CS276109B6 - A method of stabilizing a lyophilized vaccine from a Propionibacterium acnes 26/78 strain - Google Patents

Abstract

The method of stabilizing the lyophilized vaccine Propionibacterium acnes 26/78 consists in homogenizing a suspension of bacterin Propionibacterium acnes 26/78 with an antiseptic, glycine and human albumin, wherein the weight ratio of the dry matter of the suspension of bacterin and human albumin is 1: 2.85 to 5 and the weight ratio of glycine and human albumin is 1: 2 to 4 and the resulting mixture is supplemented with water for injection in a weight ratio to the dry matter of bacterin of 55 to 60: 1, after which this mixture is subjected to lyophilization.

Description

The invention relates to a method for stabilizing a lyophilized vaccine from the strain Propionibacterium acnes 26/78. ,.

Stabilizers of various compositions are used in the preparation of lyophilized vaccines. These are usually certain sugars, such as sucrose, and gelatin or bovine albumin. The amount of stabilizing agent added varies. Many different substances are often used to achieve the desired stabilizing effect. Their ratio is determined empirically individually for each different drug.

The lyophilised Propionibacterium acnes vaccine must contain, in addition to the active substance and preservative, a suitable stabilizer to ensure the good appearance of the final product, its good solubility and its long-term stability. Various stabilizers and mixtures thereof have been tested for the newly developed Propionibacterium acnes 26/78 vaccine. The results were variable. The biggest drawback was the poor solubility of the product and its short-term stability. A method for stabilizing a similar vaccine by adding glycine in a concentration of 2.3 .mu.m is known from the company literature. However, this method of stabilization results in imperfect solubility of the lyophilizate and a decrease in the stability of the formulation at room temperature.

These shortcomings are overcome by the method for stabilizing the lyophilized vaccine Propionibacterium acnes 26/78 according to the invention. Its essence lies in the fact that the culture after fermentation and thermal inactivation, the bacterium Propionibacterium acnes 26/78, is homogenized with antiseptic, glycine and human albumin, the weight ratio of dry matter of bacterin and human albumin being 1: 2.85 to 5 and the weight ratio glycine and human albumin is 1: 2 to 4 and the resulting mixture is supplemented with water for injection in a weight ratio of bacterin dry matter of 55 to 60: 1. Sodium merthiolate is suitable as antiseptic. After adding the antiseptic and stabilizing additives to the bacterin suspension, the mixture is subjected to lyophilization.

These stabilizing additives added in a specified ratio protect the individual lyophilized cells from sticking to each other. By using human albumin, complete solubility of the lyophilisate and long-term stability of the preparation at room temperature are achieved. This allows the product to be stored outside the refrigerator without the risk of losing its effect. The use of human albumin does not present a risk of sensitization to treated subjects. .

The lyophilized vaccine Propionibacterium acnes 26/78 stabilized by the method according to the invention is characterized by the following properties:

- solubility within 1 minute after the addition of saline

- the dissolved vaccine is completely homogeneous even under microscopic examination; the microscopic appearance of the dissolved vaccine is identical to that of the same vaccine before lyophilization

- the biological activity of the vaccine is at a very good level

- all the above properties do not change even after 20 months of storage of the lyophilised vaccine at 37 ° C.

The method of stabilizing a lyophilized vaccine from Propionibacterium acnes 26/78 is further illustrated by the following examples.

Example 1

The cultivation of Propionibacterium acnes 26/78 was terminated after reaching the stationary phase of the growth curve, then the temperature of the fermentation vessel was raised to 60 ° C sg for 1 h and subsequently cooled to room temperature. The obtained bacterium was centrifuged, washed with distilled water and resuspended in distilled water.

The method for stabilizing the 1st active bacterin prepared from Propionibacterium acnes strain 26/78 was as follows. Amount of individual components of the mixture before lyophilization: bacteria 7 mg dry weight, sodium merthiolate 0.04 mg, glycine 9.2 mg. The mixture was made up to 400 mg with water for injections. Sodium merthiolate is added as an antiseptic. The mixture was lyophilized to a residual moisture of 2.1% by weight, sealed under nitrogen and stored at 37 ° C.

CS 276109 B 6 2

The quality of the stabilized lyophilized vaccine from Propionibacterium acnes 26/78 was monitored over a period of 12 months by evaluating the solubility of the lyophilisate in seconds (R), the maximum number of bacteria in the microagglutinates (A) and the relative immunostimulatory activity in ¾ (I) relative to baseline ( M = 0).

Evaluation of the quality of a vaccine stabilized by method 1.

M 0 1 3 6 12 R 12 30 30 30 35 AND 20 50 100 100 100 AND 100 101 96 80 84 where M is the length storage in months.

Example 2

The cultivation was carried out as in Example 1.

Stabilization was performed according to the method of the invention. The stabilization method before lyophilization 2. consisted in homogenizing the active bacterin prepared from Propionibacterium acnes 26/78 in an amount of 7 mg dry weight, sodium merthiolate as an antiseptic additive in an amount of 0.04 mg, 9.2 mg glycine and 25 mg human albumin. The other conditions for lyophilization, storage and evaluation of the stabilized vaccine were identical to Example 1.

Evaluation of vaccine quality stabilized by method 2.

M 0 1 3 6 12 R 30 25 30 30 30 AND 5 5 5 5 15 AND 100 105 104 99 101

where M is the length of storage in months

R - solubility time of the lyophilisate in seconds

A - maximum number of bacteria in microagglutinates

I - relative immunostimulatory activity in% relative to the initial value (M = 0)

Example 3

The procedure was the same as in Example 2. The stabilization was carried out according to Method 3 according to the invention, except that the mixture contained 35 mg of human albumin before lyophilization. The other conditions were identical to Example 2.

Evaluation of vaccine quality stabilized by method 3.

M 0 1 3 6 12 R 40 35 40 40 35 AND 5 5 5 ⁵ . 15 AND 100 97 102 106 103 where M is the storage length in i months

R - solubility time of the lyophilisate in seconds

A - maximum number of bacteria in microagglutinates I - relative immunostimulatory activity in ¾ in relation to the initial value (M = 0)

CS 276109 B 6

Example 4

The procedure was the same as in Example 1. The stabilization method 4 consisted in homogenizing a mixture consisting of 7 mg of dry weight of active bacterin prepared from strain Propionibacterium acnes 26/78, sodium merthiolate as an antiseptic additive in an amount of

0.04 mg and 35 mg of human albumin. The other conditions were the same. Evaluation of vaccine quality stabilized by method 4. M 0 1 3 6 12 R 120 140 180 180 180 A 5 10 5 10 20 I 100 89 103 93 92 wherein M, R, A and I are as defined in the previous examples. The evaluation shows that the method of stabilization 1, where the homogenised mixture before lyophilisation

Although it did not contain human albumin, good solubility resulted in storage problems where immunostimulatory activity decreased. By the stabilization method 4, where the homogenized mixture does not contain glycine, a vaccine is obtained, the quality of which does not change significantly with storage, but the lyophilisate is very poorly soluble. These properties are reconciled by the method for stabilizing the lyophilized vaccine 2, 3 according to the invention.

The method for stabilizing a lyophilized Propionibacterium acnes 26/78 vaccine according to the invention is intended for the preparation of a medicament produced from a Propionibacterium acnes strain. However, it can also be used for the preparation of similar corynebacterial vaccines from other strains.

Claims (1)

PATENT CLAIMS Process for stabilizing a lyophilized vaccine of Propionibacterium acnes 26/78 by adding an antiseptic and glycine to a bacterin, characterized in that the bacterium suspension of Propionibacterium acnes 26/78 is homogenized with an antiseptic, glycine and human albumin, the dry weight ratio of the bacterin to human albumin suspension being 1: 2.85 to 5 and the weight ratio of glycine to human albumin is 1: 2 to 4, and the resulting mixture is supplemented with water for injection in a weight ratio of 55 to 60: 1 dry matter of bacterin, followed by lyophilization. '