Multi-Detection Size Exclusion Chromatography as an Advanced Tool for Monitoring Enzyme–Antibody Conjugation Reaction and Quality Control of a Final Product

Abstract

The multi-detection size exclusion chromatography (SEC) has been recognized as an advanced analytical technique for the characterization of macromolecules and process control, as well as the manufacturing and formulation of biotechnology products. It reveals reproducible molecular characterization data, such as molecular weight and its distribution, and the size, shape, and composition of the sample peaks. The aim of this work was to investigate the potential and suitability of the multi-detection SEC as a tool for surveillance over the molecular processes during the conjugation reaction between the antibody (IgG) and horseradish peroxidase (HRP), and demonstrate the plausibility of its application in the quality control of the final product, the IgG-HRP conjugate. Guinea pig anti-Vero IgG-HRP conjugate was prepared using a modified periodate oxidation method, based on periodate oxidation of the carbohydrate side chains of HRP, followed by the formation of Schiff bases between the activated HRP and amino groups of the IgG. The quantitative molecular characterization data of the starting samples, intermediates, and final product were obtained by multi-detection SEC. Titration of the prepared conjugate was performed by the ELISA and its optimal working dilution was determined. This methodology proved to be a promising and powerful technology for the IgG-HRP conjugate process control and development, as well as for the quality control of the final product, as verified by the analysis of several commercially available reagents.