CN106237320A - A kind of preparation method of A group's C meningococcal polysaccharide combined vaccine - Google Patents
A kind of preparation method of A group's C meningococcal polysaccharide combined vaccine Download PDFInfo
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- CN106237320A CN106237320A CN201610746652.9A CN201610746652A CN106237320A CN 106237320 A CN106237320 A CN 106237320A CN 201610746652 A CN201610746652 A CN 201610746652A CN 106237320 A CN106237320 A CN 106237320A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/095—Neisseria
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/385—Haptens or antigens, bound to carriers
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6031—Proteins
- A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
Abstract
The invention discloses the preparation method of a kind of A group's C meningococcal polysaccharide combined vaccine, comprise the following steps: the preparation of (1) A group C group meningitis cocci capsular polysaccharide purification；(2) C group's epidemic encephalitis polysaccharide is prepared solution, the most under agitation add CDAP, add ADH reaction after standing and obtain reactant solution, after being dialysed by reactant solution, obtain A group's C group's epidemic encephalitis Polysaccharide A DH derivant；(3) A group's C group's epidemic encephalitis Polysaccharide A DH derivant and tetanus toxoid being mixed for 1:1 in mass ratio, add EDAC, stirring reaction is dialysed to obtain reactant again, and chromatography is purified, and collects void volume eluting component, after aseptic filtration stock solution；(4) frozen-dried protective and get final product.The present invention effectively avoids the use avoiding purge process Pyrogentisinic Acid, and the preparation derivant process use to Bromine cyanide., so that whole preparation process is without toxicity damage, it is possible to reduce environmental pollution, environment plays certain protective effect.
Description
Technical field
The present invention relates to biomedicine field, a kind of A group's C meningococcal polysaccharide combines epidemic disease
The preparation method of Seedling.
Background technology
Epidemic cerebrospinal meningitis (being called for short epidemic encephalitis, lower same) is a kind of by neisseria meningitis inflammation coccus
(Neisseriameningitidis) Acute respiratory infectious disease that causes.Over more than 100 year, always the most popular or
It is dispersed in generation, septicemia, meningitis after infection pathogen, can be caused.Susceptible population is mainly child, with fulminant type case fatality rate
Height, up to 40%～60%.Each continent, world today sickness rate is 1/,100,000～10/,100,000, and total case fatality rate is 5%～15%, up to
The meningitis patient of 20% has nervous system sequela, including intellectual impairment and deafness etc..Blood is carried out according to capsular polysaccharide type
Clear credit class can divide 13 serotypes, and wherein A group, B group, C group account for the 90% of Epidemic bacterial flora.A group meningitis cocci is bigger stream
The principal causative sero-group of row, particularly at so-called Africa " the popular band of epidemic encephalitis ", arises that the biggest every 7-14
Popular.
China once occurred 5 national epidemic encephalitiss popular in, nineteen fifty-nine, 1967 and in 1938 in 1949 in 1977；Its
In with spring in 1967 popular the most serious, sickness rate is up to 4,03/,100,000, and case fatality rate is 5.49%.China's past more than 90%
Case is that A group pathogenic bacteria is caused a disease, and present B group or C group pathogenic bacteria the most also cause epidemic encephalitis to break out.2003 start, the morbidity of C group's epidemic encephalitis
Rate substantially rises.A group and C group account for more than the 50% of all sero-groups altogether at present, and C group still has the trend increased further.Cause
This, the prevention epidemic encephalitis work of current China it is important that based on prevention A group and C group.
Capsular polysaccharide is macromolecular substances, isolated and purified more complicated than monosaccharide or little molecule oligosaccharide.Separation at capsular polysaccharide
In purification, the impurity such as nucleic acid, protein, endotoxin need to be removed.A group's C group meningitis cocci capsular polysaccharide of current domestic listing
It is all conventionally to be prepared from.The i.e. method initially with liquid fermentation prepares bacterial cultures, after then sterilizing
Culture fluid utilize centrifuging remove thalline.Complex is formed again with cetyl trimethylammonium bromide (CTAB) and polysaccharide, then
Being purified process, due to relatively costly, purification process mainly uses phenol extraction, and therefore phenol extraction becomes production scale
The main method of purified capsular polysaccharide.But phenol extraction method, needs to use substantial amounts of phenol, to the harm of human body and environment very
Greatly, containing a small amount of phenol residue and in final polysaccharide preparation.
It addition, A group's polysaccharide vaccine is poor to the immunogenicity of less than 2 years old child, the protection period is short, C group's polysaccharide vaccine to 2 years old with
Lower child does not produce immunity.Therefore, World Health Organization (WHO) does not recommend A group's C group's epidemic encephalitis polysaccharide vaccine for the routine of infant
Immunity inoculation.Epidemic encephalitis polysaccharide can induce the highest bactericidin in bigger child and adult, but to the children of baby below 18 monthly ages
Youngster can not induce effective bactericidin, can not induce immunological memory；Its reason is that polysaccharide belongs to T cell independent antigen,
In 2 years old Infants Below that immune system physiogeny is not perfect, polysaccharide antigen can not stimulate body to produce potent antibodies,
So this high-risk group can not be played effective protective effect by polysaccharide vaccine.In order to change the non-T cell dependency of polysaccharide,
Polysaccharide covalent is coupled on a kind of protein carrier by people, is allowed to be changed into T cell dependence antigen, thus solves at 2 years old
The problem of Infants Below immunogenicity difference.This new generation combined vaccine not only all can induce out in any age bracket crowd
The protection antibody based on IgG of high concentration, and significantly immunity anamnesis reaction can be produced.Presently commercially available epidemic encephalitis polysaccharide knot
Conjunction vaccine includes, C group's epidemic encephalitis polysaccharide conjugate vaccine, A group's C group's epidemic encephalitis polysaccharide conjugate vaccine.
At present, domestic all combined vaccines listed are (such as b type hemophilus influenza combined vaccine, A group's C mass-brain
Meningococcus combined vaccine) all use same chemical conjugation methods to be prepared from, i.e. with Bromine cyanide. (CNBr) activated polysaccharide
Vicinal hydroxyl groups, the polysaccharide after activation and adipic dihydrazide (ADH) react generation polysaccharide-ADH derivant.Then at carbodiimide
(EDAC) under catalytic action, polysaccharide-ADH derivant and carrier protein covalent bond.This technique have a biggest shortcoming be
The activation process of polysaccharide can use Bromine cyanide..Bromine cyanide. is extremely toxic and that character is active material, is heated, meets water releasing play
Poisonous gas such as Blausure (German) and Blausure (German), meet acid and easily set off an explosion.Along with the enhancing to environmental consciousness, and to the working environment of workers
The attention of situation, people always search for suitable method and avoid using as Bromine cyanide. in vaccine manufacture process hypertoxic
Chemical reagent.Use modern times technology traditional production technology is improved with reduce or abandon toxic chemical for
Protection human health and minimizing environmental pollution all have great importance.
Summary of the invention
The present invention provides the preparation method of a kind of safer and more effective A group's C meningococcal polysaccharide combined vaccine, the party
Method effectively avoids the use avoiding purge process Pyrogentisinic Acid, and the preparation derivant process use to Bromine cyanide., so that
Obtain whole preparation process without toxicity damage, it is possible to reduce environmental pollution, environment is played certain protective effect.
For solving above-mentioned technical problem, the present invention by the following technical solutions:
The preparation method of a kind of A group's C meningococcal polysaccharide combined vaccine, comprises the following steps:
(1) meningococcus is carried out fermentation culture and carries out sterilization processing, the culture fluid sterilized；
(2) prepared by meningococcal capsular polysaccharide
The medium centrifugal that sterilized is collected supernatant, adds cetyl trimethylammonium bromide to final concentration 1.0g/L, fill
Stand overnight after dividing stirring, centrifugal collection polysaccharide precipitation thing, then with calcium chloride solution, polysaccharide precipitation thing is sufficiently stirred for 3-5 hour
Centrifugal collection supernatant I after dissociating to polysaccharide and CTAB, in supernatant I, addition ethanol is to final concentration 25%v/v, at 2-8 DEG C
Stand overnight；Recentrifuge collects supernatant II, adds ethanol at supernatant II and makes polysaccharide precipitation to final concentration 75%v/v, shaking
Get off to stand more than 18 hours, centrifugal collecting precipitation, more i.e. obtain rough polysaccharide by after precipitation clean dry；
(3) being dissolved in buffer A by rough polysaccharide, the concentration of ordinary dissolution of rough polysaccharide is 5～10mg/ml, rough after dissolving
After the clarified filtration of polysaccharide, it is splined on the DEAE-Sepharose Fast Flow chromatographic column good by buffer pre-balance, receives
Collect unadsorbed and flow through peak, be then splined on the Capto Adhere chromatographic column good with buffer B pre-balance, with buffer B
Carry out eluting mutually as flowing, collect the target peak containing polysaccharide, then by the polysaccharide solution of collection with water for injection as ultrafiltrate,
Carrying out desalination with ultrafilter membrane bag, the solution after desalination is A group's C meningococcal polysaccharide solution, and wherein buffer A is 20mM
Tris-HCl, 0.5% NaTDC, pH 8.0；Buffer B is 20mM Tris-HCl, pH 8.0；
(4) by A group's C meningococcal polysaccharide solution of 5mg/mL, CDAP, wherein A group C group meningitis ball are under agitation added
The mass ratio of granulose and CDAP is 1:0.5-1.5, and to adjust the pH value range of mixed solution be 9.0-11.0, stands 10-40min
Rear addition ADH reaction 15min obtains reactant solution, and wherein the mass ratio of A group C meningococcal polysaccharide and ADH is 1:
3.5, obtain A group's C meningococcal polysaccharide-ADH derivant after being dialysed by reactant solution；
(5) A group's C meningococcal polysaccharide-ADH derivant and tetanus toxoid are mixed for 1:1 in mass ratio, adjust pH value
Scope is 5.5-5.7, adds EDAC, and after 2-8 DEG C of stirring reaction 5-6h, then carry out reactant of dialysing to obtain, then by reactant
It is purified by chromatography, collects void volume eluting component, after aseptic filtration, obtain stock solution；
(6) in stock solution add 0.2mol/L sodium chloride solution and add distilled water adjust mixed liquor concentration meet injection
Amount, adds freeze drying protectant and i.e. obtains A group's C meningococcal polysaccharide combined vaccine.
Step (1) is specially and carries out meningococcus kind inoculation on epidemic encephalitis half synthetic medium, temperature 35～37 DEG C, training
Support 16～24 hours, after three generations's amplification cultivation, be seeded to seed tank culture, temperature 35～37 DEG C, cultivate preparation in 4～6 hours
The production seed that quantity is suitable, is then seeded to seed tank culture thing 500L fermentor cultivation, temperature 35～37 DEG C, cultivates
Formaldehyde sterilization is added after time 6～12 hours.
In step (3), the concentration of ordinary dissolution of rough polysaccharide is 6～8mg/ml.
Rough polysaccharide after dissolving in step (3) need to carry out clarification filtration through 0.45 m filter membrane.
In step (3), the molecular cut off of ultrafilter membrane bag is 300KD.
In step (4), the mass ratio of A group's C meningococcal polysaccharide and CDAP is 1:0.5.
Step (4) adjusts the pH value range of mixed solution after adding CDAP be 11.0 ± 0.2.
After step (4) addition CDAP, time of repose is 25-30min
Compared with prior art, the invention has the beneficial effects as follows:
The preparation method of A group's C meningococcal polysaccharide combined vaccine of the present invention, the method effectively avoids avoids purification mistake
The use of journey Pyrogentisinic Acid, and the preparation derivant process use to Bromine cyanide., so that whole preparation process is without toxicity damage, energy
Enough reduce environmental pollution, environment is played certain protective effect.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is further illustrated.Embodiments of the present invention include but not limited to following
Embodiment.
All products in following example are commercially available prod.
Embodiment 1
Prepared by A meningococcal polysaccharide
Open A group meningitis cocci working seed lots strain 1, be seeded to 2 epidemic encephalitis doups and close culture medium test tubes, temperature 35～
37 DEG C, cultivate 16～24 hours, for first generation strain.Every 1 first generation strain is propagated in 2 bottles of epidemic encephalitis half synthetic mediums,
Temperature 35～37 DEG C are cultivated 16～24 hours, for second filial generation strain.Every 1 bottle of second filial generation strain is propagated comprehensive in 6 bottles of epidemic encephalitiss half
Culture medium, temperature 35～37 DEG C are cultivated 16～24 hours, for third generation strain.Three generations's strain is collected 500ml normal saline
Solution mixes, is inoculated in 50L seed tank, temperature 35～37 DEG C, incubation time 4 hours.Then 50L seed tank culture thing is connect
Kind to 500L fermentor cultivation, temperature 35～37 DEG C, incubation time adds final concentration of 1%(v/v after 8 hours) formalin
Sterilization.
The medium centrifugal sterilized is collected supernatant.It is subsequently adding cetyl trimethylammonium bromide (CTAB) to the denseest
Degree 1.0g/L, stands overnight after being sufficiently stirred for, centrifugal collection polysaccharide precipitation thing.It is little that polysaccharide precipitation thing calcium chloride solution stirs 3
Time make polysaccharide and CTAB dissociate.Centrifugal collection supernatant I, supernatant I adds ethanol extremely final concentration of 25%(v/v), 2～8 DEG C are quiet
Put overnight, centrifugal collection supernatant II, supernatant II adds ethanol extremely final concentration of 75%(v/v), shake well makes polysaccharide sink
Form sediment, stand more than 18 hours, centrifugal collecting precipitation, then respectively wash three times with dehydrated alcohol and acetone, be rough many after drying
Sugar.
Being dissolved in by raw sugar in buffer (20mM Tris-HCl, 0.5% NaTDC, pH 8.0), concentration of ordinary dissolution is
5mg/ml.Sample after dissolving is after 0.45 m filter membrane clarification filtration, and (20mM Tris-HCl, 0.5% takes off to be splined on use buffer
Oxycholic acid sodium, pH 8.0) DEAE-Sepharose Fast Flow chromatographic column that pre-balance is good, collect unadsorbed flowing through
Peak, is then splined on the Capto Adhere chromatographic column good with buffer (20mM Tris-HCl, pH 8.0) pre-balance, with
20mM Tris-HCl, pH 8.0 buffer carry out eluting mutually as flowing, collect the target peak containing polysaccharide.Then will collect
Polysaccharide solution with water for injection as ultrafiltrate, carry out desalination with the ultrafilter membrane bag that molecular cut off is 300KD.Molten after desalination
Liquid is A group's C meningococcal polysaccharide solution, sampling detection quality index.From following table, the three batches of C group's polysaccharide prepared
Every quality index all meet pharmacopoeial requirements.
A group's epidemic encephalitis polysaccharide is prepared solution, the most under agitation adds CDAP, wherein A group's epidemic encephalitis polysaccharide and the mass ratio of CDAP
For 1:0.5, and to adjust the pH value range of mixed solution be 11.0 ± 0.2, adds ADH reaction 15min and obtain after standing 25-30min
Reactant solution, wherein the mass ratio of A group's epidemic encephalitis polysaccharide and ADH is 1:3.5, obtains A group's epidemic encephalitis many after being dialysed by reactant solution
Sugar-ADH derivant；
A group's epidemic encephalitis polysaccharide-ADH derivant and tetanus toxoid being mixed for 1:1 in mass ratio, tune pH value range is 5.5-
5.7, add EDAC, and after 2-8 DEG C of stirring reaction 5-6h, then carry out reactant of dialysing to obtain, then reactant chromatography is entered
Row purification, collects void volume eluting component, obtains stock solution after aseptic filtration；
In stock solution add 0.2mol/L sodium chloride solution and add distilled water adjust mixed liquor concentration meet injection dosage, then
Add freeze drying protectant and i.e. obtain A meningococcal polysaccharide combined vaccine.
Embodiment 2
Open C group meningitis cocci working seed lots strain 1, be seeded to 2 epidemic encephalitis doups and close culture medium test tubes, temperature 35～
37 DEG C, cultivate 16～24 hours, for first generation strain.Every 1 first generation strain is propagated in 2 bottles of epidemic encephalitis half synthetic mediums,
Temperature 35～37 DEG C are cultivated 16～24 hours, for second filial generation strain.Every 1 bottle of second filial generation strain is propagated comprehensive in 6 bottles of epidemic encephalitiss half
Culture medium, temperature 35～37 DEG C are cultivated 16～24 hours, for third generation strain.Three generations's strain is collected 500ml normal saline
Solution mixes, is inoculated in 50L seed tank, temperature 35～37 DEG C, incubation time 4 hours.Then 50L seed tank culture thing is connect
Kind to 500L fermentor cultivation, temperature 35～37 DEG C, incubation time adds final concentration of 1%(v/v after 8 hours) formalin
Sterilization.
The medium centrifugal sterilized is collected supernatant.It is subsequently adding cetyl trimethylammonium bromide (CTAB) to the denseest
Degree 1.0g/L, stands overnight after being sufficiently stirred for, centrifugal collection polysaccharide precipitation thing.It is little that polysaccharide precipitation thing calcium chloride solution stirs 3
Time make polysaccharide and CTAB dissociate.Centrifugal collection supernatant I, supernatant I adds ethanol extremely final concentration of 25%(v/v), 2～8 DEG C are quiet
Put overnight, centrifugal collection supernatant II, supernatant II adds ethanol extremely final concentration of 75%(v/v), shake well makes polysaccharide sink
Form sediment, stand more than 18 hours, centrifugal collecting precipitation, then respectively wash three times with dehydrated alcohol and acetone, be rough many after drying
Sugar.
Being dissolved in by raw sugar in buffer (20mM Tris-HCl, 0.5% NaTDC, pH 8.0), concentration of ordinary dissolution is
5mg/ml.Sample after dissolving is after 0.45 m filter membrane clarification filtration, and (20mM Tris-HCl, 0.5% takes off to be splined on use buffer
Oxycholic acid sodium, pH 8.0) DEAE-Sepharose Fast Flow chromatographic column that pre-balance is good, collect unadsorbed flowing through
Peak, is then splined on the Capto Adhere chromatographic column good with buffer (20mM Tris-HCl, pH 8.0) pre-balance, with
20mM Tris-HCl, pH 8.0 buffer carry out eluting mutually as flowing, collect the target peak containing polysaccharide.Then will collect
Polysaccharide solution with water for injection as ultrafiltrate, carry out desalination with the ultrafilter membrane bag that molecular cut off is 300KD.Molten after desalination
Liquid is A group's C meningococcal polysaccharide solution, sampling detection quality index.From following table, the three batches of C group's polysaccharide prepared
Every quality index all meet pharmacopoeial requirements.
C group's epidemic encephalitis polysaccharide is configured to the solution of 5mg/mL, the most under agitation adds CDAP, wherein C group's epidemic encephalitis polysaccharide and
The mass ratio of CDAP is 1:1.0-1.5, and to adjust the pH value range of mixed solution be 9.0-10.0, adds after standing 10-40min
ADH reaction 15min obtains reactant solution, and wherein the mass ratio of C group's epidemic encephalitis polysaccharide and ADH is 1:3.5, and reactant solution is saturating
C group's epidemic encephalitis polysaccharide-ADH derivant is obtained after analysis；
C group's epidemic encephalitis polysaccharide-ADH derivant and tetanus toxoid being mixed for 1:1 in mass ratio, tune pH value range is 5.5-
5.7, add EDAC, and after 2-8 DEG C of stirring reaction 5-6h, then carry out reactant of dialysing to obtain, then reactant chromatography is entered
Row purification, collects void volume eluting component, obtains stock solution after aseptic filtration；
In stock solution add 0.2mol/L sodium chloride solution and add distilled water adjust mixed liquor concentration meet injection dosage, then
Add freeze drying protectant and i.e. obtain C meningococcal polysaccharide combined vaccine.
According to the above embodiments, can well complete the present invention.
It is embodiments of the invention as mentioned above.The present invention is not limited to above-mentioned embodiment, and anyone should learn
The structure change made under the enlightenment of the present invention, every have same or like technical scheme with the present invention, each falls within this
Within the protection domain of invention.
Claims (8)
1. the preparation method of A group's C meningococcal polysaccharide combined vaccine, it is characterised in that comprise the following steps:
(1) meningococcus is carried out fermentation culture and carries out sterilization processing, the culture fluid sterilized；
(2) prepared by meningococcal capsular polysaccharide
The medium centrifugal that sterilized is collected supernatant, adds cetyl trimethylammonium bromide to final concentration 1.0g/L, fill
Stand overnight after dividing stirring, centrifugal collection polysaccharide precipitation thing, then with calcium chloride solution, polysaccharide precipitation thing is sufficiently stirred for 3-5 hour
Centrifugal collection supernatant I after dissociating to polysaccharide and CTAB, in supernatant I, addition ethanol is to final concentration 25%v/v, at 2-8 DEG C
Stand overnight；Recentrifuge collects supernatant II, adds ethanol at supernatant II and makes polysaccharide precipitation to final concentration 75%v/v, shaking
Get off to stand more than 18 hours, centrifugal collecting precipitation, more i.e. obtain rough polysaccharide by after precipitation clean dry；
(3) being dissolved in buffer A by rough polysaccharide, the concentration of ordinary dissolution of rough polysaccharide is 5～10mg/ml, rough after dissolving
After the clarified filtration of polysaccharide, it is splined on the DEAE-Sepharose Fast Flow chromatographic column good by buffer pre-balance, receives
Collect unadsorbed and flow through peak, be then splined on the Capto Adhere chromatographic column good with buffer B pre-balance, with buffer B
Carry out eluting mutually as flowing, collect the target peak containing polysaccharide, then by the polysaccharide solution of collection with water for injection as ultrafiltrate,
Carrying out desalination with ultrafilter membrane bag, the solution after desalination is A group's C meningococcal polysaccharide solution, and wherein buffer A is 20mM
Tris-HCl, 0.5% NaTDC, pH 8.0；Buffer B is 20mM Tris-HCl, pH 8.0；
(4) by A group's C meningococcal polysaccharide solution of 5mg/mL, CDAP, wherein A group C group meningitis ball are under agitation added
The mass ratio of granulose and CDAP is 1:0.5-1.5, and to adjust the pH value range of mixed solution be 9.0-11.0, stands 10-40min
Rear addition ADH reaction 15min obtains reactant solution, and wherein the mass ratio of A group C meningococcal polysaccharide and ADH is 1:
3.5, obtain A group's C meningococcal polysaccharide-ADH derivant after being dialysed by reactant solution；
(5) A group's C meningococcal polysaccharide-ADH derivant and tetanus toxoid are mixed for 1:1 in mass ratio, adjust pH value
Scope is 5.5-5.7, adds EDAC, and after 2-8 DEG C of stirring reaction 5-6h, then carry out reactant of dialysing to obtain, then by reactant
It is purified by chromatography, collects void volume eluting component, after aseptic filtration, obtain stock solution；
(6) in stock solution add 0.2mol/L sodium chloride solution and add distilled water adjust mixed liquor concentration meet injection
Amount, adds freeze drying protectant and i.e. obtains A group's C meningococcal polysaccharide combined vaccine.
Preparation method the most according to claim 1, it is characterised in that step (1) is specially at epidemic encephalitis half synthetic medium
On carry out meningococcus kind inoculation, temperature 35～37 DEG C, cultivate 16～24 hours, be seeded to after three generations's amplification cultivation plant
Sub-tank is cultivated, temperature 35～37 DEG C, cultivates 4～6 hours suitable production seeds of preparation quantity, then by seed tank culture thing
It is seeded to 500L fermentor cultivation, temperature 35～37 DEG C, incubation time 6～addition formaldehyde sterilization after 12 hours.
Preparation method the most according to claim 1, it is characterised in that in step (3) concentration of ordinary dissolution of rough polysaccharide be 6～
8mg/ml 。
Preparation method the most according to claim 1, it is characterised in that the rough polysaccharide after dissolving in step (3) need through
0.45 m filter membrane carries out clarification filtration.
Preparation method the most according to claim 1, it is characterised in that in step (3), the molecular cut off of ultrafilter membrane bag is
300KD。
Preparation method the most according to claim 1, it is characterised in that in step (4) A group's C meningococcal polysaccharide and
The mass ratio of CDAP is 1:0.5.
Preparation method the most according to claim 1, it is characterised in that step (4) adjusts mixed solution after adding CDAP
PH value range is 11.0 ± 0.2.
Preparation method the most according to claim 1, it is characterised in that after step (4) addition CDAP, time of repose is 25-
30min。
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102813917A (en) * | 2012-08-17 | 2012-12-12 | 武汉生物制品研究所有限责任公司 | Diphtheria, tetanus and acellular pertussis/Haemophilus influenzae type b - group A and C meningococcus combined vaccine |
| CN102911285A (en) * | 2012-11-16 | 2013-02-06 | 罗益（无锡）生物制药有限公司 | Process for refining group C/Y/W135 meningococcal polysaccharides |
| CN105031634A (en) * | 2015-08-31 | 2015-11-11 | 成都欧林生物科技股份有限公司 | A-group C-group Neisseria meningitidis polysaccharide conjugate vaccine activating process |
-
2016
- 2016-08-29 CN CN201610746652.9A patent/CN106237320A/en not_active Withdrawn
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102813917A (en) * | 2012-08-17 | 2012-12-12 | 武汉生物制品研究所有限责任公司 | Diphtheria, tetanus and acellular pertussis/Haemophilus influenzae type b - group A and C meningococcus combined vaccine |
| CN102911285A (en) * | 2012-11-16 | 2013-02-06 | 罗益（无锡）生物制药有限公司 | Process for refining group C/Y/W135 meningococcal polysaccharides |
| CN105031634A (en) * | 2015-08-31 | 2015-11-11 | 成都欧林生物科技股份有限公司 | A-group C-group Neisseria meningitidis polysaccharide conjugate vaccine activating process |
Non-Patent Citations (1)
| Title |
|---|
| 熊慧玲等: "C群脑膜炎球菌多糖结合疫苗制备工艺的优化", 《中国生物制品学杂志》 * |
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Application publication date: 20161221 |