Rational development of a serum-free medium and fed-batch process for a GS-CHO cell line expressing recombinant antibody

Abstract

A serum-free medium (CHO-SFM) together with a fed-batch process was developed for the cultivation of a recombinant GS-CHO cell line producing TNFR-Fc. According to the metabolic characteristics of GS-CHO cell, a basal medium was prepared by supplementing DMEM:F12:RPMI1640 (2:1:1) with amino acids, insulin, transferrin, Pluronic F68 and some other ingredients. Statistical optimization approaches based on Plackett-Burman and central composite designs were then adopted to identify additional positive determinants and determine their optimal concentrations, which resulted in the final CHO-SFM medium formulations. The maximum antibody titer reached was 90.95 mg/l in the developed CHO-SFM, which was a 18 % and 10 fold higher than that observed in the commercial EX-CELL™ 302 medium (76.95 mg/l) and basal medium (8.28 mg/l), respectively. Subsequently, a reliable, reproducible and robust fed-batch strategy was designed according to the offline measurement of glucose, giving a final antibody yield of 378 mg/l, which was a threefold improvement over that in conventional batch culture (122 mg/l) using CHO-SFM. In conclusion, the use of design of experiment (DoE) method facilitated the development of CHO-SFM medium and fed-batch process for the production of recombinant antibody using GS-CHO cells.

Fig. 1

Amino acid utilization profiles of recombinant CHO cells grown in EX-CELL™ 302 medium. CHO cells were seeded at 2–3 × 10⁵ cells/ml and conditioned medium was sampled after 5 days of incubation

Fig. 2

Effects of FAC on the growth and productivity of CHO cells. The basal medium was the formulation obtained from Plackett–Burman design

Fig. 3

Response surface plot showing the effects of 2 different variables and their interaction on the response (Antibody production) while the third variable was held at zero level

Fig. 4

Comparison between the optimized CHO-SFM and EX-CELL™ 302 medium in shake flasks for a 5 day batch culture. a Cell growth and TNFR-Fc production; b cell viability

Fig. 5

Functional assays of cells cultured in the developed SFM. Cells were inoculated at a density of 3 × 10⁵ cells/ml and subcultured every few days

Fig. 6

Batch culture. GS-CHO cells were cultured using CHO-SFM in a 2 L bioreactor with an inoculation of 2.3 × 10⁵ cells/ml. a Cell growth; b cell viability; c antibody production; d metabolism of glucose and lactate; e concentration profile of glutamine, glutamic acid and ammonia; f concentrations of amino acids at 0, 24, 48, 72, 96, 120 and 144 h

Fig. 7

Fed-batch culture. GS-CHO cells were cultured in a 2-L bioreactor using the designed CHO-SFM and feed medium. a Cell growth; b cell viability; c antibody production; d metabolism of glucose and lactate; e concentration profile of glutamine, glutamic acid, and ammonia; f osmolality