CN106220717A - A kind of purification process of diphtheria toxoid - Google Patents
A kind of purification process of diphtheria toxoid Download PDFInfo
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- CN106220717A CN106220717A CN201610622889.6A CN201610622889A CN106220717A CN 106220717 A CN106220717 A CN 106220717A CN 201610622889 A CN201610622889 A CN 201610622889A CN 106220717 A CN106220717 A CN 106220717A
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- diphtheria toxoid
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- 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 the purification process of a kind of diphtheria toxoid, it comprises the following steps: centrifugal, be concentrated by ultrafiltration, one section saltout, two-stage nitration is saltoutd, ultrafiltration desalination, detoxification and chromatography.The purification process of a kind of diphtheria toxoid that the present invention provides, diphtheria toxoid stock solution purity prepared by the method is high, up to 2500Lf/mg, thus the generation of side reaction after reducing inoculation, safety is higher；The inventive method is simple to operate, purification is convenient, low cost, be applicable to industrialization large-scale production.
Description
Technical field
The invention belongs to biological technical field, be specifically related to the purification process of a kind of diphtheria toxoid.
Background technology
Diphtheria always most makes us one of terrified childhood disease, is by producing ectotoxic diphtheria corynebacterium
The acute disease that (hereinafter referred to as diphtheria corynebacterium) causes, the outburst of this disease has destructiveness.Bacteriotoxin can cause in generation exhales
Inhale road obstructive pseudomembrane (croup) or injury of myocardium and hetero-organization thereof, although great majority infect for recessive or clinical symptoms relatively
Gently, but many patients die from the respiratory tract obstruction or toxic myocarditis caused by diphtheria.
Many countries all occurred the diphtheria based on child to be very popular in history.It is endemoepidemic state in diphtheria
Family, this disease mostly is Sporadic cases or outburst on a small scale.Although most diphtheria corynebacterium infects for recessiveness or clinical symptoms relative
Relatively light, but high (> 10% of its case fatality rate), even if being also such in nearest outburst.Make in a large number in the eighties in 20th century according to estimates
Before diphtheria toxoid, there are about 1,000,000 cases every year in developing country, 50,000～60,000 examples are dead.Even if in recent years,
Endemic area, the report case fatality rate of diphtheria is still above 10%.
People is the only naturally host of diphtheria corynebacterium.Diphtheria is only propagated by the spittle and close contact.Highly infective skin
Diphtheria is common in some places of torrid areas.Local majority of cases in temperate climate occurred in cold season, and in warm
Place with weather the most all has propagation to occur.In diphtheria still in endemoepidemic country, this disease mainly affects school age
Front child and school age population.In most industry country diphtheria without endemic conditions or the rarest.But, keep child
The most particularly significant with the vaccine height rate of vaccination of adult, this point is confirmed from the outburst of many places, world diphtheria,
The diphtheria in the former Soviet Union is occurred for especially 20 nineties in century to be very popular.
Traditional diphtheria vaccine production procedure is the diphtheria corynebacterium cultivated with solution culture fermentation and produce poison, to containing extracellular toxin
Culture supernatant centrifugal segregation thalline, then refine with ammonium sulfate two-stage nitration salting out method, refined after toxin formaldehyde carry out
Detoxification becomes toxoid stock solution.But there is the shortcoming that purity is the highest in the toxoid stock solution that traditional method produces, adds and connect
The generation of side reaction after Zhong, safety has much room for improvement.
Summary of the invention
It is an object of the invention to overcome the shortcoming of prior art, it is provided that the purification process of a kind of diphtheria toxoid, the party
Method is simple to operate, purification is convenient, low cost, be applicable to industrialization large-scale production.
The purpose of the present invention is achieved through the following technical solutions: the purification process of a kind of diphtheria toxoid, it include with
Lower step:
S1. it is centrifuged: use formaldehyde to sterilize the diphtheria culture fluid of cultivation, be then centrifuged with continuous centrifuge degerming
Body, controls liquid feeding end flow velocity to centrifuged supernatant clarification, without opalescence；
S2. it is concentrated by ultrafiltration: by the filter element pre-filtering that above-mentioned centrifugal liquid employing aperture is 0.45～0.65 μm, then use molecular cut off
Ultrafilter membrane for 30KD is concentrated by ultrafiltration to the 1/10 of original volume, then with water for injection, concentrated solution is diluted one times, then ultrafiltration is dense
It is reduced to dilute the volume of front concentrated solution, repetition aforesaid operations 3～5 times, obtains whole concentrated solution；
S3. saltout for one section: whole concentrated solution adds ammonium sulfate and saltouts, then add by the 1% of the supernatant volume of centrifugal collection
Enter activated carbon, continue stirring 10～30min, add sodium bicarbonate make its final concentration of 0.5%, continue stirring 30～60min,
Centrifugal collection saltout after supernatant；
S4. two-stage nitration is saltoutd: adds ammonium sulfate in the supernatant after saltouing and carries out two-stage nitration and saltout, centrifugal collecting precipitation after saltouing；
S5. ultrafiltration desalination: use sodium bicarbonate solution dissolution precipitation, the filter element pre-filtering using aperture to be 0.45～0.65 μm, use
Sodium bicarbonate solution by trapped fluid dilute one times, be concentrated into original volume with the ultrafiltration through membranes that molecular cut off is 30KD, repeat on
State operation until ammonium sulphate content ＜ 0.1%；
S6. detoxification: add formalin in the diphtheria toxoid after ultrafiltration desalination, be placed in 35～39 DEG C of detoxifications 28～34d；
S7. chromatography: the diphtheria toxoid after detoxification is splined on SephacrylS-300 chromatographic column, washes with the flow velocity of 1ml/min
De-, collect monomer peak, aseptic filtration is placed on 2～8 DEG C of preservations.
Further, the rotating speed of centrifuge is 18000～22000g.
Further, addition quality is supernatant volume 18～25% of ammonium sulfate described in step S3 and step S4.
Further, the mass percent concentration of sodium bicarbonate solution described in step S5 is 0.1～0.5%.
Further, in step S7, chromatographic column uses the sodium chloride balance of 0.15mol/L.
The invention have the advantages that the purification process of a kind of diphtheria toxoid that the present invention provides, prepared by the method
Diphtheria toxoid stock solution purity is high, up to 2500Lf/mg, thus the generation of side reaction after reducing inoculation, safety is higher；This
Inventive method is simple to operate, purification is convenient, low cost, be applicable to industrialization large-scale production.
Accompanying drawing explanation
Fig. 1 is the thin layer chromatography figure of eluent of the present invention.
Detailed description of the invention
Below in conjunction with the accompanying drawings and embodiment the present invention will be further described, protection scope of the present invention be not limited to
Lower described.
Embodiment 1: the purification process of a kind of diphtheria toxoid, it comprises the following steps:
S1. being centrifuged: use formaldehyde to sterilize the diphtheria culture fluid of cultivation, continuous centrifuge is centrifuged removing thalline, first
Starting centrifuge, after centrifuge speed is 18000g, opening peristaltic pump regulation rotating speed is 100rad/min, controls liquid feeding end stream
Speed is to centrifuged supernatant clarification, without opalescence；
S2. it is concentrated by ultrafiltration: by the filter element pre-filtering that above-mentioned centrifugal liquid employing aperture is 0.45 μm, then be 30KD with molecular cut off
Ultrafilter membrane be concentrated by ultrafiltration to the 1/10 of original volume, then with water for injection, concentrated solution is diluted one times, then is concentrated by ultrafiltration to the dilutest
Release the volume of front concentrated solution, repeat aforesaid operations 3 times, obtain whole concentrated solution；
S3. saltout for one section: whole concentrated solution adds the ammonium sulfate that quality is supernatant volume 18% and saltouts, then by centrifugal
Collect supernatant volume 1% addition activated carbon, continue stirring 10min, add sodium bicarbonate make its final concentration of 0.5%,
Continue stirring 30min, centrifugal collection saltout after supernatant；Wherein, the rotating speed of described centrifuge is 18000g；
S4. two-stage nitration is saltoutd: adds the ammonium sulfate that quality is supernatant volume 18% in the supernatant after saltouing and carries out two-stage nitration salt
Analysis, centrifugal collecting precipitation after saltouing；Wherein, the rotating speed of described centrifuge is 18000g；
S5. ultrafiltration desalination: use sodium bicarbonate solution dissolution precipitation, the filter element pre-filtering using aperture to be 0.45 μm, use bicarbonate
Trapped fluid is diluted one times by sodium solution, is concentrated into original volume with the ultrafiltration through membranes that molecular cut off is 30KD, repeats aforesaid operations
Until ammonium sulphate content ＜ 0.1%；Wherein, the mass percent concentration of described sodium bicarbonate solution is 0.1%；
S6. detoxification: add formalin in the diphtheria toxoid after ultrafiltration desalination, be placed in 35 DEG C of detoxification 28d；
S7. chromatography: the diphtheria toxoid after detoxification is splined on the Sephacryl S-of the sodium chloride balance using 0.15mol/L
300 chromatographic columns, with the flow velocity eluting of 1ml/min, collect monomer peak, as it is shown in figure 1, aseptic filtration is placed on 2 DEG C of preservations.
Embodiment 2: the purification process of a kind of diphtheria toxoid, it comprises the following steps:
S1. being centrifuged: use formaldehyde to sterilize the diphtheria culture fluid of cultivation, continuous centrifuge is centrifuged removing thalline, first
Starting centrifuge, after centrifuge speed is 22000g, opening peristaltic pump regulation rotating speed is 220rad/min, controls liquid feeding end stream
Speed is to centrifuged supernatant clarification, without opalescence；
S2. it is concentrated by ultrafiltration: by the filter element pre-filtering that above-mentioned centrifugal liquid employing aperture is 0.65 μm, then be 30KD with molecular cut off
Ultrafilter membrane be concentrated by ultrafiltration to the 1/10 of original volume, then with water for injection, concentrated solution is diluted one times, then is concentrated by ultrafiltration to the dilutest
Release the volume of front concentrated solution, repeat aforesaid operations 5 times, obtain whole concentrated solution；
S3. saltout for one section: whole concentrated solution adds the ammonium sulfate that quality is supernatant volume 25% and saltouts, then by centrifugal
Collect supernatant volume 1% addition activated carbon, continue stirring 30min, add sodium bicarbonate make its final concentration of 0.5%,
Continue stirring 60min, centrifugal collection saltout after supernatant；Wherein, the rotating speed of described centrifuge is 22000g；
S4. two-stage nitration is saltoutd: adds the ammonium sulfate that quality is supernatant volume 25% in the supernatant after saltouing and carries out two-stage nitration salt
Analysis, centrifugal collecting precipitation after saltouing；Wherein, the rotating speed of described centrifuge is 22000g；
S5. ultrafiltration desalination: use sodium bicarbonate solution dissolution precipitation, the filter element pre-filtering using aperture to be 0.65 μm, use bicarbonate
Trapped fluid is diluted one times by sodium solution, is concentrated into original volume with the ultrafiltration through membranes that molecular cut off is 30KD, repeats aforesaid operations
Until ammonium sulphate content ＜ 0.1%；Wherein, the mass percent concentration of described sodium bicarbonate solution is 0.5%；
S6. detoxification: add formalin in the diphtheria toxoid after ultrafiltration desalination, be placed in 39 DEG C of detoxification 34d；
S7. chromatography: the diphtheria toxoid after detoxification is splined on the Sephacryl of the sodium chloride balance using 0.15mol/L
S-300 chromatographic column, with the flow velocity eluting of 1ml/min, collects monomer peak, as it is shown in figure 1, aseptic filtration is placed on 8 DEG C of preservations.
Embodiment 3: the purification process of a kind of diphtheria toxoid, it comprises the following steps:
S1. being centrifuged: use formaldehyde to sterilize the diphtheria culture fluid of cultivation, continuous centrifuge is centrifuged removing thalline, first
Starting centrifuge, after centrifuge speed is 19000g, opening peristaltic pump regulation rotating speed is 150rad/min, controls liquid feeding end stream
Speed is to centrifuged supernatant clarification, without opalescence；
S2. it is concentrated by ultrafiltration: by the filter element pre-filtering that above-mentioned centrifugal liquid employing aperture is 0.52 μm, then be 30KD with molecular cut off
Ultrafilter membrane be concentrated by ultrafiltration to the 1/10 of original volume, then with water for injection, concentrated solution is diluted one times, then is concentrated by ultrafiltration to the dilutest
Release the volume of front concentrated solution, repeat aforesaid operations 4 times, obtain whole concentrated solution；
S3. saltout for one section: whole concentrated solution adds the ammonium sulfate that quality is supernatant volume 20% and saltouts, then by centrifugal
Collect supernatant volume 1% addition activated carbon, continue stirring 18min, add sodium bicarbonate make its final concentration of 0.5%,
Continue stirring 40min, centrifugal collection saltout after supernatant；Wherein, the rotating speed of described centrifuge is 20000g；
S4. two-stage nitration is saltoutd: adds the ammonium sulfate that quality is supernatant volume 20% in the supernatant after saltouing and carries out two-stage nitration salt
Analysis, centrifugal collecting precipitation after saltouing；Wherein, the rotating speed of described centrifuge is 20000g；
S5. ultrafiltration desalination: use sodium bicarbonate solution dissolution precipitation, the filter element pre-filtering using aperture to be 0.52 μm, use bicarbonate
Trapped fluid is diluted one times by sodium solution, is concentrated into original volume with the ultrafiltration through membranes that molecular cut off is 30KD, repeats aforesaid operations
Until ammonium sulphate content ＜ 0.1%；Wherein, the mass percent concentration of described sodium bicarbonate solution is 0.2%；
S6. detoxification: add formalin in the diphtheria toxoid after ultrafiltration desalination, be placed in 36 DEG C of detoxification 30d；
S7. chromatography: the diphtheria toxoid after detoxification is splined on the Sephacryl S-of the sodium chloride balance using 0.15mol/L
300 chromatographic columns, with the flow velocity eluting of 1ml/min, collect monomer peak, as it is shown in figure 1, aseptic filtration is placed on 4 DEG C of preservations.
Embodiment 4: the purification process of a kind of diphtheria toxoid, it comprises the following steps:
S1. being centrifuged: use formaldehyde to sterilize the diphtheria culture fluid of cultivation, continuous centrifuge is centrifuged removing thalline, first
Starting centrifuge, after centrifuge speed is 21000g, opening peristaltic pump regulation rotating speed is 190rad/min, controls liquid feeding end stream
Speed is to centrifuged supernatant clarification, without opalescence；
S2. it is concentrated by ultrafiltration: by the filter element pre-filtering that above-mentioned centrifugal liquid employing aperture is 0.58 μm, then be 30KD with molecular cut off
Ultrafilter membrane be concentrated by ultrafiltration to the 1/10 of original volume, then with water for injection, concentrated solution is diluted one times, then is concentrated by ultrafiltration to the dilutest
Release the volume of front concentrated solution, repeat aforesaid operations 5 times, obtain whole concentrated solution；
S3. saltout for one section: whole concentrated solution adds the ammonium sulfate that quality is supernatant volume 23% and saltouts, then by centrifugal
Collect supernatant volume 1% addition activated carbon, continue stirring 25min, add sodium bicarbonate make its final concentration of 0.5%,
Continue stirring 30～60min, centrifugal collection saltout after supernatant；Wherein, the rotating speed of described centrifuge is 21000g；
S4. two-stage nitration is saltoutd: adds the ammonium sulfate that quality is supernatant volume 23% in the supernatant after saltouing and carries out two-stage nitration salt
Analysis, centrifugal collecting precipitation after saltouing；Wherein, the rotating speed of described centrifuge is 21000g；
S5. ultrafiltration desalination: use sodium bicarbonate solution dissolution precipitation, the filter element pre-filtering using aperture to be 0.60 μm, use bicarbonate
Trapped fluid is diluted one times by sodium solution, is concentrated into original volume with the ultrafiltration through membranes that molecular cut off is 30KD, repeats aforesaid operations
Until ammonium sulphate content ＜ 0.1%；Wherein, the mass percent concentration of described sodium bicarbonate solution is 0.4%；
S6. detoxification: add formalin in the diphtheria toxoid after ultrafiltration desalination, be placed in 38 DEG C of detoxification 32d；
S7. chromatography: the diphtheria toxoid after detoxification is splined on the Sephacryl of the sodium chloride balance using 0.15mol/L
S-300 chromatographic column, with the flow velocity eluting of 1ml/min, collects monomer peak, as it is shown in figure 1, aseptic filtration is placed on 6 DEG C of preservations.
Claims (5)
1. the purification process of a diphtheria toxoid, it is characterised in that it comprises the following steps:
S1. it is centrifuged: use formaldehyde to sterilize the diphtheria culture fluid of cultivation, be then centrifuged with continuous centrifuge degerming
Body, controls liquid feeding end flow velocity to centrifuged supernatant clarification, without opalescence；
S2. it is concentrated by ultrafiltration: by the filter element pre-filtering that above-mentioned centrifugal liquid employing aperture is 0.45～0.65 μm, then use molecular cut off
Ultrafilter membrane for 30KD is concentrated by ultrafiltration to the 1/10 of original volume, then with water for injection, concentrated solution is diluted one times, then ultrafiltration is dense
It is reduced to dilute the volume of front concentrated solution, repetition aforesaid operations 3～5 times, obtains whole concentrated solution；
S3. saltout for one section: whole concentrated solution adds ammonium sulfate and saltouts, then add by the 1% of the supernatant volume of centrifugal collection
Enter activated carbon, continue stirring 10～30min, add sodium bicarbonate make its final concentration of 0.5%, continue stirring 30～60min,
Centrifugal collection saltout after supernatant；
S4. two-stage nitration is saltoutd: adds ammonium sulfate in the supernatant after saltouing and carries out two-stage nitration and saltout, centrifugal collecting precipitation after saltouing；
S5. ultrafiltration desalination: use sodium bicarbonate solution dissolution precipitation, the filter element pre-filtering using aperture to be 0.45～0.65 μm, use
Sodium bicarbonate solution by trapped fluid dilute one times, be concentrated into original volume with the ultrafiltration through membranes that molecular cut off is 30KD, repeat on
State operation until ammonium sulphate content ＜ 0.1%；
S6. detoxification: add formalin in the diphtheria toxoid after ultrafiltration desalination, be placed in 35～39 DEG C of detoxifications 28～34d；
S7. chromatography: the diphtheria toxoid after detoxification is splined on SephacrylS-300 chromatographic column, washes with the flow velocity of 1ml/min
De-, collect monomer peak, aseptic filtration is placed on 2～8 DEG C of preservations.
The purification process of a kind of diphtheria toxoid the most as claimed in claim 1, it is characterised in that the rotating speed of centrifuge is
18000～22000g.
The purification process of a kind of diphtheria toxoid the most as claimed in claim 1, it is characterised in that institute in step S3 and step S4
State addition quality is supernatant volume the 18～25% of ammonium sulfate.
The purification process of a kind of diphtheria toxoid the most as claimed in claim 1, it is characterised in that bicarbonate described in step S5
The mass percent concentration of sodium solution is 0.1～0.5%.
The purification process of a kind of diphtheria toxoid the most as claimed in claim 1, it is characterised in that in step S7, chromatographic column uses
The sodium chloride balance of 0.15mol/L.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113980104A (en) * | 2021-10-25 | 2022-01-28 | 北京智飞绿竹生物制药有限公司 | Process method for purifying 15-valent pneumococcal polysaccharide protein conjugate by tangential flow ultrafiltration |
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| 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 |
| CN104027797A (en) * | 2014-06-19 | 2014-09-10 | 山东亦度生物技术有限公司 | Preparation method of diphtheria vaccine |
-
2016
- 2016-08-02 CN CN201610622889.6A patent/CN106220717A/en active Pending
Patent Citations (3)
| 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 |
| CN104027797A (en) * | 2014-06-19 | 2014-09-10 | 山东亦度生物技术有限公司 | Preparation method of diphtheria vaccine |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113980104A (en) * | 2021-10-25 | 2022-01-28 | 北京智飞绿竹生物制药有限公司 | Process method for purifying 15-valent pneumococcal polysaccharide protein conjugate by tangential flow ultrafiltration |
| CN113980104B (en) * | 2021-10-25 | 2023-08-22 | 北京智飞绿竹生物制药有限公司 | Process method for purifying 15-valent pneumococcal polysaccharide protein conjugate by tangential flow ultrafiltration |
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