WO2016192487A1 - 不含明胶的疫苗保护剂组合物及流感减毒活疫苗 - Google Patents

不含明胶的疫苗保护剂组合物及流感减毒活疫苗 Download PDF

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WO2016192487A1
WO2016192487A1 PCT/CN2016/080226 CN2016080226W WO2016192487A1 WO 2016192487 A1 WO2016192487 A1 WO 2016192487A1 CN 2016080226 W CN2016080226 W CN 2016080226W WO 2016192487 A1 WO2016192487 A1 WO 2016192487A1
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vaccine
type
live attenuated
composition
attenuated influenza
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French (fr)
Inventor
申镇维
陈晓辉
徐菲
孙瑶
程晓耕
朱昌林
姜春来
孔维
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CHANGCHUN BCHT BIOTECHNOLOGY Co
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CHANGCHUN BCHT BIOTECHNOLOGY Co
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Priority to EP16802410.7A priority Critical patent/EP3305328A4/en
Priority to US15/578,124 priority patent/US10463741B2/en
Publication of WO2016192487A1 publication Critical patent/WO2016192487A1/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • 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
    • A61K39/12—Viral antigens
    • 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
    • A61K39/12—Viral antigens
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
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    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5254—Virus avirulent or attenuated
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/543—Mucosal route intranasal
    • 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
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16211—Influenzavirus B, i.e. influenza B virus
    • C12N2760/16234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to the field of vaccine production technology, and in particular, to a gelatin-free liquid vaccine protectant composition, and a live attenuated influenza vaccine comprising the liquid vaccine protectant composition.
  • Influenza is an acute respiratory infection caused by influenza viruses that is a serious hazard to human health.
  • the influenza virus is divided into three types: A (A), B (B), and C (C).
  • A A
  • B B
  • C C
  • the antigenicity of the influenza A virus often mutates, and has caused a worldwide pandemic many times in history.
  • Influenza A is also spread in pigs and poultry and causes influenza epidemics, causing a large number of animal deaths. People B and C are only broadcast between people.
  • Influenza B virus is less pathogenic to humans.
  • Influenza C virus mainly affects infants and young children and generally does not cause influenza epidemics.
  • WHO estimates in the case of an explosive epidemic, influenza viruses can spread rapidly around the world, affecting 10% to 20% of the total population. Even in non-explosive years, the flu causes 3 to 5 million serious cases and 250,000 to 500,000 deaths per year.
  • flu vaccination is the most effective means of combating influenza.
  • Vaccination can not only reduce the morbidity and mortality of high-risk groups, but also reduce symptoms, reduce the incidence of complications, and reduce the probability of continued transmission.
  • the live attenuated influenza vaccine is immunized with the nasal mucosal route and has the advantage of simulating natural infection.
  • a live attenuated influenza vaccine can induce local neutralizing antibodies as well as cellular immune responses, and the immune response lasts for a longer period of time than inactivated vaccines.
  • live attenuated influenza vaccines are easy to use and easy to perform large-scale immunization.
  • the live attenuated influenza vaccine is generally a freeze-dried powder dosage form.
  • the freeze-drying technology has matured, there are still some shortcomings: First, the freeze-dried powder injection needs to be reconstituted during use, which is inconvenient to use and increases the chance of contamination. Secondly, the freeze-drying process of biological products is complicated and the process consumes The duration and the flu virus titer loss are large. Third, the vacuum freeze-drying equipment has large investment, high energy consumption, high production cost, and limited vaccine production capacity, especially in the case of a major influenza outbreak, it is difficult for vaccine manufacturers to be in a short time. Produce a sufficient dose of vaccine.
  • the product is a cold-adapted trivalent virus vaccine, which is a water injection dosage form.
  • the product needs to be stored and transported at about 15 ° C and below, but since the pharmacy or school usually does not have equipment for handling frozen products, FluMist Can not be used on a large scale.
  • the inventors have invented a composition for use as a protective agent for live attenuated influenza vaccines, which contains no gelatin component, is less irritating to humans, and is a live attenuated influenza vaccine prepared using the composition. Good stability and long shelf life can be maintained under refrigeration conditions.
  • the present invention provides a composition for use as a protective agent for a live attenuated influenza vaccine, comprising a component and a concentration thereof: human serum albumin 1.0 to 15.0 g/L, preferably 1.5 to 12.0 g/L, more Preferably, it is 2.5 to 10.0 g/L; sucrose is 15.0 to 95.0 g/L, preferably 40.0 to 85.0 g/L, more preferably 50.0 to 75.0 g/L; sodium glutamate is 0.5 to 15.0 g/L, preferably 0.5 to 12.0 g/ L is more preferably 0.5 to 10.0 g/L; wherein the composition has a pH of 5.0 to 9.0, preferably pH 5.5 to 8.5, more preferably pH 6.0 to 8.0, and most preferably pH 6.0 to 7.4.
  • the composition of the present invention further comprises urea in a concentration of 0 to 8.0 g/L, preferably 0.5 to 6.0 g/L, more preferably 1.0 to 4.0 g/L; and arginine at a concentration of 0 to 10.0 g/L.
  • urea in a concentration of 0 to 8.0 g/L, preferably 0.5 to 6.0 g/L, more preferably 1.0 to 4.0 g/L; and arginine at a concentration of 0 to 10.0 g/L.
  • it is 1.0 to 10.0 g/L, more preferably 1.4 to 10.0 g/L
  • histidine has a concentration of 0 to 2.0 g/L, preferably 0.5 to 1.5 g/L
  • sorbitol and its concentration is 0 to 70.0.
  • g/L preferably 15.0-60.0 g/L, more preferably 25.0-50.0 g/L; glycine, the concentration is 0-20.0 g/L, preferably 3.0-15.0 g/L; mannitol, the concentration is 0-30.0 g/L is preferably 10.0 to 20.0 g/L.
  • the invention also provides the use of a composition of the invention for increasing the stability and safety of a vaccine and for the preparation of a live attenuated influenza vaccine.
  • the present invention provides a method for producing a live attenuated influenza vaccine, which comprises using the composition of the present invention as a protective agent.
  • the present invention provides a composition for use as a protective agent for a live attenuated influenza vaccine, comprising a component and a concentration thereof: human serum albumin 1.0 to 15.0 g/L, preferably 1.5 to 12.0 g/L, more Preferably it is 2.5 to 10.0 g/L; sucrose is 15.0 to 95.0 g/L, preferably 40.0 to 85.0 g/L, more preferably 50.0 to 75.0 g/L; sodium glutamate 0.5 to 15.0 g/L, preferably 0.5 to 12.0 g/L, more preferably 0.5 to 10.0 g/L; wherein the composition has a pH of 5.0 to 9.0, preferably pH 5.5 to 8.5, more preferably pH 6.0 to 8.0. Most preferably, it is pH 6.0-7.4.
  • the composition of the present invention further comprises urea in a concentration of 0 to 8.0 g/L, preferably 0.5 to 6.0 g/L, more preferably 1.0 to 4.0 g/L; and arginine at a concentration of 0 to 10.0 g/L.
  • urea in a concentration of 0 to 8.0 g/L, preferably 0.5 to 6.0 g/L, more preferably 1.0 to 4.0 g/L; and arginine at a concentration of 0 to 10.0 g/L.
  • it is 1.0 to 10.0 g/L, more preferably 1.4 to 10.0 g/L
  • histidine has a concentration of 0 to 2.0 g/L, preferably 0.5 to 1.5 g/L
  • sorbitol and its concentration is 0 to 70.0.
  • g/L preferably 15.0-60.0 g/L, more preferably 25.0-50.0 g/L; glycine, the concentration is 0-20.0 g/L, preferably 3.0-15.0 g/L; mannitol, the concentration is 0-30.0 g/L is preferably 10.0 to 20.0 g/L.
  • the invention also provides the use of a composition of the invention for the preparation of a live attenuated influenza vaccine.
  • the present invention also provides a method for preparing a live attenuated influenza vaccine, which comprises using the composition of the present invention as a protective agent.
  • the method for preparing a live attenuated influenza vaccine of the present invention comprises the steps of: sequentially dissolving the respective components of the composition of the present invention in a pH buffer; adjusting the pH to a specified value; filtering and sterilizing Adding a virus stock solution, which is a live attenuated influenza vaccine.
  • the liquid influenza attenuated live vaccine prepared according to the method of the invention has a pH of from 5.0 to 9.0, preferably from pH 5.5 to 8.5, more preferably from pH 6.0 to 8.0, most preferably from pH 6.0 to 7.4.
  • a phosphate buffer is used as the pH buffer.
  • the invention also provides a live attenuated influenza vaccine prepared using the composition of the invention and/or the method according to the invention.
  • the live attenuated influenza vaccine of the invention is an injection or a nasal spray.
  • the pH was determined according to Appendix V of the Pharmacopoeia of the People's Republic of China (2010 Edition) (Part 3).
  • MDCK cells Madin to Daby canine kidney cells, purchased from ATCC.
  • H1N1 influenza virus strain A/17/California/2009/38 (H1N1), provided by WHO;
  • H3N2 influenza virus strain A/17/Perth/1987 (H3N2), provided by WHO;
  • Influenza B virus strain B/56/Brisbane/60/08, provided by WHO.
  • TPCK-Trypsin purchased from Sigma, article number: T1426, solution;
  • DMEM cell culture solution (purchased from Sigma, article number: D6546);
  • Fetal bovine serum purchased from Hyclone, article number: SH30070.03
  • Streptomycin (purchased from Gibco, article number: 15140);
  • Anti-Influenza A Virus Nucleoprotein antibody purchased from Abcam, article number: [AA5H]ab20343;
  • Anti-Influenza B Virus Nucleoprotein antibody purchased from Abcam, article number: [B017]ab20711;
  • Alexa 488 Goat Anti-Mouse IgG (H+L) Antibody purchased from Lifetechnologies, Cat. No. A-11001;
  • Anti-A1 standard antibody Influenza Anti-A/California/7/2009 (H1N1)-HA serum, purchased from NIBSC, article number: 12/108;
  • Anti-A3 standard antibody Influenza Anti-A/Perth/16/2009-Like HA Serum, purchased from NIBSC, article number: 11/206;
  • Anti-B standard antibody Influenza Anti-B/Brisbane/60/2008 HA serum, purchased from NIBSC, article number 11/136.
  • the inactivated standard antibodies were divided into three groups, namely:
  • G1 anti-A3 standard antibody 1 part + anti-B standard antibody 1 part + PBS 3 parts;
  • G2 1 part of anti-A1 standard antibody + 1 part of anti-B standard antibody + 3 parts of PBS;
  • G3 1 part anti-A1 standard antibody + 1 part anti-A3 standard antibody + 3 parts of PBS.
  • the three groups of antibodies G1, G2, and G3 were separately mixed with the sample to be tested in a 1:1 ratio, and allowed to stand in a water bath at 32 ° C for 30 minutes, and used as a virus sample.
  • MDCK cells in good growth state were plated in 96-well plates and used after the cells were spread over 96-well plates. Discard the cell growth solution in the plate, add DMEM to wash the plate, and use it; dilute the virus sample 10 times to the appropriate concentration, inoculate it in 96-well plate, incubate in 37 ° C, 5% CO 2 cell incubator, incubate Take out after 17 ⁇ 20h.
  • the cells were fixed with 80% (v/v) acetone at about 20 ° C; washed twice with 0.5% TPBS; then added with primary antibody, incubated with cells at 37 ° C, 5% CO 2 incubator for 1 h, removed and washed with PBS The plate was added; the secondary antibody was added, and the cells were further incubated with the cells at 37 ° C in a 5% CO 2 incubator for 1 hour. After taking out, the plate was washed with PBS, and the fluorescent spot was observed with a fluorescence microscope.
  • the vaccine of the invention is formulated according to the following formulation:
  • Vaccines A1 to A7 were stored at 25 ° C, and samples were taken on days 0, 14, 21, and 28, respectively, and the appearance of the samples was examined, and the pH and virus titer were measured according to the method of Example 1.
  • Table 1 Stability results of the vaccines A1 to A7 placed at 25 ° C
  • the vaccine of the invention is formulated according to the following formulation:
  • the vaccines B1 to B9 were stored at 25 ° C, and samples were taken on days 0, 14, 21, and 28, respectively, and the appearance of the samples was examined, and the pH and virus titer were measured according to the method of Example 1.
  • the vaccine of the invention is formulated according to the following formulation:
  • Vaccines C1 to C9 were stored at 25 ° C, and samples were taken on days 0, 14, 21, and 28, respectively, and the appearance of the samples was examined, and the pH and virus titer were measured according to the method of Example 1.
  • sucrose 0.1g of sodium glutamate, 3.0ml of human serum albumin, 0.5g of arginine, 1.0g of mannitol, 3.5g of sorbitol, 0.4g of urea, 0.3g of glycine, and buffer with appropriate amount of phosphate.
  • the volume was adjusted to 70 ml, the pH was adjusted to 6.3 with 0.1 N HCl, and the bacteria were removed by filtration using a 0.22 ⁇ m filter, and 10 ml of each of the H1N1 type, H3N2 type and B type influenza virus stock solution was added to prepare a D5 vaccine;
  • Vaccines D1 to D9 were stored at 25 ° C, and samples were taken on days 0, 14, 21, and 28, respectively, and the appearance of the samples was examined, and the pH and virus titer were measured according to the method of Example 1.
  • Vaccines D1 to D9 were stored at 33 ° C, and samples were taken on days 0 and 10, respectively, and the appearance of the samples was examined, and the pH and virus titer were determined according to the method of Example 1.
  • the vaccines D1 to D9 were stored at 2 to 8 ° C, and the appearance of the samples was examined at the end of the 0, 1, 3, and 6 months, respectively, and the pH and virus titer were measured according to the method of Example 1.

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Abstract

本发明提供一种用作流感减毒活疫苗保护剂的组合物，其中包含的组分及其浓度为：人血清白蛋白1.0～15.0g/L、蔗糖15.0～95.0g/L、谷氨酸钠0.5～15.0g/L。本发明还提供一种用本发明的组合物制备流感减毒活疫苗的方法，其包括以下步骤：将本发明的组合物的各个组分依次溶于pH缓冲液中；将pH调节到指定值；过滤除菌；加入病毒原液，即得所述流感减毒活疫苗。本发明还提供用作注射剂或鼻喷雾剂的流感减毒活疫苗。

Description

不含明胶的疫苗保护剂组合物及流感减毒活疫苗 技术领域
本发明属于疫苗生产工艺技术领域，具体地，本发明涉及一种不含明胶的液体疫苗保护剂组合物，以及包含所述液体疫苗保护剂组合物的流感减毒活疫苗。
背景技术
流行性感冒是由流感病毒引起的一种严重危害人类健康的急性呼吸道感染疾病。流感病毒分为甲(A)、乙(B)、丙(C)三个型别，其中甲型流感病毒的抗原性经常出现变异，在历史上曾多次引起世界性大流行。甲型流感也在猪和禽类中传播，并引起流感流行，造成大量动物死亡。而人乙、丙型流感仅在人之间转播。乙型流感病毒对人类致病性较低。丙型流感病毒主要侵袭婴幼儿，一般不引起流感流行。据WHO估计，在爆发性流行时，流感病毒可在全世界迅速传播，影响总人口的10％～20％。即使在非爆发性流行的年份，流感每年也造成300万～500万例严重病例和25万～50万例死亡。
迄今为止，接种流感疫苗是防治流感的最有效的手段。疫苗接种不仅可以降低高危人群的发病率和死亡率，也可减轻症状，降低并发症的发生、降低继续传播的机率。
流感减毒活疫苗采用鼻粘膜途径进行免疫，具有模拟天然感染的优点。与灭活疫苗相比，流感减毒活疫苗除了能诱导体液免疫外，还能诱导产生局部中和抗体以及细胞免疫应答，并且免疫应答持续时间持久。另外流感减毒活疫苗使用方便，易于进行大规模免疫。
流感减毒活疫苗一般为冻干粉针剂型。虽然冷冻干燥技术已经成熟，但还存在一些缺点：首先，冻干粉针剂在使用过程中需要复溶，不便于使用并且增加了污染的机会；其次，生物制品的冷冻干燥工艺复杂、工艺过程耗时长、流感病毒滴度损失较大；第三，真空冷冻干燥设备投资大、能耗高、生产成本高，疫苗生产能力有限，尤其是在流感大爆发时，疫苗生产厂商很难在短时间内生产出足够剂量的疫苗。
2003年，MedImmune公司的流感减毒活疫苗FluMist获得了FDA的批准。该产品是一种冷适应三价病毒疫苗，为水针剂型。该产品需要在约15℃及以下储藏、运输，但由于药房或学校通常都没有处理冷冻产品的设备，使得FluMist 不能大范围使用。
2005年，MedImmune公司提交了名为“冷藏温度稳定的流感疫苗组合物”的专利(专利申请号PCT/US2005/035614)，提供了一个可使流感疫苗在4～8℃保持稳定的、含有水解明胶组分的液体制剂组合物。然而，该组合物中的明胶或明胶衍生物可直接引起使被接种者过敏的变态反应和非细胞介导免疫反应。
因此，为克服冷冻干燥技术成本高和水解明胶组分易引起变态反应和免疫反应的缺点，需要开发出不含明胶的疫苗液体制剂保护剂以及包含其的流感减毒活疫苗。
发明内容
发明人经过研究，发明了一种用作流感减毒活疫苗保护剂的组合物，该组合物中不含明胶成分，对人体的刺激性小，并且用该组合物制备的流感减毒活疫苗在冷藏条件下可保持较好的稳定性和较长有效期。
因此，本发明提供一种用作流感减毒活疫苗保护剂的组合物，其中包含的组分及其浓度为：人血清白蛋白1.0～15.0g/L，优选1.5～12.0g/L，更优选2.5～10.0g/L；蔗糖15.0～95.0g/L，优选40.0～85.0g/L，更优选50.0～75.0g/L；谷氨酸钠0.5～15.0g/L，优选0.5～12.0g/L，更优选0.5～10.0g/L；其中，所述组合物的pH为5.0～9.0，优选为pH 5.5～8.5，更优选为pH 6.0～8.0，最优选为pH 6.0～7.4。
本发明的组合物中还包括尿素，其浓度为0～8.0g/L，优选0.5～6.0g/L，更优选1.0～4.0g/L；精氨酸，其浓度为0～10.0g/L，优选1.0～10.0g/L，更优选1.4～10.0g/L；组氨酸，其浓度为0～2.0g/L，优选为0.5～1.5g/L；山梨醇，其浓度为0～70.0g/L，优选15.0～60.0g/L，更优选25.0～50.0g/L；甘氨酸，其浓度为0～20.0g/L，优选3.0～15.0g/L；甘露醇，其浓度为0～30.0g/L，优选10.0～20.0g/L。
此外，本发明还提供了本发明的组合物用于提高疫苗稳定性和安全性的用途以及用于制备流感减毒活疫苗的用途。
此外，本发明还提供一种流感减毒活疫苗的制备方法，其特征在于，使用本发明的组合物作为保护剂。
具体实施方式
因此，本发明提供一种用作流感减毒活疫苗保护剂的组合物，其中包含的组分及其浓度为：人血清白蛋白1.0～15.0g/L，优选1.5～12.0g/L，更优选2.5～10.0g/L；蔗糖15.0～95.0g/L，优选40.0～85.0g/L，更优选50.0～75.0g/L；谷氨酸钠 0.5～15.0g/L，优选0.5～12.0g/L，更优选0.5～10.0g/L；其中所述组合物的pH为5.0～9.0，优选为pH 5.5～8.5，更优选为pH 6.0～8.0，最优选为pH 6.0～7.4。
本发明的组合物中还包括尿素，其浓度为0～8.0g/L，优选0.5～6.0g/L，更优选1.0～4.0g/L；精氨酸，其浓度为0～10.0g/L，优选1.0～10.0g/L，更优选1.4～10.0g/L；组氨酸，其浓度为0～2.0g/L，优选为0.5～1.5g/L；山梨醇，其浓度为0～70.0g/L，优选15.0～60.0g/L，更优选25.0～50.0g/L；甘氨酸，其浓度为0～20.0g/L，优选3.0～15.0g/L；甘露醇，其浓度为0～30.0g/L，优选10.0～20.0g/L。
本发明还提供了本发明的组合物用于制备流感减毒活疫苗的用途。
本发明还提供一种流感减毒活疫苗的制备方法，其特征在于，使用本发明的组合物作为保护剂。
在一个优选实施方案中，本发明的流感减毒活疫苗的制备方法包括以下步骤：将本发明的组合物的各个组分依次溶于pH缓冲液中；将pH调节到指定值；过滤除菌；加入病毒原液，即得流感减毒活疫苗。
在一个优选实施方案中，根据本发明的方法制备的液体流感减毒活疫苗的pH为5.0～9.0，优选为pH 5.5～8.5，更优选为pH 6.0～8.0，最优选为pH 6.0～7.4。
在一个优选实施方案中，使用磷酸盐缓冲液作为pH缓冲液。
本发明还提供一种使用本发明的组合物和/或根据本发明的方法制备的流感减毒活疫苗。
在一个优选实施方案中，本发明的流感减毒活疫苗为注射剂或鼻喷雾剂。
以下通过实施例对本发明进行进一步说明。
实施例1：
1.pH的测定
pH的测定根据《中华人民共和国药典(2010年版)》(三部)附录V A进行。
2.病毒滴度的测定
细胞系：
MDCK细胞：犬肾上皮传代细胞系(Madin～Daby canine kidney cells)，购自ATCC。
病毒毒株：
H1N1型流感病毒毒株：A/17/California/2009/38(H1N1)，由WHO提供；
H3N2型流感病毒毒株：A/17/Perth/09/87(H3N2)，由WHO提供；
B型流感病毒毒株：B/56/Brisbane/60/08，由WHO提供。
试剂：
TPCK-Trypsin(购自Sigma，货号：T1426)溶液；
DMEM细胞培养液(购自Sigma，货号：D6546)；
胎牛血清(购自Hyclone，货号：SH30070.03)；
L-谷氨酰胺(购自Gibco，货号：25030)溶液；
青链霉素(购自Gibco，货号：15140)；
HEPES(购自吉诺生物，货号：GNM11344)溶液；
一抗：Anti-Influenza A Virus Nucleoprotein antibody，购自Abcam，货号：[AA5H]ab20343；
Anti-Influenza B Virus Nucleoprotein antibody，购自Abcam，货号：[B017]ab20711；
二抗：Alexa
Figure PCTCN2016080226-appb-000001
488Goat Anti-Mouse IgG(H+L)Antibody，购自Lifetechnologies，货号：A-11001；
标准抗体：
抗A1标准抗体：Influenza Anti-A/California/7/2009(H1N1)-HA serum，购自NIBSC，货号：12/108；
抗A3标准抗体：Influenza Anti-A/Perth/16/2009-Like HA Serum，购自NIBSC，货号：11/206；
抗B标准抗体：Influenza Anti-B/Brisbane/60/2008HA serum，购自NIBSC，货号：11/136。
方法：
(1)样品处理
取供试品3瓶混合在一起，作为待检样品。
将灭活后的标准抗体分为3组，分别为：
G1：抗A3标准抗体1份+抗B标准抗体1份+PBS 3份；
G2：抗A1标准抗体1份+抗B标准抗体1份+PBS 3份；以及
G3：抗A1标准抗体1份+抗A3标准抗体1份+PBS 3份。
将3组抗体G1、G2、G3分别与待检样品按1:1比例混合，在32℃水浴静置30分钟，作为病毒样品待用。
(2)滴度检测
将生长状态良好的MDCK细胞铺于96孔板中培养，待细胞铺满96孔板后使用。弃去板中细胞生长液，加入DMEM洗板，待用；将病毒样品10倍系列稀释至合适浓度，接种于96孔板中，放入37℃、5％CO2细胞培养箱中孵育，孵育17～20h后取出。用约20℃的80％(v/v)丙酮固定细胞；用0.5％TPBS洗涤两次；之后加入一抗，与细胞在37℃、5％CO2培养箱中孵育1h，取出后用PBS洗板；加入二抗，与细胞在37℃、5％CO2培养箱中再孵育1h，取出后用PBS洗板，用荧光显微镜观察荧光灶。
计算病毒滴度：在100×镜下观察，病毒滴度(lg FFU/0.2ml)＝2.8×10×病毒稀释度×该浓度各平行孔中荧光灶平均数。
除非另外特别说明，以下实施例中使用的物质或材料、仪器的来源和规格均与实施例1相同。
实施例2
根据下列处方配制本发明的疫苗：
分别称取5.0g蔗糖、0.5g谷氨酸钠、1.5ml人血清白蛋白(质量分数20％)，用适量磷酸盐缓冲液溶解后，再定容至70ml，用0.1N HCl将pH调到7.2，并使用0.22μm滤器过滤除菌，加入H1N1型、H3N2型及B型流感病毒原液各10ml，配制成A1号疫苗。
分别称取5.0g蔗糖、0.5g谷氨酸钠、1.5ml人血清白蛋白、0.2g精氨酸，用适量磷酸盐缓冲液溶解后，再定容至70ml，用0.1N HCl将pH调到7.2，并使用0.22μm滤器过滤除菌，加入H1N1型、H3N2型及B型流感病毒原液各10ml，配制成A2号疫苗。
分别称取5.0g蔗糖、0.5g谷氨酸钠、1.5ml人血清白蛋白、0.3g甘氨酸，用适量磷酸盐缓冲液溶解后，再定容至70ml，用0.1N HCl将pH调到7.2，并使用0.22μm滤器过滤除菌，加入H1N1型、H3N2型及B型流感病毒原液各10ml，配制成A3号疫苗。
分别称取5.0g蔗糖、0.5g谷氨酸钠、1.5ml人血清白蛋白、0.2g组氨酸，用适量磷酸盐缓冲液溶解后，再定容至70ml，用0.1N HCl将pH调到7.2，并使用0.22μm滤器过滤除菌，加入H1N1型、H3N2型及B型流感病毒原液各10ml，配制成A4号疫苗。
分别称取5.0g蔗糖、0.5g谷氨酸钠、1.5ml人血清白蛋白、1.0g山梨醇，用适量磷酸盐缓冲液溶解后，再定容至70ml，用0.1N HCl将pH调到7.2，并 使用0.22μm滤器过滤除菌，加入H1N1型、H3N2型及B型流感病毒原液各10ml，配制成A5号疫苗。
分别称取5.0g蔗糖、0.5g谷氨酸钠、1.5ml人血清白蛋白、1.0g甘露醇，用适量磷酸盐缓冲液溶解后，再定容至70ml，用0.1N HCl将pH调到7.2，并使用0.22μm滤器过滤除菌，加入H1N1型、H3N2型及B型流感病毒原液各10ml，配制成A6号疫苗。
分别称取5.0g蔗糖、0.5g谷氨酸钠、1.5ml人血清白蛋白、0.5g尿素，用适量磷酸盐缓冲液溶解后，再定容至70ml，用0.1N HCl将pH调到7.2，并使用0.22μm滤器过滤除菌，加入H1N1型、H3N2型及B型流感病毒原液各10ml，配制成A7号疫苗。
将A1～A7号疫苗保存于25℃，分别在第0、14、21、28天取样，检查样品外观，并根据实施例1的方法测定其pH和病毒滴度。
表1：A1～A7号疫苗在25℃放置的稳定性结果
Figure PCTCN2016080226-appb-000002
Figure PCTCN2016080226-appb-000003
实施例3
根据下列处方配制本发明的疫苗：
分别称取2.5g蔗糖、0.1g谷氨酸钠、1.5ml人血清白蛋白、0.2g精氨酸，用适量磷酸盐缓冲液溶解后，再定容至70ml，用0.1N HCl将pH调到7.2，并使用0.22μm滤器过滤除菌，加入H1N1型、H3N2型及B型流感病毒原液各10ml，配制成B1号疫苗；
分别称取2.5g蔗糖、0.5g谷氨酸钠、3.0ml人血清白蛋白、0.5g精氨酸，用适量磷酸盐缓冲液溶解后，再定容至70ml，用0.1N HCl将pH调到7.2，并使用0.22μm滤器过滤除菌，加入H1N1型、H3N2型及B型流感病毒原液各10ml，配制成B2号疫苗；
分别称取2.5g蔗糖、1.0g谷氨酸钠、5.0ml人血清白蛋白、1.0g精氨酸， 用适量磷酸盐缓冲液溶解后，再定容至70ml，用0.1N HCl将pH调到7.2，并使用0.22μm滤器过滤除菌，加入H1N1型、H3N2型及B型流感病毒原液各10ml，配制成B3号疫苗；
分别称取5.0g蔗糖、0.1g谷氨酸钠、3.0ml人血清白蛋白、1.0g精氨酸，用适量磷酸盐缓冲液溶解后，再定容至70ml，用0.1N HCl将pH调到7.2，并使用0.22μm滤器过滤除菌，加入H1N1型、H3N2型及B型流感病毒原液各10ml，配制成B4号疫苗；
分别称取5.0g蔗糖、0.5g谷氨酸钠、5.0ml人血清白蛋白、0.2g精氨酸，用适量磷酸盐缓冲液溶解后，再定容至70ml，用0.1N HCl将pH调到7.2，并使用0.22μm滤器过滤除菌，加入H1N1型、H3N2型及B型流感病毒原液各10ml，配制成B5号疫苗；
分别称取5.0g蔗糖、1.0g谷氨酸钠、1.5ml人血清白蛋白、0.5g精氨酸，用适量磷酸盐缓冲液溶解后，再定容至70ml，用0.1N HCl将pH调到7.2，并使用0.22μm滤器过滤除菌，加入H1N1型、H3N2型及B型流感病毒原液各10ml，配制成B6号疫苗；
分别称取7.5g蔗糖、0.1g谷氨酸钠、5.0ml人血清白蛋白、0.5g精氨酸，用适量磷酸盐缓冲液溶解后，再定容至70ml，用0.1N HCl将pH调到7.2，并使用0.22μm滤器过滤除菌，加入H1N1型、H3N2型及B型流感病毒原液各10ml，配制成B7号疫苗；
分别称取7.5g蔗糖、0.5g谷氨酸钠、1.5ml人血清白蛋白、1.0g精氨酸，用适量磷酸盐缓冲液溶解后，再定容至70ml，用0.1N HCl将pH调到7.2，并使用0.22μm滤器过滤除菌，加入H1N1型、H3N2型及B型流感病毒原液各10ml，配制成B8号疫苗；
分别称7.5g蔗糖、1.0g谷氨酸钠、3.0ml人血清白蛋白、0.2g精氨酸，用适量磷酸盐缓冲液溶解后，再定容至70ml，用0.1N HCl将pH调到7.2，并使用0.22μm滤器过滤除菌，加入H1N1型、H3N2型及B型流感病毒原液各10ml，配制成B9号疫苗。
将B1～B9号疫苗保存于25℃，分别在第0、14、21、28天取样，检查样品外观，并根据实施例1的方法测定其pH和病毒滴度。
表2：B1～B9号疫苗在25℃放置的稳定性结果
Figure PCTCN2016080226-appb-000004
Figure PCTCN2016080226-appb-000005
Figure PCTCN2016080226-appb-000006
实施例4
根据下列处方配制本发明的疫苗：
分别称取7.5g蔗糖、0.1g谷氨酸钠、3.0ml人血清白蛋白，0.5g精氨酸，用适量磷酸盐缓冲液溶解后，再定容至70ml，用0.1N HCl将pH调到6.8，并使用0.22μm滤器过滤除菌，加入H1N1型、H3N2型及B型流感病毒原液各10ml，配制成C1号疫苗。
分别称取7.5g蔗糖、0.1g谷氨酸钠、3.0ml人血清白蛋白，0.5g精氨酸、2.5g山梨醇、1.0g甘露醇、0.4g尿素，用适量磷酸盐缓冲液溶解后，再定容至70ml，用0.1N HCl将pH调到6.8，并使用0.22μm滤器过滤除菌，加入H1N1型、H3N2型及B型流感病毒原液各10ml，配制成C2号疫苗。
分别称取7.5g蔗糖、0.1g谷氨酸钠、3.0ml人血清白蛋白，0.5g精氨酸、5.0g山梨醇、2.0g甘露醇、0.8g尿素，用适量磷酸盐缓冲液溶解后，再定容至70ml，用0.1N HCl将pH调到6.8，并使用0.22μm滤器过滤除菌，加入H1N1型、H3N2型及B型流感病毒原液各10ml，配制成C3号疫苗。
分别称取7.5g蔗糖、0.1g谷氨酸钠、3.0ml人血清白蛋白，0.5g精氨酸、1.0g甘露醇、0.8g尿素，用适量磷酸盐缓冲液溶解后，再定容至70ml，用0.1 N HCl将pH调到7.3，并使用0.22μm滤器过滤除菌，加入H1N1型、H3N2型及B型流感病毒原液各10ml，配制成C4号疫苗。
分别称取7.5g蔗糖、0.1g谷氨酸钠、3.0ml人血清白蛋白，0.5g精氨酸、2.5g山梨醇、2.0g甘露醇，用适量磷酸盐缓冲液溶解后，再定容至70ml，用0.1N HCl将pH调到7.3，并使用0.22μm滤器过滤除菌，加入H1N1型、H3N2型及B型流感病毒原液各10ml，配制成C5号疫苗。
分别称取7.5g蔗糖、0.1g谷氨酸钠、3.0ml人血清白蛋白，0.5g精氨酸、5.0g山梨醇、0.4g尿素，用适量磷酸盐缓冲液溶解后，再定容至70ml，用0.1N HCl将pH调到7.3，并使用0.22μm滤器过滤除菌，加入H1N1型、H3N2型及B型流感病毒原液各10ml，配制成C6号疫苗。
分别称取7.5g蔗糖、0.1g谷氨酸钠、3.0ml人血清白蛋白，0.5g精氨酸、2.0g甘露醇、0.4g尿素，用适量磷酸盐缓冲液溶解后，再定容至70ml，用0.1N HCl将pH调到7.8，并使用0.22μm滤器过滤除菌，加入H1N1型、H3N2型及B型流感病毒原液各10ml，配制成C7号疫苗。
分别称取7.5g蔗糖、0.1g谷氨酸钠、3.0ml人血清白蛋白，0.5g精氨酸、2.5g山梨醇、0.8g尿素，用适量磷酸盐缓冲液溶解后，再定容至70ml，用0.1N HCl将pH调到7.8，并使用0.22μm滤器过滤除菌，加入H1N1型、H3N2型及B型流感病毒原液各10ml，配制成C8号疫苗。
分别称取7.5g蔗糖、0.1g谷氨酸钠、3.0ml人血清白蛋白，0.5g精氨酸、5.0g山梨醇、1.0g甘露醇，用适量磷酸盐缓冲液溶解后，再定容至70ml，用0.1N HCl将pH调到7.8，并使用0.22μm滤器过滤除菌，加入H1N1型、H3N2型及B型流感病毒原液各10ml，配制成C9号疫苗。
将C1～C9号疫苗保存于25℃，分别在第0、14、21、28天取样，检查样品外观，并根据实施例1的方法测定其pH和病毒滴度。
表3：C1～C9号疫苗在25℃放置的稳定性结果
Figure PCTCN2016080226-appb-000007
Figure PCTCN2016080226-appb-000008
Figure PCTCN2016080226-appb-000009
实施例5
分别称取7.5g蔗糖、0.1g谷氨酸钠、3.0ml人血清白蛋白、0.5g精氨酸、1.0g甘露醇、2.5g山梨醇、0.1g尿素、0.3g甘氨酸，用适量磷酸盐缓冲液溶解后，再定容至70ml，用0.1N HCl将pH调到6.0，并使用0.22μm滤器过滤除菌，加入H1N1型、H3N2型及B型流感病毒原液各10ml，配制成D1号疫苗；
分别称取7.5g蔗糖、0.1g谷氨酸钠、3.0ml人血清白蛋白、0.5g精氨酸、1.0g甘露醇、3.5g山梨醇、0.2g尿素、0.9g甘氨酸，用适量磷酸盐缓冲液溶解后，再定容至70ml，用0.1N HCl将pH调到6.0，并使用0.22μm滤器过滤除菌，加入H1N1型、H3N2型及B型流感病毒原液各10ml，配制成D2号疫苗；
分别称取7.5g蔗糖、0.1g谷氨酸钠、3.0ml人血清白蛋白、0.5g精氨酸、1.0g甘露醇、5.0g山梨醇、0.4g尿素、1.5g甘氨酸，用适量磷酸盐缓冲液溶解后，再定容至70ml，用0.1N HCl将pH调到6.0，并使用0.22μm滤器过滤除菌，加入H1N1型、H3N2型及B型流感病毒原液各10ml，配制成D3号疫苗；
分别称取7.5g蔗糖、0.1g谷氨酸钠、3.0ml人血清白蛋白、0.5g精氨酸、1.0g甘露醇、2.5g山梨醇、0.2g尿素、1.5g甘氨酸，用适量磷酸盐缓冲液溶解后，再定容至70ml，用0.1N HCl将pH调到6.3，并使用0.22μm滤器过滤除菌，加入H1N1型、H3N2型及B型流感病毒原液各10ml，配制成D4号疫苗；
分别称取7.5g蔗糖、0.1g谷氨酸钠、3.0ml人血清白蛋白、0.5g精氨酸、1.0g甘露醇、3.5g山梨醇、0.4g尿素、0.3g甘氨酸，用适量磷酸盐缓冲液溶解后，再定容至70ml，用0.1N HCl将pH调到6.3，并使用0.22μm滤器过滤除菌，加入H1N1型、H3N2型及B型流感病毒原液各10ml，配制成D5号疫苗；
分别称取7.5g蔗糖、0.1g谷氨酸钠、3.0ml人血清白蛋白、0.5g精氨酸、1.0g甘露醇、5.0g山梨醇、0.1g尿素、0.9g甘氨酸，用适量磷酸盐缓冲液溶解后，再定容至70ml，用0.1N HCl将pH调到6.3，并使用0.22μm滤器过滤除菌，加入H1N1型、H3N2型及B型流感病毒原液各10ml，配制成D6号疫苗；
分别称取7.5g蔗糖、0.1g谷氨酸钠、3.0ml人血清白蛋白、0.5g精氨酸、1.0g甘露醇、2.5g山梨醇、0.4g尿素、0.9g甘氨酸，用适量磷酸盐缓冲液溶解后，再定容至70ml，用0.1N HCl将pH调到6.7，并使用0.22μm滤器过滤除菌，加入H1N1型、H3N2型及B型流感病毒原液各10ml，配制成D7号疫苗；
分别称取7.5g蔗糖、0.1g谷氨酸钠、3.0ml人血清白蛋白、0.5g精氨酸、1.0g甘露醇、3.5g山梨醇、0.1g尿素、1.5g甘氨酸，用适量磷酸盐缓冲液溶解后，再定容至70ml，用0.1N HCl将pH调到6.7，并使用0.22μm滤器过滤除菌，加入H1N1型、H3N2型及B型流感病毒原液各10ml，配制成D8号疫苗；
分别称取7.5g蔗糖、0.1g谷氨酸钠、3.0ml人血清白蛋白、0.5g精氨酸、1.0g甘露醇、5.0g山梨醇、0.2g尿素、0.3g甘氨酸，用适量磷酸盐缓冲液溶解后，再定容至70ml，用0.1N HCl将pH调到6.7，并使用0.22μm滤器过滤除菌，加入H1N1型、H3N2型及B型流感病毒原液各10ml，配制成D9号疫苗。
将D1～D9号疫苗保存于25℃，分别在第0、14、21、28天取样，检查样品外观，并根据实施例1的方法测定其pH和病毒滴度。
表4：D1～D9号疫苗在25℃放置的稳定性结果
Figure PCTCN2016080226-appb-000010
Figure PCTCN2016080226-appb-000011
Figure PCTCN2016080226-appb-000012
将D1～D9号疫苗保存于33℃，分别在第0天和第10天取样，检查样品外观，并根据实施例1的方法测定其pH和病毒滴度。
表5：D1～D9号疫苗在33℃放置的稳定性结果
Figure PCTCN2016080226-appb-000013
Figure PCTCN2016080226-appb-000014
Figure PCTCN2016080226-appb-000015
将D1～D9号疫苗保存于2～8℃，分别在第0、1、3、6个月末时，检查样品外观，并根据实施例1的方法测定其pH和病毒滴度。
表6：D1～D9号疫苗在2～8℃放置的稳定性结果
Figure PCTCN2016080226-appb-000016
Figure PCTCN2016080226-appb-000017
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以进行其它多种形式的修改、替换或变更。本领域人员能够理解，本申请所描述的本发明技术方案的各个特征均可根据需要进行适当的组合。

Claims (10)

  1. 一种用作流感减毒活疫苗保护剂的组合物，其中包含的组分及其浓度为：人血清白蛋白1.0～15.0g/L，优选1.5～12.0g/L，更优选2.5～10.0g/L；蔗糖15.0～95.0g/L，优选40.0～85.0g/L，更优选50.0～75.0g/L；谷氨酸钠0.5～15.0g/L，优选0.5～12.0g/L，更优选0.5～10.0g/L；其中，所述组合物的pH为5.0～9.0，优选为pH 5.5～8.5，更优选为pH 6.0～8.0，最优选为pH 6.0～7.4。
  2. 根据权利要求1所述的组合物，其中还包括尿素，其浓度为0～8.0g/L，优选0.5～6.0g/L，更优选1.0～4.0g/L；精氨酸，其浓度为0～10.0g/L，优选1.0～10.0g/L，更优选1.4～10.0g/L；组氨酸，其浓度为0～2.0g/L，优选为0.5～1.5g/L；山梨醇，其浓度为0～70.0g/L，优选15.0～60.0g/L，更优选25.0～50.0g/L；甘氨酸，其浓度为0～20.0g/L，优选3.0～15.0g/L；甘露醇，其浓度为0～30.0g/L，优选10.0～20.0g/L。
  3. 根据权利要求1或2所述的组合物用于制备流感减毒活疫苗的用途。
  4. 一种流感减毒活疫苗的制备方法，其特征在于，在配制过程中采用权利要求1或2所述的组合物作为保护剂。
  5. 根据权利要求4所述的方法，其包括以下步骤：
    1)将权利要求1或2的组合物的各个组分依次溶于pH缓冲液中；
    2)将pH调节到指定pH值；
    3)过滤除菌；
    4)加入病毒原液，即得流感减毒活疫苗。
  6. 根据权利要求5所述的方法，其中使用磷酸盐缓冲液作为pH缓冲液。
  7. 根据权利要求5或6所述的方法，其中所述指定pH值为5.0～9.0，优选为pH 5.5～8.5，更优选为pH 6.0～8.0，最优选为pH 6.0～7.4。
  8. 一种流感减毒活疫苗，其特征在于，包含权利要求1或2的组合物。
  9. 根据权利要求8所述的流感减毒活疫苗，其特征在于，其pH为5.0～9.0，优选为pH 5.5～8.5，更优选为pH 6.0～8.0，最优选为pH 6.0～7.4。
  10. 根据权利要求8或9所述的流感减毒活疫苗，其可用作注射剂或鼻喷雾剂。
PCT/CN2016/080226 2015-06-02 2016-04-26 不含明胶的疫苗保护剂组合物及流感减毒活疫苗 Ceased WO2016192487A1 (zh)

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