Incremental Improvement in Bioengineering of Coagulation Factor VIII for Efficient Expression: Elimination of a Dispensable Disulfide Loop Enhances Secretion
Authors: Selvaraj Sundar Rajan , Kaufman Randal J , Pipe Steven W. 
Published: April 2020

Coagulation factor VIII (FVIII), a glycoprotein cofactor, performs a critical function in the intrinsic blood coagulation pathway and a quantitative or qualitative deficiency of this protein results in Hemophilia A. The primary obstacle to producing low cost replacement therapy is the inherent limitation of commercial recombinant FVIII (rFVIII) production since its expression is 2 to 3 orders of magnitude lower than that of other comparably sized proteins. Inefficient mRNA expression, protein misfolding & chaperone mediated retention and the requirement for facilitated transport from the Endoplasmic Reticulum (ER) to Golgi have been identified as the major bottlenecks to efficient expression of rFVIII. Bioengineering strategies aimed at clearing each of these hurdles have resulted in the generation of several rFVIII variants with more efficient expression. This study focused on the effect of each of FVIII’s eight disulfide bonds on its secretion efficiency and functionality. Elimination of specific disulfide loops has previously been shown to confer a secretion advantage in certain proteins, e.g. lysozyme and human chorionic gonadotropin (hCG). The FVIII disulfide bonds were eliminated by replacing the corresponding cysteine residues with glycine residues by a PCR-based site-directed mutagenesis protocol using mutagenic primers. The disulfide mutants were created both in the full length FVIII as well as a previously bioengineered FVIII variant (226/N6) with enhanced (5–10 fold) secretion efficiency. All the mutants were characterized by restriction enzyme digestion and DNA sequencing. The secretion efficiency of all the mutants was studied by transient transfection in Chinese Hamster Ovary (CHO) and COS-1 cells. FVIII activity was measured by one-stage and two-stage clotting assays and the antigen levels were quantified by anti-FVIII light chain sandwich enzyme-linked immunosorbent assay. Seven of the eight disulfide bonds in factor VIII were found to be indispensable for the correct folding and secretion since all these disulfide mutants were retained intracellularly. A single mutant with substitution of glycine for cysteine at position 2326 in the C2 domain resulted in secretion of a functionally inactive protein. Elimination of the disulfide loop between cysteine residues at positions 1899 and 1903 in the A3 domain resulted in a 1.5- to 2-fold increase in secretion of both the full length FVIII and the already efficiently expressed 226/N6 rFVIII variant. This enhanced secretion was reflected in both the antigen levels and the activity of the protein. Also, incorporation of these mutations in 226/N6 F309S, yet another efficiently secreted rFVIII variant, further improved secretion with a synergistic effect, resulting overall in a 25- to 30-fold higher expression than full length FVIII in CHO and COS-1 cells. Interestingly, coagulation factor V (FV), that shares considerable homology with FVIII and is secreted much more efficiently, lacks a disulfide loop in the homologous position to the 1899–1903 bridge with FVIII. The other seven disulfide bonds are conserved between these two proteins. Such combined targeted bioengineering strategies may facilitate more efficient production of rFVIII toward lower cost replacement therapy.