Identification and classification of host cell proteins during biopharmaceutical process development.

As significant improvements in volumetric antibody productivity have been achieved by advances in upstream processing over the last decade, and harvest material has become progressively more difficult to recover with these intensified upstream operations, the segregation of upstream and downstream processing has remained largely unchanged. By integrating upstream and downstream process development, product purification issues are given consideration during the optimization of upstream operating conditions, which mitigates the need for extensive and expensive clearance strategies downstream. To investigate the impact of cell culture duration on critical quality attributes, CHO-expressed IgG1 was cultivated in two 2 L bioreactors with samples taken on days 8, 10, 13, 15, and 17. The material was centrifuged, filtered and protein A purified on a 1 ml HiTrap column. Host cell protein (HCP) identification by mass spectrometry (MS) was applied to this system to provide insights into cellular behavior and HCP carryover during protein A purification. It was shown that as cultivation progressed from day 8 to 17 and antibody titer increased, product quality declined due to an increase in post-protein A HCPs (from 72 to 475 peptides detected by MS) and a decrease in product monomer percentage (from 98% to 95.5%). Additionally, the MS data revealed an increase in the abundance of several classes of post-protein A HCPs (e.g., stress response proteins and indicators of cell age), particularly on days 15 and 17 of culture, which were associated with significant increases in total overall HCP levels. This provides new insight into the specific types of HCPs that are retained during mAb purification and may be used to aid process development strategies.

Identification of upstream culture conditions and harvest time parameters that affect host cell protein clearance.

During early stage bioprocess development, characterizing interactions between unit operations is a key challenge. Such interactions include the release of host cell enzymes early in the process causing losses in product quality downstream. Using a CHO-expressed IgG1 system, the impact of cell culture duration was investigated using a 50 L bioreactor and performing scale-down protein A purification. While antibody titer doubled during the last week of culture, the post-protein A host cell protein (HCP) levels increased from 243 to 740 ppm. Effects of pH and temperature were then explored using fed-batch ambr250 bioreactors, and parameters enabling higher titers were linked to a decrease in post-protein A product purity. These trade-offs between titer and product quality were visualized using a window of operation. The downstream space was explored further by exposing shake flask material to shear representative of disc stack centrifugation, prior to purification, and by adding polishing chromatography. While product quality decreased with progressing cultivation, cells became more shear resistant. Polishing chromatography resulted in product fragmentation which increased fourfold from Day 10 to 24, adding constraint to achieving both efficient HCP clearance as well as high monomer purities. These examples highlight the importance of adopting integrated approaches to upstream and downstream development strategies to enable whole process optimization.