JP2000507963A - Lyophilized vaccine stabilizer - Google Patents

Abstract

  (57) [Summary] Disclosed are vaccine stabilizers, vaccine compositions, and lyophilized vaccines having improved heat stability. The vaccine composition may include a high amount of a C6 polyhydric alcohol (eg, sorbitol), a high amount of a disaccharide (eg, sucrose) and an amount of physiology to adjust the pH to about 6.0 to about 7.0. Contains an active buffer.

Description

DETAILED DESCRIPTION OF THE INVENTION                     Lyophilized vaccine stabilizer Field of the invention   The present invention relates to an extended polyhydric alcohol having 6 carbon atoms, a disaccharide and a pH of about 6.0. Vaccine stabilizer comprising a physiologically active buffer in an amount to adjust to about 7.0 , Vaccine compositions and lyophilized vaccines.Background of the Invention   Measles virus is a minus-strand RNA virus belonging to the genus Morbillivirus . The measles virus is highly contagious in human hosts, causing infected hosts to cough and sneeze It spreads by doing. The virus enters the bloodstream, spreads throughout the body, Attacks spheroid tissue. The infection period lasts about 6-7 days, after which a rash appears, Lasts a few days. For about 10 days after contact, signs of fever and malaise appear. afterwards Symptoms of sputum, cough, nasal cold, conjunctivitis and sometimes photophobia. Rash appears About 2 days ago, Koplik spots appear. At the worst stage of infection, the patient has a headache, May complain of pain, vomiting, diarrhea and / or muscle pain.   Mumps virus is a negative strand RNA virus belonging to the genus Paramyxovirus. Ruth. The mumps virus incubation period is usually 17-21 days, It can be as long as ~ 37 days. After infection and multiplication in the respiratory tract, the virus enters the bloodstream and Spread systemically in various body tissues. The characteristic symptoms of mumps are parotid gland and sometimes Swelling and compression of other salivary glands. Before swelling appears, the patient should be behind the chin and just behind the ear. I feel pain underneath and I feel that the pain increases when I push or move my chin. More severe disease In examples, precursor symptoms such as anorexia, headache, vomiting, myalgia and high fever may appear.   Rubella is a plus-strand RNA virus, a member of the Togaviridae family that causes rubella. One member. Rubella virus is usually spread by airborne infection of the infected droplets. Subclinical : Overt illness is about 2: 1 ratio and many rubella infections are asymptomatic. Rubella Will Incubation period is 14-21 days, characteristic of adenopathy, rash and low fever Indicates a pattern. Rubella in early pregnancy often infects the fetus, Is a spectrum of a disease that can be chronic and known as congenital rubella syndrome (CRS) Can occur.   Varicella (varicella-shingles) virus can be latently transmitted for several years after initial infection Herpes wisdom of nuclear double-stranded DNA viruses Ruth. Chickenpox is one of the most common and highly contagious diseases, mainly in childhood Suffer from After an incubation period of 14-17 days, a rash appears throughout the body, usually with bouts of fever You. The disease forms pustules in many cases and leaves scars in severe cases A possible muscle eruption occurs. Chickenpox can cause central nervous system disorders, myelitis and neuritis It is also known that other such complications occur. Live attenuated vaccine for chickenpox and Methods for making such vaccines are known and are disclosed in U.S. Pat. No. 85,615.   During the past few decades, a common immunization program for infants and children has been around 15 years of age. Measles, mumps and wind at the age of months and at the age of 4-6 or at secondary school Immunization with a live attenuated triple vaccine for rash. In addition, various Unit price (eg, measles, mumps, rubella or varicella) vaccine, binary (eg For example, measles-mumps vaccine and quaternary (eg, measles-mumps) (Zero-rubella-varicella) vaccines are also commonly used.   Vaccine stabilizers increase vaccine stability during cold storage or storage after lyophilization. Compound added to the vaccine composition to It is known in the art as an object.   One chemical stabilizer is called SPGA and is described by Bovarnick et al. Bact . 59: 509-522 (1950). 1 liter SPGA Is sucrose 0.218M (74.62g), KH_TwoPO_Four0.00376M (0 . 52g), K_TwoHPO_Four0.0071M (1.25g), potassium glutamate Note that it contains 0.0049M (0.912 g) and 1% serum albumin (10 g). It is listed.   U.S. Pat. No. 3,783,098 to Calnek et al. Modification of SPGA in which monopotassium glutamic acid is replaced by monosodium glutamate Odds are described. Also, instead of all or part of sucrose, starch Hydrolysates such as glucose or dextrose may be used. Finally, note As noted (US Patent 3,915, issued to Zygrschich et al.) No. 794) All or part of albumin is converted to casein or PVP. May be substituted.   US Patent No. 4,000,256 to Hilleman et al. 74.62 g of sucrose per liter of sterilized distilled water, KH_TwoPO_Four0.45g , K_TwoHPO_Four1.35 g, L-g 0.956 g of monosodium glutamic acid and 25% human serum albumin solution 40 ml of SPGA stabilizer is described.   Generally, SPGA stabilizers comprise about 2 to about 10% of a particular sugar (eg, sucrose). From about 0.05 to about 0.3% of a monobasic or dibasic alkali metal phosphate. Or a mixture thereof (for example, KH_TwoPO_Four, K_TwoHPO_Four, NaH_TwoPO_FourOr Na_Two HPO_Four), About 0.05 to about 0.2% alkali metal glutamic acid (e.g., Sodium glutamate or potassium glutamate), and about 0.5 to about 2% Serum albumin (eg, bovine serum albumin or human albumin) .   Other chemical stabilizers known in the art include hydrolyzed gelatin, Medium O and sorby Including Toll. This chemical stabilizer is disclosed in U.S. Pat. No. 147,772, about 3.5% hydrolyzed gelatin, 3.5 % Sorbitol, 1.0% Medium O and a small amount of sodium bicarbonate and Contains phenol red.   U.S. Pat. No. 4,273,762 to McAleer et al. A modified version of the stabilizer described in U.S. Pat. No. 4,147,772. Tin stabilizers have been described. This stabilizer is in addition to the components described in U.S. Pat. No. 4,147,772. And small amounts, especially cysteine, glutathione, ascorbic acid, vitamin A and Contains DPG solution containing USP.   As mentioned above, despite the advancement of vaccine compositions, improved heat Live vaccine compositions with qualitative and shelf-life, especially measles, mumps and rubella There remains a clear need for live vaccines. In conventional stabilizers, the desired It does not provide a high level of stability. The present invention has improved thermal stability Meeting long-standing requirements for stabilizers and subsequently freeze-dried live vaccine compositions You.Summary of the Invention   The present invention provides vaccine stabilizers, vaccine compositions and vaccine compositions that provide improved heat stabilizers. A live attenuated lyophilized vaccine.   The vaccine composition of the present invention comprises a virus and a stabilizer component, wherein the stabilizer component comprises g From about 15 to about 90 g / l on a carbon / polyhydric alcohol basis (but not limited to, Rubitol, mannitol and dulcitol), from about 10 to about 70 g / l disaccharides (including but not limited to sucrose, lactose, maltose and Rehalose), and an amount to adjust the pH to about 6.0 to about 7.0. Consists of a physiologically active buffer. In the present invention, polyhydric alcohols having 6 carbon atoms are Preferably, the rubitol or disaccharide is sucrose   In another embodiment of the present invention, the vaccine composition is a C6 polyhydric alcohol. About 15 to about 90 g / l of sorbitol and about 10 to about 90 g of sucrose as a disaccharide. 70 g / l, and the pH of the vaccine composition ranges from about 6.0 to about 7.0. In order to ensure buffering action, the citrate-phosphate mixture is used to It is adjusted using one of the approaches. One of the approaches described above is about 7.5 mM Phosphate at a concentration of about 75 mM is added, and another approach is from about 7.5 mM to about 7 mM. Citrate phosphate at a concentration of 5 mM and a concentration of about 30 mM to about 0.4 M : Add citrate mixture.   In another embodiment of the present invention, the vaccine composition is a C6 polyhydric alcohol. About 15 to about 90 g / l of sorbitol and about 10 to about 90 g of sucrose as a disaccharide. 70 g / l and the pH of the vaccine composition is in the preferred pH range of about 6.0 to about 7.0. Adjusted by adding phosphate buffer to ensure buffering over You.   Preferably, the vaccine composition of the present invention comprises a vaccine having improved thermostability. It may comprise one or more additional components provided alone or in a biologically effective combination . The additional components include, but are not limited to, about 10-50 g / l hydrolyzed gelatin, about 1 To about 6 g / l sodium chloride, up to about 1.5 g / l, preferably about 0.2 g / l 1 to about 1.2 g / l sodium bicarbonate, about 0.5 to 1.0 g / l, or frozen About 0.3 to about 1.0% of the dry weight of the vaccine in dry form, human serum albumin; And cell culture medium, which is a nutrient medium that promotes cell growth in vitro (without limitation) , Solution 199, Medium T, Medium O, Dulbet Known cell cultures such as modified Eagle's medium, minimal essential medium, basic Eagle medium Media). A preferred medium component is a biologically effective amount of Medium. O, Medium T and Solution 199. Vaccine of the present invention Other components of the composition include, but are not limited to, biologically active amounts of antibiotics (eg, Isine) and a pH indicator (eg, phenol red).   Thus, the vaccine composition of the present invention comprises from about 15 to about 90 g / l. Sorbitol as a polyhydric alcohol having 6 carbon atoms, about 10 to about 70 g / l Sucrose as saccharide, biologically effective concentration of cell culture medium (preferably Me dium O), a biologically effective concentration of a salt (preferably NaCl), Effective concentration of bicarbonate (preferably NaHCO_Three), Buffering in preferred pH range Citrate-phosphate mixture to ensure the presence of several additional components (non-limiting (Including neomycin and phenol red). Different amounts of bicarbonate Salts can be added to change the pH of the composition to within a biologically acceptable range. it can.   The vaccine composition of the present invention may comprise from about 15 to about 90 g / l of a C6 polyvalent alcohol. Sorbitol as a disaccharide, sucrose as a disaccharide at about 10 to about 70 g / l, Biologically effective concentration of cell culture medium (preferably, Medium O), biological An effective concentration of salt (preferably NaCl), a biologically effective concentration of bicarbonate (preferably Or NaHCO_Three), A phosphate buffer to ensure a preferred pH range, and And several additional ingredients (including but not limited to neomycin and phenol red) May be included. Add different amounts of bicarbonate to adjust the pH of the composition to biologically acceptable Range Can be changed within the box.   In a preferred vaccine composition of the invention, both sucrose and sorbitol are present. Is essential. Sorbitol in an amount of about 15 g / l to about 90 g / l Sucrose is present in an amount from about 10 g / l to about 70 g / l. Vaccine of the present invention The preferred range of sorbitol in the composition is from about 35 to about 75 g / l. Book A particularly preferred range of sorbitol in the vaccine composition of the invention is from about 45 to about 6 0 g / l. The preferred range of sucrose in the vaccine composition of the present invention is From about 15 to about 55 g / l. Characteristics of sucrose in the vaccine composition of the present invention A preferred range is from about 20 to about 45 g / l.   Particularly preferred compositions are shown in Table 1 as Compositions 1 to 12, in these compositions The skilled person, based on the presence of both sucrose and sorbitol, To produce another vaccine composition. Accordingly, the preferred component ranges described herein The heat stability improved among other properties as compared to vaccine compositions known in the art. A qualitative vaccine composition can be produced.BRIEF DESCRIPTION OF THE FIGURES   Figure 1 shows raw lyophilized sorbitol and sucrose concentrations Figure 2 shows the effect on heat stability of measles vaccine.   Fig. 2 shows raw freeze-dried mumps wax of various sorbitol and sucrose concentrations. 2 shows the effect on the thermal stability of tin.   FIG. 3 shows the M-M- No. 4,273,762 to Leer et al. Known stabilizers. This control stabilizer is available from McAleer et al. Components described in Patent No. 4,147,772 and a small amount of DPG solution (50 mg of ascorbic acid, 100 mg of L-cysteine, 50 mg of glutathione Then double distilled H_TwoO900ml, 95% ethyl alcohol 10m, poly Add 5ml of sorbate 80NF and 25mg of vitamin A (crystalline alcohol) Then double distilled H_TwoO85ml and adonosine triphosphate 10mg Addition). Said control stabilizer (and stabilizer with different osmolality) Add 3: 1 stabilizer: MMR vaccine ratio. Composition A is composed of Medium O This is a control stabilizer from which the amount was removed. Composition B contains 50% Medium O component Control stabilizer. Composition C is a control stabilizer in 75 mM NaCl. You. Composition D is a control stabilizer adjusted to 4.5% sucrose. Composition E is 1 Control stabilizer in 50 mM NaCl.   FIG. 4 shows the lyophilization yield of measles virus for the composition shown in FIG.   FIG. 5 shows measles virus, hydrolyzed gelatin concentration (1.5-4.5% w / w), Fig. 2 shows the effect on the stability of mumps virus and rubella virus.   Figures 6A-B show buffering measles virus, mumps virus and rubella virus. Shows the effect on Luss. Panel A achieves 1M phosphate buffer and desired pH Using different concentrations (0.06-0.40 M) of citrate for Shows Ilus stability. In panel B, phosphate (to achieve the desired pH value) 0.66-0.91M) and citrate (0.03-0.07M) change.   Figures A7-C show virus stabilization of buffer concentration in stabilizer without bicarbonate. Indicates effects on gender. The pH of the combination was 6.2-6.4. All concentrations The concentration of the stock solution before dilution in the final vaccine composition. Panel A-Measles Will , Panel B-mumps virus, panel C-rubella virus.   FIG. 8 shows the effect of replacing the cell culture medium with the virus stabilizer. Control stabilizer Is the stabilizer described with respect to FIG. Composition A is a stabilizer as described with respect to FIG. It is. Composition F replaced Medium O with Solution 199. Control stabilizer. Composition G is a composition contained in Medium O of the control composition. Control stabilizer containing a mixture of amino acids similar (but not identical) to amino acids.   FIG. 9 shows that the control stabilizer and Compositions 1 to 8 of Table 1 were lyophilized for 1 week at 37 ° C. 9 shows the thermostability of measles virus after storage during storage. log TCID₅₀Measured as The improved heat stability is indicated by the reduced potency loss. Bars represent standard error of the mean.   FIG. 10 shows that the control stabilizer and Compositions 1 to 8 of Table 1 were lyophilized at 37 ° C. for 1 hour. 2 shows the thermal stability of mumps virus after storage for weeks. log TCID₅₀ It can be seen from the decrease in the titer loss measured that the thermal stability was improved. bar Represents the standard error of the mean.   FIG. 11 shows that the control stabilizer and Compositions 1 to 8 of Table 1 were lyophilized at 37 ° C. for 1 hour. The heat stability of rubella virus after storage for weeks Show. log TCID₅₀As the titer loss measured as You can see the improvement in qualification. Bars represent standard error of the mean.   FIG. 12 shows the control stabilizer (pH 6.6) and the compositions 2 (pH 6.8) and Measles virus after freeze-drying and storing composition 9 (pH 6.2) at 37 ° C. for 1 week 2 shows the heat stability of mumps virus, mumps virus and rubella virus. log TCI D₅₀It can be seen from the decrease in the titer loss measured that the thermal stability was improved. Bars represent standard error of the mean.   FIG. 13 shows G in Table 1 as composition 12 compared to GOS (control stabilizer). 10 shows that the OS33 composition and S12 in Table 2 improve virus stability. 0.5 ml of GOS33 was filled in the experimental run, and 0.7 ml of GOS in the control run. Filled. log TCID₅₀Because the titer loss measured as It can be seen that the thermal stability is improved. Bars represent standard error of the mean.   FIG. 14 shows G in Table 1 as composition 12 compared to GOS (control stabilizer). 10 shows that the OS33 composition and S12 in Table 2 improve virus stability. 0.7ml for experimental run Filled. log TCID₅₀Because the titer loss measured as It can be seen that the thermal stability is improved. Bars represent standard error of the mean.Detailed description of the invention   The present invention relates to vaccine stabilizers, vaccine compositions and attenuators that confer high heat stability. It relates to a live lyophilized vaccine. The initial vaccine of the present invention is a virus and stabilizer component And the stabilizer component is the number of grams per liter of the final vaccine before lyophilization. About 15 to about 90 g / l of a C6 polyhydric alcohol (including, but not limited to, Toll, mannitol and dulcitol), from about 10 to about 70 g / l. Disaccharides (including but not limited to sucrose, lactose, maltose and trehalose) And an amount of physiologically active to adjust the pH to about 6.0 to about 7.0. Consisting of a neutral buffer.   In a particular embodiment of the invention, the vaccine composition is a C6 polyhydric alcohol. Sorbitol from about 15 to about 90 g / l.   In another particular embodiment of the invention, the vaccine composition comprises sucrose as a disaccharide. About 10 to about 70 g / l.   In a preferred embodiment of the invention, the vaccine composition has 6 carbon atoms. Sorbitol as a polyhydric alcohol from about 15 to about 90 g / l, and disaccharide as sugar Containing about 10 to about 70 g / l of sucrose.   In another aspect of the invention, the vaccine composition comprises sorbic acid as a C6 polyhydric alcohol. About 15 to about 90 g / l of tall, and about 10 to about 70 g / of sucrose as a disaccharide. The vaccine composition has a buffering effect in the pH range of about 6.0 to about 7.0. One of the two approaches described below with a citrate-phosphate mixture to preserve Adjusted using one. One of the approaches described above is from about 7.5 mM to about 75 mM. Another approach is to add about 7.5 mM to about 75 mM phosphoric acid. Citrate phosphate at a concentration of about 30 mM to about 0.4 M: citrate Add the mixture.   In another aspect of the invention, the vaccine composition is a sol. About 15 to about 90 g / l of bitol and about 10 to about 70 g of sucrose as a disaccharide / L, and the pH of the vaccine composition is moderate in the preferred pH range of about 6.0 to about 7.0. It is adjusted by adding a phosphate buffer to ensure the impact.   Preferably, the vaccine composition of the present invention provides a vaccine with high thermostability One or more additional ingredients, alone or It may be included in a chemically effective combination. The additional components described above include, but are not limited to, about 10 to 5 0 g / l hydrolyzed gelatin, up to about 10 g / l, preferably about 1 to about 6 g / l salt Sodium chloride, up to about 1.5 g / l, preferably from about 0.2 g / l to about 1.2 g / l l sodium bicarbonate, up to about 1.5 g / l, preferably about 0.5-1.0 g / l l, ie, about 0.3 to about 1.0% of the human dry weight of the vaccine in lyophilized form Serum albumin and cell culture, a nutrient medium that promotes cell growth in vitro Medium (including, but not limited to, Solution 199, Medium T, Medium mO, Dulbecco's Modified Eagle Medium, Minimum Essential Medium, Basic Eagle Medium Known cell culture media). Preferred media components are biologically effective Medium O, Medium T and Solution 199. Other components of the vaccine composition of the present invention include, but are not limited to, a biologically effective amount of an antibiotic. (Eg, neomycin) and pH indicator (eg, phenol red) It is.   Medium O contains 6.2 ml / l of 10 × Solution 199,6 80 ul / l of Solution DPG (1 liter of Solution DPG) Ascorbin per ter Acid 50 mg, L-cysteine 100 mg, glutathione 50 mg, then steam twice Retained H_TwoO900ml, 95% ethyl alcohol 10ml, polysorbate 8 Add 5 ml of 0NF and 25 mg of vitamin A (crystalline alcohol) and then twice Distilled H_TwoO85 ml and adonosine triphosphate 10 mg), 30 . 7 ml of 2.8% sodium bicarbonate solution and 340 ul of 2.0% phenol Contains red solution.   Medium T is 10 ml of 25% human serum albumin per liter. , 112 mg of (monobasic) potassium phosphate, 338 mg of (dibasic) phosphoric acid Potassium, 239 mg L-glutamate monosodium monohydrate, 18.6 g Sucrose and then 842 ml of double distilled H_TwoO, 75 ml of 10 × So solution 199, 750 u / l Solution DPG, 60 ml 2.8% sodium bicarbonate solution and 420 ul of 2.0% phenol red solution Is added.   Accordingly, the vaccine composition of the present invention comprises from about 15 to about 90 g / l of a polyvalent alcohol having 6 carbon atoms. Sorbitol as rucol, sucrose as a disaccharide of about 10 to about 70 g / l Biologically effective concentration of cell culture medium (preferably, Medium O), biology Effective Concentration of salt (preferably NaCl), biologically effective concentration of bicarbonate (preferably , NaHCO_Three) And ensures a buffering action in the preferred pH range of 6.0 to 7.0. Citrate-phosphate mixtures or phosphates alone. Different quantity Of bicarbonate is added to the phosphate-citrate buffer mixture or phosphate buffer Change the pH of the composition to a biologically acceptable range when adding the liquid alone Can be   The vaccine composition of the present invention contains about 15 to about 50 g / l of hydrolyzed gelatin. part Hydrolyzed gelatin has an average molecular weight of about 3,0, as inferred from its name. Partially hydrolyzed to obtain 00Da partially hydrolyzed gelatin I have. This gelatin hydrolyzate has about the same amino acid composition as gelatin. Get Unlike gelatin, which forms gels but does not dissolve in cold water, hydrolyzed gelatin gels And can be dissolved in cold water and other common liquids such as milk and orange juice . Aqueous solutions containing up to about 10% hydrolyzed gelatin will increase appreciable viscosity Not shown. At concentrations above about 10%, the viscosity increases gradually. At about 50% concentration Solution is quite viscous. Typical amino acid composition of hydrolyzed gelatin is known . Partially hydrolyzed gelatin Is available from a number of manufacturers, for example, sold under the trade name Dynagel. Have been. Partially hydrolyzed gelatin converts gelatin into proteolytic enzymes, e.g. For example, it can be obtained by enzymatic hydrolysis using papain, chymopapain and bromelain. It is also possible to use other known hydrolysis means, for example acid hydrolysis. Wear. The preferred range of hydrolyzed gelatin in the vaccine composition of the present invention is about 20 g / L to about 35 g / l. Characteristics of hydrolyzed gelatin in the vaccine composition of the present invention A preferred range is from about 25 g / l to about 30 g / l.   In a preferred vaccine composition of the invention, both sucrose and sorbitol are present. Is essential. Sorbitol in an amount of about 15 g / l to about 90 g / l Sucrose is present in an amount from about 10 g / l to about 70 g / l. Vaccine of the present invention The preferred range of sorbitol in the composition is from about 35 to about 75 g / l. Book A particularly preferred range of sorbitol in the vaccine composition of the invention is from about 45 to about 6 0 g / l. The preferred range of sucrose in the vaccine composition of the present invention is From about 15 to about 55 g / l. Characteristics of sucrose in the vaccine composition of the present invention A preferred range is from about 20 to about 45 g / l.   C6 Polyhydric Alcohols and Disaccharides in Vaccine Stabilizers and Compositions of the Invention Depending on the combination (sorbitol + sucrose) and the total concentration of both components, measles virus Stability is dramatically improved, not seen with commercially available stabilizers, Moderately improved cold virus stability; significant stability of rubella virus after lyophilization Does not change. pH 6.2 is advantageous for measles virus stability but is known Dramatically affects mumps virus stability unlike stabilizer pH 6.5 It is stated that there is nothing. Compared to 400-600 mOsm of known stabilizers It appears that the thermal stability is not affected. Removal of tissue culture components from various stabilizers of the present invention. On drying, the drying characteristics during freeze-drying are improved and the high sugar content vaccine compositions of the invention The overall osmolality of the product is reduced.   Therefore, in the present invention, the sugar concentration in the vaccine composition before freeze-drying is substantially substantially reduced. It is essential to increase it. As described in this section and the examples below And a bulk virus composition to produce a lyophilized vaccine composition. A substantially higher amount of 6 carbons to produce a combined vaccine stabilizer. Polyhydric alcohols (eg, sorbitol) and disaccharides (eg, sucrose) Added, which improves the thermal stability.   Due to the preferred ranges of the components described herein, the known vaccine compositions Produce vaccine compositions that exhibit particularly improved thermostability among other properties be able to. One of ordinary skill in the art will be able to disclose and write in compositions 1-12 as exemplified herein. Based on the presence of both sucrose and sorbitol within the listed A vaccine composition can be manufactured. Compositions 1 to 12 contain neomycin and phenolic Additional components, such as a pad, may be included or omitted. In other words, Variations in the proportions, concentrations and presence of additional ingredients for each composition are also contemplated. .   In an example of the present invention, a live attenuated measles-mumps-rubella virus triple vaccine Has been tested for stability. However, the present invention includes, but is not limited to, unit prices (eg, For example, measles, mumps, rubella, chickenpox) Mumps vaccine, ternary (eg, measles-mumps-rubella) And quaternary (eg, measles-mumps-rubella-varicella) vaccines You. C which can constitute the vaccine of the present invention. Examples of ills include, but are not limited to, measles, mumps, rubella, varicella-shingles, poly E, hepatitis, type 1 herpes simplex, type 2 herpes simplex, or a mixture of various 2 types, a mixture of 3 types or Included are such combinations, such as quadruple vaccines.   The range of various stabilizers and the final vaccine composition is limited to 1 liter of the final vaccine formulation. It is shown in terms of the number of grams. As those skilled in the art are aware, the present invention may be practiced. Different volumes of stabilizer may be added to apply, in which case the stabilizer component The concentration must be changed. These changes are the final live vaccine 1 before freeze-drying. By using effective concentrations of various chemical components on the basis of grams per liter, Included in the invention. In an example of the invention, to produce a final vaccine for lyophilization Shows the use of a 3: 1 stabilizer: virus combination, but never this combination It is not limited to. However, those skilled in the art will be able to select different ratios and May be used at different concentrations. Therefore, those skilled in the art Stabilizer containing the above components (eg, sucrose, sorbitol, hydrolyzed gelatin, etc.) (1) the presence of the main components mentioned above in the virus preparation and (2) the final vaccine Stabilizers for viral preparations used in manufacturing It is manufactured in consideration of the design ratio.   For example, a preferred vaccine of the present invention is a measles-mumps-rubella triple vaccine. Chin. Preferred measles-mumps-rubella vaccines of the invention are It contains at least the main components of one composition 1 to 12. Alternatively, hydrolyzed gelatin, Such as loin, sorbitol, phosphate or phosphate-citrate mixtures The major components are added to the vaccine composition at the respective concentration ranges described herein. Can be added. A measles-mumps-rubella triple vaccine is usually 3: 1. Of stabilizer: virus combination. These three types of mixture In a composition, about 2.1 g / l hydrolyzed vaccine, 2.1 g / l sorbitol and 1.54 g / l NaCl is present in the viral medium. Furthermore, vaccine composition 67.5% of the final volume of the phosphate buffer or vaccine composition at 67.5% of the final volume of the product. At 7.5% a phosphate-citrate mixture may be added to the stabilizer. Therefore, Stabilizer / Buffer: The components of the preferred stabilizer solution used in the virus combination are Easily determined based on the initial content of the components from both the medium and the buffer You.   Table 2 shows that a 3: 1 stabilizer / buffer: virus ratio was prepared. 2 shows the main components of the stabilizers associated with compositions 1 to 12 of the present invention. Stabilizer and final The concentration ranges for the major components of the vaccine composition are approximately the same. Therefore, the present invention Vaccine stabilizers having at least about 15 to about 90 g / l on a g / l basis having 6 carbon atoms Polyhydric alcohols (including but not limited to sorbitol, mannitol and dulcitol) About 10 to about 70 g / l of disaccharides (including, but not limited to, sucrose, Sesame, maltose and trehalose). The specific stabilizer of the present invention is Sorbitol as a polyhydric alcohol having 6 carbon atoms of about 15 to about 90 g / l; And about 10 to about 70 g / l of sucrose. This particular stabilizer also contains about 15 To about 50 g / l, preferably about 20 to about 30 g / l, especially about 25 to about 30 g / l Containing hydrolyzed gelatin. As in the case of the final vaccine composition of the invention, Preferred stabilizers are about 35 to about 75 g / l, particularly preferably about 40 to about 60 g / l Sorbitol. The preferred range of sucrose in the stabilizer of the present invention is It is about 15 to about 55 g / l, particularly preferably about 15 to about 45 g / l.   The present invention is illustrated by the following examples, which do not limit the invention. Not.Example 1   The control stabilizers used in Examples 1-3 were obtained from McAlee et al., US Pat. It is a known stabilizer described in Japanese Patent No. 4,273,762. This stability The agent comprises a stabilizer component as described in U.S. Pat. No. 4,147,772 and A small amount of DPG solution (50 mg of ascorbic acid, 100 mg of L-cysteine, Tathione 50 mg, double distilled H_TwoO900ml, 95% ethyl alcohol 10ml, Polysorbate 80NF 5ml, Vitamin A (crystalline alcohol) 25 mg were added and then double distilled H_TwoO85ml and Adonosine Triphos 10 mg of fate). U.S. Pat. No. 4,273,762 and U.S. Pat. No. 4,147,772 is hereby incorporated by reference. . In Examples 1-3, the measles virus (More attenuated Ender's Edmo) nston strain; minimum dose = 3.0 TCID₅₀Target filling = 3.8 TCID₅₀) Mumps virus (Jery Lynn strain; minimum release = 4.3 TCID)₅₀ / Dose; target loading = 5.0 TCID₅₀/ Dose) and rubella virus (Wista) r RA 27/3 strain; minimum release = 3.0 TCID₅₀/ Dose; target Filling = 3.8 TCID₅₀/ Dose) used as control vaccine28.9 g of hydrolyzed gelatin and 28.9 g of sorbitol per liter of final vaccine , Phosphate 10.59 g, NaCl 4.9 g, sucrose 3.74 g, sodium bicarbonate 0.9 g of thorium, 0.66 g of glucose and 0.62 g of human serum albumin Including. The composition of the control vaccine composition is based on the volume of the control stabilizer before freeze-drying. 5%, 1M phosphate 7.5%, Measles virus bulk / Mumps virus bulk Medium T (Medium T is 0.45 g of phosphate based on g / l, 6 g of NaCl, 18.7 g of sucrose, 1.68 g of sodium bicarbonate, glucose 0.75 g, human serum albumin 2.5 g and phenol red 8.4 mg 20% composition and rubella virus bulk / rubella diluent [eg rubella diluent Is 9.6 ml of 25% human serum albumin per liter, hydrolyzed gelatin 42.9 g, Eagle MEM 5.6 g, sorbitol 42.9 g, NaCl 6 . 8, glucose 1 g, human serum albumin 2.4 g and phenol red 12 mg (2.0% phenol red solution 600 ul), but in no way limited to 5%.Method   Various stabilizers: up to 3 lots of bulk will for MMR vaccine composition Tests were performed on a laboratory scale and on a production scale. Lyophilized measles virus After 1 week at 37 ° C. in the control stabilizer, about 1.0 log of infectious titer, ie 90%, lose. The stabilizer of the present invention: the virus composition may be mumps virus or rubella virus. Fever of freeze-dried measles vaccine without unacceptable compromise on ils stability Stability characteristics must be improved. Titer Assay (TCID)₅₀) Based on performance Therefore, the thermostability of measles virus observed in the experiment was less after one week at 37 ° C. Must also be 0.7 log loss (> 22% survival). Hydrolyzed gelatin and All three viruses were titered in a buffer concentration experiment. TCID₅₀Assay Is in 1x6 format (ie, 6 unique configurations, usually 1 (Vial). Other experimental conditions (pH, sugar concentration, ionic strength, Mediu mO exchange) was tested using a plaque assay in a 1 × 6 format. Assayed by incubating for 1 week at -70 ° C and stored at -70 ° C. Compared to Iar. Samples incubated at 30 ° C are usually incubated at 37 ° C. However, the difference between the compositions is large. Liquid The sample was also collected and frozen without lyophilization, and then the lyophilization yield was determined. Assayed. A total of 3960 vials were assayed.   The moisture content of the lyophilized vial was determined by Aquatest IV (Kalfischer). -Method) and shown as an average of four vials. And freeze-dried. Initial shelf temperature during initial drying to increase product temperature rapidly After the temperature was ramped up to -15C, the shelf temperature was lowered to -25C for the remainder of the initial drying. In this way, the product temperature may be reduced so that the freeze-dried cake may physically collapse. Hold during initial drying near the estimated Tg 'of the control stabilized vaccine You. In addition, a two-slot stopper (West 4405) was used in all experiments. Used and pre-dried in a vacuum oven at 140 ° C. for at least 6 hours. Used within hours. Freeze-dryer shelf with all compositions pre-cooled to -45 ° C before filling into the freeze-dryer Frozen on top. For compositions with high amounts of sugar, to ensure lower water content The final shelf temperature and holding time were extended.Effect of sugar concentration   In this example, the sucrose and sorbitol concentrations in the vaccine composition were substantially Increases the heat stability of measles virus and mumps virus. And To determine the optimal concentration and combination of sorbitol and sucrose, Various unions Incl. When only sucrose is added, the final sucrose concentration is up to 6% The increase does not affect the thermal stability of MeV. However, the addition of sorbitol Addition has a significant effect on the thermal stability of MeV, and the thermal stability of MeV It is directly related to the final concentration of Toll. Surprisingly, it contains sorbitol The addition of sucrose to certain compositions has a dramatic stabilizing effect. FIG. 1 shows a high amount of Measles with sucrose or sorbitol and sucrose in sorbitol Irs (MeV) thermal stability has been shown to increase equally. Mumps C The stability of virus (MuV) shows a similar tendency, but the change is usually small (FIG. 2). ). Rubella virus (RuV) stability is not significantly affected by changes in sugar concentration. No.Influence of penetration and ionic strength   Measles of the ionic and osmotic strength of various compositions (compositions A to E)-mumps-wind The effect on the thermal stability of the rash vaccine was determined by Me before lyophilizing the vaccine composition. By adjusting the concentration of Dium O or by adding Medium O to water or It was evaluated by substitution with sarin. Furthermore, they have the same penetration strength but Semi-normal sarin or 4.5% sulphate instead of Medium O Manufactured using sucrose. As shown in FIG. 3, MeV and M after one week at 37 ° C. There was no appreciable tendency for the virus stability of uV at 37 ° C. RuV Stability is more variable and there are indications that stability will increase with increasing osmotic strength. I got it. The titer after lyophilization ranges from 440 to 600 mOsm for all viruses. Unaffected by changes in intensity. Lyophilization yield of MeV depends on penetration strength (FIG. 4), but with a decrease in the titer before freeze-drying, The titers were even across the range of penetrating strength. MuV also Did not have a clear tendency in the lyophilization yield of RuV. In the latter two viruses> The 100% yield was observed, indicating liquid stability or liquid stability of these compositions. Sample handling suggests lower titers than lyophilized samples (The yield was 52-184% for MuV and 102-218% for RuV) . The residual water content of the composition was 1.0-1.4%.Effect of hydrolyzed gelatin concentration   Tests the effect of virus stability on hydrolyzed gelatin concentration (1.5-4.5% w / w) did. As shown in FIG. 5, the hydrolyzed gelatin concentration was changed (from the control level of 2.5%). The heat stability at 37 ° C. was not adversely affected for all viruses. 1. Lyophilized vaccine containing 5% hydrolyzed gelatin is incubated at 37 ° C for 1 week It shrank slightly. Because all samples contained 1.0-1.5% moisture Shows that changing the hydrolyzed gelatin concentration does not interfere with the drying behavior of the control composition. You. Therefore, changes in the hydrolyzed gelatin concentration may improve the integrity of the freeze-dried cake. Can be used for The lyophilization yield of MeV is 63-104%, The more enriched compositions showed lower yields. MuV And RuV were not observed, and the lyophilization yield was 54 ９３93%.Effect of pH   The citrate-phosphate buffer combination was tested. Manufacture various pH buffers Two approaches were tested for this. The first approach is to keep the phosphate concentration constant To change the citrate concentration (differences in ionic strength) and a second approach H changes the concentration of both buffers (the change in ionic strength is small, Change the concentration). Using a constant stock solution of 1.0 M sodium phosphate, add sodium citrate The concentration of the thorium stock solution is varied (0.06 to 0 to achieve the desired pH). . 40M) In the composition produced, MeV has the highest thermal stability at pH 6.3 Looks like. However, the pH dependence is small (FIG. 6A). Citrate stock solution And changing the concentration of the phosphate stock solution to reach the desired pH (phosphate 0. 66-0.91M and citrate 0.03-0.07M), pH max 6.2, which is better for the thermal stability of MeV at lower and higher pH. Dramatic reduction (FIG. 6B). No clear trend was observed in MuV stability However, the data is the highest among the three viruses. Also fluctuate greatly. RuV is the combination of 1.0M phosphate and citrate Shows maximum stability at pH 6.2, but higher with various concentrations of phosphate. At higher pH, the stability appears to improve. High phosphate concentrations may cause It can be assumed that it adversely affects the loose stability. Residual moisture content for all samples 1.1 to 1.6%. In the case of a 1M phosphate-containing composition, the lyophilization yield is pH In the pH series with different phosphate concentrations, the yield was not affected by pH. Received. Although the data is not accurate (lyophilization yield was 59-98%), M The eV yield appeared to increase with increasing pH. MuV (58-284%) Or RuV (89-131%; titer decreases, but higher yield at lower pH) (Observed) did not show a clear tendency in the lyophilization yield.Effect of buffer concentration   The sodium bicarbonate was removed from Medium O and pH was reduced at lower buffer concentrations. To determine whether it is possible to regulate or adversely affect viral thermostability Various concentrations of phosphate buffer and phosphate / citrate buffer were tested for this. p Thermal stability of MeV at various buffer concentrations when H is maintained between 6.2 and 6.4 Are not significantly different (FIG. 7A). However, MuV has a high citrate concentration. As shown in FIG. The thermal stability of RuV is phosphoric acid There is no clear trend with salt or citric acid concentration (FIG. 7C). Freeze-dried product water content The amounts ranged from 0.9 to 1.9%. Lyophilization yield and buffer for all viruses No correlation was observed between the solution concentration and the yield was 50 to 158%.Effect of cell culture medium replacement Tests were performed on various compositions that had been removed or that had Medium O completely removed. M Edium O was replaced with water (composition A), Medium 199 (composition F) or Me. A mixture of amino acids similar (but not identical) to those contained in Dium O (Composition G). Removing Medium O from the control stabilizer, Medium O component. Lyophilize and incubate at 37 ° C for 1 hour After vating, making the above changes to the control stabilizer will result in less heat stability for all viruses. (FIG. 8). Removal of Medium O has minimal impact on stability In pairs The ionic and osmotic strength of the product can be reduced. It also maintains the integrity of the freeze-dried product, Lower salt concentrations are needed to reduce the likelihood of stinging of the composition when injected Can get. No trend was observed in the lyophilized yields of the three viruses (yield 51 １４141%).Effect of stopper on residual water content and stability   (1) For the water content of the lyophilized product before and after incubation at 37 ° C. Influence of various stoppers, (2) Influence of water content on the appearance of freeze-dried product, And (3) various forms of storage to determine the effect of water content on virus titer. The par and the treatment were evaluated. Stopper in a vacuum oven at 140 ° C before use. Allow time to dry. The water content of a lyophilized vaccine with a dry stopper is It did not increase after incubation at 37 ° C. However, did not dry With stoppers, the water content of the lyophilized vaccine increased after incubation . The high sugar content compositions of the present invention were also tested because of their high hygroscopicity. This composition is The final concentrations of bitol and sucrose were 4.9% and 4.4%, respectively. Consisted of a control stabilizer supplemented with sugar. In this case, freeze-dry the cake Dry stopper Incubation at 37 ° C. using a plate showed significant shrinkage in all cakes. Was. The water content and physical stability of the lyophilized product are affected by stopper treatment. However, there was no clear trend in virus thermostability. Therefore, according to the present invention, With low sugar concentrations, virus stability cannot be affected in the water content ranges described above. I However, shrinkage can adversely affect product reconstitution, Incomplete rings may result in low titers in the assay. Similarly, If the reconstitution of Kuching is difficult, lowering the dose will create a clinical setting. obtain.   Based on the results obtained from the laboratory scale experiments described above, the 11 compositions shown in Table 1 were Example 2 (compositions 1 to 9), Example 3 (compositions 10 to 11) and Example 4 (compositions) Product 12). In the vaccine composition of the present invention, sorbitol concentration and Substantially increasing the concentration of sucrose dramatically improves the heat stability of measles virus. And to prove. The seven vaccines of the present invention to reduce the total solids content in the composition The cell culture medium was removed from the cell composition. Removing the cell culture medium from the stabilizer, Reducing the total solids content improves the drying characteristics of high sugar content compositions . In addition, salts, especially sodium chloride, substantially reduce the glass transition temperature of sugars, The likelihood of the product disintegrating during lyophilization can be increased. The male of the composition Reducing the molarity can reduce the likelihood of piercing at the time of injection. Finally, The correlation between pH and the thermal stability of MeV has been demonstrated herein. preferable Modifying the pH of Compositions 1-12 to minimize the possibility of high pH excursions Using sodium buffer or sodium phosphate / sodium citrate buffer 6.2 for MeV and 6. for measles-mumps-rubella vaccine. Adjust to 2-6.8.                               Example 2   Table 1 shows the concentrations of the main components constituting the final vaccine compositions 1 to 12 of the present invention. . The thermal stability of each final vaccine composition was tested. Compositions 1-11 are real Tests were performed as described in Examples 1 and 3, and Example 12 was described in Example 4. Tested asTable 1 Components of Compositions 1 to 12 (g / l) Table 2 3: 1 = stabilizer / buffer: viral MMR vaccine composition Stabilizers corresponding to 1 to 12 (g / l) (S1 to S12   A preferred vaccine of the present invention is a measles-mumps-rubella combination vaccine. You. The preferred measles-mumps-rubella combination vaccine of the present invention has the composition shown in Table 1. It contains the main components of products 1 to 12. Alternatively, hydrolyzed gelatin, sucrose, sorbitol The main components, such as, phosphate or phosphate: citrate mixtures are described herein. Can be added to the vaccine composition in the range described in (1). These preferred measles -Mumps-Rubella is mixed in a 3: 1 ratio of stabilizer / buffer: virus . In an exemplary triad composition, about 2.1 g / l hydrolyzed gelatin, sorbitol 2. 1 g / l, 3.7 g / l sucrose and 1.54 g / l NaCl Exists inside. Furthermore, phosphates that make up 7.5% of the final dose of the vaccine composition Final dose of vaccine composition with addition of buffer or phosphate: citrate mixture 67.5% of the stabilizer may be added. Therefore, the stabilizer / buffer: The components of the preferred stabilizer solution used in the virus combination include a virus-containing medium and a buffer. It is easily determined from the impingement solution based on the initial content of the components. Table 2 shows the composition of the present invention. 1 shows the main components of vaccines related to 1-12.   Neomycin is present in a biologically active amount, preferably about 0,000 USP stock of neomycin. . 34 ml can be added to the composition of the present invention. Phenol red is good Preferably, an amount of about 0.01 g / l can be added. Solution DP G and / or Solution 199 at the biological activity level, preferably Add about 0.1 to about 2.0 ml of each stock solution. Can be. Human serum albumin may be replaced by available biological equivalents .   Table 1 shows the main components of the freeze-dried vaccine of the present invention and the dry weight of the freeze-dried vaccine. % Can be varied. In compositions 1 to 12, the hydrolyzed gelatin About 20% to about 30% of the total dry weight, and sorbitol is present at about 35% to about 5% of the total dry weight. 0%, phosphate is present at about 7.5 to about 10% of the total dry weight, sodium chloride About 1% to about 4% of the total dry weight, and sodium bicarbonate is about 0.1% of the total dry weight. 2 to about 0.7%, up to about 3% sodium citrate, human serum Bumin is present at about 0.4 to about 0.7% of the total dry weight.   The final vaccine composition, after lyophilization, is more effective against measles virus than control stabilizer. Improved thermal stability (Fig. 9), comparable thermal stability to mumps virus (Fig. 9) FIG. 10) and slightly lower thermostability against rubella virus (FIG. 11).   The thermal stability of composition 9 was compared to composition 2. In this experiment, additional The effect of sodium bicarbonate on the thermal stability of the lyophilized composition was compared. Figure 12 shows remarkable thermostability of measles virus in composition 9 or composition 2 (Fig. 9 also reference). FIG. 12 shows the heat resistance of mumps virus in composition 9 and composition 2. Indicates qualitative or equivalent (see also FIG. 10 for composition 2). Finally, FIG. 2 shows the same heat stability of rubella virus in the control stabilizer, composition 9 and composition 2. Etc. (see FIG. 11 for composition 2).   Compositions 1-9 illustrate the range of each compound described in the present invention. Compositions 1-9 are hemp Measles virus or measles virus, showing increased thermostability of measles virus It does not adversely affect the thermal stability of the steel.                                 Example 3   The filling and lyophilization of composition 10, composition 11 and the control stabilizer are essentially as described herein. Performed as described in the book. Compositions 10 and 11 are control stabilizers 0.9 log loss (after 1 week at 37 ° C) to 0.6 log loss The thermal stability of Lus was improved. Of mumps virus component and rubella virus component Thermal stability was within 0.1 log loss of the control stabilizer. Thermal stability is on the shelf Position or position within each tray. Freeze drying cabinet Adjusting the position of the thermocouple inside makes the temperature reading more uniform and the freeze drying cycle Several freeze-drying parameters were monitored that could clearly determine the end of initial drying during . TCID₅₀Absolute titers differed between assay and plaque assay, but similar The relative titers (ie, log loss) of the samples were comparable in both assays. Vaccines frozen by different methods (shelf or liquid nitrogen tunnel) have different drying rates And microscopic morphology.Method-Composition   Three stabilizer compositions were selected for the extended run. The first stabilizer is a control stabilizer Met. The second and third stabilizers were in composition 10 and composition 11 (Table 1), respectively. there were. Each of these compositions contains 4.9% sorbitol in the final vaccine. , The compositions have different osmolality and solids content. Composition 11 is the final wax 4.9% sorbitol and 4.4% sucrose. Composition 10 Is a control containing 4.9% sorbitol in the final vaccine but without the cell culture medium. Consists of a fixed agent. Composition 10 has a low osmolality, thereby causing a stinging upon injection The lyophilization process can be accelerated due to the lower salt concentration. Contrast To adjust the pH of the stabilizer to 6.5, a sodium phosphate buffer (1M, pH 6) . 2) was used. Composition Sodium phosphate / citric acid to maintain pH of 9 and composition 10 at 6.2 Sodium buffer (142 g / l anhydrous sodium phosphate dibasic + 50.0 g / L of anhydrous citric acid).   Stoppers used to seal product vials provide moisture to dry product Was dried further (6 hours at 140 ° C.) to prevent desorption of 816 stopper. Each of the three stabilizers described above was replaced with a stabilizer, buffer and Manufactured using virus bulk. Fill each vial with eight vials of perforated tray Three in a freeze-dried cabinet packed and frozen in a liquid nitrogen tunnel and pre-cooled. Placed on one of the middle shelves. Lyophilization parameters make the current aggressive manufacturing cycle Minimizes the potential physical effects of sucrose on high sugar content compositions did. After the freezing process, raise the shelf temperature to -15 ° C to increase the product temperature rapidly. The temperature of the shelf during the remaining initial drying was then reduced to -25 ° C. In this way, the product Keep product temperature close to -40 ° C during the initial period to minimize the possibility of product collapse. Was. After the initial drying is completed, raise the shelf temperature to 30 ° C. at a rate of 3 ° C./hour. And maintained for 10 hours. Shelf temperature from about 3 ° C / hour to about 6 ° C / It may be raised at the rate of time. The above-mentioned temperature period is a period of water content in products with a high sugar content. Required for very low volume, during lake incubation at 37 ° C Essential to maintain physical integrity. Freeze to monitor freeze-drying process A total of 12 thermocouples were used in the drying cabinet. In addition, place the thermocouple vial Separated from Leh ring (approximately 12 rows from front, at least 6 rows from side) . Filling the pre-lyophilized (liquid) sample to test for reduced vial titer Collected at the beginning, midway and end. Pre-lyophilization (liquid) sample in the usual way It was frozen in a liquid nitrogen tunnel according to the method. Lyophilize two samples and tray Recovered from The first set of samples, called "edge" samples, is adjacent to the edge of the ring Vials selected from two rows. Another set of samples called "random" Were vials randomly collected from rows two or more rows away from the end of the ring. Samples were also collected from different trays on the same freeze dryer shelf. Tray 1-4 Are located at the rear of the shelf from left to right, and trays 5-8 are located in the front half of the shelf.   TCID for three virus titers₅₀Assay or plaque assay Was. These assays are 1x6 format I.e. six unique configurations, eg one via at different times and days Was carried out. Water content of freeze-dried product is measured using Karl Fischer method And the average of four vials is shown.Water content and pH   The water content of each composition was fairly uniform across the tray. This large la The water content achieved in the process is similar to that obtained with samples manufactured on a laboratory scale. The water content in all compositions ranged from 0.4 to 0.8%. 1 at 37 ° C No significant water absorption or cake collapse was observed before or after weeks (Table 3) . Therefore, the method of drying the stopper sufficiently prevents moisture transfer to the freeze-dried product I do. PH of final vaccine was 6.5 for control stabilizer, 6.2 for composition 10, composition 11 was 6.3.                                  Table 3 Before and after incubation at 37 ° C for 1 week Freeze-dried product moisture content (%) Thermal stability and lyophilization yield   Measure thermal stability 90 times on average (90 non-incubated vials + 1 week at 37 ° C) 90 vials incubated between). Example 1 for compositions 1-9 and As shown in the second embodiment, It shows improved measles virus stability (Table 4). In the case of compositions 10 and 11, Iar has a mumps virus titer of 0.9 log or less after the incubation period. Did not lose. Heat stability of mumps virus and measles virus composition Seems relatively unaffected by change. Absolute force in the three compositions tested The titer and lyophilization yield were comparable (Table 5).                                   Table 4 Log loss of virus titer after one week at 37 ° C (And range of measured values) Table 5 Average titer of lyophilized vaccine (TCID ₅₀ / ml) and Loss observed during lyophilization and filling   The liquid samples collected at the beginning, middle and end of filling are equivalent in titer. It is suggested that no significant degradation occurs during filling (about 1 hour at 4 ° C.). Freeze dryer Average loss of titer for the position of the tray on the shelf and the position of the vial in the tray Was uniform.   Table 6 shows the log loss in potency of the control composition, composition 10 and composition 11.                                 Table 6 In TCID ₅₀ assay and plaque (PFU) assay Comparison of observed vial titer losses Values are 1 week at 37 ° C in 3 different trays and 2 vial positions in the trays. The average value of log loss after incubation is shown.   Measles virus in the control stabilizer was liquid nitrogen compared to vaccine frozen on the shelf. The method frozen in is more stable. However, composition 10 and composition The MeV stability of product 11 is comparable for both freezing methods. Any freeze There is no significant difference in MuV and RuV stability using the method (Table 7). Power loss The standard deviation of the values obtained for the loss is 0.1 for MeV, 0.2 for MuV, and 01 for RuV. Met.                                   Table 7 Potency loss after 1 week at 37 ° C (and range of measured values) Example 4   Particularly preferred compositions of the invention are listed in Table 1 as composition 12 and in Table 2 as S12. Have been. The materials and methods of Examples 1 to 3 were used to create the data of this example. Can be used. The lyophilization cycle is as follows. Vaccine stabilizers, buffers and And virus bulk, place 0.5 ml in 3 ml glass vial Loosen stopper to allow water vapor to escape during lyophilization in vial I fastened it. Place vial on perforated tray and freeze through liquid nitrogen tunnel Let it. The tray is then placed on a freeze dryer shelf that has been previously cooled to about -50C. After placing in a lyophilization cabinet and initiating the lyophilization cycle, Raise to 25 ° C. and maintain for remaining initial drying. In this way, throughout the initial drying Product temperature is kept close to -40 ° C to minimize the risk of product collapse. Was. At the end of the initial drying, the bulk ice sublimes with a thermocouple placed in the vaccine vial. After the temperature is observed to rise rapidly. Then the shelf temperature is increased at a rate of 6 ° C / hour. The temperature was raised to 32 ° C. and maintained at that temperature for 15 hours. During this temperature the high sugar content The product has a very low water content and the cake object Physical integrity is maintained. As those skilled in the art are aware, this freeze-drying method is The compositions of this example are compatible with the methods described in Examples 1-3, and It can be used in combination with one to three freeze-drying methods. Viral stability is the present composition 13 (filling 0.5 ml) and FIG. 14 (0. (7 ml filling).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 39/245 A61K 39/245 39/25 39/25 39/29 39/29 A61P 31/12 A61P 31 / 12 C12N 7/04 C12N 7/04 (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AU, AZ, BA, BB, BG, BR, BY, CA, CN, CU, C Z, EE, GE, GW, HU, ID, IL, IS, JP, KG, KR, KZ, LC, LK, LR, LT, LV, MD, MG, MK, MN, MX, NO, NZ, PL , RO, RU, SG, SI, SK, SL, TJ, TM, TR, TT, UA, US, UZ, VN, YU. East Lincoln Ave. 126 (72) Inventor Shieu, Suppiy New Jersey 07065, United States, Lowway, East Lincoln Ave. 126

Claims (1)

[Claims] 1. Inactivated or attenuated virus, and about 20 to about 90 g / l on a g / l basis C6 polyhydric alcohol, about 20 to about 70 g / l disaccharide, about 10 to about 50 g / l hydrolyzed gelatin and amount of physiology to adjust the pH to about 6.0-7.0 A vaccine comprising a stabilizer consisting of a buffer that is actively active. 2. 2. The polyhydric alcohol having 6 carbon atoms is sorbitol. Vaccine. 3. 3. The vaccine according to claim 2, wherein the disaccharide is sucrose. 4. If the virus is measles, mumps, rubella, varicella-zoster, polio, hepatitis, 4. Herpes simplex type 1, herpes simplex type 2, or a combination thereof. vaccine. 5. 5. The method according to claim 4, wherein the buffer is a phosphate-citrate buffer. Kuching. 6. The vaccine according to claim 4, wherein the buffer is a phosphate buffer. 7. Claims where the cell culture medium is added at a biologically effective concentration Item 6. The vaccine according to Item 5. 8. 7. The method according to claim 6, wherein the cell culture medium is added at a biologically effective concentration. Kuching. 9. The vaccine according to claim 7, wherein the cell culture medium is Medium O. N. 10. 9. The vaccine according to claim 8, wherein the cell culture medium is Medium O. Chin. 11. Claims selected from the group consisting of Composition 1 and Composition 5 shown in Table 1. 6. The vaccine according to item 5. 12. Claims selected from the group consisting of Composition 3 and Composition 6 shown in Table 1. 6. The vaccine according to item 5. 13. Claims selected from the group consisting of Composition 4 and Composition 7 shown in Table 1. Item 7. The vaccine according to Item 6. 14． Claims selected from the group consisting of Composition 1 and Composition 5 shown in Table 1. 6. The vaccine according to item 5. 15. Claims selected from the group consisting of Composition 2 and Composition 8 shown in Table 1. Item 7. The vaccine according to Item 6. 16. A lyophilized vaccine according to claim 12. 17． A lyophilized vaccine according to claim 13. 18. A lyophilized vaccine according to claim 14. 19. A lyophilized vaccine according to claim 15. 20. The lyophilized vaccine according to claim 16. 21. By reconstituting the lyophilized vaccine according to claim 16 The resulting stabilized vaccine. 22. By reconstituting the lyophilized vaccine according to claim 17 The resulting stabilized vaccine. 23. Reconstituting the lyophilized vaccine according to claim 18 The resulting stabilized vaccine. 24. By reconstituting the freeze-dried vaccine according to claim 19, The resulting stabilized vaccine. 25. By reconstituting the lyophilized vaccine according to claim 20, The resulting stabilized vaccine. 26. g / l, from about 20 to about 90 g / l of a C6 polyhydric alcohol, A wax consisting of 20 to about 70 g / l disaccharide and about 10 to about 50 g / l hydrolyzed gelatin; Kuching stabilizer 27. The polyhydric alcohol having 6 carbon atoms is sorbitol according to claim 26. A vaccine stabilizer as described. 28. 28. The disaccharide is sucrose according to claim 27. Vaccine stabilizer. 29. S1, S2, S3, S4, S5, S6, S7, S8, S9 shown in Table 2 29. The shoe according to claim 28, which is selected from the group consisting of S10 and S11. Stabilizer. 30. A lyophilized form that is effectively reconstituted for host inoculation. The method for producing a vaccine according to the item, a) Dry the vial stopper, b) sealing the vial containing the vaccine composition with a stopper on the vial; c) The sealed vial containing the vaccine composition is lyophilized to a water content of about 0.4 % To about 0.8% of the lyophilized form of the vaccine, d) resuspend the vaccine in lyophilized form The method comprising: 31. Freeze drying step a) pre-cooled to about -45 ° C, b) first increasing the temperature of the vaccine composition to about -15 ° C and then increasing the temperature to about -25 ° C. Dried by lowering to c) Raise the shelf temperature to 30 ° C at a rate of about 3 ° C to 6 ° C / hour, d) Maintain shelf temperature at 30 ° C. for at least about 10 hours 31. The method of claim 30, comprising: 32. The vaccine was composed of composition 1, composition 2, composition 3, composition 4, and the composition described in Table 1. Group consisting of composition 5, composition 6, composition 7, composition 8, composition 9 and composition 10 31. The method of claim 30, wherein the method is selected from: 33. The vaccine was composed of composition 1, composition 2, composition 3, composition 4, and the composition described in Table 1. Group consisting of composition 5, composition 6, composition 7, composition 8, composition 9 and composition 10 32. The method of claim 31 selected from: