Quality by design approach for viral clearance by protein a chromatography

Abstract

Protein A chromatography is widely used as a capture step in monoclonal antibody (mAb) purification processes. Antibodies and Fc fusion proteins can be efficiently purified from the majority of other complex components in harvested cell culture fluid (HCCF). Protein A chromatography is also capable of removing modest levels of viruses and is often validated for viral clearance. Historical data mining of Genentech and FDA/CDER databases systematically evaluated the removal of model viruses by Protein A chromatography. First, we found that for each model virus, removal by Protein A chromatography varies significantly across mAbs, while remains consistent within a specific mAb product, even across the acceptable ranges of the process parameters. In addition, our analysis revealed a correlation between retrovirus and parvovirus removal, with retrovirus data generally possessing a greater clearance factor. Finally, we describe a multivariate approach used to evaluate process parameter impacts on viral clearance, based on the levels of retrovirus-like particles (RVLP) present among process characterization study samples. It was shown that RVLP removal by Protein A is robust, that is, parameter effects were not observed across the ranges tested. Robustness of RVLP removal by Protein A also correlates with that for other model viruses such as X-MuLV, MMV, and SV40. The data supports that evaluating RVLP removal using process characterization study samples can establish multivariate acceptable ranges for virus removal by the protein A step for QbD. By measuring RVLP instead of a model retrovirus, it may alleviate some of the technical and economic challenges associated with performing large, design-of-experiment (DoE)—type virus spiking studies. This approach could also serve to provide useful insight when designing strategies to ensure viral safety in the manufacturing of a biopharmaceutical product.

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

“…Protein A chromatography, in contrast, is not expected to bind significant amounts of virus since it is an affinity resin. Virus partitioning studies show that significant levels of virus are found in the protein A flowthrough and wash fraction as expected, however virus is also detected in the elution pool (Brorson et al, ; Lute et al, ; Zhang et al, ).…”

Characterizing and enhancing virus removal by protein A chromatography

“…For certain feedstocks tested, LRVs were lower without a clear correlation with any processrelated factors, including equilibration and wash buffers, resin, and elution conditions. 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). 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 results indicate that the non-mAb HCCF components in mAbs 1, 2, and 3 were not the main cause of low LRVs, but appeared to contribute to impact the final HCCF LRVs based on the fact that the HCCF LRVs were higher than those with the pure mAbs, especially in the cases of mAbs 1 and 2 ( Figure 1). Zhang et al, 2014). Moreover, it was the mAb itself, not the other HCCF impurities in the feed, that caused reduced LRV.…”

“…However, a small fraction of the virus is retained on the column and detected in the elution pool (M. Zhang et al, 2014). It is a regulatory expectation for the biopharmaceutical industry to demonstrate sufficient removal capacity of endogenous and adventitious viruses during manufacturing to ensure virus safety of the products (ICH Q5A).…”

“…These methods have been used widely to study the separation of the product-and processrelated impurities Kelley et al, 2008;Sisodiya, Lequieu, Rodriguez, McDonald, & Lazzareschi, 2012;Wensel, Kelley, & Coffman, 2008). The limited application of HTS for virus clearance studies is likely due to the cost, complexity, and need for specialized labs, all of which can be overcome in scale-down protein A models using quantitative methods to measure CHO endogenous, noninfectious RVLP (M. Zhang et al, 2009Zhang et al, , 2014 | 847 hydrogen-bond, or combinations of these functional groups that reverse the effect. The limited application of HTS for virus clearance studies is likely due to the cost, complexity, and need for specialized labs, all of which can be overcome in scale-down protein A models using quantitative methods to measure CHO endogenous, noninfectious RVLP (M. Zhang et al, 2009Zhang et al, , 2014 | 847 hydrogen-bond, or combinations of these functional groups that reverse the effect.…”