Effect Of Temperature On Recombinant TNFR-Fc Aggregation In CHO Cell

Recombinant human Tumor necrosis factor receptor Ⅱ-Fc(TNFR-Fc) is an therapeutic protein which is expressed in Chinese hamster ovary(CHO) cells. The desired TNFR-Fc 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.In our study, we investigated the effect of different culture temperatures on the aggregation of recombinant TNFR-Fc. 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. In the condition of 31°C, the degree of TNFR-Fc aggregation was lowest with only 12.71%, while the aggregation was more than 60% in 37°C. Temperature shift could reduce the degree of TNFR-Fc aggregation. In addition, we found that TNFR-Fc aggregation had a negative relationship with the anti-TNFα activity. Namely, the more TNFR-Fc aggregation, the lower anti-TNFα activity. We also found that the cell size could be increased greatly by reducing the culture temperature and lowering the culture temperature improved assembly rate and secretion rate of TNFR-Fc, which was good for CHO cells to transfer TNFR-Fc out of cells, improving the efficiency of TNFR-Fc post-translational modification.Three unfolded protein response(UPR) proteins were tested by Q-PCR. It was found that the mRNA of PERK was much higher at 31°C than 37°C, while IRE-1 and ATF-6 showed no difference at different culture temperature. Moreover, it was identified by western blot that PERK and phosphorylation of eIF2 a were higher at 31°C than 37°C, the latter could slow down protein synthesis to reduce the accumulation of unfolded protein or misfolded protein, which was very important for cells to survive.Both intracellular and extracellular TNFR-Fc increased when PERK inhibitor was added into the medium, which inferred that PERK inhibition could accelerate the synthesis of TNFR-Fc, but not effectively alleviated the intracellular accumulation of TNFR-Fc. PERK inhibition also decreased the phosphorylation of eIF2 a. The percentage of TNFR-Fc aggregation increased from 4.81% to 6.14% and 46.94% to 55.35% at 31°C and 37°C respectively. Overexpression of PERK could increase phosphorylation of e IF2 a, reducing the aggregation of TNFR-Fc. When PERK was overexpressed, there was no TNFR-Fc polymer detected. And dimeric TNFR-Fc was about 91.98%, compared with the control with 4.65% of aggregation and 85.54% of dimeric TNFR-Fc.In conclusion, culture temperature has an effect on TNFR-Fc synthesis and secretion. And TNFR-Fc aggregation decreases at lower temperature. Moreover, PERK plays a pivotal role in TNFR-Fc aggregation.