PEGylation of Blood Coagulation Factor VIII

A recent paper on the third PEGylated factor VIII (1) prompted me to prepare a short note on PEGylation and the development of extended  half-life factor VIII products.

Hemophilia A is a monogenic deficiency of blood coagulation factor VIII resulting in hemophilia A.   Individuals with hemophilia A require periodic infusions of factor VIII.  While the sophistication of the factor VIII drug has improved since the first therapeutic intervention in 1840, there was a necessity for frequent infusions of factor VIII.  Over the past decade. several ‘long-acting’ factor VIII concentrates have been developed which have resulted in a modest (approximately 2-fold) extension of circulatory half-life (2); similar modifications of factor IX resulted in great extension of half-life.  The majority of products have resulted from the covalent attachment of a poly(ethylene)glycol (PEGylation) chain to the factor VIII protein.  There is one extended half-life factor product which is fusion protein of B-domain deleted factor VIII and immunoglobulin Fc domain (3).  I should add that PEGylation of proteins is thought to extend circulatory half-life by inhibiting normal  clearance pathways.  In the case of Factor FVIII, it is assumed that PEGylation sterically blocks interaction with low-density lipoprotein receptor protein(4) although I could find a specific statement to that effect.   PEGylation is also thought to decrease immunogenicity of proteins by possibly sterically blocking recognition of epitopes (5) although the effect may not be consistent (6).  PEGylation extends the circulatory half-life of low molecular proteins by blocking renal clearance (7).
PEGylation was first some 40 years ago to produce a preparation of bovine serum albumin (8) and catalase (9)  with altered immunogenicity.   Subsequently, PEGylation was used produce a L-asparaginase with markedly reduced immunogenicity (10,11).  Ralph Bradshaw and I wrote an article on the use of chemical modification in the manufacture of therapeutic proteins (12) and did include a study on the one published work on the PEGylation of factor VIII(13); in this case,.  a B-domain deleted factor VIII preparation (14,15). It this work, a B-domain-deleted factor VIII was modified with three different derivatives of poly(ethylene)glycol.  A PEGylated B-domain-deleted factor VIII was obtained which had a extended half-life in a primate model.  While, as far as I know, this particular product did not make to an approved biologic, three quite different PEGylated are either approved products or on track for approval. I have to say that I tried to modify factor VIII while at the University of North Carolina with marginal success. It is my understanding that others had also tried without success. Studies on the chemical modification of factor VIII (12) have yielded conflicting results possibly resultng from the use of impure preparations of factor VIII. The current work has been peformed using highly purified preparations of factor VIII.





A full-length recombinant factor VIII modified with a ‘unique’ poly(ethylene)glycol derivative.  Data suggests selective modification is obtained at lysine residues.


BAY 94-9027

A B-domain-deleted factor VIII containing an engineered cysteine residues is modified with a poly(ethylene)glycol derivative specific for reaction with cysteine residues (likely a maleimide derivative but not stated)



A 40 kDa PEG chain is attached to a B-domain-deleted factor VIII containing a truncated B-domain by a process described as glycopegylation.  Briefly, an N-acetygalactose is enzymatically attached to a serine or threonine residue .   This is followed by the enzymatic attachment of a sialic acid conjugated PEG derivative.  



  1. Shah, A., Coyle, T., Lalezari, S., et al., BAY 94-9027, a PEGylated recombinant factor VIII, exhibits a prolonged half-life and higher area under the curve in patients with severe hemophilia A: comprehensive assessment from clinical studies, Haemophilia 24, 733-740. 2018.
  2. Mancuso, M.E. and Santagastino, E., Outcome of clinical trials with new extended half-life  FVIII/FIX concentrates, J.Clin.Med. 6(4):E39, 2017.
  3.   Leksa, N.C., Chiu, P.L., Bou-Assaf, G.M., et al., The structural basis for functional comparability of factor VIII and the long-acting variant recombinant factor VIII Fc fusion product, J.Thromb.Haemost. 15, 1167-1179, 2017.
  4.     Young, P.A., Migliorini, M., and Strickland, D.K., Evidence that Factor VIII forms a bivalent complex with the low density lipoprotein (LCL)receptor-related protein 1(LRP1): Identification of cluster IV on LRP1 as the major binding site, J.Biol.Chem. 291, 26035026044, 2016.
  5.   Xu, Y., Shi, Y., Zhou, J. et al., Structure-based antigenic epitope and PEGylation improve the efficacy of staphylokinase, Microb.Cell Fact. 16(1):197, 2017
  6. Gefen, T., Vaya, J., Khatib, S., et al., The impact of PEGylation on protein immunogenicity, Int.Immunopharmacol. 15, 254-259, 2013.
  7. Benincasa, M., Zahariev, S.,  Pelillo, C., et al., PEGylation of the peptide Bac7(1-35) reduces renal clearance while retaining antibacterial activity and bacterial cell penetration capacity, Eur.J.Med.Chem. 95, 210-219, 2015.
  8. Abuchowski, A., van Es, T., Palczuk, N.C., and Davis, F.F., Alteration of immunological properties of bovine serum albumin by covalent attachment of polyethylene glycol, J.Biol.Chem. 252, 3578-3581, 1977.
  9. 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 half-life of bovine liver catalase, J.Biol.Chem. 252, 3582-3586, 1977.
  10. Ashihara, Y., Kono, T., Yamazaki, S., and Inada, Y., Modification of E.coli L-asparaginase with polyethylene glycol: disappearance of binding ability to anti-asparaginase serum, Biochem.Biophys.Res.Commun. 83, 385-391, 1978.
  11.  Abuchowski, A., van Es, T., Palczuk, N.C., McCoy, J.R., and Davis, F.F., Treatment of L5178Y tumor-bearing BDF1 mice with a nonimmunogenic L-glutaminase-L-asparaginase, Cancer Treat.Rep. 63, 1127-1132, 1979.
  12. Lundblad, R.L., and Bradshaw, R.A., Addressing product improvement using chemical modification in biopharmaceutical manufacturing, Biopharm International, September, 30-36, 2006.
  13. Röstin, J., Smeda, A.-L., and Akerlbom, E., B-domain deleted recombinant coagulation factor VIII modified with polyethylene glycol, Bioconjug.Chem. 11, 387-396, 2000.
  14.  Webb, E., Tkalcevic, J., Edwards, S., Hocking, D., and Nisbet, I., Expression of biologically active human factor VIII using a baculovirus vector, Biochem.Biophys.Res.Commun. 190, 536-543, 1993.
  15. Pittman, D.D., Alderman, E.M., Tomkinson, K.N., et al., Biochemical, immunological, and in vivo functional characterization of B-domain-deleted factor VIII, Blood 81, 2925-2935, 1993.
  16. Turecek, P.L., Bossard, M.J. Graninger, M., et al., BAX 855, a PEGylated FVIII product with prolonged half-life . Development, functional and structural characterization, Haemstaseologie 32 (Suppl 1), 529-536, 2012.
  17.  Dunn, A.L., Ahuja, S.P., and Mullins, E.S., Real-world experience with use of antihemophilic factor (recombinant), PEGylated for prophylaxis in severe haemophilia A, Haemophilia 24(3):e84-e92, 2018.
  18. Mei, B., Pan, C., Jiang, H., et al., Rational design of a fully active, long-acting PEGylated factor VIIII for hemophilia A treatment, Blood 116, 270-279, 2010.
  19. Defrees, S., Wang, Z.G., Xing, R., et al., GlycoPEGylation of recombinant therapeutic proteins produced in Escherichia coli, Glycobiology 16, 833-844, 2006.
  20.  Thim, L., Vandahl, B., Karlsson, J. et al., Purification and characterization of a new recombinant factor VIII (N8), Haemophilia  16, 349-359, 2010.
  21. Rode, F., Almholt, K., Petersen, M., et al., Preclinical pharmacokinetics and biodistribution of subcutaneously administered glycoPEGylated recombinant factor VIII (N8-GP) and development of a human pharmacokinetic prediction model, J.Thromb.Haemost. 16, 1141-1152, 2018.

Copywright November, 2018

Roger L Lnndblad

Chapel Hill, North Carolina

919 929 5082