus filters. Circular dichroism showed that enriched proteoforms in the last sub‐pool possessed a higher percentage of bends. Most secondary structures did not vary significantly between sub‐pools. Diffusion interaction parameter was highly negative across all sub‐pools and the bulk unfractionated pool. These results provide a design space for identifying and depleting problematic mAb variants before the crucial virus filtration step.