title: Process development of a recombinant antibody/interleukin-2 fusion protein expressed in protein-free medium by BHK cells.
abstract: The production, purification and stability of quality (in terms of integrity and glycosylation) of an antibody/interleukin-2 fusion protein with potential application in tumour-targeted therapy expressed in BHK21 cells are described. Consistency of the product throughout time was determined by analysis of glycosylation of the fusion protein using MALDI-TOF mass spectroscopy and HPAEC-PAD combined with product integrity studies by SDS-PAGE and Western blotting. These investigations showed consistent expression in terms of integrity and of three major oligosaccharide structures of the fusion protein after 62 generations. The data obtained at this stage indicated the suitability of the cell line for production purposes. Different approaches for the production of this protein were subsequently carried out. The relative productivity of the recombinant fusion protein and general performance of the cells in two different protein-free medium (PFM) culture systems, continuous chemostat and continuous perfusion using a Centritech centrifuge as a cell retention device, were studied. The results indicate that the chemostat culture resulted in more stable and controllable nutrient environment, which could indicate better product consistency, in accordance with what has been observed under serum-containing conditions, in relation to the perfusion culture. Finally, product obtained from the chemostat culture was analysed and purified. The purification process was optimised with an increase in the overall yield from 38 to 70% being obtained, a significant improvement with important consequences for the implementation of an industrial-scale culture system. In conclusion, it was possible to produce and purify the recombinant antibody/interleukin-2 fusion protein assuring the quality and stability of the product in terms of integrity and glycosylation. Therefore, a candidate production process was established.
authors.affiliation: IBET/ITQB-Instituto de Biologia Experimental e Tecnológica/Instituto de Tecnologia Química e Biológica, Apartado 12, P-2781-901 Oeiras, Portugal.
authors.forename: H J
authors.initials: HJ
authors.lastname: Cruz
authors.forename: H S
authors.initials: HS
authors.lastname: Conradt
authors.forename: R
authors.initials: R
authors.lastname: Dunker
authors.forename: C M
authors.initials: CM
authors.lastname: Peixoto
authors.forename: A E
authors.initials: AE
authors.lastname: Cunha
authors.forename: M
authors.initials: M
authors.lastname: Thomaz
authors.forename: C
authors.initials: C
authors.lastname: Burger
authors.forename: E M
authors.initials: EM
authors.lastname: Dias
authors.forename: J
authors.initials: J
authors.lastname: Clemente
authors.forename: J L
authors.initials: JL
authors.lastname: Moreira
authors.forename: E
authors.initials: E
authors.lastname: Rieke
authors.forename: M J T
authors.initials: MJ
authors.lastname: Carrondo
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000737:chemistry / Q000493:pharmacokinetics; D019149:Bioreactors; D018929:Cell Culture Techniques / Q000379:methods; D002455:Cell Division; D002460:Cell Line / Q000378:metabolism*; D002474:Cell-Free System; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D007376:Interleukin-2 / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism / Q000493:pharmacokinetics; D007668:Kidney; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009844:Oligosaccharides / Q000096:biosynthesis / Q000737:chemistry / Q000493:pharmacokinetics; D010477:Perfusion; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism / Q000493:pharmacokinetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014407:Tumor Cells, Cultured
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007376:Interleukin-2; D009844:Oligosaccharides; D011993:Recombinant Fusion Proteins