Early Stage Pharmaceutical Development, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080.
Late Stage Pharmaceutical Development, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080.
J Pharm Sci. 2018 Feb;107(2):550-558. doi: 10.1016/j.xphs.2017.09.022. Epub 2017 Oct 5.
Protein oxidation is a major pathway for degradation of biologic drug products. Past literature reports have suggested that 2,2-azobis (2-amidinopropane) dihydrochloride (AAPH), a free radical generator that produces alkoxyl and alkyl peroxyl radicals, is a useful model reagent stress for assessing the oxidative susceptibility of proteins. Here, we expand the applications of the AAPH model by pairing it with a rapid peptide map method to enable site-specific studies of oxidative susceptibility of monoclonal antibodies and their derivatives for comparison between formats, the evaluation of formulation components, and comparisons across the stress models. Comparing the free radical-induced oxidation model by AAPH with a light-induced oxidation model suggests that light-sensitive residues represent a subset of AAPH-sensitive residues and therefore AAPH can be used as a preliminary screen to highlight molecules that need further assessment by light models. In sum, these studies demonstrate that AAPH stress can be used in multiple ways to evaluate labile residues and oxidation sensitivity as it pertains to developability and manufacturability.
蛋白质氧化是生物药物产品降解的主要途径。过去的文献报道表明，2,2-偶氮双（2-脒基丙烷）二盐酸盐（AAPH）是一种产生烷氧基和烷基过氧基自由基的自由基引发剂，是评估蛋白质氧化易感性的有用模型试剂。在这里，我们通过将 AAPH 模型与快速肽图方法相结合来扩展其应用，以实现单克隆抗体及其衍生物的氧化易感性的位点特异性研究，用于不同形式之间的比较、制剂成分的评估以及不同应激模型之间的比较。通过 AAPH 的自由基诱导氧化模型与光诱导氧化模型进行比较表明，光敏感残基代表 AAPH 敏感残基的一个子集，因此 AAPH 可以用作初步筛选，以突出需要通过光模型进一步评估的分子。总之，这些研究表明，AAPH 应激可以以多种方式用于评估不稳定残基和氧化敏感性，以涉及可开发性和可制造性。
