# US 10,308,694 B2 · ProNGF mutants and uses thereof in the production of beta-NGF

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- Number: US 10,308,694 B2
- Document ID: US10308694B2
- Status: Granted
- Kind: B2
- Application number: 14/978,407
- Filed: 2015-12-22
- Published: 2016-08-25
- Granted: 2019-06-04
- Earliest claimed: 2011-12-19
- Assignees: Wacker Chemie AG
- Applicants: Susan Lorey; Bernhard Janowski; Heiko Pultke; Daniela Kathmann; Antje Parthier; Andreas Anton
- Inventors: Susan Lorey; Bernhard Janowski; Heiko Pultke; Daniela Kathmann; Antje Parthier; Andreas Anton
- Web page: https://patentgrep.com/patent/US10308694B2

## CPC

- C07K 14/48: Nerve growth factor [NGF]
- A61P 17/02: for treating wounds, ulcers, burns, scars, keloids, or the like
- A61P 21/00: Drugs for disorders of the muscular or neuromuscular system
- A61P 25/00: Drugs for disorders of the nervous system
- A61P 25/02: for peripheral neuropathies
- A61P 25/28: for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
- A61P 27/02: Ophthalmic agents
- A61P 27/06: Antiglaucoma agents or miotics
- 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)
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The present invention relates to a proNGF mutant and to uses thereof, in particular the use of a proNGF mutant for producing human beta-NGF. The present invention discloses a method of preparing a biologically active human beta-NGF from an inactive insoluble proNGF mutant. A proNGF mutant of the invention is substituted by any amino acid but not Arg or Lys at the native protease cleavage site R1SK3R4 at least at positions R1 and K3 corresponding to positions 101 and 103 of the human wildtype proNGF sequence.

## Claims

### Claim 1

A method of preparing a biologically active human beta-NGF comprising:
providing a proNGF mutant wherein the protease cleavage site R1SK3R4 is substituted at least at positions R1 and K3 corresponding to positions 101 and 103 of the human wild type proNGF sequence (SEQ ID NO: 1) by any amino acid selected from the group consisting of a non-basic amino acid and Histidine and wherein the amino acid at position R4 corresponding to position 104 of human wild type proNGF sequence (SEQ ID NO: 1) is selected from the group consisting of Arginine or Lysine; and
cleaving the pros-sequence of the proNGF mutant by a serine protease in order to obtain active human beta-NGF, wherein the ratio of serine protease to pro NGF mutant is 1:5,000-1:20,000 (w/w), 1:10,000 (w/w), or 1:100,000 (w/w).

### Claim 2 (depends on 1)

The method of claim 1 further comprising the steps of:
a. dissolving the proNGF mutant by solubilisation of inclusion bodies in a denaturating solution;
b. transferring the proNGF mutant into a refolding solution where the denatured proNGF assumes a biologically active conformation; and
c. purifying the refolded proNGF mutant.

### Claim 3 (depends on 2)

The method of claim 2, wherein the denaturing solution comprises a solution containing (i) a chaotropic substance, (ii) a chelator, (iii) a buffer, and (iv) a reducing agent.

### Claim 4 (depends on 3)

The method of claim 3, wherein the denaturing solution comprises:
i. 1-8 M Guanidinium-HCl,
ii. 0.01-1 M Tris,
iii. 1-50 mM EDTA,
iv. 1-100 mM selected from Gluthione (GSH) or Cysteine,
v. pH 7.0-10.0.

### Claim 5 (depends on 4)

The method of claim 4, wherein the denaturing solution comprises:
i. 4-6 M Guanidinium-HCl,
ii. 0.01-1 M Tris,
iii. 1-50 mM EDTA,
iv. 1-100 mM selected from Gluthione (GSH) or Cysteine,
v. pH 7.0-10.0.

### Claim 6 (depends on 5)

The method of claim 5, wherein the denaturing solution comprises:
i. 4 M Guanidinium-HCl,
ii. 0.1 M Tris,
iii. 10 mM EDTA
iv. 5 mM GSH or Cysteine
v. pH 8.0.

### Claim 7 (depends on 2)

The method according to claim 2 wherein the refolding is carried out as a pulse renaturation.

### Claim 8 (depends on 2)

The method of claim 2, wherein the refolding solution comprises:
i. 0.5-1.0 M of a chaperone,
ii. 1-10 mM of a metal chelator,
iii. 0.1-10 mM of a redox shuffling system,
iv. pH 8.0-pH 11.0.

### Claim 9 (depends on 8)

The method of claim 8, wherein the refolding solution comprises;
i. 0.75 M Arginine,
ii. 5 mM EDTA
iii. 1 mM L-Cystine and 5 mM L-Cysteine, or 1 mM GSSG (oxidized glutathione) and 5 mM GSH (reduced glutathione),
iv. pH 9.5.

### Claim 10 (depends on 2)

The method according to claim 2, wherein the proNGF mutant is purified via mixed mode chromatography.

### Claim 11 (depends on 10)

The method according to claim 10, wherein the proNGF mutant is purified using a mixed mode material chromatography column with a synthetic affinity ligand.

### Claim 12 (depends on 11)

The method according to claim 11, wherein the chromatography column is a column with a synthetic affinity ligand chosen from the group consisting of 4-mercapto-ethyl-pyridine (MEP), Hexylamino (HEA), Phenylpropylamino (PPA), 2-Mercapto-5-benzamidazole sulfo acid (MB I), Capto MMC (GEHC), N-benzyl-N-methyl ethanolamine (GEHC)), CHT hydroxyapatide and CHT fluoroapatide.

### Claim 13 (depends on 12)

The method according to claim 12, wherein chromatography column is a column with a synthetic affinity ligand that is 4-mercapto-ethyl-pyridine (MEP).

### Claim 14 (depends on 2)

The method of claim 2 further comprising an additional step of purifying the active beta-NGF.

### Claim 15 (depends on 14)

The method of claim 14, wherein the active beta-NGF is purified by column chromatography.

### Claim 16 (depends on 15)

The method of claim 15, wherein active beta-NGF is purified using a SP Sepharose HP column.

### Claim 17

A method for preparing a biologically active human beta-NGF comprising:
providing a proNGF mutant wherein the protease cleavage site R1 SK3 R4 is substituted at least at positions R 1 and K 3 corresponding to positions 101 and 103 of the human wild type proNGF sequence (SEQ ID NO: 1) by an amino acid selected from the group consisting of a non-basic amino acid and Histidine; and
cleaving the proNGF mutant to obtain active human beta-NGF.

### Claim 18 (depends on 17)

The method of claim 17 further comprising the steps of:
dissolving the proNGF mutant wherein the protease cleavage site R1 SK3 R4 is substituted at least at positions R 1 and K 3 corresponding to positions 101 and 103 of the human wild type proNGF sequence (SEQ ID NO: 1) by an amino acid selected from the group consisting of a non-basic amino acid and Histidine by solubilization of inclusion bodies in a denaturing solution;
transferring the proNGF mutant into a refolding solution where the denatured proNGF assumes a biologically active conformation;
purifying the refolded proNGF mutant; and
cleaving the pro-sequence of the proNGF mutant to obtain the active beta-NGF.

### Claim 19 (depends on 17)

The method according to claim 17, wherein the pro-form of the proNGF mutant is cleaved by a protease.

### Claim 20 (depends on 19)

The method according to claim 19, wherein the pro-form of the proNGF mutant is cleaved by a serine protease.

### Claim 21 (depends on 20)

The method according to claim 20, wherein the pro-form of the proNGF mutant is cleaved by trypsin.

### Claim 22 (depends on 21)

The method of claim 21, wherein the ratio of trypsin to proNGF mutant is from 1:200-1:100,000 (w/w).

### Claim 23 (depends on 22)

The method of claim 22, wherein the ratio of trypsin to proNGF mutant is from 1:5,000-1:20,000 (w/w).

### Claim 24 (depends on 23)

The mothod of claim 23, wherein the ratio of trypsin to proNGF mutant is form 1:10,000 (w/w).

### Claim 25

A method for preparing a biologically active human beta-NGF comprising:
providing a proNGF mutant wherein the protease cleavage site R1 SK3 R4 is substituted at least at positions R 1 and K 3 corresponding to positions 101 and 103 of the human wild type proNGF sequence (SEQ ID NO: 1) by an amino acid selected from the group consisting of a non-basic amino acid and Histidine; and
cleaving the pro-sequence of the proNGF mutant to obtain active human beta-NGF.

### Claim 26 (depends on 25)

The method of claim 25 further comprising the steps of:
dissolving the proNGF mutant wherein the protease cleavage site R1 SK3 R4 is substituted at least at positions R 1 and K 3 corresponding to positions 101 and 103 of the human wild type proNGF sequence (SEQ ID NO: 1) by an amino acid selected from the group consisting of a non-basic amino acid and Histidine by solubilization of inclusion bodies in a denaturing solution;
transferring the proNGF mutant into a refolding solution where the denatured proNGF assumes a biologically active conformation; and
purifying the refolded proNGF mutant.