DE2747662A1 - POLIOMYELITIS VACCINE - Google Patents

Description

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DESCRIPTION

Poliomyelitis Vaccine

The invention relates to a new stabilizing liquid, specifically designed to stabilize poliovirus vaccines to improve their viability and stability during the Processing and storage is suitable, as well as another liquid, which is suitable for the preservation of samples which are intended for analysis for polioviruses.

The protection of humans and animals against viral diseases is often achieved by vaccination with inactivated antigens or live, attenuated virus strains. In both cases the integrity of the virus antigen must be preserved in the vaccines. However, when using live attenuated virus strains, the effectiveness of the vaccines depends entirely on viability of viruses after processing, transport and storage.

In order to achieve a certain stability, some vaccines have to be shipped in a frozen state, for example freeze-dried, or certain additives are used to reduce or eliminate the sensitivity of the vaccine virus. Examples of such additives are calcium lactobionate, as described in US-PS 3 186 9o8, dextran, as described in CA-PS 943,461, phosphate buffer, as described, or in Canadian Patent 952 429 MgCl _2, as described in US PS 3,128,229. Sometimes carbohydrates such as sorbitol or mannitol are also used, especially as anti-freeze agents during freezing and drying. Sucrose or magnesium chloride are used as stabilizers, especially for live vaccines for oral poliomyelitis vaccination according to Sabin. However, there are several drawbacks to their use. The high viscosity of the sucrose solutions makes handling and dosing difficult. Magnesium chloride has an unpleasant taste and causes difficulties in vaccinating children.

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It is known that L-cystine has a stabilizing effect on the thermal inactivation of poliomyelitis viruses. In 1961, Pohjanpelto was able to show that the speed of the cystine reaction, ie the time it takes for the cystine to combine with the virus to protect against its thermal inactivation, depends on the different types and strains of enteroviruses. Furthermore, it has been found (Ikegami et al., Jap. JM Sc. & Biol. J ^, 325-342, 1963; Jap. JMSc. & Biol. 17 , 13-22, 1964) that this effect is a genetically stable property and the response of the cystine is a function of stabilizing the protein structure of the virus. The purpose of these earlier studies was not to develop a stabilizing medium for these viruses, but to study genetic markings. For this purpose, the authors mentioned used L-cystine dissolved in 1 η HCl. Ikegami (1963) (1964) prepared his cystine solution with 0.05 molar tris (hydroxymethyl) aminomethane buffer (hereinafter TRIS buffer) as the solvent. TRIS buffer is generally used as a diluent in biochemistry. However, experiments have shown that at this low concentration, TRIS does not have a sufficient stabilizing effect on poliomyelitis viruses. Pohjanpelto (Virology J_5 »225-230, 1961) neutralized his L-cystine stock solution with sodium hydroxide. The presence of sodium ions has been found to inadvertently impair the stability of poliomyelitis viruses during storage at 25 ° C and below.

Given that currently on the market Poliovirus vaccines have a half-life of only 7 days at 25 ° C, there is thus a great need for one Extension of the storage period, especially in countries where the health regulations in the event of a decrease in the titer of over 50 percent stipulate that the product be removed from the market.

It has now been found that an aqueous medium containing 0.3 to 1.0 molar TRIS in combination with 100 ^ q L-cystine per ml of medium is excellently suited for stabilization or preservation.

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tion of samples at room temperature to be tested for the presence or absence of poliovirus.

It was also found that the stabilizing aqueous medium, that of 0.3 to 1, omolar TRIS and about 100 ng L-cystine per ml Medium has been prepared excellent for stabilizing poliomyelitis vaccines for oral vaccination for a period of time up to 4 weeks at room temperature, i.e. 25 ° C. This makes it unnecessary to have the entire in one Vaccine contained in the ampoule must be used if one is working at room temperature, or the remaining amount must be discarded if only one part of the vaccine used.

Furthermore, it was found according to the invention that can be improved the stability of poliovirus vaccines when stored at temperatures from -70 to 4 ⁰ C to a surprising degree, when using the vaccines with an aqueous stabilization medium, from about 0.3 to 1 0 molar TRIS, about 100 μg of L-cystine and about 0.08% of acid hydrolyzed gelatin per ml of vaccine has been produced.

The surprisingly improved stability is due to the fact that, in contrast to the prior art, the solubilization of L-cystine does not take place by means of hydrochloric acid and subsequent neutralization with sodium hydroxide, since it has been found that this medium leads to relatively large losses of activity, especially when stored at 25 ^° C. and below. The L-cystine must therefore be solubilized in distilled water, heating under slight pressure to reach a temperature of 105 ° C. After the L-cystine has dissolved, the solution is cooled to 60 ° C. and powdered TRIS buffer and acidic hydrolyzed gelatin (10 percent solution) are added. This solution is cooled and sterilized by filtration through a 0.22 µm membrane filter.

It is known that L-cystine is practically insoluble in water, however is relatively soluble in aqueous solutions below pH 2 or in alkaline solutions above pH 8. These extremes

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However, pH values are not suitable for virus samples to be analyzed or for poliovirus vaccines. The stabilization medium is therefore prepared as described above and the pH is established with an organic, non-toxic buffer such as TRIS buffer. The samples or vaccines can then be stored at room temperature if necessary. No more than 50 percent of the original viruses are lost within a period of 14 to 21 days. If it is otherwise desirable to store the vaccines for extended periods of time at temperatures of -70 to about 4 ⁰ C, acid hydrolysed gelatin is added to a suspending medium consisting of L-cystine and TRIS. The stabilizing, suspending effect of the new medium is achieved at a concentration of about 100 µg / ml L-Cystm in about 0.8 µg / ml acidic hydrolyzed gelatin and 0.3 to 1.0 molar TRIS. The stabilizing suspending medium is adjusted to a pH of 6.5 to 8.5, the preferred pH range being 7.0 to 7.5.

As an example of poliovirus vaccines that can be stabilized according to the invention, live attenuated Sabin poliovirus vaccines are produced from the following strains: LSc, 2 ab (type 1), P 712, CH, 2 ab (type 2) and Leon a ₂ b (type 3). Cell cultures and virus vaccine fluids are produced using known methods, which can be briefly described as follows: Monkey kidney cell cultures or diploid human cell cultures (WI-38) are grown in a cell culture medium (CMRL-1969) supplemented with bovine serum. The culture medium is removed at optimal growth (monolayers) and replaced with the virus inoculum and Earle's lactalbumin hydrolyzate medium, which is added to maintain the tissue culture. The infected tissue cultures are incubated until about 50 percent of the cells show viral cytopathic effects. The virus fluids are then harvested and filtered through a 0.45 μm membrane filter. After adding the stabilizing medium according to the invention, dilution takes place. After the suspension has been filled into glass ampoules, it can be stored. In all stages of vaccine production, the absence of minor

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products and toxic substances. The virus fluids are tested for safety, potency and effectiveness in various stages of production.

The examples illustrate the invention.

example

Sabin poliovirus fluids are in a ratio of Diluted 1:10 in 1 molar TRIS buffer. This is where the influence becomes the addition of 100 pg / ml L-cystine on virus stability certainly. The virus levels of the various suspensions of each of the three poliovirus types are initially and weekly after four Intervals determined for storage at 25 ° C. The results show that in all three Sabin polioviruses by addition of L-cystine an increased viability can be determined. In a similar experiment it was found that too L-cysteine increases the stability. However, this compound has an unpleasant odor and is used as an additive for oral vaccines not suitable. The results are shown in Table I.

Table I.

1) 2)

Stabilizing effect of cystine on Sabin polioviruses

when stored at 25 ^° C

Poliovirus L-cystine Survival Rate (%)
after χ weeks χ = 2 X at 25 ° C I. Half- ₃₎
value time i ryp added x = 0 19th = 4 (Days) 1 _ 1OO 59 5 10 2 + 1OO 2O 25th 17th 3 - 100 55 5 10 + 1OO 45 17th 16:
j 1OO 72 6th 11 ♦ 100 16 19th

100 pg / ml L-cystine;

2)

Diluent 1, Omolarer TRIS buffer

Number of days «until 50 percent of the viruses are inactivated .

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Example 2

Virus fluid (Sabin poliovirus type 1) is suspended in imolar TRIS buffer plus 100 μ9 / πι1 L-cystine. After adjustments to pH 6.4, 7.4 and 8.5, the virus stabilities are determined at the three pH values at 4 ^° C. over a period of 4 months. The experiment confirms the earlier results that the rate of inactivation for Sabin poliovirus is not affected by pH over a pH range of 6.8 to 8.5. The results are shown in Table II.

Table II

Influence of pH on the stability of Sabin polio virus (type 1) during storage at 4 ⁰ C using Imolarem TRIS buffer plus 100 ug / ml L-cystine as a stabilizer

Dilution
medium pH Survival rate (%) at 4 ⁰ C after χ months χ = 1 χ = 2 χ = 4 6.4 x = 0 79
89
83 74 47 7.4
8.5 100
100
100 - 66 74 25th

Example 3

A storage experiment is carried out at 25 ° C with all three poliomyelitis viruses, suspended in TRIS buffer of different concentrations plus 100 μg / ml L-cystine, over a 4 week period carried out. This shows that 0.1 molar TRIS buffer does not have a sufficiently high concentration, but sufficient Virus stabilities at TRIS buffer concentrations of 0.3 can graze to 1, omolar reached. An imolar TRIS buffer solution is slightly bitter; the lower concentration was selected for the standard manufacture of the stabilizer preparation. However, under certain conditions it may seem beneficial be to use the higher TRIS buffer concentration. The results are summarized in Table III.

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Table III

Influence of the different concentrations of TRIS buffer plus 100 ug / ml L-cystine on the stability of Sabin polioviruses when stored at 25 ° C

Poliovirus, !
i 3 Molar
ity of ■ χ = Survival rate
after χ weeks χ = 2 in% at 25 ° C
I. Type TRIS buffer 1OO O 40 χ = 4 1 0.1 100 32 3 0.3 1OO 40 16 I. 2 1.0 1OO 36 23 0.1 100 58 6th 0.3 100 40 45 1/0 1OO 70 32 '0.1 1OO 100 7th 0.3 1OO 79 56 1/0 50

Example 4

The three Sabin poliovirus samples (oral) are placed in a stabilization medium from 0.3 molar TRIS buffer and 100 µg / ml L-cystine suspended. One half of these preparations is then mixed with 0.08 mg / ml acidic hydrolyzed (stabilized) gelatin. Both samples are stored at -20 ° C and the virus stability is determined after 4 and 6 months. The results are summarized in Table IV. You can see that after 6 months Storage the survival rate of vaccines containing TRIS buffer, L-cystine and acid hydrolyzed gelatin is 100 percent amounts to.

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Table IV

Influence of the addition of gelatine on the stability of Sabin polioviruses in TRIS buffer and cystine when stored at -20 ° C

Poliovirus,
Type Encore
of 0.08%
gelatin Survival rate (%) at -20 ° C
after χ months χ = 4 χ = 6 1 - x = 0 56 89 2 + 1OO - 100 3 - 100 56 63 + 1OO - 100 - 100 40 40 + 100 - 100 100

Diluent: 0.3 molar TRIS buffer + 100 µg / ml L-cystine

Example 5

Each of the three Sabin poliovirus samples (oral) is suspended in a stabilizing medium made up of 0.3 molar TRIS buffer, 100 µg / ml L-cystine, and 0.08 µg / ml acid hydrolyzed gelatin. Equal amounts of the three Sabin poliovirus samples (oral) are suspended in distilled water. The different samples are stored at 25 ° C, and are carried out every week stability tests over a period of 4 weeks. The results shown in Table V show the great stability of the stabilizing medium according to the invention.

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Table V

Comparison of stabilizer and distilled water on the survival rate of polioviruses when stored at 25 ° C

Poliovirus,
Type Dilution
middle Survival rate (%) at 25 ⁰ C
in χ weeks x = 1 χ = 2 χ = 3 χ = 4 1 water X = O 71 24 17th 5 2 stabilizer 1OO 69 47 20th 13th 3 water 1OO 54 37 13th 10 stabilizer 1OO 72 72 36 23 water 100 74 30th 22nd 7th stabilizer 100 100 69 32 44 100

Stabilizer = 100 µg / ml L-cystine, 0.08% gelatin, 0.3 molar TRIS buffer, pH 7.4

Example 6

Each of the three Sabin poliovirus samples (oral) are suspended in stabilization medium made from 0.3 molar TRIS buffer, 100 µg / ml L-cystine and 0.08 µg / ml acid hydrolyzed gelatin. The stabilized poliovirus vaccines stored at 4 ⁰ C. The same subsamples are examined every month for a period of 6 months. The results, compiled in Table VI, show that the loss rate is less than 50 percent.

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' Table VI

Influence of the stabilizer on the survival rate of Sabin polio viruses during storage at 4 ⁰ C

Poliovirus, 1 Survival rate χ = 1 (%) at 4 ⁰ C after χ months χ = 4 χ = 6 Type 2 x = 0 100 χ = 2 74 53 3 100 100 100 83 74 100 1OO 100 100 50 100 -

Example 7

Several samples of oral poliovirus vaccines, made according to Example 6, freeze-thaw cycles are subjected, and although 5 cycles and 10 cycles. The results, compiled in Table VII, show that there is no loss of titer.

Table VII

Influence of the stabilizer on the survival rate of polioviruses after freezing and thawing

Poliovirus,
Type Survival rate (%) after χ cycles χ = 5 χ = 10 1 x = 0 100 100 2 100 100 1OO 3 100 100 100 100

Number of cycles (freezing and thawing) using dry ice / acetone and a water bath of 25 ⁰ C.

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example

A sample of Sabin poliovirus vaccine type 1 is mixed with a medium stabilized, the 1OO μς / ιηΐ L-cystine, dissolved in the required Contains amount of NaOH and HCl. Another sample at 100 µg / ml L-cystine is dissolved in boiling water. Both samples are stored at 25 ° C; the titer is determined after two and four weeks. The results are shown in Table VIII.

Table VIII

Influence of sodium hydroxide on the stability of type 1 Sabin polioviruses

Cystine, dissolved
in Survival rate |%) at 25 ° C after χ weeks χ β 2 χ = 4 HCl and NaOH
boiling water x = O 13th
59 3
25th 1OO
1OO

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Claims (7)

PATENT ADVOCATE DIPL.-PHYS. LUTZ H. PRÜFER · D-8000 MÜNCHEN 9O GM 5-812 CONNAOGHT LABORATORIES LIMITED, Willowdale, Prov. Ontario, Canada claims 1. Poliomyelitis vaccine containing virus from one live attenuated poliomyelitis virus types 1, 2 and / or 3, suspended in an aqueous stabilizing medium of about 0.3 to 1.0 molar tris (hydroxymethyl) aminomethane and about 100 µg / ml L-cystine. 2. Poliomyelitis vaccine according to claim 1, characterized by the additional 0.8 µg / ml acid hydrolyzed gelatin content. 3. Poliomyelitis vaccine according to at least one of the claims 1 and 2, characterized in that the pH of the stabilizing Suspending medium is 6.5 to 8.5. 4. poliomyelitis vaccine according to claim 3, characterized in that the pH of the stabilizing suspending medium Is 7.0 to 7.5. PATENT ADVERTISER OIPL.-PHVS. LUTZ M. PRÜFERO-8O0O MUNICH 0O. WILLROIOERSTR. β - TEL. (Οββ) 04ΟΟ4Ο 809822/0587 5. Suspending medium for the stabilization of attenuated poliomyelitis live vaccines of type 1, 2 and / or 3, characterized by the content of 0.3 to 1, omolar tris- (hydroxyme thy 1) -aminome than, 100 μg / ml L-cystine and 0.8 pg acid hydrolyzed Gelatin per ml. 6. A process for the preparation of a medium for stabilizing poliomyelitis vaccines 1, 2 and / or 3, characterized in that about 100 mg of L-cystine per ml of distilled water
dissolves by heating to about 10 5 ° C under slight pressure, and
about 0.3 to 1.0 molar tris (hydroxymethyl) aminomethane is added. 7. The method according to claim 6, characterized in that one
additionally about 0.8 mg / ml acid hydrolyzed gelatin is added. 809822/0587