Lee Hoon-Min, Park Jong-Ho, Kim Tae-Ho, Kim Hyun-Seung, Kim Dae Eung, Lee Mi Kyeong, You Jungmok, Lee Gyun Min, Kim Yeon-Gu
Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon, Korea.
Department of Bioprocess Engineering, KRIBB School of Biotechnology, University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon, Korea.
Appl Microbiol Biotechnol. 2024 Feb 20;108(1):224. doi: 10.1007/s00253-024-13059-9.
The occurrence of autophagy in recombinant Chinese hamster ovary (rCHO) cell culture has attracted attention due to its effects on therapeutic protein production. Given the significance of glycosylation in therapeutic proteins, this study examined the effects of autophagy-inhibiting chemicals on sialylation of Fc-fusion glycoproteins in rCHO cells. Three chemical autophagy inhibitors known to inhibit different stages were separately treated with two rCHO cell lines that produce the same Fc-fusion glycoprotein derived from DUKX-B11 and DG44. All autophagy inhibitors significantly decreased the sialylation of Fc-fusion glycoprotein in both cell lines. The decrease in sialylation of Fc-fusion glycoprotein is unlikely to be attributed to the release of intracellular enzymes, given the high cell viability and low activity of extracellular sialidases. Interestingly, the five intracellular nucleotide sugars remained abundant in cells treated with autophagy inhibitors. In the mRNA expression profiles of 27 N-glycosylation-related genes using the NanoString nCounter system, no significant differences in gene expression were noted. With the positive effect of supplementing nucleotide sugar precursors on sialylation, attempts were made to enhance the levels of intracellular nucleotide sugars by supplying these precursors. The addition of nucleotide sugar precursors to cultures treated with inhibitors successfully enhanced the sialylation of Fc-fusion glycoproteins compared to the control culture. This was particularly evident under mild stress conditions and not under relatively severe stress conditions, which were characterized by a high decrease in sialylation. These results suggest that inhibiting autophagy in rCHO cell culture decreases sialylation of Fc-fusion glycoprotein by constraining the availability of intracellular nucleotide sugars. KEY POINTS: • The autophagy inhibition in rCHO cell culture leads to a significant reduction in the sialylation of Fc-fusion glycoprotein. • The pool of five intracellular nucleotide sugars remained highly abundant in cells treated with autophagy inhibitors. • Supplementation of nucleotide sugar precursors effectively restores decreased sialylation, particularly under mild stress conditions but not in relatively severe stress conditions.
自噬在重组中国仓鼠卵巢（rCHO）细胞培养中的发生因其对治疗性蛋白质生产的影响而受到关注。鉴于糖基化在治疗性蛋白质中的重要性，本研究考察了自噬抑制化学物质对rCHO细胞中Fc融合糖蛋白唾液酸化的影响。已知能抑制不同阶段的三种化学自噬抑制剂分别与两种产生源自DUKX - B11和DG44的相同Fc融合糖蛋白的rCHO细胞系进行处理。所有自噬抑制剂均显著降低了两种细胞系中Fc融合糖蛋白的唾液酸化。鉴于细胞活力高且细胞外唾液酸酶活性低，Fc融合糖蛋白唾液酸化的降低不太可能归因于细胞内酶的释放。有趣的是，在用自噬抑制剂处理的细胞中，五种细胞内核苷糖仍然丰富。使用NanoString nCounter系统对27个N - 糖基化相关基因的mRNA表达谱进行分析，未发现基因表达有显著差异。由于补充核苷糖前体对唾液酸化有积极作用，因此尝试通过供应这些前体来提高细胞内核苷糖的水平。与对照培养相比，向用抑制剂处理的培养物中添加核苷糖前体成功提高了Fc融合糖蛋白的唾液酸化。这在轻度应激条件下尤为明显，而在以唾液酸化高度降低为特征的相对严重应激条件下则不明显。这些结果表明，在rCHO细胞培养中抑制自噬会通过限制细胞内核苷糖的可用性而降低Fc融合糖蛋白的唾液酸化。要点：• rCHO细胞培养中的自噬抑制导致Fc融合糖蛋白的唾液酸化显著降低。• 在用自噬抑制剂处理的细胞中，五种细胞内核苷糖库仍然高度丰富。• 补充核苷糖前体可有效恢复降低的唾液酸化，特别是在轻度应激条件下，但在相对严重应激条件下则不能。
