R&DB Center, LOGONE Bio-convergence Research Foundation, Seoul, Republic of Korea.
IFEZ Bio Analysis Center, Binex Co., Ltd., Incheon, Republic of Korea.
Sci Rep. 2020 May 21;10(1):8412. doi: 10.1038/s41598-020-65495-y.
A hyperglycosylated recombinant human interferon-β (rhIFN-β) R27T mutant was established to improve relapsing-remitting multiple sclerosis (RRMS) in our previous study. We focused on the stability of the R27T mutant throughout its production lifetime, including culture, purification, and storage before formulation prior to clinical use. Herein, we address the stability of this protein during optimized culture and purification processes. Additionally, we employed artificial stress conditions during culture and purification to characterize R27T instability. Although, among total R27T, relative native R27T ratio displayed transiently low even under optimized production process, the ratio was recovered by the end of the overall production process, suggesting that culture and purification processes are optimized. Artificial stress during culture and purification processes resulted in degradation of R27T acidic and basic variants, and mismatched disulfide bonds in no-aggregated forms as well as in the aggregated form. The presence of disulfide bond exchange without aggregation in the unfolded/misfolded state could be a novel finding for rhIFN-β products. The results provide meaningful information for the comprehensive evaluation of the stability of the R27T variant.
在我们之前的研究中，构建了一种高糖基化的重组人干扰素-β（rhIFN-β）R27T 突变体，以改善复发性多发性硬化症（RRMS）。我们关注的是该 R27T 突变体在整个生产寿命期间的稳定性，包括临床使用前的培养、纯化和制剂前的储存。在此，我们解决了在优化的培养和纯化过程中该蛋白的稳定性问题。此外，我们在培养和纯化过程中采用人工应激条件来表征 R27T 的不稳定性。尽管在优化的生产工艺下，总 R27T 中相对天然 R27T 的比例会短暂降低，但在整个生产过程结束时，该比例会恢复，这表明培养和纯化工艺得到了优化。在培养和纯化过程中，人工应激导致 R27T 酸性和碱性变体以及非聚集形式和聚集形式中的错配二硫键降解。在未聚集的展开/错误折叠状态下发生二硫键交换而不聚集可能是 rhIFN-β 产品的一个新发现。这些结果为 R27T 变体的稳定性综合评估提供了有意义的信息。
