CN102908368A - Preparation method of virus lyophilized preparation - Google Patents
Preparation method of virus lyophilized preparation Download PDFInfo
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- CN102908368A CN102908368A CN2011102193768A CN201110219376A CN102908368A CN 102908368 A CN102908368 A CN 102908368A CN 2011102193768 A CN2011102193768 A CN 2011102193768A CN 201110219376 A CN201110219376 A CN 201110219376A CN 102908368 A CN102908368 A CN 102908368A
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- stock solution
- lyophilized formulations
- encephalitis virus
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- lyophilization additive
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Abstract
The invention discloses a preparation method of a virus lyophilized preparation, and belongs to the field of virus lyophilized preparations and preparation methods thereof. The invention aims at providing a virus lyophilized preparation. A technical scheme of the invention is that: the virus lyophilized preparation comprises a Newcastle disease virus stock solution and a lyophilization additive; and the lyophilization additive is substantially composed of human serum albumin and hydroxyethyl starch. The method for producing the virus lyophilized preparation comprises the steps that: (1) the virus stock solution and the lyophilization additive are well mixed; the mixture is subjected to sterile filtration; and the mixture is sub-packaged into vials; (2) the vials are placed into a lyophilizing device, and are respectively equilibrated for 1h under temperatures of 5 DEG C, 0 DEG C, and -6 DEG C; (3) rapid freezing is carried out, wherein a product temperature is reduced to -50 DEG C, and the temperature is maintained for 3h; (4) vacuum drying is carried out; (5) heating drying is carried out, wherein a temperature is maintained for 3h when the product temperature reaches 30 DEG C; and (6) the products are plugged and sealed.
Description
Technical field
The present invention relates to a kind of preparation method of viral lyophilized formulations, particularly relate to a kind of preparation method of Avian pneumo-encephalitis virus lyophilized formulations.
Background technology
Avian pneumo-encephalitis virus belongs to the secondary sticking coe virus of animal, the method of production Avian pneumo-encephalitis virus lyophilized formulations is with standard virus liquid such as Avian pneumo-encephalitis virus La Sota strain, clone30 strain, HBi strain, F strain or N79 strains at present, be inoculated in the chick embryo allantois intracavity of 10 ages in days, 37 ℃ of hatching breedings.Regularly collect qualified chick embryo allantoic liquid, with an amount of lyophilization additive mix homogeneously, quantitative separating is made lyophilized formulations through the pre-freeze vacuum and heating drying in aseptic cillin bottle.
Have following problem in the process with said method production lyophilized formulations: 1, the lyophilized formulations shelf-life is shorter; 2, the biologically active labile of lyophilized formulations.
Summary of the invention
The purpose of this invention is to provide long Avian pneumo-encephalitis virus lyophilized formulations of a kind of shelf-life.
A kind of preparation method of viral lyophilized formulations comprises Avian pneumo-encephalitis virus stock solution and lyophilization additive, and described lyophilization additive is comprised of human albumin and hetastarch basically.In order to keep better and protect the activity of stock solution biology, also contain sodium ascorbate, arginine in the described lyophilization additive.The w/v of described each component of lyophilization additive is:
The ratio of described Avian pneumo-encephalitis virus stock solution and described lyophilization additive is Avian pneumo-encephalitis virus stock solution: lyophilization additive=1: 1～5: 1.
A kind of method of producing viral lyophilized formulations may further comprise the steps:
(1) Avian pneumo-encephalitis virus stock solution is crossed 0.45 μ m filter element, add the aseptic filtration of lyophilization additive mixing through behind concentrated, the purification, be sub-packed in the cillin bottle, partly jump a queue, wherein, described lyophilization additive is comprised of human albumin and hetastarch basically;
(2) cillin bottle is put into freeze dryer, respectively 4.8～5.2 ℃, 0.2～0.8 ℃ ,-5.8～-6.2 ℃ each balances 1 hour;
(3) quick freezing is down to products temperature-48～-52 ℃ and was kept 2.5～3.5 hours;
(4) vacuum drying;
(5) heat drying reaches under 25.5～37.5 ℃ of conditions at products temperature and to continue 2.5～3.5 hours;
(6) all jump a queue the press seal aluminium lid.
Described virus stock solution used is that Avian pneumo-encephalitis virus is inoculated in 10 age in days chick embryo allantois intracavity, and chick embryo allantoic liquid is collected in 36 ℃ of hatching breedings.Also have sodium ascorbate, arginine in the described lyophilization additive.
The w/v of described each component of lyophilization additive is:
The ratio of described virus stock solution used and described lyophilization additive is Avian pneumo-encephalitis virus stock solution: lyophilization additive=1: 1～5: 1.
Therefore the present invention has the following advantages owing to adopted technique scheme:
1. in decompression one intensification one dry run of lyophilized formulations, suitable heating schedule is important to the biological activity of keeping biological product, the mode of appearance of preparation.The dry terminal point that heats up is controlled at 30 ℃ and lasting 3 hours, residual moisture in the goods is existed with quantitative non-covalent bond form, with the form of keeping stock solution, active stablizing, reduce finished product lyophilized formulations tiring in the preservation process and fall after rise rapidly, and prolong the holding time.
2, lyophilization additive of the present invention and Avian pneumo-encephalitis virus stock solution have splendid biocompatibility.Hetastarch is a kind of water solublity modified starch, belongs to polysaccharide, can either well protect virus activity, is again a kind of good freeze-dried excipient; Arginine is a kind of good protein protection material; Sodium ascorbate is the vitamin of anti-cell film oxidation; Particularly when adding the human albumin, they have consisted of the bionical system that is suitable for Avian pneumo-encephalitis virus stock solution jointly, are conducive to dormancy and the recovery of stock solution, and lyophilized formulations of the present invention can be preserved 14 months under 4 ℃ of conditions at least.
3, the adaptability distortion from the macroscopic view to the microcosmic can occur, to keep its biological activity in microorganism in the process of i.e. liquid cooling one an icing formation vitrification crystalline state in the liquid freezing process.Adopting 5 ℃, 0 ℃ ,-6 ℃ each balances 1 hour in the freezing process that lyophilized formulations is produced, be conducive to stock solution to the adaptability distortion of temperature, is necessary to the biological value of keeping lyophilized formulations.
The present invention will be further described below in conjunction with specific embodiment.
The specific embodiment
Embodiment 1, production lyophilized formulations of the present invention
The production of Avian pneumo-encephalitis virus lyophilized formulations of the present invention may further comprise the steps:
1, producing with kind of a poison is the clone30 strain;
2, hatching: get fertile egg, hatched 10 according to 37 ℃ of the methods in " People's Republic of China's biological product quality standard " (hereinafter to be referred as " standard ").With normal saline dilution kind poison, making virus titer is 91og2, and every chick embryo allantoic cavity is inoculated the malicious 0.1ml of this kind, puts 36 ℃ and hatches 72 hours;
3, results stock solution: discard Embryo Gallus domesticus dead before 24 hours, remaining Embryo Gallus domesticus is put 4 ℃ of cold preservations and is spent the night.Draw allantoic fluid under aseptic condition, mixing filters, airtight preservation;
4, with the stock solution of results with 0.45 μ m filter element filtering, concentrate with ultrafilter, use the chromatographic column purification;
5, additive preparation
(1) gets sodium ascorbate 108.7 grams, hetastarch 198.2 grams, arginine 19.6 grams, be dissolved in 600 ml distilled waters, add 20% human albumin 20ml, filter, sterilized 40 minutes for 115 ℃;
(2) with (1) solution, with the pH value to 6.8 of 10% aseptic sodium hydrogen phosphate regulator solution ± 0.1, add distilled water to 1000 milliliter, mixing;
6, preparation lyophilized formulations liquid: the stock solution and the ratio mixing of additive solution with 1: 1 that will tire and be adjusted into 9log2, the injection cillin bottle is partly jumped a queue;
7, freeze: will divide the lyophilized formulations liquid that installs to put into the lyophilization work box, and preset cooling process, and respectively 5 ℃, 0 ℃ ,-6 ℃ each balances 1 hour, start the quick freezing program, and temperature of charge will be down to-50 ℃, keep 3 hours;
8, vacuum drying: start the vacuum work program, treat that relative pressure is down to 5Pa in the freeze dryer, default heating schedule was kept 30 minutes with 15 ℃ of each intensifications, and lasting decompression is warming up to 30 ℃ and kept 3 hours;
9, all jump a queue the press seal aluminium lid.
The finished product lyophilized formulations is by " standard " listed method calibrating, and is qualified.
Embodiment 2, the shelf-life of lyophilized formulations of the present invention under 2～8 ℃ of preservation conditions
3 batches under 2～8 ℃ of conditions, have been measured respectively with the lyophilized formulations of the inventive method preparation half infection rate (EID at different times
50), the result is as shown in table 1:
Table 1: the EID of lyophilized formulations under 2～8 ℃ of preservation conditions
50
Table 2: the virus titer of lyophilized formulations under 2～8 ℃ of preservation conditions
Result from table can find out that lyophilized formulations of the present invention can be preserved 14 months at least under 2～8 ℃ of conditions.
Embodiment 3, the shelf-life of lyophilized formulations under high temperature, high humidity
The EID of observation different preservation lyophilized formulations in period under 37 ℃, the condition of relative humidity 75%
50, the results are shown in Table 3.
Table 3: the EID of lyophilized formulations under 37 ℃ of relative humidity 75% preservation conditions
50
Result from table can find out that the heat stability of lyophilized formulations of the present invention meets FDA about the pertinent regulations of the stability assessment standard (FDA) of Avian pneumo-encephalitis virus lyophilized formulations alive.
Variation before and after the embodiment 43 batch sample lyophilizing
Observe appearance character, pH value, virus titer variation and moisture, the water-soluble situation of 3 batch sample lyophilizing front and back, the results are shown in Table 4.
Table 4 three batch sample freeze-dried test results
In the experiment of as can be seen from Table 4 hetastarch, arginine, three kinds of substrate of albumin being carried out as the lyophilizing adjuvant in proportion, the lyophilizing sample presents the loose agglomerate of milky, can dissolve rapidly, and significant change does not occur in hemagglutinative titer, pH before and after the lyophilizing, and effect is ideal.
Above-mentioned detailed description of the preparation method of this kind cancer therapy drug being carried out with reference to embodiment; illustrative rather than determinate; can list several embodiment according to institute's limited range; therefore in the variation and the modification that do not break away under the general plotting of the present invention, should belong within protection scope of the present invention.
Claims (7)
1. the preparation method of a viral lyophilized formulations comprises Avian pneumo-encephalitis virus stock solution and lyophilization additive, it is characterized in that: described lyophilization additive is comprised of human albumin and hetastarch basically.
2. viral lyophilized formulations according to claim 1 is characterized in that: also contain sodium ascorbate, arginine in the described lyophilization additive.
3. viral lyophilized formulations according to claim 2 is characterized in that the w/v of described each component of lyophilization additive is:
4. viral lyophilized formulations according to claim 1, it is characterized in that: the ratio of described Avian pneumo-encephalitis virus stock solution and described lyophilization additive is Avian pneumo-encephalitis virus stock solution: lyophilization additive=1: 1～5: 1.
5. method of producing viral lyophilized formulations may further comprise the steps:
(1) Avian pneumo-encephalitis virus stock solution is crossed 0.45 μ m filter element, add lyophilization additive mixing through behind concentrated, the purification, aseptic filtration is sub-packed in the cillin bottle, partly jump a queue, wherein said lyophilization additive is comprised of human albumin, hetastarch basically;
(2) treat that cillin bottle puts into freeze dryer, respectively 4.8～5.2 ℃, 0.2～0.8 ℃ ,-5.8～-6.2 ℃ each balances 1 hour;
(3) quick freezing is down to products temperature-48～-52 ℃ and was kept 2.5～3.5 hours;
(4) vacuum drying;
(5) heat drying reaches under 25.5～37.5 ℃ of conditions at products temperature and to continue 2.5～3.5 hours;
(6) all jump a queue the press seal aluminium lid.
6. the method for the viral lyophilized formulations of production according to claim 5, it is characterized in that: described Avian pneumo-encephalitis virus stock solution is that Avian pneumo-encephalitis virus is inoculated in 10 age in days chick embryo allantois intracavity, chick embryo allantoic liquid is collected in 36 ℃ of hatching breedings.
7. the method for the viral lyophilized formulations of production according to claim 5 is characterized in that: also have sodium ascorbate, arginine in the described lyophilization additive; The w/v of described each component of lyophilization additive is:
The ratio of described Avian pneumo-encephalitis virus stock solution and described lyophilization additive is Avian pneumo-encephalitis virus stock solution: freezing dry additive=1: 1～5: 1.
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| CN2011102193768A CN102908368A (en) | 2011-08-02 | 2011-08-02 | Preparation method of virus lyophilized preparation |
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| CN2011102193768A CN102908368A (en) | 2011-08-02 | 2011-08-02 | Preparation method of virus lyophilized preparation |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11604026B2 (en) | 2019-03-14 | 2023-03-14 | Terumo Bct Biotechnologies, Llc | Lyophilization loading tray assembly and system |
| US11634257B2 (en) | 2017-10-09 | 2023-04-25 | Terumo Bct Biotechnologies, Llc | Lyophilization container and method of using same |
-
2011
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11634257B2 (en) | 2017-10-09 | 2023-04-25 | Terumo Bct Biotechnologies, Llc | Lyophilization container and method of using same |
| US11604026B2 (en) | 2019-03-14 | 2023-03-14 | Terumo Bct Biotechnologies, Llc | Lyophilization loading tray assembly and system |
| US11609043B2 (en) | 2019-03-14 | 2023-03-21 | Terumo Bct Biotechnologies, Llc | Lyophilization container fill fixture, system and method of use |
| US11609042B2 (en) | 2019-03-14 | 2023-03-21 | Terumo Bct Biotechnologies, Llc | Multi-part lyophilization container and method of use |
| US11740019B2 (en) | 2019-03-14 | 2023-08-29 | Terumo Bct Biotechnologies, Llc | Lyophilization loading tray assembly and system |
| US11747082B2 (en) | 2019-03-14 | 2023-09-05 | Terumo Bct Biotechnologies, Llc | Multi-part lyophilization container and method of use |
| US11815311B2 (en) | 2019-03-14 | 2023-11-14 | Terumo Bct Biotechnologies, Llc | Lyophilization container fill fixture, system and method of use |
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Application publication date: 20130206 |