Native Reversed-Phase Liquid Chromatography: A Technique for LCMS of Intact Antibody-Drug Conjugates.,Analytical Chemistry

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Native Reversed-Phase Liquid Chromatography: A Technique for LCMS of Intact Antibody-Drug Conjugates.
Analytical Chemistry ( IF 6.7 ) Pub Date : 2019-02-06 , DOI: 10.1021/acs.analchem.8b04699
Tse-Hong Chen ₁ , Yun Yang ₁ , Zhaorui Zhang ₂ , Cexiong Fu ₂ , Qunying Zhang ₂ , Jon D Williams ₃ , Mary J Wirth ₁

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The synthesis of antibody-drug conjugates (ADCs) using the interchain cysteines of the antibody inherently gives a mixture of proteins with varying drug-to-antibody ratio. The drug distribution profiles of ADCs are routinely characterized by hydrophobic interaction chromatography (HIC). Because HIC is not in-line compatible with mass spectrometry (MS) due to the high salt levels, it is laborious to identify the constituents of HIC peaks. An MS-compatible alternative to HIC is reported here: native reversed phase liquid chromatography (nRPLC). This novel technique employs a mobile phase 50 mM ammonium acetate for high sensitivity in MS and elution with a gradient of water/isopropanol. The key to the enhancement is a bonded phase giving weaker drug-surface interactions compared to the noncovalent interactions holding the antibody-drug conjugates together. The hydrophobicity of the bonded phase is varied, and the least hydrophobic bonded phase in the series, poly(methyl methacrylate), is found to resolve the intact constituents of a model ADC (Ab095-PZ) and a commercial ADC (brentuximab vedotin) under the MS-compatible conditions. The nRPLC-MS data show that all species, ranging from drug-to-antibody ratios of 1 to 8, remained intact in the column. Another desired advantage of the nRPLC is the ability of resolving multiple positional isomers of ADC that are not well-resolved in other chromatographic modes. This supports the premise that lower hydrophobicity of the bonded phase is the key to enabling online nRPLC-MS analysis of antibody-drug conjugates.

中文翻译：

天然反相液相色谱：完整抗体-药物缀合物的 LCMS 技术。

使用抗体的链间半胱氨酸合成抗体-药物偶联物 (ADC) 本质上会产生具有不同药物与抗体比例的蛋白质混合物。 ADC 的药物分布曲线通常通过疏水相互作用色谱 (HIC) 进行表征。由于盐含量较高，HIC 与质谱 (MS) 不兼容，因此很难识别 HIC 峰的成分。这里报道了一种与 MS 兼容的 HIC 替代方案：天然反相液相色谱 (nRPLC)。这项新技术采用流动相 50 mM 醋酸铵，以实现 MS 的高灵敏度并使用水/异丙醇梯度洗脱。增强的关键是与将抗体-药物缀合物结合在一起的非共价相互作用相比，键合相提供更弱的药物-表面相互作用。键合相的疏水性各不相同，并且发现该系列中疏水性最低的键合相聚甲基丙烯酸甲酯可以在一定条件下解析模型 ADC (Ab095-PZ) 和商用 ADC (brentuximab vedotin) 的完整成分。 MS 兼容的条件。 nRPLC-MS 数据显示，从药物与抗体比率为 1 到 8 的所有种类均在色谱柱中保持完整。 nRPLC 的另一个理想优势是能够解析 ADC 的多个位置异构体，而这些异构体在其他色谱模式中无法很好地解析。这支持了这样一个前提：键合相的较低疏水性是实现抗体-药物缀合物在线 nRPLC-MS 分析的关键。

更新日期：2019-01-21

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