Polysaccharide-protein conjugates reversibly coupled via imine bonds
View Patent ↗Method for preparing an oxidized polysaccharide-protein composition, by (a) oxidizing a polysaccharide with an oxidizing agent to form an oxidized polysaccharide where less than 20% of the oxidized units are comprised of alpha-hydroxy aldehyde units, (b) reacting the oxidized polysaccharide with a protein to form a composition comprising an oxidized polysaccharide-protein conjugate, and (c) maintaining the oxidized polysaccharide-protein conjugate composition by placing it in an environment where the temperature is less than 8° C. The oxidized polysaccharide and the protein are conjugated via one or more imine bonds, the oxidized polysaccharide-protein composition is soluble in aqueous solvent, and the composition is capable of releasing the protein.
1. A method of preparing an oxidized polysaccharide-protein composition comprising the steps of:
(a) oxidizing a polysaccharide with an alkali metal periodate compound to form an oxidized polysaccharide where less than 20% of the oxidized units are comprised of alpha-hydroxy aldehyde units;
(b) reacting the oxidized polysaccharide with a protein in the absence of a reducing agent to form a composition comprising an oxidized polysaccharide-protein conjugate,
(c) subjecting the oxidized polysaccharide-protein conjugate to dialysis;
(d) subjecting the dialyzed polysaccharide-protein conjugate to sterile filtration to allow for administration to a human or animal;
the oxidized polysaccharide and the protein being conjugated via one or more imine bonds which are not subjected to reduction;
the oxidized polysaccharide-protein composition being soluble in aqueous solvent;
the composition being capable of releasing the protein.
2. A method according to claim 1 , wherein the polysaccharide is selected from cellulose, pectin, starch and hydroxyhydrocarbyl derivatives thereof.
3. A method according to claim 1 , wherein the polysaccharide is selected from cellulose, pectin, starch, hydroxyalkyl cellulose and hydroxyalkyl starch.
4. A method according to claim 1 , wherein the polysaccharide is hydroxyethyl starch.
5. A method according to claim 1 , wherein the degree of oxidation of the oxidized polysaccharide is from 1 to 100%.
6. A method according to claim 1 , wherein the weight average molecular weight of the polysaccharide is from 1 to 2000 kDa.
7. A method according to claim 1 , wherein the ratio of oxidized polysaccharide to protein is from 0.1:1 to 20:1.
8. A method according to claim 1 , wherein the oxidized polysaccharide is reacted with a protein in step (b) in the presence of at least one further protein such that the oxidized polysaccharide-protein composition comprises more than one protein and each protein is conjugated to the oxidized polysaccharide via one or more imine bonds.
9. A method according to claim 1 , wherein the or each protein is selected from antibodies, cytokines, enzymes, growth factors and regulatory proteins.
10. A method according to claim 1 , wherein the or each protein is selected from erythropoietin (EPO), granulocyte-colony stimulating factor (G-CSF), uricase, beta-glucocerebrosidase, alpha-galactosidase, C-1 inhibitor, streptokinase, DNAseI, alpha-1 antitrypsin, asparaginase, arginine deiminase, Factor IX, Factor VIIa, Factor VIII, Factor IIa (thrombin), anti-TNF-alpha antibody, tissue plasminogen activator, human growth hormone, superoxide dismutase, catalase, CD163 antibody, anti-VEGF, anti-thrombin antibody, anti-CD20 antibody, anti-IgG1 antibody, anti-HER2 antibody, anti-CD33 antibody, anti-IgG2a antibody, anti-EGFR antibody, histone, interferon, insulin, albumin and mixtures thereof.
11. A method according to claim 1 , wherein the oxidized polysaccharide-protein conjugate is less than 0.1 μm in size.
12. A method according to claim 1 , wherein the protein has a weight average molecular weight of greater than about 1 kDa.
13. A method according to claim 1 , wherein the oxidized polysaccharide is reacted with a protein in step (b) in the absence of a molecular crowding agent.
14. A method according to claim 1 , wherein the oxidized polysaccharide-protein composition further comprises nonconjugated protein.
15. A method of reversibly conjugating a protein to an oxidized polysaccharide comprising the steps of preparing an oxidized polysaccharide-protein composition according to claim 1 , and dissolving the oxidized polysaccharide-protein composition in a solvent.
16. A method according to claim 1 , wherein the polysaccharide has less than 15% 1,2,3-triol content.
17. A method according to claim 1 wherein the polysaccharide has less than 5% 1,2,3-triol content.
18. A method according to claim 1 wherein the polysaccharide has less than 1% 1,2,3-triol content.
19. A method according to claim 1 , wherein the ratio of oxidized polysaccharide to protein is 1:1 molar equivalents of polysaccharide relative to protein.
20. A method according to claim 1 , wherein the ratio of oxidized polysaccharide to protein is 0.1:1 molar equivalents of polysaccharide relative to protein.
21. A method according to claim 1 , wherein the ratio of oxidized polysaccharide to protein is 10:1 molar equivalents of polysaccharide relative to protein.
22. A method according to claim 1 , wherein the degree of oxidation of the oxidized polysaccharide is 100%.
23. A method according to claim 1 , wherein step (b) is carried out at a pH of 6-8.
24. A method according to claim 1 , wherein step (a) is carried out at a temperature of less than 20° C. and step (b) is carried out at a temperature of at least 5° C.