# US 10,307,468 B2 · Manufacture of di-chain recombinant Clostridium botulinum neurotoxins using Lys-C and a hydrophobic surface

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- Number: US 10,307,468 B2
- Document ID: US10307468B2
- Status: Granted
- Kind: B2
- Application number: 15/306,006
- Filed: 2015-04-29
- Published: 2018-05-03
- Granted: 2019-06-04
- Earliest claimed: 2014-04-29
- Assignees: Ipsen Bioinnovation Limited
- Applicants: Ipsen Bioinnovation Limited
- Inventors: Shilpa Palan; Sai Man Liu; Gavin Stephen Hackett
- Web page: https://patentgrep.com/patent/US10307468B2

## CPC

- A61K 38/4893: {Botulinum neurotoxin (3.4.24.69)}
- A61K 47/02: Inorganic compounds
- A61K 47/26: Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
- A61P 1/04: for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
- A61P 3/04: Anorexiants; Antiobesity agents
- A61P 7/00: Drugs for disorders of the blood or the extracellular fluid
- A61P 9/10: for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
- A61P 11/04: for throat disorders
- A61P 13/08: of the prostate
- A61P 13/10: of the bladder
- A61P 15/02: for disorders of the vagina
- A61P 17/00: Drugs for dermatological disorders
- A61P 19/08: for bone diseases, e.g. rachitism, Paget's disease
- A61P 21/00: Drugs for disorders of the muscular or neuromuscular system
- A61P 21/02: Muscle relaxants, e.g. for tetanus or cramps
- A61P 25/02: for peripheral neuropathies
- A61P 25/06: Antimigraine agents
- A61P 25/08: Antiepileptics; Anticonvulsants
- A61P 25/14: for treating abnormal movements, e.g. chorea, dyskinesia
- A61P 25/16: Anti-Parkinson drugs
- A61P 27/02: Ophthalmic agents
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 31/04: Antibacterial agents
- A61P 31/10: Antimycotics
- A61P 31/12: Antivirals
- A61P 33/00: Antiparasitic agents
- A61P 35/00: Antineoplastic agents
- C12N 9/6489: {Metalloendopeptidases (3.4.24)}
- C12P 21/06: produced by the hydrolysis of a peptide bond, e.g. hydrolysate products (preparing foodstuffs by protein hydrolysis A23J3/00)
- C12Y 304/24069: Bontoxilysin (3.4.24.69), i.e. botulinum neurotoxin
- 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
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12Y: ENZYMES
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

Methods for producing di-chain BoNT/A from single chain BoNT/A using Lys-C followed by purification using a hydrophobic surface.

## Claims

### Claim 1

A method for producing soluble di-chain BoNT/A protein, said method comprising:
a) providing a soluble single-chain BoNT/A protein;
b) contacting said BoNT/A protein with endoproteinase Lys-C (Lys-C) in solution; and
c) separating the soluble BoNT/A protein from the Lys-C by contacting the solution containing soluble BoNT/A protein and Lys-C with a hydrophobic surface, wherein the soluble BoNT/A protein binds with preference to the hydrophobic surface.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the soluble single-chain BoNT/A protein is produced (i) in a host cell, by expressing a nucleic acid encoding said single-chain BoNT/A protein in an expression system; or (ii) in an in vitro expression system.

### Claim 3 (depends on 2)

The method according to claim 2, wherein the host cell is a bacterial host cell and the expression system is a bacterial expression system.

### Claim 4 (depends on 3)

The method according to claim 3, wherein the bacterial expression system is an E. coli expression system and the host cell is an E. coli cell.

### Claim 5 (depends on 2)

The method according to claim 2, wherein said soluble single-chain BoNT/A protein is expressed in the cytoplasm of said host cell.

### Claim 6 (depends on 2)

The method according to claim 2, wherein said soluble single-chain BoNT/A protein is expressed at a level of at least 5 mg/L.

### Claim 7 (depends on 2)

The method according to claim 2, comprising lysis of the bacterial or E. coli host cell to provide a bacterial or E. coli host cell homogenate containing said soluble single-chain BoNT/A protein.

### Claim 8 (depends on 1)

The method according to claim 1, wherein the hydrophobic surface is an inert matrix to which a ligand consisting of aryl or alkyl groups is attached.

### Claim 9 (depends on 8)

The method according to claim 8, wherein the ligand is selected from the group consisting of: butyl, phenyl and octyl ligands.

### Claim 10 (depends on 9)

The method according to claim 9, wherein a high performance hydrophobic surface is used.