Development of chromatographic and electrochemical methods for trastuzumab determination

Today, the use of monoclonal antibodies in the treatment of a variety of diseases has made them a pioneering product in the biopharmaceutical market. In present thesis, analytical methods for bioequivalence studies of Trastuzumab, which is a monoclonal antibody in IgG structure, affects human epidermal growth factor, and used in breast cancer treatment are examined. For this purpose, three different methods were used. Initial studies included Trastuzumab analysis using HPLC-FLD system in serum samples. For total Trastuzumab determination, purification was performed from serum using a Protein G column and was analyzed by HPLC-FLD system. In the second part, considering the mechanism of action of Trastuzumab, it is examined by using electroanalytical methods whether Trastuzumab analysis can be performed by means of HER2 immobilized electrode surfaces. For this purpose, bare PGE, graphene oxide and multi-walled carbon nanotubes and their gold nanoparticle electrodes were used. After each modification, CV and EIS measurements were taken. The third part includes LC-MS/MS analysis of Trastuzumab on selected peptides after tryptic digestion. The m/z 543.2929 for the IYPTNGYTR signature peptide and m/z 402.3566 for the PTNGYTR fragment were selected and analyzed by the LC-Q-TOF-MS system. In HPLC-FLD studies, although the linearity was excellent (R=0.999) in the 5.0-120 µg/mL range, peak resolution was not sufficient. Furthermore, the selectivity of the method is limited due to multiple peak formation and poor resolution. Compared to other analytical methods, the working time is long and time consuming. When electroanalytical methods were evaluated, the best bonding was obtained with multi-walled carbon nanotubes according to the ΔRct values calculated according to the obtained data. This technique provides a great advantage to get immediate results compared to other methods. In addition, it has been found that the determination of Trastuzumab can be done by using HER2-Trastuzumab interaction for the first time electrochemically. In the LC-MS/MS studies, it was found that the quantitative analysis of Trastuzumab was possible using the LC-Q-TOF-MS system on the selected peptide. Keywords: Monoclonal Antibody (mAbs), Trastuzumab, HPLC-FLD, Elektrokimyasal Empedans Spektroskopisi, LC-Q-TOF-MS