NO20130773L - Method of preparing a vaccine - Google Patents

Abstract

The present invention relates to a method for preparing a stable immunogenic hepatitis B vaccine without traces of thiomersal.

Description

The present invention relates to a new process for the production of a hepatitis B vaccine for use in the treatment or prevention of hepatitis B virus (HBV) infections.

Chronic hepatitis B virus (HBV) infection, for which there is currently limited treatment, represents a global public health problem of enormous proportions. Chronic carriers of HBV, estimated to number more than 300 million worldwide, are at risk of developing chronic active hepatitis, cirrhosis and primary hepatocellular carcinoma.

Many vaccines currently available require a preservative to prevent deterioration. A commonly used preservative is thiomersal, which is a mercury-containing compound. Some concern has been expressed about the use of mercury in vaccines, although commentators have emphasized that the potential dangers of thiomersal-containing vaccines should not be exaggerated (Offit; P.A. JAMA Vol,283; No: 16). Still, it would be beneficial to find new and potentially safer methods for the production of vaccines to replace the use of thiomersal in the production process. There is thus a need to develop vaccines that are thiomersal-free, especially hepatitis B vaccines.

In a first aspect, the present invention provides a method for producing a hepatitis B antigen suitable for use in a vaccine, the method comprising purifying the antigen in the presence of a reducing agent comprising a free SH group.

The present invention preferably provides a method for producing a stable hepatitis B antigen without traces of thiomersal which comprises purification of the antigen in the presence of a reducing agent having a free -SH group.

The antigen preparation is generally free of traces of thiomersal when thiomersal is not detectable in the purified antigen product using absorption spectrophotometry for mercury, as described herein.

The hepatitis antigen preparation preferably comprises less than 0.0251 mercury per 201 protein, conveniently as measured by absorption spectrophotometry.

Preferably, the purification is carried out in the absence of thiomersal, and the purified antigen is completely free of thiomersal.

Preferably, the antigen is stable, suitably substantially as stable as a hepatitis antigen purified in the presence of thiomersal, as for example described in Example 1 herein.

Preferably, the hepatitis antigen is immunogenic.

Preferably, reducing agent is added during the antigen purification process, preferably after growth of cells expressing the antigen.

Preferably, the reducing agent is cysteine, dithiothreitol, Hnercaptoethanol or glutathione, cysteine being most preferred.

Accordingly, the present invention preferably provides a method for producing a stable immunogenic hepatitis B antigen without traces of thiomersal which comprises purification of the antigen in the presence of cysteine.

Preferably, purification is carried out in the presence of a cysteine solution.

Preferably, the cysteine, in solution or powder form, is added during the process to a final concentration of between 1 and 10 mM, preferably 1 to 5 mM. More preferably, the cysteine is added to a final concentration of approx. 2 mM.

Preferably, the cysteine is L-cysteine.

The invention further provides a method for producing a stable hepatitis B antigen without traces of thiomersal where the crude antigen is subjected to gel permeation chromatography, subjected to ion exchange chromatography and mixed with a reducing agent having a free -SH group.

Preferably, ion exchange chromatography is anion exchange chromatography.

The invention further provides a hepatitis B antigen free of thiomersal which can be obtained by the method of production according to the present invention where the antigen is at least as immunogenic and antigenic as the hepatitis B antigen produced in the presence of thiomersal.

The invention further provides an immunogenic hepatitis B antigen having an average ELISA protein ratio greater than 1.5 and an RF1 content with at least a 3-fold lower IC50 value than that of hepatitis B surface antigen produced in the presence of thiomersal.

In a further aspect, the invention relates to a method for producing a hepatitis antigen suitable for use in a vaccine, where the method comprises purification of the antigen in the presence of thiomersal and subsequent treatment of the antigen in the presence of a reducing agent comprising a free -SH group.

Suitably, the treatment is followed by a purification step such as a dialysis step to remove thiomersal.

Preferably, the reducing agent is cysteine, DTT, glutathione or 2-mercaptoethanol.

The hepatitis B antigen according to the present invention can be used either for the treatment or prevention of hepatitis B infections, in particular the treatment or prevention of, for example, chronic hepatitis B infections.

The present invention further provides a vaccine preparation comprising a hepatitis B antigen according to the present invention together with an adjuvant. Preferably, the adjuvant is an aluminum salt or a preferential stimulator of TH1 cell response.

Preferably, the antigen is a hepatitis B surface antigen.

Production of hepatitis B surface antigen is well documented. See for example Harford et. eel. in Develop. Biol. Standard 54, page 125 (1983), Gregg et. eel. in Biotechnology, 5, page 479 (1987), EP-A-0 226 846, EP-A-0 299 108 and references therein.

As used herein, the term "hepatitis B surface antigen" or "HBsAg" includes any HBsAg antigen or fragment thereof that exhibits the antigenicity of HBV surface antigen. It will be understood that in addition to the 226 amino acid sequence of HBsAg S antigen (see Tiollais et. al. Nature, 317, 489 (1985) and references therein) HBsAg as described herein may, if desired, contain all or part of a pre-S sequence as described in the references above and in EP-A-0 278 940. HBsAg as described herein may also refer to variants, for example the "escape mutant" described in WO 91/14703.

HBsAg can also refer to polypeptides described in EP 0 198 474 or EP 0 304 578.

Normally, HBsAg will be in particulate form. In a particularly preferred embodiment, HbsAg will consist essentially of the HbsAg S antigen mentioned above.

The vaccine may advantageously comprise a pharmaceutically acceptable additive such as a suitable adjuvant. Suitable adjuvants are commercially available such as Freund's incomplete adjuvant and complete adjuvant (Difco Laboratories, Detroit, MI); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ); AS-2 (SmithKline Beecham, Philadelphia, PA); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationic or anionic derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A. Cytokines, such as GM-CSF or interleukin-2, -7, or

-12, can also be used as adjuvants.

In the preparations according to the present invention, it is preferred that the adjuvant preparation induces an immune response predominantly of the TH1 type. High levels of Th1-type cytokines (eg, IFN-BtNfH'L-2 and IL-12) tend to favor the elicitation of cell-mediated immune responses to an administered antigen. In a preferred embodiment, where a response is predominantly Th1-type, the level of Th1-type cytokines will increase to a greater extent than the level of Th2-type cytokines. The levels of these cytokines can be easily assessed using standard assays. For an overview of the families of cytokines, see Mosmann and Coffman, Ann. Fox. Immunol. 7:145-173, 1989.

Accordingly, suitable adjuvants for use in eliciting a predominantly Th1-type response include, for example, a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL) together with an aluminum salt. Other known adjuvants that preferentially elicit a TH1-type immune response include CpG-containing oligonucleotides. The oligonucleotides are characterized in that the CpG dinucleotide is unmethylated. Such oligonucleotides are well known and are described in, for example, WO 96/02555. Immunostimulatory DNA sequences are also described, for example by Sato et al., Science 273:352, 1996. Another preferred adjuvant is a saponin, preferably QS21 (Aquila Biopharmaceuticals Inc., Framingham, MA), which can be used alone or in combination with other adjuvants. For example, an improved system involves the combination of a monophosphoryl lipid A and saponin derivative, such as the combination of QS21 and 3D-MPL as described in WO 94/00153, or a less reactogenic preparation where QS21 is dampened with cholesterol, as described in WO 96/33739. Other preferred preparations include an oil-in-water emulsion and tocopherol. A particularly potent adjuvant preparation comprising QS21, 3D-MPL and tocopherol in an oil-in-water emulsion is described in WO 95/17210.

A particularly powerful adjuvant formulation comprising QS21, 3D-MPL & tocopherol in an oil-in-water emulsion is described in WO 95/17210 and is a preferred formulation.

Accordingly, in one embodiment of the present invention, there is provided a vaccine comprising a hepatitis B surface antigen according to the present invention, which additionally comprises TH-1 inducing adjuvant. A preferred embodiment is a vaccine where the TH-1 inducing adjuvant is selected from the group of adjuvants comprising: 3D-MPL, QS21, a mixture of QS21 and cholesterol, and a CpG oligonucleotide. Another preferred embodiment is a vaccine comprising a hepatitis B surface antigen added with monophosphoryl lipid A or derivative thereof, QS21 and tocopherol in an oil-in-water emulsion.

Preferably, the vaccine additionally comprises a saponin, more preferably QS21. Another particularly suitable adjuvant formulation comprising CpG and a saponin is described in WO 00/09159 and is a preferred formulation. Most preferred is saponin in the special formulation QS21. Preferably, the formulation additionally comprises an oil-in-water emulsion and tocopherol.

The present invention further provides a vaccine preparation comprising a hepatitis B surface antigen according to the present invention together with an adjuvant and additionally comprising one or more antigens selected from the group consisting of: diphtheria toxoid (D), tetanus toxoid (T) acellular pertussis antigens (Pa ), inactivated poliovirus (IPV), haemophilus influenzae antigen (Hib), hepatitis A antigen, herpes simplex virus (HSV), chlamydia, GBS, HPV, streptococcus pneumoniae and neisseria antigens. Antigens that provide protection for other diseases can also be included in the vaccine preparation according to the present invention.

In one particular embodiment, the vaccine preparation comprises a hepatitis B surface antigen which can be obtained by the method of production according to the present invention together with an adjuvant and an inactivated poliovirus.

The present invention also provides a method for the treatment and/or prevention of hepatitis B virus infections, which comprises administering to a human or animal, suffering from or susceptible to, hepatitis B virus infection, a safe and effective amount of a vaccine according to present invention for the prevention and/or treatment of hepatitis B infection.

The invention further provides the use of a hepatitis B surface antigen according to the present invention for the manufacture of a medicament for the treatment of patients suffering from a hepatitis B virus infection, such as chronic hepatitis B virus infection.

The vaccine according to the present invention will contain an immunoprotective amount of the antigen and can be produced by conventional methods.

Vaccine manufacturing is generally described in Pharmaceutical Biotechnology, Vol. 61 Vaccine Design - the subunit and adjuvant approach, ed. Powell and Newman, Plenum Press, 1995. New Trends and Developments in Vaccines, ed. Voller et al., University Park Press, Baltimore, Maryland, U.S.A. 1978. Encapsulation in liposomes is described, for example, by Fullerton, U.S. patent 4,235,877. Conjugation of proteins to macromolecules is described, for example, by Likhite, U.S. patent 4,372,945 and by Armor et al., U.S. Pat. patent 4,474,757. Use of Quil A is described by Dalsgaard et al., Acta Vet Scand. 18:349 (1977). 3D-MPL is available from Ribi Immunochem, USA, and is described in British Patent Application No. 2220211 and US patent 4912094. QS21 is described in US patent no. 5057540.

The present invention is illustrated, but not limited, by the following examples, where:

Figure 1 illustrates the thiomersal-free production process for Engerix B

Figure 2 illustrates SDS-PAGE analysis of bulk antigen lots; and

Figure 3 illustrates residual yeast proteins in bulk antigen lots produced by the thiomersal-free process.

Example 1:

Production process for Hepatitis B surface antigen in the presence of thiomersal

The hepatitis B surface antigen (HBsAg) in SB Biological's hepatitis B monovalent vaccine (Engerix B™) is expressed as a recombinant protein in Saccharomyces cerevisiae (see Harford et. al. joe. eit.). The 24 kD protein is produced intracellularly and accumulated in the recombinant yeast cells. At the end of fermentation, the yeast cells are harvested and disrupted in the presence of a mild surfactant such as Tween 20 to release the desired protein. Next, the cell homogenate, containing the soluble surface antigen particles, is pre-purified by a series of precipitations and then concentrated by ultrafiltration.

Further purification of the recombinant antigen is carried out by subsequent chromatographic separations. In a first step, the crude antigen concentrate is subjected to gel permeation chromatography on Sepharose 4B medium. Thiomersal is present in the elution buffer at the 4B gel permeation chromatography step. The elution buffer has the following composition: 10 mM Tris, 5% ethylene glycol, pH 7.0, 50 mg/l thiomersal. Thiomersal is included in this buffer to control bioburden. Most of this thiomersal is removed during the subsequent purification steps comprising ion exchange chromatography, ultracentrifugation and desalting (gel permeation) so that purified bulk antigen preparations produced by the original process contain approx. 1.2 ug and less than 2 ug thiomersal per 20 ug protein.

An ion exchange chromatography step is performed using a DEAE matrix and this pool is then subjected to a cesium gradient ultracentrifugation on 4 pre-established layers of different cesium chloride concentrations. The antigen particles are separated from contaminating cell constituents according to their density in the gradient and eluted at the end of the centrifugation process. Cesium chloride is then removed from this pool by a second gel permeation on Sepharose gel.

When HBsAg is prepared by the method containing thiomersal in 4B gel permeation buffer, protein concentrations in excess of 30 mg/ml are recovered in the pooled HBsAg-containing fractions from the CsCl gradient, corresponding to an equivalent concentration of HBsAg as analyzed with the AUSZYME kit from Abbott Laboratories.

The CsCl ultracentrifugation step conveniently removes residual lipids, DNA and minor protein contaminants from the HBsAg preparation. It is carried out by zone centrifugation in a Ti 15 rotor from Beckman Instruments, Fullerton, California at a speed of 30,000 rpm for about 40 to 60 hours. The sample to be purified is applied in layers of CsCI solution with final concentrations of 0.75, 1.5, 2.5 and 3.25 M CsCI. After the centrifugation, the gradient is eluted in fractions. Fractions containing HBsAg can be identified by UV absorbance at 280 nm or by testing dilutions of the fractions with the AUSZYME kit. The HbsAg band has a density of 1.17 to 1.23 g/cm<3>.

The solution containing the purified HBsAg is sterile filtered before it is used to prepare a vaccine preparation.

Purification from yeast cell lysate is complex as the antigen is produced intracellularly and a series of separation techniques designed to remove different types of (yeast) contaminants are necessary to obtain pure bulk antigen. The steps of purification are important, as the product to be purified is a lipoprotein particle containing multiple copies of surface antigen polypeptide and this structure must be maintained throughout the purification process. A distinctive feature of this process is that it provides surface antigen particles that are fully immunogenic without the need for additional chemical treatment to enhance immunogenicity (compare EP0135435).

Details of the manufacturing process are further described in European patent 0199698.

Example 2

Production and characterization of yeast-derived HBsAg by a thiomersal-free process.

1. Production and purification of yeast-derived HBsAg

1.1 Overview of the production process

Hepatitis B surface antigen can be produced by fermentation of a suitable strain of Saccharomyces cerevisiae, for example that described in Harford et. eel.

(loe. eit.).

After large-scale fermentation of the recombinant yeast strain, the cells are harvested and broken up in the presence of a mild surfactant such as Tween 20. The surface antigen is then isolated by a multi-step extraction and purification procedure exactly as described above in Example 1 until step with the first gel permeation on Sepharose 4B.

1.2 Thiomersal-free cleaning process

In the thiomersal-free process, the following two changes are made compared to the method described in Example 1. 1. Elution buffer for the 4B gel permeation chromatography step no longer contains thiomersal. 2. Cysteine (2mM final concentration) is added to the eluate pool from the anion exchange chromatography step.

It was found that omission of thiomersal from 4B gel permeation buffer can result in precipitation of the HBsAg particles during the CsCl density gradient centrifugation step with loss of product and aggregation or clumping of the recovered antigen.

Addition of cysteine at 2 mM final concentration to the eluate pool from the preceding anion exchange chromatography step prevents precipitation and loss of antigen during CsCl density centrifugation.

Cysteine is a preferred substance for this treatment as it is a naturally occurring amino acid and can be removed by the subsequent desalting step on a gel permeation column using Sepharose 4BCLFF as the column matrix.

There are no other changes in the manufacturing process compared to the method described in Example 1.

The thiomersal-free process provides bulk antigen with a purity and with properties comparable to the antigen from the method according to Example 1.

1.2a

Thiomersal added to 4B buffer at 50 ug/ml is assumed to decompose and the resulting ethylmercury can bind covalently to free sulfhydryl groups on cysteine residues in the protein. The protein contains 14 cysteine residues, 7 of which are located between positions 101 and 150.

This region of the protein is believed to be located on the surface of the particle and contain the larger antigenic region of HBsAg comprising the immunodominant a region and the recognition site for RF1 monoclonal antibody (Waters J et al, Postgrad. Med. J., 1987:63 ( Suppl. 2): 51-56 and Ashton-Rickardt and Murray J. Med. Virology, 1989: 29: 196). Antigen purified with thiomersal present in 4B gel permeation buffer contains approximately 0.5-0.6 µg of mercury at the end of the purification process. This mercury is not completely removed by simple dialysis.

In one experiment, 0.56 ug of mercury per 20 ug protein measured in the bulk antigen preparation. This preparation was dialyzed for 16 hours at room temperature against 150 mM NaCl, 10 mM NaPO4 buffer pH 6.9. At the end of the dialysis, a concentration of 0.33 ug Hg per 20 ug protein measured.

In contrast, dialysis in the presence of a reducing agent such as L-cysteine at 0.1 to 5.0 mg/ml, DTT at 50 mM, or 2-mercaptoethanol at 0.5 M, followed by a second dialysis to remove the reducing agent, results in in reducing the mercury content in the antigen preparation to less than 0.025 ug of mercury per 20 ug protein. This is the lowest limit of detection by the method.

The mercury content was determined by absorption spectrophotometry. The antigen is diluted in a solution containing 0.01% weight/volume of potassium bichromate (K2Cr207) and 5% volume/volume of nitric acid. Standard solutions are prepared with thiomersal as mercury source. Atomic absorption of sample and standard solutions is measured after evaporation in a vapor generator, with a mercury-specific cathode at 253.7 nm. Atomic absorption of the diluent is measured as blank. Mercury content of the sample is calculated by calibration curves obtained from standard solutions. Results are expressed as µg of mercury per 20 ug protein.

1.3 Production of thiomersal-free bulk antigen

Process steps for purification of bulk antigen are shown in Figure 1.

1.4 Composition of vaccine formulated without thiomersal.

A typical quantitative composition of a hepatitis B vaccine without preservative and formulated from antigen prepared by the thiomersal-free process is given in Table 1.

The preparation can be varied by adding 3D-MPL and/or other adjuvants.

2. Characterization of bulk antigen and vaccine produced by the thiomersal-free process

2.1. Tests and experiments with purified bulk antigen

2. 1. 1 Basis for comparison

Three batches of bulk antigen were prepared by the thiomersal-free process according to this example (Example 1.2) and are identified as HEF001, HEF002 and HEF003. These were compared with a batch of bulk antigen (HEP2055) prepared by the previous process (as described in Example 1) in the presence of thiomersal.

2.12 Tests and experiments with bulk antigen

The three bulk antigen lots produced by the thiomersal-free process were tested and the results are summarized in Table 2.

Protein content was measured by the method according to Lowry et al (J. Biol. Chem. 1951:193:265).

Endotoxin content was measured by a Limulus gel coagulation technique using a commercially available kit from Cape Cod Associates, 704 Main St., Falmouth, MA 02540, USA. The reagent is standardized against US Pharm. Endotoxin Reference Standard.

Tween 20 was measured by the method of Huddleston and Allred (J. Amer. Oil ChemistSoc, 1965:42:983).

HbsAg content was measured with the commercially available AusZYME kit from Abbott Laboratories, One Abbott Park Road, Abbott Park, IL 60064, USA. Experimental procedure B from the manufacturer was used. A portion of bulk antigen purified by the method containing thiomersal was used as a standard to establish a dose-response curve.

Polysaccharides were measured by the method of Dubois et al (Anal. Chem. 1956:28:350).

Lipids were measured using a commercially available kit (Merkotest Total Lipids 3321) from E.Merck, B.P. 4119, Darmstad D-6100, Germany.

DNA content was measured by the threshold method using apparatus and reagents available from Molecular Devices Corp., GutenbergstraBe 10, Ismaning, Munich, Germany.

The values found in the tests and trials are in the range seen for bulk antigen batches prepared using thiomersal in the elution buffer in the Sepharose 4B gel permeation step, with the exception of the antigen activity by ELISA. The values for this measurement for the three HEF preparations are higher (1.63-2.25) than that found for the bulk antigen lot HEP2055 which has an ELISA/protein ratio of 1.13. The ELISA/protein ratio measured with the AUSZYME kit for thiomersal-containing portions of bulk antigen is generally about 1.0 and within the range of 0.8-1.2 and very rarely exceeds 1.4.

2. 1. 3 SDS-PAGE gel analysis

The bulk antigen preparations were examined by SDS-PAGE analysis under reducing conditions and Coomassie blue labeling. All samples showed a major band at 24K with traces of a dimer protein. The samples were judged to be of high purity (>99% pure) as judged by the absence of visible bands from contaminating proteins.

Samples of the bulk antigen preparations were examined by SDS-PAGE under reducing and non-reducing conditions and silver labeling (Figure 2). Under reducing conditions, the samples showed an intense band migration at 24K with traces of dimeric and multimeric forms. The gel patterns are indistinguishable from that of HEP2055 as a comparator. The tests were also run under non-reducing conditions. Under these conditions, less of the material migrates at 24K and the amount of polypeptide migration at dimeric and multimeric positions is increased. The thiomersal-free bulk antigen batches appear to have a somewhat higher degree of polymerization than the comparator HEP2055 batch.

The identity of the 24K polypeptide revealed by Coomassie blue or silver labeling was confirmed by Western blotting with rabbit polyclonal antibodies raised against plasma HBsAg. The bulk antigen preparations show a major band at 24K along with dimeric and trimeric forms. The technique reveals minor traces of degradation products of surface antigen protein. There are no differences between bulk antigen prepared by the thiomersal-free process and the HEP2055 batch.

The presence of residual yeast proteins was examined by SDS-PAGE analysis under reducing conditions and Western blotting with rabbit polyclonal antiserum raised against yeast proteins (Figure 3). The technique is qualitative and does not allow quantification of impurities.

A constant band pattern is shown across the three bulk antigen lots prepared by the thiomersal-free process and the HEP2055 lot with one exception.

A strong labeling band present at B^K for HEP2055 bulk antigen is virtually absent for the 3 HEF preparations. Western blotting shows that the thiomersal-free purification process results in a purer antigen product.

2. 1. 4 Other biochemical tests and trials

2.1.4.1 DNA content

DNA content of the 3 bulk antigen lots was measured by the threshold method (Molecular Devices Corp). The measured amounts were less than 10 pg DNA per 201 proteins (Table 2); same level of DNA content seen in bulk antigen produced by the current approved process.

2.1.4.2 Amino acid composition

The amino acid composition of the three HEF bulk antigen lots was determined after acid hydrolysis with 6N HCI by chromatography of the amino acids on an ion exchange column with post-column ninhydrin detection. Proline and tryptophan were not determined. The results are given in Table 3.

The composition found is in good agreement with that determined for HEP2055 and with the expected composition derived from the DNA sequence. Although the number of glycine residues measured for HEP2055 is close to the expected composition, a value of 16 to 17 residues is more commonly measured for bulk antigen preparations. The average number of cysteine residues found is the expected 14, showing that no additional cysteines are bound to the particle as a result of the treatment in the CsCI gradient step.

2.1.4.3 Quantification of free cysteine

The amount of free cysteine present in bulk antigen preparations obtained according to the method described was measured after oxidation of the particles with permauric acid without previous acid hydrolysis. Oxidized free cysteine residues were separated on an ion exchange column with post-column detection with ninhydrin. The limit for detection of cysteine by this method is 1 ug per ml.

No free cysteine could be measured in the 3 HEF antigen preparations when tested at the initial protein concentrations given in Table 2.

The technique measures both free cysteine residues present in the buffer and cysteine residues which are bound to the HBsAg protein by disulfide bonding, but which do not form part of the polypeptide sequence.

2.1.4.4 N-terminal sequence analysis

The presence of possible protein contaminants and degradation products in the three bulk antigen batches produced by the modified process was assessed by N-terminal sequence analysis based on Edman degradation. The N-terminal sequence MENITS... of the HBsAg protein was detected without any interference from other sequences. The N-terminal methionine was also confirmed to be 60-75% blocked by acetylation, as observed previously for HbsAg polypeptide produced by the routine process.

2.1.4.5 Laser light scattering analysis

Particle size comparisons were performed by laser light scattering between HBsAg particles produced using the modified process and the HEP2055 reference lot (Table 4).

The average molecular weights determined show good consistency between the preparations.

2.1.4.6 Electron microscopy

The bulk antigen preparations were examined by electron microscopy after fixation and labeling with uranyl acetate.

The articles observed were similar in all samples and were consistent with 20 nm subspherical or cobblestone-like particles typical of HBsAg. The particles observed in the 3 HEF batches could not be distinguished from HEP2055.

2. 1. 5 Immunological analyses

2.1.5.1 Reactivity with RF1 monoclonal antibody

The three bulk antigen preparations were tested for their reactivity with RF1 monoclonal antibody by ELISA inhibition assay. RF1 monoclonal antibody has been shown to protect chimpanzees against challenge with HBV and is thought to recognize a protective conformational epitope on the HBsAg particle (Iwarson S et al, 1985, J.Med, Virol., 16: 89-96).

The RF1 hybridoma can be propagated in the peritoneal cavity of BalbC mice or in tissue culture.

Ascites fluid diluted 1/50000 in saturation buffer (PBS containing 1% BSA, 0.1% Tween 20) was mixed 1:1 with various dilutions in PBS of HBsAg samples to be tested (final concentrations ranging between 100 µg and 0.05 ug/ml).

Mixtures were incubated in Nunc Immunoplates (96U) for 1 hour at 37°C before being transferred for 1 hour at 37°C onto plates coated with a standard preparation of HBsAg. The standard HBsAg preparation was a batch of bulk antigen (Hep 286) purified by the thiomersal-containing process. After a washing step with PBS containing 0.1% Tween 20, biotin-conjugated sheep anti-mouse IgG diluted 1/1000 in saturation buffer was added and incubated for 1 hour at 37°C. After a washing step, streptavidin-biotinylated peroxidase complex diluted 1/1000 in saturation buffer was added to the same wells and incubated for 30 min. at 37°C. The plates were washed and incubated with a solution of OPDA 0.04%, H2020.03% in 0.1 M citrate buffer pH 4.5 for 20 min. at room temperature. The reaction was quenched with 2N H 2 SO 4 and the optical densities (O.D.) were measured at 490/630 nm and plotted graphically.

IC50, defined as the concentration of antigen (inhibitor concentration) that inhibits 50% of antibody binding to coated HBsAg was calculated using a 4 parameter equation and expressed in ng/ml.

A series of HEP antigen lots comprising HEP2055 were also tested, along with Herpes simplex gD antigen as a negative control. The experiment measures the ability of each test antigen to inhibit binding of RF1 to a standard antigen preparation (HEP286) bound to microtiter plates.

Table 5 gives the concentrations of each antigen found to inhibit 50% of RF1 binding to the fixed antigen.

<*>IC50 = antigen concentration (ng/ml) that inhibits 50% of RF1 binding to fixed antigen

The results show that 4 to 7 times less HEF antigen is required to inhibit RF1 binding (Table 5). This shows that antigen produced by the modified process has an increased presentation of the RF1 epitope compared to HEP bulk antigen.

The same type of inhibition assay was performed using human sera from Engerix B™ vaccines instead of the RF1 mAb and showed no differences between the HEP antigen lots and the HEF antigens.

2.1.5.2. Affinity of binding to monoclonal RF1

The kinetic parameters of RF1 monoclonal antibody binding to the 3 HEF antigen moieties and to HEP2055 were measured by surface plasmon resonance using a Biacore 2000 apparatus from Amersham Pharmacia Biotech, Amersham Place, Little Chalfont, Bucks, UK.

The measured kinetic parameters were:

ka: association rate constant (M"<1>S"<1>)

kd: dissociation rate constant (S"<1>)

Ka: equilibrium or affinity constant (M"<1>)

The values found are given in Table 6.

The three HEF antigen lots gave similar association/dissociation constants and binding affinity values. In contrast, HEP2055 has a weaker affinity for binding to RF1.

This is in accordance with the results from ELISA inhibition experiments which showed that antigen produced by the thiomersal-free process had an increased presentation of the RF1 epitope.

2. 2. Test and trial with vaccine formulated with antigen produced by the modified process

The three HEF antigen lots were adsorbed on aluminum hydroxide and formulated as a vaccine according to the composition shown in Table 1. The presentation is a dose for adults in a medicine glass (201 antigen protein in 1 ml). The batches are identified as DENS001A4, DENS002A4 and DENS003A4.

Vaccine potency was measured by an in vitro antigen content assay using the Abbott Laboratories AUSZYME ELISA kit and a classic lot of vaccine formulated with 50 µ/ml thiomersal as a standard. Vaccine potency was measured using method B as described in PharmaEuropa Special Issue Bio97-2 (December 1997). The three HEF lots give high values for antigen content, almost twice the stated content of 201 antigen protein.

2.2. 1 Reactivity to DENS vaccine with RF1 monoclonal antibody

The antigenicity of the adsorbed vaccine was further tested in an inhibition experiment with RF1 monoclonal antibody. The experiment measures the ability of the vaccine sample to inhibit RF1 binding to fixed bulk antigen (HEP286).

Ascites fluid diluted 1/50000 in saturation buffer (PBS containing 1% BSA, 0.1% Tween 20) was mixed 1:1 with different dilutions in PBS of vaccine samples to be tested (concentration in the range between 20 ug and 0.05 ug/ml ).

Mixtures were incubated in Nunc Immunoplates (96U) for 2 hours at 37°C with agitation before being transferred onto HBsAg-coated plates. The HBsAg preparation used for plating was a batch of bulk antigen (Hep 286) purified by the thiomersal-containing process. These plates are then incubated for 2 hours at 37°C with agitation. After a washing step with PBS containing 0.1% Tween 20, biotin-conjugated sheep anti-mouse IgG diluted 1/1000 in saturation buffer was added and incubated for 1 hour at 37°C. After a washing step, streptavidin-biotinylated peroxidase complex diluted 1/1000 in saturation buffer was added to the wells and incubated for 30 min. at 37°C. The plates were washed and incubated for 20 min. at room temperature with a solution containing OPDA 0.04%, H2020.03% in 0.1M citrate buffer pH 4.5. The reaction was quenched with 2N H 2 SO 4 and optical densities (O.D.) were measured at 490/630 nm and plotted graphically.

The IC50, defined as the concentration of antigen (inhibitor concentration) that inhibits 50% of the antibody binding to coated HBsAg, was calculated using a 4 parameter equation and expressed in ng/ml.

Vaccine prepared from bulk antigen produced by the modified process was compared with Engerix B™ vaccine formulated from classic HEP bulk antigen and without thiomersal as a preservative.

The experiments were run in triplicate.

The results are given in Table 7 and show that approximately half the amount of DENS vaccine is required to achieve 50% inhibition of RF1 binding compared to the preservative-free Engerix B™ vaccine. This reflects an increased presentation of RF1 epitope on HEF/DENS antigen and is consistent with the tests performed with RF1 antibody on the purified bulk antigen.

(1) concentration of vaccine that inhibits 50% of RF1 antibody binding to fixed antigen

2.2.2 Immunogenicity of DENS vaccine in mice

A study was conducted in Balb/C mice to compare the immunogenicity of the three DENS consistency lots with Engerix B™ manufactured according to the prevailing antigen manufacturing process and formulated with thiomersal.

The following batches were tested:

#DENS001A4

# DENS002A4

# DENS003A4

# ENG2953A4/Q for reference

Groups of 12 mice were immunized intramuscularly twice at 2-week intervals with vaccine doses corresponding to 1/10 (21) or 1/50 (0.41) of the adult human dose. Antibody response to HBsAg and the isotypic profile elicited by vaccination were monitored from sera taken on day 28.

EXPERIMENTAL DESIGN

Groups of 12 Balb/C mice were immunized intramuscularly in both legs (2 x 50 µl) on days 0 and 15 with the following vaccine doses:

On days 15 (2 weeks after I) and 28 (2 weeks after II), blood was taken from the retroorbital sinus. For the design of this experiment (4 preparations x 2 doses with 12 mice per group), the potency was calculated a priori with the PASS statistical program. The PASS (Power and Sample Size) statistical program was obtained from NCSS, 329 North 1000 East, Kaysville, Utah 84037. For 2-way analysis of variance, a 2.5-fold difference in GMT between preparations with an alpha error of 5% should be detected with a strength > 90%.

RESULTS

Serology:

Humoral responses (Total lg and isotypes) were measured by ELISA assay using HBsAg (Hep286) as coating antigen and biotin-conjugated anti-mouse antibodies to reveal anti-HBs antibody binding. Only after II were sera analyzed. Table 9 shows mean and GMT anti-HBs lg antibody responses measured in individual sera 2 weeks after II

Comparable antibody responses are elicited with DENS and classic hepatitis B preparations: GMT ranging between 2304 and 3976 EU/ml for the DENS batches compared to 2882 EU/ml for SB Biologicals hepatitis B monovalent vaccine (Engerix B™) at 2ug dose , and GMT ranged between 696 and 1182 EU/ml for the DENS lots compared to 627 EU/ml for SB Biologicals hepatitis B monovalent vaccine (Engerix B™) at 0.4 ug dose.

I As expected, a clear antigen dose-dependent effect is observed for all preparations

with 21 and 0.4 ug doses with a 3- to 6-fold difference in GMT.

I Four non-responding mice (titer < 50 EU/ml) were observed without clear relationships to the antigen doses or lots used for the injection (Groups 1, 2, 3 and 8; one mouse per group). Based on statistical analysis (Grubb's Test), these mice were excluded from further analysis.

Statistical analysis:

A 2 way analysis of variance was performed on anti-HBs titers after log transformation of post II data, using the vaccines (4 lots) and the antigen doses (2 µg and 0.4 µg) as factors. This analysis confirmed that a statistically significant difference was observed between the two antigen doses (p value < 0.001) and showed no significant difference between the vaccine lots (p value = 0.2674). As previously mentioned, the strength was calculated a priori and the design of the experiment was such that a 2.5-fold difference in GMT with an alpha error of 5% could be detected between preparations with a strength >90%.

Isotypic profile:

Table 10 shows the isotypic distribution (lgG1, lgG2a and lgG2b) calculated from an analysis of pooled sera after II.

As expected, a clear TH2 response is elicited by these alum-based vaccines

as mainly IgG1 antibodies are observed.

No difference has been observed between the DENS batches or SB Biologicals hepatitis B monovalent vaccine in terms of isotypic profile.

Example 3 Formulation of combined vaccines

Bulk antigen according to the present invention is particularly suitable for formulation in a combined vaccine comprising IPV.

Stability studies carried out on original batches of a combined DTPa-HBV-IPV vaccine showed a decrease in the potency of the IPV component, especially of the type 1 poliomyelitis antigen, using an in vitro immunoassay (determination of D-antigen content by ELISA) and an in vivo strength test. No strength loss was observed for type 3. For type 2, the strength loss was within the expected range (no more than 10% loss per year of storage).

Investigations were initiated to determine the cause of this loss of potency in the combined DTPa-HBV-IPV vaccine. From the observation that the stability of IPV in SB Biological's DTPa-IPV vaccine is satisfactory (no more than 10% antigen content loss per year of storage), it was concluded that the HBV component was probably responsible for the instability of IPV in DTPa-HBV - The IPV vaccine.

The HBV component used in the original DTPa-HBV-IPV formulation is purified r-DNA, yeast-derived HBsAg also used for the production of SB Biological's hepatitis B monovalent vaccine and prepared as described in Example 1.

As a first attempt to determine which element of the HBV component was detrimental to IPV, bulk HBsAg was analyzed for the presence of thiomersal. It has previously been found (Davisson et al., 1956, J. Lab. Clin. Med 47: 8-19) that thiomersal used as a preservative in DTP vaccines "was harmful to poliomyelitis virus" in a DTP-IPV combination. This observation was taken into account by vaccine manufacturers who have replaced thiomersal with other preservatives for the formulation of their IPV-containing vaccines. Later, the effect of thiomersal on IPV strength under conditions of long-term storage at + 4 °C has been re-examined. Loss of potency of type 1 poliovirus antigen to undetectable levels after 4-6 months was reported (Sawyer, L.A. et al. 1994, Vaccine 12: 851-856).

When using atomic adsorption spectroscopy, approximately 0.5 ug of mercury (Hg) per 20 µg of HBsAg detected in antigen purified according to Example 1.

This amount of mercury (as thiomersal and ethylmercuric chloride, thiomersal breakdown product) can be reduced to undetectable levels ELISA response for D-antigen type 1 content in an IPV bulk concentrate incubated at 37°C for 7 days.

A method was established for the release of mercury present in HBsAg bulk. It was postulated that mercury could be bound to thiol groups on the HBsAg particle and could therefore be released in the presence of reducing agents. After experimentation with other reducing agents, L-cysteine was chosen as the agent for releasing mercury from the HBsAg particle. After dialysis of HBsAg bulk against saline solution containing 5.7 mM L-cysteine, no mercury was detected in the retentate (detection limit for the testing method: 25 ng Hg/20 ug HBsAg). The dialyzed antigen was mixed with IPV bulk concentrate and the stability of type 1 virus was assessed by measuring the D-antigen content after incubation at 37°C for 7 days. IPV bulk concentrate that was unmixed and mixed with HBsAg not treated with cysteine was used as controls. Reference ELISA titers were obtained for samples stored at +2°C to +8°C for 7 days.

The results are summarized in Table 11:

The data obtained for these laboratory preparations clearly demonstrate that the stability of type 1 poliovirus is significantly improved if HBsAg is treated with cysteine to remove residual mercury before mixing with IPV.

The data presented above also show a loss of D-antigen content of 23% for the reference IPV preparation after incubation for 7 days at 37°C. This confirms the inherent instability of type 1 Mahoney poliovirus, as previously reported (Sawyer, L.A. et al. (1994), Vaccine 12: 851-856).

Although commercial batches of DTPa-HBV-IPV and DTPa-HBV-IPV/Hib vaccines are prepared using a dialysis process with 5.7 mM L-cysteine to remove residual mercury and maintain the stability of IPV, the dialysis process is not suitable for large-scale production and involves a series of additional steps to produce thiomersal- or mercury-free HBsAg. In contrast, HBsAg according to the present invention, produced without thiomersal, can be used directly in preparations of combined vaccines, especially those containing IPV.

4. Summary

The previously used process for purification of yeast-derived surface antigen involves a gel permeation step where the mercury-containing anti-microbial compound thiomersal is included in the elution buffer to control bioburden.

Thiomersal is not completely excreted during the subsequent steps of the method, so that approx. 1.21 thiomersal per 201 protein is present in the purified bulk antigen.

To produce a completely thiomersal (mercury) free bulk antigen, the purification process is changed in two steps.

I Thiomersal is omitted from the elution buffer at the 4B gel permeation step.

I Cysteine (2 mM final concentration) is added to the eluate pool from the anion-exchange chromatography step. This prevents precipitation of antigen during CsCI density gradient centrifugation.

There are no other changes in the production process.

Bulk antigen produced by the modified process has been characterized. Physico-chemical tests and trials show that the thiomersal-free antigen cannot be distinguished in its properties from antigen produced by the previously used process. The antigen particles have the same components.

Identity and integrity of HBsAg polypeptide is unaffected by the modified process as judged by SDS-PAGE analysis, Western blotting using polyclonal anti-HBsAg antibodies, N-terminal sequence analysis and amino acid composition. Electron microscopy and laser light scattering analysis show that the particles have the typical shape and size expected for yeast-derived HBsAg. Analysis by Western blotting with anti-yeast protein serum shows that the antigen produced by the thiomersal-free process has a similar pattern to contaminating yeast proteins. However, the amount of a contaminating band migrating at 23K is greatly reduced in the 3 HBsAg batches produced using the modified process.

Immunological analyzes show that the thiomersal-free particles have an increased antigenicity. The particles are more reactive with the Abbott AUSZYME kit (containing a mixture of monoclonal antibodies) giving ELISA/protein ratios of 1.6 to 2.25. This increased antigenicity is also shown with the protective RF1 monoclonal antibody. Approximately 4- to 7-fold less thiomersal-free antigen is required to inhibit RF1 binding to a standard fixed antigen. The thiomersal-free and classical antigen inhibition binding curves fall into two distinct families. This difference is also shown by measuring the binding affinity constant for RF1 using surface plasmon resonance. Binding affinities of the thiomersal-free preparations are 3 to 4 times higher compared to the lot with classic bulk antigen.

The bulk antigen preparations were formulated as a vaccine by adsorption on aluminum hydroxide and without preservative.

Testing for in vitro potency using the Abbott AUSZYME ELISA kit and thiomersal-containing SB Biologicals hepatitis B monovalent vaccine as a standard showed that high in vitro potency values were achieved. Antigen content measured by this test was almost double the stated value of 201 protein per ml.

An increased reactivity of vaccine prepared from thiomersal-free antigen was also seen in an inhibition experiment with RF1 monoclonal antibody for binding to fixed antigen. Approximately half the amount of the thiomersal-free vaccine was required to provide 50% inhibition of RF1 binding to fixed antigen compared to antigen purified by the previously used process and formulated without preservative.

This increased antigenicity of the thiomersal-free vaccine with respect to RF1 is consistent with the results of the in vitro potency test (antigen content) and with RF1 antibody tests performed on the bulk antigen preparations.

A mouse immunogenicity test was performed using priming and booster vaccinations two weeks apart and doses of 2 and 0.41 antigen. The mice were bled on day 28, 14 days after the booster vaccine. Sera were analyzed for antibody titer and isotype composition. A clear antigenic dose effect was observed for the two doses administered, but there was no statistically significant difference in response in terms of antibody titers (GMT) between thiomersal-free and preservative-free vaccines.

No significant differences were observed in the isotype profiles.

Claims (10)

1. Method for producing a stable, immunogenic hepatitis B vaccine without traces of thiomersal, comprising: (a) expressing hepatitis B surface antigen (HBsAg) as a recombinant protein in Saccharomyces cerevisiae; (b) processing the yeast cells to provide a purified antigen preparation; (c) subjecting the crude antigen preparation to gel permeation chromatography, wherein the elution buffer used in the gel permeation chromatography does not contain thiomersal; (d) subjecting the antigen-containing eluate from step (c) to ion exchange chromatography; (e) adding cysteine to the antigen-containing eluate pool obtained after step (d); (f) subjecting the preparation of step (e) to cesium chloride ultracentrifugation; and (g) combining the purified HBsAg with a pharmaceutically acceptable excipients for preparing a stable, immunogenic hepatitis B vaccine; where no thiomersal is added to the vaccine obtained. 2. Method according to claim 1, where the cysteine is added to a final concentration of between 1 and 10 mM. 3. Method according to claim 2, where the cysteine is added to a final concentration of approximately 2 mM. 4. A method according to any one of the preceding claims, wherein the ion exchange chromatography is anion exchange chromatography. 5. Method according to any one of the preceding claims, wherein the vaccine is further combined with an adjuvant. 6. Method according to claim 5, where the adjuvant is an aluminum salt. 7. Method according to claim 6, where the adjuvant is aluminum hydroxide gel or aluminum phosphate. 8. Method according to claim 8, where the adjuvant is a Th-1 inducing adjuvant. 9. Method according to claim 9, wherein the Th1-inducing adjuvant is 3D-MPL; QS21; 3D-MPL and QS21; or a CpG oligonucleotide. 10. Method according to claim 9, where the adjuvant is 3D-MPL and an aluminum salt.