WO2000037104A1 - Improved recombinant hepatitis b surface antigen - Google Patents
Improved recombinant hepatitis b surface antigen Download PDFInfo
- Publication number
- WO2000037104A1 WO2000037104A1 PCT/US1999/030770 US9930770W WO0037104A1 WO 2000037104 A1 WO2000037104 A1 WO 2000037104A1 US 9930770 W US9930770 W US 9930770W WO 0037104 A1 WO0037104 A1 WO 0037104A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- rhbsag
- hepatitis
- glutathione
- hours
- disulfide
- Prior art date
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55505—Inorganic adjuvants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2730/00—Reverse transcribing DNA viruses
- C12N2730/00011—Details
- C12N2730/10011—Hepadnaviridae
- C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
- C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2730/00—Reverse transcribing DNA viruses
- C12N2730/00011—Details
- C12N2730/10011—Hepadnaviridae
- C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
- C12N2730/10134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the invention relates to recombinant hepatitis B surface antigen (rHBsAg), prophylactic and therapeutic vaccines containing HBsAg and methods of preparing HBsAg and vaccines.
- rHBsAg hepatitis B surface antigen
- the major surface antigen of the hepatitis B virus is a 25 kDa protein, HBsAg.
- the protein is known in three forms: preSl, preS2 and S.
- the preSl and preS2 forms include 14 and 39 amino acids that are cleaved from their N-termini in vivo to yield the 226 amino acid S form.
- hepatitis B surface antigen expressed as the S form in yeast cells has superseded plasma-derived antigen as a vaccine against hepatitis B infection (Valenzuela et al., 1982; McAleer et al., 1984).
- the surface antigen protein assembles into 22 nm particles comprising lipids and HBsAg.
- the assembly of the yeast produced rHBsAg into these virus-like particles has remained poorly understood.
- the recombinant proteins are expected to assume certain sub-optimal conformations as a result of mismatched disulfide bond pairings.
- cysteine-rich proteins or peptides are reported to have strong propensity to form the correct disulfide bond pairings during oxidative refolding (Moroder et al., 1996; Mosiol et al., 1994), productive folding is always in competition with nonproductive folding. These latter pathways lead to either wrong disulfide bond pairings or aggregation of polypeptides.
- GSSG/GSH mixture a redox buffer with both forms of glutathione, i.e., GSSG/GSH mixture, might facilitate the conformational searching by promoting the formation of the correct pairings and unlocking the mismatched ones.
- GSSG/GSH mixtures were only demonstrated for some small proteins and peptides (Moroder, et al., 1996).
- the nascent HBsAg molecules need to find a lipid environment for the formation of certain intramolecular and/or intermolecular disulfide bonds that lead to proper folding into the native conformation of the protein. In the natural system, this process leads to the formation of particles of approximately 22 nm in diameter made up predominantly of HBsAg protein associated with a lipid membrane. Similarly, when expressed in yeast cells or other expression host , it is believed that the nascent rHBsAg needs to find a lipid environment prior to the spontaneous folding into membrane-embedded structures.
- the rHBsAg is over-expressed in a non-natural system using a host cell. Insect, yeast and CHO cells are commonly used although other cell types may be employed.
- the rHBsAg is an assortment of aggregations of scrambled forms and non-native conformations due to mismatched disulfide bond pairings. These artifacts of over- expression yield molecules locked into conformations of low antigenicity. Therefore, once produced, the over-expressed rHBsAg is typically processed outside a cellular environment to eliminate some of the undesired artifacts of over-expression.
- the antigen is treated with thiocyanate in an oxidative step to induce a conformational search and yield the form of rHBsAg known as Form III (Wampler, et al, 1985) (See FIG. 1). Thereafter, formalin treatment is used to lock the rHBsAg into whatever conformation it has assumed under the chaotropic, partially denaturing conditions of the thiocyanate treatment. Finally, the rHBsAg it is precipitated with adjuvant.
- the present invention provides an improved recombinant hepatitis B surface antigen, rHBsAg, that has a higher specific antigenicity than previously known rHBsAg.
- the invention also provides for the use of this improved rHBsAg in prophylactic and therapeutic vaccines and in combination vaccines.
- FIG. 1. shows a schematic of the theory of conformational search as applied to rHBsAg.
- FIG. 2A Glutathione-mediated conformational search maturation of rHBsAg at 37°C . Synergistic effects of GSH and GSSG. Measured by surface plasmon resonance in reference to a stable rHBsAg preparation and graphed using arbitrary units.
- FIG. 2B Glutathione-mediated conformational search/maturation of rHBsAg at 37°C. A more antigenic conformation of HBsAg can be achieved by higher concentrations of GSH. Measured by surface plasmon resonance in reference to a stable rHBsAg preparation and graphed using arbitrary units .
- FIG. 3 The nucleotide and protein sequences of the variable regions of the mouse monoclonal antibody A1.2 (from Figures 1 and 2 of Lohman et al 1993).
- FIG. 7 A graph of in vitro relative potency (IVRP) versus the relative antigenicity measured by surface plasmon resonance.
- the present invention provides an improved recombinant hepatitis B surface antigen protein, "rHBsAg,” that exhibits a higher antigenicity than previously known rHBsAg and a method of making the improved rHBsAg.
- the invention also provides for the use of the improved rHBsAg in prophylactic and therapeutic vaccines, and in combination vaccines.
- the present invention employs a discovery that an optimized process of refolding rHBsAg can be used to achieve a product having a higher level of immunogenicity than previous rHBsAg products.
- the immunogenicity is measured by determining the amount of rHBsAg required to induce an immune response in a mouse model system.
- the optimized process preferably includes a redox buffer step to prepare the improved rHBsAg. It has been discovered that oxidative refolding of rHBsAg in a redox buffer produces a rHBsAg product of higher antigenicity than previously known in the art.
- antigenicity of rHBsAg refers to the reactivity of the rHBsAg in an in vitro test or system.
- the immunogenicity of rHBsAg refers to the ability of the rHBsAg to induce an immune reaction in an in vivo animal model or in a patient in a clinical setting.
- a redox buffer enables the rHBsAg polypeptides to undergo a conformational search in a more controlled manner over a protracted period of time.
- the higher immunogenicity of the rHBsAg produced by this process may therefore be due to the preparation of polypeptides having a greater percentage of correct disulfide bond pairings.
- the rHBsAg protein is a large polypeptide having 14 cysteines and a complex, membrane embedded three dimensional structure, these hypothetical statements may not represent a complete explanation of the factors that lead to the improved immunogenicity of the rHBsAg products taught herein.
- rHBsAg The interplay between polypeptides in a particle of rHBsAg may also play an important role.
- the process of making rHBsAg taught herein may produce particles with more, or properly folded, multimers of polypeptides.
- the improved rHBsAg produced as taught herein is due to improved dimerization of the polypeptides in the particles to yield conformations that are more immunogenetic. Therefore, theoretically, it is possible that the improved immunogenicity of the rHBsAg taught herein is due to more proper intra-molecular bonds, inter-molecular bonds, or both.
- a variety of thiol containing compounds can be used to make a redox buffer for use in the method of making improved rHBsAg.
- the basic requirements are that the thiol of the compound can interact with cysteine in rHBsAg and that the compound can exist in an oxidized form consisting of a dithiol linkage between two molecules of the compound.
- Examples of thiol compounds that can be made in reduced and oxidized states are cysteine, 2-mercaptoenthanol, thiothreitol, dithiothreitol ("DTT”), glutathione ("GSH”) and diglutathione (oxidized glutathione "GSSG”).
- our most preferred redox buffer is composed of a mixture of GSH and GSSG due to their abundance in all mammalian cells for redox regulation. It is preferred that one adds the reduced and oxidized compounds (e.g.: GSH/GSSG) at a molar ratio from about 30:1 to about 1:1, preferably, a ratio of about 25:1, 20:1, 10:1, 10:4, 5:1, 2:1 or 1:1, and most preferably at a ratio of about 5:1.
- the final concentration of the compounds should be between about 0.05 to 5.00 nM, preferably about 0.20 to about 3.0 mM, more preferably about 0.5 to about 1.5 mM, and most preferably about 1.0 mM.
- ratios and concentrations in this specification, we use the term "about" to acknowledge that the invention involves applying reagents to a complex biochemical system. Strict adherence to an absolute, time, concentration or ratio as explicitly recited herein is not frequently required. Moreover, one of skill in the art can use routine empirical testing of ratios and concentrations within 10%, 20% 25% or even 50% of more of the values explicitly stated herein to determine the quality of the product produced by the process when a particular thiol compound is employed in the conformational search step. Similarly, the times required for incubations can vary from those explicitly stated herein.
- any of these variations can be determined routinely by testing the rHBsAg product, (e.g.: using a standard mouse potency assay) to determine whether the variation yields a product having the improved antigenicity produced when using the conditions and reagents explicitly recited herein.
- this specification teaches that one can in fact produce an improved rHBsAg through the use of a conformational search step in the presence of a redox buffer. Therefore, one who practices in this field can use that teaching to determine the parameters appropriate for employing various particular redox buffers under a variety of conditions to achieve the improved rHBsAg.
- the redox buffer is used to induce what is believed to be a protracted conformational search, especially under warm physiological conditions.
- FIG. 1 The concept of conformational search, i.e., the evolution of the latent epitopes into fully functioning or mature ones through the optimal pathways, and the chemical cross- linking of the rHBsAg polypeptide chains are shown in FIG. 1.
- the redox buffer is believed to prevent premature locking of conformation, as well as the covalent disulfide bonding coupled to undesired aggregation.
- the reducing power of the redox buffer is also believed to unlock the mismatched or scrambled disulfide bonds. These disulfide bonds are much more susceptible to reduction than the correct disulfide pairings.
- FIGS. 2 A and 2B show the dramatic effects of both forms of glutathione on the enhancement of antigenicity of rHBsAg, particularly their synergistic effects in refolding this cysteine-rich protein.
- the most advantageous use of the redox step is to use conditions under which the presence of the redox buffer compounds is influential enough to disrupt the less stable mismatched disulfides but not so influential as to constantly disrupt both mismatched and correctly paired disulfide bonds.
- the preferred and most preferred conditions described herein are appropriate when using GSH/GSSG.
- the conditions appropriate for the use of other thiol compounds can be determined simply by empirical testing of conditions coupled with monitoring of the antigenicity and immunogenicity of the rHBsAg produced.
- the redox buffer step is added to existing processes for the production of rHBsAg at the point where the protein is purified from the cellular expression system in which it is produced.
- the rHBsAg is processed as described up to the point of producing the sterile filtered product, or SFP.
- SPF should be held at 4°C until use in the redox step.
- the SPF is placed in an appropriate vessel such as a glass lined tank. A means of mixing the contents of the tank should be provided. If appropriate, multiple batches of SFP can be combined. If that is the case, we prefer to combine all batches to be treated together in a redox step and to blend them gently and briefly before adding the redox buffer.
- the redox step can be performed at a variety of temperatures.
- the step should be conducted at between 4°C and 55°C. However, certain considerations lead one to conduct the step at between 18°C and 45°C.
- the rHBsAg polypeptides are embedded in a lipid membrane.
- the fluidity of the membrane should be considered. For example, while one can choose to conduct the step cold temperatures, 4°C to 10°C, that is not preferred because the thermodynamics of the process would slow to the point where an enormous amount of time would be required to complete the step. At the upper end of the range, higher temperatures lead to more flexibility in the rHBsAg polypeptide and fluidity within the lipids.
- the protein can denature at high temperatures.
- the temperature should be high enough to allow movement of the polypeptides within the membrane. Therefore, we prefer to use temperatures high enough to produce a result in a reasonable amount of time but not so high as to lead to instability in correctly paired cysteines of protein denaturation.
- Our preferred temperature ranges are 20°C to 45°C, more preferably 30°C to 40°C and most preferable 34°C to 38°C. Temperature can be controlled by a variety of means including the use of a jacketed vessel.
- a dip-tube or other appropriate means of adding the redox buffer can be used with gentle and brief agitation sufficient to distribute the buffer through the batch. Thereafter, the batch is allowed to stand for a period of time.
- This oligomerization is likely to comprise several molecular events including (1) approximation of the subunits facilitated by lipid fluidity, (2) formation of inter- and intradisulfides, (3) disulfide exchange or re-shuffling, and (4) final stabilization of the tertiary and quaternary structure.
- the present invention has revealed that rHBsAg with even higher in vitro relative potency values can be made when an optimized folding step is conducted after conversion of the rHBsAg to form 111.
- Immunoassays are used to illustrate how the rHBsAg product's structural properties impact the immunogenic response. Examples of various assay values from different Hepatitis B vaccines will be used to illustrate the use of the assay.
- a standard ELISA assay has about a two day turnaround and multiple sample manipulations needed. This analytical method is useful for process for assessing antigen maturation. The process is advantageously conducted on rHBsAg that has been formulated on an aluminum adjuvant.
- the monoclonal H166 would be useful for capture because of its IgM nature (providing multiple Fab binding sites per molecule) and because it recognizes a linear or continuous epitope (Cys[121]-Lys-Thr-Cys[124]) of the rHBsAg (Chen et al.).
- the monoclonal H35 is believed to recognize a truly conformational or epitope since it binds to 13 residues located on two discontinuous regions of the rHBsAg molecule (Cys[121] ⁇ - Leu[175]) (Chen et al. 1996).
- this antibody could be conjugated with HRP and used as a reporting antibody to monitor the formation of a conformational epitope.
- the amino acid sequences of these epitopes were derived from affinity- enrichment experiments (known as "biopanning") using a filamentous phage display peptide library. (Chen et al. 1996) In addition to biopanning, the site for HI 66 binding was also determined by cyclic peptide competition (L. Mimms et al. 1989. Second generation assays for the detection of antibody to HBsAg using recombinant DNA-derived HBsAg. J. Viral. Methods 25:211-232.) The presence of a linear amino acid sequences of these epitopes were derived from affinity- enrichment experiments (known as "biopanning") using a filamentous phage display peptide library. (Chen et al. 1996) In addition to biopanning, the site for HI 66 binding was also determined by cyclic peptide
- HI 66 epitope is also supported by the observation that HI 66 binds to monomeric p24 in Western blots (L. Mimms et al 1989).
- Enzyme immunoassays known in the art can be used to assess the improved rHBsAg.
- the AUZYME monoclonal enzyme immunoassay commercially available from Abbott Laboratories, is preferred. To employ the AUZYME assay, one can follow the manufacturer's instructions for a two step procedure. This assay has been found to be quantitative in our hands. Briefly, beads coated with mouse monoclonal antibodies to hepatitis B surface antigen are incubated with the rHBsAg product sample and then with a mouse monoclonal Anti-HBsAg peroxidase conjugate (Anti-HBsAg:HRPO). Unbound material is washed off, and color is developed by adding a O-phenylenediamine (OPD) solution containing hydrogen peroxide. Development of a yellow-orange color is proportional to the antigen concentration.
- OPD O-phenylenediamine
- the rHBsAg sample is at 5mcg/ml
- the sample should be diluted 1:10, 1:100, 1 :500, 1:1000, 1:2000, 1:4000 and 1 :8000.
- Samples from 1 :500 to 1 :8000 are prepared and tested at least as duplicates or triplicates.
- samples at 10 mcg/ml the sample is diluted further to 1:16000 and tested at 1 :100-1 :16000.
- Diluent controls should be run along with the positive and negative controls provided in the AUZYME kit.
- the average of three OD492 absorbance values for the negative control should be in the range of -0.006 to 0.001.
- the diluent controls should be in the range of at OD492 0+/-0.02.
- An OPD substrate control can be run and should read less than at OD492 0.006 and -0.100.
- the units of measurement for the immunogenicity of a preparation of rHBsAg are grounded in ED50 measured per microgram of protein in the mouse potency assay. However, that assay takes almost two months to generate a result.
- the ELISA assay taught herein and to correlate the results of that assay to the mouse potency assay.
- the correlation is monitored by assaying a reference preparation of rHBsAg as a control with each run of the ELISA where the ED50 of the control is known.
- the correlation was established as follows. The initial rHBsAg reference material was tested in an in vivo mouse potency model and generated an ED50 value of 0.26 meg protein. It was decided that the OD492 of this material in the in vitro ELISA would be assigned an arbitrary in vitro potency value of 1.0. Thereafter, numerous tests were performed on various batches of rHBsAg in both the ELISA and mouse potency model.
- IVRP in vitro relative potency
- ED50 ED50
- FIG. 5 Each data point represents a sample of rHBsAg that was tested in both the mouse model and by ELISA. These data points were used to generate an equation that related the mouse potency value to the relative potency value obtained by ELISA. Once one has established the correlation of the ED50 and ELISA output scales in this manner, one can now perform ELISA tests and expect to see an ED50 value in line with the output of the regression equation:
- Plasma-derived Dane particles were tested in the ELISA assay.
- the Dane particles showed a higher response, of about 5.0. Therefore, the high values for rHBsAg product produced by the present process, i.e., values greater than 2.5, indicates that the rHBsAg particles are approaching an immunogenicity level close to that of the natural product of Hepatitis B virus infection, the Dane particle.
- particles of HbsAg produced by recombinant methods do not contain Hepatitis DNA.
- the BIAcore assay provides quantitative estimate of the rHBsAg' s relative immunogenicity under controlled conditions where many factors, such as protein concentrations, temperature, and "contact" time (related to the injection volume and flow rate) among others, are maintained identical for samples and reference.
- This technique has been used successfully for the epitope mapping of HBsAg (J-S Tung et al. 1998. Characterization of Recombinant Hepatitis B Surface Antigen Using Surface Plasmon Resonance. J. Pharmaceutical Sciences, 87:76-80).
- the assay is performed in the following format.
- Biomolecular interaction assay (BIA) is a technology developed by BIAcore, Inc., for monitoring biomolecular binding in real time.
- SPR surface plasmon resonance
- the SPR response reflects a change of refractive index as a result of mass deposition at a derivatized metallic (gold) sensor surface as molecules bind or dissociate. Because molecules are brought to the surface and the unbound molecules are washed-off by a micro fluidic pumping system, the interactions measured are actually binding kinetics. No labeling of the interacting components is required, and the technology is applicable to a wide range of experimental situations. As in any immunoassay, the proper reagents are important to the method's response and performance.
- the antibody When using the BIAcore technique to monitor the formation of the proper conformation of the improved HBsAg it is most useful to use an antibody that recognizes a conformational epitope. It is also preferable that the antibody binds to an epitope related to the neutralization of the invading hepatitis B virus.
- An example of such an antibody is a monoclonal antibody called MAb Al .2. It is also believed that the H35 monoclonal antibody of Chen et al 1996 would be appropriate.
- variable regions of the MAb A1.2 monoclonal antibody were published in Lohman et al, 1993. Molecular Characterization and Structural Modeling of Immunoglobin Variable Regions from Murine Monoclonal Antibodies Specific for Hepatitis B Virus Surface Antigen. Molecular Immunol. 30:1295-1306, and are shown in FIG. C. Using these sequences one can construct an appropriate antibody for use in the surface plasmon resonance assay. Alternatively, from the understanding of the conduct of the assay gained from the descriptions provided herein, one can screen monoclonal antibodies to find one with an appropriate epitope specificity that performs appropriately in the assay.
- the procedure involved the following steps as outlined as follows: (1) chemical immobilization of rat anti- mouse FCgamma on the sensor chip, (2) addition of MAb Al .2 anti-HBsAg which is captured by the rat anti-mouse antibody; and (3) injection of antigen product which is finally captured by MAb A1.2. (4) the antigenicity for the rHBsAg particles is expressed as the ratio between the amount of bound rHBsAg and the amount of bound MAb A1.2. For a normalized protein concentration in compared samples, the difference in the measured binding represents the difference in the HBsAg antigenicity due to the conformation of the rHBsAg.
- rHBsAg bound in the surface plasmon resonance assay one can monitor the accumulation of rHBsAg having a conformation associated with antigenicity.
- the units used to describe the antigenicity or the rHBsAg are arbitrary and relate only to the control run for the particular assay.
- By collecting a series of data points in each assay for different concentrations of a number of preparations of rHBsAg one can generate a date set useful to correlate the responses obtained from an ELISA, the surface plasmon resonance assay and the mouse potency assay.
- Vaccines including HBsAg from plasma and rHBsAg expressed in a variety of host cells are in common use in the art.
- the improved rHBsAg of this invention can be made by adding the redox step to the present processes used to make rHBsAg from the various host cells. Thereafter, the improved rHBsAg can be used to make any of the vaccines in which the presently made rHBsAg is used.
- compositions comprising the improved rHBsAg can be formulated according to known methods such as by the admixture of a pharmaceutically acceptable carrier.
- the improved rHBsAg can be adjuvanted and formulated using the usual practices of the pharmaceutical arts as exemplified by practice manuals and texts including Remington's Pharmaceutical Sciences.
- compositions will contain an effective amount of the improved rHBsAg.
- compositions can be made by adjuvanting the improved rHBsAg.
- Many adjuvants are known in the art. Aluminum adjuvants are frequently used because they are presently approved for use in human patients. Aluminum adjuvants can be provided as amorphous or crystalline forms. Common aluminum adjuvants include aluminum phosphate, aluminum hydroxide, and aluminum hydroxyphosphate. All of these can be used with the improved rHBsAg.
- adjuvanting used in the art can be applied to the improved rHBsAg and include co-precipitation of the adjuvant and rHBsAg and adsorption of the rHBsAg onto the adjuvant.
- Other adjuvants can be used and include saponins, lipids including cationic lipids, oligonucleotides having a CpG motif, muramyl dipeptides and DNA vaccines (Donnelly, J. J., Friedman, A., Deck, R. R., DeWitt, C. M., Caulfield, M. J., Liu, M. A., and Ulmer, J. B. 1997. Adjuvant effects of DNA vaccines. In Vaccines 97. F. Brown, F. Burton, P. Doherty, J. Mekalanos, and E. Norrby, eds. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY., p. 105-111).
- Therapeutic or prophylactic vaccines according to this invention include the improved rHBsAg and are administered to an individual in amounts sufficient to treat or prevent disorders associated with infection by hepatitis virus.
- vaccines of the present invention can be administered in a single dose, or the total dosage can be administered in divided doses of two, three or more doses over a period of time as determined to by effective by a skilled physician. The number of doses will be influenced by whether the rHBsAg containing vaccine is being administered for prophylactic or therapeutic treatment of a patient.
- the dosage amount and regimen utilizing vaccines that include the improved rHBsAg of the present invention are selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal, hepatic and cardiovascular function of the patient; and the particular vaccine employed.
- a physician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition and determine an appropriate dosage regimen.
- active immunogens can be in separate dosage formulations, they can be administered concurrently, or they each can be administered at separately staggered times.
- improved rHBsAg can be formulated in a combination vaccine with other active immunogens.
- the improved rHBsAg can be formulated with immunogens such as, and without limitation, Hemophilus influenza polyribitol phosphate that has been conjugated to a toxiod or a protein such as the outer membrane protein complex ofNeiserria meningitis; Measles, Mumps and Rubella immunogens; Diptheria toxoid; Tetanus toxoid; Pertussis, either whole cell or a-cellular [a-cellular meaning at least one of the following Pertussis immunogens: Pertussis Toxoid (also called Lymphocytosis Promoting Factor), Filimentous Hemagglutinin, 69kd Protein (also called Pertactin), Agglutinogen 2 and Agglutinogen 3]; inactivated or attenuated live Varicella zoster; Streptococcus pneumonia capsular polysaccharide conjugated to a toxiod or a protein such as Neiserria menningit
- the rHBsAg can be formulated with a DNA vaccine(s) encoding an immunogen(s) alone or in addition to other immunogens listed above.
- An advantage of a vaccine formulated with the improved rHBsAg is that one can use a lower amount of the immunogen and achieve the same efficacy as the vaccines presently available in the art.
- An additional advantage is that one can formulate a vaccine that includes an amount of improved rHBsAg equal to an amounts used in present formulations and produce a stronger response in a patient.
- SFP Sterile filtered product
- phosphate buffered saline (6 mM phosphate, 0.15 M NaCl) was manufactured as known in the art (Wampler 1985) and stored at 4°C. Thereafter, to a solution of 200 mL SFP in a Pyrex glass bottle, glutathione (GSH) and oxidized glutathione (GSSG) were introduced to the final concentrations of 1.0 mM and 0.2 mM, respectively. Once the mixing was completed, the batch temperature was ramped up to 37°C in an incubator.
- GSH glutathione
- GSSG oxidized glutathione
- the batch was incubated further for another 60 hrs. It is preferable that the total time from the beginning of the heating step through the end of this incubation should be between 100 to 105 hours. At the end of that time period the batch is cooled to 2 - 8°C without mixing. When batch temperature reaches 2 - 8°C, agitation was begun and
- BAP Bulk Alum Product
- In vitro relative potency values of more than 3.0 can be obtained.
- In vitro relative potency values can be expected to range from about 2.5 to about 4.0, more usually, from about 2.75 to about 3.5 or 3.75, and commonly from about 2.75 or 3.0 to about 3.25 or 3.5. Higher values can also be achieved if the formalin step is omitted. In that case, values can range as above and up to about 4.25, 4.5, 4.75, 5.0, 5.25, 5.5, 5.75 or 6.0.
- the incubation in the presence of GSH/GSSG in this Example was conducted for 44 hours but the incubation can be conducted longer as described above.
- An incubation of about 100 hours can be preferable to one of 40 hours. However, while one can incubate for less than 40 hours, at least 40 hours is preferred.
- rHBsAg An accepted measurement of the immunogenicity of rHBsAg which will lead to an appropriate immune response in an animal is the efficacy of rHBsAg in a live mouse model.
- the assay is conducted to measure the dose of a preparation of rHBsAg that is effective in producing an immune response in the animal. This test is sometimes referred to in the art as a mouse potency test.
- the mouse potency (MP) test is performed by preparing a series of 2- fold dilutions of the rHBsAg product using the adjuvant solutions (lx Alum with antigen, other components are the same with rHBsAg preparations) are injected into adult Balb/C mice.
- An appropriate format is to use five groups of eight mice. Each group is tested at one of five concentrations of rHBsAg, e.g. 1.0 mcg/mL to 0.0625 mcg/mL.
- concentrations of rHBsAg e.g. 1.0 mcg/mL to 0.0625 mcg/mL.
- mice After injection, the mice are held for 6 weeks, bled and the individual sera are tested for anti-HBsAg using "AUSAB-EIA" diagnostic kit (Abbott Lab) following the manufacturer's instructions to determine the immuno-response by measuring the total anti-HBsAg B antibodies.
- the potency of the rHBsAg is reported as the dose which elicits antibody response in 50% of the mice (ED50).
- in vitro relative potency ,in vivo mouse potency and clinical performance of bulk alum formulated rHBsAg can be monitored by appropriate tests as taught herein or as are in practice in the art.
- the surface plasmon resonance (SPR) technology embodied in the BIAcore equipment is useful for monitoring the real time maturation of conformationally appropriate rHBsAg.
- This Example describes how to use the technology by correlating the SPR measurement of a liquid sample taken during a process step and to a reference standard of rHBsAg.
- the binding surface of the SPR detector is made of a derivatized metal, preferably gold.
- a rat anti-mouse FCgamma is covalently coupled to the derivatized gold surface through an amine coupling.
- a mouse anti-rHBsAg monoclonal antibody that binds to a conformational epitope is contacted with the detector and bound to the surface by the rat anti-mouse antibody.
- the detector surface is then washed with a buffer appropriate to the binding of the rHBsAg by the mouse monoclonal antibody.
- an antibody that binds a conformational epitope It is important to use an antibody that binds a conformational epitope. Techniques for testing an antibody for binding to a conformational epitope are known. Several antibodies that bind conformational epitopes of rHBsAg are also known and include H35 (Chen et al, 1996) and MAb A1.2 (Lohman et al, 1993). The MAb Al .2 antibody is the preferred antibody.
- the detector is calibrated by binding a reference standard of rHBsAg for which the in vitro relative potency of the product formulated on alum is known. Because the SPR technology is used to measure non-adjuvanted material in a liquid system, reference rHBsAg material needs to be preserved in a non-adjuvanted form, preferably as sterile filtered product.
- the detector is calibrated by binding a reference standard of rHBsAg. Because the SPR technology is used to measure non-adjuvanted material in a liquid system, reference rHBsAg material needs to be preserved in a non-adjuvanted form, preferably as final aqueous product. It has been found that a reference standard of FAP (final aqueous product) can be stored at 4°C for over two years. The reference should be aged for at least six months and preferably aged over 2 years. The technique is conducted according to the manufacturer's directions or as described in the literature (Tung et al (1998) J. Pharm. Sci.
- Ethanolamine-HCl, NHS (N-Hydroxysuccinimide) and EDC (N-Ethyl-N'-(3- dimethylaminopropyl)-carbodiimide hydrochloride) reagents were obtained from the manufacturer (BIAcore) and used according to directions. Sample and calibration standards are diluted to 11 mcg/ml in prepared in 0.15 mM NaCl, 6 mM phosphate, and TWEEN 20 is added to 0.05%.
- Coupling antibody rat-anti-mouse Fcg (BIAcore, Inc.) 50 ⁇ g/mL in 10 mM sodium acetate buffer pH 4.8 is immobilized on the surface of sensor chip through amine coupling using the Ethanolamine-HCl reagent.
- Binding monoclonal antibody (e.g. Mab Al .2 purified at 1.0 mg/ml, and diluted 20-fold to 50 ⁇ g/ml using HBS buffer (lOmM HEPES, 0.15mM NaCl, 3mM EDTA, 0.005% TWEEN 20, pH 7.4 filtered and degassed) (Biacore) prior to assay.) is then injected and immobilized;
- HBS buffer lOmM HEPES, 0.15mM NaCl, 3mM EDTA, 0.005% TWEEN 20, pH 7.4 filtered and degassed
- Regeneration The surface of the detector is exposed briefly to acid (e.g. 20 mM HCl/10 mM aspartic acid/1 % TWEEN 20) to remove the non-covalently- immobilized monoclonal antibody and antigen to regenerate the surface for the next cycle of assay;
- acid e.g. 20 mM HCl/10 mM aspartic acid/1 % TWEEN 20
- the ratio between the amount of antigen vs. monoclonal antibody is used to represent the antigenicity of the tested aqueous preparations of rHBsAg.
- the 68 data points were accumulated during a period of several months.
- the relative antigenicity of final aqueous products at 11 mcg/ml was measured against a reference standard at the same concentration.
- the Mab A1.2 binding to rHBsAg was carried out at 20°C in 6 mM potassium phosphate and 0.15 M NaCl. However, during the assay the sampling block is cooled to about 10°C.
- the detector surface is briefly exposed to acid to remove the non-covalently-immobilized monoclonal antibody and antigen to regenerate the surface for the next cycle of assay.
- the antigenicity is expressed as the ratio between the amount of rHBsAg and the amount of Mab A 1.2 bound to the sensor chip surface. Then, this ratio was normalized to the ratio for the calibration standard.
- ELISA was carried out on the corresponding bulk Alum products and F/RP values were derived. Good correlation between Mab A1.2 binding of rHBsAg by SPR and the INRP by conventional ELISA as shown in Fig.7. In this Example, a total of sixty eight data points were collected.
- Each data point represents a sample for which the in vitro relative potency of the alum formulated rHBsAg product was determined and compared to a surface plasmon resonance measurement of the liquid non-adjuvanted rHBsAg product. From these data points, a linear fit yielded the theoretical line and parameters shown in FIG.7. The figure shows the linear relationship between IVRP of product formulated on alum versus the relative antigenicity of the rHBsAg in final aqueous form as a (%) of a reference standard as measured by SPR.
- the SPR measurements of the liquid form of the rHBsAg product correlate well with the in vitro relative potency values obtained once the rHBsAg product is adjuvanted with alum. Therefore, the SPR technology can be used to monitor the real-time formation of conformational epitopes whose presence in the rHBsAg product result in antigenicity in vitro and immunogenicity in vivo.
- a diafiltration step can be added after the incubation, if desired, to remove the residual redox buffer agents from the rHBsAg.
- FIG. 2A shows that there is a synergistic effect when GSH and GSSG are combined in a redox buffer.
- FIG. 2B shows that there is an increase in the amount of rHBsAg containing important conformational epitopes as the amount of GSH alone is increased.
- the ELISA based IVRP and mouse ED 50 results demonstrate that the rHBsAg produced by the method of this invention has very high antigenicity and immunogenicity. If the above data is compared to the historical data for rHBsAg made by prior art methods shown in FIG. 5., the present data plots lower and to the right of the historical data.
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EP99966613A EP1140155A4 (en) | 1998-12-23 | 1999-12-22 | Improved recombinant hepatitis b surface antigen |
JP2000589214A JP2002532116A (en) | 1998-12-23 | 1999-12-22 | Improved recombinant hepatitis B surface antigen |
AU22121/00A AU2212100A (en) | 1998-12-23 | 1999-12-22 | Improved recombinant hepatitis b surface antigen |
CA 2355680 CA2355680A1 (en) | 1998-12-23 | 1999-12-22 | Improved recombinant hepatitis b surface antigen |
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US11340098P | 1998-12-23 | 1998-12-23 | |
US60/113,400 | 1998-12-23 |
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EP (1) | EP1140155A4 (en) |
JP (1) | JP2002532116A (en) |
AU (1) | AU2212100A (en) |
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US6436402B1 (en) | 1999-10-15 | 2002-08-20 | Merck & Co., Inc. | Process for making human papillomavirus virus-like particles with improved properties |
JP2004505992A (en) * | 2000-08-10 | 2004-02-26 | グラクソスミスクライン バイオロジカルズ ソシエテ アノニム | Purification of HBV antigens for use in vaccines |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
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US5614384A (en) * | 1991-04-29 | 1997-03-25 | Merck & Co., Inc. | Hepatitis B virus surface proteins with reduced host carbohydrate content |
US5972346A (en) * | 1995-02-25 | 1999-10-26 | Smithkline Beecham Biologicals S.A. | Hepatitis B vaccine |
Family Cites Families (2)
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US4707542A (en) * | 1983-08-22 | 1987-11-17 | Merck & Co., Inc. | Immunogenic HbsAg derived from transformed yeast |
EP0314240A3 (en) * | 1987-10-26 | 1990-03-28 | Merck & Co. Inc. | Process for purifying recombinant hepatitis antigens |
-
1999
- 1999-12-22 WO PCT/US1999/030770 patent/WO2000037104A1/en active Application Filing
- 1999-12-22 JP JP2000589214A patent/JP2002532116A/en not_active Withdrawn
- 1999-12-22 CA CA 2355680 patent/CA2355680A1/en not_active Abandoned
- 1999-12-22 EP EP99966613A patent/EP1140155A4/en not_active Withdrawn
- 1999-12-22 AU AU22121/00A patent/AU2212100A/en not_active Abandoned
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5614384A (en) * | 1991-04-29 | 1997-03-25 | Merck & Co., Inc. | Hepatitis B virus surface proteins with reduced host carbohydrate content |
US5972346A (en) * | 1995-02-25 | 1999-10-26 | Smithkline Beecham Biologicals S.A. | Hepatitis B vaccine |
Non-Patent Citations (1)
Title |
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See also references of EP1140155A4 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6436402B1 (en) | 1999-10-15 | 2002-08-20 | Merck & Co., Inc. | Process for making human papillomavirus virus-like particles with improved properties |
JP2004505992A (en) * | 2000-08-10 | 2004-02-26 | グラクソスミスクライン バイオロジカルズ ソシエテ アノニム | Purification of HBV antigens for use in vaccines |
JP2012255015A (en) * | 2000-08-10 | 2012-12-27 | Glaxosmithkline Biologicals Sa | Purification of hbv antigen for use in vaccine |
US8624004B2 (en) | 2000-08-10 | 2014-01-07 | Glaxosmithkline Biologicals, S.A. | Purification of HBV antigens for use in vaccines |
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AU2212100A (en) | 2000-07-12 |
JP2002532116A (en) | 2002-10-02 |
EP1140155A1 (en) | 2001-10-10 |
EP1140155A4 (en) | 2004-11-03 |
CA2355680A1 (en) | 2000-06-29 |
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