Charge Variant Analysis of Monoclonal Antibodies Using Direct Coupled pH Gradient Cation Exchange Chromatography to High-Resolution Native Mass Spectrometry,Analytical Chemistry

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Charge Variant Analysis of Monoclonal Antibodies Using Direct Coupled pH Gradient Cation Exchange Chromatography to High-Resolution Native Mass Spectrometry
Analytical Chemistry ( IF 6.7 ) Pub Date : 2018-03-01 00:00:00 , DOI: 10.1021/acs.analchem.7b05241
Florian Füssl ₁ , Ken Cook ₂ , Kai Scheffler ₃ , Amy Farrell ₁ , Stefan Mittermayr ₁ , Jonathan Bones _{1,

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Affiliation

Charge variant analysis (CVA) of monoclonal antibodies (mAbs) using cation exchange chromatography is routinely used as a fingerprint of the distribution of posttranslational modifications present on the molecule. Traditional salt or pH based eluents are not suited for direct coupling to mass spectrometry due to nonvolatility or high ionic strength. This makes further analysis complicated when an alteration in the charge variant profile or the emergence of an additional peak is encountered. Here, the use of pH gradient elution using volatile, low ionic strength buffers is reported with direct coupling to high-resolution Orbitrap mass spectrometry. The development of a universal method based on pH elution was explored using a number of mAb drug products. Optimized methods facilitated the separation and identification of charge variants including individual glycoforms of the mAbs investigated using the same buffer system but with tailored gradient slopes. The developed method represents an exciting advance for the characterization of biopharmaceuticals as intact entities through the combination of native charge variant separations with high-resolution native mass spectrometry.

中文翻译：

使用直接耦合的pH梯度阳离子交换色谱法与高分辨率天然质谱法对单克隆抗体进行电荷变异分析

通常使用阳离子交换色谱法对单克隆抗体（mAb）进行电荷变异分析（CVA），作为分子上存在的翻译后修饰分布的指纹图谱。传统的基于盐或pH的洗脱液由于不挥发或离子强度高，不适合直接与质谱联用。当遇到电荷变化曲线的变化或出现另一个峰值时，这会使进一步的分析变得复杂。在此，据报道，使用挥发性低离子强度缓冲液的pH梯度洗脱可直接与高分辨率Orbitrap质谱联用。使用多种mAb药物探索了基于pH洗脱的通用方法的开发。优化的方法促进了电荷变体的分离和鉴定，包括使用相同的缓冲液系统但量身定制的梯度斜率对mAb的单个糖型进行了分离和鉴定。通过将天然电荷变体分离与高分辨率天然质谱相结合，所开发的方法代表了将生物药物表征为完整实体的令人振奋的进步。

更新日期：2018-03-01

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