Supplementation of Nucleosides During Selection can Reduce Sequence Variant Levels in CHO Cells Using GS/MSX Selection System,Biotechnology Journal

当前位置： X-MOL 学术 › Biotechnol. J. › 论文详情

Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)

Supplementation of Nucleosides During Selection can Reduce Sequence Variant Levels in CHO Cells Using GS/MSX Selection System
Biotechnology Journal ( IF 3.2 ) Pub Date : 2017-08-17 , DOI: 10.1002/biot.201700335
Danming Tang ₁ , Cynthia Lam ₁ , Salina Louie ₁ , Kam Hon Hoi ₂ , David Shaw ₁ , Mandy Yim ₁ , Brad Snedecor ₁ , Shahram Misaghi ₁

Affiliation

In the process of generating stable monoclonal antibody (mAb) producing cell lines, reagents such as methotrexate (MTX) or methionine sulfoximine (MSX) are often used. However, using such selection reagent(s) increases the possibility of having higher occurrence of sequence variants in the expressed antibody molecules due to the effects of MTX or MSX on de novo nucleotide synthesis. Since MSX inhibits glutamine synthase (GS) and results in both amino acid and nucleoside starvation, it is questioned whether supplementing nucleosides into the media could lower sequence variant levels without affecting titer. The results show that the supplementation of nucleosides to the media during MSX selection decreased genomic DNA mutagenesis rates in the selected cells, probably by reducing nucleotide mis-incorporation into the DNA. Furthermore, addition of nucleosides enhance clone recovery post selection and does not affect antibody expression. It is further observed that nucleoside supplements lowered DNA mutagenesis rates only at the initial stage of the clone selection and do not have any effect on DNA mutagenesis rates after stable cell lines are established. Therefore, the data suggests that addition of nucleosides during early stages of MSX selection can lower sequence variant levels without affecting titer or clone stability in antibody expression. In the process of generating stable monoclonal antibody (mAb) producing cell lines, using MSX as the selective agent to inhibit glutamine synthase (GS) causes both glutamine and nucleotides starvation. Nucleotides starvation can increase the possibility of having sequence variants in the expressed antibody molecules. In this study, it is shown that supplementation of nucleosides in the media during MSX selection reduces DNA mutagenesis, possibly by preventing the mis-incorporation of defective nucleotides into the DNA. Furthermore, the data suggest that nucleosides supplementation during MSX selection does not affect selection stringency but increases clone recovery rate, making it suitable for generation of stable mAb expressing cell lines.

中文翻译：

使用GS / MSX选择系统在选择过程中添加核苷可以降低CHO细胞中的序列变异水平

在产生稳定的单克隆抗体（mAb）的细胞系的过程中，经常使用诸如甲氨蝶呤（MTX）或蛋氨酸亚砜亚胺（MSX）之类的试剂。然而，由于MTX或MSX对从头核苷酸合成的作用，使用这样的选择试剂增加了在表达的抗体分子中更高发生序列变体的可能性。由于MSX抑制谷氨酰胺合酶（GS）并导致氨基酸和核苷饥饿，因此有人质疑向培养基中添加核苷是否可以降低序列变异水平而不影响效价。结果表明，在MSX选择过程中向培养基中添加核苷可降低所选细胞中基因组DNA的诱变率，这可能是通过减少核苷酸错掺入DNA引起的。此外，添加核苷可增强选择后的克隆恢复，并且不影响抗体表达。进一步观察到，核苷补充剂仅在克隆选择的初始阶段才降低了DNA诱变速率，并且在建立稳定的细胞系后对DNA诱变速率没有任何影响。因此，数据表明在MSX选择的早期添加核苷可以降低序列变异水平，而不会影响抗体表达中的效价或克隆稳定性。在产生稳定的单克隆抗体（mAb）的细胞系的过程中，使用MSX作为抑制谷氨酰胺合酶（GS）的选择剂会导致谷氨酰胺和核苷酸饥饿。核苷酸饥饿可增加在表达的抗体分子中具有序列变异的可能性。在这项研究中，表明在MSX选择过程中在培养基中添加核苷可以减少DNA诱变，这可能是通过防止将有缺陷的核苷酸错误掺入DNA来实现的。此外，数据表明在MSX选择过程中补充核苷不会影响选择严格性，但会提高克隆回收率，使其适合生成稳定的mAb表达细胞系。

更新日期：2017-12-18

点击分享查看原文

点击收藏

阅读更多本刊新发论文本刊介绍/投稿指南