CN1168503C

CN1168503C - Heat-resisting freeze-drying protective agent for attenuated freeze-dried vaccine for newcastle disease and preparation process thereof

Info

Publication number: CN1168503C
Application number: CNB011388714A
Authority: CN
Inventors: 卫广森; 王文成; 张万林
Original assignee: 卫广森
Priority date: 2001-12-21
Filing date: 2001-12-21
Publication date: 2004-09-29
Anticipated expiration: 2021-12-21
Also published as: CN1426816A

Abstract

A heat-resistant freeze-drying protective agent for a Newcastle disease attenuated freeze-drying vaccine and a preparation process thereof are characterized in that the protective agent comprises the following material components in parts by weight: 1.5-2% of gelatin, 5-6% of cane sugar, 5-10% of dextrin, 2-5% of PVP (K90), 2% of sorbitol and 1-2% of sodium glutamate; the preparation is a dry matter component, and the residual parts in percentage are distilled water. The protective agent is prepared by sterilizing the autoclavable and non-high temperature resistant effective components respectively, mixing to obtain lyophilized heat resistant protective agent, adding into virus antigen solution at a ratio of 1: 1, packaging, and freeze drying. The product is stored at 2-8 ℃ for 24 months, the freeze-dried shape is still kept, and the virus content exceeds the standard of national veterinary biological product regulation. The invention is a low-cost, long-acting product and a preparation method thereof, and solves the problem of high cost of transportation and storage for a long time for veterinary biological products.

Description

Weak malicious freeze dried vaccine heat-resisting lyophilized protecting agent of newcastle disease and preparation technology

Technical field

The present invention relates to the Freeze Drying Technique of biological product, preservation adjuvant and manufacture method thereof.

Background technology

China's veterinary biological product protective agent commonly used at present is mainly 5% sucrose skim milk and 5-10% sucrose, the 1.5-3% gelatin, and goods require cryopreservation (below 15 ℃), and the storage life of 2-8 ℃ of condition only 3-9 month must use up at normal temperatures as early as possible.This is just for the preservation of veterinary biological product, transportation, use etc. bring many difficulties and inconvenience, simultaneously owing to must cryopreservation causing energy resource consumption and storage cost big.

At present, the flourishing in the world biological freeze-dried products of national veterinary almost all is that 2-8 ℃ of preservation is more than 24 months.But the not play-by-play of square method.Chinese patent CN1176827A " a kind of manufacture method of freeze-dried live vaccine for animal use " proposes to adopt with polyvinylpyrrolidone, sodium carboxymethyl cellulose; tamarind gum; sodium glutamate; trehaloses etc. are the freeze drying protectant that the formulated of main matrix freeze drying protectant becomes; the method that freeze-drying curve that employing adapts with it and newcastle disease vaccine virus antigen are made the newcastle disease freeze-dried live vaccine, but this Seedling long preservation under 2-8 ℃ of condition.

Summary of the invention

The purpose of this invention is to provide weak malicious freeze dried vaccine heat-resisting lyophilized protecting agent of a kind of newcastle disease and preparation technology, reduce protective condition, extend using period and reduce use cost.

The preparation technology of the weak malicious freeze dried vaccine heat-resisting lyophilized protecting agent of newcastle disease, press component, mixed, it is characterized in that protectant active substance is handled degerming respectively: sterilized 30-40 minute for 116 ℃ to being dissolved in the distilled water in the preparation ratio by autoclaved material (1); (2) material with non-refractory also is dissolved in the distilled water in the preparation ratio, with aperture 0.22 μ m membrane filtration degerming; After finishing degerming respectively (1), (2) two kinds of compositions are mixed, be the lyophilizing heat resisting protective, add in the virus antigen liquid in 1: 1 ratio during use, carry out lyophilization after the packing.

The preparation of preparation technology's preparation of the present invention can be preserved more than 24 months under 2-8 ℃ of condition, can reach every index of regulation in " People's Republic of China's veterinary biological product quality standard ".

The specific embodiment

The weak malicious freeze dried vaccine heat-resisting lyophilized protecting agent of newcastle disease, comprise gelatin commonly used, sucrose, dextrin, it is characterized in that the material component proportioning of heat-resisting lyophilized protecting agent is: gelatin 1.5-2%, sucrose 5-6%, dextrin 5-10%, PVP (K90) 2-5%, sorbitol 2%, sodium glutamate 1-2%; Be the dry component in the preparation, percentage ratio residue part is distilled water.

The preparation technology of the weak malicious freeze dried vaccine heat-resisting lyophilized protecting agent of newcastle disease, comprise that the dry component with above-mentioned heat-resisting lyophilized protecting agent is mixed in proportion, it is characterized in that protectant active substance component is carried out degerming to be handled: (1) is to can autoclaved material: gelatin, sucrose, dextrin, PVP (90), be dissolved in the distilled water 116 ℃ of sterilizations 30-40 minute in ratio separately; (2) with the material of non-refractory: sorbitol, sodium glutamate also are dissolved in the distilled water, in proportion with the membrane filtration degerming of aperture 0.22 μ m; After finishing degerming respectively (1), (2) two kinds of compositions are mixed, be the lyophilizing heat resisting protective, add in the virus antigen liquid in 1: 1 ratio during use, carry out lyophilization after the packing.

Technical parameter and detection method

1. the physical behavior of product, steriling test, mycoplasma check, exogenous virus check, specificity check, safety verification, residual moisture mensuration and vacuum are measured by the method and the standard of " People's Republic of China's veterinary biological product rules " and are undertaken.

2. lyophilization cycle is no more than 48h.

3. protectant toxicity test

Bird vaccine carries out each 20 of collunarium, eye dripping, drinking-water and subcutaneous injections by containing 10 times of amounts protecting dosage in the middle of the product respectively to 7 age in days healthy chicks, observes 20d, and chickling is all strong to live; There is not any untoward reaction.

4. viral level is measured and standard

(1) assay method:

A. make 10 times of serial dilutions by the real tissue mass that contains with sterile saline, get 3 suitable dilution factors, inoculate 5 of 10 age in days SPF Embryo Gallus domesticus in the allantoic cavity respectively, every embryo 0.1mL puts 37 ℃ and continues to hatch, the dead in the past Embryo Gallus domesticus of 48h discards to be disregarded, at the Embryo Gallus domesticus of 48-120h death, take out results Embryo Gallus domesticus liquid at any time, with same dilution Embryo Gallus domesticus liquid mixed in equal amounts, measure the red cell agglutination valency respectively.To 120h, get all embryos of living, gather in the crops Embryo Gallus domesticus liquid one by one, measure the red cell agglutination valency respectively, agglutination titer 〉=160 are judged to infection, calculate EID ₅₀

B. press label and indicate plumage part, vaccine is diluted to 1 plumage part/0.1mL with sterile saline, remake 10 times of serial dilutions, other method is the same, calculates every plumage part EID at last ₅₀Content.

(2) reach standard

Viral loss rate is less than 0.4 titre after A, the lyophilizing.

B. every plumage part viral level 〉=10 after the lyophilizing ^6.7EID ₅₀

C. the product after the lyophilizing is put 37 ℃ and is preserved 10d and 30d and carry out viral level and measure, the viral level rate of descent the former within 1 titre, the latter is within 2 titres.

D. product is put 25 ℃ of preservation 30d after the lyophilizing, and the viral level rate of descent is within 0.6 titre.

E. product is put 2-8 ℃ of preservation 24 months, every plumage part viral level 〉=10 ^6.17EID ₅₀

Claims

1. A heat-resistant freeze-drying protective agent for chicken Newcastle disease attenuated freeze-dried vaccine, comprising commonly used gelatin, sucrose, and dextrin, characterized in that the material composition ratio of the heat-resistant freeze-drying protective agent is: gelatin 1.5-2%, sucrose 5-6%, 5-10% dextrin, 2-5% PVP (K90), 2% sorbitol, 1-2% sodium glutamate; the preparation is a dry matter component, and the rest of the percentage is distilled water.

2. A process for preparing the heat-resistant freeze-drying protectant for chicken Newcastle disease attenuated freeze-dried vaccine according to claim 1, comprising mixing the dry matter components of the above-mentioned heat-resistant freeze-dry protectant in proportion, which is characterized in that the protective agent is effective Substances are sterilized: (1) autoclavable substances: gelatin, sucrose, dextrin, PVP (90), dissolved in distilled water according to their respective proportions, sterilized at 116°C for 30-40 minutes; (2) Substances that are not resistant to high temperature: sorbitol, sodium glutamate; all dissolved in distilled water according to their respective proportions, and sterilized by filtration with a filter membrane with a pore size of 0.22 μm; after the sterilization is completed, the two components (1) and (2) Mixing is the freeze-dried heat-resistant protective agent, which is added to the virus antigen solution at a ratio of 1:1 when used, and freeze-dried after subpackaging.