CN105755075A - Method of preparing high-purity diphtheria toxin original liquid - Google Patents
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Abstract
The invention discloses a method of preparing high-purity diphtheria toxin original liquid, comprising eleven steps: (1), culturing viable bacteria, (2), deactivating formaldehyde, (3), separating a bacterial liquid, (4), removing bacteria and filtering, (5), ultra-filtering and concentrating, (6), ultra-filtering and dialyzing, (7), decoloring by ion exchange, (8), salting out, (9), desalting, (10), detoxifying the formaldehyde, and (11), removing bacteria and filtering.In the preparation process of the method, the technical scheme where an ion exchange unit is used to decolor articles is employed, the problems and effects that the prior art is long in preparation cycle, low in efficiency, poor in uniformity, difficult for purity improvement are overcome, and the preparation of the diphtheria toxin original liquid is imparted shortened preparation cycle, improved efficiency, improved quality uniformity and improved purity.
Description
Technical field
The preparation method that the present invention relates to a kind of diphtheria toxoid stock solution, specifically refers to the preparation method preparing a kind of high-purity diphtheria toxoid stock solution of highly purified diphtheria toxoid stock solution for high efficiency.
Background technology
Diphtheria toxoid stock solution, it it is the core material for preparing diphtheria vaccine, the preparation method of prior art diphtheria toxoid stock solution, adopt and liquid medium within is cultivated the diphtheria corynebacterium producing poison, it is purified through activated carbon decolorizing, ammonium sulfate precipitation and ultrafiltration desalting steps, diphtheria toxoid stock solution is made, below by diphtheria toxoid stock solution referred to as stock solution then through formaldehyde detoxification；In above-mentioned technique, activated carbon decolorizing process needs time of 2～3 days, and low by duration, efficiency, the concordance of activated carbon decolorizing is poor, causes that the color and luster of each batch of stock solution there are differences；Additionally, because activated carbon is difficult to remove completely from stock solution, the purity causing stock solution is difficult to improve, make that stock solution exists foreign body risk；At present, " Chinese Pharmacopoeia three " 2015 editions purity >=1500Lf/mlPN requiring diphtheria toxoid stock solution, therefore, there is manufacturing cycle length in prior art, efficiency is low, concordance is poor, purity is difficult to the problems and shortcomings that improve.
Summary of the invention
For above-mentioned prior art Problems existing with not enough, the present invention adopting viable bacteria cultivations, formalin-inactivated, bacterium solution separation, aseptic filtration, ultrafiltration concentration, ultrafiltration dialysis, ion to exchange to decolour, saltout, desalination, formaldehyde detoxification, aseptic filtration preparation technology, the technical scheme with ion exchanger, goods decoloured in preparation process, the preparation method that a kind of high-purity diphtheria toxoid stock solution is provided, it is intended to the preparation making diphtheria toxoid stock solution, reaches to shorten manufacturing cycle, improve efficiency, improve the concordance of quality, put forward highly purified purpose.
The object of the present invention is achieved like this: the preparation method of a kind of high-purity diphtheria toxoid stock solution, decolour including viable bacteria cultivation, formalin-inactivated, bacterium solution separation, aseptic filtration, ultrafiltration concentration, ultrafiltration dialysis, ion exchange, saltout, desalination, formaldehyde detoxification, aseptic filtration processing step
Described viable bacteria is cultivated as step one, adopt diphtheria corynebacterium CMCC38007 freeze-drying lactobacillus, improve the recovery of Martin's culture medium through two generations, be inoculated in improvement Lin Shi culture medium and carry out strain amplification, it is thus achieved that propagation bacterium, afterwards, described propagation bacterium is moved to and in fermentation tank, is stirred aerobic culture, speed of agitator 500 revs/min, temperature 33～36 DEG C, 40～50 hours used times, it is thus achieved that viable bacteria is starched；
Described formalin-inactivated is step 2, by the volume ratio of the 0.6% of described viable bacteria volume of slurry, adds the formalin that mass concentration is 40% and is stirred inactivation sterilization, speed of agitator 100 revs/min, 1 hour used time, it is thus achieved that inactivation slurry in fermentation tank；
Described bacterium solution is separated into step 3, with E continuous flow centrifuge, described inactivation slurry is centrifuged bacterium solution and separates, and removes major part thalline and obtains the culture fluid containing toxin；Specifically, open E continuous flow centrifuge, treat that speed reaches 10000 revs/min, stablize 2 minutes, described inactivation slurry is pumped into E continuous flow centrifuge by peristaltic pump and is centrifuged bacterium solution separation, peristaltic pump flow velocity 1.5～2 liters/min, collect effluent and obtain serosity；
Described aseptic filtration is step 4, and the residue thalline removed in described serosity with the A germ tight filter being provided with 0.22 μm of filter element obtains the degerming slurry of clarification；Specifically, described serosity is pumped into A germ tight filter, peristaltic pump flow velocity 1.5 liters/min by peristaltic pump, collect effluent and obtain degerming slurry；
Described ultrafiltration concentration is step 5, with C ultrafilter, described degerming slurry is carried out sized molecules solution separating, removes small molecule solution and obtains macromolecular solution；It is specially, described degerming slurry is moved in C ultrafilter, C ultrafilter uses the film bag of the improvement polyether sulfone matter of 30 kilodaltons, it is called for short 30kD film bag, the pump pressure pressure of the import of described 30kD film bag is 0.1MPa, the pressure of C ultrafilter refluxing opening is 0.5MPa, start, described degerming slurry is carried out sized molecules solution separating concentration, wherein small molecule solution is discharged through the outlet of C ultrafilter, macromolecular solution mixes with still unsegregated described degerming slurry in the refluxing opening return C ultrafilter of C ultrafilter, again participates in multi-cycle separation concentration；When the degerming slurry in C ultrafilter is concentrated into the 1/10～1/15 of original volume, shutting down, stop concentration, now remaining in the solution in C ultrafilter is macromolecular solution；
Described ultrafiltration dialysis is step 6, in C ultrafilter, described macromolecular solution is carried out ultrafiltration dialysis filter wash, it is thus achieved that liquid is concentrated by ultrafiltration；It is specially, phosphate buffer is injected in C ultrafilter and mix with described macromolecular solution, start, by described film bag, described macromolecular solution is carried out equal-volume dialysis filter wash, treat that filter wash is to conductance 5～10 milli Siemens/cm, during pH7.4, stop injecting phosphate buffer, shutdown, stops dialysis filter wash, it is thus achieved that liquid is concentrated by ultrafiltration；Described equal-volume dialysis filter wash is for injecting how many phosphate buffers, and how many filter wash waste liquids are discharged in the outlet of C ultrafilter, remain the original volume of described macromolecular solution in C ultrafilter；
Described ion exchange decolouring is step 7, with ion exchanger, described ultrafiltration concentration liquid is carried out dialysis and decolours, it is thus achieved that destaining solution；Specifically, described ultrafiltration concentration liquid pump is crossed ion exchanger with peristaltic pump, by ion exchange membrane, described ultrafiltration concentration liquid is decoloured；Peristaltic pump flow velocity is 500 ml/min；Obtain destaining solution；
Described saltout as step 8, with desk centrifuge, saltout thing with ammonium sulfate described destaining solution saltoutd acquisition；Specifically, move in reactor by described destaining solution, the mass ratio by 25% is slowly added to ammonium sulfate in described destaining solution, and stirs mixing, speed of agitator 100～200 revs/min, stirs 30 minutes used times, it is thus achieved that dissolved salt liquid；Afterwards, being placed in F desk centrifuge by described dissolved salt liquid and be centrifuged precipitation, F desk centrifuge rotating speed 7000 revs/min, centrifugal 15 minutes used times, remove the macromole precipitated impurities of described dissolved salt liquid, the clear liquid collecting upper strata obtains clear saline solution；Moving in reactor by described clear saline solution afterwards, the mass ratio by 20% is slowly added to ammonium sulfate in described clear saline solution, and stirs mixing, speed of agitator 100～150 revs/min, stirs 30 minutes used times, it is thus achieved that liquid of saltouing；Afterwards, described liquid of saltouing is placed in G desk centrifuge and is centrifuged liquid, G desk centrifuge rotating speed 7000 revs/min, centrifugal 15 minutes used times, the clear liquid on liquid upper strata of saltouing described in removal, collect the precipitation of lower floor, it is thus achieved that thing of saltouing；
Described desalination is step 9, uses D ultrafilter, with water for injection, described thing of saltouing is diluted and ultrafiltration desalination, it is thus achieved that the toxin thing of cotton-shaped unit 800-1500Lf/ml；Specifically, by by 100: 1 volume ratio, with the ammonium sulfate in thing of saltouing described in water for injection dissolved dilution, it is thus achieved that bath water is saltoutd thing；Afterwards, thing of being saltoutd by described bath water moves in the D ultrafilter being provided with 30kD film bag, start carries out ultrafiltration desalination, and in D ultrafilter, fill into water for injection simultaneously, when ammonium sulphate content in the waste liquid that the outlet of D ultrafilter is discharged is less than 0.01%, stop filling into water for injection, after the outlet of D ultrafilter is discharged without waste liquid, shutdown stops ultrafiltration desalination, it is thus achieved that the toxin thing of cotton-shaped unit 800-1500Lf/ml；
Described formaldehyde detoxification is step 10, with formaldehyde, described toxin thing is carried out detoxification, it is thus achieved that detoxification thing；It is specially, described toxin thing is placed in glass container, adding mass concentration by the 0.5% of described toxin thing volume ratio is the formalin of 40%, adding mass concentration by the 2% of toxin thing volume ratio again is the lysine solution of 9.125%, pH to 7.2-7.8 is adjusted in stirring, stands detoxification, dwell temperature 38～40 DEG C, stand 42 days used times, it is thus achieved that detoxification thing；
Described aseptic filtration is step 11, carries out aseptic filtration with B germ tight filter, it is thus achieved that stock solution；Specifically, be placed in glass container by described detoxification thing, add sodium chloride by the 0.85% of described detoxification thing mass ratio, pH to 6.6-7.4 is adjusted in stirring, afterwards, carries out aseptic filtration with the B germ tight filter being provided with 0.22 μm of filter element, obtain stock solution, i.e. diphtheria toxoid stock solution.
Beneficial effect
Experiment proves, the diphtheria toxoid stock solution prepared by the present invention, purity and yield rate all obtain and significantly promotes, after particularly refining the purity of stock solution all >=2300Lf/mgPN, goods purity is higher, reduce the incidence rate of the untoward reaction caused because of the foreign protein in goods, it is ensured that the safety of goods.
Above-mentioned, the present invention is adopting viable bacteria cultivation, formalin-inactivated, bacterium solution separates, aseptic filtration, it is concentrated by ultrafiltration, ultrafiltration dialysis, ion exchange decolouring, saltout, desalination, formaldehyde detoxification, the preparation technology of aseptic filtration, the technical scheme with ion exchanger, goods decoloured in preparation process, overcoming prior art, to there is manufacturing cycle long, efficiency is low, concordance is poor, purity is difficult to the problems and shortcomings improved, the preparation method of a kind of high-purity diphtheria toxoid stock solution provided, make the preparation of diphtheria toxoid stock solution, reach shortening manufacturing cycle, improve efficiency, improve the concordance of quality, put forward highly purified purpose.
Accompanying drawing explanation
Fig. 1 is the processing step block diagram of the preparation method of a kind of high-purity diphtheria toxoid stock solution of the present invention；
Fig. 2 is contrast test, the corresponding table of the lot number of the stock solution that stock solution prepared by the present invention is prepared with prior art and preparation technology；
Fig. 3 is contrast test, the sample culturing of stock solution prepared by stock solution prepared by the present invention and prior art and verification result synopsis；
Fig. 4 is contrast test, diphtheria toxoid stock solution verification result synopsis prepared by stock solution prepared by the present invention and prior art.
Embodiment explanation
Below by way of specific embodiment, the preparation method of a kind of high-purity diphtheria toxoid stock solution of the present invention is described in further detail, those skilled in the art is referred to the processing step of embodiment and prepares high-purity diphtheria toxoid stock solution goods, but should not be construed as any limitation of the invention.
Detailed description of the invention
Embodiment
The name of an article: high-purity diphtheria toxoid stock solution
Preparation
Preparation condition,
Ambient pressure a: atmospheric pressure；Ambient temperature: room temperature；
Facility: sterilizing room；
Equipment: fermentation tank, continuous flow centrifuge, desk centrifuge backman (J26XP), germ tight filter, reactor, ultrafilter, neutral boron silica glass matter container；
Consult Fig. 1, step of preparation process, be followed successively by: viable bacteria cultivates 1, formalin-inactivated 2, bacterium solution separate 3, aseptic filtration 4, be concentrated by ultrafiltration 5, ultrafiltration dialysis 6, ion exchange decolouring 7, saltout 8, desalination 9, formaldehyde detoxification 10, aseptic filtration 11；
Step one, viable bacteria cultivate 1
Adopt diphtheria corynebacterium CMCC38007 freeze-drying lactobacillus, the recovery of Martin's culture medium is improved through two generations, it is inoculated in improvement Lin Shi culture medium and carries out strain amplification, obtain propagation bacterium, afterwards, described propagation bacterium is moved to and in fermentation tank, is stirred aerobic culture, speed of agitator 500 revs/min, temperature 33～36 DEG C, 40～50 hours used times, it is thus achieved that viable bacteria is starched；
Step 2, formalin-inactivated 2
By the volume ratio of the 0.6% of described viable bacteria volume of slurry, in fermentation tank, add the formalin that mass concentration is 40% be stirred inactivation sterilization, speed of agitator 100 revs/min, 1 hour used time, it is thus achieved that inactivation slurry；
Step 3, bacterium solution separate 3
Open E continuous flow centrifuge, treat that speed reaches 10000 revs/min, stablize 2 minutes, described inactivation slurry is pumped into E continuous flow centrifuge by peristaltic pump and is centrifuged bacterium solution separation, peristaltic pump flow velocity 1.5～2 liters/min, collect effluent and obtain serosity；
Step 4, aseptic filtration 4
Described serosity is pumped into by peristaltic pump the A germ tight filter being provided with 0.22 μm of filter element, peristaltic pump flow velocity 1.5 liters/min, collects effluent and obtain degerming slurry；
Step 5, ultrafiltration concentration 5
Described degerming slurry is moved in C ultrafilter, C ultrafilter uses the film bag of the improvement polyether sulfone matter of 30 kilodaltons, it is called for short 30kD film bag, the pump pressure pressure of the import of described 30kD film bag is 0.1MPa, the pressure of C ultrafilter refluxing opening is 0.5MPa, start, described degerming slurry is carried out sized molecules solution separating concentration, wherein small molecule solution is discharged through the outlet of C ultrafilter, macromolecular solution mixes with still unsegregated described degerming slurry in the refluxing opening return C ultrafilter of C ultrafilter, again participates in multi-cycle separation concentration；When the degerming slurry in C ultrafilter is concentrated into the 1/10～1/15 of original volume, shutting down, stop concentration, now remaining in the solution in C ultrafilter is macromolecular solution；
Step 6, ultrafiltration dialysis 6
Phosphate buffer is injected in C ultrafilter and mix with described macromolecular solution, start, by described film bag, described macromolecular solution carried out equal-volume dialysis filter wash, treat filter wash to conductance 5～10 milli Siemens/cm, during pH7.4, stop injecting phosphate buffer, shutdown, stop dialysis filter wash, it is thus achieved that liquid is concentrated by ultrafiltration；Described equal-volume dialysis filter wash is for injecting how many phosphate buffers, and how many filter wash waste liquids are discharged in the outlet of C ultrafilter, remain the original volume of described macromolecular solution in C ultrafilter；
The exchange decolouring 7 of step 7, ion
With peristaltic pump, described ultrafiltration concentration liquid pump is crossed ion exchanger, by ion exchange membrane, described ultrafiltration concentration liquid is decoloured；Peristaltic pump flow velocity is 500 ml/min；Obtain destaining solution；
Step 8, saltout 8
Moving in reactor by described destaining solution, the mass ratio by 25% is slowly added to ammonium sulfate in described destaining solution, and stirs mixing, speed of agitator 100～200 revs/min, stirs 30 minutes used times, it is thus achieved that dissolved salt liquid；Afterwards, being placed in F desk centrifuge by described dissolved salt liquid and be centrifuged precipitation, F desk centrifuge rotating speed 7000 revs/min, centrifugal 15 minutes used times, remove the macromole precipitated impurities of described dissolved salt liquid, the clear liquid collecting upper strata obtains clear saline solution；Moving in reactor by described clear saline solution afterwards, the mass ratio by 20% is slowly added to ammonium sulfate in described clear saline solution, and stirs mixing, speed of agitator 100～150 revs/min, stirs 30 minutes used times, it is thus achieved that liquid of saltouing；Afterwards, described liquid of saltouing is placed in G desk centrifuge and is centrifuged liquid, G desk centrifuge rotating speed 7000 revs/min, centrifugal 15 minutes used times, the clear liquid on liquid upper strata of saltouing described in removal, collect the precipitation of lower floor, it is thus achieved that thing of saltouing；
Step 9, desalination 9
By by 100: 1 volume ratio, with the ammonium sulfate in thing of saltouing described in water for injection dissolved dilution, it is thus achieved that bath water is saltoutd thing；Afterwards, thing of being saltoutd by described bath water moves in the D ultrafilter being provided with 30kD film bag, start carries out ultrafiltration desalination, and in D ultrafilter, fill into water for injection simultaneously, when ammonium sulphate content in the waste liquid that the outlet of D ultrafilter is discharged is less than 0.01%, stop filling into water for injection, after the outlet of D ultrafilter is discharged without waste liquid, shutdown stops ultrafiltration desalination, it is thus achieved that the toxin thing of cotton-shaped unit 800-1500Lf/ml；
Step 10, formaldehyde detoxification 10
Described toxin thing is placed in glass container, adding mass concentration by the 0.5% of described toxin thing volume ratio is the formalin of 40%, adding mass concentration by the 2% of toxin thing volume ratio again is the lysine solution of 9.125%, pH to 7.2-7.8 is adjusted in stirring, stand detoxification, dwell temperature 38～40 DEG C, stands 42 days used times, it is thus achieved that detoxification thing；
Step 11, aseptic filtration 11
Being placed in glass container by described detoxification thing, add sodium chloride by the 0.85% of described detoxification thing mass ratio, pH to 6.6-7.4 is adjusted in stirring, afterwards, carries out aseptic filtration with the B germ tight filter being provided with 0.22 μm of filter element, it is thus achieved that stock solution, i.e. diphtheria toxoid stock solution.
The calibrating of diphtheria toxoid stock solution includes: Poison Reverse test, physics and chemistry shape, sterility test, Lf pH-value determination pH, purity, specific toxicities test.
The preservation of diphtheria toxoid stock solution with effect the phase: be stored in 2～8 DEG C of freezers, the effect phase from detoxification qualified from be 42 months.
Test
The diphtheria toxoid stock solution of 3 batches undertaken by the present invention produces, 60 liters every batch；
Controlled trial result, consults Fig. 2, Fig. 3, Fig. 4.
Beneficial effect
Experiment proves, the diphtheria toxoid stock solution prepared by the present invention, purity and yield rate all obtain and significantly promotes, after particularly refining the purity of stock solution all >=2300Lf/mgPN, goods purity is higher, reduce the incidence rate of the untoward reaction caused because of the foreign protein in goods, it is ensured that the safety of goods.
Claims (1)
1. the preparation method of a high-purity diphtheria toxoid stock solution, cultivate (1), formalin-inactivated (2) including viable bacteria, bacterium solution separates (3), aseptic filtration (4), (5), ultrafiltration dialysis (6) are concentrated by ultrafiltration, (7), saltout (8), desalination (9), formaldehyde detoxification (10) and aseptic filtration (11) processing step are decoloured in ion exchange, it is characterised in that:
It is step one that described viable bacteria cultivates (1), adopt diphtheria corynebacterium CMCC38007 freeze-drying lactobacillus, improve the recovery of Martin's culture medium through two generations, be inoculated in improvement Lin Shi culture medium and carry out strain amplification, it is thus achieved that propagation bacterium, afterwards, described propagation bacterium is moved to and in fermentation tank, is stirred aerobic culture, speed of agitator 500 revs/min, temperature 33～36 DEG C, 40～50 hours used times, it is thus achieved that viable bacteria is starched；
Described formalin-inactivated (2) is step 2, by the volume ratio of the 0.6% of described viable bacteria volume of slurry, in fermentation tank, add the formalin that mass concentration is 40% be stirred inactivation sterilization, speed of agitator 100 revs/min, 1 hour used time, it is thus achieved that inactivation slurry；
It is step 3 that described bacterium solution separates (3), with E continuous flow centrifuge, described inactivation slurry is centrifuged bacterium solution and separates, and removes major part thalline and obtains the culture fluid containing toxin；Specifically, open E continuous flow centrifuge, treat that speed reaches 10000 revs/min, stable (2) minute, described inactivation slurry is pumped into E continuous flow centrifuge by peristaltic pump and is centrifuged bacterium solution separation, peristaltic pump flow velocity 1.5～2 liters/min, collect effluent and obtain serosity；
Described aseptic filtration (4) is step 4, and the residue thalline removed in described serosity with the A germ tight filter being provided with 0.22 μm of filter element obtains the degerming slurry of clarification；Specifically, described serosity is pumped into A germ tight filter, peristaltic pump flow velocity 1.5 liters/min by peristaltic pump, collect effluent and obtain degerming slurry；
Described ultrafiltration concentration (5) is step 5, with C ultrafilter, described degerming slurry is carried out sized molecules solution separating, removes small molecule solution and obtains macromolecular solution；It is specially, described degerming slurry is moved in C ultrafilter, C ultrafilter uses the film bag of the improvement polyether sulfone matter of 30 kilodaltons, it is called for short 30kD film bag, the pump pressure pressure of the import of described 30kD film bag is 0.1MPa, the pressure of C ultrafilter refluxing opening is 0.5MPa, start, described degerming slurry is carried out sized molecules solution separating concentration, wherein small molecule solution is discharged through the outlet of C ultrafilter, macromolecular solution mixes with still unsegregated described degerming slurry in the refluxing opening return C ultrafilter of C ultrafilter, again participates in multi-cycle separation concentration；When the degerming slurry in C ultrafilter is concentrated into the 1/10～1/15 of original volume, shutting down, stop concentration, now remaining in the solution in C ultrafilter is macromolecular solution；
Described ultrafiltration dialysis (6) is step 6, in C ultrafilter, described macromolecular solution is carried out ultrafiltration dialysis filter wash, it is thus achieved that liquid is concentrated by ultrafiltration；It is specially, phosphate buffer is injected in C ultrafilter and mix with described macromolecular solution, start, by described film bag, described macromolecular solution is carried out equal-volume dialysis filter wash, treat that filter wash is to conductance 5～10 milli Siemens/cm, during pH7.4, stop injecting phosphate buffer, shutdown, stops dialysis filter wash, it is thus achieved that liquid is concentrated by ultrafiltration；Described equal-volume dialysis filter wash is for injecting how many phosphate buffers, and how many filter wash waste liquids are discharged in the outlet of C ultrafilter, remain the original volume of described macromolecular solution in C ultrafilter；
Described ion exchange decolouring (7) is step 7, with ion exchanger, described ultrafiltration concentration liquid is carried out dialysis and decolours, it is thus achieved that destaining solution；Specifically, described ultrafiltration concentration liquid pump is crossed ion exchanger with peristaltic pump, by ion exchange membrane, described ultrafiltration concentration liquid is decoloured；Peristaltic pump flow velocity is 500 ml/min；Obtain destaining solution；
Described saltout (8), for step 8, with desk centrifuge, saltout thing with ammonium sulfate described destaining solution saltoutd acquisition；Specifically, move in reactor by described destaining solution, the mass ratio by 25% is slowly added to ammonium sulfate in described destaining solution, and stirs mixing, speed of agitator 100～200 revs/min, stirs 30 minutes used times, it is thus achieved that dissolved salt liquid；Afterwards, being placed in F desk centrifuge by described dissolved salt liquid and be centrifuged precipitation, F desk centrifuge rotating speed 7000 revs/min, centrifugal 15 minutes used times, remove the macromole precipitated impurities of described dissolved salt liquid, the clear liquid collecting upper strata obtains clear saline solution；Moving in reactor by described clear saline solution afterwards, the mass ratio by 20% is slowly added to ammonium sulfate in described clear saline solution, and stirs mixing, speed of agitator 100～150 revs/min, stirs 30 minutes used times, it is thus achieved that liquid of saltouing；Afterwards, described liquid of saltouing is placed in G desk centrifuge and is centrifuged liquid, G desk centrifuge rotating speed 7000 revs/min, centrifugal 15 minutes used times, the clear liquid on liquid upper strata of saltouing described in removal, collect the precipitation of lower floor, it is thus achieved that thing of saltouing；
Described desalination (9) is step 9, uses D ultrafilter, with water for injection, described thing of saltouing is diluted and ultrafiltration desalination, it is thus achieved that the toxin thing of cotton-shaped unit 800-1500Lf/ml；Specifically, by by 100: 1 volume ratio, with the ammonium sulfate in thing of saltouing described in water for injection dissolved dilution, it is thus achieved that bath water is saltoutd thing；Afterwards, thing of being saltoutd by described bath water moves in the D ultrafilter being provided with 30kD film bag, start carries out ultrafiltration desalination, and in D ultrafilter, fill into water for injection simultaneously, when ammonium sulphate content in the waste liquid that the outlet of D ultrafilter is discharged is less than 0.01%, stop filling into water for injection, after the outlet of D ultrafilter is discharged without waste liquid, shutdown stops ultrafiltration desalination, it is thus achieved that the toxin thing of cotton-shaped unit 800-1500Lf/ml；
Described formaldehyde detoxification (10) is step 10, with formaldehyde, described toxin thing is carried out detoxification, it is thus achieved that detoxification thing；It is specially, described toxin thing is placed in glass container, adding mass concentration by the 0.5% of described toxin thing volume ratio is the formalin of 40%, adding mass concentration by the 2% of toxin thing volume ratio again is the lysine solution of 9.125%, pH to 7.2-7.8 is adjusted in stirring, stands detoxification, dwell temperature 38～40 DEG C, stand 42 days used times, it is thus achieved that detoxification thing；
Described aseptic filtration (11) is step 11, carries out aseptic filtration with B germ tight filter, it is thus achieved that stock solution；Specifically, be placed in glass container by described detoxification thing, adding sodium chloride by the 0.85% of described detoxification thing mass ratio, pH to 6.6-7.4 is adjusted in stirring, afterwards, carries out aseptic filtration with the B germ tight filter being provided with 0.22 μm of filter element, it is thus achieved that diphtheria toxoid stock solution.
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Cited By (1)
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| CN106046127A (en) * | 2016-08-10 | 2016-10-26 | 成都生物制品研究所有限责任公司 | Preparation method of diphtheria toxoid |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101503725A (en) * | 2009-03-19 | 2009-08-12 | 浙江天元生物药业股份有限公司 | Technique for improving purity of diphtheria toxoid |
| CN102961740A (en) * | 2012-12-10 | 2013-03-13 | 武汉生物制品研究所有限责任公司 | Preparation method of diphtheria toxoid vaccine |
-
2016
- 2016-03-04 CN CN201610131369.5A patent/CN105755075A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101503725A (en) * | 2009-03-19 | 2009-08-12 | 浙江天元生物药业股份有限公司 | Technique for improving purity of diphtheria toxoid |
| CN102961740A (en) * | 2012-12-10 | 2013-03-13 | 武汉生物制品研究所有限责任公司 | Preparation method of diphtheria toxoid vaccine |
Non-Patent Citations (3)
| Title |
|---|
| 周先碗: "《生物化学仪器分析与实验技术》", 31 January 2003, 化学工业出版社 * |
| 王永芬: "《动物生物制品技术》", 31 August 2011, 中国农业大学出版社 * |
| 白鹏: "《制药工程导论》", 31 August 2003, 化学工业出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106046127A (en) * | 2016-08-10 | 2016-10-26 | 成都生物制品研究所有限责任公司 | Preparation method of diphtheria toxoid |
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