NO171147B - PROCEDURE FOR THE PREPARATION OF A Hepatitis B vaccine - Google Patents

Abstract

Particles of hepatitis B virus surface antigens (HBsAg) are produced by expression from a culture of CHO cells transfected with a plasmid, recovering the culture supernatant which, after sterilising filtration, is concentrated and subjected to a fractional precipitation with PEG, the precipitate being taken up and subjected to a zonal velocity centrifugation and then to a zonal flotation centrifugation, following which chromatography is performed in an anion exchanger medium. The vaccine is noteworthy for its purity and its immunogenicity, in particular for its capacity for inducing anti-pre-S2 antibodies.

Description

The present invention relates to a method for producing a vaccine against hepatitis B.

All kinds of difficulties that occur in connection with the industrial production of vaccines against hepatitis B when purifying the antigen HBsAG from human blood have quite naturally led to research for obtaining antigenic proteins from microorganisms that have been transfected with genes that code for the hepatitis B virus.

M. Michel et al. have expressed hepatitis B virus envelope antigens from Chinese hamster ovary (CHO) cells

(see M. Michel (1984) Proe. Nati. Acad. Sei. USA 81,7708-7712), transfected with a plasmid carrying the complete EBsAg gene. These cells continuously secrete into the cell culture medium HBsAg particles containing both pre-S2 and S proteins.

However, during such expression, the HBsAg particles are present in the cell culture medium which contains unwanted substances such as proteins from the calf serum used for cell growth, cell proteins and DNA and retroviral particles. These retroviral particles come from the original cell before transfection.

The present invention proposes that these disadvantages are overcome by providing a method for producing a vaccine against viral hepatitis B by expression of the HBsAg gene introduced in CHO cells, which makes it possible to produce a vaccine against hepatitis B industrially.

Another object of the invention is to provide a method which makes it possible to produce a vaccine which has an extremely high degree of purity, especially with regard to the cellular DNA or DNA fragments and also with regard to the retrovirus that contaminates the CHO cells.

According to the present invention, a method is thus provided for the production of a new recombinant vaccine against hepatitis B, where antigenic surface particles of hepatitis B are produced by expression from a culture of CEO cells transfected with a plasmid carrying the EBsAg gene to thereby release the the antigenic surface particles in the culture medium, characterized in that one

recovering the supernatant culture medium from at least one culture, preferably made with a low animal serum content,

subjecting the supernatant medium to sterilizing filtration,

subjecting it to concentration, preferably approx. 50

times, preferably by ultrafiltration,

treating the concentrate with a non-degrading precipitant selected from polyethylene glycol and ammonium sulfate, under such conditions as to precipitate the heavy DNA materials, which precipitation also entrains the retroviral particles, and proteins,

carries out a new concentration of the antigenic preparation,

preferably with a non-degrading precipitant under conditions which precipitate the antigenic EBsAg surface particles, and then redissolve the precipitate in a small volume,

performs a speed-regulated zone centrifugation in a non-chaotropic density gradient for the retroviruses so that their integrity is maintained in order to thereby achieve their separation from the EBsAg particles, depending on their size and density, preferably also by achieving distinction between the antigenic EBsAg particles on on the one hand, and separation between EBsAg, DNA and proteins on the other hand, — performs an isopycnic centrifugation of

The flotation type, which eliminates the light and heavy nucleic acids and proteins, and

performs chromatography on an anion exchanger to adsorb the traces of nucleic acids and residual non-EBsAg proteins.

By non-degrading precipitating agents is meant agents which, of course, do not degrade the EBsAg antigens and especially those of type S and pre S, but also those which have no cotropic or dissociating effect for the retroviral particles. The preferred agent is polyethylene glycol (PEG). As mentioned, ammonium sulphate can also be used.

The purification steps thus do not denature the EBsAg particles and in particular they do not denature the pre S proteins, which are the most exposed. Among suitable precipitating agents, polyethylene glycol (PEG) is preferred by more particularly choosing a molecular weight of the order of 4,000-20,000, which under suitable conditions such as more particularly a PEG concentration of the order of 5$, makes it possible to precipitate the retroviral particles while also in particular, precipitation of the heavy DNA classes is enabled while the EBsAg particles are kept in the supernatant. These particles can be recovered in a very small volume, preferably after precipitating them by adding PEG to a higher concentration.

The density gradient in which the speed controlled zone centrifugation is performed is also provided so that it does not adversely affect the EBsAg antigen, including S and pre S antigenities. Furthermore, this gradient is provided so that it has no chaotropic effect on the retroviral particles so that their integrity is maintained. This gradient, which has a low ionic strength, is preferably a sucrose gradient. In a variant, other density gradients can also be used, especially glycerol.

The speed-regulated zone centrifugation carried out in such a density gradient enables the elimination of the retroviral particles in a very efficient manner. At the same time, a certain amount of DNA purification can be achieved, especially in heavy chains and in proteins.

The isopycnic zone centrifugation of the flotation type on a saline density gradient, such as KBr, is thereby performed on a preparation that is practically completely free of retroviral particles. If retoviral particles contaminate the sample to be purified, however, the chaotropic effect of the salt forming the density gradient would destroy the viral structure and would release the viral RNA genome into the dense zone of the gradient where it would stop and thereby enable separation from The HBsAg antigen.

This step enables the elimination of virtually all the proteins as well as existing light or heavy DNA traces.

In addition, the step of anion exchange chromatography ensures the adsorption of possible nucleic acid traces as well as of DNA and RNA that are still left and of proteins.

Other advantages and characteristics of the present invention will be apparent from the example below.

1. Preparation of the cell culture

A CHO cell colony clone transfected with a recombinant plasmid (FR-A 84 03564 published as FR 2 560 890) carrying the gene for the hepatitis B virus encoding the HBS antigen (pre S and S regions) is propagated from a container with cell stores, in fermentation units of increasing volumes. The last propagation is carried out in a 300 liter fermentor, with the cells being propagated on microspheres in suspension in the culture medium. For these propagation steps, the growth medium contains 4-10% CS (calf serum), preferably 5%.

2. Cultures, harvesting and concentration

The first harvest is made after 3-4 days of cultivation in the 300 liter fermenter: After decanting the spheres supporting the cells, 250 1 of supernatant is removed and replaced with an equivalent volume of new medium containing 0.2-4$ CS, preferably 1-2 $. The cell culture is continued in the fermenter for 3-4 days, after which a new harvest of 250 1 is made. This operation can be carried out with up to 10 successive harvests, and these are kept at +4°C for purification thereof. As a general rule, the first harvest and harvest No. 7 and subsequent ones have a low HBs antigen content so that they are not retained for the purification steps.

In this example, harvests 2-6 were retained for purification and contained 1% CS.

After mixing the harvest material, the preparation, which has a volume of 1,250 1, is filtered on a 0.2 micrometer membrane, then concentrated by ultrafiltration approx. 50 times on a membrane with a cutoff point of 100,000 daltons, yielding concentrated harvest material R.

3. Fractional precipitations

The concentrated harvest material R is subjected to fractionation by selective precipitation in a first step for the high molecular weight components, especially heavy classes of DNA and retroviral particles, the HBS antigen remaining in suspension, and in a second step the EBs antigen is precipitated. The precipitant is chosen so that the retroviral particles retain their original size and density on the one hand, and on the other hand so that the antigenicity of the HBs particles is not adversely affected. The preferred precipitant is polyethylene glycol, preferably of 6,000

MW.

A concentration of 5$ of PEG 6000 is used for the first precipitation, and the precipitate obtained is eliminated by centrifugation. PEG 6000 is added to the supernatant S for a final concentration of 9.5$; as the sediment after centrifugation is completely dissolved in a volume equal to approximately one 625th of the treated harvest volume, namely approx. 2 litres, of antigenic EC preparation.

At this stage, the performance capability in terms of elimination of the cellular DNA, and non-HBsAg protein given by the values in the following table:

While the recovery rate of the EBs antigens is 80$, elimination factors of 42,000 for the DNA and 38.5 for the proteins were noted.

The reverse transcriptase activity, which reflects the retrovirus content, is not detectable in the harvests, and is sometimes weakly positive in the harvest material which is concentrated 50 times, and it is always negative in the supernatant after treatment with 5$ PEG.

The effect in terms of removal of a retrovirus by this treatment step with 5$ PEG was measured by supplementing a concentrated harvest material with MuLV (murine leukemia virus), and this gave an elimination factor of 7.

4. Speed-regulated zone centrifugation

A sucrose gradient (0-40$) is formed in a K2-Electronucleonics rotor (see Progress in Separation and Purification, published by Perry and Van Oss, Copywright 1971 by John Wiley and Sons, Inc., USA.): when the rotational speed reaches 35,000 rpm. /min., first 500 ml of EC sample to be purified (concentrate) is supplied to the rotor at a continuous flow rate of 2 l/hour, then in the next 30 min. added to a phosphate buffer, 65 mm, pH 6.8. After stopping the rotor, the contents of the rotor are fractionated, and the fractions are titrated with regard to their content of HBs antigen. The HBsAg-rich fractions are also found here in the less dense layer of the gradient, which contains less than 5% sucrose; they are mixed under identical conditions to those obtained from the treatment of other 500 ml concentrate fractions.

The mixture of these fractions is dialysed to thereby remove the sucrose and then concentrated by ultrafiltration to a volume of 0.361 1. This gives a preparation Z10. The HBsAg yield is 80$. The performance in terms of removal of EDNA and non-HBsAg proteins was:

Although significant results are achieved in terms of removal r of DNA and non-HBsAg proteins, then the relevant centrifugation speed is of particular interest due to its efficacy in achieving distinction between the antigen and the retroviral particles.

Thus, when the concentrate is deliberately contaminated with a murine retrovirus (murine leukemia virus) and then subjected to centrifugation under the aforementioned conditions, the retroviral particles titrated by reverse transcriptase are recovered in the dense layer of the gradient with an enzymatic activity peak at 33-35$ sucrose .

The retrovirus particles have attached to their iso-dense zone in the gradient by moving through the majority of the gradient, while the HBsAg particles, which have a lower sedimentation constant (40s), have at the same time only moved over a small section of the gradient. Separation thereof is achieved by means of a short centrifugation time.

The reverse transcriptase activity measured in the mixture of the HBsAg-rich fractions represents 0.013$ of that found in the retrovirus peak, a removal factor of 77.

5. Isopycnic centrifugation of the flotation type

The rotor is a zonal rotor of the type TI15-Beckman zonal by Beckman, Palo Alto, California, USA, with a total capacity of approx. 1700 ml (see A. Fritsch, Les Centrifications Prépara-tives en Gradient de densità, 2nd edition). A density gradient is formed by introducing successive volumes of potassium bromide solution (KBr) at the periphery of the rotor to thereby achieve a gradient with densities between 1.17 and 1.35. One of the introduced layers is formed from the sample to which KBr has been added so that the density of the layer is equal to 1.25, so that at the beginning of the centrifugation all the impurities and the antigen are found in the dense part of the gradient.

After centrifugation at 31,000 rpm. for 20 hours and after fractionation of the rotor contents, the HBsAg particles are found in their iso-dense zone (d 1.22-1.23), while practically all the other proteins and DNA are found in the dense layer of the gradient. The mixture of the EBsAg-rich fractions is dialyzed against phosphate buffer 65 mM pH 6.8 to remove KBr and equilibrate the sample in the buffer that will be used for chromatography. Preparation Z20 is obtained.

While the HBsAg yield in this centrifugation is close to 100$, very high elimination factors for DNA and non-HBsAg protein were obtained.

They are respectively 350 for DNA and 58 for said non-HBsAg proteins.

A retrovirus removal factor could not be calculated in this step because the reverse transcriptase activity is totally destroyed in a KBr medium. This inactivation is probably due to the chaotropic effect of the salt on the virus. It is interesting to note that this chaotropic effect would release the viral genome (RNA) into the medium, which would be present after centrifugation in the dense gradients like the cellular DNA, thus separating from the antigen peak.

6. Anion exchange chromatography

The antigenic preparation Z20, dialysed after isopycnic centrifugation, is applied to Whatman DE 52 cellulose which has been previously equilibrated in a phosphate buffer 65 mM pH 6.8. Under these conditions, the HBS antigen is not adsorbed on the exchanger, while the remaining traces of DNA and the contaminating proteins are fixed on the exchanger:

A high elimination factor of 25-fold is obtained for the non-HBsAg proteins, while DNA is no longer detectable by the dot-blot technique after hybridization with a probe labeled with P32 (sensitivity limit = 12.5 pg/ml).

To measure the elimination factor for DNA, a known amount of DNA was added to a set of 20 preparations, and chromatography was performed on the anion exchanger. The amount of DNA found in the column effluent was 1000-10,000 times less than the initial amount.

The antigenic preparation thus obtained had the following properties after purification:

After electrophoresis on polyacrylamide gel 1 a dissociation medium, the polypeptides stained with silver nitrate spread over three main bands: .P22 with an apparent molecular weight of 22,000 daltons .P26 with an apparent molecular weight of 26,000 daltons .P34 with an apparent molecular weight of 34,000 daltons

all three of which are recognized by immunoprinting with an anti-S monoclonal antibody, where only the P34 polypeptide is recognized with an anti-pre S 2 monoclonal antibody.

7. Production of vaccines and immunogenicity

The chromatographed preparation is filtered on a sterilizing membrane, then heated to +60°C for 60 min. and after cooling, formaldehyde (100 µg/ml) is added and incubation is carried out for 48 hours at +30°C. This antigenic preparation is diluted in a physiological buffer, and an additive such as aluminum hydroxide is added to obtain vaccines.

Different doses of HBsAg antigens were injected subcutaneously into guinea pigs, and the antibody responses (anti-S and anti-pre S 2 ) were measured and compared with those obtained in animals injected with equal doses of antigen obtained from human plasma (Hevac B vaccine - Pasteur Vaccins Mårnes La Coquette-France).

The anti-S antibodies, generally termed anti-HBsAg, were measured by the Ausab-Abbott technique (Abbott Laboratories, USA) and the anti-pre S2 antibodies were measured by the immunoenzymatic method using a synthetic peptide of 31 amino acids on the solid phase, including the pre S2 epitope, thereby capturing the antibodies recognized in a second step, with an anti-IgG antibody conjugated with peroxidase; after addition of the substrate (H2O2) by a chromogen (orthophenylenediamine), the developed color was measured at 492 nm, the intensity of which was proportional to the amount of captured antibodies.

As the results in the table show, an equivalent anti-S response or reaction is achieved with the two types of hepatitis B vaccine, while a higher anti-pre S2 response or reaction is observed with the recombinant vaccine (CHO).

Claims (11)

1. Process for the production of a new recombinant vaccine against hepatitis B, where antigenic surface particles of hepatitis B are formed by expression from a culture of CHO cells transfected with a plasmid carrying the HBsAg gene to thereby release the antigenic surface particles in the culture medium, characterized in that the supernatant culture medium from at least one culture is recovered, in particular in a medium with a low content of animal serum, the supernatant is subjected to sterilizing filtration, the supernatant is concentrated, the concentrate is precipitated using a non-degrading precipitant selected from polyethylene glycol and ammonium sulfate, under conditions that precipitate the heavy DNA classes, the retroviral particles and proteins, a new concentration is performed, a speed-controlled zone centrifugation is carried out in a non-chaotropic density gradient for the retroviral particles and selected so as to achieve separation of these from the HBsAg particles, depending on their size and density, a flotation-type isopycnic zone centrifugation is performed, which removes the light and heavy nucleic acids and proteins, and chromatography on an anion exchange medium is performed to thereby adsorb the remaining traces of nucleic acids and residual non-HBsAg proteins. 2. Method according to claim 1, characterized in that precipitation is carried out with polyethylene glycol, especially PEG 6000. 3. Method according to claim 2, characterized in that a first precipitation is carried out with a PEG concentration of the order of magnitude 5$. 4. Method according to any one of claims 1-3, characterized in that the new concentration is carried out with a non-degrading agent which precipitates the antigenic EBsAg particles, after which the precipitate is redissolved in a small volume. 5. Method according to claim 4, characterized in that the precipitation is carried out with PEG, especially PEG 6000 at 9.5$. 6. Method according to any one of claims 1-5, characterized in that the speed-regulated zone centrifugation is carried out in a sucrose gradient. 7. Method according to claim 6, characterized in that the centrifugation is carried out in a K-2-Electronucleonic rotor. 8. Method according to any one of claims 6 and 7, characterized in that the fractions which have a sucrose degree of less than 25$ are selected, the other fractions being discarded. 9. Method according to any one of claims 1-8, characterized in that the zone centrifugation of the rotation type is carried out in a KBr gradient. 10. Method according to claim 9, characterized in that the fractions with the density peak 1.22-1.23 are collected. 11. Method according to any one of claims 1-10, characterized in that chromatography is carried out on DE 52 cellulose.