DD155875A1 - METHOD FOR PRODUCING A BALANCE-FREE INACTIVATED INFLUENZA VACCINE - Google Patents

Abstract

Area: Techn. Virology, in particular vaccine prod. D. The goal is the development of a process that makes it possible. is - without essential Change d. Procedure for the preparation of standard Inluenzavollvirusimpfstoffen- to solubilize the crucial surface antigens of the respective Inluenzaviren used u. by separating the unloesl. Phase at the same time a decisive cleaning effect by eliminating. Influenza viruses of the corresponding types are only added by addition of sodium deoxycholate as the only additive. Adjuvant u. otherwise without change. treated under environmental conditions, then a separation takes place in a loesl. and an unloesl. Phase.The unloesl. Phase contains the essential Fiber. The loesl. Phase, which primarily contains the surface antigens haemagglutinin and neuraminidase, is processed into a vaccine. Possible uses: Flu vaccine prod., Especially for use in persons at risk and children.

Description

6778

Process for the preparation of a low-fiber inactivated influenza vaccine

Field of application of the invention

The invention relates to the industrial production of low-fiber inactivated influenza vaccines by solubilizing the surface antigens hemagglutinin and neuraminidase of influenza viruses by simple treatment with sodium deoxycholate without significant alteration of the conventional technological and substrate conditions. Due to the specific dietary fiber poverty of these vaccines, which is achieved through the targeted solubilization of the surface antigens, vaccines are formed, which are particularly important for at-risk individuals or for use in childhood. ·

The field of technical application refers to establishments producing appropriate vaccines or related medicinal products for human or veterinary use. In addition, the application is useable in facilities that produce antigens or preparations for routine diagnostics and analytics. Also, this procedure can be introduced in applied research and development, where low-fiber preparations are needed.

Characteristic of the known technical solutions

Viral cleavage solutions with chemical or physicochemical (eg detergents) or biochemical (e.g., enzymes) means for the purpose of production

of subunit vaccines or special antigen preparation. are indeed known. , but ... they have z. B. the disadvantage that working with relatively high detergent concentrations, long residence or production times, sometimes low yields destroying the antigenic structures and these are not transferable to all Influenzavirusstämme. In the case of the use of free enzymes, a considerable hydrolysis and also a transfer of the enzyme proteins into the end product of the antigen can not be avoided.

For the specific use of sodium deoxycholate or Ämmoniumdesoxycholat exist solutions or are process variants, described, but which more or less have the disadvantages of the type described above. Thus, for example, desoxycholate split vaccine was already produced in Australia in 1969 (Bull WHO 41, 639 (1969)). "Influenza virus-containing allantoic fluid was concentrated, inactivated with 0.05% HCHO, sodium desoxycholate added to the final concentration of 1% and at 37 ° C 30 treated min. After low-speed centrifugation to remove the aggregates, dialysis was carried out for 3 days to a detergent concentration of less than 100 ppm. Or in another example, 0.5 ml of 5% sodium deoxycholate solution was added to 2 ml of virus suspension (J. Immunology 2JLs 596 (1966)).

Orientation towards a targeted solubilization of the surface antigens hemagglutinin and neuraminidase of

Influenza viruses using sodium desoxycholate with a high degree of elimination of interfering fiber, a widely varying sodium desoxycholate: hemagglutinin weight ratio but with a very low detergent concentration, a high or 100% adsorption rate of the solubilized surface antigens, an inactivation the influenza

Viruses without chemicals or thermal manipulations, applicable to all influenza virus strains of the

⁵ - 226 778

speaking types, high yields and a sparing use of antigen in the application, however, is technically .-- in this complexity, yet 'not reached', ', ι,.

Some disadvantages of the type described can be eliminated by adhering to or transferring the parameters laid down in the Vos. 146 386 and WP C 12 .D / 221 207.

Object of the invention

The invention is based on the effect of developing a method by means of which it is possible - without substantially changing the procedure for the preparation of conventional influenza virus vaccines - to add the decisive surface antigens hemagglutinin and neuraminidase to the influenza viruses used in each case merely by adding sodium deoxycholate Solubilize and, by separating the insoluble phase, at the same time achieve a decisive cleaning effect by eliminating unnecessary fiber and to process the solubilized antigen directly into the vaccine, in adsorbed or non-adsorbed form, which in turn due to its low fiber Use in people at risk or especially in children is suitable.

The useful effect when using the invention compared to retracted influenza virus vaccines as follows: The vaccines in the form of

, end-prepared solubilized surface antigens contain up to 90 % less fiber from proteins or other nitrogen-containing compounds compared to industrially produced whole virus vaccines. The reactogenic aluminum content may be reduced by more than or equal to 65 % in the case of the production of adsorbed surface antigens without the adsorption of

 reduction rate. A reduction of the input antigen content by z. B. half of the full-virus

-4-; 2 2 6778

vaccine contained amount is acceptable. These factors lead to the reduction of reactogenity for the immediate type hypersensitivity reaction; at least. 65% ..- .. ·. :. and after the late type by at least 50 % ..

Explanation of the essence of the invention

The invention has for its object to provide a technically straightforward leading method for the production of low-fiber inactivated influenza vaccines in the form that result by targeted solubilization of the surface antigens of influenza viruses of the corresponding types highly purified vaccines, which are also suitable for at-risk individuals and for use in children ,

The object is achieved according to the invention by treating influenza viruses of the corresponding types only by adding sodium disodium cholate as the only additional adjuvant and otherwise without changing the environmental conditions by shaking or another way of suspending, or by simply leaving it standing while substantially retaining the technology used for influenza virus vaccines and then separated into a soluble and an insoluble phase. The insoluble phase contains the essential fiber. The soluble phase, which primarily contains the surface antigens hamagglutinin and neuraminidase, is prepared for vaccine.

The advantage of these vaccines from solubilized surface antigens compared to whole virus vaccines is in particular that the fiber is not applied when using the vaccines in humans and thus the side effects are significantly reduced.

Specifically, the following can be done: Influenza viruses are isolated from virus-containing allantoic fluid by e.g. Ultracentrifugation concentrated. The virus concentrate obtained is treated with phosphate-buffered physiological

- 5 -22 6778

NaCl solution with addition of sodium deoxycholate (about 0.15 % final concentration). After a short incubation at room temperature, the virus cores are separated by ultracentrifugation. The supernatant contains: 'the surface ^:! : Antigens in relatively pure form and therefore they can be adsorbed to a significantly reduced amount of adsorbent (compared to "whole virus vaccine") .This adsorption, eg to alumina hydrate, also allows almost complete separation of the sodium deoxycholate used as well as other impurities (Sucrose, N-containing fiber, etc.) The resulting adsorbate is adjusted to the required antigen content of the vaccine.

Exemplary embodiments Example 1

To solubilize the oesophageal haemagglutinin and heuraminida.se virus-containing suspension (X-71: A / PR / 8 / 34χA / Brazi1 / 11/78-H1N1) is added after the addition of sodium deoxycholate in the ratio of 300 μg hemagglutinin. Treat 1 500 pg of sodium deoxycholate on a horizontal shaker for 30 minutes. Thereafter, the fiber is separated by ultracentrifugation (55 000 to 65 000 g, 120 min) at 8 ⁰ C as a sediment. The supernatant obtained after centrifugation is tested for the absence of viable viruses and adjusted to the required antigen content. For dilutions, phosphate-buffered physiological NaCl solution with 0.01% thiomersal is used.

This preparation results in a vaccine of the following composition per dose: 28 μg hemagglutinin, 45 μg protein, less than 0.25 μg ovalbumin, 14 μg Kjeldahl-N.

Example 2

The procedure is as in Example 1.

The supernatant prepared in this way becomes after the adjustment of the Häinagglutiningehaltes with Aluminiumoxidhydratsuspension

added. After centrifugation at 1000 g with cooling, the supernatant is discarded and the sediment is filled up to the same volume with phosphate-buffered physiological NaCl solution. All preparation steps. under aseptic conditions. ·

This vaccine has the same composition as given in Example 1 with an additional 0.25 mg of Al per dose o

Example 3

The procedure is as in Example 1.

After the haemagglutinin content has been adjusted, the supernatant prepared in this way is mixed with equimolar amounts of calcium chloride and disodium hydrogen phosphate solution. For the adsorption of the .Oberflächenantigene to the Calciumzydrogenphosphat formed occurs short-term stirring of the suspension After centrifugation at 1 500 g with cooling, a sediment is recovered, which is taken up with phosphate-buffered physiological NaCl solution and centrifuged again. The resuspension of this sediment is again with phosphate buffered physiological NaCl solution with 0.01 % thiomersal.

This vaccine has the same composition as given in Example 1 with an additional 0.5 ng Oa per dose.

Example 4

The procedure is as in Example 1.

To solubilize the surface antigens of the viruses of strain B / Singapore / 222/79, 1 000 μl of sodium deoxycholate are added per 670 μg of hemagglutinin.

Claims (13)

1. A process for the preparation of a low-fiber inactivated Influenzaimpfstoffes by treatment with sodium deoxycholate, characterized in that a solubilization of the surface antigens hemagglutinin and neuraminidase takes place and are enriched after removal in the soluble phase as vaccine antigens. 2. Method according to item 1, characterized in that the vaccines obtained with sodium deoxycholate have the same quality as when using ammonium deoxycholate as detergent, 3 * Procedure according to item 1 ,. characterized in that. an elimination of viral and non-viral Begle.itstoffen, in particular of proteins, at a weight ratio of sodium deoxycholate: hemagglutinin of 1: 1 to IcICf ' ¹ takes place. 4. The method according to item 1, characterized in that the initial or cleaning state of the influenza viruses or the classification of the solubilization step in the process without significant influence on the removal of accompanying proteins, which is between 50 to 80%, is. 5. The method according to item 1, characterized in that the arrangement of the surface antigens in their complexes a nearly 100% adsorption, for example, to alumina hydrate for biological preparations, guaranteed and thus .Begleitstoffe, for example, originating from the density gradient centrifugation, separated. 6. The method according to item 1, characterized in that the infectivity of viruses is completely or largely lost in the solubilization and a chemical inactivation of Influenzaviren can be omitted and thus formaldehyde-free vaccines can be obtained · 7. The method according to item 1, characterized in that the process conditions applicable to the previously known influenza viruses and especially for influenza A viruses of the subtype Η1ΪΓ1 are applicable to produce mono-, bi ~ and polyvalent vaccines and combine in the application »   - 7 - 22 6 778 claims 8. The method according to item 1, characterized by »that also influenza viruses of different types and Subtyperi can be treated in admixture with the detergent and then won the surface antigens together immediately v / earth. -8-226778 , '- 9 - 22 6 778 9. The method according to item 1, characterized in that, for example, can be prepared so that in HiN1 strains the following purity criteria can be achieved in the corresponding vaccine: Hemagglutinin content of 0.058 mg / ml, protein (Lowry) with 0.092 mg / ml, ovalbumin at less than 0.0005 mg / ml, carbohydrates at 0.041 mg / ml, sodium desoxycholate at less than 0.09 mg / ml and aluminum at 0.59 mg / ml. 10. The method according to item 1, characterized in that the vaccines are particularly suitable for application in children. and also without inorganic adjuvant. show an immune response. 11. The method according to item 1, characterized in that with a single immunization (0.5 ml subcutaneous / vaccine) for a significant increase of the heme agglutination inhibiting titer 20 to 50 / ig hemagglutinin / ml vaccine and strain, preferably 30 to 40 jig / ml, are sufficient » 12. The method according to item 1, characterized in that with two immunizations (0.5 ml each subcutaneously) • for a significant increase in titre, 5 to 10 μg hemagglutinin per ml and strain, preferably 10 μg / ml, are sufficient. 13. The method according to item 1, characterized in that after two immunizations (0.5 ml each subcutaneously), a significant increase in neuraminidase inhibition titers is achieved.