Companies and institutions across the biotechnology industry have been heavily investing in the development of cell culture perfusion technologies. Perfusion, part of the Next Generation of Manufacturing platforms (NGM), has demonstrated dramatic increases in productivity for the generation of therapeutic proteins compared to traditional fed-batch processes. Typically there are two modes of operation for perfusion cell cultures: steady state and dynamic. Steady state perfusion is often achieved by bleeding the culture to maintain a viable cell density target; in dynamic perfusion, however, the culture grows until it naturally reaches stationary growth phase and decay. Dynamic perfusion is advantageous due to higher cell densities, and therefore greater volumetric productivity compared to steady state perfusion. The trade-off for higher productivities in dynamic perfusion is the increased engineering complexity required to ensure optimal nutrient delivery and process control at increased cell densities. Optimal extra cellular conditions are required to ensure cell health and achieve critical product quality attribute (CQA) targets. In particular, the consistent production of target N-linked glycosylation profiles ensures robust pharmacokinetics (PK) and pharmacodynamics (PD) depending on the molecule of interest. In this study we show how control of Mn2+ bioreactor supplementation allowed for consistent production of the desired glycosylation profiles in a dynamic perfusion mode. The possibility to control and induce complex glycosylation at high cell densities had previously only been achieved for steady state perfusion processes, but here we utilized a design of experiment (DoE) to test the efficacy of dynamic Mn2+ supplementation in both steady state perfusion and dynamic perfusion processes. It was found that Mn2+ supplementation inhibits cell growth above a certain concentration, but supplementation was successful in maintaining desired levels of complex glycosylation compared to the steady state perfusion processes. Therefore, we concluded consistent complex glycosylation can be achieved in a dynamic perfusion process via Mn2+ supplementation alone.

N-linked glycosylation control for upstream next generation manufacturing (NGM) processes

ACS Spring 2022

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