Using Hydrophilic Interaction Chromatography for Heightened Product Characterization to Overcome Challenges with Hydrophobic Monoclonal Antibodies and Antibody Drug Conjugates

■ ■ Reduced temperature dependence for decreasd on-column degradation IN T RO DU C T IO N While hydrophilic interaction chromatography (HILIC) has been widely used for separating small polar compounds, its application to large biomolecules, other than released glycans, has been surprisingly limited. In large part, this can be attributed to a paucity of suitable HILIC column technology and robust separation methods. This is despite the potential for HILIC to be highly valuable for the characterization of whole proteins with and without glycosylation. IgG based monoclonal antibodies (mAbs) have two identical heavy chains and two identical light chains that are held together by both covalent disulfide bonds and non-covalent interactions. More importantly, mAbs exhibit two functionally significant subunits: two equivalent antigen binding fragments (Fab domains), and one crystallizable fragment (Fc domain), which is glycosylated with N-linked complex-type biantennary structures. Structural characterization of these subunits typically involves proteolysis by the Immunoglobulin Degrading Enzyme of S. pyogenes (IdeS) (Figure 1). IdeS has high specificity for a conserved Gly-Gly sequence motif in the lower hinge region of mAbs, and when used with disulfide bond reduction, IdeS produces three different 25 kDa mAb fragments: light chain (LC), along with two heavy chain Fd' and single chain Fc (scFc) domains. Normally, characterization of these three antibody constituents is achieved by reversed-phase liquid chromatography (RPLC) coupled to ultrahigh-resolution mass spectrometry (MS). Many times, it can be difficult to achieve complete chromatographic recovery of all three subunits/domains due to their hydrophobic nature. And while ultra-high temperatures can improve recoveries, such RPLC method conditions can become problematic because of analyte degradation and the potential to introduce method artifacts.