Charge Variant Analysis of Monoclonal Antibodies Using Direct Coupled pH Gradient Cation Exchange Chromatography to High-Resolution Native Mass Spectrometry
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- 1 March 2018
- journal article
- research article
- Published by American Chemical Society (ACS) in Analytical Chemistry
- Vol. 90 (7), 4669-4676
- https://doi.org/10.1021/acs.analchem.7b05241
Abstract
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 non-volatility 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.Funding Information
- Science Foundation Ireland (13/CDA/2196)
This publication has 39 references indexed in Scilit:
- Microheterogeneity of therapeutic monoclonal antibodies is governed by changes in the surface charge of the proteinBiotechnology Journal, 2016
- Charge heterogeneity: Basic antibody charge variants with increased binding to Fc receptorsmAbs, 2016
- Ion-exchange chromatography for the characterization of biopharmaceuticalsJournal of Pharmaceutical and Biomedical Analysis, 2015
- Characterization of cation exchanger stationary phases applied for the separations of therapeutic monoclonal antibodiesJournal of Pharmaceutical and Biomedical Analysis, 2015
- Method development for the separation of monoclonal antibody charge variants in cation exchange chromatography, Part II: pH gradient approachJournal of Pharmaceutical and Biomedical Analysis, 2015
- Method development for the separation of monoclonal antibody charge variants in cation exchange chromatography, Part I: Salt gradient approachJournal of Pharmaceutical and Biomedical Analysis, 2015
- Chromatographic analysis of the acidic and basic species of recombinant monoclonal antibodiesmAbs, 2012
- Analytical tools for characterizing biopharmaceuticals and the implications for biosimilarsNature Reviews Drug Discovery, 2012
- Charge variants in IgG1mAbs, 2010
- Identification of multiple sources of charge heterogeneity in a recombinant antibodyJournal of Chromatography B: Biomedical Sciences and Applications, 2001