The Open Pharmacology Journal

2012, 6 : 27-33
Published online 2012 May 9. DOI: 10.2174/1874143601206010027
Publisher ID: TOPHARMJ-6-27

Functional Role of Glycosylation in a Human IgG4 Antibody Assessed by Surface Plasmon Resonance Technology

Rosie B. Wong , Ming Zeng , An-Horng Lee , T. Shantha Raju and Kuang-Chuan Cheng
the Bristol-Myers Squibb, Analytical Development and Testing, 311 Pennington-Rocky Hill Road, Pennington, NJ 08534, USA;

ABSTRACT

Fc glycosylation of immunoglobulins is necessary for antibody effector functions. These glycans of immunoglobulins are often referred as glycoforms and they can be heterogeneous due to the variations in glycosylation machinery present in the endoplastic reticulum (ER) and in the Golgi. In the absence of a generic culturing protocol that can render a consistent glycosylation pattern, monitoring glycoforms of monoclonal antibodies from cultured cells is becoming essential. Accordingly, quantification of glycosylated and deglycosylated heavy chains of an IgG4 monoclonal antibody was accomplished using an Agilent Bioanalyzer Lab-On-the-Chip electrophoresis system. In addition to the native antibody, completely deglycosylated antibody prepared by treating with PNGase F and a F(ab’)2 fraction were evaluated for their antigen binding kinetics using Biacore surface Plasmon resonance (SPR). The equilibrium binding constants KD are found to be comparable at 1.81E-09M, 1.96E-09M, for the native and deglycosylated antibody, respectively, and 5.79E-10M for the F(ab’)2. An in vitro biological activity employing a competition binding assay was also developed to demonstrate the role of the Fc glycan. The results confirm that for a neutralizing antibody therapeutic the biological activity of the native MAb-1 and deglycosylated antibody are comparable, thus indicating that the Fc glycan does not contribute to the antigen binding or the biological function. The kinetics and competitive assays performed on an SPR instrument are quick and reliable. Combined with the on-chip electrophoresis method they can be used as monitoring methods for process development and quality control.

Keywords:

Surface Plasmon resonance (SPR), on-chip electrophoresis, bioanalyzer, non-glycosylated heavy chain (NGHC), equilibrium dissociation constant (KD), competitive binding assay..