Two-dimensional liquid chromatographic methods to examine phenylboronate interactions with recombinant antibodies.

A two-dimensional liquid chromatography (2D-LC) system incorporating Agilent 1100 components was set up and 2D-LC methods were developed to determine how recombinant antibodies (rAbs) interact with a commercial boronate column. Analyses of ten rAbs demonstrated that a simple boronate affinity chromatography method cannot be generalized to separate or detect the glycation level for all of rAbs as the aggregates were also retained on the boronate column. A quantitative boronate affinity chromatography method was developed to examine the extent of glycation of a recombinant humanized monoclonal antibody (rhuMAb). With the method, only the glycated components of intact or papain-treated rhuMAb were bound to the boronate column. When denatured, rhuMAb heavy chain was also bound. However, enzymatic removal of the N-oligosaccharides did not delete the binding, indicating that the interaction between the heavy chain and the phenylboronate functionality is not mediated through the N-oligosaccharides. Two-stage 2D-LC analysis with boronate affinity chromatography in the first dimension and reversed-phase chromatography in the second demonstrated that the interaction between the antibody heavy chain and boronate column is not specific, as urea is able to selectively elute retained heavy chain without disrupting the retention of glycated light chain. Quantum mechanic calculations show that the vicinal cis-diols in beta-pyranose conformations in N-glycosylated antibodies need to overcome at least 7.172kcal/mol to become co-planar (zero-dihedral angle). However, the cis-diols on beta-furanose rings of glycated species only have to overcome a 0.885kcal/mol energy barrier to achieve the co-planar configuration that facilitates its complex formation with the phenylboronate groups. This minimal energy barrier, coupled with the stabilizing effect from the electrostatic interaction between the oppositely charged phenylboronate group on the column and the fructosamine group of glycated species, explains why the boronate column exhibits high binding selectivity towards the glycated proteins.