Partial pressure of CO2 (pCO2) and osmolality as high as 150 mmHg and 440 mOsm/kg, respectively, were observed in large-scale CHO cell culture producing an antibody-fusion protein, B1. pCO2 and osmolality, when elevated to high levels in bioreactors, can adversely affect cell culture and recombinant protein production. To understand the sole impact of pCO2 or osmolality on CHO cell growth, experiments were performed in bench-scale bioreactors allowing one variable to change while controlling the other. Elevating pCO2 from 50 to 150 mmHg under controlled osmolality (about 350 mOsm/kg) resulted in a 9% reduction in specific cell growth rate. In contrast, increasing osmolality resulted in a linear reduction in specific cell growth rate (0.008 h(-1)/100 mOsm/kg) and led to a 60% decrease at 450 mOsm/kg as compared to the control at 316 mOsm/kg. This osmolality shift from 316 to 445 mOsm/kg resulted in an increase in specific production rates of lactate and ammonia by 43% and 48%, respectively. To elucidate the effect of high osmolality and/or pCO2 on the production phase, experiments were conducted in bench-scale bioreactors to more closely reflect the pCO2 and osmolality levels observed at large scale. Increasing osmolality to 400-450 mOsm/kg did not result in an obvious change in viable cell density and product titer. However, a further increase in osmolality to 460-500 mOsm/kg led to a 5% reduction in viable cell density and a 8% decrease in cell viability as compared to the control. Final titer was not affected as a result of an apparent increase in specific production rate under this increased osmolality. Furthermore, the combined effects from high pCO2 (140-160 mmHg) and osmolality (400-450 mOsm/kg) caused a 20% drop in viable cell density, a more prominent decrease as compared to elevated osmolality alone. Results obtained here illustrate the sole effect of high pCO2 (or osmolality) on CHO cell growth and demonstrate a distinct impact of high osmolality and/or pCO2 on production phase as compared to that on growth phase. These results are useful to understand the response of the CHO cells to elevated pCO2 (and/or osmolality) at a different stage of cultivation in bioreactors and thus are valuable in guiding bioreactor optimization toward improving protein production.

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During a CHO cell culture production process, important parameters are generally well controlled by a feedback mechanism (PID loop) in order to ensure consistency in both productivity and product quality. These parameters typically include pH, dissolved oxygen (DO), and temperature. While most of these parameters are very well controlled within their specific deadband, stable DO control can be challenging. Oscillations in DO concentration are not uncommon and these fluctuations can be exacerbated with an efficient mass transfer aeration strategy. In this study, where an IgG producing cell line was used, we observed increased lactate accumulation accompanied by decreased titer production in lots with fluctuations in DO concentration (DO ) when compared with lots with stable DO control (DO ). We demonstrate that DO had a greater impact on performance with respect to titer production and lactate accumulation than DO setpoint. Furthermore, we report that estimated specific oxygen uptake rates (qOURs) were lower in DO lots when compared with DO lots. We also report that purified mAb sourced from DO lots yielded lower drug-to-antibody ratio (DAR) after the sulfhydryl-targeted maleimide conjugation process when equivalent reducing agent was used. All mAb lots were within the analytical specifications for release, though a slight increase in measureable trisulfides were observed in DO mAb lots. DO control aimed to minimize fluctuations around DO setpoint was essential for us to produce consistent DAR without adjusting the conjugation process. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018.