Investigation of Post-translational Strategies to Enhance Recombinant Human Bone Morphogenetic Protein-2 Production in Mammalian Cell Culture Systems

Bone morphogenetic proteins (BMPs) have been found useful in repairing craniofacial defects, treating non-union long bone fractures, and in spinal fusion procedures. However, the widespread use of recombinant human BMPs (rhBMPs) is hindered by the difficulties in obtaining large quantities of pure and biologically active rhBMPs. Standard approaches to improve recombinant protein yields such as increasing transgene expression by having strong promoters and/or utilizing gene amplification methods still result in low yields when applied to BMPs. The impact of altering the post-translational processing on recombinant BMP-2 yields in mammalian cell expression systems was investigated in this dissertation. We attempted to modify the proteolytic cleavage of BMP-2 by 1) adding a BMP cleavage inhibitor to the cell culture medium, 2) mutating a cleavage site on BMP-2 sequence, and 3) overexpressing BMP-cleaving proprotein convertases in cells producing rhBMP-2. All three methods led to significant increases in the yield of biologically active rhBMP-2. Characterizing the rhBMP-2 produced from these methods, the data revealed that proteolysis of BMP-2 could also influence the biological activity of the mature protein. Taken together, these results suggest that targeting post-translational processing is a useful and practical strategy to improve the yield of BMP-2 and possibly other difficult to express recombinant proteins in mammalian expression systems. As an increasing number of recombinant proteins become extremely valuable therapeutic options today, the findings from this study contribute to the collective understanding of enhancing recombinant protein yield in mammalian cell expression systems.Ph.D.2019-11-20 00:00:0