Charge-based analysis of antibodies with engineered cysteines From multiple peaks to a single main peak
Charge-based analysis of antibodies with engineered cysteines From multiple peaks to a single main peak
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DOI：
10.4161/mabs.1.6.10058
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发表时间：
2009-11-01
期刊：
影响因子：
5.3
通讯作者：
Ouyang, Jun
中科院分区：
文献类型：
--
作者：
Chen, Xiaoying (Nancy);Nguyen, Mary;Ouyang, Jun
THIOMAB是在每条重链上具有工程改造的未配对半胱氨酸残基的抗体，其可用作中间体以产生抗体-药物缀合物。在几种不同THIOMAB的阳离子交换色谱（CEX）和成像毛细管等电聚焦（cIEF）分析期间观察到多个电荷变体峰。这种电荷异质性是由于通过细胞培养过程中形成的二硫键在工程化和未配对的半胱氨酸处的半胱氨酸化和/或谷胱甘肽化。半胱氨酸处理，然后使用CEX、LC/MS和电泳进行分析，证明半胱氨酸是一种温和的还原剂，可以去除与工程化半胱氨酸结合的谷胱甘肽和半胱氨酸，而不破坏抗体的链间或链内二硫键。我们进一步证明，使用半胱氨酸/胱氨酸氧化还原对（而不是单独的半胱氨酸）不仅可以有效地去除工程化半胱氨酸处的谷胱甘肽，而且还可以产生均匀的半胱氨酸化物质，这在CEX-HPLC和成像cIEF测定中均导致具有工程化和未配对半胱氨酸的抗体的一个主峰。
THIOMABs are antibodies with an engineered unpaired cysteine residue on each heavy chain that can be used as intermediates to generate antibody-drug conjugates. Multiple charge variant peaks were observed during cation-exchange chromatography (CEX) and imaged capillary isoelectric focusing (cIEF) analysis of several different THIOMABs. This charge heterogeneity was due to cysteinylation and/or glutathionylation at the engineered and unpaired cysteines through disulfide bonds formed during the cell culture process. Cysteine treatment followed by analysis using CEX, LC/MS and electrophoresis demonstrates that cysteine is a mild reductant that can remove glutathione and cysteine bound to the engineered cysteines without disrupting the inter- or intra-chain disulfide bonds of antibodies. We further demonstrated that using a cysteine/cystine redox pair (rather than cysteine alone) can not only effectively remove glutathione at the engineered cysteines, but also generate homogeneously cysteinylated species, which resulted in one main peak in both CEX-HPLC and imaged cIEF assays for antibodies with engineered and unpaired cysteines.