Detection and characterization of altered conformations of protein pharmaceuticals using complementary mass spectrometry-based approaches.
Detection and characterization of altered conformations of protein pharmaceuticals using complementary mass spectrometry-based approaches.
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DOI：
10.1021/ac801214x
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发表时间：
2008-10-01
影响因子：
7.4
通讯作者：
Kaltashov, Igor A.
中科院分区：
文献类型：
--
作者：
Bobst, Cedric E.;Abzalimov, Rinat R.;Houde, Damian;Kloczewiak, Marek;Mhatre, Rohin;Berkowitz, Steven A.;Kaltashov, Igor A.
与小分子药物不同，蛋白质生物药物在溶液中的构象特性受到多种因素的影响，这些因素不仅仅由其共价化学结构定义。由于蛋白质的构象（或更高级结构）是其生物活性的主要调节剂，因此检测由一系列外在因素诱导的蛋白质的更高级结构和构象动力学的变化的能力在生产、纯化和配制具有一致治疗性质的商业生物药物中是至关重要的。在这项研究中，我们证明了两个互补的质谱法为基础的方法（离子电荷态分布和氢/氘交换分析）可以是一个有力的工具，在蛋白质生物制药的构象变化监测。通过检测和表征生物制药产品干扰素β-1a（IFN-β1a）的构象变化，证明了这些方法的实用性。通过诱导IFN-β1a的单一化学修饰（用N-乙基马来酰亚胺烷基化其唯一的游离半胱氨酸残基）来模拟蛋白质降解过程，这导致其抗病毒活性显著降低。IFN-β1a离子电荷态分布分析明确显示降解蛋白的构象稳定性显著降低，而氢/氘交换测量结果明确表明，高级结构受到的影响远远超出共价修饰位点。重要的是，这两种技术都不要求在分析过程之前知道或在分析过程中阐明化学修饰的位置或性质。相比之下，通常用于蛋白质药物质量控制的生物物理工具的标准设备的应用在检测和表征修饰的IFN-β1a中的构象变化方面的成功非常有限。这项工作突出的作用，质谱可以和应该发挥在生物制药行业超越目前分配的任务，一级结构分析。
Unlike small molecule drugs, the conformational properties of protein biopharmaceuticals in solution are influenced by a variety of factors that are not solely defined by their covalent chemical structure. Since the conformation (or higher order structure) of a protein is a major modulator of its biological activity, the ability to detect changes in both the higher order structure and conformational dynamics of a protein, induced by an array of extrinsic factors, is of central importance in producing, purifying, and formulating a commercial biopharmaceutical with consistent therapeutic properties. In this study we demonstrate that two complementary mass spectrometry-based approaches (analysis of ionic charge state distribution and hydrogen/deuterium exchange) can be a potent tool in monitoring conformational changes in protein biopharmaceuticals. The utility of these approaches is demonstrated by detecting and characterizing conformational changes in the biopharmaceutical product interferon β-1a (IFN-β1a). The protein degradation process was modeled by inducing a single chemical modification of IFN-β1a (alkylation of its only free cysteine residue with N-ethylmaleimide), which causes significant reduction in its antiviral activity. Analysis of IFN-β1a ionic charge state distributions unequivocally reveals a significant decrease of conformational stability in the degraded protein, while hydrogen/deuterium exchange measurements provide a clear indication that the higher order structure is affected well beyond the covalent modification site. Importantly, neither technique required that the location or indeed the nature of the chemical modification be known prior to or elucidated in the process of the analysis. In contrast, application of the standard armamentarium of biophysical tools, which are commonly employed for quality control of protein pharmaceuticals, met with very limited success in detection and characterization of conformational changes in the modified IFN-β1a. This work highlights the role mass spectrometry can and should play in the biopharmaceutical industry beyond the presently assigned task of primary structure analysis.
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影响因子：
3.7
作者：
Alam, J;Goelz, S;Rogge, M
通讯作者：
Rogge, M
影响因子：
6.1
作者：
Kuhlmann, Martin;Covic, Adrian
通讯作者：
Covic, Adrian
影响因子：
7.4
作者：
Mohimen, A;Dobo, A;Kaltashov, IA
通讯作者：
Kaltashov, IA
DOI：
10.1002/prot.340170110
发表时间：
1993-09-01
期刊：
PROTEINS-STRUCTURE FUNCTION AND GENETICS
影响因子：
--
作者：
BAI, YW;MILNE, JS;ENGLANDER, SW
通讯作者：
ENGLANDER, SW
影响因子：
3.9
作者：
Hipkiss, Alan R.
通讯作者：
Hipkiss, Alan R.