Profiling of N‐glycosylation gene expression in CHO cell fed‐batch cultures
Profiling of N‐glycosylation gene expression in CHO cell fed‐batch cultures
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CHO 细胞补料分批培养物中 N-糖基化基因表达谱分析
DOI：
10.1002/bit.22828
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发表时间：
2010
影响因子：
3.8
通讯作者：
M. Yap
中科院分区：
文献类型：
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作者：
D. Wong;Niki S. C. Wong;John Soo Yang Goh;L. May;M. Yap
重组糖蛋白生产的目标之一是实现一致的糖基化。尽管许多研究已经研究了培养过程中重组蛋白糖基化质量的变化，但很少有人研究随着培养的进展糖基化基因表达的潜在变化。在本研究中，使用定量 RT PCR 检查了涉及 N-糖基化的 24 个基因的表达，以更好地了解重组糖蛋白在生产过程中的糖基化。对产生重组人干扰素-γ (IFN-γ) 的 CHO 细胞系的补料分批培养物的指数期、稳定期和死亡期进行 N-糖基化基因分析以及糖蛋白质量的同步分析。在检查的 24 个 N-糖基化基因中，随着补料分批培养从指数期、稳定期和死亡期进展，21 个基因表达显着上调或下调。随着补料分批培养的进行，唾液酸化程度较低的 IFN-γ 糖型也有所增加，导致唾液酸与重组 IFN-γ 的摩尔比下降 30%。这与 CMP 唾液酸合成相关基因表达减少以及唾液酸酶表达增加相关。与分批培养相比，低谷氨酰胺补料分批策略似乎需要 0.5 mM 谷氨酰胺阈值才能维持相似的 N 糖基化基因表达水平并实现相当的糖蛋白质量。本研究展示了使用定量实时 PCR 方法来识别 N-糖基化中可能的“瓶颈”或“受损”途径，并随后制定提高糖基化质量的策略。生物技术。生物工程。 2010；107：516-528。 © 2010 Wiley 期刊公司。
One of the goals of recombinant glycoprotein production is to achieve consistent glycosylation. Although many studies have examined the changes in the glycosylation quality of recombinant protein with culture, very little has been done to examine the underlying changes in glycosylation gene expression as a culture progresses. In this study, the expression of 24 genes involved in N‐glycosylation were examined using quantitative RT PCR to gain a better understanding of recombinant glycoprotein glycosylation during production processes. Profiling of the N‐glycosylation genes as well as concurrent analysis of glycoprotein quality was performed across the exponential, stationary and death phases of a fed‐batch culture of a CHO cell line producing recombinant human interferon‐γ (IFN‐γ). Of the 24 N‐glycosylation genes examined, 21 showed significant up‐ or down‐regulation of gene expression as the fed‐batch culture progressed from exponential, stationary and death phase. As the fed‐batch culture progressed, there was also an increase in less sialylated IFN‐γ glycoforms, leading to a 30% decrease in the molar ratio of sialic acid to recombinant IFN‐γ. This correlated with decreased expression of genes involved with CMP sialic acid synthesis coupled with increased expression of sialidases. Compared to batch culture, a low glutamine fed‐batch strategy appears to need a 0.5 mM glutamine threshold to maintain similar N‐glycosylation genes expression levels and to achieve comparable glycoprotein quality. This study demonstrates the use of quantitative real time PCR method to identify possible “bottlenecks” or “compromised” pathways in N‐glycosylation and subsequently allow for the development of strategies to improve glycosylation quality. Biotechnol. Bioeng. 2010;107: 516–528. © 2010 Wiley Periodicals, Inc.
DOI：
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发表时间：
1989-08
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
E. Lee;J. Rothb;J. Paulson
通讯作者：
E. Lee;J. Rothb;J. Paulson
糖苷酶处理对人干扰素γ理化性质和生物活性的影响。
DOI：
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发表时间：
1983
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Kelker,HC;Yip,YK;Anderson,P;Vilcek,J
通讯作者：
Vilcek,J