Physiologically active polypeptide conjugate showing prolonged half-time index in vivo

FIELD: medicine, genetic engineering.

SUBSTANCE: invention relates to a method for preparing protein conjugates. Method involves adding immunoglobulin or physiologically active polypeptide to a single end of nonpeptide polymer having two reactive terminal groups in the presence of reducing agent. From a prepared reaction mixture the complex containing nonpeptide polymer bound with immunoglobulin or physiologically active polypeptide is isolated. Then free reactive terminal group of nonpeptide polymer of the complex is added covalently to immunoglobulin or physiologically active polypeptide in the presence of reducing agent to obtain a protein conjugate. Protein conjugate containing physiologically active polypeptide bound, nonpeptide polymer and immunoglobulin that are added one to another covalently in indicated order is isolated. Invention provides preparing a protein conjugate useful for manufacturing a polypeptide drug owing to its enhanced stability in vivo, prolonged blood half-time index and decreased immunogenic properties.

EFFECT: valuable medicinal properties of conjugate, improved preparing method.

21 cl, 16 dwg, 14 tbl, 12 ex

 

The text descriptions are given in facsimile form.

1. Protein conjugate containing

i) a physiologically active polypeptide;

ii) ones polymer selected from the group consisting of poly(ethylene glycol), poly(propylene glycol), copolymers of ethylene glycol-propylene glycol, polyoxyethylene polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinylalcohol ether, poly(lactic-glycolic) acid, a biodegradable polymer, lipid is alimera, chitin and hyaluronic acid; and

iii) an immunoglobulin,

which are covalently linked to each other in the specified order,

and with a prolonged half-life in vivo physiologically active polypeptide.

2. The protein conjugate according to claim 1, ones where the polymer contains at both ends of the two reactive groups through which the polymer is covalently bonded to a physiologically active polypeptide and the immunoglobulin.

3. The protein conjugate according to claim 2, where the immunoglobulin is covalently bonded to at least two physiologically active polypeptide with polymer ones.

4. The protein conjugate according to claim 1, where the immunoglobulin is selected from the group consisting of IgG, IgA, IgD, IgE, IgM, and mixtures thereof.

5. The protein conjugate according to claim 4, in which the immunoglobulin is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, and mixtures thereof.

6. The protein conjugate according to claim 4, where the immunoglobulin is a human immunoglobulin.

7. The protein conjugate according to claim 1, where the immunoglobulin is selected from the group consisting of immunoglobulin with glycosylation of wild-type immunoglobulin with high or low degree of glycosylation applicationvantage immunoglobulin and their combinations.

8. The protein conjugate according to claim 7, where the increase or decrease in the degree of glycosylation or glycosylamine immunoglobulin the and carry out the method, selected from the group consisting of a chemical method, enzymatic method, biotechnological methods or their combinations.

9. The protein conjugate according to claim 2, where the reactive group ones polymer selected from the group consisting of aldehyde, propionic aldehyde, butyric aldehyde, maleimide and derived succinimide.

10. The protein conjugate according to claim 9, where the derived succinimide is succinimidylester, succinimidylester, hydroxysuccinimidyl or succinimidylester.

11. The protein conjugate according to claim 9, ones where the polymer contains at both ends of the aldehyde group.

12. The protein conjugate according to claim 1, ones where the polymer is covalently bonded at the ends with end amino group, a lysine residue, a histidine residue or a cysteine residue of the immunoglobulin and end amino group, a lysine residue, a histidine residue or a cysteine residue physiologically active polypeptide, respectively.

13. The protein conjugate according to claim 1, ones where the polymer is a polyethylene glycol.

14. The protein conjugate according to claim 1, where the physiologically active polypeptide is selected from the group consisting of a hormone, cytokine, enzyme, antibody, growth factor, factor regulation of transcription factor blood, vaccines, structural proteins, ligand proteins and receptor.

15. alcovy conjugate on 14 where the physiologically active polypeptide is selected from the group consisting of human growth hormone, a hormone that stimulates growth hormone, a peptide that stimulates growth hormone, interferons, colony stimulating factor, interleukins, glucocerebrosidase, factor activation of macrophages, macrophage peptide, b-cell factor, T cell factor, protein a, allergic suppressor factor, a glycoprotein necrosis of cells, immunotoxin, lymphotoxin, tumor necrosis factor, factor, inhibiting tumor growth, transforming growth factor, α-1-antitrypsin, albumin, apolipoprotein-E, erythropoietin, erythropoietin hyperglycosylated, factor VII, factor VIII, factor IX, plasminogen activator, urokinase, streptokinase, a protein, C-reactive protein, renin inhibitor, collagenase inhibitor, superoxide dismutase, platelet-derived growth factor, epidermal growth factor, osteogenic growth factor, a protein that stimulates osteogenesis, calcitonin, insulin, atriopeptin, factor, inducing the growth of cartilage, protein activator of growth of connective tissue, follicle-stimulating hormone, luteinizing hormone, FSH-releasing hormone, nerve growth factor, parathyroid hormone, relaxin, secretin, somatomedin, insulin-like growth factor, adrenocortical oplogo hormone, glucagon, cholecystokinin, pancreatic polypeptide, gastrin-releasing peptide, corticotropin-releasing factor, thyroid hormone receptor, receptor antagonist, surface antigen cells, monoclonal antibodies, polyclonal antibodies, fragments of antibodies, including Fab, Fab', F(ab')2, Fd, scFv, and derived from a virus antigen used for production of vaccines.

16. The protein conjugate according to clause 15, where the physiologically active polypeptide is a growth factor human, α-interferon, β-interferon, granulocyte colony-stimulating factor or erythropoietin.

17. A method of obtaining a protein conjugate according to claim 1, including

A. covalent joining one end of the polymer ones with two reactive terminal groups or immunoglobulin or physiologically active polypeptide in the presence of a reducing agent;

b. selection from the resulting reaction mixture complex ones containing polymer associated with immunoglobulin or physiologically active polypeptide;

C. covalent joining of the free reactive end group ones polymer complex of immunoglobulin or physiologically active polypeptide in the presence of a reducing agent with the floor is rising protein conjugate; and

d. the selection of the protein conjugate containing a physiologically active polypeptide, polymer ones and immunoglobulin, which is covalently attached to one another in the specified order.

18. The method according to 17, where the molar ratio of the physiologically active polypeptide and the ones of the polymer phase (a) is selected from the range from 1:2.5 to 1:5.

19. The method according to 17, where the molar ratio of immunoglobulin and ones of the polymer phase (a) is selected from the range from 1:5 to 1:10.

20. The method according to 17, where the molar ratio of the complex obtained in stage (b), and physiologically active polypeptide or immunoglobulin at the stage (s) selected from the range from 1:1 to 1:3.

21. The method according to 17, where the reducing agent is cyanoborohydride sodium, sodium borohydride, dimethylaminoborane or pyridinoyl.



 

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EFFECT: valuable biological and medicinal properties of antibodies.

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