Clearance of the rodent retrovirus, XMuLV, by protein A chromatography

Abstract

Protein A chromatography is the most common unit operation used in the manufacture of therapeutic monoclonal antibodies (mAbs) due to its high affinity and specificity for the IgG Fc domain. However, protein A chromatography is often not effective for viral clearance. Typical log reduction values (LRV) for the model retrovirus XMuLV range between 1 and 4 logs, while effective steps such as viral filtration can achieve 5-7 logs of clearance. XMuLV LRVs obtained on protein A are reproducible for a given mAb, but can vary widely for different mAbs, even with the same operating conditions. In order to understand the mechanism of XMuLV clearance on protein A, we have investigated its partitioning on Mabselect SuRe protein A resin and explored how the virus interacts with resin, product, and impurities. The results show that XMuLV has some interaction with the resin backbone and ligand, but also appears to bind to and coelute with the mAb. The interaction with product was further examined by evaluating the effect of feed conditions, loading, and different washes on XMuLV partitioning on the column. Understanding the mechanism of XMuLV removal on a protein A, resin provides insight into the variability and low viral clearance of this step and suggests ways in which the removal of virus by this step can be improved.

Characterizing and enhancing virus removal by protein A chromatography

“…The retained viruses act like they were bound to the adsorbed mAbs 1 and 2, consistent with the hypothesis of Bach and Connell-Crowley (2015). The results of this study indicate the major role is played by the specific mAb product molecule.…”

“…Thus the LRV trend obtained for one virus can be applied to other viruses, as shown in this study.Low LRVs on protein A was restricted to mAbs 1 and 2 with high pI and hydrophobicity compared to mAb 3. This is consistent with previous findings that wash with 1 M NaCl and 750 mM L-Arginine increased XMuLV LRV 1-2 logs on MSS(Bach & Connell-Crowley, 2015). Furthermore, in a proposed fouling mechanism of cycled protein A resin by a hydrophobic mAb, upon binding to protein A, the mAb may undergo conformational changes and expose hydrophobic patches which in turn would attract lipophilic proteins (S.. Perhaps the hydrophobic interaction is involved in a similar fashion to facilitate the retrovirus retaining on the mAb-Protein A.The non-mAb HCCF components are highly heterogeneous, and there is an interaction between mAb and HCP as shown by multiple researchers(Hogwood, Tait, Koloteva-Levine, Bracewell, & Smales, 2013;Sisodiya et al, 2012;Tran et al, 2016;Q.…”

“…These results were extended from previous observation described by Bach and Connell-Crowley (2015) studying XMuLV partitioning on MabSelect SuRe columns, providing further evidence using additional types of viruses and resin. Previous findings have pointed to the differences in feedstocks to protein A, composed of the products and other cell culture-related impurities.…”

“…For a given mAb, LRVs were generally consistent, and insensitive to process parameter changes including load density (g mAb/L resin), flow rate, temperature, intermediate wash buffer composition, pH, conductivity, and pooling within the ranges tested (M. Zhang et al, 2014). These interactions appear to have electrostatic, hydrophobic or hydrogen bonding components (Bach & Connell-Crowley, 2015). These interactions appear to have electrostatic, hydrophobic or hydrogen bonding components (Bach & Connell-Crowley, 2015).…”

“…The only notion was that these feedstocks tend to be more complex, containing either ascites fluid or "highly concentrated" cell culture fluid (Miesegaes, Lute et al, 2010). Bach and Connell-Crowley (2015) studied the behavior of XMuLV on an agarose-base-matrix protein A resin, and their findings indicate that XMuLV has some interaction with the resin backbone and ligand, and also appears to bind to and coelute with the mAb. Bach and Connell-Crowley (2015) studied the behavior of XMuLV on an agarose-base-matrix protein A resin, and their findings indicate that XMuLV has some interaction with the resin backbone and ligand, and also appears to bind to and coelute with the mAb.…”