PBA-RDPA 2013 Bologna, 30 June – 3 July 2013 P1.06 USE OF TWO-DIMENSIONAL GEL ELECTROPHORESIS TO STUDY THE QUALITY OF MEDICINAL PRODUCTS OBTAINED BY RECOMBINANT DNA TECHNOLOGY D. Nebija 1 , B. Lachmann 2 , E. Urban 2 , C.R. Noe 2 1 University of Prishtina, Prishtina, Kosovo 2 University of Vienna, Vienna, Austria The main objective of the presented study is the evaluation of two-dimensional gel electrophoresis (2- DE) in combination with matrix assisted laser desorption of ionization mass spectrometry (MALDI-TOF MS) for the identity confirmation humanized and chimeric recombinant monoclonal antibodies (rmAbs) trastuzumab and rituximab and fusion protein abatacept. Trastuzumab (rhuMAb HER2) is a humanized IgG1 rmAb isolated from Chinese hamster ovary (CHO) cells. It is indicated for the treatment of patients with metastatic breast cancer whose tumours overexpress HER2 [1]. Rituximab is a genetically engineered chimeric rmAb, a glycosilated immunoglobulin with human IgG1 constant regions and murine light-chain and heavy chain variable region sequences, produced in CHO cell suspension culture. Rituximab is indicated for the treatment of non-Hodgkin’s lymphoma and rheumatoid arthritis [2]. Abatacept (CTLA4-Ig) is a fusion protein produced by rDNA technology in CHO cells, consists of the extracellular domain of human cytotoxic T-lymphocyte associated antigen 4 linked to a modified Fc portion of human immunoglobulin G1. Abatacept is indicated for the treatment of rheumatoid arthritis [3]. In this study, one-dimensional SDS-PAGE and 2-DE were used for the assessment of molecular weight of these medicinal products. Isoelectric points (pI) have been determined by 2-DE. One dimensional SDS-PAGE revealed that, in reducing conditions, both rmAbs are resolved in two distinct Mr species which migrated in two bands confirming the migration behaviour typical for IgG antibodies: highly complex spot patterns with small differences in Mr but different pI-s. On the other hand under these conditions, reduced form of abatacept migrated as a single band: more than 15 spots were detected. For the identification, experimentally determined peptide masses, obtained from MALDI-TOF MS analysis of in gel trypsin digested abatacept, and both recombinant antibodies are compared with calculated peptide masses, by applying the enzyme cleavage rules, from three primary sequence databases, using MASCOT and PROWL search engines [4,5]. PeptideMass program was used to in silico cleave entered sequences with trypsin and to calculate generated peptide masses with their modifications [6]. Peptide mass fingerprinting analysis enabled identity confirmation of both r mAbs, trastuzumab, rituximab and fusion protein, abatacept. References [1] D.J. Slamon, W. Godolphin, L.A. Jones, J.A. Holt, S.G. Wong, D.E. Keith, W.J. Levin, S.G. Stuart, J. Udove, A. Ullrich, and M.F. Press, Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer, Science. 244 (1989), pp. 707-712. [2] M.E. Reff, K. Carner, K.S. Chambers, P.C. Chinn, J.E. Leonard, R. Raab, R.A. Newman, N. Hanna, D.R. Anderson, Depletion of B cells in vivo by a chimeric mouse human monoclonal antibody, Blood 83 (2) (1994), pp. 435-445. [3] P.S. Linsley, J.A.Ledbetter, N.K. Damle and W. Brady: CTLA4 Ig fusion proteins. United States Patent. December. 01. 1998. U.S. Patent No. 5,844,095. Washington, DC: U.S. Patent and Trademark Office. Available from: http://patft.uspto.gov/, last accessed: 18.08.2010 [4] D.N. Perkins, D.J.C. Pappin, D.M. Creasy, J.S. Cottrell, Probability-based protein identification by searching sequence databases using mass spectrometry data, Electrophoresis 20(18) (1999), pp. 3551-67. [5] http://prowl.rockefeller.edu/prowl-cgi/profound.exe. Last accessed: 18.08.2010. [6] http://www.expasy.ch/tools/peptide-mass.html, Last accessed: 12.08.2010.