Systematic development of temperature shift strategies for Chinese hamster ovary cells based on short duration cultures and kinetic modeling

Abstract

Temperature shift (TS) to a hypothermic condition has been widely used during protein production processes that use Chinese hamster ovary (CHO) cells. The effect of temperature on cell growth, metabolites, protein titer and quality depends on cell line, product, and other bioreactor conditions. Due to the large numbers of experiments, which typically last 2-3 weeks each, limited systematic TS studies have been reported with multiple shift temperatures and steps at different times. Here, we systematically studied the effect of temperature on cell culture performance for the production of two monoclonal antibodies by industrial GS and DG44 CHO cell lines. Three 2-8 day short-duration methods were developed and validated for researching the effect of many different temperatures on CHO cell culture and quality attributes. We found that minor temperature differences (1-1.5 °C) affected cell culture performance. The kinetic parameters extracted from the short duration data were subsequently used to compute and predict cell culture performance in extended duration of 10-14 days with multiple TS conditions for both CHO cell lines. These short-duration culture methods with kinetic modeling tools may be used for effective TS optimization to achieve the best profiles for cell growth, metabolites, titer and quality attributes. Although only three short-duration methods were developed with two CHO cell lines, similar short-duration methods with kinetic modeling may be applied for different hosts, including both microbial and other mammalian cells.

Keywords: CHO cell culture; kinetic modeling; process development; quality attributes; short duration method; specific productivity; temperature shift; titer.

Figure 1.

CHO1 cell growth (A), metabolite profiles: glucose (B), glutamine (C), glutamate (D), and lactate (E), and mAb1 production: titer (F) and specific productivity q_P (G) in fed-batch production in 250-mL shake flasks at different constant temperatures of 32, 33, 34, 35, and 36.5 ºC with a high initial VCD of 10 × 10⁶ cells/mL for an 8-day short duration. The dots denote the experimental data with the average of duplicate runs (n = 2) and the lines represent the simulation results.

Figure 2.

CHO1 model validation for VCD (A), titer (B) and specific productivity (C) in fed-batch production 250-mL shake flasks with TS from 36.5 to 33 or 34 °C on different days using a typical initial VCD of 0.6 × 10⁶ cells/mL for a 14-day full duration. The dots denote the experimental data with the average of duplicate runs (n = 2) and the lines represent the computing results using the kinetic models from the 8-day short-duration study.

Figure 3.

CHO2 cell growth (A) and metabolite profiles: glucose (B), glutamine (C), glutamate (D), lactate (E) and ammonium (F) in batch production 5-L bioreactors at different constant temperatures of 33, 34, 35, 36.5, 37.5 and 38.5 ºC with an initial typical VCD of 0.6 × 10⁶ cells/mL for a 4-day short duration. The dots denote the experimental data (n = 1) and the lines represent the simulation results.

Figure 4.

CHO2 model validation for cell growth (A) and metabolites: glucose (B), glutamine (C), glutamate (D), lactate (E) and ammonium (F) in fed-batch production 5-L bioreactors (n = 3) with TS from 36.5 to 35 °C on day4 using an initial typical VCD of 0.6 × 10⁶ cells/mL for a 14-day full duration. The dots denote the experimental data for a 14-day full duration and the lines represent the computing results for first 10-day duration using the kinetic models from the 4-day short duration study.

Figure 5.

VCD profiles prediction of CHO2 at different TS conditions with a typical initial VCD of 0.6 × 10⁶ cells/mL: TS from 36.5 °C to 35, 34 and 33 °C on day 4 (A); TS from 36.5 °C to 33 °C on day 4, day 6 and day 8 (B). M represents a multiple temperature shifting steps: TS from 36.5 °C to 35 °C on day 4, then to 34 °C on day 6, and finally to 33°C on day 8. The lines represent the computational prediction results using the kinetic models from the 4-day short-duration study.

Figure 6.

Effect of different constant temperatures on CHO2 for mAb2 specific productivity (A) and quality attributes: SEC profile (HMW: B) and charge variants (Acidic: C, Basic: D) in batch 125-mL shake flasks with a high initial VCD of 10 × 10⁶ cells/mL for a 2-day short duration (n = 2).

Figure 7.

Effect of different TS conditions on mAb2 specific productivity (q_P values were calculated from day6 to day10) (A) and final D14 quality attributes: SEC profile (HMW: B) and charge variants (Acidic: C, Basic: D) in fed-batch production 5-L bioreactors (n = 2) with an initial temperature of 36.5 °C, then reduced to 35, 33.5 or 32 °C from day5 to day14 using a typical initial VCD of 0.6 × 10⁶ cells/mL for a 14-day full duration.