US-12618057-B2 - Method of purifying botulinum toxin

US12618057B2US 12618057 B2US12618057 B2US 12618057B2US-12618057-B2

Abstract

Disclosed is a method of purifying a botulinum toxin including (a) pre-treating a culture solution containing a botulinum toxin, (b) purifying the pre-treated botulinum toxin using anion exchange chromatography and (c) purifying the botulinum toxin using cation exchange chromatography. The method is capable of purifying a botulinum toxin with high purity and activity using a simple process including anion exchange chromatography and cation exchange chromatography, and is thus useful for botulinum toxin production.

Inventors

Jae Young Kim
Jeong Sun NAM
Seungho KIM
Seung Kwan CHOI
Bum Jin YUN
Jin Hee Choi

Assignees

Jetema Co., Ltd.

Dates

Publication Date: 20260505
Application Date: 20200414
Priority Date: 20190415

Claims (2)

1 . A two column chromatography method of purifying a 900 kDa botulinum toxin complex, comprising: (a) pre-treating a culture solution containing a botulinum toxin; (b) preparing the pre-treated botulinum toxin from step (a) by dissolving it in 30-70 mM sodium phosphate buffer at pH 5.5-6.5 and injecting it into an anion exchange chromatography column to bind the botulinum toxin to the anion exchange chromatography medium at a pH higher than an isoelectric point (PI) of botulinum toxin, said anion exchange chromatography column comprising a column packed with a resin containing a methacrylic bead with a particle size of 65 μm comprising a quaternary ammonium (Q) functional group, wherein the ion exchange capacity of the column is 0.25±0.05 meq/mL, and the DBC (dynamic binding capacity) of the column is 149 mg/mL based on BSA, and separating the botulinum toxin, which is bound to the anion exchange chromatography medium, by elution with a 30 to 70 mM sodium phosphate buffer having a pH of 5.5 to 6.5 comprising 0.4 to 0.6M sodium chloride; and (c) preparing the botulinum toxin from step (b) by dissolving it in 15 to 25 mM sodium citrate buffer at pH 4.0 to 5.0 and injecting it into a cation exchange chromatography column to bind the botulinum toxin to the cation exchange chromatography medium, said cation exchange chromatography column comprising a column packed with a resin that contains a sulfopropyl functional group, has a 6% spherical, cross-linked agarose matrix form, and has a DBC of 55 mg/mL based on ribonuclease A and a particle size of 34 μm, and separating 900 kDa botulinum toxin complex from the solution which is bound to the cation exchange chromatography medium, by elution with a 15 to 25 mM sodium citrate buffer having a pH of 4.0 to 5.0 comprising 0.4 to 0.6M sodium chloride.
2 . The method according to claim 1 , wherein the purified botulinum toxin is botulinum toxin A having a purity of 95% or more.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This is a United States national phase under 35 USC § 371 of International Patent Application No. PCT/KR20/05041 filed Apr. 14, 2020, which in turn claims priority under 35 USC § 119 of Korean Patent Application No. 10-2019-0043868 filed Apr. 15, 2019. The disclosures of all such applications are hereby incorporated herein by reference in their respective entireties, for all purposes. BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to a method for purifying a botulinum toxin, and more specifically to a purification method capable of obtaining a botulinum toxin at high purity through a simple process of anion exchange chromatography and cation exchange chromatography. Description of the Related Art Botulinum toxin is a neurotoxic protein produced by bacteria such as Clostridium butyricum, Clostridium baratii, and Clostridium botulinum. Botulinum toxin blocks neuromuscular transmission and causes neuroparalytic diseases in humans and animals. In particular, botulinum toxin type A is known to be very fatal to humans. In addition to botulinum toxin type A, seven other botulinum toxins types, namely B, Cl, D, E, F, G and H, have been identified. Each botulinum toxin type is distinguished by a corresponding type-specific antibody, and there is a difference in the severity of the paralysis caused thereby and the animal species affected thereby. The molecular weight of the botulinum toxin protein molecule is about 150 kDa, including a light chain of about 50 kDa and a heavy chain of about 100 kDa conjugated thereto. However, botulinum toxin released from Clostridium bacteria is released in the form of a complex of a 150 kDa toxin protein with at least one non-toxin protein. For example, botulinum toxin is released as 900 kDa, 500 kDa and 300 kDa complexes. Botulinum toxin may be very fatal to humans, but botulinum toxin has recently been developed to treat a variety of symptoms including neuromuscular disorders characterized by skeletal muscle hyperactivity. For example, Botox® is a trademark of botulinum toxin A commercially developed by Allergan, Inc., which is used to alleviate or treat blepharospasm, strabismus, cervical dystonia and glabella (facial) wrinkles, and research is underway to develop applications suitable for other serotypes and clinically utilize the serotypes. Botulinum toxins for clinical use are generally isolated from cell cultures. In this case, a variety of purification methods are used. For example, botulinum toxin is purified in a complexed form by a series of precipitation and tangential flow filtration steps. [see: for example, Schantz E. J. et al., Microbiol. Rev. 1992 March 56 (1): 80-99]. However, this method typically provided a relatively low yield of less than about 10%. Other methods used include size exclusion, ion exchange and/or affinity chromatography [see, e.g., Schmidt J. J. et al., Anal. Biochem. 1986 July; 156 (1): 213-219; Kannan K. et al., Mov. Disord. 2000; 15 (Suppl 2):20 (2000); and US Patent No. 2003/0008367]. Another method is independent synthesis of one of the heavy or light chains of botulinum toxin by recombinant means, rather than a complete and biologically active botulinum toxin protein [see e.g., Zhou L. et al., Biochemistry 1995; 34 (46): 15175-81 (1995); and Johnson S. K. et al., Protein Expr. and Purif. 2003; 32: 1-9 (2003)]. However, these methods disadvantageously require an additional step of reforming a complete and biologically active botulinum toxin protein. More recent methods involve the use of hydrophobic interaction chromatography, mixed-mode and/or ion exchange chromatography to purify botulinum toxins as complexes (see, e.g., U.S. Pat. Nos. 7,452,697 and 7,354,740). However, there is still a need in the technical art for an improved purification method for isolating complete botulinum toxins that are stable and biologically active. Accordingly, as a result of extensive efforts to develop a purification method for isolating highly pure botulinum toxins in a simplified manner, the present inventors found that a highly pure botulinum toxin can be produced using a simplified process of anion exchange chromatography and cation exchange chromatography, and in particular, a botulinum toxin can be purified to a purity of 95% or more using a Q column as an anion exchange resin and using an SP column as a cation exchange resin. Based on this finding, the present invention has been completed. PRIOR ART DOCUMENT Patent Document US Patent Laid-open No. 2003/0008367U.S. Pat. No. 7,452,697U.S. Pat. No. 7,354,740 Non-Patent Document Schantz E J, et al, Properties and use of botulinum toxin and other microbial neurotoxins in medicine, Microbiol. Rev. 1992 March 56(1):80-99Schmidt J. J., et al., Purification of type E botulinum neurotoxin by high-performance ion exchange chromatography, Anal. Biochem. 1986 July; 156(1):213-219Kannan K. et al., Methods development for the biochemical assess