Department of Protein Characterization, TRION Pharma, Frankfurter Ring 193a, Munich, 80807, Germany.
J Chromatogr B Analyt Technol Biomed Life Sci. 2010 Mar 15;878(9-10):777-84. doi: 10.1016/j.jchromb.2010.01.036. Epub 2010 Jan 29.
Oxidation of methionine residues and deamidation of asparagine residues are the major causes of chemical degradation of biological pharmaceuticals. The mechanism of these non-enzymatic chemical reactions has been studied in great detail. However, the identification and quantification of oxidation and deamidation sites in a given protein still remains a challenge. In this study, we identified and characterized several oxidation and deamidation sites in a rat/mouse hybrid antibody. We evaluated the effects of the sample preparation on oxidation and deamidation levels and optimized the peptide mapping method to minimize oxidation and deamidation artifacts. Out of a total number of 18 methionine residues, we identified six methionine residues most susceptible to oxidation. We determined the oxidation rate of the six methionine residues using 0.05% H(2)O(2) at different temperatures. Methionine residue 256 of the mouse heavy chain showed the fastest rate of oxidation under those conditions with a half life of approximately 200 min at 4 degrees C and 27 min at 37 degrees C. We identified five asparagine residues prone to deamidation under accelerated conditions of pH 8.6 at 37 degrees C. Kinetic characterization of the deamidation sites showed that asparagine residue 218 of the rat heavy chain exhibited the fastest rate of deamidation with a half live of 1.5 days at pH 8.6 and 37 degrees C. Analysis of antibody isoforms using free flow electrophoresis showed that deamidation is the major cause of the charged variants of this rat/mouse hybrid antibody.
甲硫氨酸残基的氧化和天冬酰胺残基的脱酰胺作用是生物制药化学降解的主要原因。这些非酶化学反应的机制已经被深入研究。然而，在给定的蛋白质中鉴定和定量氧化和脱酰胺位点仍然是一个挑战。在这项研究中，我们鉴定并表征了一种大鼠/小鼠杂交抗体中的几个氧化和脱酰胺位点。我们评估了样品制备对氧化和脱酰胺水平的影响，并优化了肽图分析方法以最小化氧化和脱酰胺假象。在总共 18 个甲硫氨酸残基中，我们鉴定了 6 个最易氧化的甲硫氨酸残基。我们使用 0.05% H(2)O(2)在不同温度下测定了这 6 个甲硫氨酸残基的氧化速率。在这些条件下，鼠重链的甲硫氨酸残基 256 显示出最快的氧化速率，在 4°C 时半衰期约为 200 分钟，在 37°C 时半衰期约为 27 分钟。我们鉴定了 5 个在加速条件下（pH 8.6，37°C）易发生脱酰胺的天冬酰胺残基。脱酰胺位点的动力学特征表明，大鼠重链的天冬酰胺残基 218 表现出最快的脱酰胺速率，在 pH 8.6 和 37°C 时半衰期为 1.5 天。使用自由流电泳分析抗体亚型表明，脱酰胺是导致这种大鼠/小鼠杂交抗体带电荷变异体的主要原因。
