Understanding virus retention mechanisms on protein a chromatography based on using different wash buffers – Evaluating the possibility for a generic wash buffer toolbox to improve virus clearance capacity

Abstract

During manufacturing of mammalian-cell derived monoclonal antibodies (mAbs) virus clearance capacity of the downstream process has to be demonstrated. The protein A chromatography step typically achieves less than 4 log₁₀ and is not considered as a major contributing step. Having been successfully applied to host cell protein removal before, we used different wash buffers for three mAbs with two model viruses (Minute virus of mice and Murine leukemia virus) in series as well as separately to further understand major contributing interactions for virus retention and potentially design a generic toolbox of stringent wash buffers to be applied to various mAbs. Results indicate a major relevance of hydrophobic interaction for Murine leukemia virus (xMuLV) and mAb A, based on improved clearance for buffers additionally containing increased levels of hydrophobic compounds. This effect was less pronounced for Minute virus of mice (MVM), whereby hydrogen-bonds were expected to play a stronger role for this model virus. Additionally, electrostatic interactions presumably are more relevant for MVM retention compared to xMuLV under the conditions evaluated. A generic mAb and virus-independent stringent wash buffer toolbox could not be identified. However, based on our results a customized mAb and virus wash buffer design with improved virus clearance is possible, with here demonstrated log reduction increase by 1.3 log₁₀ for MVM and 2.2 log₁₀ for xMuLV for the protein A step compared to equilibration buffer alone.