FabRICATOR® in service for in-depth 2D-LC MS profiling of therapeutic mAbs

 

In an article by Stroll et al. (2018), the authors demonstrate a striking in-depth characterization of three therapeutic mAbs, achieved by combining FabRICATOR® (IdeS) digestion with an online two-dimensional LC-MS approach. The authors generate a highly resolved separation and detection of FabRICATOR-digested N-glycosylated mAb subunits by implementing Active Solvent Modulation (ASM), a method for valve-based effluent dilution between the first and second dimension separations.

Multidimensional Liquid Chromatography constitutes a powerful technology for in-depth profiling of therapeutic proteins, capable of generating rapid and highly resolved separations. The authors demonstrate the advantages of implementing ASM in an online 2D-LC system for deep profiling of antibody glycosylations, subjecting mAbs to FabRICATOR digestion followed by HILIC x RP separation and ESI Mass Spectrometry (ESI-MS) detection.

Three therapeutic antibodies displaying diverse N-glycosylation patterns were submitted to digestion using FabRICATOR for a single site-specific proteolytic cleavage below the hinge, generating Fc/2 and F(ab’)2 fragments. Further reduction of the interchain disulphide bonds of the F(ab’)2 subunit was carried out on the FabRICATOR-digested samples for the additional generation of LC and Fd fragments.

Implementing the ASM method on antibody subunits, the authors achieved a significant increase in detection sensitivity for Fc/2 and Fd fragments, without detectable breakthrough, otherwise associated with larger loading volumes in the second-dimension separation. Furthermore, the authors demonstrated the resolving power of HILIC x RP for analyzing the extent of glycosylations present in heavily glycosylated mAbs, the method showing increased separation and detection for both high and low abundant glycan species, compared to 2D-LC combining CEX and RP separations.

FabRICATOR is a protease with a single digestion site below the hinge of IgG. The enzyme is widely used in middle-level analytical workflows for characterization of antibody based biopharmaceuticals.

 
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Stoll, D.R. et al., 2018. 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. Analytical Chemistry, pp.acs.analchem.8b00776–9.