pH Condition in temperature shift cultivation enhances cell longevity and specific hMab productivity in CHO culture

Abstract

Controlling cell proliferation during cell culturing is an effective way to improve the production yield in mammalian cell culture. We examined the effect of temperature shifts (TS) under pH control conditions in Chinese hamster ovary cells. When we shifted the culture temperature from 37 degrees C to 31 degrees C before a stationary phase at pH 6.8 (TS/pH 6.8), cell viability remained high, and the final human monoclonal antibody (hMab) concentration increased to 2.3 times that in the culture remaining at 37 degrees C. However, there were no significant effects on the cell viability or production yield with the same TS at pH 7.0 (TS/pH 7.0). The average specific hMab productivity and mRNA level of TS/pH 7.0 were the same as that of TS/pH 6.8. The control of cell growth by the TS or the addition of rapamycin was effective in the maintenance of cell viability, but there was no significant increase of the average specific hMab productivity in the culture where cell proliferation was controlled with rapamycin. The hMab mRNA concentration decreased to 55%-65% at a 37 degrees C culture with the addition of actinomycin D. In contrast, actinomycin D did not affect the mRNA level in the TS culture. This result suggested that the increase in the mRNA level in the TS condition was caused by an increase in mRNA stability. In this study, we show that TS can produce two unrelated effects: a prolongation of cell longevity and an improvement in mRNA stability.

Fig. 1

The profiles of temperature shift cultures in pH 7.0 or 6.8 conditions using 2-L bioreactors. (a) VCD, (b) Viability, (c) hMab concentration, (d) hMab concentration as a function of IVCD. The average specific hMab productivity was determined by the slope of the hMab concentration versus IVCD. Results are mean of multiple experiments. (* n = 3, ** n = 4)

Fig. 2

Northern blot and Q-PCR analysis of HC and LC mRNA levels in various culture conditions. RNAs were sampled 4 days after inoculation at pH 7.0 (Lane 1) or 6.8 (Lane 2) conditions and 8 days after inoculation at 37 °C/pH 7.0 (Lane 3), TS/pH 7.0 (Lane 4) and TS/pH 6.8 (Lane 5). The percentage under the bands were the relative mRNA values against 37 °C/pH 7.0 on day 8 quantified by Q-PCR. The mRNA value of the HC and LC was normalized to β-actin

Fig. 3

Northern blot of HC and LC mRNA levels in the culture with or without rapamycin. After 4 days of cultivation, 100 ng mL⁻¹ rapamycin was added to the culture. RNAs were sampled 7 days after inoculation. Lane 1 is the culture without rapamycin, Lane 2 is the culture with rapamycin

Fig. 4

A TS affects the stability of hMab mRNA. The cells maintained 24 h at 37 °C or at 31 °C after 3 days cultivation at 37 °C were chased for 0, 12 or 24 h in the presence of 5.0 μg mL⁻¹ of actinomycin D. At the end of each chase period, the cells were counted and hMab transcripts were quantified by Q-PCR. (a) Percentage of HC against 37 °C at 0 h, (b) Percentage of LC against 37 °C at 0 h. Error bars indicate SD (n = 3)