Comprehensive characterisation of the heterogeneity of adalimumab via charge variant analysis hyphenated on-line to native high resolution Orbitrap mass spectrometry.
Comprehensive characterisation of the heterogeneity of adalimumab via charge variant analysis hyphenated on-line to native high resolution Orbitrap mass spectrometry.
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DOI：
10.1080/19420862.2018.1531664
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发表时间：
2019-01
期刊：
影响因子：
5.3
通讯作者：
Bones J
中科院分区：
文献类型：
--
作者：
Füssl F;Trappe A;Cook K;Scheffler K;Fitzgerald O;Bones J
电荷变异分析是一种广泛使用的工具，用于监测单抗生产过程中产品质量的变化。尽管它是揭示mAb异质性的一种强大技术，但一个意想不到的结果，例如以前未检测到的异构体的出现，需要进一步的、耗时的分析。识别这些未知因素的过程也可能导致分子发生不必要的变化，而这些变化不能归因于制造过程。为了克服这一问题，我们最近报道了一种将高选择性阳离子交换层析电荷变量分析与在线质谱仪(MS)检测相结合的方法。我们进一步探索和改造了色谱缓冲系统，扩大了应用范围。此外，我们在天然蛋白质上没有观察到盐加合物，这也得到了MS参数的优化选择的支持，从而提高了数据质量和质量准确性。在这里，我们通过对强制降解前后的阿达利单抗进行深入的分析，证明了这种改进方法的实用性。通过结合分子质量和保留时间信息，我们能够识别对adalimumab的多种修饰，包括赖氨酸截断、糖基化、脱酰胺化、琥珀酰亚胺形成、异构化、N-末端天冬氨酸丢失或C-末端脯氨酸酰胺化和断裂以及这些已鉴定蛋白形式的N-糖链分布。对宿主细胞蛋白(HCP)进行了液-质联用分析，证实了组织蛋白酶L的存在。基于微量具有催化活性的HCP的存在，可以怀疑裂解是由自发水解还是也可能是由酶降解驱动的。
Charge variant analysis is a widely used tool to monitor changes in product quality during the manufacturing process of monoclonal antibodies (mAbs). Although it is a powerful technique for revealing mAb heterogeneity, an unexpected outcome, for example the appearance of previously undetected isoforms, requires further, time-consuming analysis. The process of identifying these unknowns can also result in unwanted changes to the molecule that are not attributable to the manufacturing process. To overcome this, we recently reported a method combining highly selective cation exchange chromatography-based charge variant analysis with on-line mass spectrometric (MS) detection. We further explored and adapted the chromatographic buffer system to expand the application range. Moreover, we observed no salt adducts on the native protein, also supported by the optimal choice of MS parameters, resulting in increased data quality and mass accuracy. Here, we demonstrate the utility of this improved method by performing an in-depth analysis of adalimumab before and after forced degradation. By combining molecular mass and retention time information, we were able to identify multiple modifications on adalimumab, including lysine truncation, glycation, deamidation, succinimide formation, isomerisation, N-terminal aspartic acid loss or C-terminal proline amidation and fragmentation along with the N-glycan distribution of each of these identified proteoforms. Host cell protein (HCP) analysis was performed using liquid chromatography-mass spectrometry that verified the presence of the protease Cathepsin L. Based on the presence of trace HCPs with catalytic activity, it can be questioned if fragmentation is solely driven by spontaneous hydrolysis or possibly also by enzymatic degradation.
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影响因子：
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作者：
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通讯作者：
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DOI：
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发表时间：
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影响因子：
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Guillarme, Davy
DOI：
10.1016/j.jchromb.2004.12.033
发表时间：
2005-04-25
影响因子：
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作者：
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