Defining the mechanistic binding of viral particles to a multi‐modal anion exchange resin

Abstract

A multi-tiered approach to determine the binding mechanism of viral clearance utilizing a multi-modal anion exchange resin was applied to a panel of four viral species that are typically used in validating viral clearance studies (i.e., X-MuLV, MVM, REO3, and PrV). First, virus spiked buffer-only experiments were conducted to evaluate the virus's affinity for single mode and multi-modal chromatography resins under different buffer conditions in a chromatography column setting. From these results we hypothesize that the mechanisms of binding of the viruses involve binding to both the hydrophobic and anionic functional groups. This mechanistic view agreed with the general surface characteristics of the different virus species in terms of isoelectric point and relative hydrophobicity values. This hypothesized mechanistic binding was then tested with commercially relevant, in-process materials, in which competitive binding occurred between the load components (e.g., viruses, target product, and impurities) and the resin. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1019-1026, 2018.

Impact of the isoelectric point of model parvoviruses on viral retention in anion‐exchange chromatography

“…Our results are in good agreement with previous studies, reporting significant binding of viruses (Brown et al, 2018, 2017; Cai et al, 2019; Strauss, Lute, Tebaykina et al, 2009), including parvoviruses to Q sepharose below their measured pI app . This phenomenon has been explained by the clustering of charges on the virus surface and a multivalent interaction with the AEX resin (Brown et al, 2018, 2017; Cai et al, 2019).…”

“…The negatively charged protruding loop on the threefold spike of MVM should dominate the adsorption to the quaternary amines localized within a relatively thin charged double layer on the Mono Q resin (Dismer & Hubbuch, 2007;Kopaciewicz et al, 1983). This strong electrostatic interaction may induce conformational changes in the viral structure that further favor a clustering of charges and multivalent interaction as previously proposed (Brown et al, 2018(Brown et al, , 2017Cai et al, 2019;Strauss, Lute, Tebaykina et al, 2009). The structure shown in Figure 6 indicates that parvoviruses exhibit significant flexibility in their surface, which would allow conformational changes to favor multivalent attractive interactions with the Mono Q resin, while repulsive areas are buried in the rugged surface.…”

“…This phenomenon has been explained by multimeric interactions of persistent negative patches on the capsid surface (Brown et al, 2018(Brown et al, , 2017.…”

“…A better understanding of the molecular mechanisms governing viral removal by FT-AEX helps us to implement an effective and safe downstream purification process of recombinant proteins through quality by design principles and facilitate risk assessments of deviations or changes during manufacturing. Several studies indicate that enveloped viruses, and to a lesser extent also nonenveloped viruses, electrostatically bind to strong AEX resins such as Q sepharose even below their pI (Brown et al, 2018(Brown et al, , 2017Cai et al, 2019;Strauss, Lute, Tebaykina et al, 2009). However, the detailed molecular and electrostatic mechanisms responsible for this phenomenon remain elusive.…”

“…However, the detailed molecular and electrostatic mechanisms responsible for this phenomenon remain elusive. To evaluate the effectiveness of viral clearance by AEX in relation to the viral net surface charge, comparative studies have used viruses with different pI values, which also exhibited fundamental differences in size, structure, and surface charge distributions (Brown et al, 2018(Brown et al, , 2017Cai et al, 2019;Strauss, Lute, Tebaykina et al, 2009).…”