Glycopegylation and Factor VIII

There has been considerable effort to develop a factor VIII product with extended half-life for the treatment of hemophilia A (1).   Such a product would be of great value to patient community as it would require less frequent administration of product.  There are several approaches to the development of a factor VIII product with an extended half-life (1,2).  The most common approach uses the attachment of poly(ethylene)glycol (PEG)(3).  Abuchowski and coworkers at Rutgers University in 1977 developed the covalent attachment of PEG to proteins as method to reduce antigenicity and extend circulatory half-life(4,5).  Early work on the pegylation of proteins focused on the derivatization of therapeutic proteins produced in bacterial system without the post-translational step of glycosylation (6,7).  The PEG chain could be attached at glycosylation sites in these recombinant proteins and was shown to improve renal clearance (6).  These early studies used attachment at the ε-amino group of lysine or at α-amino groups.  It was quite difficult to obtain specificity of modification and/or a homogenous product.  Glycopegylation is a method by which a PEG chain may be attached to a specific site in a protein.  The method is based on the ability to couple a PEG chain to 5’-amino position of sialic acid and subsequent transfer of the sialic acid -PEG conjugate to an existing glycan chain on a protein (8.9).  This allows for the site-specific conjugation of a PEG chain to a protein.  Glycopegylation has been successfully applied to the factor VIII protein (10) resulting in a biopharmaceutical with increased circulatory half-life.  There is one other pegylated factor VIII product resulting from attachment of a PEG chain at a specific site (11).  This biopharmaceutical is obtained by coupling of a maleimide derivative of PEG to a cysteine residues which had been engineered into B-domain delated factor VIII (12).  The use of an engineered cysteine residue has precedent as an engineered cysteine residue was used for the PEGylation of recombinant interleukin-2 (IL-2) (6).  The cysteine residue was substituted for the threonine residue (T3C) at the glycosylation site of IL-2 providing rationalization for this derivative.  The mechanism(s) for the prolongation of half-life of the factor VIII product is not clearly understood.  It seems mostly likely that PEGylation influences the interaction between factor VIII and the low-density lipoprotein receptor binding protein (13,14).  The mechanism for the extension of half-life may be different for factor VIII fusion proteins (15) using binding to the neonatal Fc receptor.


1.  Mancuso, M.E. and Santagostino, E., Outcome of clinical trials with new extended half-life FVIII/IX concentrates, J.Clin.Med. 6(4):E39. doi:10.3390.jcm6040039,. 2017
2.  Leksa, N.C., Chiu, P.L., Bou-Assay, G.b., et al., The structural basis for the functional comparability of factor VIII and the long-acting variant recombinant Factor VIII Fc fusion protein, J.Thromb.Haemost. in press, 2017
3.  Pasur, G. and Veronese, F.M., Start of the art in PEGylation: The great versatility achieved after forty years of research, J.Control.Rel. 161, 461-472, 2012
4.  Abuchowski, A., van Es, T., Palczuk, N.C., and Davis, F.F., Alternation of immunological properties on bovine serum albumin by covalent attachment of polyethylene glycol, J.Biol.Chem. 252, 3578-3581. 1977
5.  Abuchowski, A., McCoy, J.R., Palczuk, N.C., van Es, T., and Davis, F.F., Effect of covalent attachment of polyethylene glycol on immunogenicity and circulating life of bovine liver catalase, J.Biol.Chem. 252, 3582-3586, 1977
6.  Goodson, R.J. and Katre,N.V., Site-directed pegylation of recombinant interleukin-2 at its glycosylation site, Bio/Technology 8, 343-346, 1990
7.  DeFrees, S., Wang,Z-G., Xing, R., et al., GlycoPEGylation of recombinant therapeutic proteins produced in Escherichia coli, Glycobiology 16, 833-843, 2006.
8.  Giorgi, M.E., Agusti, R., and de Lederkremer, R.M., Carbohydrate PEGylation, an approach to improve pharmacological potency, Beilstein J.Org.Chem. 10, 1433-1444, 2014
9.  Tarp, M.A. and Clausen, H., Mucin-type O-glycosylation and its potential use in drug and vaccine development, Biochim.Biophys.Acta 1780, 546-563, 2008
10.  Giangrande, P., Andreeva, T., Chowdary, P., et al., Clinical evaluation of glycopegylated recombinant FVIII: Efficacy and safety in severe haemophilia A, Thromb.Haemost. 117, 252-261, 2017
11.  Reding , M.T., Ng, H.J., Poulsen, L.H., et al., Safety and efficacy of BAY 94-9027, a prolonged-half-life factor VIII, J.Thromb.Haemost. 15, 411-419, 2017
12.  Mei, B., Pan., C., Jiang, H., et al., Rational design of a fully active, long-acting PEGylated Factor VIII for hemophilia A treatment, Blood 116, 270-279, 2010
13.  van den Biggelaar, M., Madsen, MJ., Faber, J.H., et al., FactorVIII interacts with the endocytic receptor low-density lipoprotein receptor=related protein 1 vai an extended surface comprising “hot-spot” lysine residues, J.Biol.Chem. 290, 16463-16476, 2015.
14. Young, P.A. Migliorini, M., and Strickland, D.K., Evidence that factor VIII forms a bivalent complex with the low density lipoprotein (LDL) receptor-related protein 1 (LRP1). Identification of cluster IV IV on LRP1 as the major binding site, J.Biol.Chem. 291, 26035-26044, 2016.
15.  van der Flier, A., Liu, Z., Tan, X., et al., FcRn rescues recombinant factor VIII Fc fusion protein from a VWF independent FVIII clearance pathway, PLoS One  10(4):e0124930, 2015