CN101204577A - Process for preparing crystal composite of BTX-A - Google Patents
Process for preparing crystal composite of BTX-A Download PDFInfo
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- CN101204577A CN101204577A CNA2006101677700A CN200610167770A CN101204577A CN 101204577 A CN101204577 A CN 101204577A CN A2006101677700 A CNA2006101677700 A CN A2006101677700A CN 200610167770 A CN200610167770 A CN 200610167770A CN 101204577 A CN101204577 A CN 101204577A
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Abstract
The invention relates to method for preparing a crystal complex of A type botulinum toxin and the preparation product is biological medicine articles, which is mainly used for curing clinical adaptive diseases such as muscular tension obstacle of facial muscles, heterotropia, eyelid convulsion as well as wrinkles-removing and face- beautifying etc.The process includes the steps: cultivating toxin production, acid-precipitating clostridium botulinum of whole culture material, extracting phosphate, precipitating ammonium sulfate, treating nuclease, filtering Sephacryl-200 gel, DEAE-Sepharose, dialyzing sulfate and forming the crystal naturally, diluting and freeze-drying the toxin.The invention is characterized by less consuming time, low cost, simple operation as well as has the advantages of good product quality, high purity etc.
Description
Technical field
The present invention relates to the microbial toxin preparation method, be specifically related to a kind of preparation method of bacteriotoxin crystalline composites.
Background technology
Botulinum neurotoxin (botulinum neurotoxin, BoNT), also claim botulinum toxin (botulinum toxin), it is the anaerobism Clostridium botulinum, be a kind of extracellular toxin that bacillus botulinus (Clostridium botulinum) produces in the growth and breeding process, belong to the protide neurotoxin.A type BoNT is the strongest biotoxin of present occurring in nature known toxicity, mouse peritoneal LD
500.001 μ g/kg is only arranged.It can act on motor nerve ending muscle contact, suppresses presynaptic membrane and discharges acetylcholine, thereby cause property paralysis of flaccid muscles.
In the world to the research of BoNT, with the A type be morning, serve as many, also solve the clearlyest, be applied to clinical treatment the earliest.Nineteen forty-six, the colleague of U.S. doctor Schantz and Ta purifies in a large number and has prepared crystalloid A type BoNT (Schantz 1994 by cultivating A type bacillus botulinus first; Schantz ﹠amp; Johnson 1997).People such as Duff has carried out the improvement (Duff et al., 1957) of purification process subsequently.These work become the important foundation for preparing A type BoNT crystalline composites afterwards.At the end of the fifties, doctor Brooks finds that injection BoNT can seal the release of the neural mould tip of hyperactivity muscle acetylcholine, is temporary paralysis, and this discovery has excited the interest as potential drug research.
In the later stage sixties 20th century, U.S. ophthalmologist Scott and doctor Schantz cooperate evidence A type BoNT by monkey and help the rectification of looking side ways, and try out in stravismus patient (Scott 1980).1978, the approval that the A type BoNT of Schantz preparation has passed through FDA (Food and Drug Adminstration) (FDA) entered clinical experimental study.In successful treatment through 7000 many cases stravismus patient, and on the basis of a large amount of experimental studies and clinical practice, in December, 1989 U.S. FDA official approval A type BoNT preparation is a new drug, is used for the treatment of diseases (http://www.garlandscience.com/cbl/pdflibrary/botox_timeline.pdf) such as the stravismus that is caused by the muscle tone obstacle more than 12 years old, blepharospasm.This is the 1st microbial toxin that is approved for medicine in the world, and Scott doctor and doctor Schantz also are described as the pioneer of BoNT clinical practice.
1991, Britain ratified the A type BoNT goods of oneself, and the medicine English name is Dysport.In December, 2000 U.S. FDAs ratify to be used for the Type B BoNT of cervical region myodystonia treatment again, and the medicine English name is Mybloc or Neurobloc.Up to the present, the BoNT goods of existing 2 types, 4 kinds appear on the market (http://www.garlandscience.com/cbl/pdflibrary/botox_timeline.pdf).
China treatment starts from twentieth century nineties with A type BoNT development, and the injection A type creotoxin of in October, 1993 Lanzhou Institute of Biological Products's development and production obtains New Drug Certificate and produces code as a trial.Obtain accurate font size in February, 1997 and produce code, and progressively promoted in fields such as ophthalmology, department of neurology, rehabilitation sections.
From 1989, after A type BoNT was become the clinical treatment medicine by official approval, A type BoNT was widely used in the treatment of multiple muscular movement disorder disease.Send out property blepharospasm, Meige syndrome, inclined to one side side masticatory spasm, writers spasm, dispel facial wrinkles etc. as stravismus, facial spasm, spasm type torticollis, the special property sent out blepharospasm, spy.These diseases relate to a plurality of subjects and comprise a plurality of fields such as ophthalmology, department of neurology, orthopaedics, rehabilitation section, Gastroenterology dept., urology department and cosmetic plastic surgery, altogether kind of clinical indication surplus in the of about 50.And, deepening continuously and expanding, the possibility that the clinical indication of BoNT treatment also has continuation to increase along with research.
Dosage according to monkey test and people's botulism calculates that A type BoNT is 0.1～1 μ g to adult lethal dose, is equivalent to 3000～30000 mice median lethal dose(LD 50)s.The therapeutic dose of BoNT is nanogram (ng) level, and (about 50～100U), particularly facial area comprises the beauty treatment reduce wrinkle, only is tens of and even hundreds of/one of people's lethal dose to be generally 2～4ng.The U.S. Committee of Experts also claims, only for sucking 0.3% of lethal dose, 0.005% of oral lethal dose belongs to atomic low dose to the BoNT therapeutic dose, is the minimum medicine (Arnon 2001) of therapeutic dose in the medicine of all listings at present.A year Clinical Experience shows surplus in the of 20, and the treatment of A type BoNT preparation is safe, effective, simple and easy to do, by extensive patients and clinicist's understanding and acceptance.The in-depth of exploring along with old mechanism of action and the discovery of new role mechanism, indication is increasing, is especially more paid close attention to and favors at the reduce wrinkle beauty treatment fields.
Summary of the invention
The invention provides a kind of preparation method of new A type BoNT crystalline composites, can obtain quality by this method, purity all can meet the requirements of A type BoNT crystalline composites treatment preparation.A type BoNT crystalline composites of the present invention can prepare by the following method:
1.A poison is produced in the cultivation of type bacillus botulinus
To carry out the A type bacillus botulinus strain that adaptability is cultivated, in 1: (50～500) preferred 1: 100 ratio is inoculated in produces in the venom body culture medium, and 30～37 ℃ (preferred 37 ℃) were cultivated 96 hours.After the 0.22 μ m membrane filtration degerming of aperture, survey virulence with white mice, the specificity virulence should be 10
5LD
50More than/the ml.
2. the thick purification of toxin
(1) with the 3N sulfuric acid solution toxin producing medium pH is transferred to 3.2～4.2 preferred (3.3～3.8), precipitation appears in the room temperature standing over night.
(2) adopt siphon to remove supernatant liquid, precipitation is moved to the 500mL centrifuge tube, in 8 ℃, the centrifugal 20min of 5000 * g, remove supernatant after the balance, stay the toxin precipitation.
(3) the sterilization water washing and precipitating of usefulness 150mL, the centrifugal 20min of the same condition, collecting precipitation;
(4) 0.2MpH 6.0 phosphate buffers with same ratio have fully hanged toxin precipitation (if pH is lower than 6.0, with 1N NaOH pH value of solution being transferred to 6.0), at room temperature gentle agitation 1h; 8 ℃, the centrifugal 30min collection of 8000 * g supernatant; The residue precipitation is with repeating step 6, and the collection supernatant.
(5) twice supernatant merged, in ice bath or 4 ℃ slowly add the ultra-pure sulfuric acid ammonium down and make and reach 60% saturated, treat that the toxic substance in the solution is separated out with precipitation; 8 ℃ then, the centrifugal 20min collecting precipitation of 5000 * g.
(6), add ribonuclease (50～100 μ g/mL) and act on 3h～5h down at 30 ℃～34 ℃ with the abundant dissolution precipitation of 0.05MpH6.0 phosphate buffer of 50mL.
(7) 8 ℃, 8000 * g are centrifugal, and 30min abandons precipitation, slowly adds ammonium sulfate to supernatant and makes and reach 60% saturated, treats that the toxic substance in the solution is separated out with precipitation.
(8) 8 ℃, the centrifugal 30min collecting precipitation of 8000 * g are with 0.05MpH5.2-5.8 sodium acetate or sodium citrate buffer dissolution precipitation.
3. the essence of toxin is purified
(1) above-mentioned thick extracting toxin is centrifugal, remove insoluble substance.
(2) above-mentioned thick toxin supernatant carries out the Sephacryl-200 gel filtration, carries out eluting with 0.05M acetate buffer or the citrate buffer solution of pH 5.2-5.8, collects first sample peak;
The gel filtration gel of the present invention range of choice is: effective protein isolate molecular weight 3 * 10
5Following gel.
Preferred scope is: effective protein isolate molecular weight 2.5 * 10
5Following gel.
Most preferred gel is Sephacryl-200.
(3) with the capable anion-exchange chromatography of the sample behind the gel filtration:, collect OD with 0.05 M acetate or citrate buffer solution (pH5.2-5.8) eluting
260/ OD
280First eluting peak between 0.5-0.6;
The gel range of choice is in the anion-exchange chromatography of the present invention: anion exchange resin.
Most preferred gel is DEAE-Sepharose.
4. the crystallization and the preservation of preparation toxin
The toxin sample concentration of collecting is transferred to 6-8mg/ml, with pH6.8, the dissolving of 0.05M phosphate buffer, adding ammonium sulfate to final concentration is 0.9M, 4 ℃ leave standstill crystallization and form.
Preparation of the present invention can prepare by the following method:
1) with phosphate buffer dissolving and percrystallization toxin, measures virulence after the filtration sterilization;
2) with phosphate buffer the toxin soiutions of known virulence is diluted to debita spissitudo (10
5-10
6LD
50/ ml);
3) the suitable dilution toxin is joined in the quantitative frozen-dried protective liquid, make content of toxins in every milliliter of frozen-dried protective liquid at the scope 100-400LD that tires and require
50/ ml);
4) packing, lyophilizing
The above-mentioned toxin dilution 0.5ml that contains frozen-dried protective liquid of the quantitative packing of each cillin bottle carries out lyophilizing with the freeze-drying curve of protein product to it, and the lyophilizing finished product is aseptic, and water content is no more than 3%.
Used bacterial strain includes but not limited to that HallA-hyper, 62A, NCTC291 etc. can produce the bacterial strain of botulinum toxin type A, preferred strain 62A in the said method.
The composition of the toxin producing medium of said method is as follows:
Enzyme hydrolysis casein or peptone 1～10%
Yeast soaks powder 1～10%
Sodium thioglycolate 0.01～0.05%
Cysteine hydrochloride 0.01～0.1%
Glucose 0.1～2%
The preferred toxin producing medium composition of the present invention is as follows:
Enzyme hydrolysis casein 1～10%
Yeast soaks powder 1～10%
Sodium thioglycolate 0.01～0.05%
Cysteine hydrochloride 0.01～0.1%
Glucose 0.1～2%
The most preferred toxin producing medium composition of the present invention is as follows:
Enzyme hydrolysis casein 1～3%
Yeast soaks powder 1～3%
Sodium thioglycolate 0.01～0.025%
Cysteine hydrochloride 0.01～0.05%
Glucose 0.1～1%
Toxin producing medium of the present invention prepares by the following method:
(1) with above-mentioned culture medium except that glucose, all add the distilled water need volume, transfer pH7.2～7.4 with sodium hydroxide;
(2) 121 ℃ of sterilizations in 30 minutes are standby.
(3) join 20% glucose solution in addition in 112 ℃ of sterilizations in 30 minutes, add according to quantity before the inoculation.
Beneficial effect of the present invention:
(1) selected for use the 62A bacterial strain to produce the botulinum toxin type A crystalline composites for the first time.
(2) use sodium thioglycolate and two kinds of Reducing agents of cysteine, consume the dissolved oxygen in the culture medium, guarantee the oxygen-free environment of bacillus botulinus growth, do not need to use specific apparatus, reduce production costs.
(3) equipment of Yao Qiuing is simple, and normal-temperature operation is simple and easy to do, good reproducibility.
(4) step that later toxin crosses the DEAE-A50 ion-exchange chromatography of will dialysing changes Sephacryl-200 gel filtration and DEAE-Sepharose chromatography into, the new technology purification botulinum toxin time foreshortens to 3 days from 7 days of old technology, reduced operating procedure, reduced the degraded of toxin protein, yield improves about 5%, and the sample purity that obtains is higher.
(5) new invention technology is compared with traditional handicraft on time and yield bigger advantage, and product quality is better than traditional handicraft, as following table.
Can be used for the myodystonia disease in fields such as department of neurology, ophthalmology and department of dermatologry by the toxin preparation of method of the present invention preparation, as blepharospasm, facial spasm, stravismus and spasmodic torticollis, and the treatment of reduce wrinkle beauty treatment etc.
The main reference document
Arnon?SS，Schecter?R，Inglesby?TV，et?al.Botulinum?toxin?as?a?biological?weapon.medical?andpublic?health?management.JAMA.2001，285：1059-1070.
DuffJT，Wright?GG，Klerer?J，et?al.Studies?on?immunity?to?toxins?of?Clostridiitln?botulinum.1.Asimplified?procedure?for?isolation?of?type?A?toxin.J.Bacteriol.1957，73：42-47.
Schantz?EJ.Historical?perspective.In：Jankovic?J，Hallet?M，eds.Therapy?with?botulinum?toxin.New?York，NY：Marcel?Dekker?Inc；1994：22-26.
Schantz，E.J.and?E.A.Johnson.Botulinum?toxin：the?story?of?its?development?for?the?treatment?ofhuman?disease.Perspectives?Biol.Med.1997，40：317-327.
Scott?AB.Botulinum?toxin?injection?into?extraocular?muscles?as?an?alternative?to?strabismus?surgery.Ophthalmology.1980，87：1044-1049.
Claims (13)
1. the preparation method of an A type BoNT crystalline composites, comprise the steps: and to produce the bacterial strain process of A type BoNT by the enzyme hydrolysis casein, yeast soaks powder, sodium thioglycolate, cysteine hydrochloride, the culture medium culturing that glucose and water are formed is then by comprising that Acid precipitation, phosphate extracting, nuclease are handled, the thick purification of ammonium sulfate precipitation, the essence that comprises gel permeation chromatography, anion-exchange chromatography is purified, and obtains A type BoNT crystalline composites then after the crystallization.
2. according to the preparation method of the A type BoNT crystalline composites of claim 1, the bacterial strain that wherein produces A type BoNT is HallA-hyper, 62A or NCTC291.
3. according to the preparation method of the A type BoNT crystalline composites of claim 1 or 2, the bacterial strain of wherein used generation A type BoNT is 62A.
4. according to the described method of claim 1, wherein medium component is:
Enzyme hydrolysis casein 1～10%
Yeast soaks powder 1～10%
Sodium thioglycolate 0.01-0.05%
Cysteine hydrochloride 0.01-0.1%
Glucose 0.1～2%
All the other are water.
5. according to claim 1 or 4 described methods, wherein medium component is:
Enzyme hydrolysis casein 1～3%
Yeast soaks powder 1～3%
Sodium thioglycolate 0.01-0.025%
Cysteine hydrochloride 0.01-0.05%
Glucose 0.1～1%
All the other are water.
6. the incubation step that produces the bacterial strain of A type BoNT in the described method of claim 1 is: will carry out the A type bacillus botulinus strain that adaptability is cultivated, be inoculated in by a certain percentage and produce in the venom body culture medium, 30-37 ℃ of cultivation, use the filtering with microporous membrane degerming then, survey virulence with white mice, the specificity virulence should be 10
5LD
50More than/the ml.
7. the thick method of purification of toxin in the described method of claim 1 comprises the steps:
(1) Acid precipitation and extraction: will produce venom body medium pH with the 3N sulfuric acid solution and transfer to 3.2-4.2, the room temperature standing over night, remove supernatant, with twice of water washing and precipitating of sterilization, fully hanged the toxin precipitate with 0.2 M pH, 6.0 phosphate buffers, at room temperature gentle agitation 1-3 hour, the residue precipitation will repeat above-mentioned steps and collect supernatant, merge supernatant twice
(2) ammonium sulfate precipitation and removal nucleic acid: slowly adding ammonium sulfate to 60% is saturated in the ice bath, 4 ℃ are spent the night, collecting precipitation, the abundant dissolution precipitation of 0.05 M pH, 6.0 phosphate buffers, add ribonuclease (50-100 μ g/mL) and act on 3～5 hours down at 30 ℃～34 ℃, collect supernatant, wherein, add 60% ammonium sulfate, after precipitation is separated out, centrifugal collecting precipitation, with 0.05 M pH 5.2-5.8 sodium acetate or citric acid solution dissolution precipitation, the centrifugal precipitation of going
8. the smart method of purification of toxin in the described method of claim 1 comprises the steps:
(1) the toxin supernatant of slightly purifying carries out gel permeation chromatography;
(2) select the scope of gel to be: effective protein isolate molecular weight 3 * 10
5Following gel;
(3) 0.05 M acetate buffer or the citric acid solution with pH 5.2-5.8 carries out eluting as elution buffer, collects first sample peak;
(4) sample behind the gel filtration is carried out anion-exchange chromatography, carry out eluting, collect first sample peak with 0.05M acetate buffer or the citric acid solution of pH 5.2-5.8.
9. described according to Claim 8 method, the scope of wherein selecting gel is effective protein isolate molecular weight 2.5 * 10
5Following gel.
10. described according to Claim 8 method, the scope of wherein selecting gel is Sephacryl-200.
11. described according to Claim 8 method, wherein the used gel of anion-exchange chromatography is an anion exchange resin.
12. described according to Claim 8 method, wherein the used gel of anion-exchange chromatography is DEAE-Sepharose.
13. according to the described method of claim 1, wherein crystallization comprises the steps: with phosphate buffer the toxin sample concentration of collecting to be transferred to 6-8mg/ml, adding ammonium sulfate to final concentration is 0.9 M, and 4 ℃ leave standstill formation to be crystallized.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102985102A (en) * | 2010-05-31 | 2013-03-20 | 魅德秀专有限公社 | Non-diffusive botulinum toxin causing local muscle paralysis, and purification method thereof |
| WO2017043796A1 (en) * | 2015-09-09 | 2017-03-16 | Hugel Inc. | Method for purifying botulinum toxin |
| CN113056478A (en) * | 2018-12-21 | 2021-06-29 | 一般财团法人阪大微生物病研究会 | Method for producing botulinum toxin |
-
2006
- 2006-12-21 CN CNA2006101677700A patent/CN101204577A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102985102A (en) * | 2010-05-31 | 2013-03-20 | 魅德秀专有限公社 | Non-diffusive botulinum toxin causing local muscle paralysis, and purification method thereof |
| US20130071331A1 (en) * | 2010-05-31 | 2013-03-21 | Medexgen Incorporated | Non-diffusive botulinum toxin causing local muscle paralysis, and purification method thereof |
| CN102985102B (en) * | 2010-05-31 | 2016-06-08 | 魅德秀专有限公社 | Cause non-diffusing type Botulinum toxin and process for purification thereof that meromyarian benumbs |
| US9598683B2 (en) * | 2010-05-31 | 2017-03-21 | Medexgen Incorporated | Non-diffusive botulinum toxin causing local muscle paralysis, and purification method thereof |
| US10369235B2 (en) | 2010-05-31 | 2019-08-06 | Medexgen Incorporated | Non-diffusive botulinum toxin causing local muscle paralysis, and purification method thereof |
| WO2017043796A1 (en) * | 2015-09-09 | 2017-03-16 | Hugel Inc. | Method for purifying botulinum toxin |
| CN113056478A (en) * | 2018-12-21 | 2021-06-29 | 一般财团法人阪大微生物病研究会 | Method for producing botulinum toxin |
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Open date: 20080625 |