Control of IgG LC:HC ratio in stably transfected CHO cells and study of the impact on expression, aggregation, glycosylation and conformational stability
Control of IgG LC:HC ratio in stably transfected CHO cells and study of the impact on expression, aggregation, glycosylation and conformational stability
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DOI：
10.1016/j.jbiotec.2013.03.019
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发表时间：
2013-06-10
影响因子：
4.1
通讯作者：
Yang, Yuansheng
中科院分区：
文献类型：
--
作者：
Ho, Steven C. L.;Koh, Esther Y. C.;Yang, Yuansheng
免疫球蛋白 G (IgG) 是最常见的一类商业单克隆抗体 (mAb)，以两条相同的轻链 (LC) 和两条相同的重链 (HC) 通过二硫桥组装在一起的多聚体形式存在。由于 mAb 组装的动力学，表明等量 LC 和 HC 的表达对于 IgG 生产来说并不是最佳的。我们设计了一组使用内部核糖体进入位点 (IRES) 元件来控制 LC 和 HC 表达的载体。稳定表达 IgG 的中国仓鼠卵巢 (CHO) 细胞库的细胞内 LC:HC 比率可以有效控制在 3.43、1.24、1.12 和 0.32 四种不同的比率。使用稳定库来研究 LC:HC 比率对 mAb 表达和质量的影响。基因扩增对于具有过量 LC 的库最为有效，并在转染库中产生最高的 mAb 滴度。当LC:HC比大于1时，97%以上的分泌产物是IgG单体。在这些比例下，这些产品还具有相似的 N-糖基化特征和构象稳定性。对于 LC:HC 比率较低（0.32）的池，单体仅占产物的一半，另一半是聚集体和 mAb 片段。在最低的 LC:HC 比率下，高甘露糖型 N-聚糖增加，而岩藻糖基化和半乳糖基化聚糖显着减少。产品稳定性也受到不利影响。获得的结果提供了不同 LC:HC 比例对稳定 mAb 生产的影响的见解，并为 mAb 生产细胞系生成过程中的载体设计提供了有用的信息。 (C) 2013 Elsevier B.V. 保留所有权利。
Immunoglobulin G (IgG), the most common class of commercial monoclonal antibodies (mAbs), exists as multimers of two identical light chains (LC) and two identical heavy chains (HC) assembled together by disulfide bridges. Due to the kinetics of mAb assembly, it is suggested that expression of LC and HC in equal amounts is not optimal for IgG production. We designed a set of vectors using internal ribosome entry site (IRES) elements to control LC and HC expression. The intracellular LC:HC ratio of stable IgG expressing Chinese hamster ovary (CHO) cell pools can be controlled effectively at four different ratios of 3.43, 1.24, 1.12, and 0.32. The stable pools were used to study the impact of LC:HC ratio on mAb expression and quality. Gene amplification was most effective for pools with excess LC and generated the highest mAb titers among the transfected pools. When LC:HC ratio was greater than one, more than 97% of the secreted products were IgG monomers. The products also have similar N-glycosylation profiles and conformational stabilities at those ratios. For pools presented a lower LC:HC ratio of 0.32, monomers only constituted half of the product with the other half being aggregates and mAb fragments. High mannosetype N-glycans increased while fucosylated and galactosylated glycans decreased significantly at the lowest LC:HC ratio. Product stability was also adversely affected. The results obtained provide insights to the impact of different LC:HC ratios on stable mAb production and useful information for vector design during generation of mAb producing cell lines. (C) 2013 Elsevier B.V. All rights reserved.