Glutathione and thioredoxin systems contribute to recombinant monoclonal antibody interchain disulfide bond reduction during bioprocessing
Glutathione and thioredoxin systems contribute to recombinant monoclonal antibody interchain disulfide bond reduction during bioprocessing
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DOI：
10.1002/bit.26278
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发表时间：
2017-07-01
影响因子：
3.8
通讯作者：
Ahuja, Sanjeev
中科院分区：
文献类型：
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作者：
Handlogten, Michael W.;Zhu, Min;Ahuja, Sanjeev
自2010年首次报道以来，生物制药生产过程中的抗体链间二硫键还原已受到越来越多的关注。抗体减少导致产物损失和产物稳定性降低。因此，了解减少的基本机制至关重要。迄今为止，硫氧还蛋白系统已被报道为生物加工过程中抗体还原的唯一贡献者。在这项工作中，我们表明，谷胱甘肽系统，除了硫氧还蛋白系统，参与减少抗体分子和两个系统的贡献可以根据细胞培养过程而变化。评价了谷胱甘肽和硫氧还蛋白系统对具有不同IgG亚类的三种分子的作用，其中在生产期间观察到还原：mAb A、mAb B和mAb C分别代表IgG（1）、IgG（2）和IgG（4）。在所有三种细胞系中证实了硫氧还蛋白和谷胱甘肽系统的酶表达。使用纯化的哺乳动物还原酶评价抑制剂以评价其特异性。优化的实验条件使得能够确定还原酶活性以及由每个酶系统还原的抗体的量。我们的研究结果表明，潜在的酶机制是不同的，这取决于细胞培养过程;两个系统之一可能是主导机制，或两个酶系统可能参与。具体而言，发现谷胱甘肽系统是mAb A还原的主要贡献者，而硫氧还蛋白系统是mAb C还原的主要贡献者。有趣的是，mAb B在两种酶系统中都经历了显著降低。总之，我们已经证明，除了硫氧还蛋白途径之外，谷胱甘肽系统是有助于抗体还原的第二个主要途径，并且可以利用这些知识来开发针对主导还原机制的更特异性的抗体还原缓解策略。Biotechnol. Bioeng. 2017;114：1469-1477. (c)2017 Wiley Periodicals，Inc.
Antibody interchain disulfide bond reduction during biopharmaceutical manufacturing has received increased attention since it was first reported in 2010. Antibody reduction leads to loss of product and reduced product stability. It is therefore critical to understand the underlying mechanisms of reduction. To date, the thioredoxin system has been reported as the sole contributor to antibody reduction during bioprocessing. In this work, we show that the glutathione system, in addition to the thioredoxin system, is involved in reducing antibody molecules and the contributions of the two systems can vary depending upon the cell culture process. The roles of the glutathione and thioredoxin systems were evaluated for three molecules with different IgG subclass where reduction was observed during manufacturing: mAb A, mAb B, and mAb C representing an IgG(1), IgG(2), and IgG(4,) respectively. The expression of enzymes for both the thioredoxin and glutathione systems were confirmed in all three cell lines. Inhibitors were evaluated using purified mammalian reductases to evaluate their specificity. The optimized experimental conditions enabled both the determination of reductase activity contributed from as well as the amount of antibody reduced by each enzymatic system. Our results demonstrate that the underlying enzymatic mechanisms are different depending upon the cell culture process; one of the two systems may be the dominant mechanism, or both enzymatic systems may be involved. Specifically, the glutathione system was found to be the major contributor to mAb A reduction while the thioredoxin system was the major contributor to mAb C reduction. Intriguingly, mAb B experienced significant reduction from both enzymatic systems. In summary, we have demonstrated that in addition to the thioredoxin pathway, the glutathione system is a second major pathway contributing to antibody reduction and this knowledge can be leveraged to develop more specific antibody reduction mitigation strategies targeted at the dominant reduction mechanism. Biotechnol. Bioeng. 2017;114: 1469-1477. (c) 2017 Wiley Periodicals, Inc.