Alsenaidy Mohammad A, Jain Nishant K, Kim Jae H, Middaugh C Russell, Volkin David B
Department of Pharmaceutical Chemistry, Macromolecule and Vaccine Stabilization Center, University of Kansas Lawrence, KS, USA.
Front Pharmacol. 2014 Mar 12;5:39. doi: 10.3389/fphar.2014.00039. eCollection 2014.
In this review, some of the challenges and opportunities encountered during protein comparability assessments are summarized with an emphasis on developing new analytical approaches to better monitor higher-order protein structures. Several case studies are presented using high throughput biophysical methods to collect protein physical stability data as function of temperature, agitation, ionic strength and/or solution pH. These large data sets were then used to construct empirical phase diagrams (EPDs), radar charts, and comparative signature diagrams (CSDs) for data visualization and structural comparisons between the different proteins. Protein samples with different sizes, post-translational modifications, and inherent stability are presented: acidic fibroblast growth factor (FGF-1) mutants, different glycoforms of an IgG1 mAb prepared by deglycosylation, as well as comparisons of different formulations of an IgG1 mAb and granulocyte colony stimulating factor (GCSF). Using this approach, differences in structural integrity and conformational stability profiles were detected under stress conditions that could not be resolved by using the same techniques under ambient conditions (i.e., no stress). Thus, an evaluation of conformational stability differences may serve as an effective surrogate to monitor differences in higher-order structure between protein samples. These case studies are discussed in the context of potential utility in protein comparability studies.
在本综述中，总结了蛋白质可比性评估过程中遇到的一些挑战和机遇，重点是开发新的分析方法以更好地监测高阶蛋白质结构。介绍了几个案例研究，这些研究使用高通量生物物理方法收集蛋白质物理稳定性数据，作为温度、搅拌、离子强度和/或溶液pH的函数。然后，这些大数据集被用于构建经验相图（EPD）、雷达图和比较特征图（CSD），以进行数据可视化以及不同蛋白质之间的结构比较。展示了具有不同大小、翻译后修饰和固有稳定性的蛋白质样品：酸性成纤维细胞生长因子（FGF-1）突变体、通过去糖基化制备的IgG1单克隆抗体的不同糖型，以及IgG1单克隆抗体和粒细胞集落刺激因子（GCSF）不同制剂的比较。使用这种方法，在应激条件下检测到了结构完整性和构象稳定性概况的差异，而在环境条件下（即无应激）使用相同技术无法分辨这些差异。因此，构象稳定性差异的评估可以作为监测蛋白质样品之间高阶结构差异的有效替代方法。在蛋白质可比性研究的潜在用途背景下讨论了这些案例研究。
