The Effect of Culture Temperature on the Aggregation of Recombinant TNFR-Fc is Regulated by the PERK-eIF2a Pathway in CHO Cells

Protein and Peptide Letters
Kai WangQiuling Xie

Abstract

Recombinant human Tumor necrosis factor receptor Ⅱ-Fc (TNFR-Fc) is a therapeutic protein which is expressed in Chinese Hamster Ovary (CHO) cells. The desired TNFRFc is a dimeric form, however, polymeric TNFR-Fc aggregation often occurs during cell culture which needs to be removed to minimize the potential risk of immunogenicity to patients. Lowering the culture temperature is useful to improve the production of many recombinant proteins in CHO cells. The effect of different culture temperatures (37°C and 31°C, or a shift from 37°C to 31°C on the 3rd day) on the aggregation of recombinant TNFR-Fc were investigated. Recombinant cells were cultivated at three different temperatures, namely consistent 37°C, 31°C and temperature shifted from 37°C to 31°C on the 3rd day. Intracellular and extracellular TNFR-Fc were quantified by ELISA. Bioactivity of TNFR-Fc was measured by WST-8. UPR sensors such as IRE1, PERK, ATF6, and BiP tested by Q-PCR and WB. The fusion protein TNFRFc was purified by affinity column Protein A. Size-Exclusion Chromatography (SEC) was used to determine the amount of dimeric and multimeric TNFR-Fc. Culture at 31°C could improve the bioactivity, assembly and secretion of TNFR-Fc, while protein aggregation decreas...Continue Reading

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