Abstract
The N-linked glycosylation in recombinant monoclonal antibodies (mAb) occurs at Asn297 on the Fc region in the CH2 domain. Glycosylation heterogeneities have been well documented to affect biological activities such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) through their interaction with Fc-receptors. Hence, it is critical to monitor and characterize the N-linked glycosylation profile in a therapeutic protein such as a mAb for product consistency. In one approach, the glycans are first released from the mAb using an enzyme specific digestion, such as Protein N-Glycosidase F (PNGase) and subsequently they are labeled using a fluorophore, for example, 8-aminopyrene-1,3,6-trisulfonic acid (APTS) . Here we have applied this approach and used Capillary Electrophoresis with Laser-Induced Fluorescence detection (CE-LIF) to analyze a recombinant mAb produced in murine myeloma (NS0) cells. The technique provides short analysis times, efficient separations, and high sensitivity. CE-LIF peak identification was done by a combination of glycan standards and treatment with various exoglycosidases. Furthermore, the APTS-labeled glycans were also analyzed using hydrophilic interaction chromatography (HILIC) high performance liquid chromatography (HPLC) to aid identification of minor peaks by sample collection and off-line mass spectrometry (MS) analysis.
Highlights
Development of monoclonal antibodies as therapeutics in the biopharmaceutical industry, including biosimilar or biobetter versions of marketed mAbs has increased tremendously over the last ten years [1,2,3]
The N-linked glycans from a mAb produced in NS0 were first released from the protein using a specific enzyme, Protein N-Glycosidase F (PNGase), which removed the glycans from the Asn297 position in the Fc region of the mAb
There are four major peaks observed in the electropherogram, G0F, G1F(1,6), G1F(1,3), and G2F which are typical for a mAb produced in either Chinese Hamster Ovary (CHO) or NS0 cell lines
Summary
Development of monoclonal antibodies (mAb) as therapeutics in the biopharmaceutical industry, including biosimilar or biobetter versions of marketed mAbs has increased tremendously over the last ten years [1,2,3]. There are known biological functions of N-linked glycosylation in a mAb which are related to the micro-heterogeneities of glycan structures. The most commonly used quantitative tools to analyze glycosylation are HPLC either with pulsed amperometric detection (PAD) [16,17] or with fluorescence detection employing fluorescently-labeled glycans [18,19,20] and CE with a LIF detector for various fluorescently-labeled glycans [21,22,23]. A peak characterization strategy for APTS labeled glycans most commonly uses a combination of glycan standards and exoglycosidase-treatments. We report characterization of N-linked glycosylation in a mAb produced in NS0 cells using a combination of CE-LIF and HILIC HPLC of APTS-labeled glycans including off-line MS analysis for confirmation
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