CN1504482A - Separation and purification process for acellular whooping cough antigen albumen - Google Patents
Separation and purification process for acellular whooping cough antigen albumen Download PDFInfo
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- CN1504482A CN1504482A CNA021536600A CN02153660A CN1504482A CN 1504482 A CN1504482 A CN 1504482A CN A021536600 A CNA021536600 A CN A021536600A CN 02153660 A CN02153660 A CN 02153660A CN 1504482 A CN1504482 A CN 1504482A
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Abstract
The invention relates to a method for further separating and purifying pertussis antigen for the supernatant fluid of pertussis bacilliculture liquid, wherein the screen selected domestic pearlstone is used as column chromatography material in the process, the sample is concentrated for conductance adjustment processing and elution condition optimized for further segregation and purification, to obtain the Filamentous hamagglutmin (FHA) and pertassis toxin (PT), the polyacrylamide gel electrophoresis (SDS-PAGE) evaluation has shown that its purity is as high as 85-95%.
Description
Technical field
The present invention relates to the antigenic processing method of a kind of column chromatography separation and purification acellular pertussis, relate in particular to the pearlite of a kind of employing as the column chromatography material, the supernatant liquor of Whooping cough bag Te Shi bacillus nutrient solution is carried out the method for a step separation and purification pertussis antigen through screening.
Background technology
Whooping cough M ﹠ M in children population below four years old is all very high, has every year 40000000 children infected according to statistics, wherein nearly 950,000 children ill death because not injecting pertussis vaccine.1,000,000,000 pertussis vaccines of approximately annual use in the global range.Wherein the acellular pertussis vaccine of purifying has more than 8,000 ten thousand.
Various countries use the existing many decades of whole cell pertussis vaccine, owing to may cause the side reaction of symptoms such as in various degree fever, vomiting, injection site redness after the vaccinate, discrete also may produce more serious cranial nerve disease, even dead.Therefore some country as: Britain, Sweden and Japan once stopped to inoculate the whole cell pertussis vaccine, but it is popular that Whooping cough has taken place subsequently, and lesson is deep.Therefore eighties of last century the eighties, Japan at first begins to manufacture experimently acellular pertussis vaccine, and it is the antigen protein that extracts from Whooping cough bag Te Shi bacillus exocytosis liquid, contains filamentous hemagglutinin (FHA) and toxicide hundred orders are coughed toxin (PT).
The purifying process of Japan comprises ammonium sulfate precipitation basically, high salt concentration extract and saccharose gradient super centrifugal (Noble et al., 1987 J.Am.Med.Assoc.257,1351-1356.).Product purity through this cover explained hereafter is not high enough, and productive rate is lower and facility investment is huge, unsuitable scale operation.Each biological products company of China still continues to use the acellular pertussis production technique of Japan at present and carries out small serial production.
In past 20 years, North America and European various countries develop acellular pertussis vaccine one after another, and preparation technology comprises following several:
1. hydroxyapatite, haptoglobin column chromatography and gel filtration method.
2. blue glue affinity chromatography and Pp63 glycophosphoproteins affinity chromatography.
3. blue glue affinity chromatography and hydroxyapatite chromatography method.
Though use these technologies all can obtain purer pertussis antigen PT and FHA, every kind of technology all has its unavoidable problem.For example: blue glue affinity chromatography, Pp63 glycophosphoproteins affinity chromatography, haptoglobin affinity chromatography, the conjugated group of chromatographic stuffing is degraded easily and is influenced vaccine quality.In addition, it is too harsh to contain toxicant or elution requirement in the elutriant, and these all can directly influence antigen yield and quality.
Canada Connaught company adopts perlite column chromatography and hydroxyapatite column chromatography to come purifying acellular pertussis antigen (U.S.Pat.No.4 the earliest, 997,915), can obtain the PT and the FHA of higher degree and productive rate, be the used preferred process of at present external each company.But the vaccine culture is behind the perlite column chromatographic isolation and purification, and the purity of PT and FHA can only reach 62%-68%, also will pass through hydroxyapatite (Hydroxyapatite) column chromatographic isolation and purification, and the purity of PT and FHA just can reach about 90%.Hydroxyapatite price very expensive (about 4,700 dollars/kilogram), and this step separation and purification PT and FHA yield be about 90%, have about 10% PT and FHA to lose.
The used perlite of external report is the product of Canadian Filchem company, and about 6 dollars/kilogram of price, as select the import perlite for use not only will spend and remit purchase outward, and the proteinic effect of separation and purification is also poor than pearlite.Though pearlite ample supply and prompt delivery, low price (1.5-3.0 yuan/kilogram), product performance are stable, only are used as flocculating aids at present, domestic still do not have a proteinic report of the perlite of utilization separation and purification.
Summary of the invention
The invention provides the antigenic easy processing method of a kind of separation and purification acellular pertussis, the present invention adopts a kind of pearlite through screening as the column chromatography material, optimize processing condition the supernatant liquor of Whooping cough bag Te Shi bacillus nutrient solution carried out a step separation and purification pertussis antigen, be a kind of simply, fast, the purification process of acellular pertussis vaccine cheaply.
The present invention realizes as follows:
(1) dress post: choose and contain SiO
270-80%, Al
2O
3The edible class middling speed type perlite of 10-20%, granulometric range is at 30-250 μ m.Scanning electron microscopic observation, screening surface imperfection have the product of multi-cellular structure.After removing suspended substance and suspension liquid, with water for injection flushing perlite chromatography column;
(2) sample preparation, application of sample: the nutrient solution of Whooping cough bag Te Shi bacillus is removed the microorganism collection supernatant liquor through high speed centrifugation, supernatant liquor is removed remaining thalline through the film Sterile Filtration, the degerming rear filtrate is through the 0.1-20% of 10-30KD film bag ultrafiltration and concentration to original volume, concentrated solution adds water for injection adjustment electricity and is directed at 2-4mS/cm, passes through the perlite chromatography column with linear rate of flow less than 300cm/hr.
(3) wash-out: at first use the Tris-HCl of 50-60mM, pH=7.0-8.0 carries out wash-out to the perlite chromatography column, is 0-0.05 until the OD of elutriant value; Second step was used the 50-60Mm Tris-HCl damping fluid that contains 0.05-0.15M NaCl, pH=7.0-8.0, the perlite chromatography column is carried out the secondary wash-out, is 0-0.05 until the OD of elutriant value, and the elutriant of collecting for second step is the Toxins, pertussis of perlite chromatography column separation and purification; The 3rd step was used the 50-60mM Tris-HCl that contains 0.4-0.7M NaCl, pH=7.0-8.0 carries out wash-out for the third time to the perlite chromatography column, until the OD of elutriant value is 0-0.05, and the elutriant of collecting for the 3rd step is the filamentous hemagglutinin of perlite chromatography column separation and purification.
Preferred perlitic granulometric range is at 50-250 μ m in the described step (1).
Filtrate is concentrated into the 10-20% of original volume in step (2) described in the preferred embodiment.
In order to make absorption of proteins effective, flow velocity is good more more slowly in the step (2), but for reducing the operating time, preventing protein coacervation, selects for use linear rate of flow 150-250cm/hr comparatively suitable.
Adopt the bordetella pertussis CS bacterial strain of domestic separation screening, it is to belong to 1,2,3 serotypes.Other Whooping cough bag Te Shi bacillus strain also can use.
We find that in experiment perlite has different chemical constitutions and physicals, and factor differences such as its granular size, surface tissue are also different with elution requirement to the adsorptive power of range protein.The present invention with garbled pearlite as chromatographic material, separation and purification pertussis antigen effectively.Pearlite (edible class), granulometric range is generally 3-250 μ m.According to the difference of performance index, the perlite model can be divided into (K) middling speed (Z) and (M) type (the building material industry standard JC849-1999 of the People's Republic of China (PRC), perlite help and consider agent) fast at a slow speed.Select for use the edible class perlite of middling speed preferably from Whooping cough bag Te Shi bacillus nutrient solution separation and purification filamentous hemagglutinin (FHA) and Toxins, pertussis (PT).Granulometric range is at 30-250 μ m, the separation and purification effect is better, removes too small and oversized particles perlite with standard sieve, makes the perlite particle homogeneous, test shows that the perlite of particle inequality is different with elution rate to absorption of proteins, thereby causes the separation and purification effect bad.To the perlite particle that sieved with deionized water or distilled water flushing, leave standstill the back and remove suspended substance and suspension liquid, the chromatography column of packing into after carrying out repeatedly repeatedly is with 2-10 water for injection (WFI) flushing perlite chromatography column doubly.
Filamentous hemagglutinin (FHA) and Toxins, pertussis (PT) antigenic solution through the separation and purification of perlite chromatography column, identify (Chinese biological goods rules through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), version in 2000, absorption acellular pertussis, diphtheria, tetanus combined vaccine are made and vertification regulation) the proteinic purity of separation and purification, purity reaches 85-95%.
The technology that the present invention relates to selects for use garbled pearlite as chromatographic material, the production bacterial strain of producing acellular pertussis with domestic current carries out liquid culture, adopts the purifying process one step separation and purification pertussis antigen that the present invention relates to obtain good effect.Compare with domestic and international similar technology and to have following advantage:
The present invention with garbled pearlite as chromatographic material, separation and purification pertussis antigen effectively.The pearlite low price, per kilogram 1.5-3.0 yuan, to compare with external equivalent material, price differs 20-30 doubly.And the effect of separation and purification pertussis antigen is better than external like product greatly.
2. carry out one step of column chromatography with the method that the present invention relates to and separate pertussis antigen, the productive rate of Whooping cough bag Te Shi bacillus nutrient solution per ton is brought up to 500,000 person-portions by domestic existing 6.7 ten thousand person-portions.Identify that through SDS-PAGE purity reaches 85-95%, the technical feature of this project product has met and exceeded external like product level.Come purifying acellular pertussis antigen method (U.S.Pat.No.4,997,915) to compare with external report perlite column chromatography and hydroxyapatite column chromatography, omitted the processing step of hydroxyapatite column chromatography secondary separation purifying.Saved the time, saved corresponding apparatus and expensive raw material, reduced production costs significantly.For manufacturing enterprise provide a kind of simply, the purification process of acellular pertussis vaccine fast.
3. carry out the method for isolating pertussis antigen of one step of column chromatography with garbled pearlite, with domestic existing ammonium sulfate precipitation, high salt concentration extracts and compares with the vaccine product of the super centrifugal method separation and purification of saccharose gradient acellular pertussis, and productive rate improves 6-10 doubly, and purity improves 5 times.For the production technique of reforming China's acellular pertussis vaccine has been established solid basis comprehensively.
4. the perlite column chromatography of the separation and purification pertussis antigen that the present invention relates to has that technology is easy, the advantage of good separating effect, and the technology whole process can be carried out in closed system continuously, but implementation procedure control automatically, be suitable for the requirement of large scale continuous prod.
5. the Whooping cough bag Te Shi bacillus strain that uses among the present invention is the CS bacterial strain that China uses already, belongs to 1,2,3 serotypes.Other Whooping cough bag Te Shi bacillus is fit to also can use as long as produce antigenic output.PT after the separation and purification and FHA can be mixed in proportion separately as acellular pertussis vaccine after detoxifcation, also can be mixed in proportion with other vaccines and make combined vaccine.
Description of drawings
Fig. 1 is the wash-out collection of illustrative plates among the embodiment 1
Fig. 2 is the wash-out collection of illustrative plates among the embodiment 2
Fig. 3 SDS-PAGE identifies the purity of separating pertussis antigen
Wherein 1 is lower molecular weight MARK protein sample, and molecular weight is respectively 116,66,45,35,25,18,14kDa;
2 is the FHA of perlite column chromatographic isolation and purification;
3 is the PT of perlite column chromatographic isolation and purification;
4 is the ultrafiltration and concentration liquid of the nutrient solution of Whooping cough bag Te Shi bacillus.
Embodiment
Embodiment 1:
Choose homemade edible class middling speed type perlite (Chifeng, Inner Mongol perlite factory), contain SiO
272%, Al
2O
310%, remove too small and oversized particles perlite with standard sieve, obtain granulometric range at 50-125 μ m.To the perlite particle that sieved with distilled water flushing, leave standstill the back and remove suspended substance and suspension liquid, packing into after carrying out repeatedly repeatedly, (Beijing Bin Daying creates Science and Technology Ltd. to chromatography column, internal diameter 210mm) loading height 300mm is with 5 times waters for injection (WFI) flushing perlite chromatography column.
Open the freeze dried peace bottle of Whooping cough bag Te Shi bacillus CS bacterial strain, inclusion is sprinkling upon in the sterilized water, and its dislocation is closed on the gac nutrient agar in containing doup, cultivated 72 hours down at 37 ℃ with transfer pipet.Organism gone down to posterity on the above-mentioned substratum of dislocation 37 ℃ cultivated 48 hours, so increase 3-4 generation.
Then these organisms are scraped in the seeding tank that contains 25L liquid ring dextrin substratum (modified Frohlichmedium), aeration-agitation was cultivated 36 hours.Be inoculated in the fermentor tank of 250L liquid ring dextrin substratum (modified Frohlich medium), aeration-agitation was cultivated 36 hours.The nutrient solution of Whooping cough bag Te Shi bacillus is removed thalline through high speed centrifugation, collects supernatant liquor.
Supernatant liquor is removed remaining thalline through 0.22 μ m film Sterile Filtration.The degerming rear filtrate through 10KD film bag ultrafiltration and concentration to 25L.The concentrated solution of pertussis antigen adds WFI adjustment electricity and is directed at 3-4ms/cm.At the uniform velocity by the perlite chromatography column, linear rate of flow is 150cm/hr.
With the Ultraviolet Detector of 280nm wavelength the elutriant of perlite chromatography column is carried out online detection, the ultraviolet detection curve that registering instrument is printed is seen Fig. 1.At first use the Tris-HCl of 51mM, pH=8.0 carries out wash-out to the perlite chromatography column, is 0-0.05 until the OD of elutriant value, sees peak 1 among Fig. 1.Second step was used the 51mM Tris-HCl that contains 0.1M NaCl, pH=8.0 carries out the wash-out second time to the perlite chromatography column, is 0-0.05 until the OD of elutriant value, the elutriant of collecting for second step is the Toxins, pertussis (PT) of perlite chromatography column separation and purification, sees peak 2 among Fig. 1.The 3rd step was used the 51mM Tris-HCl that contains 0.5MNaCl, pH=8.0 carries out wash-out for the third time to the perlite chromatography column, is 0-0.05 until the OD of elutriant value, the elutriant of collecting for the 3rd step is the filamentous hemagglutinin (FHA) of perlite chromatography column separation and purification, sees peak 3 among Fig. 1.
Embodiment 2:
Choose homemade edible class middling speed type perlite (Xinyang, Henan Province perlite factory), contain SiO
280%, Al
2O
320%, remove too small and oversized particles perlite with standard sieve, obtain granulometric range at 125-250 μ m.To the perlite particle that sieved with distilled water flushing, leave standstill the back and remove suspended substance and suspension liquid, packing into after carrying out repeatedly repeatedly, (Beijing Bin Daying creates Science and Technology Ltd. to chromatography column, internal diameter 210mm) loading height 300mm is with 5 times waters for injection (WFI) flushing perlite chromatography column.
Open the freeze dried peace bottle of Whooping cough bag Te Shi bacillus CS bacterial strain, inclusion is sprinkling upon in the sterilized water, and its dislocation is closed on the gac nutrient agar in containing doup, cultivated 72 hours down at 37 ℃ with transfer pipet.Organism gone down to posterity on the above-mentioned substratum of dislocation 37 ℃ cultivated 48 hours, so increase 3-4 generation.
Then these organisms are scraped in the seeding tank that contains 25L SSM synthetic medium (modified Stainer-Scholte medium), aeration-agitation was cultivated 36 hours.Be inoculated in the fermentor tank of 250L SSM synthetic medium (modified Stainer-Scholte medium), aeration-agitation was cultivated 36 hours.The nutrient solution of Whooping cough bag Te Shi bacillus is removed thalline through high speed centrifugation, collects supernatant liquor.
Supernatant liquor is removed remaining thalline through 0.22 μ m film Sterile Filtration.The degerming rear filtrate through 30KD film bag ultrafiltration and concentration to 25L.The concentrated solution of pertussis antigen adds WFI adjustment electricity and is directed at 2-3mS/cm.At the uniform velocity by the perlite chromatography column, linear rate of flow is 250cm/hr.
With the Ultraviolet Detector of 280nm wavelength the elutriant of perlite chromatography column is carried out online detection, the ultraviolet detection curve that registering instrument is printed is seen Fig. 1.At first use the Tris-HCl of 55mM, pH=7.5 carries out wash-out to the perlite chromatography column, is 0-0.05 until the OD of elutriant value, sees peak 1 among Fig. 2.Second step was used the 55mM Tris-HCl that contains 0.05M NaCl, pH=7.5 carries out the wash-out second time to the perlite chromatography column, is 0-0.05 until the OD of elutriant value, the elutriant of collecting for second step is the Toxins, pertussis (PT) of perlite chromatography column separation and purification, sees peak 2 among Fig. 2.The 3rd step was used the 55mM Tris-HCl that contains 0.4M NaCl, pH=7.5 carries out wash-out for the third time to the perlite chromatography column, is 0-0.05 until the OD of elutriant value, the elutriant of collecting for the 3rd step is the filamentous hemagglutinin (FHA) of perlite chromatography column separation and purification, sees peak 3 among Fig. 2.
Embodiment 3:
Collection liquid to three ultraviolet absorption peaks of perlite chromatography column elutriant is surveyed hemagglutinative vigor respectively.
Gather fresh gander blood 5ml, add 10ml 0.9%NaCl solution, evenly the centrifugal supernatant discarded in back adds 10ml 0.9%NaCl solution again and washes, and washes altogether 3 times.With 0.9%NaCl solution goose blood is made into 0.8% red blood corpuscle suspension again.
Add 40 μ l, 0.9% physiological saline on the hemagglutination plate, add 40 μ l testing samples again and do doubly dilution, add 40 μ l, 0.8% red blood corpuscle suspension at last, fully vibration was placed 30 minutes under the room temperature, and experimental result is listed in table 1.As can be seen from Table 1, the pertussis antigen peak 2 of perlite separation and purification, peak 3 are collected liquid and are had higher hemagglutination activity, and peak 2 is collected the liquid hemagglutination activity and reached 1: 2048 and 1: 1024; Peak 3 is collected the liquid hemagglutination activity and is reached 1: 8192 and 1: 2048.Provable thus peak 2, peak 3 are collected liquid and are had antigenic activity, are the purpose product pertussis antigen albumen of separation and purification.Peak 1 contains extremely low hemagglutination activity, is respectively 1: 8 and 1: 4.Provable thus peak 1 is collected liquid and is had extremely low antigenic activity, is foreign protein.
Table 1. absorption peak protein concn and hemagglutination activity thereof
| Peak 1 | | | ||||
| Protein concentration (μ g/ml) | Hemagglutination activity | Protein concentration (μ g/ml) | Hemagglutination activity | Protein concentration (μ g/ml) | | |
| Embodiment | ||||||
| 1 | ??1414.2 | ??1∶8 | ??165.6 | ??1∶2048 | ????190.4 | 1∶8192 |
| | ??1326.2 | ??1∶4 | ??105.7 | ??1∶1024 | ????118.3 | 1∶2048 |
Embodiment 4:
Each peak value protein solution that will obtain respectively is collected in one, identifies that with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) purity of separation pertussis antigen is seen Fig. 3.Compare as can be known with the proteic electrophoretogram of standard lower molecular weight MARK, the pertussis antigen absorption peak 1 of pearlite column chromatography for separation is a foreign protein; Peak 2 is the PT of perlite column chromatographic isolation and purification; Peak 3 is the FHA of perlite column chromatographic isolation and purification.Wherein the PT of separation and purification and FHA lipidated protein reach more than 90%.By each absorption peak protein concn in the table 1 as calculated as can be known, the average yield of PT is 21.93 μ g/ml; The average yield of FHA is 24.76 μ g/ml.
Claims (4)
1. the separation purification method of an acellular pertussis antigen protein comprises the steps:
(1) dress post: choose and contain SiO
270-80%, Al
2O
3The edible class middling speed type perlite of 10-20%, granulometric range are at 30-250 μ m, behind removal suspended substance and the suspension liquid, with water for injection flushing perlite chromatography column;
(2) sample preparation, application of sample: the nutrient solution of Whooping cough bag Te Shi bacillus is removed the microorganism collection supernatant liquor through high speed centrifugation, supernatant liquor is removed remaining thalline through the film Sterile Filtration, the degerming rear filtrate is through the 0.1-20% of 10-30KD film bag ultrafiltration and concentration to original volume, concentrated solution adds water for injection adjustment electricity and is directed at 2-4mS/cm, passes through the perlite chromatography column with linear rate of flow less than 300cm/hr;
(3) wash-out: at first use the Tris-HCl damping fluid of 50-60mM, pH=7.0-8.0 carries out wash-out to the perlite chromatography column, is 0-0.05 until the OD of elutriant value; Second step was used the 50-60mM Tris-HCl damping fluid that contains 0.05-0.15M NaCl, pH=7.0-8.0, the perlite chromatography column is carried out the secondary wash-out, is 0-0.05 until the OD of elutriant value, and the elutriant of collecting for second step is the Toxins, pertussis of perlite chromatography column separation and purification; The 3rd step was used the 50-60mM Tris-HCl damping fluid that contains 0.4-0.7M NaCl, pH=7.0-8.0, the perlite chromatography column is carried out wash-out for the third time, is 0-0.05 until the OD of elutriant value, and the elutriant of collecting for the 3rd step is the filamentous hemagglutinin of perlite chromatography column separation and purification.
2. the separation purification method of acellular pertussis antigen protein as claimed in claim 1 is characterized in that perlitic granulometric range is at 50-250 μ m in the step (1).
3. the separation purification method of acellular pertussis antigen protein as claimed in claim 1 is characterized in that the middle filtrate of step (2) is concentrated into the 10-20% of original volume.
4. the separation purification method of acellular pertussis antigen protein as claimed in claim 1 is characterized in that the middle concentrated solution of step (2) is 150-250cm/hr by the linear rate of flow of perlite chromatography column.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1314473C (en) * | 2004-07-16 | 2007-05-09 | 大连大学 | Gel column mounting method |
| CN102443572A (en) * | 2011-10-21 | 2012-05-09 | 武汉生物制品研究所有限责任公司 | Method for purifying detoxified pertussis vaccine antigen solution |
| CN102793915A (en) * | 2012-07-25 | 2012-11-28 | 天津康希诺生物技术有限公司 | Production method of acellular pertussis vaccine |
| CN105175517A (en) * | 2015-09-17 | 2015-12-23 | 北京民海生物科技有限公司 | Separation and purification method for pertussis antigen |
| CN105358572A (en) * | 2013-05-06 | 2016-02-24 | 赛诺菲 | Continuous multistep process for purifying antibodies |
-
2002
- 2002-12-03 CN CNA021536600A patent/CN1504482A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1314473C (en) * | 2004-07-16 | 2007-05-09 | 大连大学 | Gel column mounting method |
| CN102443572A (en) * | 2011-10-21 | 2012-05-09 | 武汉生物制品研究所有限责任公司 | Method for purifying detoxified pertussis vaccine antigen solution |
| CN102793915A (en) * | 2012-07-25 | 2012-11-28 | 天津康希诺生物技术有限公司 | Production method of acellular pertussis vaccine |
| CN102793915B (en) * | 2012-07-25 | 2013-10-23 | 天津康希诺生物技术有限公司 | Production method of acellular pertussis vaccine |
| CN105358572A (en) * | 2013-05-06 | 2016-02-24 | 赛诺菲 | Continuous multistep process for purifying antibodies |
| US10793622B2 (en) | 2013-05-06 | 2020-10-06 | Sanofi | Continuous multistep process for purifying antibodies |
| CN105175517A (en) * | 2015-09-17 | 2015-12-23 | 北京民海生物科技有限公司 | Separation and purification method for pertussis antigen |
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