Characterization of a therapeutic monoclonal antibody by orthogonal chromatographic techniques

Monoclonal antibodies are the largest class of biotechnological drugs. Therapeutic monoclonal antibodies have been used since 1986. FDA has approved more than fourty therapeuetic monoclonal antibodies in last five years. Market share of biopharmaceuticals has been increasing in pharma industry for ten years. Expiration of the original patents of biotechnological drugs has resulted in the emergence of biosimilars. Biosimilar candidate monoclonal antibodies should be characterized according to regulatory compliance. The more physicochemically biosimilar a candidate biosimilar is to the original molecule, the higher are the clinical safety, efficacy and success. Chromatographic techniques have widely been used for assessment of physicochemical properties and characterization of monoclonal antibodies. Belimumab, which is a therapeutic monoclonal antibody, was characterized with orthogonal methods by using protein A, reverse phase, hydrophobic interaction, hydrophilic interaction, size exclusion and cation exchange chromatography techniques. It was found that constant region in heavy chain (Hc) domain of monoclonal antibody Belimumab has the peptide sequence Gln-Asp-Glu-Leu-Thr-Gln-Asp-Pro-Ala-Val. Trp and Cys could not be obtained by amino acid composition analysis. The most abundant amino acids in Belimumab are methionine and cysteine is the least abundant ones. Antibody titer determination method was developed by Protein A chromatography and R2 was found 0.9994. Protein titer quantification and impurity testing methods were developed by reverse phase chromatography. According to impurity assay, percentage impurity value was found 8%. Oxidized groups were separated by hydrophobic interaction chromatography. Glycan profile was determined by hydrophilic interaction chromatography and it was found that amount of G1Fa 35%, which is the most abundant. According to the results of cation-exchange chromatography analysis, charge variants are within the range of 8-12% and there are more acidic variants than basic variants. Molecular weight was measured by SEC-MALS and mass spectrometry. Molecular weight was found between 142 kDa and 145 kDa by SEC-MALS and 147.016 kDa by mass spectrometry. In this thesis, physicochemical features and characterization profile of Belimumab monoclonal antibody were determined.