Physicochemical and Functional Comparison Between the Cetuximab Biosimilars Cetuxa® and Lupitux® and Cetuximab Originator Erbitux®

ENZ-124, the first approved cetuximab biosimilar by Indian health authorities, was developed using the Chinese hamster ovary cell line, whereas the SP2/0 cell line was used for developing cetuximab originator (Erbitux®). Hence, this assessment aimed to evaluate the physiochemical, structural, and biological characterization of four batches each of Cetuxa®, Lupitux® and cetuximab originator. Analytical methods like peptide mapping (liquid chromatography–tandem mass spectrometry [LC-MS/MS]), glycan mapping (liquid chromatography–fluorescence detection), isoform analysis (hydrophobic interaction chromatography [HIC]-high-performance liquid chromatography), charge variant analysis (capillary electrophoresis coupled with mass spectrometry), FcγRIIIa binding assays, cell-based antibody-dependent cell-mediated cytotoxicity (ADCC) measurement, and host cell protein analysis (LC-MS/MS) were utilized for analysis. Variations in physicochemical and biological attributes were observed between cetuximab originator and its biosimilars. HIC analysis revealed C-terminal heterogeneity and increased hydrophobicity in biosimilars, that with the different N-glycan profile affected charge distribution. Charge variant analysis indicated a more acidic profile for the biosimilars. The biosimilars showed higher oxidation levels and misincorporation. The biosimilars also showed an enhanced FcγRIIIa binding affinity and in vitro ADCC activation compared with cetuximab originator. The study outcomes highlight the ability to differentiate cetuximab originator from its biosimilars Cetuxa® and Lupitux® based on the observed physicochemical and biological differences.