NO773378L - CARRIER FOR MANUFACTURE OF STORAGE STABLE POLIOVAXINE - Google Patents

Description

"Carrier for the production of storage-stable polio vaccine".

The present invention relates to the production of a storage-stable polio vaccine using a special carrier which ensures the desired stability. The carrier will of course also be used as a preservative pending analysis for the detection of poliovirus.

Protection of humans and animals from virus-induced diseases is often achieved by vaccination with inactivated anti-genes or live, weakened virus strains. In both cases, the integrity of the virus antigens must be preserved in the vaccines. However, when live attenuated virus strains are used, the effectiveness of the vaccines depends entirely on the viability of the viruses after treatment, transport and storage.

To achieve a certain stability, some vaccines must be distributed in a freeze-dried state or must have agents added to reduce or eliminate the lability of the vaccine virus. Some of the materials that have been added are calcium lactobionate as referred to in US patent document 5,186,908, dextran as referred to in Canadian patent document 943,461, phosphate buffers as referred to in Canadian patent document 952,429 or MgC^ as referred to in US patent document 3,128,229. Carbohydrates such as sorbitol, mannitol and the like are sometimes used, especially as freeze-protecting agents during freezing and drying. Sucrose or magnesium chloride have been used as stabilizers especially for live, oral poliovirus vaccine (Sabin). However, their use entails certain disadvantages. The high viscosity of sucrose solutions makes the product difficult to process and use. Magnesium chloride has an unpleasant taste and causes difficulties in administration when vaccinating children.

It is also known that L-cystine has a stabilizing effect

on the thermal inactivation of polioviruses. Pohjanpelto

(1961) have shown that the rate of cystine action, that is, the time required for the cystine to combine with the virus for protection against thermal inactivation, varied with the different types and strains of enteroviruses.

Ikegami et al, Jap. J. M. See & Biol 16_, 325-342, 1963, as well as

Yap. J.M. See. & Biol. 17, 13-22, 1964 found that this effect

was a genetically stable property and that the effect of the cystine was a function of stabilizing the protein structure of the virus. The purpose of these earlier studies was not aimed at developing a stabilizing medium for these viruses, but at studying genetic markers. The above authors used L-cystine dissolved in IN HCl. Ikegami (1963) (1964) prepared his cystine storage solution with 0.05M Tris buffer as diluent. Tris buffer as a diluent is commonly used in biochemistry. Experiments have shown, however, that at this low concentration, Tris buffer does not have a sufficient stabilizing effect on polioviruses. Pohjanpelto Virology 15, 225-230, 1961 neutralized his L-cystine storage solution with sodium hydroxide. It has been shown that the presence of sodium ions will adversely affect the stability of the polioviruses during storage at 25°C or at lower temperatures.

On the basis of the fact that the currently used available poliovirus vaccines have a half-life of only 7 days at 25°c, it would be very desirable if one could achieve

a longer storage time, especially in countries where the legislation prescribes that in the event of a drop in strength of more than 50%, the product must be removed from the market.

It has now been found that a combination of an aqueous medium comprising from 0.3 to 1.3 M Tris hydroxymethylaminomethane in combination with lOO^ug L-cystine per milliliter medium is particularly suitable for stabilizing or preserving a sample at room temperature while awaiting analysis for the presence or absence of poliovirus.

It has also been found that the stabilizing aqueous solution consisting of from 0.3 to 1.0 M Tris-hydroxymethylaminomethane and approximately 100 µg of L-cystine per milliliter medium is particularly suitable for stabilizing oral poliovirus vaccines for a period of up to 4 weeks at a room temperature of 25°C, thus avoiding the need to use all the vaccine in a container when it is at room temperature or to discard any residual amounts when only part of the vaccine is used.

It has also been found that the stability of poliovirus vaccines is unexpectedly improved upon storage at temperatures of from -70°C to 4°C when such vaccines are combined with a stabilizing aqueous medium consisting of about 0.3 to 1.0 M Tris-hydroxymethylaminomethane, about lOO^ug L-cystine and approximately 0.8 mg acid hydrolyzed gelatin per ml of vaccine.

The unexpected improved stability is due to the fact that

in contrast to the previously known technique, the solubilization of the L-cystine is not carried out with hydrochloric acid followed by neutralization with sodium hydroxide, as it was found that such a mixture would entail rather high activity losses, especially when stored at 25°C and lower temperatures. The L-cystine must therefore be made soluble in distilled water by heating under a slight positive pressure to reach a temperature of 105°C. After the L-cystine has been dissolved, the solution is cooled

to 60°C and Tris buffer as a powder and acid hydrolyzed gelatin (10% solution) are added. The 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,

but is quite soluble in aqueous solutions below pH 2 or alkaline solutions above pH 8. However, these extreme pH values are not suitable for virus samples awaiting analysis or

for poliovirus vaccines, The stabilizing medium is therefore prepared as described above and the pH is adjusted with an organic, non-toxic buffer such as Tris. Samples or vaccines can then be stored if necessary at room temperature"During time periods of 14 to 21 days, no more than 50% of the original virus will be lost. On the other hand, if it is desired to store the vaccine for longer periods at a temperature of from -70°C to 4°C, acid hydrolyzed gelatin is added to a suspending medium of L-cystine and Tris-hydroxymethylaminomethane.

The stabilizing, suspending effect of the new medium is achieved with a concentration of about 100 µg milliliters of L-cystine in about 0.8 mg/ml acid hydrolyzed gelatin and 0.3 to 1.0 M Tris-hydroxymethylaminomethane. The stabilizing suspension medium is adjusted to a pH of 6.5 to 8.5, with a preferred pH range of 7.0 to 7.5.

As an example of poliovirus vaccines that can be stabilized

in accordance with the present invention, live attenuated Sabin poliovirus vaccines are derived from the following strains:

LSc, 2 ab (type 1), P 712, CH, 2 ab (type 2) and Leon a2b

(type 3). Cell cultures and virus vaccine fluids were prepared using methods known to those skilled in the art, briefly described as follows: Apenary or human diploid cell cultures (Wl-38) are grown in cell culture medium CMRL-1969 supplemented with bovine serum. The culture medium is removed at optimal growth (monolayer)

and is replaced with the virus inoculum and Earle's lactalbumin hydrolyzate medium, which is added to maintain the cell culture. The infected cell cultures are incubated until approximately 50% of the cells show viral cytopathic effects. The viral fluids are collected and further processed by filtration using a 0.45 µm membrane filter, and addition of stabilizer according to the present invention followed by dilution. The suspension is then filled into glass flasks and stored. At all stages in the production of the vaccine, precautions are taken to ensure freedom from harmful agents and toxic substances. The virus fluids are examined with regard to safety, potency and effectiveness during the various treatment

steps.

The invention will appear more clearly by reference to them

the following examples of preferred embodiments.

EXAMPLE 1

Sabin poliovirus fluids were diluted at a ratio of 1:10

in a IM Tris buffer and the effect of the addition of 100 µg/ml L-cystine on virus stability was determined. The virus contents of the different suspensions of each of the three types of poliovirus were assessed initially and at four weekly periods during storage at 25°C. The results showed that increased viability could be imparted to all three Sabin polioviruses by the addition of L-cystine. In a similar experiment, it was shown that L-cysteine also increased stability, but the latter compound has an unpleasant odor and is not a suitable additive for an oral vaccine. The results are shown in the following table 1.

(1) 100 µg/ml l-cystine

(2) Diluent 1.0M Tris buffer

(3) Number of days until 50% of virus was inactivated.

EXAMPLE 11

Sabin poliovirus type 1 virus fluid was suspended in a 1M Tris buffer plus 100 µg/ml L-cystine. After settings to pH 6.4, 7.4 and 8.5 respectively, virus stabilities at the three pH levels were examined at 4°C over a four-month period. The experiment confirms the previous results that the inactivation rate of Sabin polioviruses was not affected at pH in the range pH 6.8 to 8.5. The results are shown in the following table II.

EXAMPLE 111

A storage experiment at 25°C with each of the three polioviruses suspended in various concentrations of Tris buffer plus 100 µg/ml L-cystine was conducted over a four week period. It was shown that 0.1 M Tris buffer was not a sufficiently high concentration, but sufficient virus stability was achieved with Tris buffer between 0.3 and 1.0 M concentrations. As IM Tris buffer is slightly bitter, the lower concentration was chosen for the standard preparation of the stabilizer. However, under certain conditions it may be advantageous to use the higher Tris buffer concentration. The results are shown in the following table

III.

EXAMPLE IV

Each of the three oral S-abin polioviruses was suspended in a stabilizer prepared from 0.3 M Tris and 100 µg/ml L-cystine. 0.8 mg/ml acid stabilized gelatin was then added to half of these mixtures. Both portions were stored at -20°C and the virus stability was determined after 4 and 6 months and the results are reproduced in the subsequent Table IV. It can be seen that after 6 months of storage, the survival rate of the vaccines containing Tris, L-cystine and acid hydrolysed gelatin was 100%.

EXAMPLE V

Each of three oral Sabin polioviruses was suspended in a stabilizing medium prepared from 0.3M Tris, 100 µg/ml L-cystine and 0.8 mg/ml acid hydrolysed gelatin, while corresponding portions of three oral Sabin polioviruses were suspended in distilled water. Both portions were stored at 25°C and stability tests were carried out every week for a period of 4 weeks. The results reproduced in the following table V show the high stability achieved by the medium according to the present invention.

EXAMPLE VI

Each of the three oral Sabin polioviruses is suspended in a stabilizer prepared from 0.3 M Tris, 100 µg/ml L-cystine and 0.8 mg/ml acid hydrolysed gelatin. The stabilized poliovirus vaccines were stored at 4°C and equal portions were tested for stability monthly over a period of 6 months. The results reproduced in the following table IV show that the losses were less than 50%.

EXAMPLE VII

A number of portions of oral poliovirus vaccines prepared in accordance with Example VI were subjected to a series of 5 and 10 freeze-thaw cycles and titer determination showed no loss of potency as shown in the following Table IV.

EXAMPLE VIII

One portion of Sabin virus type 1 vaccine was stabilized with a medium containing 100 µg/ml L-cystine dissolved in the required amount of NaOH and HCl while for another portion 100 µg/ml L-cystine was dissolved in boiling water. Both portions were stored at 24°C and titrated after 2 and 4 weeks and the results are reproduced in the following table

VIII.

Claims (5)

1 Method for producing a storage-stable polio vaccine, characterized by dissolving approximately 100 mg of L-cystine per ml of distilled water by heating to approximately 105°C under a slight positive pressure, after which from 0.3 to 1.0 M tris (hydroxymethyl)aminomethane and optionally also approximately 0.8 mg/ml of acid hydrolyzed gelatin are added, the mixture being adjusted to pH 6.5 - 8.5, preferably 7.0 - 7.5, after which polio virus or virus fluid of type 1, 2 or 3 is added. 2. Carrier for the production of the storage-stable polio vaccine specified in claim 1, characterized by the fact that it comprises approximately 100 mg of L-cystine per ml of distilled water as well as from 0.3 to 1.0 M tris (hydroxymethyl)aminomethane, with pH from 6.5 to 8.5 and preferably 7.0 to 7.5. 3. Carrier as stated in claim 2, characterized in that it also comprises 0.8 mg/ml acid hydrolysed gelatin. 4. Method for producing the carrier specified in claim 2 or 3, characterized by dissolving approximately 100 mg of L-cystine per ml of distilled water at about 105°C under a slight positive pressure and adding from 0.3 to 1.0 M tris(hydroxymethyl)aminomethane, adjusting the mixture to pH 6.5 to 8.5 and preferably to pH 7.0 to 7 ,5. 5. Method as stated in claim 4, characterized in that approximately 0.8 mg/ml of acid hydrolysed gelatin is also added.