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A Novel Online Four-Dimensional SEC×SEC-IM×MS Methodology for Characterization of Monoclonal Antibody Size Variants.
- Source :
-
Analytical chemistry [Anal Chem] 2018 Dec 04; Vol. 90 (23), pp. 13929-13937. Date of Electronic Publication: 2018 Nov 12. - Publication Year :
- 2018
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Abstract
- The determination of size variants is a major critical quality attribute of a therapeutic monoclonal antibody (mAb that may affect the drug product safety, potency, and efficacy. Size variant characterization often relies on size-exclusion chromatography (SEC), which could be hampered by difficult identification of peaks. On the other hand, mass spectrometry (MS)-based techniques performed in nondenaturing conditions have proven to be valuable for mAb-related compound characterization. On the basis of the observation that limited SEC performance was observed in nondenaturing MS compatible ammonium acetate buffer compared with classical phosphate salts, a multidimensional analytical approach was proposed. It combines comprehensive online two-dimensional chromatography (SEC×SEC), with ion mobility and mass spectrometry (IM-MS) in nondenaturing conditions for the characterization of a variety of mAbs. We first exemplify the versatility of our approach for simultaneous detection, identification, and quantitation of adalimumab size variants. Benefits of the SEC×SEC-native IM×MS were further highlighted on forced degraded pembrolizumab and bevacizumab samples, for which the 4D setup was mandatory to obtain an extensive and unambiguous identification, and accurate quantitation of unexpected high/low molecular weight species (HMWS and LMWS). In this specific context, monomeric conformers were detected by IM-MS as HMWS or LMWS. Altogether, our results emphasize the power of comprehensive 2D LC×LC setups hyphenated to IM×MS in nondenaturing conditions with unprecedented performance including: (i) maintaining optimal SEC performance (under classical nonvolatile salt conditions), (ii) performing online native MS identification, and (iii) providing IM-MS conformational characterization of all separated size variants.
Details
- Language :
- English
- ISSN :
- 1520-6882
- Volume :
- 90
- Issue :
- 23
- Database :
- MEDLINE
- Journal :
- Analytical chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 30371058
- Full Text :
- https://doi.org/10.1021/acs.analchem.8b03333