Recent progress in mammalian cell culture process has resulted in significantly increased product titers, but also resulted in substantial increases in cell density as well as process and product related impurities. Due to the diverse physicochemical properties of these impurities, multiple purification techniques comprising distinct separation mechanisms are often required to meet final drug substance specifications. For monoclonal antibodies, Protein A chromatography as a capture step has traditionally been the work horse for clearing a large proportion of these impurities however, remaining levels of process and product related impurities present significant challenges on the development of further polishing steps. In this study, we have incorporated high throughput screening to evaluate and improve three areas of separation: i) Harvesting ii) Protein-A Chromatography and iii) Viral Inactivation. This is aimed at both reducing the impurities as well as enable a two-column purification process for monoclonal antibodies. Harvest evaluation included pre-treatment with a variety of flocculant/precipitants followed by a charged hybrid or uncharged depth filtration while improvements in Protein-A chromatography were achieved by evaluating a number of wash conditions aimed at disrupting the interactions between process related impurities and the monoclonal antibody. Further, we show that one can strategically use low pH viral inactivation step and filtration thereafter to further remove impurities from the product pool. Together, we demonstrate removal of process related impurities down to drug substance specifications with just one column and one hybrid depth filter. A good viral clearance achieved across these steps also indicates the robustness of these steps enabling a two column purification process.

Development of robust orthogonal methods for impurity clearance using high throughput screening

249th National Meeting (2015)

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