Method for purifying recombinant FSH
The present invention relates to a method for purifying a recombinant follicle stimulating hormone (FSH) or recombinant FSH variant. The method comprises the steps of subjecting a liquid containing a recombinant FSH or recombinant FSH variant to an anion exchange chromatography, to a hydrophobic interaction chromatography, and to a dye affinity chromatography, wherein these chromatographies may be performed in any order, and wherein the method neither comprises a weak anion exchange chromatography nor a reverse phase chromatography. The method of purification results in a high yield of recombinant FSH having a desired degree of purity. The obtained FSH is especially useful for the prophylaxis and treatment of disorders and medical indications where FSH preparations are considered as useful remedies.
1. A method of purifying a recombinant follicle stimulating hormone (FSH), comprising the steps of subjecting a liquid containing said FSH to:
an anion exchange chromatography,
a hydrophobic interaction chromatography, and
a dye affinity chromatography,
which are performed in any order, wherein the method comprises neither a weak anion exchange chromatography nor a reverse phase chromatography.
2. The method of claim 1 , wherein the steps are performed in the following order:
a) an anion exchange chromatography
b) a hydrophobic interaction chromatography, and
c) a dye affinity chromatography.
3. The method of claim 1 , wherein the anion exchange chromatography is performed using a strong anion exchange resin having —N + (CH 3 ) 3 functional groups.
4. The method of claim 1 , wherein the hydrophobic interaction chromatography is performed using a resin consisting of cross-linked agarose beads derivatized with phenyl or butyl groups.
5. The method of claim 1 , wherein the dye affinity chromatography is performed with 1-amino-4-[4-[[4-chloro-6-(2-sulfoanilino)-1,3,5-triazin-2-yl]amino]-3-sulfoanilino]-9,10-dioxoanthracene-2-sulfonic acid as the ligand, covalently coupled to any matrix.
6. The method of claim 1 , wherein the chromatography is performed using a Tris-HCl/sodium chloride buffer as eluent at a pH in the range between 7.0 and 9.0.
7. The method of claim 1 , the method further comprising a cation exchange chromatography.
8. The method of claim 7 , wherein the cation exchange chromatography is performed with a strong acidic cation exchanger sulfonic acid fixed on a membrane.
9. The method of claim 7 , wherein the steps are performed in the following order:
a) anion exchange chromatography,
b) a hydrophobic interaction chromatography,
c) a dye affinity chromatography, and
d) a cation exchange chromatography.
10. The method of claim 1 , further comprising a further anion exchange chromatography.
11. The method of claim 10 , wherein the anion exchange chromatography is performed using a strong anion exchange resin having —N + (CH 3 ) 3 functional groups, or a resin having similar characteristics.
12. The method of claim 10 , wherein the anion exchange chromatography is performed using a Tris-HCl/sodium chloride buffer as eluent at a pH in the range between 7.0 and 9.0.
13. The method of claim 10 , wherein the steps are performed in the following order:
a) a first anion exchange chromatography,
b) a hydrophobic interaction chromatography,
c) a dye affinity chromatography,
d) an optional cation exchange chromatography, and
e) a second anion exchange chromatography.
14. The method of claim 1 , the method further comprising a size exclusion chromatography.
15. The method of claim 14 , wherein the size exclusion chromatography is performed using a matrix of spherical composite of cross-linked agarose and dextran.
16. The method of claim 14 , wherein the steps are performed in the following order:
a) a first anion exchange chromatography,
b) a hydrophobic interaction chromatography,
c) a dye affinity chromatography,
d) an optional cation exchange chromatography,
e) an optional second anion exchange chromatography, and
f) a size exclusion chromatography.
17. The method of claim comprising the following steps in the following order:
a) a first anion exchange chromatography,
b) a hydrophobic interaction chromatography,
c) a dye affinity chromatography,
d) a membrane cation exchange,
e) a second anion exchange chromatography, and
f) a size exclusion chromatography.
18. The method of claim 1 , further comprising one or more ultrafiltration and/or nanofiltration steps.
19. The method of claim no metal ion affinity chromatography is performed.
20. The method of claim 1 , wherein no immunoaffinity chromatography is performed.
21. The method of claim 1 , wherein the FSH has an α-subunit according to SEQ ID NO: 1 and a β-subunit according to SEQ ID NO: 2.
22. An FSH obtained by the method according to claim 21 comprising less than 1% dimers and related substances of higher molecular mass. less than 10 ppm generic host cell protein (HCP), less than 0.006 pg/IU FSH DNA, and having a purity of more than 97%.
23. A pharmaceutical composition comprising the FSH according to claim 22 as well as a pharmaceutically acceptable excipient.
24. A method for treating fertility disorders comprising a step of administering the FSH according to claim 22 to a patient.
25. A method for treating fertility disorders comprising a step of administering the pharmaceutical composition according to claim 23 to a patient.
26. A method of producing a recombinant human FSH comprising the steps of
a) generating a CHO cell clone which produces the recombinant human FSH from one or more recombinant nucleic acid molecules which code for the α-chain and the β-chain of human FSH, thereby obtaining CHO host cells,
b) cultivating of the CHO host cells under suitable conditions, thereby obtaining a cell culture containing a recombinant human FSH, and
c) purifying the recombinant human FSH from the cell culture according to the method of claim 1 .