Method for the purification of a non-immunoglobulin protein comprising an immunoglobulin-like (ig-like) domain

• US 20070032638A1
• Filed: 08/05/2004
• Published: 02/08/2007
• Est. Priority Date: 08/07/2003
• Status: Active Grant

• Current Assignee: Merck Serono SA (Merck & Co., Inc.)
• Original Assignee: Applied Research Systems ARS Holding NV (Merck & Co., Inc.)
• Inventors: Schwartsburd, Boris, Belzer, Ilana
• Granted Patent: US 7,550,563 B2
• Time in Patent Office: Days
• US Class Current: 530/350
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 1/20 Partition-, reverse-phase or hydrophobic interaction chromatography
• CPC Class Codes: C07K 1/22 Affinity chromatography or related techniques based upon selective absorption processes
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C07K 14/5412 IL-6
• CPC Class Codes: C07K 14/7155 for interleukins [IL]
• CPC Class Codes: C07K 19/00 Hybrid peptides , i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes

Abstract

The present invention relates to a method for the purification of non-immunoglobulin proteins comprising one or more immunoglobulin-like (Ig-like) domain.

Claims

1. A method for purifying or capturing a non-immunoglobulin protein of interest having between one and ten immunoglobulin-like (Ig-like) domains from a biological fluid, comprising the steps of:

a) contacting the biological fluid containing the protein of interest with an Hydrophobic Charge Chromatography (HCIC) resin,

b) washing out the resin to remove unbound contaminants,

c) eluting the protein of interest by treating the resin with a solution having an acidic pH or with a solution comprising an organic solvent.

2. A method according to claim 1, wherein the HCIC resin used in step a) comprises a mercapto-ethyl pyridine ligand.

3. A method according to claims 1 or 2, wherein the organic solvent used in step c) is propylene glycol.

4. A method according to claim 3, wherein the concentration of propylene glycol in the solution is between about 25 and 50%.

5. A method according to claim 1, wherein step a) is carried out at acidic pH.

6. A method according to claim 5, wherein the pH used is between about 3 and 6.8.

7. A method according to claim 1, wherein the washing of step b) is carried out with a solution having an acidic pH.

8. A method according to claim 7, wherein the pH used is between about 3 and 6.8.

9. A method according to claim 1, wherein the biological fluid is selected from a cell-conditioned culture medium, cell lysate, cell extract, tissue extract, blood plasma, serum, milk, urine, ascites, cerebrospinal fluid, vegetable juice, plant extracts or a fraction derived from an earlier chromatographic separation step.

10. A method according to claim 1, wherein the protein of interest has 1 to 7 Ig-like domains.

11. A method according to claim 1, wherein the protein of interest is selected from IL-18BP, NCAM, Fibronectin type III, ICAM-1, mad CAM-1, PE CAM-1, VCAM-1, titin, cadherin, neurocan, LIFR, CNTFR, IL-1R, IL-3R, IL5R, IL-6R, IL-12R, GM-CSFR, OSMR, VEGF receptor, FGF receptor, hPDGF receptor, T cell receptor, MHC proteins, microglobulin-β

, CTLA4, B7 activation agent, neuregulin, coagulation factor XIII, NF-kB, IL6-IL6R, beta-galactosidase and superoxide dismutase or an isoform, mutein, fused protein, functional derivative or fragment thereof comprising at least one Ig-like domain.

12. A method according to claim 11, wherein the protein is IL-18 binding protein (IL-18BP).

13. A method according to claim 1 wherein the protein is IL6-IL6R chimera.

14. A method according to claim 11, wherein the protein is beta galactosidase.

15. A method according to claim 1, wherein the purification factor of the eluted protein is in the range of 11 and 94 fold.

16. A method according to claim 15, wherein the purification factor of the eluted protein is about 94 fold.

17. A method according to claim 1, wherein the concentration factor of the eluted protein is in the range of 1.5 and 3.1 fold.

18. A method according to claim 17, wherein the concentration factor of the eluted protein is about 3.1 fold.

19. A method according to claim 1, wherein the yield of the eluted protein is in the range of 73 and 98%,

20. A method according to claim 19, wherein the yield of the eluted protein is about 85%.

42. A purified protein preparation, comprising a non-immunoglobulin protein of interest having between 1 and 10 immunoglobulin-like (Ig-like) domains, purified or captured from a biological fluid by the method according to claim 1, wherein the protein of interest is selected from the group consisting of IL-18BP, NCAM, Fibronectin type III, ICAM-1, mad CAM-1, PE CAM-1, VCAM-1, titin, cadherin, neurocan, LIFR, CNTFR, IL-1R, IL-3R, IL5R, IL-6R, IL-12R, GM-CSFR, OSMR, VEGF receptor, FGF receptor, hPDGF receptor, T cell receptor, MHC proteins, microglobulin-β

, CTLA4, B7 activation agent, neuregulin, coagulation factor XIII, NF-kB, IL6-IL6R, beta-galactosidase and superoxide dismutase or an isoform, mutein, fused protein, functional derivative or fragment thereof comprising at least one Ig-like domain.

43. A protein preparation according to claim 42, wherein the protein is IL-18BP.

44. A protein preparation according to claim 42, wherein the protein is IL6-IL6R.

45. A protein preparation according to claim 42, wherein the protein is beta galactosidase.

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