Development of an enzyme-linked immunoreceptor assay (ELIRA) for quantification of the biological activity of recombinant human bone morphogenetic protein-2.

Human bone morphogenetic protein-2 is a representative of the transforming growth factor-beta (TGF-beta) superfamily of cytokines. It was produced in high-cell-density cultivations of recombinant Escherichia coli leading to the formation of inclusion bodies with aggregated inactive protein so that the protein had to be solubilized and renatured. Thus, the biological activity of the recombinant protein had to be determined. To avoid time-consuming cell-based assays or radioactive labelling of proteins enzyme-linked immunoreceptor assays were developed. They were based on the specific interaction between the biologically active protein and its receptors, of which the extracellular ligand binding domains were tagged with the Fc part of human IgG and expressed in insect cells. The amount of bound ligand, corresponding to the biologically active recombinant protein, was determined via enzyme-labelled antibodies. Application to various batches of protein showed that not only the amount of active protein could be quantified but also the quality of the protein preparations could be evaluated in significantly shorter analysis times than with conventional cell-based assays.

Application of an SPR-based receptor assay for the determination of biologically active recombinant bone morphogenetic protein-2.

Human bone morphogenetic protein-2 (hBMP-2) is a member of the human transforming growth factor-beta superfamily. Biologically active bone morphogenetic protein-2 (BMP-2) is a dimeric protein that binds in the first step of the signal transduction cascade to specific receptors on the cell-surface. This specific interaction of the dimeric protein with the extracellular ligand-binding domain (ECD) of the receptor was used to develop a receptor-based assay based on an optical biosensor system (Biacore 2000, Biacore AB, Uppsala, Sweden). The ECD of the BMP-receptor type IA, tagged with the Fc part of IgG (BMPR-IA-Fc), was immobilised on the surface of a dextran-protein A-coated sensor chip. Calibration curves were obtained with purified and biologically active recombinant hBMP-2 (rhBMP-2) that showed a linear range from approximately 5 to 250 nM rhBMP-2. Moreover, this assay was used to quantitatively follow the generation of biologically active protein during the renaturation from unfolded and reduced monomers to biologically active dimers. A refolding mixture containing renatured dimeric rhBMP-2 and not correctly folded monomers, was used as the sample solution without any further pre-treatment. It was proven that only the biologically active dimers were recognised by the immobilised receptor, so the generation of biologically active rhBMP-2 during the renaturation process could be monitored directly and rapidly. Furthermore, the results from the optical sensor obtained during the renaturation process showed a good correlation with the data obtained by non-reducing SDS-PAGE analysis carried out at the end of the renaturation process. These data show that the disulphide-bonded dimer corresponds to the biologically active protein capable of binding the BMP-receptor type IA.