Cell Sciences & Technology, Amgen, Inc., Seattle, Washington, 98119.
Biotechnol Bioeng. 2014 May;111(5):965-79. doi: 10.1002/bit.25155. Epub 2013 Dec 11.
Depletion of two nonessential amino acids, asparagine (Asn) and glutamine (Gln), occurred during a fed-batch production process with a CHO cell line expressing a recombinant antibody. This depletion coincided with growth suppression and the onset of the stationary phase. Experimental withdrawal of Asn led to cell cycle arrest of cell line A in G0/G1 phase. On a mechanistic level, withdrawal of either Asn or Gln stimulated the amino-acid response (AAR) pathway, indicating that depletion of nonessential amino acids can induce AAR in this cell line. Compared to withdrawal of an essential amino acid, leucine (Leu), withdrawal of either Asn or Gln induced fewer changes in downstream effectors of mammalian target of rapamycin (mTOR) signaling involved in regulation of global protein synthesis. Global transcriptional analysis followed by pathway analysis revealed that the cultures experienced a down-regulation of cell-cycle progression, DNA replication and nucleotide biosynthesis in an E2F-dependent manner, as well as a down-regulation of lipid metabolism in a SREBP1/2-dependent manner as a result of individual amino-acid withdrawal. Timing and magnitude of observed phenotypic and transcriptional responses to amino-acid withdrawal differed between essential (Leu) and nonessential (Asn and Gln) amino acids examined. Observed responses were similar in parental (CS9 and CHOK-1) and two other antibody-producing CHO cell lines, but the magnitude of the transcriptional response was both cell-line and amino-acid dependent. Overall, these results suggest that depletion of nonessential amino acids in cell culture plays a role in the onset of the stationary phase of production process and offer mechanistic insights into the observed growth attenuation phenotype.
在表达重组抗体的 CHO 细胞系的分批补料生产过程中，两种非必需氨基酸（天冬酰胺（Asn）和谷氨酰胺（Gln））被耗尽。这种消耗与生长抑制和静止期的开始同时发生。实验性去除 Asn 导致细胞系 A 在 G0/G1 期停滞。在机制水平上，去除 Asn 或 Gln 都会刺激氨基酸反应（AAR）途径，表明非必需氨基酸的消耗可以诱导该细胞系中的 AAR。与去除必需氨基酸亮氨酸（Leu）相比，去除非必需氨基酸 Asn 或 Gln 会导致参与调节整体蛋白质合成的哺乳动物雷帕霉素靶蛋白（mTOR）信号下游效应物的变化减少。随后进行的转录组分析和途径分析表明，培养物经历了 E2F 依赖性的细胞周期进程、DNA 复制和核苷酸生物合成的下调，以及 SREBP1/2 依赖性的脂质代谢下调，这是由于单独氨基酸的去除。对氨基酸去除的表型和转录响应的观察到的时间和幅度在检查的必需（Leu）和非必需（Asn 和 Gln）氨基酸之间有所不同。在亲本（CS9 和 CHOK-1）和另外两种产生抗体的 CHO 细胞系中观察到相似的反应，但转录反应的幅度既依赖于细胞系又依赖于氨基酸。总体而言，这些结果表明细胞培养中非必需氨基酸的消耗在生产过程静止期的开始中起作用，并为观察到的生长衰减表型提供了机制见解。
