# US 12,767,942 B2 · Method for purification of botulinum toxin complex with improved purification yield

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- Number: US 12,767,942 B2
- Document ID: US12767942B2
- Status: Granted
- Kind: B2
- Application number: 18/291,001
- Filed: 2022-07-22
- Published: 2024-07-25
- Granted: 2026-10-20
- Earliest claimed: 2021-07-22
- Assignees: Inibio Co., Ltd.
- Applicants: Inibio Co., Ltd.
- Inventors: Chung Sei Kim; Young Jun Song
- Web page: https://patentgrep.com/patent/US12767942B2

## CPC

- C12N 9/52: derived from bacteria {or Archaea}
- C07K 1/18: Ion-exchange chromatography
- C07K 14/33: from Clostridium (G)
- C12N 1/20: Bacteria; Culture media therefor
- C12R 2001/145: Clostridium
- C12Y 304/24069: Bontoxilysin (3.4.24.69), i.e. botulinum neurotoxin
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)
- C12R: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12Y: ENZYMES

## Abstract

The present invention relates to a method for purification of botulinum toxin with improved purification purity and yield. When the method for purification of botulinum toxin of the present invention is used, botulinum toxin can be purified with a very high yield.

## Claims

### Claim 1

A method for purification of botulinum toxin complex comprising the following steps:
(a) a step of preparing a strain culture solution producing botulinum toxin;
(b) a first purification step of purifying the prepared culture solution using a mixed-mode anion exchange resin; and
(c) a second purification step of purifying the culture solution that has passed the first purification step using a mixed-mode cation exchange resin,
wherein the mixed-mode anion exchange resin and the mixed-mode cation exchange resin have mixed-mode selectivity in which hydrophobic interaction and ionic interaction are mixed.

### Claim 2 (depends on 1)

The method of claim 1, wherein the strain producing botulinum toxin is Clostridium botulinum (C. botulinum) type A.

### Claim 3 (depends on 1)

The method of claim 1, wherein step (a) comprises the following steps:
(a-1) a step of culturing botulinum bacteria in a culture medium to obtain a culture solution comprising botulinum toxin;
(a-2) a first precipitation step of reacting the medium by adding sulfuric acid;
(a-3) an enzyme treatment step of reacting the medium by adding benzonase;
(a-4) a second precipitation step of recovering supernatant of the solution that has undergone the enzyme treatment step and reacting it by adding phosphoric acid; and
(a-5) a re-dissolution step of re-dissolving the precipitate using a buffer solution.

### Claim 4 (depends on 3)

The method of claim 3, further comprising the step of removing precipitates by centrifugation after step (a-5).