Method for purifying recombinant fsh

Patent metadata

Field	Value
Publication number	US-2016304575-A1
Application number	US-201615200714-A
Country	US
Kind code	A1
Filing date	Jul 1, 2016
Priority date	Apr 1, 2009
Publication date	Oct 20, 2016
Grant date	—

Abstract

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.

First claim

1 . A method of purifying a recombinant follicle stimulating hormone (FSH), comprising the steps of subjecting a liquid containing said FSH to:
an affinity chromatography step, an ultrafiltration/diafiltration step using a molecular weight cut-off of 10 kDa, an anion exchange chromatography, wherein the anion exchange chromatography is performed using a Q-resin, a hydrophobic interaction chromatography step, an ultrafiltration/diafiltration step, and a nanofiltration step, wherein the method neither comprises a weak anion exchange chromatography nor a reverse phase chromatography.

2 .- 27 . (canceled)

28 . The method of claim 1 , wherein the pH of the eluent of the affinity chromatography is at a pH at or about 7 to at or about 9, or between 7 and 8.

29 . The method of claim 1 , wherein no metal ion affinity chromatography is performed.

30 . 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, or a resin having similar characteristics.

31 . The method claim 1 , wherein the anion exchange chromatography is performed using a member selected from Q Sepharose XL, Q Sepharose FF and Q Sepharose HP.

32 . The method of claim 1 , wherein the anion exchange chromatography is performed using a Tris buffer.

33 . The method of claim 1 , wherein the anion exchange chromatography is performed using a buffer having a pH at or about 7.0 to at or about 9.0, or about 7.5 to at or about 8.5.

34 . The method of claim 1 , wherein the hydrophobic interaction chromatography is performed using a resin comprising a matrix selected from the group consisting of phenyl sepharose, butyl sepharose, propyl sepharose and octyl sepharose, preferably the matrix is phenyl sepharose.

35 . The method of claim 1 , wherein the hydrophobic interaction chromatography is performed using a buffer containing a salt selected from the group consisting of NaCl, (NH 4 ) 2 SO 4 and Na 2 SO 4 , preferably NaCl.

36 . The method of claim 1 , wherein the hydrophobic interaction chromatography is performed using an equilibration and washing buffer containing a higher concentration of NaCl than the elution buffer.

37 . The method of claim 1 , wherein the recombinant FSH has an α-subunit according to SEQ ID NO: 1 and a β-subunit according to SEQ ID NO: 2.

38 . The method of claim 1 , wherein the purified recombinant FSH has a purity of at least 99%.

39 . A recombinant FSH obtained by the method of claim 1 .

40 . The recombinant FSH according to claim 39 , 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 DNA/IU FSH, and having a purity of more than 97%.

41 . The recombinant FSH of claim 39 having a purity of at least 99%.

42 . The recombinant FSH of claim 39 , wherein the FSH is provided in a pharmaceutical composition comprising the recombinant FSH and a pharmaceutically acceptable excipient.

43 . Use of the recombinant FSH according to claim 39 for the treatment of fertility disorders.

44 . A method of producing a recombinant human FSH comprising producing the recombinant human FSH in a Chinese Hamster Ovary (CHO) cell clone transfected with one or more recombinant nucleic acid molecules which code for the α-chain of human FSH comprising an amino acid sequence as depicted in SEQ ID NO: 1 and the β-chain of human FSH comprising an amino acid sequence as depicted in SEQ ID NO: 2, and purifying the recombinant human FSH from the cell culture according to the method of claim 1 .

45 . The method according to claim 44 comprising the steps of
a) generating a CHO cell clone transfected with a vector or vectors comprising DNA coding for the human glycoprotein α-subunit and the β-subunit of FSH, either encoded by SEQ ID NO: 3 and 4 or by SEQ ID NO: 5 and 6 which produces the recombinant human FSH from one or more recombinant nucleic acid molecules which code for the α-chain of human FSH comprising an amino acid sequence as depicted in SEQ ID NO: 1 and the β-chain of human FSH comprising an amino acid sequence as depicted in SEQ ID NO: 2,
b) cultivating of the CHO host cells under suitable conditions, and
c) purifying the recombinant human FSH from the cell culture according to the method of any one of claims 1 to 12 .

46 . The method according to claim 44 , further comprising formulating the FSH in the form of a pharmaceutical composition together with a pharmaceutically acceptable excipient.

Assignees

• Ratiopharm Gmbh

Inventors

• Scheckermann Christian
• Eichinger Dietmar
• Arnold Stefan

Classifications

• A61P15/08

  for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis · CPC title

• A61P15/00

  Drugs for genital or sexual disorders (for disorders of sex hormones A61P5/24); Contraceptives · CPC title

• C07K1/22

  Affinity chromatography or related techniques based upon selective absorption processes · CPC title

• C07K14/59 Primary

  Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH] · CPC title

• C07K1/18 Primary

  Ion-exchange chromatography · CPC title