Development of Comprehensive Online Two-Dimensional Liquid Chromatography/Mass Spectrometry Using Hydrophilic Interaction and Reversed-Phase Separations for Rapid and Deep Profiling of Therapeutic Antibodies

Abstract

Monoclonal antibodies (mAb) and related molecules are being developed at a remarkable pace as new therapeutics for the treatment of diseases ranging from cancer to inflammatory disorders. However, characterization of these molecules at all stages of development and manufacturing presents tremendous challenges to existing analytical technologies because of their large size (ca. 150 kDa) and inherent heterogeneity resulting from complex glycosylation patterns and other post-translational modifications. Multidimensional liquid chromatography is emerging as a powerful platform technology that can be used to both improve analysis speed for these molecules by combining existing one-dimensional separations into a single method (e.g., Protein A affinity separation and size-exclusion chromatography) and increasing the resolving power of separations by moving from one dimension of separation to two. In the current study, we have demonstrated the ability to combine hydrophilic interaction (HILIC) and RP separations in an online comprehensive 2D separation coupled with high resolution MS detection (HILIC × RP-HRMS). We find that active solvent modulation (ASM) is critical for coupling these two separation modes, because it mitigates the otherwise serious negative impact of the acetonitrile-rich HILIC mobile phase on the second dimension RP separation. The chromatograms obtained from these HILIC × RP-HRMS separations of mAbs at the subunit level reveal the extent of glycosylation on the Fc/2 and Fd subunits in analysis times on the order of 2 h. In comparison to previous CEX × RP separations of the same molecules, we find that chromatograms from the HILIC × RP separations are richer and reveal separation of some glycoforms that coelute in the CEX × RP separations.

Evaluation of LC-MS and LC×LC-MS in analysis of zebrafish embryo samples for comprehensive lipid profiling

“…3 The surface coverage values of the C18×HILIC (a), the HILIC×C18 (b), and the HILIC×PFP (c) separations, which were estimated using all annotated lipid species from the zebrafish sample. Several vectors were introduced to simply the calculation process which have a broad range of polarities but a short span of hydrophobic properties, such as metabolites [32], polymers [21], and therapeutic antibodies [49].…”

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

“…More recently, Stoll et al developed a new comprehensive HILIC Â RPLC approach for rapid and deep characterization of mAbs [120]. By introducing active solvent modulation (ASM), the peak distortion effect caused by the large acetonitrile proportion during the transfer of the HILIC to RPLC separation could be reduced.…”

Hydrophilic interaction chromatography for the analysis of biopharmaceutical drugs and therapeutic peptides: A review based on the separation characteristics of the hydrophilic interaction chromatography phases

“…Analysis of labeled N ‐glycans from mAbs, (Top) Atezolizumab, (Middle) Obinutuzumab, and Cetuximab (Bottom) in HILIC‐FL . Copyright 2018 with permission of American Chemical Society.…”