MXPA98002766A - Vaccine against influe - Google Patents

Abstract

The present invention relates to an influenza surface antigen vaccine obtainable by production from influenza viruses propagated in animal cell culture and having a host cell DNA content equal to or less than 25 pg per dose. The present invention also relates to a method for the preparation of surface antigen protein from influenza viruses propagated in animal cell cultures comprising the subsequent steps of: a. treatment of the whole virus containing fluid obtained from cell culture with an enzyme that digests DNA, and b. add a cationic detergent, followed by isolation of surface antigen proteins

Description

INFLUENZA VACCINE DESCRIPTIVE MEMORY The present invention relates to influenza surface antigen vaccines obtainable by the production of influenza viruses propagated in animal cell culture and with a method for the preparation of surface antigen proteins of influenza viruses propagated in culture of animal cells. The body of the influenza virus has a size of approximately 125 nm and consists of a ribonucleic acid (RNA) center associated with the nuci eoprotein, surrounded by a viral envelope with a lipid bilayer structure. The inner layer of the viral envelope is composed predominantly of dp matrix proteins and the outer layer contains most of the lipid material derived from the host. The so-called "surface proteins" neuraminidase (NA) and haemagglutinin (HA) appear as spikes on the surface of the viral body. Most commercially available inactivated influenza vaccines are called "divided vaccines" or "subunit vaccines." "Split vaccines" are prepared by treating the entire influenza virus with Concentrations of sun, detergent and subsequent removal of the detergent and the mass of the viral lipid material. "Subunit vaccines" against influenza, unlike "split vaccines," do not contain all viral proteins. Instead, "subunit vaccines" are enriched in surface proteins responsible for the induction of neutralizing (and thus protective) antibodies to viruses desired with vaccination. Most commercially available influenza vaccines are derived from influenza viruses grown in chicken embryos. However, it is widely recognized that the production derived from eggs of an influenza virus for vaccine purposes has several disadvantages: 1. This production procedure is rather vulnerable due to the variable microbiogenic quality of the eggs. 2. The procedure completely lacks flexibility if it suddenly increases demand, that is, in the case of a severe epidemic or pandemic, due to the logistical problems that result from the unavailability of large quantities of suitable eggs. 3. Vaccines thus produced are contraindicated for people with known sensitivity to chicken and / or egg proteins.

A solution to these problems may lie in the tissue-derived production of influenza virus. This production method is considered to have many advantages: 1. Tissue culture cell lines are available in well-defined cell banking systems free of icrobiological contaminants, so consistency from one batch to another is greatly improved and you get an ayor quality product. 2. It will increase the changes to have enough available vaccine in case of threat of epidemic or severe pandemic. 3. The resultant influenza virus material will be better adapted for alternative routes of administration (oral, nasal, inhaled). . From the point of view of WHO, the technology will postpone the recommendation of annual vaccine composition (from mid-February to mid-March), increasing the equalization of the vaccine with circulating strains. However, an important problem persists in relation to the culture of influenza virus tissue, as a genetic material from continuous cell lines that may be present in vaccines. This problem has a risk, which, if it is not remedied, can lead the regulatory authorities to declining the requirements for market permission for such influenza vaccines for safety reasons. For example, l U.S. The Food and Drug Administration (US Food and Drug Administration) demands that biotechnological products for human use contain no more than 100 pg of host cell DNA per dose. Thus, the present invention provides a method for the preparation of influenza virus surface antigen for vaccine purposes that is safe and does not contain unacceptable amounts of deleterious genetic material, and satisfies the requirements set forth by the regulatory authorities. However, it was considered desirable and also surprisingly achievable to prepare influenza vaccines with a host cell DNA content considerably less than 100 pg / dose. Accordingly, the present invention relates to an anti-influenza antigen vaccine obtainable by the production of influenza viruses propagated in animal cell culture and having a host cell DNA content equal to or less than 25 pg. per dose. In a specific embodiment, the present invention provides a method for the preparation of surface antigen proteins useful for preparing said low DNA influenza vaccine from influenza viruses propagated in animal cell culture comprising the steps of Subsequent to: a) treating the whole virus containing fluid obtained from the cell culture with an enzyme that digests DNA, and b) adding a cationic detergent, followed by isolation of the surface antigen proteins. The method according to the present invention can be applied during the application of vaccines containing various strains of influenza viruses such as viruses typical for human influenza, swine influenza, pneu influenza and poultry influenza. The culture of animal cells according to the present invention may contain either primary cells, such as chicken embryo fibroblasts or a continuous cell line, such as dog kidney cells (MDCK) Chinese hamster ovary cells (CHO) and Vero cells. The treatment of the whole virus containing fluid with enzyme that digests DNA can be carried out directly in the fermenter, optionally already during the cell culture and the viral propagation process. Suitable examples of enzymes that digest DNA are DNase (for example classified according to EC 3.1.21 and EC 3.1.22) and nucleases (e.g., classified according to EC 3.1.30 and 3.1.31). Suitable cationic detergents according to the present invention consist predominantly of a composed of the general formula Ri «^ ^? > R3 R2 - ^^^^^ * where R1, R2 and R3 are the same and different and each signifies alkyl or aryl, or Ri and R2 together with the nitrogen atom to which they are attached form a saturated heterocyclic ring of 5 or 6 members, and R3 means alkyl or aryl, or Ri, R2 and R3 together with the nitrogen atom to which they are attached, means a 5- or 6-membered heterocyclic ring, unsaturated at the nitrogen atom, R means alkyl or aryl, and X means an anion. Examples of said cationic detergents are salts of cetyltrimethylammonium, such as cetyltrimethylammonium bromide (C.T.A.B.), and salt of my ristiltimammonium. The appropriate detergents are also lipofectin, lipofectamine, DOTMA. Optionally these cationic detergents can be supplemented with a nonionic detergent, such as Tueen. The isolation of the surface antigen proteins subsequent to the step of the detergent treatment, for example, may comprise the steps of: 1. Separation of the particle from the RNP particle (body) dp the proteins of the antigen of supprficie, for example, by centrifugation or ultratification, and 2. Removal of the detergent from proteins of the surface antigen, for example, by hydrophobic interaction of the detergent with a suitable resin (such as Anderlite XAB-4) and / or by ultradiafiltration. Surprisingly, the method according to the present invention gives a product that PS ex purely low in its content of DNA derived from animal cells. DNA concentrations as low as 25 pg / dose and in many cases even as low as 10 pg / dose are easily achievable. The surface antigen proteins can be processed to prepare the influenza vaccine, for example by adding pH buffer (for example PBS) and / or mixing with agents from other stereotypes of influenza viruses. Optionally, the concentration of the surface antigen is required for subsequent vaccine preparation.

EXAMPLE 1 A. Virus multiplication 1.- Influenza virus antigen type B / Yamagata multiplies in Madin canine kidney cells Darby Canine Kidney (MDCK) (ATCC CCL34.) In a fermentor incubating the seed virus with the cells for 2 days at 35 ° C. 2.- Next, the pH of the fermenter fluid is increased to 8.0 mp by the addition of dilute sodium hydroxide and Benzon nuclease is added to the final concentration of 1000 units (1 μg) per liter. 3.- Incubation is carried out at 35 ° C for another four hours.

B. Isolation of the virus 1.- The virus is filtered through a depth filter with a nominal pore size of 0.5 microns to remove the cellular debris. 2.- Subsequently, the influenza virus is concentrated and purified by ultrafiltration using a membrane with a molecular weight cutoff of 300,000. 3.- Sucrose is added to the concentrate at a final concentration of 30% (w / v) after which formaldehyde is added to a final concentration of 0.015% (w / v). This mixture is stirred at 2-8 ° C for 72 hours. 4.- Next, the virus concentrate is diluted 5 times with saline regulated in its pH with phosphate and loaded onto an affinity column containing ami sulfate with celluin (Amicon Cellufine Sulphate). After removing the impurities by washing with saline regulated in its pH with phosphate, the virus is eluted with a 1.5 molar solution of sodium chloride in an outlet solution regulated in its pH with phosphate. The diluted product is concentrated and desalted by ultrafiltration using a 300,000 molecular weight cutoff membrane.

C. Insulation of subunits 1. - Add non-ionic detergent Tween-80 to a final concentration of 300 μg / ml and cetyl trimethylamine bromide is added to a final concentration of 750 μg / ml. This mixture is stirred at ° C for 3 hours after which the RNP particle is separated from the surface antigen proteins by centrifugation. 2.- The supernatant is stirred with Amberlite XAD-overnight at 2-8 ° C to remove the detergents. The berry is removed by filtration and the filtrate is subsequently subjected to sterile filtration by passing it through a 0.22 micron filter. Throughout the above procedure, the DNA content of the sample cells was analyzed according to a valid test, based on slotted blot hybridization using a canine DNA probe labeled with 32 P. the results of the test of DNA made after several steps are given in the following table (in this table, the amount of DNA per IVV dose is expressed in picograms per 50 μg HA).

EXAMPLE 2 The mu tylication, purification, inactivation and cutting of the virus are affected as in example 1. The treatment with Benzon nuclease of the fermenter fluid during the last hours of the multiplication of the virus (steps A2 and A3), using the pH of the medium of the multiplication (step Al), gives similar results with respect to DNA removal. DNase I, although it is required in concentrations larger, is equally effective in removing DNA from host cells (during steps A1-A3). Similar results can be obtained using other endonucleases.

EXAMPLE 3 The procedure is carried out as in Example 1 or 2 except that it was removal of cell debris (step Bl) is effected by centrifugation.

EXAMPLE 4 The procedure is carried out as in Example 1, 2 or 3 except that the virus is concentrated and purified (step B2) from the fermenter fluid by centrifuging in a continuous flow zonal centrifuge, for example, Electro-Nucleonics (model RK ) using a gradient of sucrose in the saline regulated at its pH with phosphate.

EXAMPLE 5 The procedure is carried out as in example 1 to 4, except that the addition of cetyl trimethyl ammonium bromide solution in step Cl is replaced by the addition of a solution of cetylpiperidium bromide, myristyltrimonylammonium bromide, chloride of benzetonium, chloride methylbenzetonium, decamethonium chloride or stearyl dimethyl Ibenci lamonium bromide. Similar results are obtained with respect to DNA removal from host cells.

EXAMPLE 6 The procedure is carried out as described in example 1, except that the addition of sucrose is carried out after affinity column chromatography and formaldehyde is added at a maximum of 0.05% (w / v) for 120 hours.

EXAMPLE 7 The steps in accordance with examples 1, 2, 3, 4 and 6 were successfully applied for e for low DNA vaccines from influenza viruses of B / Harbin strains, B / Panama, A / Texas (H1 1), A / Taiuan (H1N1), A / Johannesburg (H3N2) and A / Wuhan (H3N2).

Claims (9)

NOVELTY OF THE INVENTION CLAIMS

1. - An influenza surface antigen vaccine obtainable by production from influenza viruses propagated in animal cell cultures and having a host cell DNA content equal to or less than 25 pg per dose.

2. An influenza surface antigen vaccine according to claim 1, further characterized in that it has a host cell DNA content of less than 10 pg per dose.

3. A method for the preparation of surface antigen proteins from influenza viruses propagated in an animal cell culture comprising the following steps of: • treatment of the whole virus containing Fluid obtained from the culture of cells with a enzyme that digests DNA, and b add a cationic detergent, followed by the isolation of surface antigen proteins.

4. The method according to claim 3, further characterized in that the treatment with enzymes that digest DNA takes place during the propagation of the influenza viruses in the cell culture.

5. A method according to claim 3, further characterized in that the cationic detergent consists of First of all a compound of the generic formula Ri R3 \ /. N + X-R2 XR where Ri R2 and 3 are the same or different and each means alkyl or aryl or Ri and R2, together with the nitrogen atom to which they are attached form a saturated 5- or 6-membered heterocyclic ring and R3 means alkyl or aryl or Ri, 2 and R3 together with the nitrogen atom to which they are attached mean a 5 or 6 membered heterocyclic ring, unsaturated at the nitrogen atom, R /, means alkyl or aryl, and X means an anion .

6. A method according to claim 3, further characterized in that the cationic detergent comprises predominantly ceti 1trimeti lamonium glomide.

7. A method according to claim 3, further characterized in that the cationic detergent is complemented with a non-ionic detergent.

8. A method according to claim 3, further characterized in that the influenza viruses are propagated in a line of animal cells.

9. A method according to rei indication 3, characterized in that influenza viruses are propagated in MDCK cells.