Using high throughput screening to define virus clearance by chromatography resins

Abstract

High throughput screening (HTS) of chromatography resins can accelerate downstream process development by rapidly providing information on product and impurity partitioning over a wide range of experimental conditions. In addition to the removal of typical product and process-related impurities, chromatography steps are also used to remove potential adventitious viral contaminants and non-infectious retrovirus-like particles expressed by rodent cell lines used for production. This article evaluates the feasibility of using HTS in a 96-well batch-binding format to study removal of the model retrovirus xenotropic murine leukemia virus (xMuLV) from product streams. Two resins were examined: the anion exchange resin Q Sepharose Fast Flow™ (QSFF) and Capto adhere™, a mixed mode resin. QSFF batch-binding HTS data was generated using two mAbs at various pHs, NaCl concentrations, and levels of impurities. Comparison of HTS data to that generated using the column format showed good agreement with respect to virus retentation at different pHs, NaCl concentrations and impurity levels. Results indicate that NaCl concentration and impurity level, but not pH, are key parameters that can impact xMuLV binding to both resins. Binding of xMuLV to Capto adhere appeared to tolerate higher levels of NaCl and impurity than QSFF, and showed some product-specific impact on binding that was not observed with QSFF. Overall, the results demonstrate that the 96-well batch-binding HTS technique can be an effective tool for rapidly defining conditions for robust virus clearance on chromatographic resins.

Characterizing and enhancing virus removal by protein A chromatography

“…(Zhang et al, 2009;2014 through but decreased during washes. In addition, the application of HTS can reach beyond the protein A step, such as an ion exchange or mixedmode chromatography shown by Connell-Crowley, as long as a HTS method is optimized to represent the flow condition on columns (Connell-Crowley, et al, 2013). Further evidence is needed to demonstrate whether the size or the hydrophobicity of a virus contribute to the differential retaining on the column before the elution phase.…”

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

“…Previous studies had claimed that a multi‐modal anion exchange resin's characteristic binding behavior was similar to that of a single mode AEX resin, but was able to capture viral particles in higher conductivity solutions which suggests against a single mechanism of adsorption. Our group followed up these studies to further elucidate the virus binding mechanism.…”

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

“…Improved clearance of impurities and model viruses at low pH and/or high conductivity had previously been reported for mixed mode anion exchange resins (Connell-Crowley et al, 2013). A titration of the molecule from pH 5.5 to pH 7.0 below 4 mS/cm leads to precipitation this approach was impractical for the two-step process as addition of salt would negatively impact AIX.…”

“…A screening DOE explored a pH range from 5.5 to 7.2 and conductivity ranges from 3 to 10 mS/cm. Based on previously published observations (Connell-Crowley et al, 2013;Toueille et al, 2011), viral clearance is expected to be met as well, though it was not tested as the three-step process was selected for FIH production. 6).…”