A Novel Online Four-Dimensional SEC×SEC-IM×MS Methodology for Characterization of Monoclonal Antibody Size Variants

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.

Multi-dimensional LC-MS: the next generation characterization of antibody-based therapeutics by unified online bottom-up, middle-up and intact approaches

“…phosphate for SEC) used with these LC separations. Numerous 2D-LC applications for biopharmaceutical proteins have been described, where RPLC was used for desalting of samples coming from the first dimension separations that involve the use of non-volatile salts or high volatile salt concentrations (59)(60)(61)(62). In particular, SEC-RPLC and IEX-RPLC methods have been published for the characterization of size and charge variants of antibody at intact level using denaturing conditions in the second dimension (63,64).…”

Recent progress in the molecular imaging of therapeutic monoclonal antibodies

“…The molecular mass of mAb can be indicative of its primary structure, and mass spectrometry (MS) alone [ 8 , 9 ] or coupled with efficient separation methods like liquid chromatography (LC; including reverse-phase [ 10 , 11 ], size-exclusion [ 12 , 13 ], and ion-exchange chromatography [ 14 ]) and capillary electrophoresis (CE) [ 15 ] can therefore be used to identify the primary structure of mAbs. To further identify the peptide sequence, the amide bond between a specific amino acid sequence is cleaved by chemical or enzymatic methods, followed by analysis with MS [ 16 ].…”

Glycosylation of biosimilars: Recent advances in analytical characterization and clinical implications

“…More interestingly is the coupling of IM-MS to 2D-LC techniques previously described, to further improve the separation of glycoforms and PTMs at subunit level [119,137,138]. A comparable approach was recently demonstrated by Ehkirch et al that coupled SECxSEC-native IM-MS for the comprehensive analysis of mAb size variants [139]. The online 2D-LC setup consisting of SEC in both dimensions allowed the use nonvolatile salts for optimal separation performance prior to native IM-MS [140].…”