Particulate formation was observed during the downstream development of an intensified monoclonal antibody (mAb) process at harvest and low pH virus inactivation. The intensified process is operated at high cell density leading to higher titer and elevated levels of impurities. To debottleneck the downstream process, high capacity protein A resin and flow through polishing steps are utilized. Particulates observed during processing decreased filter loading causing significant risk for fit to plant. The cause of particulate formation during processing is either the intrinsic solubility of the mAb itself, or the particulate consists of, or its formation is catalyzed, by an impurity. Workflows to determine root cause and mitigation across the process based on these findings are presented. To determine root cause, process steps that triggered particulates were repeated with varying buffer, environmental stress, and purity of the starting material. For virus inactivation of the particular mAb of this study, varying buffer or environmental stress did not impact particulate formation. However, increasing purity of the starting material, by buffer exchanging drug substance to VI starting material or flocculation upstream of the step, significantly decreased impurities and eliminated particulate formation. Based on the determined root cause, process alterations to improve impurity removal through adjusting centrifuge conditions, adding flocculation during harvest, and alternative protein A washes are compared for effectiveness in decreasing particulate generation. Flocculation by pH adjustment of the cell broth or centrate was found to increase the particulate size from &lt;1 µm to ~ 10 µm in diameter facilitating its removal by centrifugation or depth filtration. Alterations to process steps increased filter loading and derisked fit to plant for tech transfer.

Strategies for root cause analysis and mitigation for particulate formation during harvest and downstream purification for a monoclonal antibody

ACS Spring 2022

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