CN108676829A - A method of removal acellular pertussis components endotoxin in vaccine - Google Patents
A method of removal acellular pertussis components endotoxin in vaccine Download PDFInfo
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- CN108676829A CN108676829A CN201810131774.6A CN201810131774A CN108676829A CN 108676829 A CN108676829 A CN 108676829A CN 201810131774 A CN201810131774 A CN 201810131774A CN 108676829 A CN108676829 A CN 108676829A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
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- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
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- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/18—Ion-exchange chromatography
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Abstract
The present invention provides a kind of methods of removal acellular pertussis components endotoxin in vaccine, the method utilizes new type compound anion chromatography filler Capto adhere, pertussis toxin (Pertussistoxin is removed by chromatographic purifying,) and the endotoxin in filamentous hemagglutinin (Filamentous hemagglutinin, FHA) PT.The method can be under the premise of not bringing allogenic material into, while keeping active ingredient PT and FHA bioactivity and the rate of recovery, by the endotoxin removal in PT and FHA to 100 EU/mg or less.The filler Capto adhere require the conductance range of buffer solution wider when handling sample, reduce the process of buffer exchange.The use of Capto adhere chromatography medias removal endotoxin is a kind of reasonable and efficient method when being prepared on a large scale PT and FHA for other methods.
Description
Technical field
The invention belongs to biopharmaceutical technology, more particularly to endogenous toxic material in a kind of removal acellular pertussis components vaccine
The method of element.
Background technology
Bordetella pertussis is the pathogen of pertussis, hemophilus, is in Gram-negative, rod-short or ellipse, hundred
Day cough bacterium can generate exotoxin and endotoxin and other many antigenic bioactive substances.Wherein endotoxin results from
The mantle of Gram-negative bacteria, is the substance for causing the vaccines side reactions such as fever, and the improvement of new antigen purification method makes
Endotoxic removal problem is obtained to display,《British Pharmacopoeia》The endotoxin of combined vaccine DTaP requires<100 EU/ agent, not
The regulation can not be less than by carrying out the requirement of domestic quality standard induced by endotoxin.Therefore, it is answered in acellular pertussis vaccine production
Endotoxin content is reduced as possible, to ensure production of vaccine and the use of high quality.
Endotoxic essence is lipopolysaccharides, is the active part of heat source, contains 3 different chemical areas：Drawing including internal layer
Play the areas lipoid A, the core oligosaccharide area in middle level and the specific polysaccharide sequence of outer layer of toxic reaction.Endotoxin relative molecular mass
From thousands of to tens of thousands of etc., there is heat resistance and chemical stability, property is extremely inhomogenous, it is difficult to find it is a kind of highly effective and versatile
Endotoxin removal method, by its disposable thoroughly removal, some after multiple step process, does not still reach requirement even.
Especially in biological products production process, needs stringent technology controlling and process induced by endotoxin and be removed or running water.
The protein purification of pertussis component vaccine needs to consider endotoxic removal problem.There is producer to use more classical
Nonionic surfactant, such as TritonX, to remove endotoxin, although in actual use we have found that this method is simple, only
The surfactant of suitable dose need to be added in purification process, but can make to increase in vaccine simultaneously it is unrelated with antigen extra
Ingredient increases the calibrating project work in later stage.
Invention content
In view of the above-mentioned problems, the object of the present invention is to provide a kind of removal acellular pertussis components endotoxin in vaccine
Method, and it is endotoxic meanwhile, it is capable to obtain the higher target protein rate of recovery removing.
The purpose of the present invention is what is realized by following scheme：
A method of removal acellular pertussis components endotoxin in vaccine includes the following steps：
1）Bordetella pertussis zymotic fluid is centrifuged, supernatant is collected and is fermented through 0.45 μm of membrane filtration, then through being concentrated by ultrafiltration
Supernatant adjusts the pH and conductance of fermentation supernatant with urea-containing phosphoric acid equilibration buffer, and sample is splined on Capto SP after adjustment
In ImpRes columns, elutes and collect PT components and FHA components respectively；
2）By step 1）Obtained PT components continue to be splined on Capto MMC columns, afford the PT components after being further purified；
3）With urea-containing phosphoric acid equilibration buffer regulating step 2）Collect be further purified after PT components and step 1）It receives
The pH and conductance of the FHA components of collection are then splined in Captoadhere columns and elute, endogenous toxic material is removed by chromatographic purifying respectively
Element collects sample penetration peak to get to destination protein PT and FHA.
Further, the urea-containing phosphoric acid equilibration buffer is that the phosphoric acid that urea concentration is 1.5 ~ 2.5 mol/L is flat
Weigh buffer solution.
Further, step 1）Described in be adjusted to pH5.0 ~ 6.0, conductance before fermentation supernatant loading<8ms/cm.
Further, step 1）Described in elution be respectively with contain 0.11 mol/L, 0.15 mol/L and 0.35 mol/
The elution of L NaCl.
Further, step 2）Described in elution be to be eluted with Tris-HCl buffer solutions.
Further, step 3）Described in step 2）Collect be further purified after PT components and step 1）It collects
PH5.3 ~ 6.3,10 ~ 18ms/cm of conductance are adjusted to before FHA loadings.
It is another object of the present invention to what is realized by following scheme：
A kind of pertussis toxin and Filamentous haemagglutinin being prepared using the above method.
The present invention having the beneficial effect that compared with prior art：
1, the method for removal acellular pertussis components endotoxin in vaccine of the present invention, according to endotoxin internal layer and middle level
Containing multiple phosphates and acidic group, have the characteristics that lower isoelectric point, negatively charged under general condition, uses Capto
This novel compound anion chromatography fillers of adhere remove endotoxin, the new type compound by chromatographic purifying
Anion chromatography filler Capto adhere require the conductance range of buffer solution wider, reduce buffer solution when handling sample
The process of displacement, when being prepared on a large scale PT and FHA, uses Capto adhere chromatography medias for other methods
Removal endotoxin is a kind of reasonable and efficient method；
2, the method for removal acellular pertussis components endotoxin in vaccine of the present invention, can not bring allogenic material into
Under the premise of, while keeping active ingredient PT and FHA bioactivity and the rate of recovery, extremely by the endotoxin removal in PT and FHA
100 EU/mg or less.
Specific implementation mode
Embodiment 1
A kind of method of removal acellular pertussis components endotoxin in vaccine is present embodiments provided, is as follows：
1）1 Bordetella pertussis working seed lots strain is opened in half synthetic medium of activated carbon, 36 DEG C are cultivated, and strain reaches one
Pertussis liquid synthetic medium is inoculated in after fixed number amount（SSM）After 2 generations, as fermentation tank seed, fermentation tank culture is put into.Training
Object is supported to harvest in exponential phase later stage or early period resting stage.52L Bordetella pertussis zymotic fluids are taken to centrifuge thalline and supernatant,
Supernatant is collected through 0.45 μm of membrane filtration, then is concentrated by ultrafiltration to obtain fermentation supernatant 2.5L, with the phosphoric acid of the urea containing 1.5mol/L
The pH6.0 of fermentation supernatant, conductance described in equilibration buffer tune<8ms/cm；8L samples are splined on Capto SP ImpRes after adjustment
In column, after Equilibration buffer wash is not associated with sample, respectively with containing 0.11 mol/L, 0.15 mol/L and 0.35 mol/L
The elution of NaCl collects PT component 260ml and FHA components 180ml, spare；
2）By step 1）Obtained PT components, which continue to be splined on CaptoMMC columns, is further purified PT, and PT is adsorbed on column in loading
On, it is eluted by containing Tris-HCl buffer solutions, collects the PT components 55ml after being further purified；
3）With the phosphoric acid equilibration buffer pacing rapid 2 of the urea containing 1.5mol/L）The 55ml of collection be further purified after PT components
PH5.5, conductance 16ms/cm；240ml samples are splined in Captoadhere columns after adjustment, collect sample peak 254ml to get
To destination protein PT components；
Take 50ml steps 1）The FHA components of collection, with the phosphoric acid equilibration buffer tune pH5.3 containing 1.5 mol/L urea, conductance
13ms/cm；100ml samples are splined in Captoadhere columns after adjustment, collect sample peak 147ml to get to destination protein
FHA components；
4）Destination protein PT components, the FHA components of collection are detected endotoxin content and protein content, and be calculated as follows respectively
Protein recovery：
Protein recovery（%）=（It harvests refined solution albumen concentration * and harvests volume）/（Loading sample protein concentration * loading volumes）*
100%
The step 1）Pertussis strain used in middle fermentation process is I phase CS bacterial strains of pertussis（Bacterium number is 58003-3）,
Purchased from National Institute for Food and Drugs Control's serum room.Used medium ferment by Chinese Academy of Medical Sciences's medical biotechnology research
It is prepared and provide.
Capto adhere, CaptoSP ImpRes, Capto MMC and chromatographic column described in the present embodiment are purchased from the U.S.
GE companies.
Ultrafiltration concentration buffer solution described in the present embodiment is standby simultaneously by Institute of Medical Biology of the Chinese Academy of Medical Sciences
It provides.
The step 4）, the detection of endotoxin content, according to《Chinese Pharmacopoeia》Three（2015 editions）1143 bacterium of general rule
Bacterial endotoxins test in detecting method --- gel method requirement is detected using gel limiting test method.Bacterial endotoxin standard items and
Reagents is purchased from Zhanjiang Andusi Biology Co., Ltd., 0. 25 EU/ml of sensitivity.
The step 4）, the detection of protein content, according to《Chinese Pharmacopoeia》Three（2015 editions）0731 protein of general rule
Content determination the second method Forint phenol method（Lowry methods）It is measured.
The step 4）, the detection of antigenic content, the ELISA methods survey recommended according to National Institute for Food and Drugs Control
Determine PT and FHA antigenic contents.Mono-mouse-anti-PT and Mono-mouse-anti-FHA is examined purchased from Chinese food drug
Determine research institute.
Embodiment 2
A kind of method of removal acellular pertussis components endotoxin in vaccine is present embodiments provided, agents useful for same, equipment are equal
It is same as Example 1, it is as follows：
1）1 Bordetella pertussis working seed lots strain is opened in half synthetic medium of activated carbon, 36 DEG C are cultivated, and strain reaches one
Pertussis liquid synthetic medium is inoculated in after fixed number amount（SSM）After 2 generations, as fermentation tank seed, fermentation tank culture is put into.Training
Object is supported to harvest in exponential phase later stage or early period resting stage.51L Bordetella pertussis zymotic fluids are taken to centrifuge thalline and supernatant,
Supernatant is collected through 0.45 μm of membrane filtration, then is concentrated by ultrafiltration to obtain fermentation supernatant 2L, is balanced with the phosphoric acid of the urea containing 2mol/L
The pH6.0 of fermentation supernatant, conductance described in buffer solution tune<8ms/cm；8L samples are splined on Capto SP ImpRes columns after adjustment
In, after Equilibration buffer wash is not associated with sample, respectively with containing 0.11 mol/L, 0.15 mol/L and 0.35 mol/L NaCl
Elution, collect PT component 310ml and FHA components 205ml, it is spare；
2）By step 1）Obtained PT components, which continue to be splined on CaptoMMC columns, is further purified PT, and PT is adsorbed on column in loading
On, it is eluted by containing Tris-HCl buffer solutions, collects the PT components 48ml after being further purified；
3）With the phosphoric acid equilibration buffer pacing rapid 2 of the urea containing 2mol/L）The 48ml of collection be further purified after PT components
PH5.3, conductance 15ms/cm；240ml samples are splined in Captoadhere columns after adjustment, collect sample peak 252ml to get to
Destination protein PT components；
Take 50ml steps 1）The FHA components of collection, with the phosphoric acid equilibration buffer tune pH5.5 containing 2 mol/L urea, conductance
16ms/cm；95ml samples are splined in Captoadhere columns after adjustment, collect sample peak 123ml to get to destination protein FHA
Component；
4）Destination protein PT components, the FHA components of collection are detected into endotoxin content and protein content respectively, and calculates albumen and returns
Yield.
Embodiment 3
A kind of method of removal acellular pertussis components endotoxin in vaccine is present embodiments provided, agents useful for same, equipment are equal
It is same as Example 1, it is as follows：
1）1 Bordetella pertussis working seed lots strain is opened in half synthetic medium of activated carbon, 36 DEG C are cultivated, and strain reaches one
Pertussis liquid synthetic medium is inoculated in after fixed number amount（SSM）After 2 generations, as fermentation tank seed, fermentation tank culture is put into.Training
Object is supported to harvest in exponential phase later stage or early period resting stage.50L Bordetella pertussis zymotic fluids are taken to centrifuge thalline and supernatant,
Supernatant is collected through 0.45 μm of membrane filtration, then is concentrated by ultrafiltration to obtain fermentation supernatant 2L, it is flat with the phosphoric acid of the urea containing 2.5mol/L
Weigh the pH5.9 of fermentation supernatant, conductance described in buffer solution tune<8ms/cm；8L samples are splined on Capto SP ImpRes after adjustment
In column, after Equilibration buffer wash is not associated with sample, respectively with containing 0.11 mol/L, 0.15 mol/L and 0.35 mol/L
The elution of NaCl collects PT component 270ml and FHA components 162ml, spare；
2）By step 1）Obtained PT components, which continue to be splined on CaptoMMC columns, is further purified PT, and PT is adsorbed on column in loading
On, it is eluted by containing Tris-HCl buffer solutions, collects the PT components 40ml after being further purified；
3）With the phosphoric acid equilibration buffer pacing rapid 2 of the urea containing 2.5mol/L）The 40ml of collection be further purified after PT components
PH5.5, conductance 13ms/cm；240ml samples are splined in Captoadhere columns after adjustment, collect sample peak 260ml to get
To destination protein PT components；
Take 50ml steps 1）The FHA components of collection, with the phosphoric acid equilibration buffer tune pH5.3 of the urea containing 2.5mol/L, conductance
15ms/cm；86ml samples are splined in Captoadhere columns after adjustment, collect sample peak 119ml to get to destination protein FHA
Component；
4）Destination protein PT components, the FHA components of collection are detected into endotoxin content and protein content respectively, and calculates albumen and returns
Yield.
The quality testing of each embodiment：
Each acellular pertussis components vaccine prepared by embodiment 1-3 is pressed respectively《Chinese Pharmacopoeia》Three（2015 editions）It is required that will
The PT and FHA of collection detect endotoxin content and protein content respectively, and calculate protein recovery, the results are shown in Table 1.
The endotoxin value of destination protein PT in 1 to 3 each sample of embodiment can be down to 50EU/mg using Captoadhere
Hereinafter, the endotoxin value of FHA is down to 100EU/mg hereinafter, and under conditions of current optimization, the PT destination protein rate of recovery
Up to 95% or more, the FHA destination protein rate of recovery up to 75% or more.
Claims (8)
1. a kind of method of removal acellular pertussis components endotoxin in vaccine, which is characterized in that the method includes following
Step：
1）Bordetella pertussis zymotic fluid is centrifuged, supernatant is collected and is fermented through 0.45 μm of membrane filtration, then through being concentrated by ultrafiltration
Supernatant adjusts the pH and conductance of fermentation supernatant with urea-containing phosphoric acid equilibration buffer, and sample is splined on Capto SP after adjustment
In ImpRes columns, elutes and collect PT components and FHA components respectively；
2）By step 1）Obtained PT components continue to be splined on Capto MMC columns, afford the PT components after being further purified；
3）With urea-containing phosphoric acid equilibration buffer regulating step 2）Collect be further purified after PT components and step 1）It receives
The pH and conductance of the FHA components of collection are then splined in Captoadhere columns and elute, endogenous toxic material is removed by chromatographic purifying respectively
Element collects sample penetration peak to get to destination protein PT and FHA.
2. the method for removal acellular pertussis components endotoxin in vaccine according to claim 1, which is characterized in that institute
It is the phosphoric acid equilibration buffer that urea concentration is 1.5 ~ 2.5 mol/L to state urea-containing phosphoric acid equilibration buffer.
3. the method for removal acellular pertussis components endotoxin in vaccine according to claim 1, which is characterized in that step
Rapid 1）Described in be adjusted to pH5.0 ~ 6.0, conductance before fermentation supernatant loading<8ms/cm.
4. the method for removal acellular pertussis components endotoxin in vaccine according to claim 1, which is characterized in that step
Rapid 1）Described in elution be to be washed respectively with the eluent containing 0.11 mol/L, 0.15 mol/L and 0.35 mol/L NaCl
It is de-.
5. the method for removal acellular pertussis components endotoxin in vaccine according to claim 1, which is characterized in that step
Rapid 2）Described in elution be to be eluted with Tris-HCl buffer solutions.
6. the method for removal acellular pertussis components endotoxin in vaccine according to claim 1, which is characterized in that step
Rapid 3）Described in step 2）Collect be further purified after PT components and step 1）Be adjusted to before the FHA loadings of collection pH5.3 ~
6.3,10 ~ 18ms/cm of conductance.
7. a kind of endotoxic pertussis toxin of removal, which is characterized in that the endotoxic pertussis toxin of removal is by right
It is required that any one of 1-6 the methods are prepared.
8. a kind of endotoxic Filamentous haemagglutinin of removal, which is characterized in that the endotoxic Filamentous haemagglutinin of removal is by right
It is required that any one of 1-6 the methods are prepared.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111298109A (en) * | 2020-03-09 | 2020-06-19 | 辽宁成大生物股份有限公司 | Method for removing residual host DNA in Japanese encephalitis vaccine product by using multi-mode chromatography medium Capto adhere |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2008073620A2 (en) * | 2006-11-02 | 2008-06-19 | Neose Technologies, Inc. | Manufacturing process for the production of polypeptides expressed in insect cell-lines |
| WO2014188313A1 (en) * | 2013-05-20 | 2014-11-27 | Shantha Biotechnics Limited | Purification of polysaccharide protein conjugates |
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2018
- 2018-02-09 CN CN201810131774.6A patent/CN108676829A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008073620A2 (en) * | 2006-11-02 | 2008-06-19 | Neose Technologies, Inc. | Manufacturing process for the production of polypeptides expressed in insect cell-lines |
| WO2014188313A1 (en) * | 2013-05-20 | 2014-11-27 | Shantha Biotechnics Limited | Purification of polysaccharide protein conjugates |
Non-Patent Citations (1)
| Title |
|---|
| 田阳等: "百日咳毒素、丝状血凝素和黏附素的纯化_", 《中国生物制品学杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111298109A (en) * | 2020-03-09 | 2020-06-19 | 辽宁成大生物股份有限公司 | Method for removing residual host DNA in Japanese encephalitis vaccine product by using multi-mode chromatography medium Capto adhere |
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