Exploring cellular behavior under transient gene expression and its impact on mAb productivity and Fc-glycosylation.
Exploring cellular behavior under transient gene expression and its impact on mAb productivity and Fc-glycosylation.
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DOI：
10.1002/bit.26456
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发表时间：
2018-03
影响因子：
3.8
通讯作者：
Kontoravdi C
中科院分区：
文献类型：
--
作者：
Sou SN;Lee K;Nayyar K;Polizzi KM;Sellick C;Kontoravdi C
瞬时基因表达(TGE)是一种用于快速提供重组蛋白材料的生物处理方法。经常引入亚低温来克服TGE通常获得的低产量，并提高特定蛋白质的生产率。因此，研究亚低温对瞬时表达蛋白质的产量和质量的影响以及与细胞过程和代谢变化的关系是很有意义的。在这项研究中，我们重点研究了中国仓鼠卵巢细胞系对重组单抗(MAb)产物半乳糖化的能力。通过实验和通量平衡分析，我们的结果表明，在我们的系统中，在轻度低温条件下，与36.5℃下的TGE相比，TGE使QP增加了76%。这种增加伴随着营养素和氨基酸的消耗增加，以及细胞内核苷酸糖种的产生增加，以及mAb半乳糖化的速度增加，尽管细胞生长速度降低。生物量积累的减少使细胞能够将其能量和资源重新分配到mAb合成和Fc糖基化上。有趣的是，细胞在32℃下半乳糖化TGE中重组产物的较高能力似乎并不是由于半乳糖基转移酶(Galts)的上调，而是由于N-乙酰氨基葡萄糖转移酶II(GnTII)在该细胞系中表达的增加，这有助于产生用于进一步加工的双触角糖链结构。
Transient gene expression (TGE) is a methodology employed in bioprocessing for the fast provision of recombinant protein material. Mild hypothermia is often introduced to overcome the low yield typically achieved with TGE and improve specific protein productivity. It is therefore of interest to examine the impact of mild hypothermic temperatures on both the yield and quality of transiently expressed proteins and the relationship to changes in cellular processes and metabolism. In this study, we focus on the ability of a Chinese hamster ovary cell line to galactosylate a recombinant monoclonal antibody (mAb) product. Through experimentation and flux balance analysis, our results show that TGE in mild hypothermic conditions led to a 76% increase in qP compared to TGE at 36.5°C in our system. This increase is accompanied by increased consumption of nutrients and amino acids, together with increased production of intracellular nucleotide sugar species, and higher rates of mAb galactosylation, despite a reduced rate of cell growth. The reduction in biomass accumulation allowed cells to redistribute their energy and resources toward mAb synthesis and Fc‐glycosylation. Interestingly, the higher capacity of cells to galactosylate the recombinant product in TGE at 32°C appears not to have been assisted by the upregulation of galactosyltransferases (GalTs), but by the increased expression of N‐acetylglucosaminyltransferase II (GnTII) in this cell line, which facilitated the production of bi‐antennary glycan structures for further processing.
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影响因子：
14.9
作者：
Starega-Roslan J;Krol J;Koscianska E;Kozlowski P;Szlachcic WJ;Sobczak K;Krzyzosiak WJ
通讯作者：
Krzyzosiak WJ
影响因子：
2.9
作者：
Cain, Katharine;Peters, Shirley;Dickson, Alan
通讯作者：
Dickson, Alan
影响因子：
3.4
作者：
Codamo, Joe;Hou, Jeff Jia Cheng;Munro, Trent P.
通讯作者：
Munro, Trent P.
影响因子：
4.1
作者：
Wingens, Marc;Gaetgens, Jochem;Hoffrogge, Raimund
通讯作者：
Hoffrogge, Raimund
影响因子：
2.9
作者：
Wulhfard, Sarah;Tissot, Stephanie;Wurm, Florian M.
通讯作者：
Wurm, Florian M.