Development and use of specific ELISA methods for quantifying the biological activity of bevacizumab, cetuximab and trastuzumab in stability studies.
Bevacizumab (BVZ), cetuximab (CTX) and trastuzumab (TTZ) are monoclonal antibodies (mAbs) used worldwide for the treatment of several widespread kinds of cancer. They are marketed as medicines under their respective tradenames: Avastin(®), Erbitux(®) and Herceptin(®). The aim of this research was to develop in-house specific enzyme-linked immunosorbent assays (ELISA) to assess the long-term stability of these three mabs. These assays assess the biological functionality of the mAbs by quantifying their biological activity. For this purpose, we developed an indirect ELISA procedure whereby the specific antigens against which the mAbs are directed are used as specific "capturing" antibodies on the ELISA plates. We therefore used vascular endothelial growth factor (VEGF) in the ELISA for BVZ; human epidermal growth factor receptor (hEGFR) in the ELISA for CTX and human receptor HER2 (hHER2) in the ELISA for TTZ. After the mAbs had attached to their antigen, we used an anti-human IgG (whole molecule) peroxidase-conjugate and o-phenylenediaminedihydrochloride substrate. The reaction was stopped using sulphuric acid and absorbance was recorded at a wavelength of 450nm. The three ELISA methods were validated in terms of calibration models, range of the assay, limits of detection and quantitation, intra and interday precision and accuracy, and specificity by cross reactions. Forced degradation studies were also conducted on the medicines, providing useful information. Finally, the proposed ELISA were successfully used in a long-term stability study to quantify the remaining biological activity in medicines that had been opened and then stored under two different storage conditions, i.e. refrigerated at 4°C and frozen at -20°C. Results indicated that BVZ (Avastin(®)) is the most stable of the three in terms of its biological functionality.
Department of Analytical Chemistry, University of Granada, Faculty of Science, Campus Fuentenueva s/n, E-18071 Granada, Spain.
Inmaculada
I
Suárez
UGC Intercentro Interniveles Farmacia Granada, "San Cecilio Hospital", Biomedical Research Institute ibs.Granada, Hospitales Universitarios de Granada, University of Granada, E-18012 Granada, Spain.
Antonio
A
Salmerón-García
UGC Intercentro Interniveles Farmacia Granada, "San Cecilio Hospital", Biomedical Research Institute ibs.Granada, Hospitales Universitarios de Granada, University of Granada, E-18012 Granada, Spain.
José
J
Cabeza
Department of Analytical Chemistry, University of Granada, Faculty of Science, Campus Fuentenueva s/n, E-18071 Granada, Spain.
Luis Fermín
LF
Capitán-Vallvey
Department of Analytical Chemistry, Science Faculty, Biomedical Research Institute ibs.Granada, University of Granada, Campus Fuentenueva s/n, E-18071 Granada, Spain. Electronic address: natalia@ugr.es.
Natalia
N
Navas
D000970:Antineoplastic Agents / Q000737:chemistry*; D000068258:Bevacizumab / Q000737:chemistry*; D000068818:Cetuximab / Q000737:chemistry*; D004355:Drug Stability; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D055550:Protein Stability; D000068878:Trastuzumab / Q000737:chemistry*
D000970:Antineoplastic Agents; D000068258:Bevacizumab; D000068878:Trastuzumab; D000068818:Cetuximab
Bevacizumab; Cetuximab; ELISA; Monoclonal antibody based-medicines; Stability study; Trastuzumab