Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Patent
1998-05-21
2000-04-11
Carlson, Karen Cochrane
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
530383, 530815, 530812, 530813, 514 8, 514834, C12N 950, A61K 3514, A61K 3816
Patent
active
060487208
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to a process for improving the in-vivo function of a polypeptide by shielding exposed targets of said polypeptide, by immobilizing the polypeptide to a group-specific adsorbent carrying ligands manufactured by organic-chemical synthesis, activating the biocompatible polymer, conjugating the thus activated biocompatible polymer to the immobilized polypeptide, and thereafter eluting the conjugate from the adsorbent. The present invention further relates to conjugates of a polypeptide and a biocompatible polymer obtainable by the present process, and use of said conjugates as medicaments. Specifically, the polypeptide is factor VIII, the von Willebrand factor or factor IX. The invention is particularly advantageous for conjugates where the polypeptide is factor VIII with a high specific activity using monomethoxy polyalkyleneoxide (mPEG) as the biocompatible polymer.
BACKGROUND OF THE INVENTION
It is well known, that the in-vitro stability and in-vivo half-life of polypeptides can be increased by covalent attachment of biocompatible polymers (in the following referred to as conjugation or modification). Modification of the polypeptide surface also has the advantage of decreasing the immunogenicity exhibited by the polypeptide.
Pegylation, i.e. coupling of various polyethyleneglycols (PEG) to a polypeptide, is a technique widely used for increasing the in-vitro stability and in-vivo half-life of e.g. proteins. In pegylation, many techniques have been proposed over the years. Reference is here made to Zalipsky, S. et al in Poly(Ethylene Glycol) Chemistry, Biotechnical and Biomedical Applications, Plenum, N.Y. (1992), and Katre N. V., Adv. Drug Deliv. Rev., 10 91-114 (1993).
For some polypeptides, a loss of activity or function has been recognized as a consequence of this conjugation, an effect that increases by degree of modification (Inada, Y. et al, Trends in Biotechnology, 13, 86-91 (1995)). Methods have been developed for making coupling more selective, to circumvent this problem. Site-directed mutagenesis for instance, has been applied for recombinant interleukin-2 (rIL-2). A specific target can be created by insertion of a cystein (Goodson, R. J. and Katre, N. V., Bio/Technology 8, 344-346 (1990); Katre 1993, see above). Such a route is not generally applicable for therapeutic proteins, since amino acid substitution may change the original characteristics of the molecule, and is therefore controversial. Alternatively, the polymer can be directed towards glycosylated sites of a protein, e.g. factor IX as disclosed in WO 94/29370 (Enzon). This involves, however, oxidation of the carbohydrate moieties which can be achieved by reacting the glycoprotein with sodium periodate or enzymatically by galactose oxidase. These conditions are often detrimental for the molecule. In this particular case, the glycosylated sites intended for conjugation were located at a factor IX peptide sequence which is removed during proteolytic activation in-vivo. Hence, the biocompatible polymer does not influence the function of the active polypeptide.
In WO 94/13322 (Farmitalia Carlo Erba) it is shown that pegylation can be carried out without impairing the function of certain sites essential for the function of the particular protein ("first substance"). This is achieved by protecting the sites by contacting the first substance with a second substance which specifically binds to the said sites. More particularly, the pegylation is carried out by immobilizing the particular protein on a resin with ligands having specific affinity to the said protein. Second substances are for instance complementary biological molecules. Examples of couples disclosed in WO 94/13322 are antibody (first substance)--corresponding antigen (second substance); specific inhibitor (first substance)--enzyme (second substance); growth factor (first substance)--corresponding receptor (second substance), or the reverse of each of these couples.
In a process intended for pharmaceutical production it is how
REFERENCES:
patent: 4904584 (1990-02-01), Shaw
patent: 4970300 (1990-11-01), Fulton et al.
S. Zalipsky et al, Poly(Ethylene Glycol) Chemistry, Bioctechnical and Biomedical Applications, Plenum, New York (1992).
N.V. Katre, Adv. Drug Deliv. Rev., (1993) 10:91-114.
Y. Inada et al, Trends in Biotechnology, (1995) 13:86-91.
G.E. Francis et al, Stability of Protein Pharmaceuticals/Series: Pharmaceutical Biotechnology (1992) 3:235-263.
S. Dreborg et al, Crit. Rew. Therap. Drug Carr. Sys., (1990) 6(4):315-365.
M.S. Hershfield et al, New Eng. J. Med., (1987)316:589-596.
J. Gitschier et al, Nature, (1984) 312:326-330.
W.I Wood et al, Nature, (1984) 312:330-337.
J.J. Toole, Nature, (1984) 312:342-347.
R.J. Kaufman et al, J. Biol. Chem., (1988) 263(13):6352-62.
L.O. Anderson et al, Proc. Natl. Acad. Sci., (1986) 83:2979-83.
P. Lind et al, Eur. J. Biochem, (1995) 232:19-27.
J.A. Koedam et al, Doctoral Thesis, ICG Printing, Dordrecht (1986) 74-90.
E.G.D. Tuddenham et al, Br. J. Haematol., (1982) 52:259-267.
K.M. Brinkhous et al, Proc. Natl. Acad. Sci., (1985) 82:8752-8756.
R.G. DiScipio et al, Biochemistry, (1977)16:698-705.
K. Nilsson et al, Methods in Enzymology, (1984)104:56-69.
A. Garman et al, Febs Letters, (1987) 223(2):361-365.
J.C. Jansson et al, Protein Purification, Principles, High Resolution Methods and Applications, VCH Publishers (1989) 274-285.
J.J. Morgenthaler et al, Thromb. Haemostas., (1982) 47(2):124-127.
M. TeBooy et al, J. Chrom., (1990)503:103-114.
M. Mikaelsson et al, Blood, (1983)62:1006-1015.
C. Fulcher et al, Blood, (1983)61:807-811.
D. Eaton et al, Biochemistry (1986)25:505-512.
F. Rotblat et al, Biochemistry (1985)24:2494-4300.
Dalborg Johanna
kerblom Eva
Sandberg Helena
Smeds Anna-Lisa
Carlson Karen Cochrane
Pharmacia & UpJohn AB
Schnizer Holly
LandOfFree
Conjugates of a polypeptide and a biocompatible polymer does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Conjugates of a polypeptide and a biocompatible polymer, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Conjugates of a polypeptide and a biocompatible polymer will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-1175660