IE46717B1 - Vaccine stabilizer - Google Patents

Description

This invention, relates to vaccines and their stabilization. The present invention provides a vaccine in lyophilised or liquid form and comprising an inactivated or attenuated live virus and a stabiliser 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.

Examples of suitable viruses are Herpes simplex 1 or 2, measles, mumps, r.ubella, varicella, polio, and hepatitis, and mixtures of any two or more of such viruses. Hydrolysed gelatin is used to provide a soluble, non-gelling proteinaceo15 us matrix with little or no pyrogenicity or antigenicity.

The partially hydrolysed gelatin has approximately the same amino acid composition as gelatin. 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 composi25 tion of hydrolysed gelatin is as follows : i ϋ i i ϊ Alanine 8.5 % Arginine 7.9 % Aspartic Acid 5.7 % Cystine 0.08% Glutamic Acid 9.5 % Glycine 22.8 % Histidine 0.77% Hydroxy Proline 13-14% Xsoleucine 1.3 % Leucine 2.9 % Lys ine 4.2 % Methionine 0.78% Phenyl Alanine 2.0 % Proline 13.a % Serine 3.3 % Threonine 1.9 % Tyrosine 0.40 °A Valine 2.4 % Partially hydrolyzed 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 employed, e.g. acid hydrolysis. A suitable hydrolyzed 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. 6'« 17 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., 21=I-8» 1950; Basal Medium Eagle, Eagle, Science, 122, 501-504, 1955; In Vitro, Vol. 6, No. 2, 1970; Dulbecoo’s Modified Eagle's Medium,' Dulbeooo et al., Virology, 8, 395, 1959; Smith et al. , J. Virol., 12, 185-195, 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 about the amounts indicated: Ingredient Partially hydrolysed gelatin Polyhydric alcohol Nutrient medium (solids) Physiologically acceptable buffer to adjust pH to 6.0 - 6.5 Parts by weight 2-5 - 55 0.5 - 1.7 quantity sufficient With a liquid vaccin 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 30 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: Partially hydrolysed gelatin Sorbitol Medium 199 (solids content) Sodium phosphate buffer, M, pH 6.0 Distilled water A 1 35.7 g. 35.7 g. 526 g. 35.7 g. 11.06g, 11.06g. 100 ml 100 ml to 1 liter to 1 liter In addition the stabilizer optionally but preferably contains small amounts of NaHCO and of phenol red. In the case of the foregoing formulation the NaHCO^ 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 tollowing examples illustrate the present invention and conventional vaccines for comparative purposes. example: 1 ml. of measles viral concentrate which has been » stored at -70° is thawed in a water bath at 25°C and then kept at 4-8°. The liquid viral concentrate is then split into two aliquot portions each at 40 ml. 4ό'«1 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 .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 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: ’ Titers of Liquid Vaccines Stored at 2-8°C Stabilizer of Time Formulation B 3.4 months 3.2 SPGA Stabilizer 3.6 0.6 (1)Titers are expressed as TCID /0.1 ml. i 20 EXAMPLE 2 ml. of measles viral concentrate which has been stored at -70° is thawed in a water bath at 25°C and then kept at 4-8°. 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 'ί ΰ , ΐ ϊ vaccine is dispensed into 2 ml glass ampoules (0.7 ml vaccine per ampoule) which are immediately flame sealed and stored at 37°C. b) The second aliquot portion is handled as the first, except that, instead of the stabilizer of formulation B,· the stabilizer of formulation k is used. The storage stability of the vaccines is described in the following table: ci C Titers of Liquid Vaccines Score! Time 0 Stabilizer of Formulation 3 Stabilizer of Formulation A i, · J 2.7 24 Hours 1.6 2.1 48 Hours 1.2 1.8 72 Hours 0.6 1.4 (1) Titers are expressed as TCIDgQ/0.1 ml.

EXAMPLE 3 ml. of measles viral concentrate which has been stored at -70°C is thawed in a water bath at 20°C. and then kept at 4-8°C. 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 Q stored at 37' c. - 8b) 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: Titers (1) of Lyophilized Vaccine Stored at 37°C Time Days Stabilizer of Formulation B 3.5 3.6 SPGA Stabilizer 3.5 0.6 (1) Titers are expressed as log TCID^^/O.1 ml.

EXAMPLE Lyophilized vials prepared as in Example 3 are reconstituted in distilled water (0.7 ml per vial) and stored at 2-8°C.

The storage stability of these vaccines is described in the following table: Titers of Reconstituted Vaccine Stored at 2-8°C Time Stabilizer of Formulation B SPGA Stabilizer 0 3.70 3.43 4 days 3.17 2.20 1 week 3.23 2 8 weeks 3.03 - Loss (log/week) 0.030 0.649 (33 Titers are expressed as log ΤΟΙϋ^θ/Ο.Ι ml.

Claims (11)

1. CLAIMS :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, a^l parts being by weight.

6. A vaccine according to Claim 1, in which the stabilizer consists essentially 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 to from 6.0 to 6.5, all parts being by weight. - 10 * 1

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 to Claim 1. 5

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

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. 10

11. A vaccine as claimed in Claim 1 substantially as hereinbefore described in any one of the Examples. F. R. KELLY & CO. AGENTS FOR THE APPLICANTS.