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Accelerating process development of recombinant protein production in perfusion cultures of Chinese hamster ovary cells Angepat, Sumitra


Perfusion processes provide high productivity, low product residence times and consistent culture conditions, but require long process development times. It can require a week or more to reach each steady state result for process optimization, slowing process development and causing increased production cost. The objective of this work was to accelerate process development in perfusion cultures. Recombinant CHO cells producing t-PA were used, and the effects of temperature, pH and dissolved oxygen were investigated in pseudo steady state perfusion cultures at ~10 cells/mL. It was found that the t-PA concentration increased over 50% at temperatures lower than 37 °C, with concentrations over 200 mg/L produced at 33 °C. Cell specific t-PA productivity also increased at low temperatures, reaching a maximum at 33 °C. A low DO concentration of 16% air saturation had a detrimental effect on t-PA productivity and concentration compared to 70%, while 100% air saturation had no significant effect. Cell specific t-PA production and titre were also negatively affected by pH values < 6.85. To decrease the time needed to achieve steady state, a method to use non-steady state transient responses was developed to qualitatively predict steady state production performance. The method was tested using 3 day temperature shifts in perfusion cultures scanned down by 2 °C from 37 °C to 31 °C, then scanned up to 37 °C. Transient results showed that higher t-PA concentrations could be predicted at lower temperatures, as pseudo steady state results confirmed. In most cases, transient values on the 3rd day were also in close concordance with pseudo steady state values. To further accelerate process development, transient scanning was applied to small scale, non-instrumented cultures. Similar results were produced, although quantitative t-PA values were 15-30 times lower than in perfusion culture. The method was further refined by investigating 1 day transient shifts in temperature. More variability was observed in the 1 day transient response, suggesting that cells are still adapting to the new environment, but the overall response still qualitatively predicted the pseudo steady state results. By using a 1 day transient scanning method in small scale culture, process development time could be reduced by as much as 90% when compared to steady state evaluation.

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