Efren Pacis

The glycosylation profile of therapeutic antibodies is routinely analyzed throughout development to monitor the impact of process parameters and to ensure consistency, efficacy, and safety for clinical and commercial batches of therapeutic products. In this study, unusually high levels of the mannose-5 (Man5) glycoform were observed during the early development of a therapeutic antibody produced from a Chinese hamster ovary (CHO) cell line, model cell line A. Follow up studies indicated that the antibody Man5 level was increased throughout the course of cell culture production as a result of increasing cell culture medium osmolality levels and extending culture duration. With model cell line A, Man5 glycosylation increased more than twofold from 12% to 28% in the fed-batch process through a combination of high basal and feed media osmolality and increased run duration. The osmolality and culture duration effects were also observed for four other CHO antibody producing cell lines by adding NaCl in both basal and feed media and extending the culture duration of the cell culture process. Moreover, reduction of Man5 level from model cell line A was achieved by supplementing MnCl2 at appropriate concentrations. To further understand the role of glycosyltransferases in Man5 level, N-acetylglucosaminyltransferase I GnT-I mRNA levels at different osmolality conditions were measured. It has been hypothesized that specific enzyme activity in the glycosylation pathway could have been altered in this fed-batch process.

One of the major goals in cell culture process development for therapeutic antibody production is to develop methods to reach high titer in classical fed-batch processes. This goal is often achieved through the optimizations of expression vector, cell line, media and cell culture process controls to increase cell specific productivity, viable cell density, and culture longevity. During process optimization for a selected production cell line, cell specific productivity (qP) can vary significantly with culture conditions. Therefore, identifying strategies to maintain maximal specific productivity throughout the entire fed-batch culture and to eliminate cellular/process bottlenecks that prevent high levels of antibody production would be crucial for further advancements in this area. In this work, specific productivity was increased and maintained at high level throughout the course of the culture by the optimization of feed media and feeding strategy. Through the enhancement of nutrient feeding, final titer was increased by 2.5-fold from the platform fed-batch process and reached 7.5 g/L. In addition, further insight upon possible cellular bottlenecks in high yield antibody production was obtained by comparing the levels of heavy chain (HC) and light chain (LC) mRNA and the levels of intracellular antibody between the non-optimized and optimized feeding processes. The mRNA levels of the two processes were measured and exhibited no significant difference suggesting that transcription is not the bottleneck. When intracellular antibody level was studied, the relatively constant level of HC, LC, and intact antibody between days 9 and 14 suggested that translation could be the rate-limiting step under the non-optimized nutrient feeding condition due to the dramatic drop of qP to roughly zero which correlated with the depletion of tyrosine as one of the key amino acids for protein synthesis. Finally, accumulation of unassembled HC but not intact antibody was observed at days 14-18 under the enhanced feeding condition, implying that folding and assembly may be the bottleneck toward the end of the culture.