DK150814B - PROCEDURE FOR STABILIZING A VACCINE CONTAINING A LIVE VIRUS AND STABILIZER FOR USING THE PROCEDURE - Google Patents

Description

150814

The present invention relates to a method for stabilizing a vaccine as set forth in the preamble of claim 1.

The process according to the invention is characterized by the characterizing part of claim 1.

As mentioned, the process according to the invention uses sorbitol, mannitol or dulcitol. It is preferred to use sorbitol.

The partially hydrolyzed gelatin having a molecular weight of approx. 3000 has about the same amino acid composition as gelatin. Unlike gelatine, which forms gels but is insoluble in cold water, hydrolyzed gelatine does not form gels but is soluble in cold water and in other ordinary liquids such as milk and orange juice.

Aqueous solutions containing up to approx. 10% hydrolyzed gelatin does not significantly increase their viscosity.

Above this concentration, the viscosity slowly increases.

At about. At 50% concentration, the solution is quite viscous.

The following is the typical amino acid composition of hydrolyzed gelatin:

Alanine 8.5%

Arginine 7.9%

Aspartic acid 5.7%

Cystine 0.08%

Glutamic Acid 9.5%

Glycine 22.8%

Histidine 0.77%

Hydroxyproline 13-14%

Isoleucine 1.3%

Leucine 2.9%

Lysine 4.2%

Methionine 0.78¾

Phenylalanine 2.0¾

Proline 13.8¾

Serine 3.3¾

Threonine 1.9¾

Tyrosine 0.40¾

Selected 2.4¾

Partially hydrolyzed gelatin can be prepared by enzymatic hydrolysis of gelatin by a proteolytic enzyme such as, for example, papain, chymopapain and bromelin, although other known methods of hydrolysis may be used, for example, acid hydrolysis. Appropriate hydrolyzed gelatin is available from Wilson and Co., Inc. of Calumet City,

Illinois under the trade mark S0L-U-PRO0.

The acidic buffer may be any physiologically acceptable buffer which will maintain the desired pH value of approx. 6 to approx. 6.5, e.g., a phosphate buffer, acetate buffer or citrate buffer. Preferably a phosphate buffer.

For lyophilized vaccines, the weight ratio of gelatin, sorbitol to cell culture substrate, such as medium 199, relative to the weight of the sodium phosphate buffer, is from ca. 3 to approx. 8, preferably approx. 5.5.

A cell culture substrate is meant a nutrient substrate that allows growth of cells in vitro. Particular nutrient substrates are, for example, Medium 199, Morgan et al., Proc.

Soc. Exp. Biol. & Med., 7_3: 1-8. 1950; Basal Medium Eagle,

Eagle, Science, 122, 501-504, 1955; In Vitro, Vol. 6, 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, Volume 6, No. 2. 1970; Minimum Essential Medium (Eagle), Science, 130, 432 (1959) and RPMI Media, Moore et al., 199, 519-524, 1967; In Vitro, Volume 6, No. 2, 1970.

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Medium 199 has the following composition:

Catalog No. 12-109 with NaHCO ^

Catalog No. 12-110 without NaHCO ^

Catalog No. 12-119 (Earle's BSS) with NaHCO3

Amino Acids: mq / liter L-Alanine 25.0 L-Arginine HCl 70.0 L-Aspartic acid 30.0 L-Cysteine HCl 0.1 L-Cystine 20.0 L-Glutamic acid 67.0 'L-Glutamine 100 , 0 L-Glycine 50.0 L-Histidine-HCl-H2 O 22.0

Hydroxy-L-proline 10.0 L-Isoleucine 20.0 L-Leucine 60.0 L-Lysine-HCl 70.0 L-Methionine 15.0 L-Phenylalanine 25.0 L-Proline 40.0 L-Serine 25 0 L-Threonine 30.0 L-Tryptophan 10.0 L-Tyrosine 40.0 L-Valine 25.0

Vitamins mq / liter p-Aminobenzoic acid 0.050

Ascorbic Acid 0.050 D-Biotin 0.010 4 150814 (continued)

Vitamins mq / liter

Calciferol 0.100 D-Ca pantothenate 0.010

Cholesterol 0.200

Choline chloride 0.500 Γ oleic acid 0.010 i-Inositol 0.050

Menadione 0.010

Nicotinamide 0.025

Nicotinic acid 0.025

Pyridoxal-HCl 0.025

Pyridoxine HCl 0.025

Riboflavin 0.010

Thiamine HCl 0.010 DL-ce-Tocopherol Phosphate (Na2) 0.010

Tween 80 5,000

Vitamin A acetate 0.140

Other mq / liter

Adenine HCl-2H 2 O 12.10

Adenosine 5'-Monophosphoric Acid, Dihydrate (AMP) (Muscle Adenyl Acid) 0.20

Adenosine 5'-triphosphate, disodium, tetrahydrate (ATP) 1.08

Deoxyribose 0.50

Dextrose 1000.00

Glutathione (reduced) 0.05

Guanine HCl-H 2 O 0.33

Hypoxanthine 0.30

Ribose 0.50 5 150814 (continued)

Other mg / liter

Sodium acetate-3H 2 O 83.00

Thymin 0.30

Uracil 0.30

Xanthine 0.34

Inorganic salts (12-119) (12-109)

CaCl2-2H2O 265.0 186.0

Fe (NO3) 3-9H20 0.7 0.7 KC1 400.0 400.0 KH2 PO4 --- 60.0

MgSO4-7H2O 200.0 200.0

NaCl 6800.0 8000.0

NaHCO 3 2200.0 1400.0

Na2HPO4-7H2O - 90.0

NaH 2 PO 4 -H 2 O 140.0

Phenol red 10.0 20.0

A particularly preferred embodiment of the method according to the invention is characterized in that the stabilizer used consists mainly of approx. 3.6 parts by weight of partially hydrolyzed gelatin, approx. 3.6 parts by weight of sorbitol, approx. 1.1 parts by weight of "Medium 199" and such an amount of phosphate buffer sufficient to adjust the pH to approx. 6.0 to approx. 6.5.

A particularly preferred embodiment of the method according to the invention is characterized in that the stabilizer used consists mainly of approx. 3.6 parts by weight of partially hydrolyzed gelatin, approx. 53 parts by weight of sorbitol, approx. 1.1 parts by weight of "Medium 199" and such an amount of phosphate buffer sufficient to adjust the pH to approx. 6.0 to approx. 6.5.

A particularly preferred embodiment of the method of claim 1 is peculiar to the fact that about 2 to approx. 12 volume stabilizer per volume of vaccine.

The invention further relates to a stabilizer for use in the practice of the invention of the kind set forth in the preamble of claim 7. The stabilizer according to the invention is characterized by the characterizing part of claim 7.

A stabilizer composition according to the invention may contain the following ingredients in the following approximate amounts:

Ingredient Weight Parts

Partially hydrolyzed gelatin 2-5

Sorbitol, mannitol or dulcitol 2-55 Nutrient Substrate 0.5 - 1.7 Physiologically Acceptable Buffer for Adjusting pH to 6.0 - 6.5 Sufficient Amount In the case of a liquid vaccine, sorbitol is usually present in an amount towards the upper part of the range, whereas in the case of a freeze-dried vaccine, sorbitol is usually present in an amount towards the lower part of the range.

The detailed composition of the virus vaccine stabilizer according to the invention is set out below. Formulation B is preferred for a freeze-dried vaccine.

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A B

Partially hydrolyzed gelatin 35.7 g 35.7 g

Sorbitol 526 g 35.7 g

Medium 199 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

The stabilizer may optionally, but preferably, contain a small amount of NaHCO 3 and phenolic red. In the case of the above formulations, NaHCO 3 may be present in an amount of about 1.2 g and phenol red in an amount of approx. 0.01 g. While detailing the above formulations, it is to be understood that the variation in the amount ratio and concentration of each component may be conceivable.

A volume of vaccine in bulk is usually diluted by approx. 2 to approx. 12 volume stabilizer.

U.S. Patent No. 2,879,202 discloses the stabilization of live viral vaccines by the addition of an alcohol of 6 hydroxyl groups to the vaccine. However, nothing herein anticipates the stabilizer described above and its use. The advantage of the invention is that an improved stabilizer is provided which retains the vaccine titer for longer than has hitherto been possible with the aid of state-of-the-art stabilizers. The examples of the present invention, for example, show that the vaccine titer was substantially unchanged after storage for 4 months for liquid vaccines (Example 1 below) and after storage for 1 week at 27 ° C in the case of lyophilized vaccine (Example 3 ). In each case, the titer was essentially unchanged at the end of storage time. In contrast, the titers dropped substantially at the end of the storage period in the case of a stabilizer known from the above-mentioned U.S. patent.

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The following examples illustrate the present invention.

EXAMPLE 1 80 ml of measles virus concentrate, which has been stored at -70 ° C, is thawed in a water bath at 25 ° C and then kept at 4-8 ° C. The liquid virus concentrate is then divided into two equal portions.

a) Dilute one portion of this viral fluid with 210 ml of the previously described sterile stabilizer of formulation B. The mixing is carried out under aseptic conditions and in sterile working area of the so-called clean-bench type. To prevent microbial growth, add 10.5 neomycin to the mixture. The diluted vaccine is transferred to 2 ml vials (0.7 ml vaccine per vial), which are immediately sealed after flame and stored at 4-8 ° C.

b) the second portion is treated like the first, except that a standard, commercially available vaccine diluent (SPGA containing sucrose, phosphate, glutamine and albumin) is used in place of Formula B. The shelf life of the vaccines is described in the following table: 9 150814

Time Titer ^^ for liquid vaccines stored at 2-8 ° C

Stabilizer according to SPGA

formulation B_ Stabilizer 0 3.4 3.6 4 months 3.2 0.6

Titers are expressed as TCID µg / 0.1 ml.

Example 2 32 ml of measles virus concentrate stored at -70 ° C is thawed in a water bath at 25 ° C and then kept at 4-8 ° C. The liquid virus concentrate is then divided into two equal portions.

a) Dilute one portion of this viral fluid with 48 ml of the previously described sterile stabilizer of formulation B. The mixing is carried out under aseptic conditions in sterile clean-bench type work area. To prevent microbial growth, add 2.5 mg of neomycin to the mixture. The diluted vaccine is transferred to 2 ml vials (0.7 ml vaccine per vial) which are immediately sealed and stored at 37 ° C after flame.

b) the second portion is treated like the first except that instead of the stabilizer of Formulation B, the stabilizer of Formulation A. is used. The shelf life of the vaccines is described in the following table:

Time Titer ^ · * · ^ for liquid vaccines stored at 37 ° C_

Stabilizer with Stabilizer formulation B of formula

Teaching A

0 2.9 2.7 24 hours 1.6 2.1 48 hours 1.2 1.8 72 hours 0.6 1.4 ^^ Titer is expressed as TCID ^ g / 0.1 ml.

EXAMPLE 3 80 ml of measles virus 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 virus concentrate is divided into two equal portions.

a) one portion of this viral fluid is diluted in 210 ml of the previously described sterile stabilizer of formulation B. The mixing is carried out under aseptic conditions in sterile clean-bench type work area. To prevent microbial growth, add 10.5 mg of neomycin to the mixture. The diluted vaccine is transferred to 3 ml glass containers (0.7 ml vaccine per container) which are then freeze-dried, capped and stored at 37 ° C.

b) the second portion of viral fluid is treated as the first except that a standardized, commercially available diluent (Medium 199 containing SPGA) is used in place of Formula B.

150814 π

The storage stability of these vaccines is described in the following table:

Time Titer of freeze-dried vaccine stored at 37 ° C

Stabilizer with SPGA

formulation B Stabilizer 0 3.5 3.5 7 days 3.6 0.6

Titers are expressed as log TCID µg / 0.1 ml.

EXAMPLE 4

Freeze-dried vaccine as prepared in Example 3 is reconstituted with distilled water (0.7 ml per container) and stored at 2-8 ° C.

The shelf life of these vaccines is described in the following table: (1)

Time Titer reconstituted vaccine stored at 2 - 8 ° C_

Stabilizer with SPGA

Formulation B Stabilizer 0 3.70 3.43 4 days 3.17 2.20 1 week 3.23 2 8 weeks 3.03 loss (I0g / week) 0.030 0.649

Titers are expressed as log TCID µg / 0.1 ml.

Claims (5)

150814 A method of stabilizing a vaccine containing a live virus, especially a measles, mumps, rubella, chickenpox, polio or hepatitis, herpes simplex 1, herpes simplex 2 virus or combinations thereof, wherein a stabilizer containing sorbitol, mannitol or dulcitol is added to the vaccine, characterized in that the amount of sorbitol, mannitol or dulcitol is approx. 2 to approx. 55 parts by weight, and that the stabilizer further mainly consists of approx. 2 to approx. 5 parts by weight of partially hydrolyzed gelatin with a molecular weight of approx. 3000, ca. 0.5 to approx. 1.7 parts by weight of cell culture substrate and a physiologically acceptable buffer in an amount such that the pH is adjusted to approx. 6.0 to approx. 6.5. Process according to claim 1, characterized in that the stabilizer contains sorbitol. Process according to claim 1, characterized in that the buffer used is a phosphate buffer. Method according to claim 1, characterized in that the stabilizer used consists mainly of approx. 3.6 parts by weight of partially hydrolyzed gelatin, approx. 3.6 parts by weight of sorbitol, approx. 1.1 parts by weight of the "Medium 199" defined in the specification as cell culture substrate constituent and such amount of phosphate buffer sufficient to adjust the pH to ca. 6.0 to approx. 6.5. Method according to claim 1, characterized in that the stabilizer used mainly consists