CN103301443A - Preparation method of stable chicken lyophilized alpha-interferon dosage form - Google Patents
Preparation method of stable chicken lyophilized alpha-interferon dosage form Download PDFInfo
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- CN103301443A CN103301443A CN201210059441XA CN201210059441A CN103301443A CN 103301443 A CN103301443 A CN 103301443A CN 201210059441X A CN201210059441X A CN 201210059441XA CN 201210059441 A CN201210059441 A CN 201210059441A CN 103301443 A CN103301443 A CN 103301443A
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Abstract
The invention discloses a chicken lyophilized recombinant alpha-interferon preparation which uses gelatin and cane sugar as a stabilizing agent, and glycine as a freeze-drying protective agent. The gelatin, the cane sugar and the glycine solution are added into an interferon original liquid, and then are uniformly mixed and filtered for sterilization, and the products are freeze-dried to preserve. The stabilizing agent and the protective agent used in the invention are all nontoxic and harmless substances, and are good in stability and obvious in protective effect. After preserved for one year under 4 DEG C, the alpha-interferon dosage form is good in effect through determining a protection rate for chick embryo and an interferon activity by using a cytopathic effect inhibition method. Clinic test proves that the chicken lyophilized interferon dosage form has good treatment effect on newcastle disease, and by using the preparation which is a novel poultry antiviral drug, economic loss can be reduced for the majority of farmers.
Description
Technical field
The present invention relates to a kind of technology of preparing of bio-pharmaceutical, particularly a kind of preparation method of chicken alpha interferon freeze-dried formulation.
Background technology
Interferon is the extensive bioactive albumen that has that exists in one group of body, has the multiple effects such as antiviral, antitumor and adjusting body's immunity.Studies have shown that, chicken interferon has good therapeutic effect to chicken viral diseases.But interferon is as a kind of protein, and what easily be subject to the many factors such as temperature, illumination, jolting, pH, heavy metal ion, enzyme, microorganism in aqueous solution affects generation oxidation, degraded, polymerization and inactivation.Therefore; in order better to keep the biologic activity of goods, add stabilizing agent and freeze drying protectant in the chicken genetic engineering interferon solution behind purification, adopt vacuum freeze-drying method to make lyophilized formulations; thereby avoided liquid drugs injection dosage form poor stability, the shortcomings such as storage and transport condition harshness.
Summary of the invention
The present invention discloses a kind of preparation method of chicken alpha interferon freeze-dried formulation, this dosage form prescription is simple, preparation stabilization, application are convenient, effect significantly, be easy to storage and transport.By Embryo Gallus domesticus protective rate mensuration, cytopathic-effect inhibition assay being measured its activity and clinical therapeutic efficacy observation, confirm that this interferon dosage form has good inhibitory action to Avian pneumo-encephalitis virus and vesicular stomatitis virus.
Component of the present invention is as follows:
The solvent of freeze-dried drug of the present invention is 0.9%Nacl.
Concrete preparation method is as follows:
Getting chicken alpha interferon stock solution, to adjust its final concentration with 0.9%Nacl be 0.2mg/ml, adds gelatin, sucrose and the glycine for preparing in interferon stock solution, and Entkeimung is sub-packed in the penicillin bottle under aseptic condition.Penicillin bottle is put into-80 ℃ of refrigerator pre-freeze 4h, opened freezer dryer, insulation 1-2h when temperature is down to-50 ℃, then evacuation sublimes up into the complete lyophilizing of sample.After lyophilizing finishes, sample is placed 4 ℃ of preservations.
The present invention compared with prior art by rational prescription, makes freeze-dried formulation with interferon, can keep for a long time the stability of interferon.Have following advantage: (1) freeze-dried formulation moisture is low, interferon good stability (2) is easy to transportation and preservation (3) raiser (4) easy to use technique is simple, required cost is low, toxicity is little, oral safe, stable, the Degradation of the reasonable inhibition gastrointestinal enzyme of energy when drinking-water, collunarium, and easily absorb.
Description of drawings
Fig. 1: the protective rate of recombined chicken alpha interferon affinitive layer purification albumen after to Embryo Gallus domesticus counteracting toxic substances Avian pneumo-encephalitis virus.Different broken lines represent respectively different interferon additions among the figure.
Fig. 2: cytopathic-effect inhibition assay is surveyed the negative control hole of interferon activity: only add interferon, do not add VSV
Fig. 3: cytopathic-effect inhibition assay is surveyed the positive control hole of interferon activity: only add VSV, do not add interferon
Fig. 4: the interferon that cytopathic-effect inhibition assay is surveyed interferon activity is diluted to 10
5Inhibition to VSV
Fig. 5: the interferon that cytopathic-effect inhibition assay is surveyed interferon activity is diluted to 10
6Inhibition to VSV
Fig. 6: the interferon that cytopathic-effect inhibition assay is surveyed interferon activity is diluted to 10
7Inhibition to VSV
Specific embodiments
Below in conjunction with embodiment, to details are as follows according to the specific embodiment provided by the invention:
Embodiment 1
Preparation and formulation and technology
Interferon 100mg, gelatin 2g, sucrose 5g, glycine 1g adds sterile saline and is dissolved to 500ml.After the dissolving, with 0.22 μ m filter membrane Entkeimung, be divided in the 10ml penicillin bottle by every 0.5ml, with sealed membrane sealing, totally 1000.Penicillin bottle is put into-80 ℃ of refrigerator pre-freeze 4h, opened freezer dryer, insulation 1-2h when temperature is down to-50 ℃, then evacuation sublimes up into the complete lyophilizing of sample.After lyophilizing finishes, sample is placed 4 ℃ of preservations.
Embryo Gallus domesticus analysis of experiments interferon anti-reflecting virus effect
After interferon protein utilized the affinity chromatography purification, make curve with the time of Embryo Gallus domesticus survival rate after to counteracting toxic substances, Analysis interference element antiviral effect, the results are shown in Figure 1: giving 18 μ g protein groups Embryo Gallus domesticus hatchabilities the highest, is 80%.All the other are respectively organized the Embryo Gallus domesticus survival rate and all are higher than and do not add the interferon group, illustrate that interferon has good inhibition to Avian pneumo-encephalitis virus.
Embodiment 3
Adopt " the few cells pathological changes suppresses method " to measure interferon activity
With CEF cell/VSV system of determination.The CEF cell is about 40,000 cells (approximately 6-9h) in the every hole of adherent growth on 96 orifice plates, and (Fig. 4, Fig. 5, Fig. 6 are respectively 10 to remove the interferon sample to be measured that adds 10 times of gradient dilutions behind the nutritional solution
5, 10
6, 10
7Dilution) cultivates again 15-18h, remove interferon solution, use 100TCID
50Vesicular stomatitis virus (VSV) is attacked, set up simultaneously negative control hole (only to add interferon, do not add virus, the results are shown in Figure 2), the positive control hole (does not add interferon, only adds virus, the results are shown in Figure 3), approximately after 12-24 hour under inverted microscope observed result, in the time of the complete pathological changes in positive control hole, judged result.Be defined as a unit interferon with the interferon dilution factor that can press down 50% cytopathic sample.
The result is as follows:
Embodiment 4
The chicken interferon freeze-dried formulation is to the therapeutical effect of newcastle
The SPF chicken of 200 plumages, 20 ages in days is made the chickling morbidity with the method for artificial infection chicken's Avian pneumo-encephalitis virus.The morbidity chicken is divided into 2 groups, every group of 100 plumages at random.The A group is " chicken interferon freeze-dried formulation " treatment group, and the chicken interferon freeze-dried formulation adopts the preparation of embodiment 1 method; The B group is treated with normal saline for matched group.Utilize the collunarium mode simultaneously 2 groups of chickens to be contaminated the 0.2ml/ plumage with the strong poison of Avian pneumo-encephalitis virus.After clinical symptoms appearred in chicken, the A group gave " chicken interferon freeze-dried formulation " 20,000 units/plumage in the collunarium mode, every day 1 time, is used in conjunction 3 days; The B group gives normal saline 0.5ml/ plumage, is used in conjunction 3 days every day 1 time, observes simultaneously morbidity, death and the pathological change of chicken, and itemized record.
Behind the contamination 24h, chicken begins to occur clinical symptoms: lethargy, stretch the neck necking down, and feather is fluffy and disorderly, arranges green feces, and diet reduces or is absolutely useless.After the medication, A group chicken takes a turn for the better since the 2nd day state of an illness, has dead chicken to occur on the 3rd, 4 day, the afterwards basic rehabilitation of chicken of survival in 5 days.Symptom appears in B group chicken, and sb.'s illness took a turn for the worse after the 2nd day, begins to occur death, reached peak mortality in 3-5 days, and it is normal that not dead chicken recovered on the 6th day.Chicken interferon freeze-dried formulation treatment group is compared with matched group, and therapeutic effect difference is (P＜0.01) extremely significantly, illustrates that giving " chicken interferon freeze-dried formulation " in the collunarium mode has certain therapeutical effect to newcastle disease.
Claims (6)
1. stable chicken alpha interferon freeze-dried formulation is characterized in that component is as follows:
Chicken alpha interferon, 0.4% gelatin, 1% sucrose, 0.2% glycine.
2. as claimed in claim 1, used stabilizing agent and the protective agent of chicken alpha interferon freeze-dried formulation is nontoxic, innocuous substance.
3. as described in the claim 1,2, chicken alpha interferon stock solution, gelatin, sucrose, glycine be all with the 0.9%Nacl dilution, behind the mixing and Entkeimung.
4. as claimed in claim 3, the preparation method of stable chicken alpha interferon freeze-dried formulation is characterized in that: be sub-packed in the penicillin bottle under aseptic condition after the Entkeimung.Penicillin bottle is put into-80 ℃ of refrigerator pre-freeze 4h, opened freezer dryer, insulation 1-2h when temperature is down to-50 ℃, then evacuation sublimes up into the complete lyophilizing of sample.
5. as claimed in claim 4, the preparation method of stable chicken alpha interferon freeze-dried formulation is characterized in that: the vigor of every bottle of interferon is 3 * 10 after the packing
7Unit of activity.
6. stable chicken alpha interferon freeze-dried formulation is characterized in that: during clinical use, adopt the collunarium mode to treat.
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| CN201210059441XA CN103301443A (en) | 2012-03-08 | 2012-03-08 | Preparation method of stable chicken lyophilized alpha-interferon dosage form |
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| CN201210059441XA CN103301443A (en) | 2012-03-08 | 2012-03-08 | Preparation method of stable chicken lyophilized alpha-interferon dosage form |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1260171A (en) * | 2000-01-25 | 2000-07-19 | 长春长生基因药业股份有限公司 | Recombined alpha-type interferon freeze-drying protective agent |
| CN1389470A (en) * | 2002-06-21 | 2003-01-08 | 长春长生基因药业股份有限公司 | Stabilizer for recombinant alpha-interferon liquid |
| CN1861634A (en) * | 2006-04-06 | 2006-11-15 | 北京江中泽生科技有限责任公司 | Dog interferon alpha, preparation process and use thereof |
| WO2010142017A1 (en) * | 2009-06-09 | 2010-12-16 | Defyrus, Inc . | Administration of interferon for prophylaxis against or treatment of pathogenic infection |
| CN102058546A (en) * | 2009-11-12 | 2011-05-18 | 上海市农业科学院 | Freeze-drying preparation for recombinant swine alpha-interferon and preparation method and usage thereof |
-
2012
- 2012-03-08 CN CN201210059441XA patent/CN103301443A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1260171A (en) * | 2000-01-25 | 2000-07-19 | 长春长生基因药业股份有限公司 | Recombined alpha-type interferon freeze-drying protective agent |
| CN1389470A (en) * | 2002-06-21 | 2003-01-08 | 长春长生基因药业股份有限公司 | Stabilizer for recombinant alpha-interferon liquid |
| CN1861634A (en) * | 2006-04-06 | 2006-11-15 | 北京江中泽生科技有限责任公司 | Dog interferon alpha, preparation process and use thereof |
| WO2010142017A1 (en) * | 2009-06-09 | 2010-12-16 | Defyrus, Inc . | Administration of interferon for prophylaxis against or treatment of pathogenic infection |
| CN102058546A (en) * | 2009-11-12 | 2011-05-18 | 上海市农业科学院 | Freeze-drying preparation for recombinant swine alpha-interferon and preparation method and usage thereof |
Non-Patent Citations (2)
| Title |
|---|
| SARANJIT SINGH,ET AL.: "alteration in dissolution characteristics of geltatin-containing formulations", 《PHARMACEUTICAL TECHNOLOGY》, 30 April 2002 (2002-04-30), pages 36 - 58 * |
| 韦琴等: "鸡A干扰素基因的克隆、原核表达及抗病毒效果研究", 《生物工程学报》, vol. 22, no. 5, 30 September 2006 (2006-09-30), pages 737 - 743 * |
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Application publication date: 20130918 |