CN1063971C - Method for preparing freeze-dried live vaccine for animal use - Google Patents
Method for preparing freeze-dried live vaccine for animal use Download PDFInfo
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- CN1063971C CN1063971C CN97116489A CN97116489A CN1063971C CN 1063971 C CN1063971 C CN 1063971C CN 97116489 A CN97116489 A CN 97116489A CN 97116489 A CN97116489 A CN 97116489A CN 1063971 C CN1063971 C CN 1063971C
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- newcastle disease
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- 208000010359 Newcastle Disease Diseases 0.000 claims abstract description 24
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
本发明是一种动物用的冻干活疫苗的制造方法，属动物用生物制品制造领域。本发明的目的是通过多种冻干保护剂基质与鸡新城疫活疫苗制苗病毒毒株所制取的病毒抗原进行了相溶性研究，找出合适的基质，设计一种新的冻干保护剂的配方，用此配方配制成的冻干保护剂，采用与之相适应的冻干曲线，与新城疫疫苗病毒抗原制成鸡新城疫冻干活疫苗在2-8℃条件下可长期保存。The invention relates to a method for producing a freeze-dried live vaccine for animals, which belongs to the field of biological products for animals. The purpose of the present invention is to carry out compatibility research through various lyoprotectant matrices and virus antigens produced by live Newcastle disease vaccine production virus strains, find out a suitable matrix, and design a kind of new lyoprotectant The formula, the freeze-dried protective agent formulated with this formula, adopts the corresponding freeze-drying curve, and the Newcastle disease vaccine virus antigen to make chicken Newcastle disease freeze-dried live vaccine, which can be stored for a long time under the condition of 2-8°C.
Description
The present invention is a kind of manufacture method of freeze-dried live vaccine for animal use, belongs to animal and makes the field with viral lived vaccine.
Newcastle disease is a kind of acute infectious disease of chicken, all has popularly all over the world, and this disease is that the newcastle disease virus by Paramyxoviridae is caused, primary disease is propagated fast, susceptible chickling especially, and mortality rate is up to 100%, and is very harmful to poultry husbandry.
This kind viral infectious is not had special effect medicine therapeutic, and with vaccine chicken being carried out prophylactic immunization is the best way.This class vaccine has two kinds of attenuated live vaccine and inactivated vaccines, and live vaccine has attenuated live vaccines such as newcastle disease I system, II system, F system, LaSota system.The basic manufacture method of these live vaccine is that the weak seed culture of viruses with different strains is inoculated in the Embryo Gallus domesticus of the well-developed no specificity cause of disease of 10 ages in days (specific pathogen free-SPF) or does not have in the healthy chick embryo allantoic cavity of newcastle epidemic disease antibody; 37 ℃ continue hatching to 60～120 hours after the results chick embryo allantoic liquid as antigen, mixes afterwards with the freeze drying protectant of certain proportion amount, packing forms through vacuum freeze-drying.
Lyophilization microbiology class biological product for success; select the suitable freeze drying protectant that suspends to be absolutely necessary; because freeze drying protectant can make biological product keep bottom line death in freeze-drying process; alleviate the caused damage to microorganism of lyophilization, withstand higher temperatures is not damaged Products Quality when preserving.Thereby the kind of freeze drying protectant, concentration and compound method etc. have tangible influence to the preservation effect of biological product.Harrison (1963) recommends freeze drying protectant should possess following condition: 1. protective agent should contain the material that can form skeleton; 2. it should contain the buffer substance of restricted residual moisture; 3. in should containing and the active material of carboxyl; 4. electrolytical content should lack.
Animal can keep Products Quality to change in 1～1.5 year not quite, and be kept under 2～8 ℃-15 ℃ of preservations with the preservation of viral freeze-dried live vaccine goods, domestic general employing, and the storage life of goods then shortens; External many 2～8 ℃ of preservations 1.5～2 years, quality of item is more stable.Domestic and international used freeze drying protectant great majority are to be the main matrix raw material with defatted milk powder, lactose, lactoalbumin hydrolysate, sucrose, account for 10%～20% of protective agent total amount; The domestic freeze-drying curves that adopt the relative short periods in the freeze-drying process more, external then adopt the freeze-drying curve of relative long period.
The objective of the invention is to have carried out intermiscibility research by the virus antigen that multiple frozen protection agent substrate and chicken Newcastle disease live vaccine seedling virus stain are produced; find out suitable substrate; design a kind of prescription of freeze drying protectant; the freeze drying protectant that is mixed with adopts the freeze-drying curve that adapts with it.Make chicken Newcastle disease live vaccine long preservation under 2～8 ℃ of conditions that chicken Newcastle disease live vaccine is become after by this freeze-drying curve lyophilizing with this protective agent and newcastle disease vaccine virus antigen, need preservation problem under-15 ℃ of conditions to replace the present chicken Newcastle disease live vaccine of China.
Technical characterictic of the present invention is with after the dilution of newcastle disease attenuated virus kind poison; be inoculated in the Embryo Gallus domesticus of the well-developed no specificity cause of disease of 10 ages in days (specific pathogen free-SPF) or do not have in the healthy chick embryo allantoic cavity of newcastle epidemic disease antibody; the results chick embryo allantoic liquid is as antigen after 37 ℃ of continuation hatchings were to 60～120 hours, and the freeze drying protectant designed with the present invention mixes the back by a certain percentage, is divided in the live vaccine of making the prevention newcastle disease by the designed freeze-drying curve of the present invention through vacuum freeze-drying.The design of freeze drying protectant
The designed freeze drying protectant of the present invention has a very heat-stable framing structure, and is indeformable through 30 minutes these framing structures at 100 ℃; The small-molecule substance and the buffer system that have with this framing structure the virus of matching, can protect simultaneously.In this prescription, also be added with simultaneously and prevent that the anti-brilliant agent of flake-like crystal from appearring in product after the lyophilizing.For preventing that the live virus under the lyophilization state from contacting the dead material with antioxidation that increased of generation with airborne oxygen.The frozen-dried protective agent prescription is as follows
Polyvinylpyrrolidone 5～15g, sodium carboxymethyl cellulose 2～10g, tamarind gum 5～15g, sodium glutamate 20～40g, trehalose 10～15g, vitamin C 1～5g, glycyrrhizin 2～15g, MgCl
20.01～0.05g, K
2HPO
41.3g, KH
2PO
40.05g, DDW 1000ml.The design of freeze-drying curve
Be cooled to-5 ℃ in freeze drying box in advance, divide the container install Embryo Gallus domesticus liquid to enter freeze drying box, the speed with 1 ℃/min is cooled to-35 ℃ again, keep 2 hours under this temperature after, begin to be evacuated to 10
-4After the holder (1 holder=133.322368 Pa), begin to heat up, promptly require to be warming up in 16 hours-20C, continue to be warming up to 20 ℃ and keep 6 hours outlets of jumping a queue with 5 ℃ speed per hour thereafter, whole freeze-drying process is 32 hours.
Below in conjunction with animal with using maximum a kind of newcastle disease freeze-dried live vaccine production instances to specify technical characterictic of the present invention in the viral lived vaccine: 1 produces vaccine with seed culture of viruses and seed culture of viruses subculture
Producing with seed culture of viruses is that the newcastle disease low virulent strain is (as newcastle disease mesogenic strain I system-Mukteswar, newcastle disease is hanged down virulence strain II system, F system, LaSota and C-30 strain etc.), identify, take care of and supply by China Veterinary Drugs Supervisory Inst., the seed culture of viruses standard meets specified standard in " The Ministry of Agriculture of the People's Republic of China, MOA's veterinary biologics rules promulgated by the ministries or commissions of the Central Government " (version in 1992) (to call " rules " in the following text).The preparation of 2 freeze drying protectants
This protective agent is made up of A and B two parts.
A: polyvinylpyrrolidone 5～15g, sodium carboxymethyl cellulose 2～10g, tamarind gum 5～15g, sodium glutamate 20～40g, K
2HPO
41.3g, KH
2PO
40.05g, DDW 500ml.Preparation is after standby behind 121 ℃ of 15 minutes autoclavings.
B: trehalose 10～15g, vitamin C 1～5g, glycyrrhizin 2～15g, MgCl
20.05g, DDW 500ml.The degerming of various composition dissolving after-filtration is standby.
Be used to join Seedling after A and B mixed by 1: 1.The selection of 3 vaccine manufacturings and lyophilizing 3.1 eggs and hatching are selected the SPF chicken for use or are not had the egg that healthy chicken produces of newcastle disease antibody, by the Embryo Gallus domesticus of " rules " regulation hatching to 10 ages in days.Produce with kind of a poison 3.2 the seed culture of viruses inoculation is got, be diluted to 10 with sterile saline
-4Or 10
-5, inoculation 0.1ml in every embryo allantoic cavity.Sealing pin hole in inoculation back is put 36～37 ℃ and is continued to hatch.3.3 shine egg 1 every day after the egg inoculation of antigen results, will discard by dead Embryo Gallus domesticus before 60 hours.After 60 hours, the photograph egg was 1 time in per 4～8 hours, and dead Embryo Gallus domesticus takes out at any time, and until 96～120 hours, no matter embryo all takes out anyway, air chamber was upwards upright, puts 2～8 ℃ of coolings.Embryo Gallus domesticus through cooling off 4～24 hours takes out, and with iodine tincture sterilization air chamber position, removes air chamber portion eggshell with aseptic operation, tears membrana putaminis off and draws Embryo Gallus domesticus liquid, places the sterilization bottle, adds penicillin and streptomycin, puts into 2～8 ℃ of processing.Treated blastochyle carries out steriling test and viral level is measured.3.4 join Seedling the blastochyle that is up to the standards is filtered, added the freeze drying protectant that the present invention is designed and prepare, add suitable antibiotic (the same) simultaneously, fully shake up, quantitatively carry out lyophilization immediately after the packing by 1: 1.3.5 lyophilizing is cooled to-5 ℃ in advance in freeze drying box, divide the container install Embryo Gallus domesticus liquid to enter freeze drying box, the speed with 1 ℃/min is cooled to-35 ℃ again, keep 2 hours under this temperature after, begin to be evacuated to 10
-4After the holder (1 holder=133.322368 Pa), begin to heat up, promptly require to be warming up to-20 ℃ in 16 hours, continue to be warming up to 20 ℃ and keep 6 hours outlets of jumping a queue with 5 ℃ speed per hour thereafter, whole freeze-drying process is 32 hours.
The every plumage part of vaccine seedling diseases poison content answers 〉=10 after the lyophilizing
6EID
504 product inspections, 4.1 physical behaviors should be little yellow, and spongy loose agglomerate easily comes off with a bottle wall, add dissolving rapidly behind the diluent.4.2 steriling test, mycoplasma check, diagnostic test and exogenous virus check are undertaken by pertinent regulations in " rules ".4.3 the following method of safety verification is appointed and is selected one 4.3.1 and with Embryo Gallus domesticus check vaccine is diluted with sterile saline, inoculation instar chicken embryo on the 10th is 10 in the allantois, every embryo 0.1ml (containing 10 using dosages), inoculation back 24～72 hours, the chicken embryo death rate is no more than 20% for qualified.Surpass 20% as mortality rate, multiplicable heavy inspection once examines heavily as a result that mortality rate still surpasses 20%, and this vaccine is judged to dangerous.4.3.2 with 20 of 2～7 Japanese instar chicklings of true no newcastle disease antibody, be divided into two groups with chicken check, first group 10, every collunarium is inoculated the toxic blastochyle 0.05ml of 5 times of dilutions; Second group 10, do not inoculate in contrast, two groups with respectively feeding and management under the condition, observed 10, should not have abnormal response.If any non-specific death, immune group and matched group all should be above 1.4.4 the following method of efficacy test is appointed and is selected one 4.4.1 and with Embryo Gallus domesticus check vaccine is made 10 times of serial dilutions with sterile saline, get 3 dilution factors respectively in the allantois inoculation 10 ages in days do not have 5 of the Embryo Gallus domesticus of newcastle disease antibody, every embryo 0.1ml, put 37 ℃ and continue hatching, dead Embryo Gallus domesticus discarded and disregards before 48 hours, at 48～120 hours dead Embryo Gallus domesticus, at any time take out, results Embryo Gallus domesticus liquid with same dilution Embryo Gallus domesticus liquid mixed in equal amounts, is measured the red cell agglutination valency respectively.To 120 hours, take out all embryos of living, gather in the crops Embryo Gallus domesticus liquid one by one, measure the red cell agglutination valency respectively; Agglutination titer 〉=1: 160 (micromethod 1: 128) person is judged to infection, calculates median infective dose, and every plumage part answers 〉=10
6EID
50, vaccine is judged to qualified.4.4.2 (red cell agglutination inhibition valency should＜1: 4) 10, every collunarium is inoculated 1/100 using dosage, and after 10～14 days, the immunity identical together with condition contrasts 3 of chickens, each intramuscular injection 10000EID with the healthy susceptible chicken at 1～2 monthly age with chicken check
50The strong malicious 1ml of Beijing strain newcastle disease.Observed the death of should all falling ill of contrast chicken 10～14; The immunity chicken is protected 9 at least, and vaccine is judged to qualified.5 residue moisture determinations adopt the vacuum drying method or take the Xiu Shi method and measure, and the water content of every bottle product should not surpass 4%.The vacuum that 6 vacuums measure to use the high-frequency spark vacuum determinator to seal back glass container dress freeze-dried products is measured, as occurring white, pink colour and purple aura in the container, then vacuum all belong to qualified.
Embodiment 1
Present embodiment is selected for use and produced with seed culture of viruses is newcastle disease low virulent strain LaSota and C-30 strain etc.), identify, take care of and supply by China Veterinary Drugs Supervisory Inst., the seed culture of viruses standard meets specified standard in The Ministry of Agriculture of the People's Republic of China, MOA's " veterinary biologics rules " promulgated by the ministries or commissions of the Central Government (version in 1992) (to call " rules " in the following text).
Freeze drying protectant is standby by following prescription and method preparation back:
A: polyvinylpyrrolidone 15g, sodium carboxymethyl cellulose 8g, tamarind gum 13g, sodium glutamate 20g,, K
2HPO
41.3g, KH
2PO
40.05g, DDW 500ml.Preparation is after standby behind 121 ℃ of 15 minutes autoclavings.
B: trehalose 10g, vitamin C 3g, glycyrrhizin 10g, MgCl
20.05g, DDW 500ml.The degerming of various composition dissolving after-filtration is standby.
Be used to join Seedling after A and B mixed by 1: 1.
Inoculation: get production with LaSota kind poison, be diluted to 10 with sterile saline
-4Or 10
-5, inoculate 50 piece of 10 age in days SPF Embryo Gallus domesticus, inoculation 0.1ml in every embryo allantoic cavity.Sealing pin hole in inoculation back is put 36～37 ℃ and is continued to hatch.
The antigen results: shine egg 1 every day after the egg inoculation, will discard by dead Embryo Gallus domesticus before 60 hours.After 60 hours, the photograph egg was 1 time in per 4～8 hours, and dead Embryo Gallus domesticus takes out at any time, and until 96～120 hours, no matter embryo all takes out anyway, air chamber was upwards upright, puts 2～8 ℃ of coolings.Embryo Gallus domesticus through cooling off 4～24 hours takes out, and with iodine tincture sterilization air chamber position, removes air chamber portion eggshell with aseptic operation, tears membrana putaminis off and draws Embryo Gallus domesticus liquid, places the sterilization bottle, adds penicillin and streptomycin, puts into 2～8 ℃ of processing.Treated blastochyle carries out steriling test and viral level is measured.
Join Seedling: the blastochyle that is up to the standards is filtered, added the freeze drying protectant that the present invention is designed and prepare, add suitable antibiotic (the same) simultaneously, fully shake up, quantitatively carry out lyophilization immediately after the packing by 1: 1.
Lyophilizing: be cooled to-5 ℃ in freeze drying box in advance, divide the container install Embryo Gallus domesticus liquid to enter freeze drying box, the speed with 1 ℃/min is cooled to-35 ℃ again, keep 2 hours under this temperature after, begin to be evacuated to 10
-4After the holder (1 holder=133.322368 Pa), begin to heat up, promptly require to be warming up to-20 ℃ in 16 hours, continue to be warming up to 20 ℃ and keep 6 hours outlets of jumping a queue with 5 ℃ speed per hour thereafter, whole freeze-drying process is 32 hours.Embodiment 2
Present embodiment is selected for use and produced with seed culture of viruses is newcastle disease low virulent strain LaSota and C-30 strain etc.), identify, take care of and supply by China Veterinary Drugs Supervisory Inst., the seed culture of viruses standard meets specified standard in The Ministry of Agriculture of the People's Republic of China, MOA's " veterinary biologics rules " promulgated by the ministries or commissions of the Central Government (version in 1992) (to call " rules " in the following text).
Freeze drying protectant is standby by following prescription and method preparation back:
A: polyvinylpyrrolidone 15g, sodium carboxymethyl cellulose 8g, tamarind gum 13g, sodium glutamate 20g,, K
2HPO
41.3g, KH
2PO
40.05g, DDW 500ml.Preparation is after standby behind 121 ℃ of 15 minutes autoclavings.
B: trehalose 10g, vitamin C 3g, glycyrrhizin 10g, MgCl
20.05g, DDW 500ml.The degerming of various composition dissolving after-filtration is standby.
Be used to join Seedling after A and B mixed by 1: 1.
Inoculation: get production with LaSota kind poison, be diluted to 10 with sterile saline
-4Or 10
-5, inoculate 50 piece of 10 age in days SPF Embryo Gallus domesticus, inoculation 0.1ml in every embryo allantoic cavity.Sealing pin hole in inoculation back is put 36～37 ℃ and is continued to hatch.
The antigen results: shine egg 1 every day after the egg inoculation, will discard by dead Embryo Gallus domesticus before 60 hours.After 60 hours, the photograph egg was 1 time in per 4～8 hours, and dead Embryo Gallus domesticus takes out at any time, and until 96～120 hours, no matter embryo all takes out anyway, air chamber was upwards upright, puts 2～8 ℃ of coolings.Embryo Gallus domesticus through cooling off 4～24 hours takes out, and with iodine tincture sterilization air chamber position, removes air chamber portion eggshell with aseptic operation, tears membrana putaminis off and draws Embryo Gallus domesticus liquid, places the sterilization bottle, adds penicillin and streptomycin, puts into 2～8 ℃ of processing.Treated blastochyle carries out steriling test and viral level is measured.
Join Seedling: the blastochyle that is up to the standards is filtered, added the freeze drying protectant that the present invention is designed and prepare, add suitable antibiotic (the same) simultaneously, fully shake up, quantitatively carry out lyophilization immediately after the packing by 1: 1.
Lyophilizing: be cooled to-5 ℃ in freeze drying box in advance, divide the container install Embryo Gallus domesticus liquid to enter freeze drying box, the speed with 1 ℃/min is cooled to-35 ℃ again, keep 2 hours under this temperature after, begin to be evacuated to 10
-4After the holder (1 holder=133.322368 Pa), begin to heat up, promptly require to be warming up to-20 ℃ in 16 hours, continue to be warming up to 20 ℃ and keep 6 hours outlets of jumping a queue with 5 ℃ speed per hour thereafter, whole freeze-drying process is 32 hours.Embodiment 3
Present embodiment is selected for use and produced with seed culture of viruses is newcastle disease low virulent strain LaSota and C-30 strain etc.), identify, take care of and supply by China Veterinary Drugs Supervisory Inst., the seed culture of viruses standard meets specified standard in The Ministry of Agriculture of the People's Republic of China, MOA's " veterinary biologics rules " promulgated by the ministries or commissions of the Central Government (version in 1992) (to call " rules " in the following text).
Freeze drying protectant is standby by following prescription and method preparation back:
A: polyvinylpyrrolidone 15g, sodium carboxymethyl cellulose 8g, tamarind gum 13g, sodium glutamate 20g,, K
2HPO
41.3g, KH
2PO
40.05g, DDW 500ml.Preparation is after standby behind 121 ℃ of 15 minutes autoclavings.
B: trehalose 10g, vitamin C 3g, glycyrrhizin 10g, MgCl
20.05g, DDW 500ml.The degerming of various composition dissolving after-filtration is standby.
Be used to join Seedling after A and B mixed by 1: 1.
Inoculation: get production with LaSota kind poison, be diluted to 10 with sterile saline
-4Or 10
-5, inoculate 50 piece of 10 age in days SPF Embryo Gallus domesticus, inoculation 0.1ml in every embryo allantoic cavity.Sealing pin hole in inoculation back is put 36～37 ℃ and is continued to hatch.
The antigen results: shine egg 1 every day after the egg inoculation, will discard by dead Embryo Gallus domesticus before 60 hours.After 60 hours, the photograph egg was 1 time in per 4～8 hours, and dead Embryo Gallus domesticus takes out at any time, and until 96～120 hours, no matter embryo all takes out anyway, air chamber was upwards upright, puts 2～8 ℃ of coolings.Embryo Gallus domesticus through cooling off 4～24 hours takes out, and with iodine tincture sterilization air chamber position, removes air chamber portion eggshell with aseptic operation, tears membrana putaminis off and draws Embryo Gallus domesticus liquid, places the sterilization bottle, adds penicillin and streptomycin, puts into 2～8 ℃ of processing.Treated blastochyle carries out steriling test and viral level is measured.
Join Seedling: the blastochyle that is up to the standards is filtered, added the freeze drying protectant that the present invention is designed and prepare, add suitable antibiotic (the same) simultaneously, fully shake up, quantitatively carry out lyophilization immediately after the packing by 1: 1.
Lyophilizing: be cooled to-5 ℃ in freeze drying box in advance, divide the container install Embryo Gallus domesticus liquid to enter freeze drying box, the speed with 1 ℃/min is cooled to-35 ℃ again, keep 2 hours under this temperature after, begin to be evacuated to 10
-4After the holder (1 holder=133.322368 Pa), begin to heat up, promptly require to be warming up to-20 ℃ in 16 hours, continue to be warming up to 20 ℃ and keep 6 hours outlets of jumping a queue with 5 ℃ speed per hour thereafter, whole freeze-drying process is 32 hours.The heat resistant test of the test 1 different protective agent freeze-drying prods of freeze-dried products
With the freeze dried product of different protective agents, place under 100 ℃, 45 ℃, 37 ℃, 2～8 ℃ conditions and preserve, viral level is measured in the different time sampling.Viral level assay method in 2 freeze-dried products
Make 10 times of serial dilutions by the real Embryo Gallus domesticus liquid measure that contains with sterile saline, get 10
-7,
-8,
-9Inoculation 10 ages in days do not have 5 of the Embryo Gallus domesticus of newcastle disease virus antibody in 3 each allantois of dilution factor, every embryo 0.1ml, put 37～38 ℃ and continue hatching, dead Embryo Gallus domesticus discards and disregards before 48 hours, at any time take out at 48～120 hours dead Embryo Gallus domesticus, results Embryo Gallus domesticus liquid, same dilution blastochyle mixed in equal amounts is measured the red cell agglutination valency respectively by dilution factor; To 120 hours, take out all embryos of living, gather in the crops Embryo Gallus domesticus liquid one by one, measure the red cell agglutination valency respectively, agglutination titer 〉=be judged to infection at 1: 128 calculates EID
50, every 0.1ml should contain virus 〉=OEID
50Viral level measurement result in 3 freeze-dried products
Be shown in the following table listed result
Table 1 freeze-dried products places 100 ℃ of physical behavior and EID of boiling indirectly after 10 minutes
50Measurement result
| The vaccine lot number | After doing | 100 ℃ of physical behaviors after 10 minutes | EID 50 |
| 9601 batches of experimental vaccines | 10 8．9 | Loose spongy contraction | 10 4．5 |
| 9602 batches of experimental vaccines | 10 8．9 | The loose spongy agglomerate that is shrunk to | 10 4．7 |
| 9603 batches of experimental vaccines | 10 8．9 | The loose spongy mushroom that is shrunk to | 10 5．4 |
| 9604 batches of experimental vaccines | 10 8．7 | Loose spongy expansion is cellular | 10 5．5 |
| The domestic goods Seedling | 10 8．9 | The loose spongy glue that is shrunk to | 10 0 |
| External commercial seedling 1 | 10 8．7 | Loose spongy contraction | 10 4．1 |
| External commercial seedling 2 | 10 8．7 | The loose spongy agglomerate that is shrunk to | 10 0 |
Vaccine is preserved different time EID in 2～8 ℃ before and after table 2 lyophilizing
50Measurement result
| The vaccine lot number | Before doing | After the lyophilizing (my god) | Descend to 1～10 day average every day | Every decline 10 1Required day | |||
| 0 | 30 | 70 | 180 | ||||
| 9601 batches of experimental vaccines | 10 9.1 | 10 8.9 | 10 8.9 | 10 8.7 | 10 8.5 | 10 0.002 | 450 |
| 9602 batches of experimental vaccines | 10 9.1 | 10 8.9 | 10 8.9 | 10 8.9 | 10 8.7 | 10 0.001 | 900 |
| 9603 batches of experimental vaccines | 10 9.1 | 10 8.9 | 10 8.9 | 108.9 | 10 8.7 | 10 0.001 | 900 |
| The domestic goods Seedling | 10 9.1 | 10 8.9 | 10 8.9 | 10 8.7 | 10 8.7 | 10 0.012 | 81 |
Vaccine is preserved different time EID in 37 ℃ before and after table 3 lyophilizing
50Measurement result
Vaccine is preserved different time EID in 45 ℃ before and after table 4 lyophilizing
50Measurement result
| The vaccine lot number | After the lyophilizing (my god) | Descend to 1～10 day average every day | Every decline 10 1 | ||
| 0 | 10 | 30 | |||
| 96017 batches of experimental vaccines | 10 8．5 | 10 7．9 | 10 6．5 | 10 0．067 | 15 |
| Test 96051 batches | 10 8．7 | 10 7．5 | 10 7．1 | 10 0．059 | 18．75 |
| 96053 batches of experimental vaccines | 10 8．7 | 10 7．5 | 10 6．3 | 10 0．08 | 12．5 |
| The domestic goods Seedling | 10 8．9 | 10 3．7 | 10 0 | 10 0．89 | 0．96* |
| The vaccine lot number | Before doing | After freezing (my god) | Descend to 1～5 day average every day | Every decline 10 1Required day | |
| 0 | 5 | ||||
| 9601 batches of experimental vaccines | 10 9.1 | 10 8.9 | 10 8.3 | 10 0.72 | 1.39 |
| 9602 batches of experimental vaccines | 10 9.1 | 10 8.9 | 10 6.1 | 10 0.56 | 1.79 |
| 9603 batches of experimental vaccines | 10 9.1 | 10 8.9 | 10 6.1 | 10 0.56 | 1.79 |
| The domestic goods Seedling | 10 9.1 | 10 8.9 | 10 0 | 10 1.78 | |
| External commercial seedling | 10 9.1 | 10 8.9 | 10 5.9 | 10 0.6 | 1.67 |
| The experimental vaccine lot number | EID after the lyophilizing 50 | The lyophilizing date | The vaccine storage life | Preserve back EID 50 |
| 960418 batches of experimental vaccines | 10 9．1 | 1996．04．18 | 11 months | 10 9．1 |
| 960430 batches of experimental vaccines | 10 9．1 | 1996．04．30 | 11 months | 10 9．1 |
| 960507 batches of experimental vaccines | 10 8．7 | 1996．05．07 | 10 months | 10 8．3 |
| 960725 batches of experimental vaccines | 10 8．9 | 1996．07．25 | 8 months | 10 8．9 |
| Experimental vaccine 960802 | 10 8．5 | 1996．08．02 | 7 months | 10 8．9 |
| Experimental vaccine is big 1 batch | 10 8．5 | 1996．11．28 | 4 months | 10 8．5 |
| Experimental vaccine is big 2 batches | 10 8．5 | 1996．11．30 | 4 months | 10 8．5 |
| Experimental vaccine is big 3 batches | 10 9．1 | 1996．12．04 | 4 months | 10 8．5 |
After table 1 shows that freeze dried vaccine with the preparation of the freeze drying protectant of formulation of the present invention is through 100 ℃ of processing, its physical behavior and EID
50Measure through identical with external vaccine.
Table 2,3,4 results show the freeze dried vaccine preservation under 2～8 ℃, 37 ℃ and 45 ℃ with the freeze drying protectant preparation of formulation of the present invention, its EID
50Valency, every decline 10
1Titre needs 450～900,15 and 1.39 days respectively approximately, than long 5～10 times of domestic goods vaccine, reaches the level of external commercially available vaccine.
Table 5 result shows with preserving nearly 1 year its EID down at 2～8 ℃ with the freeze dried vaccine of the freeze drying protectant of formulation of the present invention preparation
50Valency does not significantly decrease as yet.
Claims (3)
- The manufacture method of the freeze-dried live vaccine that 1 one kinds of animals are used; it is characterized in that: will prepare newcastle disease vaccine virus antigen that vaccine uses add the present invention designed with polyvinylpyrrolidone; sodium carboxymethyl cellulose; tamarind gum; sodium glutamate; trehalose is the formulated freeze drying protectant of main matrix; adopt the freeze-drying curve that adapts with it; promptly earlier freeze drying box is cooled in advance-5 ℃; the container that above antigen-frozen-dried protective agent composition will be housed again enters freeze drying box; speed with 1 ℃/min is cooled to-35 ℃, keep 2 hours under this temperature after, begin to be evacuated to 10 -4After the holder, 1 holder=133.322368Pa begins to heat up, and promptly requires to be warming up in 16 hours-20 ℃, continues to be warming up to 20 ℃ with 5～8 ℃ speed per hour thereafter, and keeps 6 hours outlets of jumping a queue, and whole freeze-drying process is 32 hours.
- 2 vaccine manufacture methods according to claim 1; it is characterized in that: the frozen-dried protective agent prescription is as follows: polyvinylpyrrolidone 5～15g; sodium carboxymethyl cellulose 2～10g; tamarind gum 5～15g; sodium glutamate 20～40g, trehalose 10～15g, vitamin C 1～5g; glycyrrhizin 2～15g, MgCI 20.01～0.05g, K 2HPO 41.3g, KH 2PO 40.05g, DDW 1000ml.
- 3 vaccine manufacture methods according to claim 1 is characterized in that: with the freeze-dried live vaccine of the inventive method preparation, preserve its significant change of having tired in 1 year under 2～8 ℃ of conditions.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN97116489A CN1063971C (en) | 1997-09-24 | 1997-09-24 | Method for preparing freeze-dried live vaccine for animal use |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN97116489A CN1063971C (en) | 1997-09-24 | 1997-09-24 | Method for preparing freeze-dried live vaccine for animal use |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1176827A CN1176827A (en) | 1998-03-25 |
| CN1063971C true CN1063971C (en) | 2001-04-04 |
Family
ID=5173897
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN97116489A Expired - Fee Related CN1063971C (en) | 1997-09-24 | 1997-09-24 | Method for preparing freeze-dried live vaccine for animal use |
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| CN (1) | CN1063971C (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| MX2007005379A (en) * | 2004-11-05 | 2007-07-04 | Wellstat Biologics Corp | Stable and filterable enveloped virus formulations. |
| CN100418580C (en) * | 2005-11-14 | 2008-09-17 | 中国农业科学院哈尔滨兽医研究所 | Heat-resistant freeze-drying protectant for chicken Newcastle disease live vaccine, preparation method and application thereof |
| CN102626520B (en) * | 2012-04-23 | 2013-07-03 | 青岛农业大学 | Application of heat-resisting cryoprotectant of mink canine distemper live vaccine |
| CN102671207B (en) * | 2012-05-30 | 2013-06-05 | 青岛农业大学 | Newcastle disease and avian infectious bronchitis bigeminal live vaccine heat-resisting freeze-drying protective agent and preparation method |
| CN102671208B (en) * | 2012-05-30 | 2013-07-03 | 青岛农业大学 | Heat-resisting freeze-dried protective agent for live vaccine for chicken infectious bursal diseases and preparation method for heat-resisting freeze-dried protective agent |
| CN102671209B (en) * | 2012-05-30 | 2013-06-05 | 青岛农业大学 | Heat-resistant freeze-drying protective agent for newcastle disease live vaccines and preparation method |
| CN103550401B (en) * | 2013-11-19 | 2016-06-22 | 浙江美保龙生物技术有限公司 | A kind of preparation method of composite microbial ecological agent effervescent tablet |
| CN112807423A (en) * | 2020-12-29 | 2021-05-18 | 肇庆大华农生物药品有限公司 | Freeze-drying process of chick embryo culture vaccine |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0300111A1 (en) * | 1987-07-22 | 1989-01-25 | Farvalsa AG | A moisture stable solid valproic acid formulation and a method of preparing the same |
| US5294439A (en) * | 1986-06-02 | 1994-03-15 | Nippon Chemiphar Co., Ltd. | Stabilized benzimidazole derivative and composition |
-
1997
- 1997-09-24 CN CN97116489A patent/CN1063971C/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5294439A (en) * | 1986-06-02 | 1994-03-15 | Nippon Chemiphar Co., Ltd. | Stabilized benzimidazole derivative and composition |
| EP0300111A1 (en) * | 1987-07-22 | 1989-01-25 | Farvalsa AG | A moisture stable solid valproic acid formulation and a method of preparing the same |
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