FI57534B - FOERFARANDE FOER FOERBAETTRANDET AV STABILITENEN VID RUMSTEMPERATUR HOS ETT ORALT POLIOVIRUSVACCIN AV TYPEN 1,2 ELLER 3 - Google Patents

Description

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Patant- och rcgicterstyralaan An * ek »n utiigd och uti. * KrHt * n pubiic« nd 30.05.80 (32) (33) (31) Privilege requested —Begird prlorltet 29.11.76

Canada (CA) 266768 (71) Connaught Laboratories Limited, 1755 Steeles Avenue West, Willovdale,

Ontario, Canada (CA) (72) Adolf von Seefred, Sharon, Ontario, Joong Hwan Chuun, Mississauga,

Ontario, Hildegarde MG Macmorine, Thornhill, Ontario, Canada (CA) (7 * 0 Oy Kolster Ab (5M Method for improving the shelf life of an oral poliovirus type 1, 2 or 3 vaccine at room temperature - Förfarande för förbättrandet av stabeten vid rumstemperatur hos ett oralt nitrogen 1, 2 or 3

The invention relates to a method for improving the shelf life of an oral poliovirus type 1, 2 or 3 vaccine at room temperature.

Protection of humans and animals against viral diseases is often achieved by vaccination with inactivated antigens or live, attenuated virus strains. In both cases, viral antigens must be preserved intact and therefore preservation methods must be used. However, when live, attenuated virus strains are used, the effectiveness of vaccines depends solely on whether the viruses remain alive during manufacture, transport, and storage.

In order to obtain adequate stability, some vaccines must be supplied frozen or lyophilized or mixed with substances that reduce or eliminate the instability of the vaccine virus. Such additives include calcium lactobionate disclosed in U.S. Patent 3,186,908, phosphate buffers disclosed in Canadian Patent No. 952 U29, and MgCl 2 disclosed in U.S. Patent No. 3,128,229. Carbohydrates such as sorbitol, monnitol, etc. are sometimes used, especially as cryoprotectants in freezing or lyophilization. Sucrose or magnesium 2,57534 chloride have also been used as stabilizers, especially in Poliovirus Vaccine, Live, Oral (Sabin). However, the use of these substances has certain disadvantages. The high viscosity of sucrose solutions makes the product difficult to handle and administer. Magnesium chloride has an unpleasant taste and is difficult to give to children.

It is also known that L-cystine has a stabilizing effect on heat inactivation of polioviruses. Pohjanpelto, Virology J_5, 225-230 (I960) have shown that the rate of action of cystine, i.e. the time required for cystine to join a virus to be protected against heat inactivation, varies with different enterovirus types and strains. Ikegami et al.

J. M. Sc. & Biol. j6, 325-3 ^ 2, 1963, Jap. J. M. Sc. & Biol. 17, 13-22, 196U found that this phenomenon was a genetically permanent characteristic and that the stabilizing effect of cystine was dependent on the structure of the viral protein. The purpose of these studies was not to develop a stabilizer for these viruses, but to study genetic markers. The articles mention the use of L-cystine dissolved in 1N hydrochloric acid. Ikegami (1963) (196Π) prepared his stock solution of cystine using 0.05 m Tris buffer as dilution. This buffer is commonly used in biochemistry as a diluent. However, it has been shown that such a low concentration of tris buffer does not have a sufficient stabilizing effect on polioviruses. The bottom field, Virology 15, 225-230, 1961, neutralizes a stock solution of L-cystine with sodium hydroxide. However, sodium ions have been shown to have an adverse effect on the persistence of polioviruses at or below 25 ° C.

Given that current poliovirus vaccines lose half of their potency at 25 ° C in as little as seven days, it is clear that a longer shelf life for these vaccines is highly desirable, especially in countries where health regulations have a product with a titer below 50 #: n must be withdrawn from the market.

According to the invention, it has thus been found that it is particularly advantageous to add to a test sample at room temperature for the presence of possible polioviruses as a stabilizing or preservative an aqueous medium consisting of 0.3-1.0 m tris (hydroxymethyl) aminomethane and containing 100 pg / ml. L-cystine.

It has also been found that an aqueous stabilizing composition consisting of 0.3-1.0 m tris (hydroxymethyl) aminomethane and containing about 100 pg / ml L-cystine is particularly suitable for stabilizing oral poliovirus vaccines for up to four weeks at room temperature, i.e. 25 ° C. , thus avoiding using the entire vaccine bottle at the same time as it has warmed to room temperature or discarding the remainder when only part of the vaccine has been used.

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According to the invention, it has been found that the stability of a poliovirus vaccine when stored at -70 to UC can be surprisingly improved by suspending the vaccine in an aqueous stabilizing medium having a pH of 6.5-8.5, preferably 7 → 0 ~ 7.5 and prepared at about 0 ° C. , 3-1.0 -ra tris (hydroxymethyl) aminomethane solution and contains about 100 μg / ml L-cystine and about 0.8 mg / ml acid hydrolyzed gelatin.

The unexpected improved stability is due to the fact that, contrary to previous methods, L-cystine is not dissolved by hydrochloric acid and subsequent neutralization with NaOH, as such a method has been found to cause significant losses in activity, especially when stored at 25 ° C and below. L-cystine must therefore be dissolved in distilled water by heating gently under pressure to reach a temperature of 105 ° C. After the L-cystine is dissolved, the solution is cooled to 60 ° C and the Tris buffer is added as a powder and the acid-hydrolyzed gelatin as a 10-S solution. The solution is cooled and sterilized by filtration through a 0.22 μβ membrane filter.

It is known that L-cystine is practically insoluble in water, but quite soluble in aqueous solutions below pH 2 or in temporal solutions above pH 8. However, these pH levels are not suitable for virus assay samples or poliovirus vaccines. The stabilizer is therefore prepared as described above and the pH is adjusted with an organic, non-toxic buffer such as tris buffer. The samples or vaccines can then be stored at room temperature, if necessary. In the period every Z] days. virus loss is up to 50%. On the other hand, if it is desired to store the vaccine for a longer period of time at a temperature of -70 ° to about 4 ° C, acid-hydrolyzed gelatin is added to the suspension medium containing L-cystine and tri (hydroxymethyl) aminomethane.

The stabilizing and suspending effect of the new medium according to the invention is obtained at a concentration of about 100 pg / ml of L-cystine and about 0.8 mg / ml of acid-hydrolyzed gelatin and 0.3 ~ 1.0-m tri (hydroxymethyl) aminomethane. The pH of the stabilizing suspension medium is adjusted to 6.5-8.5, preferably 7.0-7.5.

Examples of poliovirus vaccines that can be stabilized by the method of the present invention include live attenuated Sabin poliovirus vaccines prepared from the following strains: Type 1 = (LSc, 2ab.), QVype 2 = (P712, CH, 2ab.) And Type 3 = (Leon a ^ b). Cell cultures and virus vaccine fluids are prepared by methods known in the art, of which the following is a brief description: Monkey erythrocyte or human diploid cell cultures (WI-30) are grown in CMRL-1990 cell culture medium (Connaught Medical Research Laboratories) li 57534 supplemented with bovine serum. The culture medium is removed at its best growth (single cell layer) and replaced with a virus inoculum and Earle's lactalbumin hydrolyzate medium, which is added to maintain the cell culture. Infected cell cultures are incubated until about 50 3 * of the cells show the cytopathic effect of the viruses. The suspension is then filled into glass bottles and stored. Care is taken at all stages of the vaccine's preparation to ensure that no harmful or toxic substances enter the vaccine. The safety, potency, and efficacy of viral fluids are tested at various stages of manufacture.

The invention is described in more detail in the following examples.

Example 1

Sabin poliovirus fluids were diluted 1:10 with 1 M Tris buffer, and the effect of normal addition of L-cystine on stability was determined by adding 100 pg / ml with gentle agitation. The virus content of the different suspensions of each of the three polio virus types was determined at the beginning of the experiment and then every four weeks when the suspensions were stored at 25 ° C. The results show that the number of live viruses of each of the three Sabin polioviruses was improved by the addition of L-cystine. In a similar experiment, it was shown that L-cystine similarly improved stability, but this compound has an unpleasant odor and is thus not suitable as an additive to an oral vaccine. The results are shown in Table I.

Table I

Stabilizing effect of cystine on Sabin polioviruses when stored at 25 ° C

Poliovirus type L-cystine Live viruses Arena at 25 ° C Half-life ^ added '(weeks) (days)

0 2 U

1 - 100 19 5 10 + 100 59 25 17 2 - 100 20 5 10 + 100 55 17 16 3 - 100 U5 6 11 + 100 72 16 19 1) 100 pg / ml L-cystine 2) Diluent 0.1 m tris buffer 3) Number of days for 50-Ji inactivation of viruses

Example 2 5 57534

Sabin poliovirus (type 1) fluid was suspended in 1 μm Tris buffer with 100 pg / ml L-cystine. The pH was adjusted to 6.4, 7.4, and 8.5, and the persistence of the viruses was determined at these three pH levels at 4 ° C for four months. The experiment confirmed previous results that pH does not affect the inactivation of Sabin polioviruses in the pH range of 6.8 to 8.5. The results are shown in Table II.

Table II

Effect of pH on the stability of Sabin poliovirus type 1 when stored at 4 ° C using 1-m Tris buffer + 100 pg / ml L-cystine for stabilization

Suspension Live viruses in% at 4 ° C (months) pH 0124 6.4 1 00 79 74 47 7.4 100 89 - 66 8.5 100 83 74 25

Example 3

Three types of poliovirus were subjected to a four-week storage experiment at 25 ° C with the viruses suspended in different concentrations of Tris-buffer solutions supplemented with 100 pg / ml L-cystine. It turned out that the concentration of tris buffer was not high enough, but sufficient virus stability was obtained with concentrations of tris buffer of 0.3 to 1.0 m. Because the 1-m Tris buffer is somewhat bitter-tasting, a lower concentration was chosen as the standard for the stabilizer. However, it may be advantageous in some circumstances to use a higher concentration of tris buffer. The results are shown in Table III.

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Table III

Effect of different concentrations of Tris buffer + 100 pg / ml L-cystine on the stability of Sabin polioviruses when stored at 25 ° C

Poliovirus Tris buffer Live viruses in% at 25 ° C (weeks) type molarity 02 4 0.1 100 40 3 1 0.3 100 32 16 1.0 100 40 23 0.1 100 36 6 2 0.3 100 58 45 1.0 100 40 32 0.1 1 00 70 7 3 0.3 100 100 56 1, 0 1 00 79 50

Example 4

Each of the three oral Sabin virus types was suspended in stabilization medium prepared from 0.3 m Tris buffer containing 100 pg / ml L-cystine. To half the amount of each preparation was added about 8 mg / ml of acid hydrolyzed gelatin. Both batches were stored at -20 ° C and virus persistence was determined after four and six months. The results are shown in Table IV. From them, it can be seen that after six months of storage, the ratio of live viruses in vaccines containing tris buffer, L-cystine and acid-hydrolyzed gelatin was 100%.

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Table IV

Effect of gelatin addition on the persistence of Sabin polioviruses in tris buffer-cystine medium when stored at -20 ° C

Poliovirus- 0,08% ge- Live viruses in% at -20 ° C (monthly type Latin 4 4) u 4 6 _added _______ - 100 56 μg 1 + Ί00 - 100 1 00 56 ~ 7ä 2 + 100 - 100 100 40 40 3 + 100 - 100 1) Diluent: 0.3 m Tris buffer +100 pg / ml L-cystine Example 5

Each of the three oral Sabin poliovirus types was suspended in a stabilization medium prepared from 0.3 μm Tris buffer containing 100 μg / ml L-cystine and 0.4 mg / ml acid hydrolyzed gelatin. Similarly, three oral Sabin poliovirus types were suspended in distilled water. Both batches were stored at 25 ° C and shelf life tests were performed once a week for four weeks. The results are shown in Table V and show that the stability was clearly improved with the use of the stabilizing medium according to the invention.

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Table V

Comparative test on the effect of stabilizer and distilled water on the number of live polioviruses when stored at 25 ° C

Poliovi- Diluent Live viruses in% at 25 ° C rust type- ___) 0__ 3 • 0 12 3 4

Ho0 100 71 24 17 5

1 L

Stabilizer 100 69 47 20 13 H90 100 54 37 1 3 10 2 1

Stabilizer 100 72 72 36 23

Ho0 100 74 30 22 7 3 1

Stabilizer 100 100 69 32 44

Note: 1) Stabilizer = 100 pg / ml L-cystine, 0.08% gelatin, 0.3 m Tris buffer, pH 7.4

Example 6

Each of the three oral Sabin poliovirus types was suspended in a stabilization medium prepared from 0.3 μm Tris buffer containing 100 pg / ml L-cystine and 0.8 μg / ml acid hydrolyzed gelatin. Stabilized poliovirus vaccines were stored at 4 ° C, and stability was determined for each sample once a month for six months. The results are shown in Table VI and show that the loss was less than 50%.

Table VI

Effect of stabilizer on the amount of live Sabin polioviruses when stored at 4 ° C

Poliovirus- Live viruses in% at 4 ° C (months) type__0__1__2__4__6_ 1 1 00 1 00 1 00 74 53 2 1 00 1 00 1 00 83 74 3 100 100 - 100 50

Example 7 9 57534

Several batches of oral poliovirus vaccine were prepared according to Example 6. These vaccines were subjected to a series of five and ten freeze-thaw cycles, and titers were then determined to show that the vaccines had not lost any efficacy. The results are shown in Table VII.

Table VII

Effect of stabilizer on the number of live polioviruses after freeze-thaw treatments

Poliovirus _Live viruses,% _ type__0__5__10 cycles 1 100 100 100 2 100 100 100 3 100 100 100

1) Number of freeze-thaw cycles Freezing: dry ice + acetone Defrost: water bath, 25 ° C

Example 8

One batch of Sabin poliovirus vaccine (type 1) was stabilized with medium containing 100 pg / ml L-cystine dissolved in the required amount of NaOH and HCl. In another batch, L-cystine (100 pg / ml) was dissolved in boiling water. Both batches were stored at 25 ° C and their titers were determined after two and four weeks. The results are shown in Table VIII.

Table VIII

Effect of NaOH on the persistence of Sabin poliovirus (type 1)

Cystine dissolved Live viruses in% at 25 ° C (weeks) __0__2__M_ HCl + NaOH 100 13 3

In boiling water 100 59 25