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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 cells. 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.

Elevated serum uric acid is detected in pulmonary arterial hypertension (PAH) and is associated with poor patient outcomes. High serum uric acid is an independent risk factor for cardiovascular disease and renal impairment. We analyzed the effects of endothelin receptor antagonism on serum uric acid in PAH patients participating in the Sitaxentan to Relieve Impaired Exercise (STRIDE)-2/2X trial, and the impact of uric acid on 6-minute walk distance (6MWD), time to clinical worsening (TtCW) and survival.In the 18-week, double-blind, placebo-controlled STRIDE-2 trial, 246 PAH patients were randomized and received matched placebo, sitaxentan 50 or 100 mg orally once daily, or open-label bosentan 125 mg twice daily. STRIDE-2X was a 1-year, open-label extension of STRIDE-2.Baseline serum uric acid was similar between groups. Increased serum uric acid was a significant risk factor for 1-year mortality and TtCW. Compared with placebo, sitaxentan 50 and 100 mg and bosentan all reduced serum uric acid (p0.05). Reduced serum uric acid correlated with increased 6MWD (p = 0.0037).Endothelin receptor antagonism reduces serum uric acid in PAH patients, and this reduction is associated with improved survival and longer TtCW. Further prospective studies are needed to investigate the pathogenic role of serum uric acid in PAH and its prognostic potential.