Therapeutic glycoproteins: sample preparation for N-glycan profiling of monoclonal antibodies

INTRODUCTION. Glycan profile is a critical quality parameter for therapeutic monoclonal antibodies (mAbs) that is consistently estimated during development and release of each drug batch. The need to develop a reproducible sample preparation protocol for glycan profiling brings about the relevance of the study. The profile should be adapted to the conditions of a standard physicochemical laboratory, so that it avoids using commercial preparation kits that are currently in short supply.

AIM. This study aimed to develop an alternative sample preparation procedure for quantitation of glycans in therapeutic monoclonal antibodies without using commercial kits.

MATERIALS AND METHODS. Omalizumab, ustekinumab, canakinumab, tocilizumab, natalizumab, and human anti-PD IgG2 were used as the study objects. N-glycans were released from mAbs by peptide-N-glycosidase (PNGase F) and labeled with fluorescent tags 2-aminobenzamide (2-АВ) or 2-aminobenzoic acid (2-АА) or InstantAB. Subsequently, glycan samples were cleaned from impurities. Glycan compounds were analyzed using hydrophilic interaction liquid chromatography (HILIC-FLD) on Alliance e2695 and Acquity Arc Bio chromatographic systems equipped with FLR 2475 fluorescence detector. The glycan profile was assessed by the content of functional glycan groups.

RESULTS. Conditions for preparing therapeutic mAbs N-glycans were chosen as follows: incubation with 2 mEU PNGase F per 100 µg protein in 10 mM Tris-HCl (pH 8.0) at 37 °C for 3 h without protein denaturation; derivatization of glycans with 2-AA at 65 °C for 1.5 h; extraction of 2-AA excess with acetonitrile. Centrifugation of labeled glycans with acetonitrile is suitable for purification and concentration of samples. Minor glycans with a content not more than 0.1–0.2% were determined using HILIC-FLD. Comparison with data obtained using a commercial kit for sample preparation indicated acceptable comparability of the results. When preparing the samples, we took into account structural features of specific mAbs, for instance, desialylation control during the staining was found necessary for high-sialylated mAbs.

CONCLUSIONS. The developed sample preparation procedure is suitable for the analysis of N-glycans of omalizumab, canakinumab and natalizumab and may be used to develop the analysis methods of the glycan profile of other glycoproteins using HILIC-FLD mode.

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