DD300833A7 - Method for the production of inactivated influenza full virus vaccines - Google Patents

Abstract

The invention relates to a method for obtaining highly purified influenza virus and its use for the production of influenza vaccines, which have a very good compatibility due to their degree of purity. The object of the invention is an industrially applicable method of obtaining inactivated influenza whole virus vaccines containing essentially only the influenza viruses and not egg proteins from engineered crude virus products. The essence of the invention is that by a combination of virus adsorption of calcium phosphate with a precipitate by means of ammonium sulfate in concentrations of 0.3 to 0.7 mol / l and subsequent clarification centrifugation and clarification intensive pre-purification of Rohvirusprodukte by separating in particular high molecular weight impurities achieved sedimentation properties similar to influenza viruses and a zonalzentrifugation advantageous, particle-free and purified virus solution is obtained.

Description

Field of application of the invention

The invention relates to a method for obtaining highly purified influenza virus and its use for the production of influenza vaccines, which have a very good compatibility due to their degree of purity.

Characteristic of the known state of the art

For the production of influenza vaccines embryonated chicken eggs is assumed in the known method of the virus-containing allantoic fluid. After a strain-specific duration of influenza virus proliferation, the allantoic fluid is removed from the chicken eggs. This process has been largely mechanized and semi-automated in recent years in the interests of more economical vaccine production. However, in addition to the reduction in labor and time required, the performance requirements of subsequent concentration and purification procedures have also increased, as greater contamination of crude virus products with broiler constituents in automated harvesting operations can not be avoided. In the known processes for the production of influenza vaccines, after a clarification centrifugation (about 10 ⁴ g) to remove cellular components from the allantoic fluid of the virus is concentrated and freed of dissolved impurities.

The majority of vaccine manufacturers have been using REIMER, C. B. et al. (Journal of Virology 1 [1967] 1207-1216) Zonalzentrifugation in flow rotors for the large-scale production of influenza vaccines. The capacity of the cane sugar gradients used and the purity of the density gradient enriched virus fraction can be improved by intensive pre-purification of the processed crude virus product. The preparation thus becomes more economical and leads to purer and more compatible vaccines (HINZ, J. et al., European Symposium of Zonal Centrifugation in Density Gradients, Spectra 2000, 1973, No. 4,211-214). By pre-cleaning, in particular, high-molecular contaminants with similar sedimentation properties as the influenza viruses (sedimentation coefficient, isopycnic density) should be separated while the virus activity is largely preserved. Otherwise, such contaminants completely or partially overlap the virus band in the density gradient. Importantly, the methods used for the prepurification have only minor strain-specific differences, meet the conditions of industrial vaccine production and allow the processing of engineered crude virus products (higher level of hatchery contaminants). The methods described in the patents DE 1617635 and US 3485718 virus purification by adsorption / elution of barium sulfate prove to be technologically disadvantageous because repeated sedimentation processes including the separation of sediment and supernatant time-consuming labor and a ... aseptic work difficult. The recovery of the adsorbed virus antigens is not quantitative and has according to their own studies strain-dependent differences. The purification and concentration of influenza viruses with polyethylene glycol (PEG) precipitations is based on POLSON (Preparative Biochemistry4 (1974) 435-456) and is described in detail in the patents GB 1358734, DE 2258300 and DE 2847959 and SU 543674. The cleaning effect achievable with this method is determined by the composition and concentration of contaminants that undergo considerable variation upon mechanical recovery of the raw virus products. In particular, changing concentrations of lipids and lipoproteins negatively affect PEG precipitation.

Combined methods involving Llpidextraktion with organic solvents (DE 2258300) lead according to our own investigations to significant losses of virus activity.

As a technologically disadvantageous proves associated with the PEG precipitation sedimentation of Precipitate, which is to be overcome in industrial vaccine production only in flow rotors of costly industrial separators. The purification of influenza viruses by adsorption on mineral aluminum compounds has been studied by several authors. MILLER u. SCHLESINGER (Journal of Immunology 75 (1355) 155-160) describes an adsorption chromatographic method using aluminum phosphate In addition to the difficulties of using chromatographic methods on an industrial scale (working under aseptic conditions), the method showed reduced yields in the processing of B-salts. DRESCHER (American Journal of Hygiene 74 [1961] 104-110) and SENGBUSCH (FEBS Letters 15 [1971] 78-80) used alumina or aluminum hydroxide and aluminum phosphate for adsorption in the batch process. Analogously, here are the findings made for the use of barium sulfate findings, in particular, the adsorption of mechanically recovered crude virus products proves to be not reproducible. The method described in the patent DD 114616 is not suitable for the intended use, since the adsorbed virus antigen remains on the mineral carrier and is used to increase the immunogenic effect in this form. Such vaccines have only a limited purity and effectiveness, the production of vaccines, which in addition to two influenza virus Α strains include a B strain is not possible by this method. A vortoil over the described Adsorptionsmaihoden provides the adsorption of virus antigens to calcium phosphate. The procedures are described in patents US 3197374, US 3316153 and US 3478145. Calcium phosphate gels can be dissolved in the batch process after virus adsorption by solutions of suitable complex-forming compounds (sodium salts of ethylenediaminetetraacetic acid), which allows complete recovery of the adsorbed virus antigens. The quantitative adsorption can be controlled by the amount of adsorbent and could be detected for all previously examined virus antigens of serotypes A and B in corresponding experiments. The process results in a separation of more than 90% of the low molecular weight protein contaminants. The removal of high-molecular impurities from the propagation substrate with similar sedimentation properties as the influenza viruses, however, succeeds only insufficiently with this technique. In addition to their influence on the reactogenicity of the vaccines, such contaminants cause considerable loss of activity by forming gel layers (secondary membranes) in the sterile filtration of the purified virus concentrates. Under unfavorable conditions, the yield losses can amount to up to 50% of the viral material used.

Object of the invention

The aim of the invention is an industrially applicable method for obtaining inactivated influenza whole virus vaccines containing essentially only the influenza viruses and not egg proteins from mechanically recovered crude virus products.

Explanation of the essence of the invention

The invention has for its object to develop a method for achieving the above object so that effectively removed by intensive pre-purification of Rohvirusprodukte especially high molecular weight contaminants with similar sedimentation properties as influenza viruses and thus optimal conditions for a maximum capacity of the Rohrzuckergradienten be created in Zonalzentrifugation simultaneous high purity of the virus zone in the gradient. According to the invention, it has been found that by a combination of the known adsorption on calcium phosphate with a subsequent precipitation by means of ammonium sulfate in concentrations of 0.3 to 0.7 mol / l, a highly effective pre-cleaning takes place, which leads to the separation of above all such impurities, which have the same isopyknischo density as the influenza virus.

This precipitation is combined with a subsequent clarification centrifugation by the flow-through method and clarification filtration over cellulose acetate or cellulose nitrate membranes, whereby a particle-free and purified virus solution which is advantageous for zonal centrifugation is obtained. According to the invention, it has been found that virus losses in the clarification by impregnation of the membranes with a solution of the disodium salt of ethylenediaminetetraacetic acid (EDTA) in concentrations of 0.01 to 1 mol / l and hydrolyzed gelatin in a concentration of 0.1 to 0.5% be avoided in phosphate buffered NaCl solution. The pre-purification by ammonium sulfate precipitation also leads to a complete separation of compounds of uric acid (urates) from the raw virus products, which otherwise contaminate the fractions of the cane sugar gradient due to diffusion. The processing capacity of a Zonalzentrifugation in K 3 rotors from. Electronucleonics Inc./USA can be increased as a result of the inventive pre-purification of usually 150 to 200I (based on volume of allantoic fluid) to 200 to 600I (depending on the virus concentration). The conditions for the sterile filtration of the inactivated virus fraction of the cane sugar gradient and the stabilization of the monovalent virus bulbs and the influenza virus full vaccines could be improved according to the invention by adding a stabilizer of hydrolyzed gelatin in a concentration of 0.1 to 0.5%. Compared with the use of native gelatin (CA 987230, US 3880993), this stabilizer does not affect the filtration capacity and excludes undesirable side effects in the recipients of the vaccines. The purity of influenza whole virus vaccine produced by the method described is 14 to 20 μS protein nitrogen per vaccine dose at a hemagglutinin concentration of 35pg per vaccine dose. The maximum recommended by the World Health Organization of 50pg protein nitrogen per vaccine dose (Technical Report Series 638, WHO, Geneva 1979) is well below.

Ausfuhrungsbelsplel

2001 influenzavirus Allantolsflüssigkeit are added with stirring with 5.281 Na ₂ HPO ₄ solution (0.5 mol / l) and 4.801 CaCl ₂ solution (0.5 mol / l). After sedimentation of the resulting calcium phosphate gel and pumping off the supernatant, the gel is added in the stoichiometric volume of 10.01 equivalent to the amount of calcium phosphate to a solution of the disodium salt of EDTA (0.26 mol / l) adjusted to a pH of 8.3. dissolved. From the partially purified and concentrated virus solution high molecular weight impurities and urates by adding a pH adjusted to pH 7.2 (NH ₄ ) ₂ SO ₄ solution ( ₄ mol / l) to a final concentration of 0.3 mol / l (NH ₄ I] SO _{4, followed} by clarification by centrifugation (CEPA centrifuge Z61G, 17000 g, 100 l / h) and clarifying filtration through cellulose acetate membranes rinsed with an impregnating solution, and the prepurified virus solution is converted into a particle-free, purified form suitable for subsequent flow-zonal centrifugation Phosphate-buffered NaCl solution, pH 7.2, with additions of 0.1 mol / l EDTA-Na ₂ and 0.2% hydrolyzed gelatin The ultracentrifugation is carried out in a flow-rate rotor (K 3 rotor, ultracentrifuge Model K Mark II, 3500 μm ^-1 ), which was loaded with a cane sugar gradient (0-60% sucrose) a peristaltic pump set to 12l / h. After completion of the centrifugation, the virus-containing gradient is fractionally collected.

The virus fractions are in the concentration range of 38 to 48% sucrose. For further processing to monovalent vaccine concentrate, the virus fractions are combined (about 0.5I), inactivated with formaldehyde (0.025%, 72 h, 4 ⁰ C), with phosphate-buffered NaCl solution, pH 7.2 to a concentration of about 100pg Hemagglutinin / ml diluted, treated with hydrolyzed gelatin (final concentration: 0.2%) and sterile filtered.

Claims (4)

1. A process for the preparation of inactivated influenza whole virus vaccines containing essentially only the complete, non-viable Influenzaviren and no egg proteins by adsorption of virus-containing allantoic liquid to calcium phosphate, dissolution of the resulting gel in the disodium salt of EDTA, Klärzentrifugation, Klärfiltration over with the disodium salt cellulose acetate or nitrate membranes impregnated with EDTA, flow zonal centrifugation in a cane sugar gradient, inactivation with formaldehyde solution, stabilization, adjustment of a defined virus concentration and further processing into a sterilized, monovalent virus bulker, characterized in that

after dissolution of the calcium phosphate gel, high-molecular-weight impurities and urates with 0.3 to 0.7 mol / l of ammonium sulfate fall,

the impregnation of the cellulose acetate nitrate membranes with a solution of the disodium salt of EDTA in concentrations of 0.01 to 1 mol / l and hydrolysed gelatin in a concentration of 0.1 to 0.5% in phosphate-buffered NaCl solution,

- Added to the influenza whole virus vaccine for stabilization hydrolyzed gelatin (commercially available under the name Gelafusal) in a concentration of 0.1 to 0.5%.

2. The method according to claim 1, characterized in that one adds 0.5 mol / l ammonium sulfate.

3. The method according to claim 1, characterized in that one uses for impregnation, a solution of the disodium salt of EDTA of 0.1 mol / l and hydrolyzed gelatin of 0.2%.

4. Process according to claims 1 to 3, characterized in that one uses for stabilization hydrolyzed gelatin in a concentration of 0.2%.