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Bioprocess monitoring using sequential injection capillary electrophoresis


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Alhusban, AAA 2016 , 'Bioprocess monitoring using sequential injection capillary electrophoresis', PhD thesis, University of Tasmania.

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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 μL
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 μL 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,
β-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 μL 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.

Item Type: Thesis - PhD
Authors/Creators:Alhusban, AAA
Keywords: Bioprocess, Online Monitoring, Biopharmaceuticals, Cell Culture, Sequential Injection Capillary Electrophoresis (SI-CE), Metabolic Status, Cell Density, Pharmacological Assays
Copyright Information:

Copyright 2015 the Author

Additional Information:

Chapter 2 appears to be the equivalent of a post-print version of an article published as: Alhusban, A. A., Gaudry, A. J., Breadmore, M. C., Gueven, N., Guijt, R. M., 2013. On-line sequential injection-capillary electrophoresis for near-real-time monitoring of extracellular lactate in cell culture flasks, Journal of chromatography A, 1323, 157-162. DOI: 10.1016/j.chroma.2013.11.006

Chapter 3 appears to be the equivalent of a post-print version of an article published as: Alhusban, A. A., Breadmore, M. C., Gueven, N., Guijt, R., M. 2016. Capillary electrophoresis for automated on-line monitoring of suspension cultures: correlating cell density, nutrients and metabolites in near real-time, Analytica chimica acta, 920, 94-101

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