Transient ammonia stress on Chinese hamster ovary (CHO) cells yield alterations to alanine metabolism and IgG glycosylation profiles
Transient ammonia stress on Chinese hamster ovary (CHO) cells yield alterations to alanine metabolism and IgG glycosylation profiles
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DOI：
10.1002/biot.202100098
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发表时间：
2021-06-09
影响因子：
4.7
通讯作者：
Sandoval，Nicholas R.
中科院分区：
文献类型：
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作者：
Synoground，Benjamin F.;McGraw，Claire E.;Sandoval，Nicholas R.
背景在哺乳动物细胞培养期间，氨浓度通常会增加，主要是由于谷氨酰胺和其他氨基酸的消耗。早期氨应激指标是相对于丙氨酸的代谢转变。为了确定这种代谢转变的潜在机制，在平行补料分批生物反应器中培养了两种不同年龄（标准和年轻）的中国仓鼠卵巢（CHO）细胞系，在12 h时加入0 mM或10 mM氨。观察到强制降解细胞的活细胞密度降低，而活力未受到显著影响。应激培养物具有较高的丙氨酸、乳酸和谷氨酸积累。有趣的是，到第8.5天，所有培养物的氨浓度相似。我们假设氨转化为丙氨酸是一种应对机制。有趣的是，未观察到由于细胞年龄导致的代谢产物谱的显著差异。糖基化分析显示氨强制降解降低了半乳糖基化、唾液酸化和岩藻糖基化。标准老化培养物的转录组分析表明，氨强制降解对转录组的影响有限，其中很少有显著变化与代谢物或糖基化反应直接相关。这些结果表明，用于缓解氨应激的机制最有可能是转录后控制的，这是未来研究的重点。
BackgroundAmmonia concentrations typically increase during mammalian cell cultures, mainly due to glutamine and other amino acid consumption. An early ammonia stress indicator is a metabolic shift with respect to alanine. To determine the underlying mechanisms of this metabolic shift, a Chinese hamster ovary (CHO) cell line with two distinct ages (standard and young) was cultured in parallel fed‐batch bioreactors with 0 mM or 10 mM ammonia added at 12 h. Reduced viable cell densities were observed for the stressed cells, while viability was not significantly affected. The stressed cultures had higher alanine, lactate, and glutamate accumulation. Interestingly, the ammonia concentrations were similar by Day 8.5 for all cultures. We hypothesized the ammonia was converted to alanine as a coping mechanism. Interestingly, no significant differences were observed for metabolite profiles due to cell age. Glycosylation analysis showed the ammonia stress reduced galactosylation, sialylation, and fucosylation. Transcriptome analysis of the standard‐aged cultures indicated the ammonia stress had a limited impact on the transcriptome, where few of the significant changes were directly related metabolite or glycosylation reactions. These results indicate that mechanisms used to alleviate ammonia stress are most likely controlled post‐transcriptionally, and this is where future research should focus.