Goh, Hai-Yuan; (2021) The Characterisation, Optimisation, Scalability, And Use Of A GS-CHO Cell Line To Improve Bioprocess Productivity. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Process characterisation and optimisation had been carried out for a mammalian cell culture using a GS-CHO cell line that produced the chimeric IgG4 monoclonal antibody Cb72.3. A series of experimentations in the shake flasks, bench top bioreactors and pilot scale bioreactor had been carried out. Culture additives were also used to investigate their impact on the GS-cell culture, productivity and charged variant profiles. A high throughput and automated methodology for miniature chromatography was developed and used to purify samples for product quality analysis. Optimisation of various analytical assays was successfully performed to address the assay artefacts observed during the analysis. Sodium butyrate boosted the titer to the largest extent by ~40% when compared against the control but only had a culture viability of ~10% on Day 14. Copper addition resulted in the largest increase (~6% points) in the basic isoforms of the antibody out of all the additives used compared to control culture. Spermine increased acidic isoforms by ~3% points compared to control culture. Long-R3 promoted growth rate and glucose consumption rate but had a negative impact on viability only when used with the feed media. Various process conditions like pH setpoint and temperature were also investigated to elucidate the growth and metabolism profiles. A low pH setpoint of pH 6.9 in the bioreactor prolonged the culture viability the longest with a viability of ~90% even after 21 days. The development and improvement of feeding strategy had also facilitated more robust bioreactor fed-batch cultures with reproducible metabolism profiles (especially with respect to lactate), growth and productivity compared to a previous strategy. A real-time monitoring tool for mammalian cell culture using off-gas mass spectrometry was successfully demonstrated. Process events in the bioreactors like antifoam addition and blocked venting filter were captured along with specific oxygen consumption rate (qO2) and mass transfer of oxygen (kLa). The respiration quotient (RQ) values from the off-gas analysis also correlated to different metabolic states (high and low glycolytic flux) during the fed-batch bioreactor cultures, specifically the lactate production (RQ>1) and lactate consumption phases (RQ<1). Subsequently, a whole bioprocess scale-up study had successfully demonstrated the scalability of the cell culture. The cell culture was performed in a 50L pilot scale single use bioreactor, followed by clarification, capture (Protein A) and polishing (cation exchange and anion exchange chromatography) steps at the pilot scales. It was shown that high purity Mab (>99% monomer and <51 parts per million host cell protein) could be obtained at the end of the platform process with minimal optimisation. Charged variants analysis also revealed the charged variants distribution had significant differences between a main and shoulder peak fraction pools isolated in the cation exchange (CEX) polishing step – the latter had 12% points more basic variants than the former. As the main peak fraction pool went through the final polishing step in an anion exchange (AEX) chromatography, it only had a 2-4% points difference when compared to the CEX step. For the shoulder peak fraction pool, there was only 2-4% points difference in the charge variant profile before and after the AEX step. An overall process yield of 75% was obtained.

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