Abstract

N-LINKED GLYCOSYLATION AND CHARGE VARIANT ANALYSIS OF BIOSIMILAR ANTI HER2 ANTIBODY IN TREATMENT OF BREAST CANCER

Pratik R. Gajjar*, Dasharath M. Patel, Amar K. Shrivastava, Umesh S. Shaligram, Noelle Sunstrom

Abstract

Glycosylation moiety and charge variants both are playing important role in biological activity of Anti HER2 antibody. The binding affinity of the mAb molecule always depends upon the glycosylation pattern. However the variation in glycan profile may affect the biological activity which further promotes the subsequent mechanism in ADCC as well as CDC activities, the terminal galactosylation and afucosylation of glycan residues effect on CDC and ADCC activity. Therefore, the evaluation of glycan profile was carried out by Rapid PNGaseF digestion followed by UHPLC analysis. Mostly G0, G0F, Man5, G1Fa, G1Fb, G2 and G2F residue were found in the Anti-HER2 molecule. Further, each glycan was identified based on its retention time of standard glycans library and a comparison was made with respect to originator HERCLON®. Based on the results the molecule containing predominant residues was identified and also demonstrated their role in vivo biological activity. The charge variant analysis was equally important to demonstrate the quality of the Anti-HER2 antibody. Acidic variant, main (HC-K0) and basic variants (K1, K2) were determined by cation exchange chromatography (CEX) and percentage of all variants were evaluated based on separation on Protein Pak SP column in UHPLC system. The comparisons was carried out based on response of reference peaks under identical condition and the percentage of acidic, main and basic variants was determined. Our results showed that the high degree of similarity in glycan profiling and charge variants of biosimilar Anti-HER2 antibody with respect to the innovator product may ensure in comparable biological activity.

Keywords: Biosimilar, N-glycosylation, Charge variants, Anti-HER2 antibody.