半乳糖流加对CHO细胞生长代谢及其表达的Fc融合蛋白糖基化的影响

Galactose Feeding in CHO Cell Culture Process :the Impacts on the Cell Growth,Metabolism and Glycosylation of Fc Fusion Protein

旨在深入认识补料分批培养过程中,以半乳糖替代葡萄糖作为碳源,对CHO细胞生长、代谢和产物表达的影响。通过将补料培养基中的葡萄糖用等摩尔的半乳糖进行替换,综合考察了不同比例替换条件下CHO细胞的生长代谢和Fc融合蛋白的产物合成特性。结果显示:摇瓶的数据表明60%比例以上半乳糖的替代对细胞的生长造成了不利影响,培养后期的pH出现了大幅上升。同时随着半乳糖替代比例的增加,虽然代谢副产物乳酸的浓度有明显下降,但氨的生成却显著增多;此外,培养过程中谷氨酸和丙氨酸的浓度也随着替代比例的增加而增加。产物表达方面,在较低替代比例内(0%-40%),Fc融合蛋白的表达量和总唾液酸含量都随着替代比例的增加而升高,而随着替代比例的进一步升高(60%-100%),两者都逐渐降低。最后,在反应器内通过对培养pH的稳定控制,40%半乳糖替代过程的产物表达量和总唾液酸含量分别提高了43%和37%。补料培养基中以半乳糖替代葡萄糖进行补料的方式,有效地提高了最终Fc融合蛋白的表达量和总唾液酸含量,有助于建立高产高质的CHO细胞培养过程。

This work aims to get insights into the effects of feeding galactose on the CHO cell growth,metabolism and product expression during fed-batch culture process. Through substituting glucose with equimolar galactose in the feed medium of CHO cell culture process,the impacts of feeding galactose with varied ratio on cell growth,metabolism and product characteristics of Fc fusion protein were investigated. Results showed that the data from shake flask indicated more than 60% galactose substitution adversely affected cell growth,and the pH of late culture process increased significantly. Moreover,with the increase of galactose substitution proportion in the feed medium,metabolic by- product lactate concentration rapidly decreased,while accumulation of ammonia increased significantly;in addition,the concentrations of glutamic acid and alanine also showed a gradient increase. Within low substitution percentage(0%-40%)of galactose,the titer and sialic acid content increased with the increase of substitution proportion;however,within a higher proportion(60%-100%),they both decreased with the replacement ratio increasing. Through stably controlling the culture pH in the bioreactor,by replacing the 40% of total glucose with galactose,the titer and total sialic acid content of Fc fusion protein were enhanced by 43% and 37% respectively. In sum,substitution of glucose with galactose in CHO fed-batch process may greatly enhance product expression and sialic acid content,and is conducive to establish the high-yield and high-quality CHO culture process.