CN106046127A - Preparation method of diphtheria toxoid - Google Patents
Preparation method of diphtheria toxoid Download PDFInfo
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- CN106046127A CN106046127A CN201610655056.XA CN201610655056A CN106046127A CN 106046127 A CN106046127 A CN 106046127A CN 201610655056 A CN201610655056 A CN 201610655056A CN 106046127 A CN106046127 A CN 106046127A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
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Abstract
The invention discloses a preparation method of diphtheria toxoid. The method comprises the following steps: 1, taking a Corynebacteria diphtheriae culture solution, adding ammonium sulfate until the final concentration of ammonium sulfate is 20-24% (w/v), centrifuging the obtained solution, removing obtained precipitate, clarifying the obtained solution, and filtering the clarified solution; 2, adding ammonium sulfate until the final concentration of ammonium sulfate is 21-25% (w/v), centrifuging the obtained solution, and collecting obtained precipitate; 3, dissolving the precipitate, and carrying out ultrafiltration to obtain purified toxins; and 4, taking the purified toxins, adding a formaldehyde solution, and detoxifying the obtained solution to obtain the diphtheria toxoid. The diphtheria toxoid prepared through the method has the advantages of high purity, high monomer content, high content of residual amino groups, good potence and low toxicity, and the preparation method has the advantages of realization of large scale production, stable process, easy control of the operation, low cost, high production efficiency and good application prospect.
Description
Technical field
The invention belongs to field of biological product, be specifically related to a kind of method preparing diphtheria toxoid.
Background technology
Diphtheria (Diphtheria) is by a kind of Acute respiratory infectious disease caused by diphtheria corynebacterium, to generate heat, suffocated breathing,
Hoarseness, barking cough, pharynx, tonsil and surrounding tissue thereof occur that white pseudomembrane is characterized.Severe patient systemic toxicity profiles disease
Shape is obvious, can complicated myocardial inflammation and peripheral nerve paralysis.
Diphtheria toxin, diphtherotoxin by diphtheria corynebacterium secreting, expressing, molecular weight about 58KD, comprise two fragments of A, B, B fragment be responsible for
Adhering to and penetration cell, A fragment has ADP ribosyltransferase activity, can terminate albumen synthesis and cause cell death.By diphtheria
Toxin is refined, detoxification carries out prophylactic immunization after being prepared as diphtheria toxoid list Seedling or multiple vaccines, can effectively prevent the quick-fried of diphtheria
Send out popular.According to vaccine adverse reaction monitoring, white broken (DT) bigeminy, adsorb acellular whooping cough (DTaP) triple vaccine and still deposit
In certain side reaction.Based on DTaP, with b type Haemophilus influenzae vaccine (Hib), poliomyelitis vaccine (IPV) etc.
Single Seedling associating, exploitation can reduce frequency injection, the multiple vaccines of tool high added value be current bacterial vaccine study hotspot and
Development trend.Along with the research and development of multiple vaccines, the quality of composition each to multiple vaccines is had higher requirement, diphtheria toxoid
The quality of stock solution also needs more to pay close attention to.
Toxin purity is the highest, and impurity content is the lowest, advantageously reduces side reaction.The purifying technique of current diphtheria toxin, diphtherotoxin is all adopted
The method purified with ammonium sulfate precipitation, the method is simple to operate, and flux is high, low cost.
Summary of the invention
This patent achieves diphtheria toxin, diphtherotoxin polishing purification by ammonium sulfate analysis method, but the precise controlling of technical process makes
The toxoid purity that must prepare is high, content of monomer amino content high, remaining is high, and this technique energy large-scale production, process stabilizing,
Application prospect is good.
The invention provides the preparation method of a kind of diphtheria toxoid and diphtheria toxoid that the method prepares.
The preparation method of diphtheria toxoid of the present invention, it comprises the steps:
(1) take diphtheria corynebacterium culture fluid, final concentration of 20～24% (w/v) of addition ammonium sulfate to ammonium sulfate, be centrifuged, go
Except precipitation, clarification filtration；
(2) addition ammonium sulfate is to final concentration of 21～25% (w/v) of ammonium sulfate, is centrifuged, and collects precipitation；
(3) by ultrafiltration after resolution of precipitate, it is refined toxin；
(4) refined toxin is taken, addition formalin, detoxification,.
In step (1), the cotton-shaped unit of diphtheria toxin, diphtherotoxin of described diphtheria corynebacterium culture fluid is 160～200Lf/ml.
In step (1), described centrifugal rotating speed is 13000～17000 revs/min, preferably 15000 revs/min.
In step (1), centrifugal employing continuous flow centrifuge is centrifuged；And/or, clarification filtration uses microporous membrane filter cartridge to enter
Row clarification filtration.
In step (2), adding before ammonium sulfate and first carry out ultrafiltration, described ultrafiltration is ultrafiltration to the 1/6-1/10 of original volume,
Ultrafiltration uses membrane aperture to be the ultrafilter membrane of 10kD.
In step (2), described centrifugal condition is 4000～6000 revs/min, and the centrifugal time is 40～60 minutes.
In step (3), ultrafiltration to remaining ammonium sulfate concentrations is less than one thousandth；Ultrafiltration uses membrane aperture to be 10kD ultrafiltration
Film.
In step (4), final concentration of 0.4～0.5% (v/v) of addition formalin to formaldehyde, the temperature of detoxification is
36.5～38.5 DEG C, the time of detoxification is 30～45 days.
In step (4), being first diluted refined toxin before adding formalin, the multiple of dilution is 1.5～1.7 times,
It is subsequently adding lysine, final concentration of the 0.15 of lysine～0.25% (w/v)；
Present invention also offers the diphtheria toxoid that preceding method prepares.
To sum up, the diphtheria toxoid that the inventive method prepares is superior in quality, purity at more than 2150Lf/mgPN, and
And monomer whose content is up to 90%, remaining amino content is higher than 18mol-NH/molDT, effect is good, toxicity is low.Additionally, the present invention
Preparation method energy large-scale production, process stabilizing, operate easily controllable, low cost, production efficiency is high, and application prospect is good.
Obviously, according to the foregoing of the present invention, according to ordinary technical knowledge and the customary means of this area, without departing from
Under the present invention above-mentioned basic fundamental thought premise, it is also possible to make the amendment of other various ways, replace or change.
The detailed description of the invention of form by the following examples, remakes the most specifically the foregoing of the present invention
Bright.But this should not being interpreted as, the scope of the above-mentioned theme of the present invention is only limitted to Examples below.All based on foregoing of the present invention
The technology realized belongs to the scope of the present invention.
Accompanying drawing explanation
Under 3 batches of diphtheria toxoid stock solutions 280nm of Fig. 1, HPLC analyzes peak type result, and three batches of peak figure height overlap and demonstrate,prove further
Bright product batch between concordance.
Detailed description of the invention
Bacterial strain: diphtheria PW8 strain (CMCC 38007)
Instrument and reagent:
Instrument: DHP-9272 electro-heating standing-temperature cultivator, Shanghai Yiheng Scientific Instruments Co., Ltd；1000L fermentation tank, Chengdu
Biological engineering company limited of Germany and Britain；GQLY-125N/150N continuous flow centrifuge, Liaoyang sunlight pharmaceutical machine company limited；
FLTRON ultrafilter, Amada Co., Ltd.；SASO11G23J microporous membrane filter cartridge, quite that filter (Beijing) company limited；
Ultrafilter membrane, quite that filter (Beijing) company limited.
Reagent: formalin, Guangdong Guanghua Science and Technology Co., Ltd.；Ammonium sulfate, Chengdu Ke Long chemical reagent factory.
The method that embodiment 1 present invention prepares diphtheria toxoid
One, preparation method
1. diphtheria is cultivated
Pass on through culture medium after the diphtheria corynebacterium breakdown of assay approval, after cultivate, to obtaining the cotton-shaped list of diphtheria toxin, diphtherotoxin
Position reaches the culture fluid of 160Lf/ml,.
2. one section of collection supernatant of saltouing
Adding ammonium sulfate to concentration in culture fluid is 24% (w/v), carries out one section and saltouts, continuous flow centrifuge 13000 turns/
Minute centrifugal segregation precipitation, then carry out clarification filtration through microporous membrane filter cartridge, collect supernatant.
3. two-stage nitration is saltoutd
Supernatant is concentrated by ultrafiltration to the 1/6 of original volume through 10KD ultrafilter membrane, and adding ammonium sulfate to concentration is 21% (w/v),
Carrying out two-stage nitration to saltout, 4000 revs/min are centrifuged 60 minutes, collect precipitation.
4. ultrafiltration desalination
Disalt precipitation after solution dissolves, with 10kD ultrafilter membrane ultrafiltration desalination to remaining ammonium sulphate content less than thousand/
One, it is refined toxin.
5. detoxification
After refined toxin dilutes 1.5 times with water for injection, add lysine solution to its final concentration of 0.15% (g/ml),
Addition formalin, to final concentration of 0.4% (ml/ml) of formaldehyde, puts 38.5 DEG C of detoxifications 30 days,.
Two, yield
After testing, preparation method of the present invention, in the case of culture fluid volume is 500L, prepares the yield of diphtheria toxoid
It is 54.7%.
The method that embodiment 2 present invention prepares diphtheria toxoid
One, preparation method
1. diphtheria toxoid is cultivated
Pass on through culture medium after the diphtheria corynebacterium breakdown of assay approval, after cultivate, to obtaining the cotton-shaped list of diphtheria toxin, diphtherotoxin
Position reaches the culture fluid of 180Lf/ml,.
2. one section of collection supernatant of saltouing
Adding ammonium sulfate to concentration in culture fluid is 20% (w/v), carries out one section and saltouts, continuous flow centrifuge 15000 turns/
Minute centrifugal segregation thalline, then carry out clarification filtration through microporous membrane cartridge filter, collect supernatant.
3. two-stage nitration is saltoutd
Supernatant is concentrated by ultrafiltration to the 1/8 of original volume through 10KD ultrafilter membrane, and adding ammonium sulfate to concentration is 23% (w/v),
Carrying out salt two-stage nitration analysis, 5000 revs/min are centrifuged 50 minutes, collect precipitation.
4. ultrafiltration desalination
Ultrafiltration removes ammonium sulfate with embodiment 1.
5. detoxification
After refined toxin dilutes 1.6 times with water for injection, add lysine solution to its final concentration of 0.2% (g/ml),
Addition formalin, to final concentration of 0.45% (ml/ml) of formaldehyde, puts 37 DEG C of detoxifications 40 days,.
Two, yield
After testing, preparation method of the present invention, in the case of culture fluid volume is 500L, prepares the yield of diphtheria toxoid
It is 62.1%.
The method that embodiment 3 present invention prepares diphtheria toxoid
One, preparation method
1. diphtheria toxoid is cultivated
Pass on through culture medium after the diphtheria corynebacterium breakdown of assay approval, after cultivate, to obtaining the cotton-shaped list of diphtheria toxin, diphtherotoxin
Position reaches the culture fluid of 200Lf/ml,.
2. one section of collection supernatant of saltouing
Adding ammonium sulfate to concentration in culture fluid is 22% (w/v), carries out one section and saltouts, continuous flow centrifuge 17000 turns/
Minute centrifugal segregation thalline, then carry out clarification filtration through microporous membrane cartridge filter, collect supernatant.
3. two-stage nitration is saltoutd
Supernatant is concentrated by ultrafiltration to the 1/10 of original volume through 10KD ultrafilter membrane, and adding ammonium sulfate to concentration is 25% (w/v),
Carrying out two-stage nitration to saltout, 6000 revs/min are centrifuged 40 minutes, collect precipitation.
4. ultrafiltration desalination
Ultrafiltration removes ammonium sulfate with embodiment 1.
5. detoxification
After refined toxin dilutes 1.7 times with water for injection, add lysine solution to its final concentration of 0.25% (g/ml),
Addition formalin, to final concentration of 0.5% (ml/ml) of formaldehyde, puts 36.5 DEG C of detoxifications 45 days,.
Two, yield
After testing, preparation method of the present invention, in the case of culture fluid volume is 500L, prepares the yield of diphtheria toxoid
It is 69.4%.
Quality testing:
1. by existing official method, 3 embodiment diphtheria toxoid bulk samples are examined and determine.
2.TNBS detects diphtheria toxoid residual ammonia radix amount
3.HPLC detects diphtheria toxoid content of monomer
Testing result is as shown in following table and Fig. 1:
13 embodiment diphtheria toxoid stock solution quality result statistics of table
As shown in Table 1, the three batches of diphtheria toxoid stock solution purity, content of monomer, residual ammonia base value are the highest, concordance between batch
Well.
The results show, the diphtheria toxoid stock solution quality that the inventive method is produced is high, does not easily cause inoculation secondary anti-
Should, more remaining amino explanation antigen integrity is good.Content of monomer is high, residual amino height is conducive to developing high-quality single Seedling
Or multiple vaccines.
To sum up, diphtheria toxoid prepared by the inventive method is superior in quality, and purity contains at more than 2150Lf/mgPN, monomer
Amount up to 90%, remaining amino content is higher than 18mol NH/mol TT, and preparation technology energy large-scale production, process stabilizing, behaviour
Making easily controllable, low cost, production efficiency is high, and application prospect is good.
Claims (10)
1. the preparation method of a diphtheria toxoid, it is characterised in that:
(1) taking diphtheria corynebacterium culture fluid, final concentration of 20～24% (w/v) of addition ammonium sulfate to ammonium sulfate, centrifugal, it is heavy to remove
Form sediment, clarification filtration；
(2) addition ammonium sulfate is to final concentration of 21～25% (w/v) of ammonium sulfate, is centrifuged, and collects precipitation；
(3) by ultrafiltration after resolution of precipitate, it is refined toxin；
(4) refined toxin is taken, addition formalin, detoxification,.
Preparation method the most according to claim 1, it is characterised in that: in step (1), described diphtheria corynebacterium culture fluid white
The cotton-shaped unit of larynx toxin is 160～200Lf/ml.
Preparation method the most according to claim 1, it is characterised in that: in step (1), described centrifugal rotating speed is 13000
～17000 revs/min, preferably 15000 revs/min.
Preparation method the most according to claim 1, it is characterised in that: in step (1), centrifugal use continuous flow centrifuge from
The heart；And/or, clarification filtration uses microporous membrane filter cartridge to carry out clarification filtration.
Preparation method the most according to claim 1, it is characterised in that: in step (2), first surpass before adding ammonium sulfate
Filter, described ultrafiltration is ultrafiltration to the 1/6-1/10 of original volume, and ultrafiltration uses membrane aperture to be the ultrafilter membrane of 10kD.
Preparation method the most according to claim 1, it is characterised in that: in step (2), described centrifugal condition be 4000～
6000 revs/min, the centrifugal time is 40～60 minutes.
Preparation method the most according to claim 1, it is characterised in that: in step (3), ultrafiltration is little to remaining ammonium sulfate concentrations
In one thousandth；Ultrafiltration uses membrane aperture to be 10kD ultrafilter membrane.
Preparation method the most according to claim 1, it is characterised in that: in step (4), the end of addition formalin to formaldehyde
Concentration is 0.4～0.5% (v/v), and the temperature of detoxification is 36.5～38.5 DEG C, and the time of detoxification is 30～45 days.
Preparation method the most according to claim 1, it is characterised in that: in step (4), first to refining before adding formalin
Toxin is diluted, and extension rate is 1.5～1.7, is subsequently adding lysine, final concentration of the 0.15～0.25% of lysine
(w/v)。
10. the diphtheria toxoid that prepared by method described in claim 1～9 any one.
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