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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

  • Dwight R. Stoll*
    Dwight R. Stoll
    Department of Chemistry, Gustavus Adolphus College, St. Peter, Minnesota 56082, United States
    *Mailing Address: Dwight R. Stoll, 800 West College Avenue, Saint Peter, MN 56082, United States; E-mail: [email protected]
  • David C. Harmes
    David C. Harmes
    Department of Chemistry, Gustavus Adolphus College, St. Peter, Minnesota 56082, United States
  • Gregory O. Staples
    Gregory O. Staples
    Agilent Technologies, Santa Clara, California 95051, United States
  • Oscar G. Potter
    Oscar G. Potter
    Agilent Technologies, Santa Clara, California 95051, United States
  • Carston T. Dammann
    Carston T. Dammann
    Department of Chemistry, Gustavus Adolphus College, St. Peter, Minnesota 56082, United States
  • Davy Guillarme
    Davy Guillarme
    School of Pharmaceutical Sciences, University of Geneva and University of Lausanne, Rue Michel Servet 1, 1206 Geneva 4, Switzerland
  • , and 
  • Alain Beck
    Alain Beck
    Institut de Recherche Pierre Fabre, Center of Immunology, Pierre Fabre, 5, Avenue Napoléon III, BP 60497, 74160 Saint-Julien-en-Genevois, France
    More by Alain Beck
Cite this: Anal. Chem. 2018, 90, 9, 5923–5929
Publication Date (Web):April 4, 2018
https://doi.org/10.1021/acs.analchem.8b00776
Copyright © 2018 American Chemical Society

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    Abstract

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    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.

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