Structural Characterisation of Non-Deamidated Acidic Variants of Erwinia chrysanthemi L-asparaginase Using Small-Angle X-ray Scattering and Ion-Mobility Mass Spectrometry
Structural Characterisation of Non-Deamidated Acidic Variants of Erwinia chrysanthemi L-asparaginase Using Small-Angle X-ray Scattering and Ion-Mobility Mass Spectrometry
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DOI：
10.1007/s11095-015-1722-2
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发表时间：
2015-11-01
影响因子：
3.7
通讯作者：
Smith, Stuart
中科院分区：
文献类型：
--
作者：
Gervais, David;King, Darryl;Smith, Stuart
欧文氏菌L-天冬酰胺酶（ErA）是一种常用于急性淋巴细胞白血病（ALL）治疗方案的酶。生物制药产品（如ErA）必须进行监测，以进行脱酰胺等修饰，通常使用离子交换色谱法（IEX）。使用天然IEX分析临床级ErA解决了许多酶活性，酸性变体，这些变体的特征很差。ErA IEX变体被分离并使用毛细管电泳（cIEF），蛋白水解酶的LC-MS和LC-MS/MS以及结构技术（包括圆二色谱，小角X射线散射（SAXS）和离子迁移质谱（IM-MS））进行充分表征。LC-MS，MS/MS和cIEF表明，所有ErA分离株主要由缺乏一级序列修饰（如脱酰胺）的酶组成。SAXS和IM-MS都揭示了最突出的酸性IEX峰的不同构象状态。然而，SAXS数据也表明，主峰和主要的酸性变体之间的构象差异很小，基于晶体结构的比较。生物药物如ErA的IEX数据应彻底表征，因为最常见的修饰，如脱酰胺，可能不存在。
Erwinia chrysanthemi L-asparaginase (ErA) is an enzyme commonly used in the treatment regimen for Acute Lymphoblastic Leukaemia (ALL). Biopharmaceutical products such as ErA must be monitored for modifications such as deamidation, typically using ion-exchange chromatography (IEX). Analysis of clinical-grade ErA using native IEX resolves a number of enzymatically-active, acidic variants that were poorly characterised.ErA IEX variants were isolated and fully characterised using capillary electrophoresis (cIEF), LC-MS and LC-MS/MS of proteolytic digests, and structural techniques including circular dichroism, small-angle X-ray scattering (SAXS) and ion-mobility mass spectrometry (IM-MS).LC-MS, MS/MS and cIEF demonstrated that all ErA isolates consist mainly of enzyme lacking primary-sequence modifications (such as deamidation). Both SAXS and IM-MS revealed a different conformational state in the most prominent acidic IEX peak. However, SAXS data also suggested conformational differences between the main peak and major acidic variant were minor, based on comparisons with crystal structures.IEX data for biopharmaceuticals such as ErA should be thoroughly characterised, as the most common modifications, such as deamidation, may be absent.