GB1564998A

GB1564998A - Vaccine and stabilizing solution therefor

Info

Publication number: GB1564998A
Application number: GB49172/77A
Authority: GB
Original assignee: Connaught Laboratories Ltd
Current assignee: Sanofi Pasteur Ltd
Priority date: 1975-03-21
Filing date: 1977-11-25
Publication date: 1980-04-16

Description

(54) VACCINE AND STABILIZING SOLUTION THEREFOR (71) We, CONNAUGIIT LABORATORIES LIMITED, a Canadian company of 1755 Steeles Avenue West, Willowdale, Ontario, Canada, do hereby declare the invention for which we pray that a patent may be granted to us and the method by which it is to be performed, to be particularly described in and by the following Statement: The present invention relates to a novel suspending medium particularly useful in stabilizing polio-virus vaccines to enhance their viability and stability during processing and storage and which is also useful in preserving specimens while awaiting analysis for the detection of poliovirus. The invention also relates to stabilized poliovirus vaccines.

Protection of man and other animals from viral diseases is frequently achieved by vaccination with inactivated antigens or live, attenuated virus strains. In both cases, the integrity of the viral antigens must be preserved in the vaccines. However, when live attenuated virus strains are used the efficacy of the vaccines depends entirely on the viability of the viruses after processing, transport and storage.

To achieve a measure of stability some vaccines must be distributed in a frozen state, lyophilized, or have agents added to them to decrease or eliminate lability of the vaccine virus. Some of the materials which have been added are calcium lactobionate as disclosed in U.S. Patent 3,186,908, dextran as disclosed in Can. Patent 943,461, phosphate buffers as dis closed in Can. Patent 952,429, or Mg Cl2 as disclosed in U. S. Patent 3,128,229. Carbo hydrates, such as sorbitol, mannitol etc., are .sometimes employed, especially as cryo protective agents during freezing and drying.

,sucrose or magnesium chloride have been used is stabilizers, particularly for Poliovirus Vaccine, Live, Oral (Sabin). However, their use has certain disadvantages. The high viscosity of sucrose solutions makes this product difficult to process and dispense. Magnesium chloride has an unpleasant taste and creates difficulties of administration in the vaccination of children.

It is also known that L-cystine has a stabilizing effect on the thermal inactivation of polioviruses. Pohjanpelto (1961) has shown that the rate of cystine action, i.e. the time required for cystine to combine with the virus to protect from thermal inactivation, varied with the different enterovirus types and strains. Ikegami et alia. J. M.Sc. & Biol. 16, 325-342, 1963, Jap. J. M.Sc. & Bio. 17, 13-22, 1964 found that this effect was a genetically stable characteristic and the action of cystine a function of stabilizing the protein structure of the virus. The purpose of these earlier studies was not directed towards the development of a stabilizing medium for these viruses, but to study genetic markers. The authors mentioned above used L-cystine which was dissolved in 1N ElC1. Ikegami (1963) (1964) prepared his cystine stock solution with 0.05M Tris buffer as the diluent. Tris buffer as a diluent is commonly employed in biochemistry. However, experiments have shown that at this low concentration Tris buffer does not have sufficient stabilizing effect on polioviruses. Pohjanpelto Virology 15, 225-230, 1961 neutralized his L-cystine stock solution with sodium hydroxide. It has been established that the presence of sodium ions inadvertently affected the stability of polioviruses during storage at 25 C and below.

In view of the fact that present day poliovirus vaccines available present a half-life of only 7 days at 25"C, it would appear highly desirable if a longer shelf-life could be obtained, particularly in those countries where health regulations prescribe a drop in titer of more than 50% requires that the product be removed from the market.

In accordance with the present invention, it has now been found that a combination of an aqueous medium comprising from 0.3 to 1.0M Tris (hydroxymethyl) aminomethane in combination with 80 to 120ug, preferably about 100us of L-cystine per ml of medium, and having a pH in the range from 6.5 to 8.5, is particularly useful in stabilizing or preserving a specimen at room temperature while awaiting analysis for the presence or absence of poliovirus.

It has also been found that the stabilizing aqueous composition made up of from 0.3 to 1 .0M Tris (hydroxymethyl) aminomethane and 80 to 120,us, preferably about 100pg of L-cystine per ml of medium, and having a pH in the range from 6.5 to 8.5, is particularly useful in stabilizing oral poliovirus vaccines for a period of up to 4 weeks at room temperature that is 25"C thus avoiding the necessity of using all the vaccine in a vial when it is at room temperature or of discarding any amount left over when only part of the vaccine is used.

It has also been found in accordance with the present invention that the stability of poliovirus vaccines can be unexpectedly enhanced upon storage at temperatures of from -70 to 4 C when such vaccines are combined with a stabilizing aqueous medium made up of 0.3 to 1 .0M Tris (hydroxymethyl) aminomethane, 80 to 120dung, preferably about 100,ug of L-cystine and optionally about 0.8 mg of acid hydrolyzed gelatin per ml of vaccine, and having a pH in the range from 6.5 to 8.5.

The unexpected enhanced stability is due to the fact that contrary to the prior art, solubilization of the L-cystine is not carried out with hydrochloric acid followed by neutralization with sodium hydroxide since it has been found that such a medium will cause substantially higli losses of activity, particularly upon storage at 250C and below. L-cystine must, therefore, be solubilized in distilled water with heating under slight pressure to obtain a temperature of about 105us. The preparation of stabilizing media as defined above in this manner forms a further aspect of the present invention. After L-cystine has been dissolved, the solution is preferably cooled at 600C and Tris buffer as a powder and acid hydrolyzed gelatin (10% solution) added.

The solution may then be cooled and sterilized by filtration through a 0.22u membrane filter.

It is known that L-cystine is practically in soluble in water, but is quite soluble in aqueous solutions below pH 2 or alkaline solutions above pH 8. However, these extreme pH levels are not suitable for virus specimens awaiting analyses or for poliovirus vaccines. The stabilizing medium is, therefore, prepared as described above and the pH adjusted with an organic non-toxic buffer, such as Tris. Speci mens or vaccines, can then be stored at room temperature if necessary. Within time periods of 14-21 days not more than 50% of the original virus will be lost. On the other hand, when it is desired to store the vaccine for longer periods at a temperature of from -700C to 4 C, acid hydrolyzed gelatin is added to a sus pending medium of L-cystine and Tris (hydroxymethyl) aminomethane.

The optimum stabilizing, suspending effect of the novel medium is obtained with a concentration of about 100pg/ml of L-cystine in about 0.8 mg/ml of acid hydrolyzed gelatin and 0.3 to 1 .0M Tris (hydroxymethyl) aminomethane. The stabilizing suspension medium is adjusted to a pH of from 6.5 to 8.5, with a preferred pH range being from 7.0 to 7.5.

As examples of the poliovirus vaccines which 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 in accordance with procedures known to those skilled in the art, briefly described as follows: monkey kidney or human diploid cell cultures (WI-38) are grown in 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 hydrolysate medium, which is added for cell culture maintenance. The infected cell cultures are incubated until approximately 50% of the cells show viral cytopathic effects. The virus fluids are harvested and further processed by filtration using a 0.45,u membrane filter, and addition of stabilizing medium of the present invention followed by dilution. The suspension is then filled into glass vials and stored. At all stages in the preparation of the vaccine, precautions are taken to ensure freedom from adventitious agents and toxic substances. The virus fluids are tested for safety, potency and efficacy during various stages of processing.

The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention only.

EXAMPLE I Sabin poliovirus fluids were diluted at a ratio of 1:10 in 1M Tris buffer and the effect of the addition of 100,ug/ml L-cystine on virus stability was determined. The virus contents of the various suspensions of each of the three types of poliovirus were assayed initially and at four weekly intervals during storage at 250 C.

The results indicated that increased viability could be conferred on all three Sabin polioviruses by the addition of L-cystine. In a similar experiment it was shown that L-cystine increased the stability as well, but this compound has an unpleasant odour and is not a suitable additive in an oral vaccine. The results are shown in Table I.

EXAMPLE II Sabin poliovirus type 1 virus fluid was suspended in 1M Tris buffer plus 100,ug/ml L-cystine. After adjustments to pH 6.4, 7.4, and 8.5 virus stabilities at the three pH levels were tested at 40C over a four month period.

The experiment confirmed previous results that the rate of inactivation for Sabin polioviruses was not affected by pH over a range of TABLE 1 The Stabilizing Effect of Cystine (1) on Sabin Polioviruses (2) Daring Storage at 250C

Poliovirus L-Cystine Per Cent Survival at 25 C (Weeks) 2 Half Life (3) Type Added 0 2 4 (Days) Type 19 1 5 10 + + 100 59 25 17 ~ 100 20 5 10 2 + 100 55 17 16 - 100 45 6 11 I 3 + 100 72 1 16 19 (1) 100 ugtml L-cystine (2) Diluent 1 .0M Tris Buffer (3) Number of days until 50% of virus inactivated TABLE II The Effect of pH on the Stability of Sabin Poliovirus Type 1 during Storage at 40C Using 1M Tris buffer plus 100,ug/ml L-cystine as Stabilizer

Diluent Per Cent Survival at 40C Months pH 0 1 2 4 6.4 100 79 74 47 7.4 100 89 - 66 8.5 100 83 74 25 pH 6.8-8.5. Results are shown in Table 11.

EXAMPLE III A storage experiment at 250C with each of the three polioviruses suspended in various con centrations of Tris buffer plus 100,ug/ml L-cystine was performed over a four week period. It was shown that 0.1M Tris buffer was not a sufficiently high concentration, but adequate virus stability was obtained with Tris buffer between 0.3 and 1.0M concentrations.

As 1M Tris buffer is slightly bitter, the lower concentration was selected for the standard preparation of the stabilizer preparation.

However, it may be advantageous, under certain conditions, to use the higher Tris buffer concentration. Results are shown in table Ill.

EXAMPLE IV Each of the three Sabin oral polioviruses are suspended in a stabilizing medium made up of 0.3M Tris and 100 Mg/ml of L-cystine. To one half of these preparations there was then added 0.8 malml of acid stabilized gelatin. Both lots were stored at 200C and the virus stability was determined after 4 and 6 months and the results are reported in Table IV. It is seen that after 6 months of storage the survival rate of the vaccines containing Tris, L-cystine and acid hydrolized gelatin was 100%.

EXAMPLE V Each of three Sabin oral polioviruses were suspended in a stabilizing medium made up of 0.3M Tris, 100ug/ml of L-cystine and 0.8 mg/ml TABLE III The Effect of Various Concentrations of Tris buffer plus 100,ug/ml of L-cystine on the Stability of Sabin Polioviruses during Storage at 250C

Poliovirus Tris Buffer. Per Cent Survival at 250C (Weeks) Type Molarity 0 2 4 0.1 100 40 3 1 G3 100 32 16 1.0 100 40 23 0.1 100 36 6 2 0.3 100 58 45 1.0 100 40 32 0.1 100 70 7 3 0.3 100 100 56 1.0 100 79 50 TABLE The Effect of Gelatin Addition on the Stability of Sabin Polioviruses in Tris buffer and Cystine During Storage at -200C

Polio Addition (1) of Per Cent Survival at -200C Months Virus 0.08% gelatin 0 4 6 Type 100 56 89 + 100 - 100 100 56 63 2 + 100 - 100 3 100 40 40 + 100 - 100 (1) Diluent: 0.3M Tris buffer plus 100 g/ml of L-cystine of acid hydrolyzed gelatin, while equivalent lots of three Sabin oral polioviruses were suspended in distilled water. Both lots were stored at 25 C and stability tests were performed each week for a period of four weeks. The results reported in Table V illustrated the high stability of the medium of the present invention.

EXAMPLE VI Each of the three Sabin oral polioviruses are suspended in a stabilizing medium made up of 0.3M Tris, 100,ug/ml of L-cystine and 0.8 mglml of acid hydrolyzed gelatin. The stabilized poliovirus vaccines were stored at 4 C and equal lots were tested for stability each month for a period of six months. Results reported in Table VI show that the loss was less than 50%.

EXAMPLE VH Several lots of oral poliovirus vaccines pre TABLE V Comparison of Stabilizer (1) and Distilled Water on the Survival of Polioviruses During Storage at 250C

Polio Polio Per Cent Survival Virus Diluent ~ at 25 C (Weeks Type 0 1 1 2 3 4 H2O 100 71 24 17 5 1 Stabilizer 100 69 47 20 13 H2 O 100 54 37 13 10 2 Stabilizer 100 72 72 36 23 H2 O 100 74 30 22 7 3 Stabilizer 100 100 69 32 44 Note: (1) Stabilizer = 100,ug/ml L-cystine, 0.08% gelatin, 0.3M Tris buffer, pH 7.4 TABLE VI The Effect of the Stabilizer on the Survival of Sabin Polioviruses During Storage at 40C

1Polio Polio Per Cent Survival at 40C Virus Months Type 0 1 2 4 6 1 100 100 100 74 53 2 100 100 100 83 74 3 100 100 - 100 50 TABLE VII The Effect of the Stabilizer on the Survival of Polioviruses After Freezing and Thawing

Polio Virus Per Cent Survival Type Type 0 5 10 Cycles(I)- 1 100 100 I also 2 100 100 100 3 100 100 100 (1) Number of cycles of freezing and thawing using dry ice with acetone and 25 C water bath TABLE VIII Effect of Sodium Hydroxide on the Stability of Sabin Poliovirus Type 1

Dissolved I Per Cent Survival at 25 C (Weeks) in 0 2 4 HC1 HCI andNaOH 100 13 3 Boiling Water 100 59 25 pared in accordance with Example VI were subjected to a series of five and ten cycles of freezing and thawing and the titer determination showed that no potency was lost as reported in Table VII.

EXAMPLE VIII One lot Sabin poliovirus vaccine of Type 1 was stabilized with a medium containing 100,ug/ml of L-cystine dissolved in the required amount of NaOH and MCI while in another lot 100,ug/ml of L-cystine was dissolved in boiling water. Both lots were stored at 25 C and titred after two and four weeks and the results are reported in Table VIII.

WHAT WE CLAIM IS: 1. An improved stabilized poliovirus vaccine containing a live, attenuated poliovirus of types 1, 2 or 3 (as hereinbefore defined) or mixtures thereof suspended in an aqueous stabilizing medium comprising 0.3 to 1.0M tris (hydroxy methyl) aminomethane and from 80 to 120ug/ml of L-cystine and having a pH in the range from 6.5 to 8.5.

2. An improved stabilized poliovirus vaccine according to Claim 1 wherein said aqueous stabilizing medium comprises about 100Sug/ml of L-cystine.

3. An improved stabilized poliovirus vaccine containing a live, attenuated poliovirus of type 1, 2 or 3 (as defined herein) or mixtures thereof suspended in an aqueous stabilizing medium containing 0.3 to 1.0M tris (hydroxymethyl) aminomethane, 100,ug/ml of L-cystine and 0.8 mg/ml of acid hydrolyzed gelatin and having a pH in the range from 6.5 to 8.5.

4. An improved poliovirus vaccine according to any preceding claim wherein the pH of the stabilizing suspension medium is within the range of from 7.0 to 7.5 5. An aqueous suspending medium for stabilizing live attenuated poliovirus vaccines of types 1, 2 or 3 (as defined herein) or mixtures thereof which comprises from 0.3 to 1.0M tris (hydroxymethyl) aminomethane and from 80 to 120,ug/ml of L-cystine and having a pH in the range from 6.5 to 8.5.

6. A suspending medium according to Claim 5, containing about 0.8 mg of acid hydrolyzed gelatin per ml.

7. An aqueous suspending medium for

**WARNING** end of DESC field may overlap start of CLMS **.

Claims

**WARNING** start of CLMS field may overlap end of DESC **. TABLE VII The Effect of the Stabilizer on the Survival of Polioviruses After Freezing and Thawing Polio Virus Per Cent Survival Type Type 0 5 10 Cycles(I)- 1 100 100 I also 2 100 100 100 3 100 100 100 (1) Number of cycles of freezing and thawing using dry ice with acetone and 25 C water bath TABLE VIII Effect of Sodium Hydroxide on the Stability of Sabin Poliovirus Type 1 Dissolved I Per Cent Survival at 25 C (Weeks) in 0 2 4 HC1 HCI andNaOH 100 13 3 Boiling Water 100 59 25 pared in accordance with Example VI were subjected to a series of five and ten cycles of freezing and thawing and the titer determination showed that no potency was lost as reported in Table VII. EXAMPLE VIII One lot Sabin poliovirus vaccine of Type 1 was stabilized with a medium containing 100,ug/ml of L-cystine dissolved in the required amount of NaOH and MCI while in another lot 100,ug/ml of L-cystine was dissolved in boiling water. Both lots were stored at 25 C and titred after two and four weeks and the results are reported in Table VIII. WHAT WE CLAIM IS:

1. An improved stabilized poliovirus vaccine containing a live, attenuated poliovirus of types 1, 2 or 3 (as hereinbefore defined) or mixtures thereof suspended in an aqueous stabilizing medium comprising 0.3 to 1.0M tris (hydroxy methyl) aminomethane and from 80 to 120ug/ml of L-cystine and having a pH in the range from 6.5 to 8.5.

4. An improved poliovirus vaccine according to any preceding claim wherein the pH of the stabilizing suspension medium is within the range of from 7.0 to 7.5

5. An aqueous suspending medium for stabilizing live attenuated poliovirus vaccines of types 1, 2 or 3 (as defined herein) or mixtures thereof which comprises from 0.3 to 1.0M tris (hydroxymethyl) aminomethane and from 80 to 120,ug/ml of L-cystine and having a pH in the range from 6.5 to 8.5.

7. An aqueous suspending medium for

stablizing live, attenuated poliovirus vaccines of types 1, 2 and 3 or mixtures thereof which comprises from 0.3 to 1 .0M tris (hydroxymethyl) aminomethane, 1 00ug/ml of Lcystine and 0.8mg of acid hydrolyzed gelatin per ml.

8. A method for preparing a medium for stabilizing a poliovirus vaccine of types 1, 2 and 3 (as defined herein) or mixtures thereof, which comprises dissolving from 80 to 120,ug of L-cystine per ml of distilled water by heating at about 105"C under slight pressure, and adding from 0.3 to 1.0M tris (hydroxymethyl) aminomethane to give a pH in the range as in Claim 1.

9. A method according to Claim 8 wherein about 100,ug per ml of L-cystine are dissolved.

10. A method according to Claim 8 wherein there is also added about 0.8 mg/ml of acid hydrolyzed gelatin.

11. An improved stabilized poliovirus vaccine as claimed in Claim 1 and substantially as hereinbefore described and exemplified.

12. A suspending medium for stabilizing live attenuated poliovirus vaccine as claimed in Claim 5 and substantially as hereinbefore described.

13. A method for preparing a medium for stabilizing a poliovirus vaccine as claimed in Claim 8 substantially as hereinbefore described.