2. Academic Validation
  3. Middle-up analysis of monoclonal antibodies after combined IgdE and IdeS hinge proteolysis: Investigation of free sulfhydryls

Middle-up analysis of monoclonal antibodies after combined IgdE and IdeS hinge proteolysis: Investigation of free sulfhydryls

  • J Pharm Biomed Anal. 2018 Feb 5:149:541-546. doi: 10.1016/j.jpba.2017.11.046.
Valegh Faid 1,  Yann Leblanc 1,  Nicolas Bihoreau 1,  Guillaume Chevreux 2
Affiliations

Affiliations

  • 1 Analytical Department, LFB Biotechnologies, 3 avenue des Tropiques, 91958 Courtaboeuf, Les Ulis, France.
  • 2 Analytical Department, LFB Biotechnologies, 3 avenue des Tropiques, 91958 Courtaboeuf, Les Ulis, France. Electronic address: [email protected].
PMID: 29179100 DOI: 10.1016/j.jpba.2017.11.046
Abstract

Despite significant analytical improvements during this last decade, characterizing the whole integrity of monoclonal Antibodies during their bioproduction remains a challenge. In this study, we report a new analytical approach to evaluate the overall heterogeneity/integrity of mAbs by LC-MS after combined proteolysis at their lower- and upper-hinge sites using the immunoglobulin-degrading enzymes IdeS and IgdE respectively. The whole sample preparation did not use any harsh conditions such as low pH, high temperature or reductive conditions and enables the splitting of mAbs structure into three fragments, namely the hinge dimer, Fab and Fc/2. Using the NIST mAb reference material, this method was demonstrated to be particularly suited for the analysis of mAbs disulfide bridges. The three fragments as well as their corresponding free sulfhydryl forms were well separated by chromatography and identified online by mass spectrometry. The method was then successfully applied to several mAbs of variable hydrophobicities.

Keywords

Disulfide bonds; Free sulfhydryls; IdeS; IgdE; Mass spectrometry; Monoclonal antibody.

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