CHO-K1 host cells adapted to growth in glutamine-free medium by FACS-assisted evolution
CHO-K1 host cells adapted to growth in glutamine-free medium by FACS-assisted evolution
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DOI：
10.1002/biot.201000095
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发表时间：
2010-10-01
影响因子：
4.7
通讯作者：
Borth, Nicole
中科院分区：
文献类型：
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作者：
Bort, Juan A. Hernandez;Stern, Beate;Borth, Nicole
在生物制药生产的重组细胞系优化过程中，必须考虑多种细胞特性，如抗应激能力、获得高细胞浓度和维持高活力，以最大限度地提高蛋白质产量。为了改善生长和活力，补充谷氨酰胺作为快速分裂细胞的替代能源，这些细胞氧化葡萄糖效率低下。然而，由此产生的副产物氨在高浓度下有毒，并对蛋白质糖基化产生负面影响，而蛋白质糖基化是生物制药的主要质量决定参数。在这项工作中，CHO-K1 细胞系在 3 周内适应了化学成分确定的培养基并悬浮生长。随后，谷氨酰胺浓度从8mM逐步降低至4mM和2mM。每次减少后，批次中的最终细胞浓度和活力都会下降。为了迫使细胞快速进化以达到较高的最终细胞浓度，以高密度（10(7) 个细胞/mL）接种细胞，并在活力下降至 10% 时（通常在 24 小时后）通过 FACS 或 MACS 对存活细胞进行分选。分批培养分批细胞，直至活力下降至 10%，并再次恢复活力。最终分选的群体能够达到可比甚至更好的活细胞浓度，并且与祖先相比，活力显着提高。将 2 mM 谷氨酰胺适应细胞系直接转移到不含谷氨酰胺的培养基中，并且能够以相当的速率生长，而无需进一步适应。细胞通过增加谷氨酸和天冬氨酸的消耗来补偿谷氨酰胺的缺乏。
During the process of recombinant cell line optimisation for production of biopharmaceuticals, multiple cellular properties like robustness against stress, the attainment of high cell concentrations and maintenance of high viability must be considered to maximize protein yield. To improve growth and viability, glutamine is supplemented as an alternative energy source for rapidly dividing cells that oxidize glucose inefficiently. However, the resulting by-product ammonia is toxic at high concentrations and has a negative impact on protein glycosylation, a major quality-determining parameter of biopharmaceuticals. In this work, the CHO-K1 cell line was adapted to a chemically defined medium and suspension growth within 3 weeks. Subsequently, the glutamine concentration was stepwise reduced from 8 to 4 and 2 mM. After each reduction, both the final cell concentration in the batch and the viability decreased. To force a rapid evolution of cells to achieve high final cell concentrations, cells were seeded at high densities (10(7) cells/mL) and surviving cells were sorted by FACS or MACS when viability declined to 10% (typically after 24 h). Sorted cells were grown in batch until viability declined to 10% and viable cells recovered again. The final sorted population was able to reach comparable or even better viable cell concentrations and showed a significantly improved viability compared to their ancestors. The 2 mM glutamine-adapted cell line was directly transferred into glutamine-free medium and was able to grow at comparable rates without requiring further adaptation. Cells compensated the lack of glutamine by increasing their consumption of glutamate and aspartate.