Cell Culture Development, Biogen Idec, 14 Cambridge Center, Cambridge, Massachusetts 02142, USA.
Biotechnol Bioeng. 2012 Oct;109(10):2523-32. doi: 10.1002/bit.24511. Epub 2012 Apr 9.
Molecular heterogeneity was detected in a recombinant monoclonal antibody (IgG1 mAb) due to the presence of a trisulfide linkage generated by the post-translational insertion of a sulfur atom into disulfide bonds at the heavy-heavy and heavy-light junctions. This molecular heterogeneity had no observable effect on antibody function. Nevertheless, to minimize the heterogeneity of the IgG1 mAb from run-to-run, an understanding of the impact of cell culture process conditions on trisulfide versus disulfide linkage formation was desirable. To investigate variables that might impact trisulfide formation, cell culture parameters were varied in bench-scale bioreactor studies. Trisulfide analysis of the samples from these runs revealed that the trisulfide content in the bond between heavy and light chains varied considerably from <1% to 39%. Optimizing the culture duration and feeding strategy resulted in more consistent trisulfide levels. Cysteine concentration in the feed medium had a direct correlation with the trisulfide level in the product. Systematic studies revealed that cysteine in the feed and the bioreactor media was contributing hydrogen sulfide which reacted with the IgG1 mAb in the supernatant leading to the insertion of sulfur atom and formation of a trisulfide bond. Cysteine feed strategies were developed to control the trisulfide modification in the recombinant monoclonal antibody.
由于重链-重链和重链-轻链连接处的二硫键发生了翻译后插入硫原子的反应，形成了一个三硫键，导致重组单克隆抗体（IgG1 mAb）中出现了分子异质性。这种分子异质性对抗体功能没有明显影响。然而，为了尽量减少 IgG1 mAb 在运行过程中的异质性，需要了解细胞培养工艺条件对三硫键与二硫键形成的影响。为了研究可能影响三硫键形成的变量，在台式生物反应器研究中对细胞培养参数进行了调整。对这些运行样品的三硫键分析表明，重链和轻链之间键的三硫键含量差异很大，从<1%到 39%不等。优化培养时间和补料策略可使三硫键水平更稳定。补料培养基中的半胱氨酸浓度与产物中的三硫键水平呈直接相关性。系统研究表明，补料和生物反应器中的半胱氨酸提供了硫化氢，它与上清液中的 IgG1 mAb 反应，插入硫原子并形成三硫键。制定了半胱氨酸补料策略来控制重组单克隆抗体中的三硫键修饰。
