GB1575155A - Vaccine stabilizer - Google Patents

Description

(54) VACCINE STABILIZER (71) We, MERCK & CO. INC., a corporation duly organized and existing under the laws of the State of New Jersey, United States of America, of Rahway, New Jersey, United States of America, do hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed to be particularly described in and by the following statement:- This invention relates to vaccines and their stabilization. The present invention provides a vaccine in lyophilized or liquid form and comprising an inactivated or attenuated live virus and a stabilizer consisting essentially of from 2 to 5 parts partially hydrolysed gelatin having a molecular weight of about 3000, from 2 to 55 parts of a 6-carbon polyhydric alcohol, a cell culture medium in an amount such that the content of solids from one medium is 0.5 to 1.7 parts, and sufficient physiologically acceptable buffer to adjust the pH to from 6.0 to 6.5, all parts being by weight.

Examples of suitable viruses are Herpes simplex 1 or 2, measles, mumps, rubella, varicella, polio, and hepatitis, and mixtures of any two or more of such viruses. Hydrolysed gelatin is used to provide a soluble non-gelling proteinaceous matrix with little or no pyrogenicity or antigenicity.

The partially hydrolysed gelatin has approximately the same amino acid composition as gleatin. Unlike gelatin, which forms gels but is insoluble in cold water, hydrolysed gelatin does not gel but is soluble in cold water and other common liquids such as milk and orange juice. Aqueous solutions containing up to about 10% hydrolysed gelatin do not increase appreciably in viscosity. Above about 10% concentration, viscosity increases slowly. At about 50% concentration, solutions are quite viscous. The typical amino-acid composition of hydrolysed gelatin is as follows: Alanine 8.5 % Arginine 7.9 % Aspartic Acid 5.7 % Cystine 0.08% Glutamic Acid 9.5 /O Glycine 22.8 % Histidine 0.77 % Hydroxy Proline 13-14 /,, Isoleucine 1.3 ( Leucine 2.9 % Lysine 4.2 /n Methionine 0.78 % Phenyl Alanine 2.0 % Proline 13.8 % Serine 3.3 % Threonine 1.9 % Tyrosine 0.40 % Valine 2.4 % Partially hydrolysed gelatin may be obtained by enzymatic hydrolysis of gelatin by means of a proteolytic enzyme, such as, for example, papain, chymopapain and bromelin, although other known hydrolysis means may be used, e.g., acid hydrolysis. A suitable hydrolysed gelatin is obtainable from Wilson and Co., Inc., Calumet City, Illinois under the trade name SOL-U-PRO. The 6-carbon polyhydric alcohol may be, for example, sorbitol, mannitol or dulcitol. Sorbitol is preferred.

The acidic buffer may be any physiologically acceptable buffer which will maintain the desired pH of from 6 to 6.5, for example, phosphate buffer, acetate buffer or citrate buffer. Phosphate buffer is preferred. The stabilizer is diluted with from 3 to 8 times, preferably about 5.5 times, its weight of distilled water before use.

By a cell culture medium is meant a nutrient medium which permits growth of cells in vitro. Some specific nutrient media are, for example, Medium 199, Morgan et al., Proc. Soc. Exp. Biol. & Med., 73:1-8, 1950: Basal Medium Eagle, Eagle, Science, 122, 501-504, 1955; In Vitro, Vol. 6, No. 2, 1970; Dulbecco's Modified Eagle's Medium, Dulbecco et al., Virology, 8, 396, 1959; Smith et al., J. Virol., 12, 185-196, 1960; In Vitro, Vol. 6, No. 2, 1970; Minimum Essential Medium (Eagle), Science, 130, 432 (1959) and RPMI Media, Moore et al., 199, 519-524, 1967; In Vitro, Vol. 6, No. 2, 1970.

The stabilizer composition--used in the vaccines of the present invention contains the following ingredients in the amounts indicated: Parts by Ingredient weight Partially hydrolysed gelatin 2-5 Polyhydric alcohol 2-55 Nutrient medium (solids) 0.5-1.7 Physiologically acceptable buffer to adjust pH to 6.0-6.5 quantity sufficient With a liquid vaccine sorbitol is generally present in an amount toward the upper end of the range, while with a lyophilized vaccine sorbitol is generally present in an amount toward the lower end of the range.

Specific formulations for the stabilizer for the vaccines of the present invention follow. Formulation B is preferred for use in the case of a lyophilized vaccine.

The stabilizer may consist essentially of about 3.6 parts of partially hydrolysed gelatin, about 3.6 parts of sorbitol, about 1.1 parts (solids content) of Medium 199 and an amount of phosphate buffer sufficient to adjust the pH of from 6.0 to 6.5, or of about 3.6 parts of partially hydrolysed gelatin, about 53 parts of sorbitol, about 1.1 parts (solids content) of Medium 199 and sufficient phosphate buffer to adjust the pH from 6.0 to 6.5, all parts being by weight. The second of these is preferred for use in a lyophilized vaccine.

More specific formulations are these: A B Partially hydrolysed gelatin 35.7 g 35.7 g Sorbitol 526g 35.7 g Medium 199 (solids content) 11.06 g 11.06 g Sodium phosphate buffer, 1 M, pH 6.0 100 ml 100 ml Distilled water to 1 liter to 1 liter In addition to the stabilizer optionally but preferably contains small amounts of NaHCO3 and of phenol red. In the case of the foregoing formulation the NaHCO3 may be present in an amount of about 1.2 g and the phenol red in an amount of about 0.01 g. A typical stabilized vaccine usually contains from 2 to 12 volumes of stabilizer per 1 volume of vaccine.

The following examples illustrate the present invention and conventional vaccines for comparative purposes.

EXAMPLE 1 80 ml of measles viral concentrate which has been stored at -70 is thawed in a water bath at 250C and then kept at 48 . The liquid viral concentrate is then split into two aliquot portions, each of 40 ml.

a) One aliquot portion from this virus fluid is diluted in 210 ml of the previously described sterile stabilizer of formulation B. Formulation is carried on under aseptic conditions and laminar flow hood. To prevent microbial growth 10.5 mg. Neomycin is added to the preparation. The diluted vaccine is dispensed into 2 ml glass ampoules (0.7 ml vaccine per ampoule) which are immediately flamesealed and stored at 4--8"C.

b) The second aliquot portion is handled as the first, except that, instead of the stabilizer of formulation B, a standard commercial vaccine diluent (SPGA) is used.

The storage stability of the vaccines is described in the following table: Titersl) of Liquid Vaccines Stored at 2-80C Stabilizer of SPGA Time Formulation B Stabilizer 0 3.4 3.6 4 months 3.2 0.6 11Titers are expressed as TCID5J0.1 ml.

EXAMPLE 2 32 ml of measles viral concentrate which has been stored at -70 is thawed in a water bath at 250C and then kept at 48 . The liquid viral concentrate is then split into two aliquot portions each of 16 ml.

a) One aliquot portion from this virus fluid is diluted in 48 ml of the previously described sterile stabilizer of formulation B. Formulation is carried on under aseptic conditions and laminar flow hood. To prevent microbial growth 2.5 mg Neomycin is added to the preparation. The diluted vaccine is dispensed into 2 ml glass ampoules (0.7 ml vaccine per ampoule) which are immediately flame-sealed and stored at 370C.

b) The second aliquot portion is handled as the first, except that, instead of the stabilizer of formulation B, the stabilizer of formulation A is used. The storage stability of the vaccines is described in the following table: Titfers"' of Liquid Vaccines Stored at 370C Stabilizer of Stabilizer of Time Formulation B Formulation A 0 2.9 2.7 24 Hours 1.6 2.1 48 Hours 1.2 1.8 72 Hours 0.6 1.4 "'Titers are expressed as TCID5J0.1 ml.

EXAMPLE 3 80 ml of measles viral concentrate which has been stored at -700C is thawed in a water bath at 200C and then kept at 4--80C. The liquid viral concentrate is split into two aliquot portions of 40 ml each.

a) One aliquot portion from this virus fluid is diluted in 210 ml of the previously described sterile stabilizer of formulation B. Formulation is carried on under aseptic conditions and laminar flow hood. To prevent microbial growth 10.5 mg Neomycin is added to the preparation. The diluted vaccine is dispensed into 3 ml glass vials (0.7 vaccine per vial) which are lyophilized, stoppered and stored at 370C.

b) The second viral aliquot portion is handled as the first one, except that, instead of the stabilizer of formulation B, a standard commercial diluent (Medium 199 containing SPGA) is used.

The storage stability of these vaccines is described in the following table: Titersfl) of Lyophilized Vaccine Stored at 370C Stabilizer of SPGA Time Formulation B Stabilizer 0 3.5 3.5 7 Days 3.6 0.6 "' Titers are expressed as log TCID5j0.l ml.

EXAMPLE 4 Lyophilized vials prepared as in Example 3 are reconstituted in distilled water (0.7 ml per vial) and stored at 2-80C.

The storage stability of these vaccines is described in the following table: Titfers"' of Reconstituted Vaccine Stored at 2-80C Stabilizer SPGA Time Formulation B Stabilizer 0 3.70 3.43 4 days 3.17 2.20 1 week 3.23 2 8 weeks 3.03 Loss (logiweek) 0.030 0.649 "'Titers are expressed as log TCID5j0.l ml.

WHAT WE CLAIM IS:- 1. A vaccine in lyophilized or liquid form and comprising an inactivated or attenuated live virus and a stabilizer consisting essentially of from 2 to 5 parts partially hydrolysed gelatin having a molecular weight of about 3000, from 2 to 55 parts of a 6-carbon polyhydric alcohol, a cell culture medium in an amount such that the content of solids from the medium is 0.5 to 1.7 parts, and sufficient physiologically acceptable buffer to adjust the pH to from 6.0 to 6.5, all parts being by weight.

2. A vaccine according to Claim 1, in which the 6-carbon polyhydric alcohol is sorbitol.

3. A vaccine according to Claim 1 or 2, in which the buffer is phosphate buffer.

4. A vaccine according to Claim 1, 2 or 3, in which the virus is measles, mumps, rubella, varicella, polio, hepatitis, herpes simplex 1, herpes simplex 2, or a mixture of two or more thereof.

5. A vaccine according to Claim 1, in which the stabilizer consists essentially of about 3.6 parts of partially hydrolysed gelatin, about 3.6 parts of sorbitol, about 1.1 parts (solids content) of Medium 199 and an amount of phosphate buffer sufficient to adjust the pH to from 6.0 to 6.5, all parts being by weight.

6. A vaccine according to Claim 1, in which the stabilizer consists essentially of about 3.6 parts of partially hydrolyzed gelatin, about 53 parts of sorbitol, about 1.1 parts (solid content) of Medium 199 and sufficient phosphate buffer to adjust the pH to from 6.0 to 6.5, all parts being by weight.

7. A stabilized vaccine obtained by reconstituting a lyophilized vaccine according to Claim 1.

8. A stabilized vaccine obtained by thawing a frozen liquid vaccine according

Claims (1)

1. to Claim 1.

   9. A vaccine according to Claim 1, in which the amount of stabilizer is from 2 to 12 volumes per volume of vaccine.

   10. A method for stabilizing a vaccine comprising adding to the vaccine an amount of stabilizer as defined in Claim 1 effective to stabilize the vaccine.

   11. A vaccine as claimed in Claim 1 substantially as hereinbefore described in any one of the Examples.