CN102327605B - Preparation process of meningococcal polysaccharide vaccine - Google Patents
Preparation process of meningococcal polysaccharide vaccine Download PDFInfo
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Abstract
The invention discloses preparation process of meningococcal polysaccharide vaccine, which comprises the following steps that: selecting strain; establishing and transferring seed batch; producing vaccine stock solution; acquiring and sterilizing; removing thalli; precipitating and dissociating; removing nucleic acid; precipitating and collecting polysaccharide; removing foreign protein and endotoxin during the ultrafiltration membrane process; precipitating refined polysaccharide; preparing a semi-finished product; split charging and freeze drying; and preparing the vaccine finished product. The meningococcal polysaccharide vaccine produced through the preparation process is a quadrivalent vaccine, and multiple immune functions can be realized by injecting one vaccine; and the safety and each inspection parameter of the vaccine conform to the standard.
Description
Technical field
The present invention relates to the vaccine preparation field, concrete, relate to the preparation technology of tetravalence meningococcal polysaccharide vaccine.
Background technology
Epidemic cerebrospinal meningitis is the purulent meningitis that is caused by meningococcus, is called for short epidemic encephalitis.The popular of epidemic encephalitis namely was found as far back as 16th century, and Susceptible population is mainly child and teenager.Disease symptom divides plain edition and fulminant; Plain edition shows as violent headache, frequent vomiting, dysphoria and the meningeal irritation symptoms such as stiffness of the neck Kernig sign and the Brudzinski sign positive, with breathing, circulatory failure or other complication; Fulminant is more common in child patient.Onset is rapid, and the state of an illness is dangerous, such as untimely rescue, and the threat to life in 24 hours of being everlasting.The fulminant case fatality rate reaches as high as 40
~60%.Any age all can fall ill, wherein the age below 14 years old, especially child morbidity is the highest below 7 years old.Meningococcus is hidden in patient or bacillicarrier's the nasopharyngeal secretions, mainly by cough, sneeze, speak etc. by the spittle directly from air borne, enter respiratory tract and cause infection.
From the whole world, epidemic encephalitis Sporadic cases and endemicity are popular still healthy in harm humans.Its popular Regional Distribution is extremely wide, almost spreads all over each continent.The morbidity level difference of epidemic encephalitis is very large between the various countries, and is relatively high at developing country's sickness rate, the highest at African sickness rate.Now, annual 300000～350,000 epidemic encephalitis cases that occur in the whole world.The hotspot still is Africa, Asia and South America.The sickness rate in " the meningitis area " of Africa on the south the Sahara is the highest, can be up to 400/ ten ten thousand～800/ ten ten thousand in popular year.Because geography and climate reasons, epidemic season generally by the end of November to next year by the end of June between.This area has extended to Ethiopia eastwards, has expanded westerly Senegal to, relates to 18 countries, and 300,000,000 populations are arranged approximately, and also has the trend that enlarges.From 1980, this area was regularly outbreak of epidemic.In recent years, the epidemic cerebrospinal meningitis eruption and prevalence is main by the A group in past, develops into gradually A group and other group and deposits.1996, southern the Sahara African country occured once that maximum in history epidemic encephalitis is popular, and the report case load reaches more than 180,000, dead 18000 people, and Epidemic bacterial flora is take B group and C group as main.2000 and the twice eruption and prevalence cerebrospinal meningitis in area, calendar year 2001 Saudi Arabia haji, 500 many cases of falling ill altogether, wherein 230 many cases are W135 group, all the other are A group.According to WHO report, Burkina Faso outburst spring in 2002 epidemic encephalitis, morbidity 12,587 examples, dead Isosorbide-5-Nitrae 47 examples, mortality rate 11.5%.Also mainly take W135 group as main.China 1938,1949, nineteen fifty-nine, 1967,1977 five national epidemic encephalitiss occured altogether is very popular, average about 8～10 years once popular, as mainly to cause with A group meningitis diplococcus epidemic encephalitis Flow Behavior masters.Spring in 1967, sickness rate reached summit, was 4,03/,100,000, case fatality rate 5.49%.The beginning of the eighties, A group's epidemic encephalitis polysaccharide vaccine of China's development was got permission to come into operation, and preventive effect is good, and nationwide being very popular do not occur again in China after the eighties.1985 annual morbidities are that the sickness rate in 9.9/10 ten thousand, 1986～1989 is followed successively by 7.56/10 ten thousand, 3.21/10 ten thousand, 1.97/10 ten thousand, 1.32/10 ten thousand.Epidemic peak has been controlled in the use of vaccine effectively.But Sporadic cases still exists.According to regularty of epidemic, infer that China will be the popular cycle of epidemic cerebrospinal meningitis in recent years.In recent years, the popular characteristics that also have the flora drift of epidemic cerebrospinal meningitis are that A group's epidemic cerebrospinal meningitis is popular such as the U.S. in the past, are later on that B group's epidemic cerebrospinal meningitis is popular, it is popular to transfer again afterwards C group's epidemic cerebrospinal meningitis to again, and it is popular then to transfer at present Y group to.The epidemic cerebrospinal meningitis of China is popular mainly take A group as main.Along with being widely used of A group's epidemic encephalitis polysaccharide vaccine, the crowd has certain immunity to A group's epidemic encephalitis diplococcus, the epidemic cerebrospinal meningitis ratio that is caused by A group's epidemic encephalitis diplococcus is year by year downward trend, and the epidemic cerebrospinal meningitis case that is caused by C group and other groups epidemic encephalitis diplococcus rises year by year.The local outburst idol of C group's epidemic cerebrospinal meningitis recent years has generation, 11 cities, Anhui Province in January in last Dec to this year that C group's epidemic cerebrospinal meningitis case 60 examples, dead 8 examples occur altogether.4 routine C group's epidemic cerebrospinal meningitis also occur the end of last year in Fujian Province.From fashion trend, C group's epidemic cerebrospinal meningitis is sent out the case load increasing year by year, and the morbidity scope enlarges gradually.In addition, do not get rid of the popular W135 group's epidemic cerebrospinal meningitis of African country, the probability of the epidemic cerebrospinal meningitis such as Y group input China that the U.S. is popular yet.All have the popular band of epidemic cerebrospinal meningitis in Africa and Asia, the past is also all with A group's epidemic cerebrospinal meningitis Flow Behavior master, but the popular band of African epidemic cerebrospinal meningitis has transferred W135 group's Flow Behavior master to, the phenomenon that A group's Flow Behavior is auxiliary.Along with the increase of international interchange, flow of personnel is frequent, and the probability of popular Y type and W135 type epidemic encephalitis input China is also increasing abroad.Therefore, development (A+C+Y+W135) tetravalence meningococcal polysaccharide vaccine is significant.
Summary of the invention
Technical problem to be solved by this invention provides the preparation technology of tetravalence meningococcal polysaccharide vaccine, and the tetravalence meningococcal polysaccharide vaccine that this preparation technology can prepare is tetravalent vaccine, and injecting a pin namely has panimmunity effectiveness; And this vaccine safety and every verification parameters be conformance with standard all.
The present invention solves the problems of the technologies described above the technical scheme that adopts: the preparation technology of tetravalence meningococcal polysaccharide vaccine, it is characterized in that, and comprise the steps:
(1) select strain: producing with strain is A group meningitis Neisseria gonorrhoeae strain, C group meningitis Neisseria gonorrhoeae strain, Y group meningitis Neisseria gonorrhoeae strain and W135 group meningitis Neisseria gonorrhoeae strain;
(2) foundation of seed lot and going down to posterity: after 1 primordial seed freeze-drying lactobacillus opened, be seeded on the 10% SBA culture medium, be put in 35～37 ℃, 16～20 hours biographies of 5～10% carbon dioxide environments cultivation second filial generation, second filial generation culture is adopted in the germfree defatted milk, and mixing lyophilizing preparation is main for seed lot; After 1 main seed lot strain being opened, be seeded on the 10% SBA culture medium again, be put in 35～37 ℃, 16～20 hours biographies of 5～10% carbon dioxide environments cultivation second filial generation, lyophilizing is the work seed lot; Carry out biological characteristics calibrating after described main seed lot and the lyophilizing of described work seed lot strain, it is consistent that the biological characteristics of described main seed lot and described work seed lot strain and primordial seed are criticized strain;
(3) produce vaccinogen liquid: open 1 of work seed lot strain, be inoculated in 10% SBA culture medium and prepare the first generation, with the 1st generation bacterial classification inoculation in improvement half comprehensive solid medium, continue the 2nd generation that goes down to posterity, with the 2nd generation bacterial classification inoculation in improvement half comprehensive solid medium, continued to go down to posterity the 3rd generation, with the 3rd generation bacterial classification inoculation in improvement half comprehensive fluid medium, continued to go down to posterity the 4th generation, the extremely large tank of the 4th generation bacterial classification inoculation is cultivated in continuation, it was the 5th generation that large tank is cultivated, and the large tank in the 5th generation is cultivated and is improvement half comprehensive fluid medium; In the 5th generation,, the culture of large tank cultivation was produced vaccinogen liquid, and per 1 work seed is used for 1 batch of vaccinogen liquid production; Large tank was cultivated and was adopted the culture tank liquid culture described the 5th generation;
(4) results and sterilization: stop cultivating in the later stage of exponential phase or the results in early stage of resting stage, pure bacterium test is carried out in sampling when stopping cultivating, in the culture fluid of results, add after qualified formalin to final concentration by volume mark count 1%, sterilized 30 minutes;
(5) remove thalline: the culture fluid that will sterilize is centrifugal through 14000rpm tubular type continuous flow centrifuge, and centrifugal flow velocity is 1500～1600ml/min, collects supernatant;
(6) precipitate, dissociate: the supernatant that described step (5) is collected, add 10% cetyl trimethyl ammonium bromide to final concentration by volume mark count 0.1%, abundant mixing, A group was left standstill 3～5 hours, Y group was left standstill 5～8 hours, and C, W135 group were left standstill 8～12 hours; Centrifugal with 14000rpm tubular type continuous flow centrifuge, centrifugal flow velocity is 2000～2500ml/min, centrifugal rear collecting precipitation thing; With the precipitate 1mol/LCaCl that collects
2Grind evenly, obtain the polysaccharides compound of mixing; The polysaccharides compound of described mixing is collected in the bottle, is placed on the magnetic stirring apparatus, stirred 1 hour, polysaccharide and cetyl trimethyl ammonium bromide are fully dissociated;
(7) enucleation acid: in polysaccharides compound, add 2～8 ℃ of ethanol of 95% that cooled off to final concentration by volume mark count 25%, shake up, deposit for 2～8 ℃, A group's polysaccharide leaves standstill deposits 1～3 hour, C group and W135 group are left standstill and are deposited 16～20 hours, and Y group's polysaccharide leaves standstill deposits 10～14 hours; Centrifugal 30 minutes of 4000rpm goes precipitation, collects supernatant;
(8) precipitation is collected polysaccharide: add 2～8 ℃ and cooled off 95% ethanol to ultimate density and be in above-mentioned supernatant: A group by volume mark counts 80%; C group, Y group and W135 group by volume mark count 75%, and shake well left standstill 1～2 hour after precipitation occurs; Centrifugal 5 minutes collecting precipitations of 4000rpm; More than 2 times, collect polysaccharide precipitation with dehydrated alcohol and washing with acetone, compressed air is air-dry; Dry sediment is the polysaccharide semifinished product; Be kept at-20 ℃ or following, to be purified;
(9) the ultrafilter membrane process is removed foreign protein and endotoxin: the polysaccharide semifinished product that described step (8) is obtained is dissolved in the 1/10 saturated neutral sodium acetate solution, A group makes its concentration reach 15～20mg/ml, and C group, Y group and W135 group make its concentration reach 10～15mg/ml; Be that 6000 daltonian ultrafilter membranes carry out ultrafiltration with molecular cut off, be removed thereby foreign protein and endotoxin are trapped within on the ultrafilter membrane that the collection ultrafiltrate is harvest liquid;
(10) precipitate smart polysaccharide: add 95% ethanol to final concentration in the harvest liquid that step (9) is collected and be A group by volume mark count 80%, C group, Y group and W135 group by volume mark count 75%; Fully shake up, put 2～8 ℃ in freezer 1 hour, 10 minutes centrifugal collecting precipitates of 4000rpm; More than 2 times, collect polysaccharide precipitation with dehydrated alcohol and washing with acetone, compressed air is air-dry, and dry sediment is smart polysaccharide, is kept at-20 ℃ or following; Leaching process is carrying out below 15 ℃.Dissolve smart polysaccharide with sterile water for injection, be stock solution after aseptic filtration, 0.2 μ m integrity of filtration membranes pass the test before and after requiring to filter is examined and determine after the stock solution sampling;
(11) semi-finished product preparation: be semi-finished product after merging the stock solution more than single batch or two batches, be diluted to aseptic apyrogeneity lactose and sterilized water for injection and contain A group, C group, Y group, W135 group's polysaccharide and be respectively 100.0 μ g/ml, lactose is 20mg/ml; Filter through 0.2 μ m immediately after the dilution, the filter samples censorship is in 2～8 ℃ of preservations;
(12) packing, lyophilizing, preparation vaccine finished product: above-mentioned semi-finished product adopt the packing of washing, drying and pouring linkage unit, injection bottles made of glass tubes splendid attire, the packing specification is the 0.5ml/ bottle, divide automatic bittern adding butyl rubber bung in the process of assembling, after packing is finished, lyophilizing in 6 hours after packing; Lyophilizing is finished and is namely carried out the vacuum tamponade and roll lid; Finish the goods that roll lid and carry out fluoroscopic examination and labeling; The goods of finishing labeling are the freeze dried vaccine finished product, send 2～8 ℃ of freezers to preserve.
Further, to reach the determination methods in the early stage of later stage of exponential phase or resting stage be that bacterial concentration no longer increases, and reaches 16,000,000,000/ml or pH bottom out to 7.0～7.5 to described step (4) yeast culture.
Further, the condition of culture of the large tank cultivation of described step (3): the cultivation base unit weight is 300～600L, and ventilation is 10～30m
3/ h, mixing speed is 200～350rpm, keeps pH 6.70～7.40 with 20%NaOH solution; In incubation, carry out bacterial concentration and measure and gram stain microscopy, as finding pollution microbes, 121 ℃, discarded after the 30min sterilization treatment.
Further, the prescription of described 10% SBA culture medium is: in 100ml, contain Sanguis caprae seu ovis 10ml; Agar culture medium 100 ml.
Further, the prescription of described improvement half comprehensive solid medium is: in 1000ml, contain
50% hydrochloric acid casein hydrolyzed solution or hydrochloric acid hydrolysis casein are calculated as 1300mg by total nitrogen; 2% hydrochloric acid casein hydrolyzed solution is calculated as 1300mg by total nitrogen; Yeast dialysis solution 50ml; Starch 5g; Potassium dihydrogen phosphate 1g; Magnesium sulfate 0.6g; Glucose 2g; Agar 26g.
Further, described improvement half comprehensive fluid medium is made of first liquid and second liquid, take the prescription of 10000ml as: described first liquid comprises that 50% hydrochloric acid casein hydrolyzed solution or hydrochloric acid hydrolysis casein are calculated as 13000mg by total nitrogen; Yeast dialysis solution 100ml; Potassium chloride 10g; Pidolidone sodium 10g; Ammonium chloride 12.5g; Disodium hydrogen phosphate dodecahydrate 7.5g; Sodium dihydrogen phosphate 2.5g; 10% cystine solution 3ml; Described second liquid comprises magnesium sulfate 6g and glucose 55g.
In sum, the present invention compared with prior art, its advantage is: the present invention adopted the ultrafilter membrane process implementation one-step method remove simultaneously except foreign protein and endotoxic purpose; Not only guarantee purity and the immunocompetence conformance with standard of the ACYW135 tetravalence meningococcal polysaccharide vaccine that the present invention prepares, reduced preparation technology's Financial cost.
The specific embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited to this.
Embodiment 1The preparation of ACYW135 tetravalence meningococcal polysaccharide vaccine
The preparation technology of tetravalence meningococcal polysaccharide vaccine comprises the steps:
(1) select strain: producing with strain is A group meningitis Neisseria gonorrhoeae strain, C group meningitis Neisseria gonorrhoeae strain, Y group meningitis Neisseria gonorrhoeae strain and W135 group meningitis Neisseria gonorrhoeae strain;
(2) foundation of seed lot and going down to posterity: produce and to set up the seed lot system with strain.Primordial seed is criticized and should be identified its record, history, source and biological characteristics.After 1 primordial seed freeze-drying lactobacillus unlatching, be seeded on the 10% SBA culture medium, being put in 35～37 ℃, 5～10% carbon dioxide environments cultivated 16～20 hours, pass the second filial generation under 35～37 ℃ of conditions, it is main for seed lot that second filial generation culture is adopted in the germfree defatted milk mixing lyophilizing preparation.After 1 main seed lot strain being opened again, adopt above-mentioned the same terms to cultivate and reach the second filial generation, lyophilizing is the work seed lot after the amplification.All must carry out the biological characteristics calibrating after main seed lot and the lyophilizing of work seed lot strain, the various biological characteristicses of main seed lot and the seed lot strain of working should to criticize strain consistent with primordial seed.Main seed lot strain is for the production of the work seed lot, and the work seed lot is for the production of vaccine.Main seed lot is criticized by primordial seed and is opened the rear 2 generations preparation that passes, biography 2 generations preparation work seed lot after main seed lot is opened; Biography 4 generations preparation was produced and is used seed after the work seed lot was opened.After opening, the work seed lot must not surpass for 5 generations to inoculation fermentation tank subculture number of times.
(3) produce vaccinogen liquid: open 1 of work seed lot strain, be inoculated in 2 of 10% SBA culture medium, put 35～37 ℃, 5～10% carbon dioxide environments were cultivated 16～20 hours, smear for microscopic examination is qualified, with the 1st generation bacterial classification inoculation in 4～6 bottles of improvement half comprehensive solid mediums, continue the 2nd generation that goes down to posterity, in 35～37 ℃, 5～10% carbon dioxide environments were cultivated 10～18 hours, after smear for microscopic examination is qualified with the 2nd generation bacterial classification inoculation in 20～25 bottles of improvement half comprehensive solid mediums, continued to go down to posterity the 3rd generation, cultivated 10～18 hours in 35～37 ℃, after smear for microscopic examination is qualified with the 3rd generation bacterial classification inoculation in improvement half comprehensive fluid medium 30～40 L, continued to go down to posterity the 4th generation, continuation is with the 4th generation, in 35～37 ℃ of deep ventilation stir culture 3～8 hours, smear for microscopic examination and bacterial concentration measure qualified after just the 4th generation strain be seeded to large tank with seed and cultivate as producing.The condition of culture that described large tank is cultivated is: adopt the culture tank liquid culture, culture medium is improvement half comprehensive fluid medium, and the cultivation base unit weight is 300～600L.After the 4th generation production of large tank inoculation of medium is with seed, the deep ventilation stir culture.Ventilation is 10～30m
3/ h, mixing speed is 200～350rpm.Keep pH in 6.70～7.40 scopes with 20%NaOH solution.In incubation, carry out that bacterial concentration is measured and gram stain microscopy, as finding pollution microbes, answer after 121 ℃ of 30min sterilization treatment discarded.The culture that large tank is cultivated is produced vaccinogen liquid, and per 1 work seed is used for 1 batch of vaccinogen liquid production.
(4) results and sterilization: stop cultivating in the later stage of exponential phase or the results in early stage of resting stage, pure bacterium test is carried out in sampling when stopping cultivating, in the culture fluid of results, add after qualified formalin to final concentration by volume mark count 1%, sterilized 30 minutes; Stop cultivating in the later stage of exponential phase or the results in early stage of resting stage, A group, C group and W135 group cultivate and are generally 6.5 ± 1 hours, Y group cultivates and is generally 11 ± 3 hours, when bacterial concentration no longer increases, reaching 16,000,000,000/ml or pH bottom out to 7.0～7.5 o'clock can stop cultivating, pure bacterium test is carried out in sampling when stopping cultivating, and adds formalin to final concentration 1%(V/V in the culture fluid of results), sterilized 30 minutes.
(5) remove thalline: the culture fluid that will sterilize is centrifugal through 14000rpm tubular type continuous flow centrifuge, and centrifugal flow velocity is 1500～1600ml/min, collects supernatant.
(6) precipitate, dissociate: the supernatant that described step (5) is collected, add 10% cetyl trimethyl ammonium bromide to final concentration by volume mark count 0.1%, abundant mixing, A group was left standstill 3～5 hours, Y group was left standstill 5～8 hours, and C, W135 group were left standstill 8～12 hours; Centrifugal with 14000rpm tubular type continuous flow centrifuge, centrifugal flow velocity is 2000～2500ml/min, centrifugal rear collecting precipitation thing; With the precipitate 1mol/LCaCl that collects
2Grind evenly, obtain the polysaccharides compound of mixing; The polysaccharides compound of described mixing is collected in the bottle, is placed on the magnetic stirring apparatus, stirred 1 hour, polysaccharide and cetyl trimethyl ammonium bromide are fully dissociated.
(7) enucleation acid: in polysaccharides compound, add 2～8 ℃ of ethanol of 95% that cooled off to final concentration by volume mark count 25%, shake up, deposit for 2～8 ℃, A group's polysaccharide leaves standstill deposits 1～3 hour, C group and W135 group are left standstill and are deposited 16～20 hours, and Y group's polysaccharide leaves standstill deposits 10～14 hours; Centrifugal 30 minutes of 4000rpm goes precipitation, collects supernatant;
(8) precipitation is collected polysaccharide: add 2～8 ℃ and cooled off 95% ethanol to ultimate density and be in above-mentioned supernatant: A group by volume mark counts 80%; C group, Y group and W135 group by volume mark count 75%, and shake well left standstill 1～2 hour after precipitation occurs; Centrifugal 5 minutes collecting precipitations of 4000rpm; More than 2 times, collect polysaccharide precipitation with dehydrated alcohol and washing with acetone, compressed air is air-dry; Dry sediment is the polysaccharide semifinished product; Be kept at-20 ℃ or following, to be purified.
(9) the ultrafilter membrane process is removed foreign protein and endotoxin: the polysaccharide semifinished product that described step (8) is obtained is dissolved in the 1/10 saturated neutral sodium acetate solution, A group makes its concentration reach 15～20mg/ml, and C group, Y group and W135 group make its concentration reach 10～15mg/ml; Be that 6000 daltonian ultrafilter membranes carry out ultrafiltration with molecular cut off, be removed thereby foreign protein and endotoxin are trapped within on the ultrafilter membrane that the collection ultrafiltrate is harvest liquid.
Described ultrafiltration operating pressure is 200kPa, the ultrafilter membrane process implementation one-step method remove foreign protein and endotoxic effect, so processing step is less, and has reduced the destruction to antigen.Foreign protein content difference＜10mg/g in A, the C group's polysaccharide after the ultrafilter membrane process is processed, foreign protein content＜35.6mg/g in Y group's polysaccharide, foreign protein content＜35.2mg/g in W135 group's polysaccharide, A, C, Y and W135 group's polysaccharide endotoxin content be＜12 EU/ μ g respectively, the antigen response rate: A group's polysaccharide＞65.2%, C group's polysaccharide＞75.5%, Y and W135 group's polysaccharide difference＞80%.
(10) precipitate smart polysaccharide: add 95% ethanol to final concentration in the harvest liquid that step (9) is collected and be A group by volume mark count 80%, C group, Y group and W135 group by volume mark count 75%; Fully shake up, put 2～8 ℃ in freezer 1 hour, 4000rpm, 10 minutes centrifugal collecting precipitates; More than 2 times, collect polysaccharide precipitation with dehydrated alcohol and washing with acetone, compressed air is air-dry, and dry sediment is smart polysaccharide, is kept at-20 ℃ or following; Leaching process is carrying out below 15 ℃.Dissolve smart polysaccharide with sterile water for injection, be stock solution after aseptic filtration, 0.2 μ m integrity of filtration membranes pass the test before and after requiring to filter is examined and determine after the stock solution sampling;
(11) semi-finished product preparation: be semi-finished product after merging the stock solution more than single batch or two batches, be diluted to aseptic apyrogeneity lactose and sterilized water for injection and contain A group, C group, Y group, W135 group's polysaccharide and be respectively 100.0 μ g/ml, lactose is 20mg/ml; Filter through 0.2 μ m immediately after the dilution, the filter samples censorship is in 2～8 ℃ of preservations;
(12) packing, lyophilizing, preparation vaccine finished product: above-mentioned semi-finished product adopt the packing of washing, drying and pouring linkage unit, injection bottles made of glass tubes splendid attire, the packing specification is the 0.5ml/ bottle, divide automatic bittern adding butyl rubber bung in the process of assembling, after packing is finished, lyophilizing in 6 hours after packing; Lyophilizing is finished and is namely carried out the vacuum tamponade and roll lid; Finish the goods that roll lid and carry out fluoroscopic examination and labeling; The goods of finishing labeling are the freeze dried vaccine finished product, send 2～8 ℃ of freezers to preserve.Described fluoroscopic examination adopts ocular estimate by thoroughly inspection of bottle, rejects outward appearance substandard products such as taking off lid, loading amount, empty bottle, broken bottle, foreign body, atrophy.The fluoroscopic examination ambient temperature must not be higher than 30 ℃, has checked in time to carry out labeling.Adopt labelling machine to carry out labeling, every bottle of vaccine sticks 1 on (ACYW135) tetravalence meningococcal polysaccharide vaccine label, and the goods of finishing labeling in time send 2～8 ℃ of freezers preservations to be packaged.Whole fluoroscopic examination and labeling process environment temperature must not be higher than 30 ℃.
Vaccine preparation technology's of the present invention stock solution is produced yield: open 1 work seed and can produce rough polysaccharide by the large tank cultivation of every 100L amount: A group 16～51g, C group 9～29g, Y group 2～6g, W135 group 3～9g.
The yield of every batch of smart polysaccharide of vaccine preparation technology of the present invention=(receiving to get smart polysaccharide amount)/(the crude polysaccharides amount that drops into before refining) * 100%, each group of yield of the extraction purge process from crude polysaccharides to smart polysaccharide is respectively: A group 35～59%, C group 41～57%, Y group 35～79%, W135 group 29～77%.
Embodiment 2The production culture medium
The culture medium that above-mentioned preparation process uses is as follows:
The preparation 1st generation of producing with seed is 10% SBA culture medium, and 2nd generation to the 3 is on behalf of improvement half comprehensive solid medium, and it is improvement half comprehensive fluid medium that the production in the 4th generation is cultivated with the large tank in the preparation of seed strain and the 5th generation.The improvement of production usefulness half comprehensive fluid medium does not contain the composition that can form with the cetyl trimethyl ammonium bromide that adds precipitation, does not contain harmful or other anaphylactogen materials.
A 10% SBA culture medium
| Name of material | Prescription (take 100ml as example) |
| Sanguis caprae seu ovis | 10ml |
| Agar culture medium | 100 ml |
The prescription (take 1000ml as example) of b improvement half comprehensive solid medium sees the following form:
| Name of material | Prescription (take 1000ml as example) |
| 50% hydrochloric acid casein hydrolyzed solution or hydrochloric acid hydrolysis casein | 1300mg (pressing total nitrogen calculates) |
| 2% hydrochloric acid casein hydrolyzed solution | 1300mg (pressing total nitrogen calculates) |
| The yeast dialysis solution | 50ml |
| Starch | 5g |
| Potassium dihydrogen phosphate | 1g |
| Magnesium sulfate | 0.6g |
| Glucose | 2g |
| Agar | 26g |
The prescription (take 10000ml as example) of c epidemic encephalitis half comprehensive fluid medium sees the following form:
Embodiment 3The calibrating of seed lot
All must carry out the biological characteristics calibrating after the main seed lot of technique of the present invention and the lyophilizing of work seed lot strain, the various biological characteristicses of main seed lot and the seed lot strain of working should to criticize strain consistent with primordial seed.Described biological characteristics calibrating content and correlation method are as follows:
A form and cultural character: bacterial classification inoculation is containing on the 10% SBA culture medium, and meningococcus is not grown at 25 ℃.Cultivated 16～20 hours under 35～37 ℃, 5～10% carbon dioxide environments, grow smooth, moistening, linen bacterium colony, lawn easily takes off, and presents even suspension in 0.85% sodium chloride solution.Dyeing microscopic examination is gram-negative diplococci, monococcus.
B biochemical characteristic: be inoculated in glucose, maltose, lactose, mannitol, fructose and sucrose biochemical reaction pipe, under 35～37 ℃ of conditions, cultivated 5～7, A, C, Y, W135 group's strain fermentation glucose, maltose produce not aerogenesis of acid, azymic lactose, mannitol, fructose and sucrose.
C serological test: will be suspended in 0.85% sodium chloride solution (containing 0.5% formaldehyde) at 35～37 ℃ of lawns of cultivating 16～20 hours, or after 56 ℃ of heating sterilization in 30 minutes, make every l ml contain bacterium 1.0 * 10
9～2.0 * 10
9Do quantitative agglutination with same group's serum, place 35～37 ℃ and spend the night, place again 2 hours observed results of room temperature next day, take the high dilution of the serum of the visible clear agglutination phenomenons of naked eyes (+) as agglutination titer, described agglutination titer must reach former serum titer half can.
Embodiment 4The calibrating of vaccine finished product
(1) discrimination test: adopt immune agar double immunodiffusion method, the vaccine finished product that the present invention prepares forms obvious precipitation line with A, C, Y, W135 group meningitis cocci antibody respectively.
(2) physical appearance: be the white loose body, add rapidly dissolving behind the following PBS, should be clear liquid after the dissolving, foreign.Described PBS prescription sees the following form:
| Name of material | Prescription (take 1000ml as example) |
| Sodium chloride | 8.5g |
| Sodium hydrogen phosphate | 0.5g |
| Potassium dihydrogen phosphate | 0.17g |
(3) chemical assay:
A moisture: be not higher than 3.0%.
The b determination of polysaccharide: adopt quantitative immuno-chemical method to measure, per 1 human dosage (0.5ml) contains A group's polysaccharide 35～66 μ g, and C group's polysaccharide is 35～65 μ g, and Y group's polysaccharide is 35～65 μ g, and W135 group's polysaccharide is 35～65 μ g.
The c molecular size is measured
Per 5 batches of vaccines are taken out at least 1 batch and are checked the polysaccharide molecule size.A, C, Y and W135 group's polysaccharide Kd value difference＜0.4.The Kd value is less than 0.5 eluent polysaccharide recovery: A group's polysaccharide＞65.2%, C group's polysaccharide＞75.5%, Y and W135 group's polysaccharide respectively＞80%.
The d sterility test
Adopt the growing state of direct inoculation viewing test bacterium.
The e abnormal toxicity tests
The Cavia porcellus injected dose is 5ml (10 personal dosage), and injected in mice dosage is the personal dosage of 0.5 ml(1), up to specification.
Experiment 1
Therefore, the vaccine of the present invention's preparation is qualified.
Experiment 2
Result's demonstration of experiment 1 and experiment 2, therefore the observation period judges that the vaccine that the present invention prepares is safe to all healthy survival and the body weight increases of rear animal.
The f pyrogen test
Injected dose is injected 0.1 μ g polysaccharide by the every 1Kg of rabbit body weight, and is up to specification.
Experiment 3
Therefore, the vaccine of the present invention's preparation is qualified.
As mentioned above, just can realize preferably the present invention.
Claims (6)
1. the preparation technology of tetravalence meningococcal polysaccharide vaccine is characterized in that, comprises the steps:
(1) select strain: producing with strain is A group meningitis Neisseria gonorrhoeae strain, C group meningitis Neisseria gonorrhoeae strain, Y group meningitis Neisseria gonorrhoeae strain and W135 group meningitis Neisseria gonorrhoeae strain;
(2) foundation of seed lot and going down to posterity: after 1 primordial seed freeze-drying lactobacillus opened, be seeded on the 10% SBA culture medium, be put in 35～37 ℃, 16～20 hours biographies of 5～10% carbon dioxide environments cultivation second filial generation, second filial generation culture is adopted in the germfree defatted milk, and mixing lyophilizing preparation is main for seed lot; After 1 main seed lot strain being opened, be seeded on the 10% SBA culture medium again, be put in 35～37 ℃, 16～20 hours biographies of 5～10% carbon dioxide environments cultivation second filial generation, lyophilizing is the work seed lot; Carry out biological characteristics calibrating after described main seed lot and the lyophilizing of described work seed lot strain, it is consistent that the biological characteristics of described main seed lot and described work seed lot strain and primordial seed are criticized strain;
(3) produce vaccinogen liquid: open 1 of work seed lot strain, be inoculated in 10% SBA culture medium and prepare the first generation, with the 1st generation bacterial classification inoculation in improvement half comprehensive solid medium, continue the 2nd generation that goes down to posterity, with the 2nd generation bacterial classification inoculation in improvement half comprehensive solid medium, continued to go down to posterity the 3rd generation, with the 3rd generation bacterial classification inoculation in improvement half comprehensive fluid medium, continued to go down to posterity the 4th generation, the extremely large tank of the 4th generation bacterial classification inoculation is cultivated in continuation, it was the 5th generation that large tank is cultivated, and the large tank in the 5th generation is cultivated and is improvement half comprehensive fluid medium; In the 5th generation,, the culture of large tank cultivation was produced vaccinogen liquid, and per 1 work seed is used for 1 batch of vaccinogen liquid production; Large tank was cultivated and was adopted the culture tank liquid culture described the 5th generation;
(4) results and sterilization: stop cultivating in the later stage of exponential phase or the results in early stage of resting stage, pure bacterium test is carried out in sampling when stopping cultivating, in the culture fluid of results, add after qualified formalin to final concentration by volume mark count 1%, sterilized 30 minutes;
(5) remove thalline: the culture fluid that will sterilize is centrifugal through 14000rpm tubular type continuous flow centrifuge, and centrifugal flow velocity is 1500～1600ml/min, collects supernatant;
(6) precipitate, dissociate: the supernatant that described step (5) is collected, add 10% cetyl trimethyl ammonium bromide to final concentration by volume mark count 0.1%, abundant mixing, A group was left standstill 3～5 hours, Y group was left standstill 5～8 hours, and C, W135 group were left standstill 8～12 hours; Centrifugal with 14000rpm tubular type continuous flow centrifuge, centrifugal flow velocity is 2000～2500ml/min, centrifugal rear collecting precipitation thing; With the precipitate 1mol/LCaCl that collects
2Grind evenly, obtain the polysaccharides compound of mixing; The polysaccharides compound of described mixing is collected in the bottle, is placed on the magnetic stirring apparatus, stirred 1 hour, polysaccharide and cetyl trimethyl ammonium bromide are fully dissociated;
(7) enucleation acid: in polysaccharides compound, add 2～8 ℃ of ethanol of 95% that cooled off to final concentration by volume mark count 25%, shake up, deposit for 2～8 ℃, A group's polysaccharide leaves standstill deposits 1～3 hour, C group and W135 group are left standstill and are deposited 16～20 hours, and Y group's polysaccharide leaves standstill deposits 10～14 hours; Centrifugal 30 minutes of 4000rpm goes precipitation, collects supernatant;
(8) precipitation is collected polysaccharide: add 2～8 ℃ and cooled off 95% ethanol to ultimate density and be in above-mentioned supernatant: A group by volume mark counts 80%; C group, Y group and W135 group by volume mark count 75%, and shake well left standstill 1～2 hour after precipitation occurs; Centrifugal 5 minutes collecting precipitations of 4000rpm; More than 2 times, collect polysaccharide precipitation with dehydrated alcohol and washing with acetone, compressed air is air-dry; Dry sediment is the polysaccharide semifinished product; Be kept at-20 ℃ or following, to be purified;
(9) the ultrafilter membrane process is removed foreign protein and endotoxin: the polysaccharide semifinished product that described step (8) is obtained is dissolved in the 1/10 saturated neutral sodium acetate solution, A group makes its concentration reach 15～20mg/ml, and C group, Y group and W135 group make its concentration reach 10～15mg/ml; Be that 6000 daltonian ultrafilter membranes carry out ultrafiltration with molecular cut off, be removed thereby foreign protein and endotoxin are trapped within on the ultrafilter membrane that the collection ultrafiltrate is harvest liquid;
(10) precipitate smart polysaccharide: add 95% ethanol to final concentration in the harvest liquid that step (9) is collected and be A group by volume mark count 80%, C group, Y group and W135 group by volume mark count 75%; Fully shake up, put 2～8 ℃ in freezer 1 hour, 10 minutes centrifugal collecting precipitates of 4000rpm; More than 2 times, collect polysaccharide precipitation with dehydrated alcohol and washing with acetone, compressed air is air-dry, and dry sediment is smart polysaccharide, is kept at-20 ℃ or following; Leaching process is carrying out below 15 ℃; Dissolve smart polysaccharide with sterile water for injection, be stock solution after aseptic filtration, 0.2 μ m integrity of filtration membranes pass the test before and after requiring to filter is examined and determine after the stock solution sampling;
(11) semi-finished product preparation: be semi-finished product after merging the stock solution more than single batch or two batches, be diluted to aseptic apyrogeneity lactose and sterilized water for injection and contain A group, C group, Y group, W135 group's polysaccharide and be respectively 100.0 μ g/ml, lactose is 20mg/ml; Filter through 0.2 μ m immediately after the dilution, the filter samples censorship is in 2～8 ℃ of preservations;
(12) packing, lyophilizing, preparation vaccine finished product: above-mentioned semi-finished product adopt the packing of washing, drying and pouring linkage unit, injection bottles made of glass tubes splendid attire, the packing specification is the 0.5ml/ bottle, divide automatic bittern adding butyl rubber bung in the process of assembling, after packing is finished, lyophilizing in 6 hours after packing; Lyophilizing is finished and is namely carried out the vacuum tamponade and roll lid; Finish the goods that roll lid and carry out fluoroscopic examination and labeling; The goods of finishing labeling are the freeze dried vaccine finished product, send 2～8 ℃ of freezers to preserve.
2. the preparation technology of tetravalence meningococcal polysaccharide vaccine according to claim 1, it is characterized in that, the determination methods that described step (4) yeast culture reaches the early stage of later stage of exponential phase or resting stage is, bacterial concentration no longer increases, and reaches 16,000,000,000/ml or pH bottom out to 7.0～7.5.
3. the preparation technology of tetravalence meningococcal polysaccharide vaccine according to claim 2 is characterized in that,
The condition of culture that the large tank of described step (3) is cultivated: the cultivation base unit weight is 300～600L, and ventilation is 10～30m
3/ h, mixing speed is 200～350rpm, keeps pH 6.70～7.40 with 20%NaOH solution; In incubation, carry out bacterial concentration and measure and gram stain microscopy, as finding pollution microbes, 121 ℃, discarded after the 30min sterilization treatment.
4. the preparation technology of tetravalence meningococcal polysaccharide vaccine according to claim 3 is characterized in that, the prescription of described 10% SBA culture medium is: in 100ml, contain Sanguis caprae seu ovis 10ml; Agar culture medium 100 ml.
5. the preparation technology of each described tetravalence meningococcal polysaccharide vaccine is characterized in that according to claim 1-4,
The prescription of described improvement half comprehensive solid medium is: in 1000ml, contain
50% hydrochloric acid casein hydrolyzed solution or hydrochloric acid hydrolysis casein are calculated as 1300mg by total nitrogen;
2% hydrochloric acid casein hydrolyzed solution is calculated as 1300mg by total nitrogen;
Yeast dialysis solution 50ml;
Starch 5g;
Potassium dihydrogen phosphate 1g;
Magnesium sulfate 0.6g;
Glucose 2g;
Agar 26g.
6. the preparation technology of each described tetravalence meningococcal polysaccharide vaccine is characterized in that according to claim 1-4, and described improvement half comprehensive fluid medium is made of first liquid and second liquid, take the prescription of 10000ml as:
Described first liquid comprises
50% hydrochloric acid casein hydrolyzed solution or hydrochloric acid hydrolysis casein are calculated as 13000mg by total nitrogen;
Yeast dialysis solution 100ml;
Potassium chloride 10g;
Pidolidone sodium 10g;
Ammonium chloride 12.5g;
Disodium hydrogen phosphate dodecahydrate 7.5g;
Sodium dihydrogen phosphate 2.5g;
10% cystine solution 3ml;
Described second liquid comprises magnesium sulfate 6g and glucose 55g.
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| CN2011102457183A CN102327605B (en) | 2011-08-25 | 2011-08-25 | Preparation process of meningococcal polysaccharide vaccine |
| PCT/CN2012/080398 WO2013026385A1 (en) | 2011-08-25 | 2012-08-21 | Preparation method for quadrivalent meningococcal polysaccharide vaccine |
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| CN102327605B (en) * | 2011-08-25 | 2013-01-30 | 成都康华生物制品有限公司 | Preparation process of meningococcal polysaccharide vaccine |
| CN102631673A (en) * | 2012-04-17 | 2012-08-15 | 金宇保灵生物药品有限公司 | Method for foot-and-mouth disease vaccine concentration and purification |
| CN103721249B (en) * | 2014-01-16 | 2015-06-24 | 华兰生物工程股份有限公司 | Meningitis vaccine and preparation method thereof |
| CN105039227A (en) * | 2015-08-31 | 2015-11-11 | 成都欧林生物科技股份有限公司 | A-group Neisseria meningitidis cultivation method |
| CN105641690A (en) * | 2016-02-03 | 2016-06-08 | 成都康华生物制品有限公司 | Preparation method of tetravalent epidemic meningitis-Hib combined vaccine |
| CN106191164A (en) * | 2016-08-02 | 2016-12-07 | 成都欧林生物科技股份有限公司 | A kind of C group meningitis cocci high density produces the method for sugar |
| CN106367450A (en) * | 2016-08-29 | 2017-02-01 | 成都欧林生物科技股份有限公司 | Group A meningococcal capsular polysaccharide preparation method capable of reducing phenol dosage in purification step |
| CN106191166A (en) * | 2016-08-29 | 2016-12-07 | 成都欧林生物科技股份有限公司 | The technique being beneficial to Polysaccharide A DH derivant quality control |
| CN106282262A (en) * | 2016-08-29 | 2017-01-04 | 成都欧林生物科技股份有限公司 | A kind of preparation method improving epidemic encephalitis A group's capsular polysaccharide productivity |
| CN106318992A (en) * | 2016-08-29 | 2017-01-11 | 成都欧林生物科技股份有限公司 | Process beneficial for quality control of polysaccharide-SPH derivative |
| CN106350553A (en) * | 2016-08-29 | 2017-01-25 | 成都欧林生物科技股份有限公司 | Process for reducing content of impurities in meningococcal group A bacteria fermentation liquor |
| CN107058421B (en) * | 2017-05-15 | 2021-01-26 | 中国农业科学院兰州畜牧与兽药研究所 | Method for extracting and purifying capsular polysaccharide in 336 type staphylococcus aureus |
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| BRPI0516372A (en) * | 2005-12-22 | 2008-09-02 | Sanofi Pasteur Inc | multivalent meningococcal derivatized polysaccharide-protein conjugates and vaccine |
| US7491517B2 (en) * | 2006-07-19 | 2009-02-17 | Jeeri R Reddy | Method of producing meningococcal meningitis vaccine for Neisseria meningitidis serotypes A,C,Y, and W-135 |
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