Characterization of Nonenzymatic Glycation on a Monoclonal Antibody

Abstract

We present here an improved analytical method for the analysis of glycation events in proteins. Nonenzymatic glycation of an IgG2 monoclonal antibody was studied using affinity chromatography, mass spectrometry, and chemical derivatization. Analysis of both forced-degraded and bulk-drug substance (BDS) samples showed the presence of glycated protein. A new peptide mapping procedure, incorporating derivatization using sodium borohydride, allowed the development of a sensitive method for detecting and identifying the sites of modification. When combined with tandem mass spectrometry, peptides glycated by glucose showed dramatically improved MS/MS spectra as compared to underivatized controls. Using these methods we were able to map a number of glycation sites in both forced-degraded and BDS samples that were distributed across both light and heavy chain subdomains. The combination of affinity chromatography, high-resolution mass spectrometry, and a simple derivatization procedure should allow the facile analysis of glycation for other antibody and protein samples.

Glycation of antibodies: Modification, methods and potential effects on biological functions

“…Top-down mass spectrometry of the intact antibody or enzyme-cleaved mAb fragments can also be used to determine glycation level, 31 either by matrix-assisted laser desorption/ionization (MALDI) 48 or electrospray ionization (ESI). 8,15,49 As each glycation site shows a C162 Da mass shift, the top-down approach can be used as a quick estimation of glycation level in the antibody.…”

“…53,54,[56][57][58] These issues often require extra experiments to confirm or detect glycated peptides. 31 Another way of improving the sensitivity and reducing the neutral loss of the glycated peptide is by using sodium borohydride or sodium cyanoborohydride reduction followed by trypsin cleavage and peptide map analysis with MS/MS detection. 31,37,59 In this approach, the bond between the carbohydrate and peptide is stabilized due to the reduced glycated sugar moiety, which results in higher quality MS/MS spectra.…”

“…30 The types of sugars present, such as hexose sugars, for example, and the specific reactivity of the accessible amino groups affect the protein glycation, and therefore increase the heterogeneity of therapeutic proteins. 31 During storage of therapeutic mAbs, glycation can be introduced by reducing sugars in the formulation. The presence of reducing sugars in the formulation has produced protein glycation even in the solid state after lyophilization.…”

“…44 For antibody glycation analysis, BAC is a common method of identification, quantitation and isolation of glycated antibodies because it requires minimum sample preparation and uses native running conditions. 5,15,31 As an example, a normal boronate affinity chromatogram of the therapeutic antibody is shown in Fig. 2.…”

“…5,45 Careful optimization of the concentration of shielding agent, pH and buffer salt composition allows the quantitation of the glycation level of the bulk-drug substance. 31 BAC could also be coupled with other methods by offline fraction collection for further characterization of the glycated species. Although BAC has been widely used for enrichment of glycated species, it has some limitations.…”