Tools for the development of intensified perfusion processes for mammalian cell culture

QC 2022-05-19

Abstract

Recombinant protein therapeutics have become an indispensable part of modern medicine to treat a wide variety of diseases. Their manufacture is mostly based on mammalian cells with fed-batch production bioreactors and downstream batch purification unit operations. Although significant improvements have been made over the last decades to yield high production titers, concerns about their high capital investment costs and limitations in production flexibility have been raised. As a result, the biopharmaceutical industry is slowly transitioning to continuous biomanufacturing with perfusion bioreactors and integrated continuous downstream processes. The potential to maintain very high cell densities over extended process times in a perfusion culture offers higher volumetric productivities in comparison to fed-batch. This intensified production of recombinant proteins can be performed in smaller volumes, thus reducing equipment size and capital costs. However, new challenges are emerging from this shift to continuous bioprocessing. These include the lack of available scale-down models to streamline process development programs, limited knowledge on the optimization of culture medium and perfusion feed strategy, and shortage of tools for the in-line monitoring of relevant culture parameters to ensure stable perfusion operation with consistent product quality.

This thesis presents tools for the development of intensified perfusion processes with mammalian cells. Monoclonal antibody producing Chinese Hamster Ovary (CHO) cells or erythropoietin producing Human Embryonic Kidney (HEK293) cells were used as model organisms in the studies. Efforts were made to develop a small-scale perfusion system of 200 mL optimized for long-term steady-state cultivations with achievable cell densities of at least 10⁸ cells/mL. It was shown that volumetric productivities increased linearly with the cell density, thus demonstrating successful process intensification by high cell density perfusion. The small scale of this perfusion bioreactor enabled the conduction of a larger number of experiments with reduced workload and material consumption in a shorter timeframe in the following studies. To control the formation of byproducts, such as lactate, and the N-glycosylation of antibodies, which is an important quality attribute, a novel feed strategy for sugars including glucose, mannose and galactose was developed and implemented. With this feeding strategy, the sugars are delivered to match a target cell specific consumption rate, independent of the cell specific perfusion rate (CSPR). Furthermore, a high cell density perfusion process at a CSPR of 15 pL cell^-1 day^-1 was developed with high specific antibody productivity after sequential scanning of multiple steady states with various cell densities and perfusion rates. The dynamics in culture parameters from these development runs allowed the calibration of predictive models based on Raman spectroscopy, to enable real-time monitoring of multiple parameters in a perfusion culture. It was shown for example that the N-glycosylation profile was predicted with sufficient accuracy in validation experiments. Moreover, the perfusion process developed on small scale was transferred to a 30 L pilot-scale process, where it was successfully operated for 20 days. The steady state operation ensured a consistent product quality and the integrated continuous downstream process removed most impurities, while ensuring a high recovery yield. Finally, a strategy for perfusion medium optimization in CHO cell cultures operated with very low CSPR was presented by utilizing microbioreactors in combination with design of experiments methodology.

To conclude, the strategies presented in this thesis provide a new toolbox for the development and control of intensified perfusion processes, and supports the biopharmaceutical industry in their efforts to swiftly adapt continuous bioprocessing in commercial manufacturing of recombinant proteins.

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