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UBC Theses and Dissertations

Increasing the performance of mammalian perfusion culture system Drouin, Hans

Abstract

Perfusion culture processes are potentially the most efficient way of producing large quantities of biopharmaceuticals. However, these processes are not the most commonly used by industry in part due to a lack of simple solutions to perfusion challenges. This thesis has investigated recombinant protein production instability, a strategy to improve low perfusion rate culture performances and the complications due to cell aggregate formation. Human embryonic kidney 293 (HEK293) cells producing recombinant human interferon-alpha2b (IFN-α2b) were investigated as a model recombinant cell line. These cells also were maintained for more than 4 weeks in batch cultures using three media with different osmolalities in order to evaluate production stability. Exposure to high osmolality (~ 375 mOsm kg­‾¹) gradually decreased the yield of IFN-α2b secreted by the viable cells. Perfusion cultures validated the batch cultures with results showing that it was not possible to maintain stable production at elevated osmolality whereas at normal osmolality (~ 300 mOsm kg­‾¹), the titer was maintained at 250 mg L­‾¹. A reduced perfusion rate strategy was explored to increase the product titer in a perfusion bioreactor using enriched media. The HEK293 cell line was found to have a growth-associated production. By increasing the bleed rate in order to increase growth, the perfusion process yielded an up to 35% increased IFN-α2b concentration. Several modified medium conditions were investigated in batch cultures to help identify the main mechanism of aggregation observed in perfusion cultures. The addition of dead cells in the batch case was found to yield cellular aggregation that was most similar to perfusion cultures. The presence of aggregation did not affect the on-line monitoring of the viable cell concentration using a permittivity signal. However, if cells in aggregates are neglected, this can result in major cell specific-rate calculation errors. The image analysis used to estimate the cellular content of aggregates was an efficient method of improving viable cell estimates and cell specific-rate analyses. Overall, advances in the methods to more efficiently monitor and operate high performance perfusion-culture processes should expand their potential to fulfill the increasing demand for recombinant protein products from biotechnology.

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