Bioprocess monitoring using sequential injection capillary electrophoresis

Bioprocess monitoring has received significant interest over the past few years. The production of biopharmaceuticals synthesized by living cells during fermentation or cell culture processes is a rapidly growing field. Additionally, cell based assays have replaced many in vivo assays because of ethical and regulatory restrictions on working with laboratory animals. Biological processes are naturally susceptible to variability because living cells consume substrates and produce metabolites and products in a dynamic way with variations in metabolic rate across short time intervals. For the production of biopharmaceuticals, the FDA recommends documentation of nutrient and metabolite time profiles in the process analytical technology (PAT) policy to ensure product quality. At present, the majority of cell culture based monitoring is restricted to a few end point based assays that do not reflect the dynamic metabolic processes in cells that influence the final product. Therefore, a detailed and continuous monitoring of the bioprocesses in each production batch would significantly help manufacturers to control product quality, increase production yields and reduce production costs. At the same time, online monitoring of bioprocesses will also significantly enhance our understanding of fundamental dynamic cellular metabolic reactions that cannot be easily ascertained by end point measurements, and in turn facilitate pharmacological and biotechnological studies employed for screening and compounds testing. This thesis explores the potential of capillary electrophoresis (CE) for bioprocess monitoring. CE is a powerful and high resolution separation technique with the potential to provide detailed chemical information quickly using small sample volumes. First, the potential of Sequential injection capillary electrophoresis (SI-CE) for monitoring lactate production, an important metabolic indicator, during adherent mammalian cell culture, was examined. A new sampling interface was developed to sample from the medium covering a culture of human embryonic kidney cell line HEK293 and mouse fibroblast cell lines. Changes in lactate concentration in the cell culture medium were measured every 20 minutes over 3 days, requiring only 8.73 ˜í¬¿L of sample per analysis. Second, a SI-CE system was developed for automated, online, near real-time monitoring of suspension cultures by integrating microfluidic components for cell counting and analyte extraction with the high-resolution separation technique. The correlation of cell growth of a human lymphocyte cell line with changes in the essential metabolic markers including glucose, glutamine, leucine/isoleucine and lactate provided new insights in the metabolic changes over time. Using only 8.1 mL of media (41 ˜í¬¿L per analysis), the metabolic status and cell density were recorded every 30 minutes over 4 days. This system provides a promising new solution to meet the future demands in process monitoring in the biopharmaceutical industry. The developed platform for monitoring suspension cultures was extended to simultaneous monitoring of five parallel suspension cultures, capable of conducting cell density measurement and a high-resolution separation every 12 minutes for 4 days. This system was applied to study the metabolic effects of the drugs rotenone, ˜í‚â§-lapachone and clioquinol on metabolism using lactate as indicator. For each drug, suspension culture experiments for three drug concentrations and two controls were monitored in parallel. Over the 4 days, 5.78 mL of media was consumed from each culture, equating to 60 ˜í¬¿L per analysis. The fully automated system offers high sample throughput, good temporal resolution and low sample consumption combined with robustness, sensitivity and flexibility which provides a promising new platform for pharmacological and biotechnological studies.