Pharmaceutical Development, Genentech, South San Francisco, CA 94080, USA.
Oceanside Pharmaceutical Technical Development, Genentech, Oceanside, CA 92056, USA.
Int J Mol Sci. 2021 Apr 12;22(8):3952. doi: 10.3390/ijms22083952.
Oxidation is an important degradation pathway of protein drugs. The susceptibility to oxidation is a common concern for therapeutic proteins as it may impact product efficacy and patient safety. In this work, we used 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) as an oxidative stress reagent to evaluate the oxidation of therapeutic antibodies. In addition to the oxidation of methionine (Met) and tryptophan (Trp) residues, we also observed an increase of protein aggregation. Size-exclusion chromatography and multi-angle light scattering showed that the soluble aggregates induced by AAPH consist of dimer, tetramer, and higher-order aggregate species. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that inter-molecular disulfide bonds contributed to the protein aggregation. Furthermore, intrinsic fluorescence spectra suggested that dimerization of tyrosine (Tyr) residues could account for the non-reducible cross-links. An excipient screening study demonstrated that Trp, pyridoxine, or Tyr could effectively reduce protein aggregation due to oxidative stress. This work provides valuable insight into the mechanisms of oxidative-stress induced protein aggregation, as well as strategies to minimize such aggregate formation during the development and storage of therapeutic proteins.
氧化是蛋白质药物的重要降解途径。对于治疗性蛋白质，氧化的易感性是一个共同关注的问题，因为它可能会影响产品的疗效和患者的安全。在这项工作中，我们使用 2,2'-偶氮双（2-脒基丙烷）二盐酸盐（AAPH）作为氧化应激试剂来评估治疗性抗体的氧化。除了甲硫氨酸（Met）和色氨酸（Trp）残基的氧化外，我们还观察到蛋白质聚集的增加。尺寸排阻色谱和多角度光散射表明，AAPH 诱导的可溶性聚集体由二聚体、四聚体和更高阶的聚集体组成。十二烷基硫酸钠聚丙烯酰胺凝胶电泳表明，分子间二硫键有助于蛋白质聚集。此外，内源荧光光谱表明，酪氨酸（Tyr）残基的二聚化可以解释不可还原的交联。辅料筛选研究表明，色氨酸、吡哆醇或酪氨酸可以有效地减少由于氧化应激引起的蛋白质聚集。这项工作为氧化应激诱导的蛋白质聚集的机制以及在治疗性蛋白质的开发和储存过程中减少这种聚集形成的策略提供了有价值的见解。
