S.A. Shestopal, J.‐J. Hao, E. Karnaukhova, Y. Liang, M.V. Ovanesov, M. Lin, J.H. Kurasawa, T.K. Lee, J.H. Mcvey, A.G. Sarafanov

Production of recombinant factor VIII (FVIII) is challenging due to its low expression. It was previously shown that codon-optimization of a B domain-deleted FVIII (BDD-FVIII) cDNA resulted in increased protein expression. However, it is well-recognized that synonymous mutations may affect the protein structure and function. To compare biochemical properties of a BDD-FVIII expressed from codon-optimized (CO) and the wild-type (WT) cDNAs. Each variant of the BDD-FVIII was expressed in several independent CHO cell lines, generated using a lentiviral platform. The proteins were purified by two-step affinity chromatography and analyzed in parallel by: PAGE-Western blot (including thrombin cleavage), mass-spectrometry, circular dichroism, surface plasmon resonance, chromogenic, clotting and thrombin generation assays. The average yield of the CO was 7-fold higher than WT, while both proteins were identical in the amino acid sequences (99% coverage) and very similar in patterns of the molecular fragments (before and after thrombin cleavage), glycosylation and tyrosine sulfation, secondary structures and binding to von Willebrand factor and to a fragment of the low-density lipoprotein receptor-related protein 1. The CO preparations had averagely 1.5-fold higher FVIII specific activity (activity normalized to protein mass) than WT preparations, which was attributed to better preservation of the CO structure due to considerably higher protein concentrations during the production. We concluded that the codon-optimization of the BDD-FVIII resulted in significant increase of its expression and did not affect the structure-function properties. This article is protected by copyright. All rights reserved.