CN1073626C - Productive technology for A type botulism crystalline toxin and frozen-dried protective liquid needed for producing said toxin - Google Patents

Abstract

The present invention relates to a microbial toxin, and an obtained product belongs to a new biological product. The microbial toxin is used for curing the myodystonia diseases, such as blepharospasm, facial spasm, strabismus, spasmodic torticollis, etc., in the fields of departments of neurology, ophthalmology, etc. The technology of the present invention comprises the steps of preparation of a toxin producing culture medium, cultivation for producing toxins, sterilization, filtration, refining and purification of the toxins, and dilution and freeze drying of the toxins. The product prepared by the present invention has the advantages of high toxin yield, good quality, long validity period, low cost, etc.

Description

The production technique of A type meat poison crystalline toxin and produce the required frozen-dried protective liquid of this toxin

The present invention relates to microbial toxin, resulting product belongs to new biological products, is used for the myodystonia disease in fields such as neurology department, ophthalmology, as: the treatment of blepharospasm, mimic convulsion, stravismus and spasmodic torticollis etc.

Botulinum toxin type A is the extremely strong neurotoxin of a kind of virulence that produces in the A type Clostridium botulinum growth and breeding process, and it can act on the motor nerve ending myoneural junction, suppresses presynaptic membrane and discharges vagusstoff, thereby cause the flaccidity paralysis.The pharmacological action of this uniqueness, listed in clinical the beginning of the eighties by American scholar, diseases such as stravismus, blepharospasm, mimic convulsion and spasmodic torticollis are carried out therapeutic trial, and obtain satisfied curative effect.In December, 1989, U.S. FOA official approval botulinum toxin type A (trade(brand)name Oculinum) is gone into operation as new drug and is gone on the market.In February, 1989, " injection botulinus toxin " that I developed goes through to carry out clinical trial, and be new drug by Ministry of Health's approval in October, 1993, formally transfers accurate font size in February, 1997 to | and (97) defend the accurate word of medicine (orchid) S-01 number | go into operation.

The U.S. adopts at present; Clostridium botulinum is cultivated Acid precipitation entirely, and--the toxin phosphate buffered saline buffer extracts the concentrated spontaneous nucleation supervisor that reaches of--cold ethanol precipitation--ammonium sulfate and prepares A type meat poison crystalline toxin.All higher with botulinum toxin type A purity, quality that this kind method is produced, but have certain problem and limitation in technology, consumption, safety, aspect stable, be listed below:

1, toxin producing medium composition N-2Amine (Sheffied), Yeast extract (Oifco) etc. all is the dry powder finished product of single-minded manufacturer production, costs an arm and a leg.

2, cultivate the purification toxin entirely with thalline, easily cause brood cell's (belonging to two bacterioids) of laboratory environment and facility to pollute, have serious risk and harm.

3, one of step of purification botulinum toxin type A, the cold ethanol precipitation needs specific low temperature factory building and facility.The pure toxin yield rate of this technology is lower in addition, only is 17%.

4, product is made the dry toxin of protection liquid cooling freeze-drying with human serum albumin, the danger that exists the blood source to pollute, and toxin power in the freeze-drying process (being unit) is lost up to 50-90%; The poor stability of freeze-drying finished product, validity period is short.Merge it;

3, enzyme is handled and concentrated: extracting solution press 100ug/ml and is added rnase, 35 ℃ of incubations 3 hours, and it is saturated to add ammonium sulfate to 60%, and 2-8 ℃ is spent the night, centrifugal, and precipitation is dialysed after dissolving with an amount of PH5.5,0.05M citrate buffer solution;

4, chromatography: dialyzate is collected, is merged 00 through the OEAE-ASO ion exchange chromatography ₂₆₀～00 ₂₈₀Post liquid excessively for 0.5-0.6;

5, crystallization: it is saturated that amalgamation liquid adds ammonium sulfate to 60%, and 2-8 ℃ is spent the night, centrifugal, precipitation is dialysed in containing the above-mentioned damping fluid of 0.9M ammonium sulfate with an amount of PH6.8, the dissolving of 0.05M phosphate buffered saline buffer again, forms crystallization naturally, also can repeat said process, to form crystallization for the second time;

The dilution of VI, toxin, freeze-drying

1, dilution

A, with phosphate buffered saline buffer dissolving and percrystallization toxin, measure virulence after the Sterile Filtration;

B, the toxin soiutions of known virulence is diluted to proper concn (10 with phosphate buffered saline buffer ⁵-10 ⁶L0 ₅₀/ ml);

C, an amount of dilution toxin is joined in the quantitative frozen-dried protective liquid, make in every milliliter of frozen-dried protective liquid content of toxins at the scope (50-100L0 that tires and require ₅₀/ ml);

2, packing, freeze-drying

Every peace bottle is 1 milliliter of the above-mentioned toxin dilution that contains frozen-dried protective liquid of packing quantitatively, with the freeze-drying curve of protein articles it is carried out freeze-drying, and the freeze-drying finished product should be aseptic, water content is no more than 3%.

2, a kind of required frozen-dried protective liquid of A type meat poison crystalline toxin of producing is characterized in that:

1, composition

Gelatin 1%

Dextran 5%

Sucrose 5%

Distilled water adds to 100ml

2, compound method

A, weigh gelatin by amount of preparation, back high pressure (121 ℃) sterilization 30 minutes is dissolved in heating;

B, add dextran and sucrose in proportion, treat off-bottom after, filter bulb filters, BP (113 ℃) sterilization 30 minutes is stand-by.

A, Acid precipitation; Parent toxin liquid adds Sodium hexametaphosphate 99, makes ultimate density reach 0.2%, transfers about PH to 3.5 with 3 equivalent hydrochloric acid, and 2-8 ℃ is spent the night;

B, toxin extract; Abandon supernatant, precipitation adds an amount of PH6.0,0.2M phosphate buffered saline buffer with distillation washing 1 time, divides 2 dissolvings, extraction, puts room temperature 1 hour at every turn, gets supernatant liquor respectively after centrifugal and merges it;

C, enzyme are handled and are concentrated; Extracting solution is pressed 100ug/ml and is added rnase, 35 ℃ of incubations 3 hours, it is saturated to add ammonium sulfate to 60%, 2-8 ℃ is spent the night, centrifugal, precipitation with an amount of PH5.5, the dissolving of 0.05M citrate buffer solution after, dialyse;

D, chromatography: dialyzate is collected, is merged 00 through the OEAE-ASO ion exchange chromatography ₂₆₀/ 00 ₂₈₀Post liquid excessively for 0.5-0.6;

E, crystallization: it is saturated that amalgamation liquid adds ammonium sulfate to 60%, and 2-8 ℃ is spent the night.Centrifugal, precipitation is dialysed in containing the above-mentioned damping fluid of 0.9M ammonium sulfate with an amount of PH6.8, the dissolving of 0.05M phosphate buffered saline buffer again, forms crystallization naturally.Also can repeat said process, to form crystallization for the second time;

(6), the dilution of toxin, freeze-drying

(1), dilution

A, with phosphate buffered saline buffer dissolving and percrystallization toxin, measure virulence after the Sterile Filtration;

6, with phosphate buffered saline buffer the toxin soiutions of known virulence is diluted to proper concn (10 ⁵-10 ⁶L0 ₅₀/ ml);

C, an amount of dilution toxin is joined in the quantitative frozen-dried protective liquid, make in every milliliter of frozen-dried protective liquid content of toxins at the scope (50-100L0 that tires and require ₅₀/ ml);

(2), packing, freeze-drying

Every peace bottle is 1 milliliter of the above-mentioned toxin dilution that contains frozen-dried protective liquid of packing quantitatively, with the freeze-drying curve of protein articles it is carried out freeze-drying.The freeze-drying finished product should be aseptic, moisture content is no more than 3%.

Required its composition of frozen-dried protective liquid in the above-mentioned A type meat poison crystalline toxin production process;

Gelatin 1%

Dextran 5%

Sucrose 5%

Distilled water adds to 100ml

Compound method:

(1), weigh gelatin, back high pressure (121 ℃) sterilization 30 minutes is dissolved in heating by amount of preparation;

(2), in proportion add dextran and sucrose, treat off-bottom after, filter bulb filters, BP (113 ℃) sterilization 30 minutes is stand-by.

Beneficial effect of the present invention:

1, the toxin yield rate brings up to 33% by 17% in the background technology, thereby has reduced cost effectively, has kept the low price of finished product, is fit to the national conditions and the level of consumption.

2, quality contrasts the single pin of the project U.S. single needle crystal of resulting product form of the present invention, bar-shaped crystallization purity 3.0 * 10 well ⁷L0 ₅₀(± 20%)/mgpt 2.5-3.0 * 10 ⁷L0 ₅₀/ mgpt ※ 00 ₂₆₀/ 00 ₂₈₀＜0.6≤0.55

The low more toxin of this number of ※ is pure more

3, the application of frozen-dried protective liquid makes in the freeze-drying process virulence loss be reduced to 0-20%, and has prolonged the validity period (goods of the present invention are 3 years, and the U.S. is 2 years) of goods;

4, factory building, facility, the equipment of the present invention's requirement are simpler, and toxin is purified and can be carried out at normal temperatures, and simple and easy to do, good reproducibility;

5, purify with removing verticillium toxin, cultivate entirely with thalline than background technology and purify saferly, avoided the danger of Clostridium botulinum brood cell contaminate environment;

6, produce poison and cultivate available domestic raw material, self-control, both can reduce import, save foreign exchange, can suit measures to local conditions again, guarantee supply.

Claims (2)

1, a kind of production technique of A type meat poison crystalline toxin is characterized in that:

I, toxin producing medium composition

Casein trysinization liquid 50%

Yeast dialyzate 20%

Distilled water 30%

Wherein: casein trysinization liquid composition

Casein C, P 100g

Distilled water 1000ml

Pancreas enzyme powder (unit of activity is more than 1: 100) 8.8 unit of activity/lg casein

II, toxin producing medium form

(1), casein trysinization liquid is mixed with distilled water, heat 50-60 ℃,, boil the back and add the yeast dialyzate, boiled again 10 minutes, transfer PH to 7.4 again with 20% sodium hydroxide with 20% sodium hydroxide solution levelling PH7.0;

(2), boil, filter paper filtering, the upright bottle of packing, standby in 113 ℃ of sterilizations in 30 minutes;

(3), in addition join 50% glucose solution in 113 ℃ of sterilizations in 30 minutes, add during inoculation, ultimate density reaches 1%;

III, product poison are cultivated

To be inoculated in the toxin producing medium of 18.000-36.000ml through adapting to A type Clostridium botulinum (Hall strain) bacterial classification after cultivating, cultivated 5 days for 35 ℃;

IV, Sterile Filtration

After microscopy is pure culture, with no asbestos degerming plate or degerming membrane filtration, get parent toxin liquid, measure virulence with small white mouse, virulence should be 10 ⁶L0 ₅₀About/ml;

The refining purification of V, toxin

(1), Acid precipitation; Parent toxin liquid adds Sodium hexametaphosphate 99, makes ultimate density reach 0.2%, transfers about PH to 3.5 with 3 equivalent hydrochloric acid, and 2-8 ℃ is spent the night;

(2), toxin extracts; Abandon supernatant, precipitation adds an amount of PH6.0,0.2M phosphate buffered saline buffer with distillation washing 1 time, dissolves at twice, extracts, and puts room temperature 1 hour at every turn, gets supernatant liquor respectively after centrifugal and merges it;

(3), enzyme is handled and concentrate: extracting solution press 100ug/ml and is added rnase, 35 ℃ of incubations 3 hours, it is saturated to add ammonium sulfate to 60%, 2-8 ℃ is spent the night, centrifugal, precipitation is dialysed after dissolving with an amount of PH 5.5,0.05M citrate buffer solution;

(4), chromatography: dialyzate is collected, is merged 00 through the 0AEE-ASO ion exchange chromatography ₂₆₀～002 ₂₈₀Post liquid excessively for 0.5-0.6;

(5), crystallization: it is saturated that amalgamation liquid adds ammonium sulfate to 60%, and 2-8 ℃ is spent the night, centrifugal, precipitation is dialysed in containing the above-mentioned damping fluid of 0.9M ammonium sulfate with an amount of PH6.8, the dissolving of 0.05M phosphate buffered saline buffer again, forms crystallization naturally, also can repeat said process, to form crystallization for the second time;

The dilution of VI, toxin, freeze-drying

(1), dilution

A, with phosphate buffered saline buffer dissolving and percrystallization toxin, measure virulence after the Sterile Filtration;

B, the toxin soiutions of known virulence is diluted to proper concn (10 with phosphate buffered saline buffer ⁵-10 ⁶L0 ₅₀/ ml);

C, an amount of dilution toxin is joined in the quantitative frozen-dried protective liquid, make in every milliliter of frozen-dried protective liquid content of toxins at the scope (50-100L0 that tires and require ₅₀/ ml);

(2), packing, freeze-drying

Every peace bottle is 1 milliliter of the above-mentioned toxin dilution that contains frozen-dried protective liquid of packing quantitatively, with the freeze-drying curve of protein articles it is carried out freeze-drying, and the freeze-drying finished product should be aseptic, water content is no more than 3%.

2, a kind of required frozen-dried protective liquid of A type meat poison crystalline toxin of producing is characterized in that:

(1), composition

Gelatin 1%

Dextran 5%

Sucrose 5%

Distilled water adds to 100ml

(2), compound method

A, weigh gelatin by amount of preparation, back high pressure (121 ℃) sterilization 30 minutes is dissolved in heating;

B, add dextran and sucrose in proportion, treat off-bottom after, filter bulb filters, BP (113 ℃) sterilization 30 minutes is stand-by.

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