Modification of Coagulation Factors to Extend Half-Life

One of the considerable benefits of being a football official at some time in one's life is ability to know that you can make mistakes and have the films to prove it.   Being wrong, admitting same and moving forward is a valuable experience not shared, of course, by the political class.  In this case, the success of Biogen in launching two engineered coagulation proteins with improved pharmacokinetics (1) is not something that I thought would happen.    A factor IX product (2) and factor VIII (3) product have been developed.   The extended half-life of Fc fusion proteins is thought to reflect binding to the neonatal Fc  receptor (FcRn) (4,5) but other factors are also likely important (5).  Regardless of the underlying basis for the improvement in pharmacokinetics, the use of these products should reduce number of injections required.   Cost issues will need to be balanced against improved clinical performance and outside the scope of the current work.   Other fusion partners such as albumin (6-9) and a proline-alanine-serine copolymer (PAsylation)(10) are being used.    The use of albumin fusion proteins is based on interaction with the neonatal Fc receptors (6) described above for Fc fusion partners.

 The manufacture of Fc fusion proteins has an advantage over other approaches to half-life extension in that an additional process  step is required in these other approaches ;  the additional process step is also complicated in that it requires the chemical modification of proteins.  Notwithstanding that the chemical modification of proteins is close to my heart (11), I would approach the use of such technology in biopharmaceutical manufacturing process with considerable trepidation.  I would also note that a quick and admittedly incomplete  PubMed search showed approximately 600 citations for "Fc fusion protein" and 125 citation for "protein pegylation." 
There are several other approaches to the extension of the half-life of therapeutic proteins in the vascular space (12,13).    While the majority of these approaches use the attachment of a poly(ethylene) glycol (PEG) chain to a protein (14-16), there are examples of formulating therapeutic proteins  by binding to to pegylated liposome (17,18).   
To the best of my knowledge, the use of covalent modification of proteins to improve pharmacokinetic performance dates to the work of Kosower (19,20) some fifty years ago.  Kosower proposed the use of N-carboxyanhydride derivatives for the modification of tyrosyl residues in proteins for reducing the antigenicity of proteins and tissues.   As far I can tell, this approach was not the subject of further investigation.    There was previous work which showed that harsh chemical treatment such as acid or base could destroy antigenic determinants (21,22) and formaldehyde could convert toxins to toxoids (21) providing the basis for vaccine development (23). 
The successful covalent modification of proteins with PEG to extend half-life in the circulation date back to the observations of Abuchowski and coworkers (24,25).  This early work was directed at reducing the antigenicity of proteins by blocking antigenic sites (epitopes) on "foreign" proteins.  While this was an important goal, work over the past 30+ years has shown that PEGylation reduces renal clearance, increases solubility and protects against proteolytic degradation in addition to reducing antigenicity (26).
It is not clear as to what lead Davis and his colleagues to the use of PEG to modify proteins.  If one likens PEG to a polysaccharide chain, then while there is recent work (27) suggesting that glycosylation blocks antigenic sites on proteins, early work with dextran coupling to protein showed that dextran acquired the properties of a hapten (28).   It is of interest that the N-glycans of Factor X have been suggested to influence the half-life of this protein by interactions with monocytes/macrophages (29)
 While not directly related to the current topic, I like to mention the work on Baynes and Wold (30) on the observed half-life the four forms of bovine pancreatic ribonuclease (RNase) in the rat.   Nephrectomized rats were used because of the rapid renal clearance of a small protein such as RNAse.  RNase A (no glycosylation) had an observed half-life of 528-582 minutes while a half-life of 700-900 minutes was observed for RNasc C  or RNase D (both complex with terminal sialic acid). RNAse B(terminal mannose) was cleared in 15 minutes; removal of three mannose residues leaving a trisaccharide with a terminal β-mannosyl residue provided a derivative with a half-life of 616-733 minutes.    There was not a second adminisration to seeing RNAse would have elicited an antibody response.    



1.  Mancuso, M.E., and Mannucci, P.M., Fc-fusion technology and recombinant FVIII and FIX in the management of the hemophilias, Drug.Des.Devel.Ther. 8, 365-371, 2014.

2. Powell, J.S., Pasi, K.J., Rangi, M.V., et al., Phase 3 study of recombinant factor IX Fc fusion protein in hemophilia B, N.Engl.J.Med. 369, 2313-2323, 2013.

3.  Mahlangu, J., Powell, J.S., Ragni, M.V., et al., Phase 3 study of recombinant factor VIII Fc fusion protrein i severe hemophilia A., Blood 123, 317-325, 2014.

4.  Valentino, L.A., Recombinant FIXFc: a novel therapy for the royal disease?, Expert Opin Biol.Ther. 11, 1361-1368, 2011.

5.   Suzuki, T., Ishii-Watabe, A., Tada, M., et al., Importance of neonatal FcR in regulating the serum half-life of therapeutic proteins containing the Fc domain of human IgG1: a comparative study of the affinity of monoclonal antibodies and Fc-fusion proteins to human neonatal FcR, J.Immunol. 184, 1968-1976, 2010.

6.  Sleep,D., Cameron, J., and Evans, L.R., Albumin as a versatile platform for drug half-life extension, Biochim.Biophys.Acta 1830, 5526-5534, 2013.

7.  Kodama, A., Watanabe, H. Tanaka, R., et al., Albumin fusion render thioredoxin an effective anti-oxidative and anti-inflammatory agent for preventing cisplatin-induced nephrotoxicity, Biochim.Biophys.Acta  1840, 1152-1162, 2014.

8. Zollner, S.,Schuermann, D., Raquet, E., et al., Pharmacological characteristics of a novel, recombinant fusion protein linking coagulation factor VIIa with albumin (rVIIa-FP), J.Thromb.Haemost. 12, 220-228, 2014.

9.  Herzog, E., Harris, S., Henson, C., et al., Biodistribution of the recombinant fusion protein linking coagulation factor IX with albumin (rIX-FP) in rats, Thromb.Res. 133, 900-907, 2014.

10.  Schlapschy, M., Binder, U., Börger, C., et al., PASylation: a biological alternatives to PEGylation for extending the plasma half-life of pharmaceutically active proteins, Protein Eng.Des.Sel. 26, 489-501, 2013.

11.  Lundblad, R.L., Chemical Reagents for the Modification of Proteins., 4th edn,  Taylor and Francis/CRC Press, Boca Raton, Florida, USA, 2014.

12.   Pipe, S.W., The hope and reality of long-acting hemophilia products, Am.J.Hematol. 87(Suppl 1), S33-S39, 2012.

13.  Carcao,M., Changing paradigm of prophylaxis with longer acting factor concentrates, Haemophiiia 20 (Suppl 4), 99-105, 2014.

14.  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.

15.  Turecek, P.L., Bossard, M.J., Graninger,M., et al., BAX 855, a PEGylated rFVIII product with prolonged half-life.  Development, functional and structural characterisation, Hamostasseologie 32 (Suppl 1), S29-S38, 2012.

16.  Pastoft, A.E., Exgan, M., Tranholm, M., et al., Prolonged effect of a new O-glycoPEGylated FVIII (N8-GP) in a murine saphenous vein bleeding model, Haemophilia 19, 913-919, 2013.

17.  Spira, J., Plyushch,O.P., Andreeva, T.A., and Andreev, Y., Prolonged bleeding-free period following prophylatic infusion of recombinant factor VIII reconstituted with pegylated liposomes, Blood 108, 3668-3673, 2006.

18.  Yatuv, R., Robinson, M., Dayan, I., and Baru, M., Enhancement of the efficacy of therapeutic proteins by formulation with PEGylated liposomes: a case of FVIII, FVIIa, and G-CSF,  Expert Opin.Drug.Deliv. 7, 187-201, 2010.

19. Kosower, E.M., Decreased tyrosine hydroxyl acidity through polyalanylation, Proc.Natl.Acad.Sci. USA 51, 1141-1146, 1964.

20.  Kosower, E.M., The therapeutic possibilities arising from the chemical modification of proteins, Proc.Natl.Acad.Sci. USA 53, 897-901, 1965.

21.  Boyd, W.C., The proteins of immune reactions, in The Proteins, ed. H.Neurath and K.Bailey, Vol. II, Pt. B, Chapter 22, pps. 756-844, Academic Press, New York, New York,  USA, 1954.

22.  Singer, S.J., Structure and function of antigen and antibody proteins, in The Proteins, 2nd edn., ed. H.Neurath, Vol.III, Chapter 15, pps. 270-307, Academic Press, New York, New York, USA, 1965.

23.  Lundblad, R.L., Applications of Solution Protein Chemistry to Biotechnology, CRC  Press, Boca Raton, Florida, USA, 2009.

24.  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.

25.   Abuchowski, A., McCoy, J.R., Palczuk, N.C., et al., Effect of covalent modification of polyethylene glycol on immunogenicity and circulating life of bovine liver catalase, J.Biol.Chem. 252, 3582-3586, 1977.

26. Ikeda, Y. and Nagasaki, Y., PEGyation technology in nanomedicine, Adv.Polymer Sci. 247, 115-140, 2012).

27.  Fairlie-Clarke, K.J., Lamb, T.J.,Langhorne, J..,  et al., Antibody isotype anlaysis of malaria-nematode co-infection: problems and solutions associated with cross-reactivity, BMC Immunol. 11:6, 2010.

28.  Evans, T.H., Hawkins, W.L., and Hibbert, H., Studies on reactions related to carbohydrates and polysaccharides : LXIV. Antigenicity of dextran produced by Leuconostoc mesenteroides, J.Exp.Med. 74, 511-518, 1941.

29.  Kurdi, M., Cherel, G., Lenting, P.J. et al., Coagulation factor X interaction with macrophages through its N-glycans protects it from a rapid clearance, PLoS One 7(():e45111, 2012.

30.  Baynes, J.W. and Wold, F.,  Effect of glycosylation on the in vivo  circulating half-life of ribonuclease, J.Biol.Chem.251, 6016-6024, 1976.