A step‐wise approach to define binding mechanisms of surrogate viral particles to multi‐modal anion exchange resin in a single solute system

Abstract

Multi-modal anion exchange resins combine properties of both anion exchange and hydrophobic interaction chromatography for commercial protein polishing and may provide some viral clearance as well. From a regulatory viral clearance claim standpoint, it is unclear if multi-modal resins are truly orthogonal to either single-mode anion exchange or hydrophobic interaction columns. To answer this, a strategy of solute surface assays and High Throughput Screening of resin in concert with a scale-down model of large scale chromatography purification was employed to determine the predominant binding mechanisms of a panel of bacteriophage (i.e., PR772, PP7, and ϕX174) to multi-modal and single mode resins under various buffer conditions. The buffer conditions were restricted to buffer environments suggested by the manufacturer for the multi-modal resin. Each phage was examined for estimated net charge expression and relative hydrophobicity using chromatographic based methods. Overall, PP7 and PR772 bound to the multimodal resin via both anionic and hydrophobic moieties, while ϕX174 bound predominantly by the anionic moiety. Biotechnol. Bioeng. 2017;114: 1487-1494. © 2017 Wiley Periodicals, Inc.

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

“…Due to the similarities in physical and chemical characteristics (i.e., hydrodynamic radius, isoelectric point, and relative hydrophobicity), bacteriophage should theoretically be a representative surrogate for estimation models of mammalian viruses while having the advantage of ease of propagation and speed of titer determination (i.e., bacteriophage require overnight incubation while mammalian viruses can take up to 10 days). Specifically, the bacteriophage buffer‐only studies were able to predict both the failure point of the single mode AEX resin as a function of pH and NaCl concentration, as well as the salt tolerant behavior of the multi‐modal AEX resin as a function of relative hydrophobicity. For the in‐process testing, the bacteriophage were able to estimate the loading density where viral breakthrough occurred for all three mAb species and the lack of LRV decline for the reduced impurity loads.…”

“…This information provides a basis for predicting the propensity of the virus to interact with the individual moieties of the multi‐modal resin under various chemical compositions of the feed material. Previous work, which used bacteriophage as surrogates, focused on the bacteriophage's ability to adsorb to single mode and multi‐modal resin in isolation as a function of mobile phase chemical composition (i.e., pH and NaCl concentration). These studies determined if varied mobile phases produced characteristic binding responses due to a dominating AEX or HIC functional group.…”

Polysaccharide-based chromatographic adsorbents for virus purification and viral clearance

“…Zhao et al [76] exploited this property by introducing a Capto adhere MMC step in the multistep chromatography purification process of EV71 VLPs produced from insect cells, following negative CC700 MMC. The Capto adhere-based AEC-HIC step may also contribute to viral clearance by removing adventitious viruses from product streams in downstream purification processes of mAbs and other biopharmaceuticals [209,246,247]. In recent studies, Brown et al [246,247] investigated the binding mechanisms of model virus surrogates (i.e., bacteriophages displaying different surface charge characteristics and hydrophobicities) to the Capto adhere resin.…”

“…The Capto adhere-based AEC-HIC step may also contribute to viral clearance by removing adventitious viruses from product streams in downstream purification processes of mAbs and other biopharmaceuticals [209,246,247]. In recent studies, Brown et al [246,247] investigated the binding mechanisms of model virus surrogates (i.e., bacteriophages displaying different surface charge characteristics and hydrophobicities) to the Capto adhere resin. They first compared the efficiencies of the MMC resin and purely anionic (Capto Q) or hydrophobic (Capto Phenyl) Seph-based resins to remove phages from spiked buffers with varying pH and salt concentrations [246].…”

“…In recent studies, Brown et al [246,247] investigated the binding mechanisms of model virus surrogates (i.e., bacteriophages displaying different surface charge characteristics and hydrophobicities) to the Capto adhere resin. They first compared the efficiencies of the MMC resin and purely anionic (Capto Q) or hydrophobic (Capto Phenyl) Seph-based resins to remove phages from spiked buffers with varying pH and salt concentrations [246]. The most hydrophobic and negatively charged phages were adsorbed to Capto adhere by synergistic binding to the quaternary amine and phenyl groups, yielding a better phage removal efficiency of the MMC resin (LRV > 3in all tested buffer conditions) compared to single mode ones.…”