In this work we demonstrate how counterflow centrifugation can be developed in order to double depth filtration performance for cell culture clarification. Clarification and cell harvest steps initiate the purification process for biologicals. Depth filtration and combined centrifugation/ depth filtration steps are the two primary approaches in mammalian fed batch processes. As process intensification is explored in the future, clarification will be challenged in two key ways: increased biomass and smaller batch size. To address these challenges, different techniques have been adopted such as flocculation, body feed filtration or acoustic wave separation. Here we investigate counterflow centrifugation as another option and show how to systematically develop the process.<br/>First we evaluated the key centrifugation parameters expected to affect cell removal while using the kSep counterflow centrifuge. We used an industrial mAb producing CHO fed batch process with ~5g/l product titer, ~8% biomass, and ~ 90% viability. The key parameters investigated in detail were: 1. flow rate during culture loading 2. flow rate during wash steps of the fluidized bed, and 3. composition of the wash buffer. We evaluated centrifugation performance using cell counting, turbidity measurement and particle size analysis.<br/>Following optimization of centrifugation parameters, different combinations of available and new double layered depth filters were tested in-line with 0.2 micron filters. Assessment of the filter trains was achieved by monitoring the blocking of each single layer filter and overall capacity of the filter trains. This approach identified the limiting filter stage. Filter optimization resulted in doubling the overall capacity of the filter train. The results are discussed with respect to the data found for cell count, turbidity and particle size after the centrifugation step.<br/>Furthermore the data are compared to standard clarification methods run in parallel

Counterflow-centrifugation and depth filtration optimization: Systematic approach to develop CHO harvest process

2019 Spring National Meeting

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