Understanding the mechanism of virus removal by Q sepharose fast flow chromatography during the purification of CHO‐cell derived biotherapeutics

Abstract

During production of therapeutic monoclonal antibodies (mAbs) in mammalian cell culture, it is important to ensure that viral impurities and potential viral contaminants will be removed during downstream purification. Anion exchange chromatography provides a high degree of virus removal from mAb feedstocks, but the mechanism by which this is achieved has not been characterized. In this work, we have investigated the binding of three viruses to Q sepharose fast flow (QSFF) resin to determine the degree to which electrostatic interactions are responsible for viral clearance by this process. We first used a chromatofocusing technique to determine the isoelectric points of the viruses and established that they are negatively charged under standard QSFF conditions. We then determined that virus removal by this chromatography resin is strongly disrupted by the presence of high salt concentrations or by the absence of the positively charged Q ligand, indicating that binding of the virus to the resin is primarily due to electrostatic forces, and that any non-electrostatic interactions which may be present are not sufficient to provide virus removal. Finally, we determined the binding profile of a virus in a QSFF column after a viral clearance process. These data indicate that virus particles generally behave similarly to proteins, but they also illustrate the high degree of performance necessary to achieve several logs of virus reduction. Overall, this mechanistic understanding of an important viral clearance process provides the foundation for the development of science-based process validation strategies to ensure viral safety of biotechnology products.

Strategies for the purification and characterization of protein scaffolds for the production of hybrid nanobiomaterials

“…Accordingly, SDS-PAGE of peaks 1 and 2 of the AEX chromatography showed that gp64 was absent in peak 1 and abundant in peak 2, and that most VP6 did not bind to the column. AEX resins have been used to bind viruses (X-MuLV, MMV and SV40) and gp64 through electrostatic interactions [20][21][22]24]. The recovery yield of the AEX step was 65.8%, and the overall yield was 48.1% to this step (Table 1).…”

“…The loss of VP6 in the concentration step was possibly a result of the removal of unassembled VP6 ( Table 1). The next step in the purification scheme was anion exchange (AEX) chromatography, which is a common step used for the effective recovery, removal or inactivation of viruses in biotechnology products [20][21][22][23]. Fig.…”

Suitability of a generic virus safety evaluation for monoclonal antibody investigational new drug applications

“…Another well‐studied unit operation that is effective at separating viruses from process streams is anion exchange chromatography . Typical model viruses used in early phase clearance studies have more acidic isoelectric points—6.2 for minute virus of mice (MVM) and 5.8 for MuLV . Because IgG monoclonal antibodies often have basic isoelectric points, there is a large net charge difference between model viruses and many antibody products at neutral pH.…”

“…This charge difference is such that antibody flows through the column while viruses adsorb onto the AEX column. When this charge difference is maintained, AEX has been shown to provide robust viral clearance of at least four LRVs across isoelectric points from 7.3 to 8.8 …”

Development and scale‐up of the recovery and purification of a domain antibody Fc fusion protein‐comparison of a two and three‐step approach

“…While viral clearance is highest in the neutral to alkaline pH regime at low conductivity (Strauss et al,2009(Strauss et al, , 2010, a wider design space was initially explored due to the relatively low pI of the molecule. While viral clearance is highest in the neutral to alkaline pH regime at low conductivity (Strauss et al,2009(Strauss et al, , 2010, a wider design space was initially explored due to the relatively low pI of the molecule.…”