Merck Research Laboratories, Union, NJ 07083, USA.
Anal Biochem. 2013 May 15;436(2):93-100. doi: 10.1016/j.ab.2013.01.020. Epub 2013 Jan 29.
Confirmation of the correct disulfide linkage and demonstration of the lack of a significant level of scrambled disulfide bonds are critical to ensure the appropriate folding and structure of recombinant monoclonal antibodies. Currently these are typically achieved by carrying out multiple experiments, most commonly via the comparison of the samples before and after reduction by LC-MS and MS/MS. The data are then analyzed by searching across all the possible disulfide linkages manually or with the aid of computer algorithms. To eliminate the need of multiple experiments and complicated data analysis, a simple LC-MS-based method coupled with post-column partial reduction was developed. Using a recombinant monoclonal IgG1 antibody as an example, this method demonstrates the ability to confirm the correct disulfide linkage and the ability to detect scrambled disulfide bonds from a single experiment with a simple data analysis strategy.
确认正确的二硫键连接并证明没有大量错配的二硫键对于确保重组单克隆抗体的适当折叠和结构至关重要。目前，这通常通过进行多个实验来实现，最常见的方法是通过 LC-MS 和 MS/MS 比较还原前后的样品。然后，通过手动或借助计算机算法搜索所有可能的二硫键连接来分析数据。为了消除多次实验和复杂数据分析的需要，开发了一种简单的基于 LC-MS 的方法，结合柱后部分还原。使用重组单克隆 IgG1 抗体作为示例，该方法证明了从单个实验中仅使用简单的数据分析策略即可确认正确的二硫键连接以及检测错配的二硫键的能力。
