Characterization of glycosylation in monoclonal antibodies and its importance in therapeutic antibody development

Abstract

Glycosylation is one of the structurally diverse and complex forms of post translational modifications observed in proteins which influence the effector functions of IgG-Fc. Although the glycosylation constitutes 2–3% of the total mass of the IgG antibody, a thorough assessment of glycoform distribution present on the antibody is a critical quality attribute (cQA) for the majority of novel and biosimilar monoclonal antibody (mAb) development. This review paper will highlight the impact of different glycoforms such as galactose, fucose, high mannose, NANA (N-acetylneuraminic acid), and NGNA (N-glycoylneuraminic acid) on the safety/immunogeneicity, efficacy/biological activity and clearance (pharmacodynamics/pharmacokinetic property (PD/PK)) of biological molecules. In addition, this paper will summarize routinely employed reliable analytical techniques such as hydrophilic interaction chromatography (HILIC), high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and mass spectrometry (MS) for characterizing and monitoring glycosylation in monoclonal antibodies (mAbs). The advantages and disadvantages of each of the methods are addressed. The scope of this review paper is limited to only N-linked and O-linked glycosylation.

Keywords:

• Glycosylation
• monoclonal antibodies
• ADCC
• CDC
• therapeutic
• N-linked glycans
• O-linked glycans
• HILIC
• HPAEC
• mass spectrometry

Disclosure statement

The author is employed by Aurobindo Biologics. There is no conflict of interest.

Glossary

1. Peeling: Phenomenon of degradation of terminal carbohydrates particularly N-acteylglucosamine.

2. Hemiacetal or hemiketal: Hemiacetal or a hemiketal is a compound that results from the addition of an alcohol to an aldehyde or a ketone.

3. Anomers: Anomers are Anomers are stereoisomers of cyclic sugars that differ in configuration only at the hemiacetal or hemiketal carbon.

4. Antibody Dependent Cell Mediated Cytotoxicity (ADCC): ADCC is an immune mechanism where the Fc receptor bearing effector cells kill the target cells that have antigen complexed with the antigen.

5. Complement Dependent Cytotoxcity (CDC): CDC is an immune mechanism where the binding of the antibody to the antigen present on the target cells triggers the complement pathway resulting in the formation of the major histocompatibility complex (MHC) and target cell lysis.

6. Reference Standard (RS): A reference standard is defined as a substance that is prepared from lot(s) representative of the production and clinical materials and which has been well characterized using an extensive set of analytical tests [93].