表达rhTNFR-Fc的CHO细胞的氨基酸代谢特征及氨基酸流加培养工艺研究

为提高表达重组人肿瘤坏死因子受体-Fc融合蛋白(rhTNFR-Fc)的CHO工程细胞的培养密度和目的蛋白表达,研究了葡萄糖和谷氨酰胺流加-批式培养工艺的细胞氨基酸代谢特征及游离氨基酸和大豆蛋白水解物的补加策略。通过补加葡萄糖和谷氨酰胺浓缩液分别维持其浓度为10 mmol/L和2 mmol/L左右,在不同阶段定期补加特定的氨基酸浓缩液或大豆蛋白水解物补充氨基酸消耗,检测细胞培养上清中rhTNFR-Fc的表达。谷氨酸、天冬酰胺、天冬氨酸、脯氨酸等4种非必需氨基酸快速消耗,是CHO工程细胞增殖和目的蛋白表达的限制性底物。以游离氨基酸、游离氨基酸合并大豆蛋白水解物补充氨基酸消耗可分别使细胞生长密度提高50%、100%,目的蛋白表达增加60%、125%,培养时程延长44%、66%,同时葡萄糖比消耗率、乳酸比生成率降低,谷氨酰胺的利用效率提高。

Optimization of Propionic Acid Feeding for Production of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in Fed-Batch Culture of Ralstonia eutropha

The feeding method of propionic acid for production ofpoly(3-hydroxybutyrate-co-3-hydro xyvalerate) [P(3HB-co-3HV)] by fed-batch culture of Ralstoniaeutropha was optimized to achieve high cell density and high 3HV yield. Effects of different feedingstrategies of propionic acid on the production of P(3HB-co-3HV) were investigated. A decline ofspecific synthesis rate of copolymer and the yield of 3HV unit from propionic acid were observed dueto the propionic acid accumulation in culture broth when the feeding solution with highP/G(propionic acid to glucose) ratio was employed. It was further confirmed by controlling propionicacid concentration at a low level in the separate feeding of propionic acid. An optimal feedingstrategy was demonstrated to reduce the propionic acid accumulation. The cell concentration,P(3HB-co-3HV) productivity and 3HV unit fraction reached to 163.9kg·m^(-3), 1.8kg·m^(-3)·h^(-1),and 10.6%(by mass), respectively, resulting in a yield of 0.33g HV per g propionic acid.