Analytical Characterization of Monoclonal Antibody IgG2

Haiying Chen, Sepax Technologies, Inc., USA
James Xu, Sepax Technologies, Inc., USA
Ke Yang, Sepax Technologies, Inc., USA
Yair Shachar, Yair Technologies, Israel

Monoclonal antibodies have been the fastest growing protein therapeutics. Due to the molecular complexity of monoclonal antibodies, the characterization remains a challenge and required step throughout the development and manufacturing process. In order to determine the efficacy of the molecules, aggregation, heterogeneity such as charge variants, C-terminal lysine processing, deamidation, glycosylation must be screened for their structural and biological changes. Analytical techniques are employed for these product and process characterizations at different stages of product cycles such as in process monitoring, lot release and product stability studies.

In this poster, we would like to present the IgG2 Vectibix (panitumumab) characterization in a few different chromatographic areas. Vectibix is a fully human mAb IgG2 specific to the epidermal growth factor receptor (EGFR). At first we apply size exclusion chromatography (SEC) with 1.8 µm particle size, 300 Å modified resin surface for Vectibix IgG2 aggregate, monomer and fragment analysis. With added MALS detector, molecular weight of different species can be determined in the same SEC separation. The second characterization method is the strong cation exchange chromatography. It provides the charge variants separation which may be due to IgG2’s major disulfide-mediated structural isoforms. Fractions of the charge variants separation can be collected for further characterization. In the third chromatographic method, Proteomix HIC butyl provides an orthogonal analysis of Vectibix variants under the native running condition based upon the different species’ hydrophobicity. Lastly Intact IgG2 and DTT reduced subunits can be analyzed with reversed phase chemistry. Polymer based Proteomix RP- 1000 provides excellent evaluation of IgG2 subunit heterogeneity. In conclusion, these four chromatographic methods provide a comprehensive characterization of IgG2 Vectibix heterogeneity.