Characterization and engineering of proprotein convertases in recombinant CHO cells for efficient BMP-7 processing

Abstract

Recombinant glycoprotein therapeutics has proven to be invaluable pharmaceuticals for the treatment of chronic and life-threatening diseases. Since 2001, Recombinant human BMP-7 (rhBMP-7) has used as an alternative to autograft in bone non unions such as the tibial non union and spinal fusions. Although these are extraordinarily efficacious, high dosage requirements of several hundred milligrams of protein for each ad- ministration, often makes the treatment expensive. The major challenge currently lies in the develoment of large-scale, cost-effective manufacturing processes for BMP-7 molecules. BMP-7, a complex glycoprotein, is synthesized as a large precursor and requires proteolytic cleavage in the late secretory pathway for its maturation into functionally active form. Thus rhBMP-7 is routinely pro- duced in CHO cells, as they provide efficient platform for protein synthesis, post translational modification and secretion. Inspite of this, BMP-7 production in CHO cells yields low titers for a number of reasons. In order to understand the bottleneck which limits the production of BMP-7 in CHO cells, a compre- hensive study of the BMP-7 producing CHO cells (CHO BMP-7) were undertaken. A suspension batch cul- ture of the CHO BMP-7 resulted in the production of $3-5 \mu g/ mL$ which is atleast 10 times lesser than the pro- tein of similar size produced in CHO cells. On analysis of the culture supernatants, it was found that a significant amount of unwanted precursor forms of rhBMP-7 (ca. 69% of total rhBMP-7), along with the mature form of rhBMP-7, was secreted into the culture medium, likely due to the insufficient amount of the proprotein convertases (PC) within the secretory pathway. Additionally, the secreted BMP-7 in the medium was unstable and underwent degradation when the viability dropped below 90%. As the first step to solve the precursor accumulation, the characterization of proprotein convertases in CHO BMP-7 was undertaken. Inves- tigation of the P...