Patent EA-004385-B1 (EA)
Title: Process for purification of luteinizing hormone
Inventor: PARADISI GIANFRANCO
IPC: A61K47/26
Filed: 2001-01-22
Abstract: l. A process for purification of LH from a sample comprising the use of ion exchange chromatography and reverse phase HPLC. 2. A process according to claim 1 comprising the steps of: (a) subjecting the sample to ion-exchange chromatography to produce a first eluate; (b) subjecting the first eluate to reverse phase HPLC, to produce a second eluate; and (c) subjecting the second eluate to gel permeation chromatography. 3. A process according to claim 1 or 2, wherein the ion-exchange chromatography and the reverse phase HPLC are performed twice. 4. A process according to claim 3, wherein the ion-exchange chromatography and the reverse phase HPLC are performed twice under different matrix and/or operative conditions. 5. A process according to any one of claims 1 to 4, wherein a step of ion exchange chromatography is carried out using DEAE Sepharose as solid support. 6. A process according to any one of claims 1 to 5, wherein a step of ion exchange chromatography is carried out using Q-Sepharose as solid support. 7. A process according to any one of claims 1 to 6, wherein a step of reverse phase HPLC is carried out with silica C 18 as solid support. 8. A process according to any one of claims 1 to 4, comprising the steps of: (a) eluting the sample through a DEAE Sepharose ion-exchange chromatography column; (b) eluting through a Q-sepharose ion-exchange chromatography column; and (c) eluting through a Silica C18 reverse phase HPLC column. 9. A process according to claim 8, comprising the additional step of: (d) again eluting through a Silica C 18 reverse phase HPLC column. 10. A process according to any one of claims 5 to 9, wherein elution through DEAE Sepharose ion-exchange chromatography is carried out in sodium phosphate buffer at about pH 8. 11. A process according to any one of claims 5 to 10, wherein elution through Q Sepharose ion-exchange chromatography is carried out in ammonium acetate buffer at about pH 7. 5. 12. A process according to claim 8, wherein step (c) is carried out with 2 propanol/ammonium acetate as mobile phase. 13. A process according to claim 9, wherein step (d) is carried out with 2 propanol/Tris-HCl as mobile phase. 14. A process according to any one of claims 1 to 13, wherein the LH is human LH. 15. A process according to any one of claims 1 to 14, wherein the LH is recombinant LH. 16. A process according to any one of claims 1 to 15, wherein the sample is a culture medium from CHO cells. 17. A pharmaceutical composition comprising a therapeutically effective amount of the recombinant LH as prepared by the process of claim 15 and suitable excipients thereof. 18. A pharmaceutical composition according to claim 17, wherein the excipient is sucrose. 19. A pharmaceutical composition according to claim 17 and 18 for subcutaneous administration. 20. A recombinant LH having specific bioactivity amounting from 20522 to 31229 IU/mg. 21. A recombinant LH having specific bioactivity amounting to about 25000 IU/mg. 22. A recombinant LH according to claim 20 or 21, wherein the LH is human LH.