Browsing by Subject "SDS-PAGE"

Monoclonal antibodies (mAbs) are widely used in the treatment of several diseases such as cancer and autoimmune diseases. Due to their high prices and growing consumption, therapeutic mAbs have become potential targets of falsification. This generates a demand for quick and efficient analytical procedures for identifying and characterizing mAbs in a case of suspected falsification. The structure of therapeutic mAbs consists of human or murine IgG framework, where unique complementarity determining regions (CDRs) are engineered with different recombinant techniques. Given the complex nature of the mAbs, they must be identified using multiple complementary analytical methods. Ten full-sized therapeutic mAbs, Fab-fragment abciximab and CTLA4-Fc-fusion protein belatacept were studied in order to find analytical methods for efficient characterization and identification. All studied antibodies were characterized by their charge and molecular weight by isoelectric focusing (IEF) in polyacrylamide gels, native and reduced SDS-PAGE, and size exclusion chromatography (SEC). Six mAbs, abciximab and belatacept were digested with trypsin, and the cleaved peptides were further analysed by RPLC-MS. In addition, quantification methods including SEC peak area measurements and Bradford protein assay were performed for all antibodies. As expected, SDS-PAGE of non-reduced and reduced mAbs gave little distinction between the mAbs. Both methods were however shown to be useful in the identification of the mAb nature, as they confirmed the presence of heavy chains, light chains, and disulfide bonds. IEF showed potential in mAb identification, as clear, partly distinguished patterns of charge variants were obtained. However, some improvements to the pH gradient are needed to enable better separation and pI estimation of basic variants. Determination of molecular size with SEC was found to be difficult, as there seemed to be no consistency between the calculated molecular weights based on measured elution times, and the theoretical molecular weights. Nevertheless, SEC brings added value in mAb quantification and detection of protein aggregation and fragmentation. Finally, RPLC-MS analysis of tryptic peptides resulted in mAb identification, with the measured sequence coverage of 87-97 %. Identification process may be enhanced by focusing on the known CDR-peptides prior the constant frame peptides. Given the structural similarity of therapeutic mAbs, identification of an unknown mAb requires combination of multiple analytical methods. If available, the use of reference mAb product obtained from a reliable source is recommended, as the identification may be based on comparative analyses using simpler analytical steps, e.g. IEF, SDS-PAGE and SEC. If no reference product is available, identification of the mAb requires peptide mapping and determination of the CRD sequences by RPLC-MS analysis. Further research is needed to find a suitable set of analytical methods for identification of all therapeutic mAbs.