Background: 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
Objective: 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.
Methods: 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.
Results: Culture at 31°C could improve the bioactivity, assembly and secretion of TNFR-Fc, while
protein aggregation decreased. The temperature shift led to a high TNFR-Fc titer, decreased aggregation,
and increased anti-TNF-α activity. Surprisingly, PERK mRNA and protein levels were
higher at 31°C than at 37°C, and phosphorylation of eIF2a increased. PERK inhibition and overexpression
experiments confirmed its regulatory role in protein aggregation and the overall reduced
protein production at low temperatures (31°C).
Conclusion: Culture temperature affects TNFR-Fc folding and secretion. Protein aggregation was
partly reduced in a PERK-dependent way at 31°C.