Osmolality Effects on CHO Cell Growth, Cell Volume, Antibody Productivity and Glycosylation

Abstract

The addition of nutrients and accumulation of metabolites in a fed-batch culture of Chinese hamster ovary (CHO) cells leads to an increase in extracellular osmolality in late stage culture. Herein, we explore the effect of osmolality on CHO cell growth, specific monoclonal antibody (mAb) productivity and glycosylation achieved with the addition of NaCl or the supplementation of a commercial feed. Although both methods lead to an increase in specific antibody productivity, they have different effects on cell growth and antibody production. Osmolality modulation using NaCl up to 470 mOsm kg^-1 had a consistently positive effect on specific antibody productivity and titre. The addition of the commercial feed achieved variable results: specific mAb productivity was increased, yet cell growth rate was significantly compromised at high osmolality values. As a result, Feed C addition to 410 mOsm kg^-1 was the only condition that achieved a significantly higher mAb titre compared to the control. Additionally, Feed C supplementation resulted in a significant reduction in galactosylated antibody structures. Cell volume was found to be positively correlated to osmolality; however, osmolality alone could not account for observed changes in average cell diameter without considering cell cycle variations. These results help delineate the overall effect of osmolality on titre and highlight the potentially negative effect of overfeeding on cell growth.

Keywords: Chinese hamster ovary cells; antibody glycosylation; cell volume; hyperosmolality; monoclonal antibody.

Figure A1

Culture viability profile across different osmolality values. The cultures were harvested on the day after the final measurement reported herein, when the viability in one of the biological duplicates dropped below 80%.

Figure A2

RNA (y axis) and DNA (x axis) distribution on days 4 and 9 of the bioreactor run at physiological temperature.

Figure A3

Forward scatter height versus area for single cells on days 2 and 14 from the bioreactor run at physiological temperature.

Figure 1

Maximum growth rate of Chinese hamster ovary (CHO) cells as a function of extracellular osmolality. The first point at 320 mOsm kg⁻¹ represents the control condition.

Figure 2

Time evolution of cell culture parameters under different osmolality conditions. (A) Viable cell density; (B) Osmolality value; (C) Average cell diameter; (D) Extracellular ammonia concentration; (E) Extracellular lactate concentration; (F) Extracellular glucose concentration; (G) Extracellular Na⁺ concentration; (H) Extracellular K⁺ concentration; (I) Extracellular glutamate concentration; (J) Extracellular glutamine concentration. Error bars represent the standard deviation of two biological replicates, except for the average cell diameter, which shows standard errors.

Figure 2

Time evolution of cell culture parameters under different osmolality conditions. (A) Viable cell density; (B) Osmolality value; (C) Average cell diameter; (D) Extracellular ammonia concentration; (E) Extracellular lactate concentration; (F) Extracellular glucose concentration; (G) Extracellular Na⁺ concentration; (H) Extracellular K⁺ concentration; (I) Extracellular glutamate concentration; (J) Extracellular glutamine concentration. Error bars represent the standard deviation of two biological replicates, except for the average cell diameter, which shows standard errors.

Figure 3

Specific uptake/production rates of (A) ammonia, (B) glucose, (C) glutamate, (D) glutamine and (E) lactate under different osmolality conditions. The control cultures produced no detectable levels of glutamine. Error bars represent the standard deviation of two biological replicates. For the analysis of significance (* p < 0.05), equal variance of each variable between different experiments was assumed.

Figure 4

Final mAb titre (bars) and integral viable cell density (green points) for control, NaCl and Feed C experiments. Error bars represent the standard deviation of two biological replicates.

Figure 5

Specific antibody productivity for control, NaCl and Feed C experiments. Error bars represent the standard deviation of two biological replicates. For the analysis of significance (* p < 0.05), equal variance of each variable between different experiments was assumed.

Figure 6

Glycosylation profile for control, NaCl and Feed C experiments at harvest. Error bars represent the standard deviation of two biological replicates. For the analysis of significance (* p < 0.05), equal variance of each variable between different experiments was assumed.

Figure 7

(a) Osmolality profile for the bioreactor runs at 36.5 °C (blue) and with a temperature shift to 32° C on day 5; (b) Average cell volume against culture osmolality for both bioreactor datasets.

Figure 8

(a) Cell cycle distribution on day 4 of culture; (b) Cell cycle distribution on day 9 of culture as determined by a Flowtop multicycle DNA analysis.