A Simple Strategy to Eliminate Hexosylation Bias in the Relative Quantification of N‐Glycosylation in Biopharmaceuticals

Abstract

N‐glycosylation may affect the safety and efficacy of biopharmaceuticals and is thus monitored during manufacturing. Mass spectrometry of the intact protein is increasingly used to reveal co‐existing glycosylation variants. However, quantification of N‐glycoforms via this approach may be biased by single hexose residues as introduced by glycation or O‐glycosylation. Herein, we describe a simple strategy to reveal actual N‐glycoform abundances of therapeutic antibodies, involving experimental determination of glycation levels followed by computational elimination of the “hexosylation bias”. We show that actual N‐glycoform abundances may significantly deviate from initially determined values. Indeed, glycation may even obscure considerable differences in N‐glycosylation patterns of drug product batches. Our observations may thus have implications for biopharmaceutical quality control. Moreover, we solve an instance of the problem of isobaricity, which is fundamental to mass spectrometry.

Unambiguous Identification of Glucose-Induced Glycation in mAbs and other Proteins by NMR Spectroscopy

“…Denosumab was earlier reported to be glycated [38], but in our case the spectra lacked any signs of glycation, suggesting a glycation level below our NMR detection limit, or a modification with another reducing sugar other than glucose. However, it is also possible, that the glycation was lost during the sample preparation due to its reversibility [28].…”

“…Despite the high complexity of the 1 H- 13 C HSQC spectrum and many correlations of the containing glycans, the typical regions for C5-H5 (71.8/4.01 ppm), C6-H6 (66.7 /4.01 ppm) and C6-H6′ (66.7 /3.76 ppm) were empty. Denosumab was described to be glycated [38], but from our NMR data (see Fig. 5C), we concluded that either the glycation level was below the detection limit, or another form of modification was present, different from glycation with glucose.…”

“…None of the therapeutic mAbs showed any signs of the Amadori product (Fig. 5C - E ), even for a mAb that was reported to be glycated [ 38 ]. Despite the high complexity of the 1 H- 13 C HSQC spectrum and many correlations of the containing glycans, the typical regions for C5-H5 (71.8/4.01 ppm), C6-H6 (66.7 /4.01 ppm) and C6-H6′ (66.7 /3.76 ppm) were empty.…”

“…Because glycation is considered a critical quality attribute (CQA) in therapeutic mAbs, especially in the antigen-binding region of the Fab part [ 36 , 37 ], it is important to monitor if this modification is present and if it stays within a certain tolerance range. The mass difference for glycation is isobaric to an additional hexose attached to a glycan [ 38 ]. For very complicated glycosylation patterns, it is especially challenging to distinguish between glycation and an additional hexose attached to a glycan.…”

High-Resolution Native Mass Spectrometry

“…Non-enzymatic attachment of a hexose to a backbone residue results in a mass shift identical to that for a mannose or antennal galactose extension of an N -glycan, making it indiscernible on the intact-protein level. Recently, Esser-Skala et al , presented a method to assign possible PTM compositions and to eliminate hexosylation bias in the analysis of N-glycosylation patterns by native MS. When measuring the antibody in its N -deglycosylated form, satellite peaks at intervals of 162 Da indicated the presence of nonspecifically glycated isoforms.…”