Molecular and Analytical Development, Bristol-Myers Squibb, 311 Pennington Rocky Hill Road, Pennington, New Jersey 08534.
Molecular and Analytical Development, Bristol-Myers Squibb, 311 Pennington Rocky Hill Road, Pennington, New Jersey 08534.
J Pharm Sci. 2019 Jun;108(6):1944-1952. doi: 10.1016/j.xphs.2018.12.027. Epub 2019 Jan 11.
Tryptophan (Trp) oxidation in proteins leads to a number of events, including changes in color, higher order structure (HOS), and biological activity. We describe here a number of new findings through a comprehensive characterization of 6 monoclonal antibodies (mAbs) following selective oxidation of Trp residues by 2,2'-azobis(2-amidinopropane) dihydrochloride. Fluorescence spectroscopy, in combination with second derivative analysis, demonstrates that the loss of Trp fluorescence intensity is a sensitive indicator of Trp oxidation in mAbs. Size-exclusion chromatography with UV and intrinsic Trp fluorescence detection was demonstrated to be a useful method to monitor Trp oxidation levels in mAbs. Furthermore, the Trp oxidation levels measured by size-exclusion chromatography with UV and intrinsic Trp fluorescence detection were found to be in agreement with the values obtained from tryptic peptide mapping by liquid chromatography with mass spectrometric detection and correlate with the total solvent accessible surface area of the exposed Trp residues from in silico modeling. Finally, near-UV circular dichroism and Raman spectroscopy were used to evaluate the impact of Trp oxidation on HOS and identify specific oxidation products, respectively. This work demonstrates that protein HOS is altered on Trp oxidation in mAbs and multiple spectroscopic markers can be used to monitor the molecule-dependent Trp oxidation behavior.
色氨酸（Trp）在蛋白质中的氧化会导致许多事件的发生，包括颜色变化、高级结构（HOS）变化和生物活性变化。我们在这里描述了通过对 2,2'-偶氮双（2-脒基丙烷）二盐酸盐选择性氧化 Trp 残基的 6 种单克隆抗体（mAb）进行全面表征所获得的一些新发现。荧光光谱学结合二阶导数分析表明，Trp 荧光强度的丧失是 mAb 中 Trp 氧化的敏感指标。用紫外和本征 Trp 荧光检测的凝胶过滤色谱法被证明是监测 mAb 中 Trp 氧化水平的有用方法。此外，用紫外和本征 Trp 荧光检测的凝胶过滤色谱法测量的 Trp 氧化水平与通过液相色谱与质谱检测的胰蛋白酶肽图获得的值一致，并与从计算机建模获得的暴露 Trp 残基的总溶剂可及表面积相关。最后，近紫外圆二色性和拉曼光谱分别用于评估 Trp 氧化对 HOS 的影响，并确定特定的氧化产物。这项工作表明，mAb 中的 Trp 氧化会改变蛋白质的 HOS，并且可以使用多种光谱标记物来监测分子依赖性的 Trp 氧化行为。
