Human bone morphogenetic protein (hBMP)-2 characterization by physical chemical, immunological and biological assays

Abstract

Commercial preparations of human-met-BMP-2 (GenScript) and of CHO-derived hBMP-2 (Infuse-Medtronic) provided a complete characterization of this protein, which belongs to the “transforming growth factors β” superfamily, via SDS-PAGE, Western blotting, reversed-phase HPLC, high-performance size-exclusion chromatography and MALDI-TOF-MS. E.coli-derived met-hBMP-2 has shown a large presence of dimer (MM= 26,054 Da), versus a theoretic value of 26,072 Da. More complex was the distribution of the CHO-derived product, whose exact MM has never been reported due to variable glycosylation: via MALDI-TOF-MS a dimer (28,732 Da) and a large amount of monomer (14,377 Da) were found. A novel method based on RP-HPLC was also validated for hBMP-2 qualitative and quantitative analysis directly in ongoing culture media. The classical “in vitro” bioassay, via alkaline phosphatase induction in murine myoblastic cells C2C12, confirmed that hBMP-2 bioactivity is mostly related to the dimer, being ∼6-fold higher for the CHO-derived glycosylated form. Considering that hBMP-2 is a highly effective osteoinductors, plays an important role during bone regeneration and repair, as well as during embryonic development, and presents an extremely high aggregate value, we believe that these data pave the way to the characterization of this important factor when obtained by DNA recombinant techniques in different host cells.