WO2017043796A1

WO2017043796A1 - Method for purifying botulinum toxin

Info

Publication number: WO2017043796A1
Application number: PCT/KR2016/009597
Authority: WO
Inventors: Chang-Jin Lee; Kyeong-Yeop MOON
Original assignee: Hugel Inc.
Priority date: 2015-09-09
Filing date: 2016-08-29
Publication date: 2017-03-16
Also published as: KR20170030176A; KR101853463B1

Abstract

The present invention relates to a method of purifying botulinum toxin using a heparin-affinity column. It is expected that the method for purifying botulinum toxin according to the present invention can greatly contribute to the production of high-purity botulinum toxin.

Description

METHOD FOR PURIFYING BOTULINUM TOXIN

The present invention relates to a method for purifying botulinum toxin.

Clostridium strains which secrete neurotoxins have been found since the 1890s up to the present time, and the characteristics of toxins which are secreted by these strains have been studied over the past 70 years (Schant, E. J. et al., Microbiol. Rev. 56; 80; 1992). Among these toxins, particularly botulinum toxin is a neurotoxin which is produced by the growth of Clostridium botulinum in either can contents which were not properly sterilized or foods which were not properly stored, and the botulinum toxin is known to cause food poisoning, vomiting, visual impairment, movement disorders, etc. When this toxin is taken, it incubates for 12-72 hours, and then blocks secretion of the neurotransmitter “acetylcholine” at the area where a motor nerve meets a muscle, thereby causing muscle paralysis. Because the botulinum toxin is fatal to the human body even in small amounts and can be easily produced in large quantities, it constitutes four major bio-terror weapons together with Bacillus anthracis, Yersinia pestis, and smallpox virus. However, when botulinum toxin type A among botulinum toxin types is injected at a dose that has does not systemically affect the human body or lower, it can paralyze local muscle in the injected site. Based on this characteristic, botulinum toxin type A can be used in a wide range of applications, including wrinkle-removing agents, agents for treating spastic hemiplegia and cerebral palsy, etc., and thus is a very useful toxin.

For the reason as described above, the demand for the botulinum toxin is increasing rapidly, and studies on methods for purifying the botulinum toxin have been conducted. In the prior art, ion-exchange chromatography was used as a method for purifying the botulinum toxin, but there was a problem in that the purified active ingredient is not clearly isolated and identified, and thus contains impurities. Accordingly, in an effort to increase the purity of purified botulinum toxin, technologies were developed, including a method of treating with DNase and RNase during purification (Korean Patent No. 1,025,617), a method of preparing crystalline botulinum type A toxin by acid precipitation, extraction, nuclease addition and crystallization steps (Japanese Unexamined Patent Application Publication No. 1994-192296), a method of using ion exchange and lactose columns (US Patent No. 6,818,409), a method of using anion-exchange and cation-exchange chromatography processes (US Patent Publication No. 2013-0156756), and a method of treating with a protease inhibitor (Korean Patent No. 1,339,349). However, these technologies have problems in that it is hard to guarantee a purity of 98% or higher, and these technologies are difficult to apply commercially because the purification process is complicated and the purification period is long (about 10-14 days).

Accordingly, the present invention is directed to a method of purifying botulinum toxin using a heparin-affinity column, and it is expected that the method for purifying botulinum toxin according to the present invention can significantly contribute to the production of high-purity botulinum toxin.

The present invention has been made in order to solve the above-described problems occurring in the prior art, and it is an object of the present invention to provide a method of purifying botulinum toxin using a heparin-affinity column.

However, the technical object to be achieved by the present invention is not limited to the above technical object, and other objects that are not mentioned above can be clearly understood by those skilled in the art from the following description.

Hereinafter, various embodiments described herein will be described with reference to figures. In the following description, numerous specific details are set forth, such as specific configurations, compositions, and processes, etc., in order to provide a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or in combination with other known methods and configurations. In other instances, known processes and preparing (or manufacturing) techniques have not been described in particular detail to prevent vagueness in the present invention. Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrase "in one embodiment" or "an embodiment" in various places throughout this specification are not necessarily referring to the same embodiment of the present invention. Additionally, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

Unless otherwise specified in the specification, all the scientific and technical terms used in the specification have the same meanings as commonly understood by those skilled in the technical field to which the present invention pertains.

In one embodiment of the present invention, botulinum toxin is a neurotoxin which is produced by the growth of Clostridium botulinum in either can contents which were not properly sterilized or foods which were not properly stored, and the botulinum toxin is known to cause food poisoning, vomiting, visual impairment, movement disorders, etc. When this toxin is taken, it incubates for 12-72 hours, and then blocks secretion of the neurotransmitter “acetylcholine” at the area where a motor nerve meets a muscle, thereby causing muscle paralysis.

The botulinum toxin is classified into seven types (types A to G) according to their serological characteristics. Each of the toxins has about 150 kDa toxin protein and naturally contains a complex of several non-toxic proteins. A medium complex (300 kDa) is composed of a toxin protein and a non-toxic non-hemagglutinin protein, and a large complex (450 kDa) and a very large complex (900 kDa) are composed of the medium complex bound to hemagglutinin (Sugiyama, H., Microbiol. Rev ., 44: 419, 1980). Such non-toxic hemagglutinin proteins are known to protect the toxin from low pH and various proteases in the intestines. Furthermore, each of the toxins is cleaved at a position of 1/3 starting from the N-terminal end by the action of intracellular protease or treatment with an artificial enzyme such as trypsin into two units: a light chain (L; molecular weight: 50 kDa) and a heavy chain (H; molecular weight: 100 kDa). The cleaved toxin has greatly increased toxicity compared to the single polypeptide. The two units are linked to each other by a disulfide bond and have different functions. The heavy chain binds to a receptor of a target cell (Park. M. K., et al., FEMS Microbiol . Lett ., 72:243, 1990) and functions to interact with a biomembrane at low pH (pH 4) to form a channel (Mantecucco, C. et al., TIBS ., 18:324, 1993), and the light chain has pharmacological activity, and thus interferes with the secretion of a neurotransmitter when introduced into cells by, for example, detergent or electroporation (Poulain, B. et al., Proc . Natl . Acad . Sci . USA., 85:4090, 1988). In addition, the toxin inhibits the exocytosis of acetylcholine at the cholinergic presynapse of a neuromuscular junction to cause asthenia. It has been considered that even treatment with a very small amount of the toxin shows toxicity, suggesting that the toxin has any enzymatic activity (Simpson, L. L. et al., Ann . Rev . Pharmaeol . Toxicol ., 26:427, 1986).

According to a recent report, the toxin has metallopeptidase activity, and its substrate is composed of synaptobrevin, syntaxin, a synaptosomal associated protein of 25 kDa (SNAP25) or the like, which are the unit proteins of an exocytosis machinery complex. Each type of toxin uses one of the above-described three proteins as its substrate, and it is known that type B, D, F and G toxins cleave synaptobrevin at a specific site, type A and E toxins cleave SNAP25 at a specific site, and type C cleaves syntaxin at a specific site (Binz, T. et al., J. Biol . Chem ., 265:9153, 1994). Particularly, type A botulinum toxin is known to be soluble in a dilute aqueous solution at a pH of 4.0-6.8. It is known that the neurotoxin is isolated as a stable non-toxic protein at a pH of about 7 or higher, and as a result, the toxicity is gradually lost. Particularly, it is known that the toxicity decreases as pH and temperature increase.

In one embodiment of the present invention, heparin is a kind of acidic polysaccharide having sulfate groups, which is a strong inhibitor of blood coagulation. Heparin was discovered in 1922 and is found in the capillary-rich organs (such as livers or lungs) and blood of vertebrates. Heparin has a molecular weight of about 10,000-20,000. Heparin is produced from mast cells around capillaries, binds to proteins in tissue to produce mucoproteins, and is obtained by removing proteins with alkali, enzyme or the like. Chemically, heparin consists of alternating chains of D-glucosamine and D-glucuronic acid linked together by α-1,4 bonds. It is thought that the N and 6-position of glucosamine are sulfated and the 2-position of glucuronic acid is alternately sulfated. Heparin inhibits thromboplastin production and thrombin activity to inhibit blood coagulation, and thus is used to prevent blood coagulation and thrombosis in medical practice recently. In addition, heparin has the property of binding to hemagglutinin. Hemagglutinin, also known as phytoagglutinin or lectin, is a toxic substance that naturally found in many plants belonging to the family Fabaceae, and is known to coagulate erythrocytes.

In one embodiment of the present invention, purification means an operation of removing impurities from any substance to increase purity. For purification, various methods may be used, including a precipitation or fractional precipitation method based on a difference in solubility, a distillation or fractional distillation method based on vapor pressure, distribution, adsorption, ion-exchange chromatography, electrophoresis, gel filtration, etc. As used herein, the term “purification” means isolating botulinum toxin, produced by the death of Clostridium botulinum after overgrowth, from a culture of the Clostridium botulinum. In the present invention, a heparin-affinity column may be used to increase the botulinum toxin purity in a purification process. Heparin has the property of binding to hemagglutinin (Pethe K., et al., J Biol Chem. 2000 May 12; 275(19):14273-80.), and a protein complex having botulinum toxin bound thereto contains hemagglutinin, and thus it is expected that high-purity botulinum toxin can be purified using the heparin-affinity column. In the present invention, the heparin-affinity chromatography is conducted using a heparin-Sepharose column, but is not limited thereto.

In accordance with one embodiment of the present invention, there is provided a method for purifying botulinum toxin, comprising the steps of: (a) culturing Clostridium botulinum in a culture medium to produce botulinum toxin; (b) precipitating the botulinum toxin in the culture having the botulinum toxin produced therein; (c) adding a buffer to the precipitate, followed by removal of nucleic acid; (d) purifying the botulinum toxin by anion-exchange chromatography; and (e) isolating the botulinum toxin by heparin-affinity chromatography. In the method for purifying botulinum toxin, the purified botulinum toxin may be a botulinum toxin type A protein having at least 98% purity. The medium in step (a) may contain casein digest, yeast extract and glucose. The precipitating in step (b) may be carried out with an acid precipitation, and the acid precipitation may be performed using sulfuric acid. The removal of the nucleic acid in step (c) may be performed using protamine sulfate, and the protamine sulfate may be added to a final concentration of 0.01-0.2% (w/v). The anion-exchange chromatography in step (d) may be performed using a diethylaminoethyl column. The heparin-affinity chromatography in step (e) may be performed using a heparin-Sepharose column. In addition, the method for purifying botulinum toxin according to the present invention may further comprise, after step (e), a step of fractionating the botulinum toxin according to protein size. The step of fractionating the botulinum toxin according to protein size may be performed using a Superose 6-column.

In accordance with another embodiment of the present invention, there is provided a method for preparing a botulinum toxin solution for practical use, comprising the steps of: (a) purifying botulinum toxin by the above-described method; and (b) diluting the purified botulinum toxin with phosphate buffer. Herein, the concentration of the botulinum toxin in the botulinum toxin solution for practical use may be 0.01-1 mg/ml.

Hereinafter, each step of the method according to the present invention will be described in detail.

The present invention is directed to a method of purifying botulinum toxin using a heparin-affinity column. It is expected that the method of purifying botulinum toxin according to the present invention can greatly contribute to the production of botulinum toxin with high-purity.

FIG. 1 is a schematic view showing a process for purifying botulinum toxin according to one embodiment of the present invention.

The present invention is provided a method for preparing a botulinum toxin solution for practical use, comprising the steps of: (a) purifying botulinum toxin by the above-described method; and (b) diluting the purified botulinum toxin with phosphate buffer. Herein, the concentration of the botulinum toxin in the botulinum toxin solution for practical use may be 0.01-1 mg/ml.

Hereinafter, the present invention will be described in further detail with reference to examples. It will be obvious to those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1: Culture of Clostridium botulinum

Clostridium botulinum (accession number in the Korean Centers for Disease Control and Prevention: 4-003-CBB-CO-038), stored at -80°C, was thawed and cultured in a cooked meat medium (CMM) at 37°C under anaerobic conditions for 22-26 hours to increase the number of the Clostridium botulinum bacteria. The culture with an increased number of the bacteria was further cultured in a toxin production medium (TPM; containing 2% casein digest, 0.5% yeast extract and 0.5% glucose) at 35°C under anaerobic conditions for 93-99 hours, thereby producing toxin from the Clostridium botulinum dead after overgrowth.

Example 2: Purification of Botulinum Toxin

The botulinum toxin produced by the method of Example 1 was purified.

FIG. 1 shows an overall process for purifying botulinum toxin.

2-1: Sulfuric Acid Precipitation and Toxin Extraction

To the botulinum toxin production-induced culture of Example 1, 3.7N H₂SO₄was added until a pH of 3.2-3.5 was reached, and precipitation was performed at a temperature of 2 to 8°C for 16-24 hours. The sulfuric acid precipitate solution was centrifuged to collect the precipitate, after which a solution of 1M NaCl (pH 6.0) in 200 mM phosphate buffer was added to the precipitate, and the precipitate solution was rotated for 1 hour to dissolve protein from the precipitate, followed by centrifugation to separate the supernatant. The above-described procedure was repeated three times.

2-2: Treatment with Protamine Sulfate and Ammonium Sulfate

To the supernatant separated by the method of Example 2-1, 9% protamine sulfate was added to a final concentration of 0.09%. The protamine sulfate, an extract from salmon sperm, shows a strong positive charge, and thus functions to bind to the nucleic acid of the supernatant to thereby remove nucleic acid from the Clostridium botulinum precipitate. The aqueous solution to which the protamine sulfate was added was centrifuged to separate the supernatant, and ammonium sulfate was added to the separated supernatant to a final saturation concentration of 80% or higher, followed by a precipitation reaction at a temperature of 2 to 8°C for 12-24 hours. The resulting material was centrifuged to collect the precipitate.

2-3: DEAE Column Purification

The precipitate obtained by the method of Example 2-2 was dissolved in 50 mM phosphate buffer (pH 6.0), and the solution was added to a membrane and dialyzed in 50 mM citrate buffer (2 to 8°C, pH 5.5). The dialysis was performed a total of three times (the first-step and second-step dialysis processes were performed for 2 hours or more, and the third-step dialysis process was performed for 12 hours or more). The dialyzed solution was centrifuged to separate the supernatant, and the separated supernatant was injected into fast protein liquid chromatography (FPLC) connected with a DEAE (diethylaminoethyl Sepharose, GE-Healthcare, 17-0709-01) column. To the liquid that passed through the column, ammonium sulfate was added to a final saturation concentration of 40%, and the resulting solution was precipitated for 3 days, and then centrifuged to collect the precipitate.

2-4: Heparin-Sepharose Column Purification

The precipitate obtained by the method of Example 2-3 was dissolved in 50 mM phosphate buffer (pH 6.0), and the solution was added to a membrane and dialyzed in 10 mM citrate buffer (2 to 8°C; pH 5.5). The dialysis was performed a total of three times (the first-step and second-step dialysis processes were performed for 2 hours or more, and the third-step dialysis process was performed for 12 hours or more). The dialyzed solution was centrifuged to separate the supernatant, and the separated supernatant was injected into fast protein liquid chromatography (FPLC) connected with a heparin-Sepharose (GE Healthcare, 17-0407-03) column. Protein was eluted with 0-0.3 M NaCl, and a fraction containing a botulinum toxin was separated by SDS-PAGE. Ammonium sulfate was added to the fraction to a final saturation concentration of 80%. Next, the fraction was precipitated for 12-24 hours, and then centrifuged to collect the precipitate.

2-5: Superose-6 Column Purification

The precipitate obtained by the method of Example 2-4 was dissolved in 50 mM phosphate buffer (pH 6.0) and centrifuged, and the supernatant was injected into FPLC connected with Superose-6 (GE Healthcare, 17-5172-01). The Superose-6 is a column for separation based on particle size and is a column capable of separating and purifying proteins according to their size. Protein was eluted with a solution of 500 mM NaCl in 50 mM phosphate buffer (pH 6.0), and fractions containing a botulinum toxin was identified by SDS-PAGE. The fractions were added to a membrane and dialyzed in a solution of 250 mM NaCl (2 to 8°C; pH 6.0) in 50 mM phosphate buffer for 12 hours or more. The protein solution resulting from the dialysis process was named “undiluted botulinum toxin solution”.

2-6: Dilution of Botulinum Toxin

The undiluted botulinum toxin solution obtained by the method of Example 2-5 was diluted with 50 mM phosphate buffer (pH 6.0), thereby preparing a usable concentration of a botulinum toxin solution (botulinum toxin concentration: 0.1 mg/ml).

Example 3: Analysis of Purification Efficacy

The purification efficacy was analyzed according to the following criteria.

Table 1: Purity

	Column used for purification
Control	Anion exchange column
Preparation Example 1	Anion exchange column + heparin affinity column
Preparation Example 2	Anion exchange column + heparin affinity column + protein size column

The purity of botulinum toxin in each of Preparation Examples 1 and 2 increased by 40% or more compared to that of the control.

Table 2: Biological Activity

The biological activity of botulinum toxin in each of Preparation Examples 1 and 2 was increased by 2-fold or more compared to that of the control.

Although the present invention has been described in detail with regard to certain preferred methods, other embodiments, versions and modifications are possible without departing from the scope of the present invention. For example, a wide variety of neurotoxins can be effectively used in the method of the present invention. In addition, the present invention encompasses an oral formulation for concurrently or consecutively administering two or more neurotoxins, such as two or more botulinum toxins. For example, botulinum toxin type A can be administered until a loss of clinical response or neutralizing antibodies develop, followed by administration of an oral formulation of botulinum toxin type B or E. Alternately, a combination of any two or more of the botulinum serotypes A-G can be locally administered to control the onset and duration of the desired therapeutic result. Furthermore, non-neurotoxin compounds can be administered prior to, concurrently with or subsequent to administration of the neurotoxin to obtain a proved adjunct effect such as enhanced or a more rapid onset of denervation before the neurotoxin, such as a botulinum toxin, begins to exert its therapeutic effect.

A pharmaceutical composition according to the present invention may be varied depending on various factors, including the activity of a certain compound used, the patient’s age, weight, general health, sex and diet, the time of administration, the route of administration, excretion rate, drug combination, and the severity of a certain disease to be prevented or treated. The dose of the pharmaceutical composition varies depending on the patient’s condition and weight, the severity of the disease, the form of drug, the route of administration, and the period of administration, but may be suitably selected by those skilled in the art, and may be 0.0001-50 mg/kg/day or 0.001-50 mg/kg/day. The composition may be administered once or several times a day. The dose is not intended to limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated as pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries, or suspensions.

All references, articles, patents, applications and publications set forth above are incorporated herein by reference in their entireties. Accordingly, the spirit and scope of the following claims should not be limited to the description of the preferred embodiments set forth above.

The present invention is directed to a method of purifying botulinum toxin using a heparin-affinity column, and it is expected that the method for purifying botulinum toxin according to the present invention can greatly contribute to the production of high-purity botulinum toxin.

Claims

A method for purifying botulinum toxin, comprising the steps of:

(a) culturing Clostridium botulinum in a culture medium to produce botulinum toxin;

(b) precipitating the botulinum toxin in the culture having the botulinum toxin produced therein;

(c) adding a buffer to the precipitate, followed by removal of nucleic acid;

(d) purifying the botulinum toxin by anion-exchange chromatography; and

(e) isolating the botulinum toxin protein by heparin-affinity chromatography.
The method of claim 1, wherein the purified botulinum toxin is a botulinum toxin type A protein having at least 98% purity.
The method of claim 1, wherein the culture medium in step (a) contains casein digest, yeast extract, and glucose.
The method of claim 1, wherein the precipitating in step (b) is carried out with an acid precipitation.
The method of claim 4, wherein the acid precipitation is performed using sulfuric acid.
The method of claim 1, wherein the removal of the nucleic acid in step (c) is performed using protamine sulfate.
The method of claim 6, wherein the protamine sulfate is added to a final concentration of 0.01-0.2% (v/v).
The method of claim 1, wherein the anion-exchange chromatography in step (d) is performed using a diethylaminoethyl column.
The method of claim 1, wherein the heparin-affinity chromatography in step (e) is performed using a heparin-Sepharose column.
The method of claim 1, further comprising, after step (e), a step of fractionating the botulinum toxin according to protein size.
The method of claim 10, wherein the step of fractionating the botulinum toxin according to protein size is performed using a Superose 6-column.
A method for preparing a botulinum toxin solution for practical use, comprising the steps of:

(a) purifying botulinum toxin by a method set forth in any one of claims 1 to 11; and

(b) diluting the purified botulinum toxin with phosphate buffer.
The method of claim 12, wherein a concentration of the botulinum toxin in the botulinum toxin solution for practical use is 0.01-1 mg/ml.