HK1186969A

HK1186969A - Stable formulations of neisseria meningitidis rlp2086 antigens

Info

Publication number: HK1186969A
Application number: HK13114428.8A
Authority: HK
Inventors: L．汉德科; R．阿鲁穆加姆; B.劳恩
Original assignee: 惠氏有限责任公司
Priority date: 2010-08-23
Filing date: 2011-08-22
Publication date: 2014-03-28

Description

Stable formulations of neisseria meningitidis rLP2086 antigen

Technical Field

The present invention relates to formulations of Neisseria meningitidis (Neisseria meningitidis) rp2086 subfamily B antigen in the form of an immunogenic composition as described herein. The invention also relates to methods of maintaining the conformation of the neisseria meningitidis rLP2086 antigen, and methods of determining the efficacy of the neisseria meningitidis rLP2086 antigen.

Background

rLP2086 is a 28kDa recombinant lipoprotein that elicits cross-reactive bacterial antibodies to a variety of neisseria meningitidis strains. Based on putative amino acid sequence homology, two distinct rLP2086 subfamilies were identified: a and B. These two subfamilies were used to formulate MnB-rLP2086 vaccine samples of each subfamily containing 20, 60, 120, and 200. mu.g/mL in 10mM histidine (pH6.0), 150mM NaCl, and 0.5mg/mL aluminum with different levels of polysorbate 80 (PS-80). Polysorbate 80, also known as TWEEN80, is a nonionic surfactant and emulsifier derived from sorbitol and is frequently used in pharmaceutical formulations as an emulsifier, solubilizer and stabilizer. The presence of polysorbate 80 in the MnB rLP2086 immunogenic composition is believed to prevent aggregation during formulation, processing, filtration, filling, and transportation, reduce filter membrane uptake, and reduce tube uptake.

Summary of The Invention

In some embodiments, the present invention provides a stable immunogenic composition wherein the potency of the LP2086 subfamily B polypeptide is maintained for at least about 1-12 months, about 6-18 months, about 12-24 months, about 24-36 months, or about 36-48 months. In some embodiments, the immunogenic composition further comprises a LP2086 subfamily a polypeptide.

In some embodiments, the immunogenic composition further comprises a detergent. In some embodiments, the molar ratio of detergent to protein is from about 0.5:1 to about 10:1, from about 1:1 to about 5:1, or from about 1.4:1 to 4.2: 1. In some embodiments, the molar ratio of detergent to protein is about 2.8: 1. In some embodiments, the amount of detergent is sufficient to reduce binding of the polypeptide to silicon in a container, such as a syringe or vial. In some embodiments, the detergent is a non-ionic detergent, such as a polysorbate detergent. In some embodiments, the detergent is polysorbate-80.

In some embodiments, the immunogenic composition further comprises a multivalent cation. In some embodiments, the multivalent cation is calcium or aluminum. In some embodiments, the immunogenic composition comprises calcium phosphate. In some embodiments, the immunogenic composition comprises aluminum in the form of aluminum phosphate, aluminum hydroxide, aluminum sulfate, or alum. In some embodiments, the concentration of aluminum is about 0.1mg/mL to 1.0 mg/mL. In some embodiments, the concentration of aluminum is about 0.5 mg/mL.

In some embodiments, the immunogenic composition further comprises histidine. In some embodiments, the concentration of histidine is from about 2mM to about 20mM, alternatively from about 5mM to about 15 mM. In some embodiments, the concentration of histidine is about 10 mM. In some embodiments, the pH of histidine is from about 5.0 to about 8.0, or from about 5.8 to about 6.0. In some embodiments, histidine is at a concentration of 10mM and pH 6.0.

In some embodiments, the immunogenic composition further comprises succinate. In some embodiments, the concentration of succinate is from about 2mM to about 10mM, or from about 3mM to about 7 mM. In some embodiments, the concentration of succinate is about 5 mM. In some embodiments, the pH of the succinate salt is from about 5.0 to about 8.0, or from about 5.8 to about 6.0. In some embodiments, the succinate is at a concentration of 5mM and pH 6.0.

In some embodiments, the immunogenic composition is lyophilized. In some embodiments, the lyophilized composition is resuspended in a buffer comprising aluminum. In some embodiments, the aluminum is present as aluminum phosphate, aluminum hydroxide, aluminum sulfate, or alum.

In some embodiments, the immunogenic composition comprises polysorbate 80 at a molar ratio of about 2.8:1 to protein, 0.5mg/mL AlPO₄Form aluminum, 10mM histidine pH6.0 and 150mM NaCl. In some embodiments, the immunogenic composition consists essentially of 200ug/mL LP2086(fHBP) subfamily B polypeptide, polysorbate 80 at a molar ratio to protein of about 2.8:1, 0.5mg/mL AlPO₄Form aluminum, 10mM histidine pH6.0 and 150mM NaCl. In some embodiments, the immunogenic composition consists essentially of 200ug/mL rLP2086(fHBP) subfamily a polypeptide, 200ug/mL LP2086(fHBP) subfamily B polypeptide, polysorbate 80 in a molar ratio to protein of about 2.8:1, 0.5mg/mL @mL AlPO₄Form aluminum, 10mM histidine pH6.0 and 150mM NaCl.

In some embodiments, the immunogenic composition comprises polysorbate 80 at a molar ratio of about 2.8:1 to protein, 0.5mg/mL AlPO₄Form aluminum, 5mM succinate pH6.0 and 150mM NaCl. In some embodiments, the immunogenic composition consists essentially of 200ug/mL LP2086(fHBP) subfamily B polypeptide, polysorbate 80 at a molar ratio to protein of about 2.8:1, 0.5mg/mL AlPO₄Form of aluminum, 5mM succinate pH6.0 and 150mM NaCl. In some embodiments, the immunogenic composition consists essentially of 200ug/mL of rLP2086(fHBP) subfamily a polypeptide, 200ug/mL of LP2086(fHBP) subfamily B polypeptide, polysorbate 80 at a molar ratio to protein of about 2.8:1, 0.5mg/mL of AlPO 80₄Form of aluminum, 5mM succinate pH6.0 and 150mM NaCl.

In another aspect, the invention provides a method of stabilizing the potency of a LP2086 subfamily B polypeptide in an immunogenic composition by: the LP2086 subfamily B polypeptide is stored in a buffer having a detergent to protein molar ratio of about 0.5:1 to 10:1, about 1:1 to about 5:1, or about 1.4:1 to about 4.2: 1. In some embodiments, the molar ratio of detergent to protein is about 2.8: 1. In some embodiments, the amount of detergent is sufficient to reduce binding of the polypeptide to silicon in a container, e.g., a syringe or vial. In some embodiments, the detergent is a nonionic detergent, such as a polysorbate detergent. In some embodiments, the detergent is polysorbate-80.

In some embodiments, the buffer further comprises a multivalent cation. In some embodiments, the multivalent cation is calcium or aluminum. In some embodiments, the buffer comprises calcium phosphate. In some embodiments, the buffer comprises aluminum in the form of aluminum phosphate, aluminum hydroxide, aluminum sulfate, or alum. In some embodiments, the concentration of aluminum is about 0.1mg/mL to 1.0 mg/mL. In some embodiments, the concentration of aluminum is about 0.5 mg/mL.

In some embodiments, the buffer further comprises histidine. In some embodiments, the concentration of histidine is from about 2mM to about 20mM or from about 5mM to about 15 mM. In some embodiments, the concentration of histidine is about 10 mM. In some embodiments, the pH of histidine is from about 5.0 to about 8.0 or from about 5.8 to about 6.0. In some embodiments, histidine is at a concentration of 10mM and pH 6.0.

In some embodiments, the buffer further comprises succinate. In some embodiments, the concentration of succinate is from about 2mM to about 10mM or from about 3mM to about 7 mM. In some embodiments, the concentration of succinate is about 5 mM. In some embodiments, the pH of the succinate salt is from about 5.0 to about 8.0 or from about 5.8 to about 6.0. In some embodiments, the succinate is at a concentration of 10mM and pH 6.0.

In some embodiments, the buffer consists essentially of polysorbate 80, 0.5mg/mL AlPO at a molar ratio to protein of about 2.8:1₄Form aluminum, 10mM histidine pH6.0, and 150mM NaCl. In some embodiments, the immunogenic composition consists essentially of 200ug/mLLP2086(fHBP) subfamily B polypeptide, polysorbate 80 at a molar ratio to protein of about 2.8:1, 0.5mg/mL AlPO₄Form aluminum, 10mM histidine pH6.0 and 150mM NaCl.

In some embodiments, the buffer consists essentially of polysorbate 80, 0.5mg/mL AlPO at a molar ratio to protein of about 2.8:1₄Form of aluminum, 5mM succinate pH6.0 and 150mM NaCl. In some embodiments, the immunogenic composition consists essentially of 200ug/mLLP2086(fHBP) subfamily B polypeptide, polysorbate 80 at a molar ratio to protein of about 2.8:1, 0.5mg/mL AlPO₄Form of aluminum, 5mM succinate pH6.0 and 150mM NaCl.

In another aspect, the invention provides a method of stabilizing the potency of LP2086 subfamily a polypeptide and LP2086 subfamily B polypeptide in an immunogenic composition by: storing the LP2086 subfamily A polypeptide and the LP2086 subfamily B polypeptide in a buffer comprising about 0.1mg/mL to about 10mg/mL aluminum and a detergent to protein molar ratio of about 0.5:1 to 10: 1. In some embodiments, the molar ratio of detergent to protein is from about 1:1 to about 5:1, or from about 1.4:1 to about 4.2: 1. In some embodiments, the molar ratio of detergent to protein is about 2.8: 1. In some embodiments, the amount of detergent is sufficient to reduce binding of the polypeptide to silicon in a container, e.g., a syringe or vial. In some embodiments, the detergent is a nonionic detergent, such as a polysorbate detergent. In some embodiments, the detergent is polysorbate 80.

In some embodiments, the aluminum is present as aluminum phosphate, aluminum hydroxide, aluminum sulfate, or alum. In some embodiments, the concentration of aluminum is about 0.5 mg/mL.

In some embodiments, the buffer consists essentially of polysorbate 80, 0.5mg/mL AlPO at a molar ratio to protein of about 2.8:1₄Form aluminum, 10mM histidine pH6.0, and 150mM NaCl. In some embodiments, the immunogenic composition consists essentially of 200ug/mL LP2086(fHBP) subfamily a polypeptide, 200ug/mL LP2086(fHBP) subfamily B polypeptide, polysorbate 80 at a molar ratio to protein of about 2.8:1, 0.5mg/mL AlPO 80₄Form aluminum, 10mM histidine pH6.0 and 150mM NaCl.

In some embodiments, the buffer consists essentially of polysorbate 80, 0.5mg/mL AlPO at a molar ratio to protein of about 2.8:1₄Form of aluminum, 5mM succinate pH6.0 and 150mM NaCl. In some embodiments, the immunogenic composition consists essentially of 200ug/mL LP2086(fHBP) subfamily a polypeptide, 200ug/mL LP2086(fHBP) subfamily B polypeptide, polysorbate 80 at a molar ratio to protein of about 2.8:1, 0.5mg/mL AlPO 80₄Form aluminum, 5mM succinate pH6.0 and 150mM NaCl.

In another aspect, the invention provides a method of determining the potency of an rLP2086 subfamily a polypeptide and/or an rLP2086 subfamily B polypeptide, comprising the steps of: (a) binding first and second functional monoclonal antibodies recognizing conformational epitopes on each subfamily of protein to the immunogenic composition, and (b) quantifying the antibodies bound to the polypeptides. In some embodiments, the quantifying is performed by electrochemiluminescence. In some embodiments, polypeptides that exhibit epitopes recognized by both antibodies are quantified. In some embodiments, the first antibody is conjugated to a label, such as biotin. In some embodiments, the first antibody is isolated from a compound bound to a conjugated label, such as streptavidin beads or a streptavidin column. In some embodiments, the second antibody is conjugated to a quantitative label. In some embodiments, the potency of the immunogenic composition is compared to the potency of a reference substance.

Brief Description of Drawings

FIG. 1: stability of subfamily B in formulations with different polysorbate 80 concentrations.

FIG. 2: accelerated stability of subfamily B with different polysorbate 80 concentrations.

FIG. 3: potency of 200. mu.g/mL of subgroup B within 28 days.

FIG. 4: 20 μ g/mL of subfamily B in 28 days of efficacy.

FIG. 5: efficacy results of 200. mu.g/mL with different molar ratios.

FIG. 6: efficacy results of 20. mu.g/mL with different molar ratios.

FIG. 7: binding of protein to aluminum phosphate at ph 6.5.

FIG. 8: binding of MnB rLP2086 subfamilies a and B as a function of pH.

FIG. 9: effect of pH, buffer and protein concentration on binding of rLP2086 subfamily a and B.

FIG. 10: appearance of rLP2086 formulation without aluminum phosphate.

FIG. 11: OD measurement for appearance samples at 2-8 ℃.

FIG. 12: with and without AlPO₄The efficacy results of subfamily a of the formulation of (a).

FIG. 13: with and without AlPO₄The efficacy results of subfamily B of the formulation of (a).

FIG. 14: polysorbate 80 results in rLP2086 placebo with 0.5mg/mL aluminum.

FIG. 15: polysorbate 80 results for subfamily a.

FIG. 16: polysorbate 80 results from subfamily B.

FIG. 17: the correlation of the potency of subfamily B with the binding molar ratio (bound molar ratio).

FIG. 18: the mole ratio of subfamily A results.

FIG. 19: the molar ratio of subgroup B results.

FIG. 20: results for a molar ratio of rLP2086 formulation of 400. mu.g/mL.

FIG. 21: polysorbate 80 results for rLP2086 drug at different time points.

FIG. 22: binding mole ratio results for rLP2086 drug at different time points.

FIG. 23: potency and binding mole ratio results for subfamily a.

FIG. 24: potency and binding mole ratio results for subgroup B.

FIG. 25: subfamily A and AlPO₄Binding in succinate and histidine buffers.

FIG. 26: subgroup B and AlPO₄Binding in succinate and histidine buffers.

FIG. 27 is a schematic view showing: binding in succinate, histidine and phosphate buffer were compared.

FIG. 28: subfamily A and AlPO₄pH dependent binding of (a).

FIG. 29: subgroup B and AlPO₄pH dependent binding of (a).

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patents, and other references mentioned herein are incorporated by reference in their entirety.

Throughout this specification, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

Definition of

As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "the method" includes one or more of the types of methods and/or steps described herein and/or apparent to one of ordinary skill in the art upon reading this disclosure, and so forth.

As used herein, the plural encompasses singular references unless the context clearly dictates otherwise. Thus, for example, reference to "the method" includes one or more of the types of methods and/or steps described herein and/or apparent to one of ordinary skill in the art upon reading this disclosure, and so forth.

As used herein, "about" means within a statistically significant range of values, such as the recited concentration range, time frame (time frame), molecular weight, temperature, or pH. Such a range may be within an order of magnitude of a given value or range, typically within 20%, more typically within 10%, and even more typically within 5%. The allowable variation (allowable variation) covered by the term "about" will depend on the particular system under study and can be readily understood by one of ordinary skill in the art. Whenever a range is recited in this application, every integer within the range is also considered an embodiment of the invention.

The term "adjuvant" refers to an enhancing pairA compound or mixture of immune responses to an antigen as further described and exemplified herein. Non-limiting examples of adjuvants that can be used in the vaccines of the present invention include RIBI adjuvant system (Ribi Inc., Hamilton, Mont.), alum, mineral gels such as aluminum hydroxide gel, oil-in-water emulsions, water-in-oil emulsions such as Freund's complete and Freund's incomplete adjuvant, block copolymers (CytRx, Atlanta Ga.), QS-21(Cambridge Biotech Inc., Cambridge Mass.), SAF-M (Chiron, Emeryville Calif.)Adjuvants, saponins, Quil a or other saponin components, monophosphoryl lipid a (monophosphoryl lipid a), and Avridine lipid-amine adjuvants (Avridine lipid-amine adjuvants).

The term "aluminum-to-protein binding" refers to the percentage of protein molecules in the composition that are bound to aluminum. Binding of aluminum to proteins can be determined using the methods disclosed herein or methods known in the art.

The term "effective immunogenic amount" as used herein refers to an amount of a polypeptide or a composition comprising a polypeptide that is effective to elicit an immune response in a vertebrate host. For example, an effective immunogenic amount of an rLP2086 protein of the invention is an amount effective to elicit an immune response in a vertebrate host. The specific "effective immunogenic dose or amount" will depend on the age, weight and medical condition of the host, and also on the method of administration. Suitable dosages are readily determined by those skilled in the art.

The term "molar ratio" as used herein refers to the ratio of the number of moles of two different ingredients in a composition. In some embodiments, the molar ratio is the ratio of moles of detergent to moles of protein. In some embodiments, the molar ratio is the ratio of moles of polysorbate 80 to moles of protein. Based on the concentration of protein and polysorbate 80, the molar ratio was calculated using the following equation:

for example, a composition comprising 0.01% polysorbate 80 and 200 μ g protein has a detergent to protein molar ratio of 10.8:1[ (0.01/0.2) x216 ]. The ratio of 3 moles polysorbate 80 to 2 moles protein can be expressed as a 3:2 molar ratio of PS80 to protein. In addition, if a molar ratio is expressed as a single number, it refers to the ratio of that single number to 1. For example, polysorbate 80 to protein ratios of 0.5, 2, and 10 refer to ratios of 0.5:1, 2:1, and 10:1, respectively. The terms "detergent to protein" molar ratio and "polysorbate 80 to protein" molar ratio as used herein generally refer to the molar ratio of detergent (or polysorbate 80) to protein antigen (particularly the P2086 antigen). Based on the teachings disclosed herein, one skilled in the art can determine how to calculate the mole ratios of the other detergents and the optimal mole ratios for formulations containing the other detergents. As used herein, a "low" molar ratio generally refers to a molar ratio of detergent to protein antigen in the immunogenic composition that is less than the "high" molar ratio. A "high" molar ratio generally refers to a molar ratio of detergent to protein antigen in the immunogenic composition that is higher than a "low" molar ratio. In some embodiments, a "high molar ratio" of detergent to protein refers to a molar ratio of greater than 10: 1. In some embodiments, a "low molar ratio" of detergent to protein refers to a 0.5:1 to 10:1 molar ratio.

The term "ORF 2086" as used herein refers to a open reading frame 2086 derived from a bacterium of the Neisseria (Neisseria) species. Neisserial ORF2086, the proteins encoded thereby, fragments of those proteins, and immunogenic compositions comprising these proteins are known in the art and are described, for example, in U.S. patent application publications US20060257413 and US20090202593 (each of which is incorporated herein by reference in its entirety). The term "P2086" generally refers to the protein encoded by ORF 2086. The P2086 proteins of the invention may be lipidated (lipidated) or non-lipidated. Generally, "LP 2086" and "P2086" refer to lipidated and non-lipidated forms of 2086 protein, respectively. The P2086 protein of the invention may be recombinant. Generally, "rLP 2086" and "rP 2086" refer to lipidated and non-lipidated forms of the recombinant 2086 protein, respectively.

The term "pharmaceutically acceptable carrier" as used herein is intended to include any and all solvents, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic and absorption delaying agents, and the like, compatible with administration to a human or other vertebrate host. Typically, a pharmaceutically acceptable carrier is approved by a regulatory agency of the federal, a state government, or other regulatory agency, or a carrier for animals (including humans as well as non-human mammals) which is set forth in the U.S. pharmacopeia or other generally recognized pharmacopeia. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin. Water, saline solutions, aqueous dextrose solutions, and glycerol solutions can be employed as the liquid carrier, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition may also contain minor amounts of wetting agents, fillers, emulsifiers or pH buffers, if desired. These compositions may be in the form of solutions, suspensions, emulsions, sustained release formulations, and the like. Examples of suitable pharmaceutical carriers are described in "Remington's pharmaceutical sciences" by e.w. martin. The formulation should be adapted to the mode of administration. Suitable carriers will be apparent to those skilled in the art and will depend greatly on the route of administration.

The term "potency" refers to the ability of an antigen to elicit an immunogenic response. In some embodiments, potency is measured by the ability of the epitope to bind to an antibody. Efficacy may be due to loss of integrity of the antigen or epitope, or a change in conformation of the antigen or epitopeAnd is lost or reduced by the passage. Efficacy may be lost or reduced due to factors including, but not limited to: light, temperature, freeze-thaw cycle, agitation, and pH. Potency can be measured by the methods disclosed herein as well as tests known in the art. Such potency assay tests include, but are not limited to, animal vaccination models, serum bactericidal tests (SBA), flow cytometry, and in vitro potency tests. Preferred methods for determining potency are SBA and in vitro potency tests. A more preferred method for determining potency is SBA. In some embodiments, the efficacy may be determined using at least one monoclonal antibody directed against at least one epitope involved in an immune response. In some embodiments, the potency of a test sample is compared to the potency of a reference standard. In some embodiments, the reference standard is T₀The test sample of (1). In some embodiments, the reference standard is an immunogenic composition without a detergent. In some embodiments, the reference standard is an immunogenic composition having a detergent to protein molar ratio of greater than 10: 1.

By "protective" immune response is meant the ability of the immunogenic composition to elicit an immune response (including humoral and cell-mediated immune responses) that serves to protect an individual from infection. If there is a statistically significant improvement compared to a population of control individuals (e.g., infected animals not administered the vaccine or immunogenic composition), the protection provided need not be absolute, i.e., the infection need not be completely prevented or eliminated. Protection may be limited to relieving the severity or the rate of onset of symptoms of infection. In general, a "protective immune response" includes eliciting an increase in the level of antibodies specific for a particular antigen (including some measurable level of functional antibody responses against each antigen) in at least 50% of individuals. In particular instances, a "protective immune response" may include eliciting a two-fold or four-fold increase in antibody levels specific for a particular antigen (including some measurable level of functional antibody responses to each antigen) in at least 50% of individuals. In certain embodiments, an opsonizing (opsonizing) antibody is associated with a protective immune response. Thus, protective immune responses can be tested by measuring the percent reduction in bacterial count in opsonophagocytosis tests (such as those described below). In some embodiments, there is a reduction in bacterial count of at least 10%, 25%, 50%, 65%, 75%, 80%, 85%, 90%, 95% or more compared to bacterial count in the absence of the immunogenic composition.

The terms "protein", "polypeptide" and "peptide" refer to a polymer of amino acid residues and are not limiting as to the shortest length of the product. Accordingly, peptides, oligopeptides, dimers, multimers, and the like are included within this definition. Both full-length proteins and fragments thereof are included within this definition. The term also includes modifications such as deletions, additions and substitutions to the native sequence (which are generally conserved in nature, but may also be non-conserved), preferably such that the protein retains the ability to elicit an immune response in the animal to which it is administered. Also included are post-expression modifications such as glycosylation, acetylation, lipidation, phosphorylation, and the like.

The term "recombinant" as used herein refers to any protein, polypeptide, or cell expressing a gene of interest produced by genetic engineering methods. The term "recombinant" as used with respect to a protein or polypeptide refers to a polypeptide produced by expression of a recombinant polynucleotide. The protein of the present invention may be isolated from natural sources or may be produced by genetic engineering methods. "recombinant" as used herein also describes nucleic acid molecules that: due to its origin or manipulation, the nucleic acid molecule is not associated with all or part of the polynucleotide with which it is associated in nature. With respect to host cells, the term "recombinant" is used to refer to host cells that comprise a recombinant polynucleotide.

The terms "stable" and "stability" refer to the ability of an antigen to maintain immunogenicity over a period of time. The stability of efficacy over time can be measured. The terms "stable" and "stability" also refer to the physical, chemical, and conformational stability of an immunogenic composition. Instability of protein compositions may be caused by the following reasons: the protein molecules are chemically degraded or aggregated to form higher order polymers; the heterodimer dissociates into monomers; deglycosylation; glycosylation modification; or any other structural modification that reduces at least one biological activity of a protein composition encompassed by the invention. Stability can be assessed by methods well known in the art, including measuring light scattering, apparent attenuation of light (absorbance or optical density), size (e.g., by size exclusion chromatography), in vitro or in vivo biological activity and/or properties of the sample (by Differential Scanning Calorimetry (DSC)). Other methods of assessing stability are also known in the art and may also be used in accordance with the present invention.

In some embodiments, an antigen in a stable formulation of the invention can maintain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% efficacy for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, 12 months, 18 months, 24 months, 30 months, 36 months, 42 months, 48 months, 54 months or 60 months as compared to a reference standard. In some embodiments, the antigen in the stable formulation of the invention may maintain at least 50% efficacy over at least 1 year, 2 years, 3 years, 4 years, or 5 years, as compared to a reference standard. The terms "stable" and "stability" also refer to the ability of an antigen to maintain an epitope or immunoreactivity over a period of time. For example, an antigen in a stable formulation of the invention may maintain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of its epitope or immunoreactivity for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, 12 months, 18 months, 24 months, 30 months, 36 months, 42 months, 48 months, 54 months or 60 months as compared to a reference standard. In some embodiments, stability is measured as a function of environmental conditions. Non-limiting examples of environmental conditions include light, temperature, freeze-thaw cycles, agitation, and pH. One skilled in the art can determine the presence of an antigenic epitope or immunoreactivity using the methods disclosed herein or other methods known in the art. See the discussion of McNeil et al,Vaccine,27:3417-3421(2009). In some embodiments, the stability of the antigen is measured from the day of its formulation. In some embodiments, the stability of the antigen is measured from the day that its storage conditions are changed. Non-limiting examples of changes in storage conditions include from freezing to refrigeration, from freezing to room temperature, from refrigeration to refrigeration, from room temperature to refrigeration, from light to no light, or the introduction of agitation.

The term "stabilizer" refers to a compound that binds to an antigen and maintains an epitope or immunoreactivity of the antigen over a period of time. Stabilizers are known in the art. Examples of stabilizers include multivalent cations such as calcium or aluminum.

The term "subject" refers to a mammal, bird, fish, reptile species, or any other animal. The term "subject" also includes humans. The term "individual" also includes domestic pets. Non-limiting examples of domestic pets include: dogs, cats, pigs, rabbits, rats, mice, gerbils, hamsters, guinea pigs, ferrets, birds, snakes, lizards, fish, turtles, and frogs. The term "subject" also includes livestock animals. Non-limiting examples of livestock animals include: alpaca, bison, camel, cow, deer, pig, horse, llama, mule, donkey, sheep, goat, rabbit, reindeer, yak, chicken, goose and turkey.

The terms "vaccine" or "vaccine composition" are used interchangeably and refer to a pharmaceutical composition comprising at least one immunogenic composition that induces an immune response in an individual.

General description

The present invention is made based on the following novel findings: rLP2086 subfamily B antigen, but not rLP2086 subfamily a antigen, loses potency over time in bivalent vaccine formulations and is therefore unstable. By varying the components in the bivalent formulation, it was determined that high detergent to protein molar ratios in the bivalent vaccine formulation resulted in specific instability of the rLP2086 subfamily B antigen. Reducing the detergent to protein molar ratio in divalent and monovalent formulations results in increased stability of the rLP2086 subfamily B antigen (as measured by maintenance of potency over time) without affecting the stability of the rLP2086 subfamily a antigen. This result is surprising because lipoproteins are generally purified and preserved using high detergent concentrations in order to prevent aggregation of their hydrophobic lipid fraction. Thus, in some embodiments, the invention provides an immunogenic composition comprising an rLP2086 subfamily B antigen and a low molar ratio of detergent to protein. In some embodiments, the present invention provides a method of maintaining the stability of an rLP2086 subfamily B antigen in an immunogenic composition, the method comprising the steps of: storing the rLP2086 subfamily B antigen in a buffer comprising a low molar ratio of detergent to protein.

Further studies have shown that low molar ratios of the formulation result in aggregation of the rLP2086 subfamily a and B antigens when the low molar ratio of the immunogenic composition is agitated. However, increasing the aluminum concentration in low molar ratio compositions prevents aggregation of rLP2086 subfamily a and B antigens, even with agitation. In addition, in the absence of aluminum, the rLP2086 subfamily a antigen is more sensitive to the effects of low detergent mole ratios. Thus, in some embodiments, the invention provides an immunogenic composition comprising an rLP2086 subfamily a antigen, an rLP2086 subfamily B antigen, a high concentration of aluminum, and a low molar ratio of detergent to protein. In some embodiments, the present invention provides methods of maintaining the stability of rLP2086 subfamily a antigen and rLP2086 subfamily B antigen in an immunogenic composition, comprising the steps of: storing the rLP2086 subfamily A antigen and rLP2086 subfamily B antigen in a buffer comprising a high concentration of aluminum and a low molar ratio of detergent to protein.

Immunogenic compositions

Immunogenic compositions comprising a protein encoded by the nucleotide sequence of neisseria meningitidis ORF2086 are known in the art. Exemplary immunogenic compositions include those disclosed in U.S. patent application publications US20060257413 and US20090202593, both of which are incorporated herein by reference in their entirety. Such immunogenic compositions described therein comprise a protein exhibiting bactericidal activity designated ORF2086 protein, immunogenic portions thereof, and/or biological equivalents thereof. The ORF2086 protein refers to the protein encoded by open reading frame 2086 of neisseria species.

The protein may be a recombinant protein, or a protein isolated from natural neisseria. For example, the neisserial ORF2086 protein may be isolated from bacterial strains, such as those of Neisseria species, including strains of Neisseria meningitidis (serogroups A, B, C, D, W-135, X, Y, Z, and 29E), Neisseria gonorrhoeae (Neisseria gonorrhoeae), and Neisseria lactamica (neisserial lactamica), as well as immunogenic portions and/or biological equivalents of the protein.

ORF2086 proteins include the 2086 subfamily a and subfamily B proteins, immunogenic portions thereof, and/or biological equivalents thereof. The ORF2086 protein, or equivalent thereof, and the like, can be lipidated or non-lipidated. Preferably, the neisserial ORF2086 protein is lipidated.

In one embodiment, the immunogenic composition comprises an isolated protein having at least 95% amino acid sequence identity to a protein encoded by a nucleotide sequence from neisserial ORF 2086.

In one embodiment, the immunogenic composition comprises an isolated protein having at least 95% amino acid sequence identity to a subfamily a protein encoded by a nucleotide sequence from neisseria ORF 2086. Preferably, the immunogenic composition comprises an isolated subfamily a protein encoded by a nucleotide sequence from neisseria ORF 2086.

In another embodiment, the immunogenic composition comprises an isolated protein having at least 95% amino acid sequence identity to a subfamily B protein encoded by a nucleotide sequence from neisseria ORF 2086. Preferably, the immunogenic composition comprises an isolated subfamily B protein encoded by a nucleotide sequence from neisseria ORF 2086. In some embodiments, the ORF2086 subfamily B protein is a B01 variant.

In yet another embodiment, the immunogenic composition comprises an isolated protein having at least 95% amino acid sequence identity to a subfamily a protein encoded by the nucleotide sequence from neisserial ORF2086, and an isolated protein having at least 95% amino acid sequence identity to a subfamily B protein encoded by the nucleotide sequence from neisserial ORF 2086. Preferably, the immunogenic composition comprises a subfamily a protein encoded by the nucleotide sequence from neisseria ORF2086 and a subfamily B protein encoded by the nucleotide sequence from neisseria ORF 2086.

In one embodiment, the immunogenic composition comprises a subfamily a protein to a subfamily B protein in a ratio of 1: 1.

The immunogenic composition may comprise a protein encoded by a nucleotide sequence from neisseria ORF2086, a polynucleotide, or an equivalent thereof, as the only active immunogen in the immunogenic composition. Alternatively, the immunogenic composition may also comprise an active immunogen, including immunogenic polypeptides of other neisserial species or an immunologically active protein of one or more other microbial pathogens (such as, but not limited to, viruses, prions, bacteria or fungi), or a capsular polysaccharide. The composition may comprise one or more desired proteins, fragments or pharmaceutical compounds as required for the chosen indication.

The present invention includes any multiple antigen or multivalent immunogenic composition. For example, the immunogenic composition can comprise a combination of two or more ORF2086 proteins, a combination of ORF2086 proteins with one or more Por a proteins, a combination of ORF2086 proteins with meningococcal (meningococus) serogroup A, C, Y and W135 polysaccharide and/or polysaccharide conjugates, a combination of ORF2086 proteins with meningococcal and pneumococcus (pneumococcus), or a combination of any of the foregoing in a form suitable for administration as desired, e.g., for mucosal delivery. One skilled in the art can readily formulate such multi-antigen or multivalent immunogenic compositions.

The invention also encompasses multiple immunization (multi-immunization) protocols in which any composition useful against a pathogen may be combined therein, or in combination with a composition of the invention. For example, but not by way of limitation, an immunogenic composition of the invention and another immunological composition for immunization against Human Papillomavirus (HPV) (e.g., HPV vaccine) may be administered to a patient) As part of a multiple immunization protocol. The person skilled in the art is readily able to select immunogenic compositions for use in combination with the immunogenic compositions of the invention to develop and implement multiple immunization protocols.

ORF2086 polypeptides, fragments, and equivalents can be used as part of a conjugated immunogenic composition, where one or more proteins or polypeptides are conjugated to a carrier to generate a composition that is immunogenic against several serotypes and/or several diseases. Alternatively, one of the ORF2086 polypeptides can be used as a carrier protein for other immunogenic polypeptides. The formulation of such immunogenic compositions is well known to those skilled in the art.

The immunogenic compositions of the invention preferably comprise a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers and/or diluents include any and all conventional solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial agents, antifungal agents, isotonic agents, absorption delaying agents, and the like. Suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof.

The pharmaceutically acceptable carrier may also contain minor amounts of auxiliary substances, such as wetting agents, emulsifying agents, preservatives or buffers, which may prolong the shelf life or effectiveness of the antibody. The preparation and use of pharmaceutically acceptable carriers are well known in the art. Except to the extent that any conventional media or agents are incompatible with the active ingredient, their use in the immunogenic compositions of the invention is contemplated.

The immunogenic compositions can be administered parenterally, for example by injection (subcutaneously or intramuscularly), as well as orally or intranasally. Methods of intramuscular immunization are described in Wolff et alBiotechniques11(4) 474-85 (1991) and Sedegah et alPNASVol.91, pp.9866-9870, (1994). For example, other modes of administration employ oral formulations, pulmonary formulations, suppositories, and transdermal administration, but are not limited thereto. For example, oral formulations include such common excipients as, but not limited to, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Preferably, the immunogenic composition is administered intramuscularly.

The immunogenic compositions of the invention may comprise one or more adjuvants. Exemplary adjuvants include, but are not limited to, aluminum hydroxide, aluminum phosphate, STIMULON^TMQS-21(AquilaBiopharmaceuticals,Inc.,Framingham，Mass.);MPL^TM(3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, Mont.), 529 (an aminoalkyl glucosamine phosphate compound, Corixa, Hamilton, Mont.), IL-12(Genetics Institute, Cambridge, Mass.), GM-CSF (Immunex Corp., Seattle, Wash.), N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP); N-acetyl-N-muramyl-L-alanyl-D-isoglutamine (N-acetyl-N-muramyl-L-alanyl-D-isoglutamine, CGP11637, referred to as N-MDP); N-acetyl-L-alanyl-D-isoglutamyl-L-alanine-2- (1 ' -palmitoyl-2 ' -diglycerol-2 ' -diacyl-2-glycerol-D-isoglutamyl-L-alanine-L-alanyl-D-isoglutamyl-2- (1 ' -palmitoyl-2 ' -diglycerol-diacyl-D-glycerol-D-isoglutamide -3-hydroxyphosphoryloxy-ethylamine) (CGP19835A, known as MTP-PE), and cholera toxin. In certain preferred embodiments, the adjuvant is QS-21.

Other exemplary adjuvants include nontoxic derivatives of cholera toxin, including the a subunit thereof, and/or conjugates or genetically engineered fusions of neisseria meningitidis polypeptides with cholera toxin or its B subunit ("CTB"), a prohodelogenin-like precursor (procholesteronoid), a fungal polysaccharide (including schizophyllan), a muramyl dipeptide ("MDP") derivative, phorbol esters, thermolabile toxins of e.

Aluminum phosphate has been used as an adjuvant in phase 1 clinical trials at a concentration of 0.125 mg/dose, well below the limits set by U.S. federal regulations [610.15(a) ], i.e., 0.85 mg/dose. Aluminum-containing adjuvants are widely used in humans to enhance the immune response of an antigen when administered intramuscularly or subcutaneously.

In certain preferred embodiments, the proteins of the invention are used in immunogenic compositions comprising mucosal adjuvants for oral administration, and for the treatment or prevention of neisseria meningitidis infection in a human host. The mucosal adjuvant may be a cholera toxin, but preferably, other mucosal adjuvants than cholera toxin that can be used according to the present invention include non-toxic derivatives of cholera holotoxin, wherein the a subunit is a mutagenized, chemically modified cholera toxin, or a related protein produced by modifying the amino acid sequence of cholera toxin. For specific cholera toxins that may be particularly useful in preparing the immunogenic compositions of the invention, see mutant cholera holotoxin E29H disclosed in published International application WO00/18434 (incorporated herein by reference in its entirety). These substances may be added to or conjugated with the polypeptides of the present invention. The same technique can be applied to other molecules with mucosal adjuvants or delivery properties, such as e.g. e. Other compounds with mucosal adjuvant or delivery activity may be used, such as bile; polycations such as DEAE-dextran and polyornithine; detergents such as sodium dodecylbenzenesulfonate, lipid-conjugated materials, antibiotics such as streptomycin, vitamin a, and other compounds that alter the structural or functional integrity of mucosal surfaces. Other compounds with mucosal activity include derivatives of microbial structures such as MDP, acridine and cimetidine. As noted above, STIMULON may also be used^TMQS-21, MPL and IL-12.

The immunogenic compositions of the invention may be delivered in the form of ISCOMS (immunostimulatory complexes), ISCOMS containing CTBs, liposomes, or encapsulated in a compound such as an acrylate or poly (DL-lactide-glycolide) to form microspheres of a size suitable for absorption. The protein of the invention may also be incorporated into an oily emulsion.

The amount (i.e., dose) of the immunogenic composition to be administered to a patient can be determined according to standard techniques known to those of ordinary skill in the art, taking into account factors such as the particular antigen, adjuvant (if present), age, sex, weight, species, physical condition, and route of administration of the particular patient.

For example, a dose for a young patient may comprise at least 0.1 μ g, 1 μ g, 10 μ g or 50 μ g of neisserial ORF2086 protein and at most 80 μ g, 100 μ g, 150 μ g or 200 μ g of neisserial ORF2086 protein. Any minimum value and any maximum value may be combined to define a suitable range.

In vitro potency assay

Potency was determined by quantifying functional epitopes in subfamily a and subfamily B proteins in immunogenic compositions using a conformation-specific monoclonal antibody to rLP2086 reference substance. Potency is determined by quantitative measurement of functional epitopes in subgroup a or subgroup BrLP2086 proteins that elicit an immune response in vivo to produce bactericidal antibodies. The selected monoclonal antibodies (mabs) were used to apply quantitative techniques for potency testing. For each subfamily of rLP2086 proteins in the immunogenic composition, two functional monoclonal antibodies, conformational and non-overlapping, were selected. In both purified monoclonal antibodies, the first antibody is conjugated to a first label, wherein the first label is used to capture the rLP2086 protein molecule. In some embodiments, the first tag is biotin, glutathione-S transferase (GST), a 6xhis tag, or a bead (e.g., carboxylated polystyrene beads or paramagnetic beads). In some embodiments, the first label is captured with streptavidin beads, streptavidin columns, nickel beads, nickel columns, by centrifugation, or with a magnetic field. The second antibody is conjugated to a second label, wherein the second label is quantifiable. In some embodiments, the second label is biotin, horseradish peroxidase (HRP), a fluorophore, or a radioactive label. In some embodiments, the second label is detected with streptavidin conjugated to a fluorophore or HRP, by electrochemiluminescence, fluorescence detection, or radioactivity detection. Only proteins exhibiting two epitopes recognized by the two monoclonal antibodies in each immunogenic composition were measured. Reflecting changes in either or both epitopes of the protein. The potency of the sample is reported relative to the potency of the reference substance.

In some embodiments, the invention includes a method of determining the potency of a 2086 protein. In some embodiments, the method comprises the steps of: (1) incubating a first monoclonal antibody and a second mAb with an immunogenic composition comprising a 2086 protein, wherein the first mAb is conjugated to a first label for capturing the mAb and the second mAb is conjugated to a second label that is detectable, and wherein the first and second mabs are directed to different conformational epitopes on a 2086 reference protein; (2) capturing 2086 protein that binds to the first mAb using the first label; and (3) detecting and quantifying the amount of captured 2086 protein bound to the second mAb using the second label. In some embodiments, the 2086 protein is a subfamily a protein. In some embodiments, the 2086 protein is a subfamily B protein. In some embodiments, the 2086 protein is lipidated. In some embodiments, the 2086 protein is non-lipidated. In some embodiments, the 2086 protein is recombinant. In some embodiments, the first tag is biotin, a 6xHis tag, or a bead (e.g., a carboxylated polystyrene bead or a paramagnetic bead). In some embodiments, the first label is captured with streptavidin beads, streptavidin columns, glutathione columns, nickel beads, nickel columns, by centrifugation, or with a magnetic field. In some embodiments, the second label is biotin, HRP, a fluorophore, or a radiolabel. In some embodiments, the second label is detected with streptavidin conjugated to a fluorophore or HRP, by electrochemiluminescence, fluorescence detection, or radioactivity detection. In some embodiments, the immunogenic composition comprises a plurality of 2086 protein variants.

Stability of potency of rLP2086 subfamily B antigens

In some embodiments, the present invention provides immunogenic compositions for stabilizing an rLP2086 subfamily B antigen over time, comprising a buffer with a low detergent to protein molar ratio.

In some embodiments, the molar ratio of detergent to protein in the immunogenic composition is from about 0.5 to about 10. In some embodiments, the molar ratio of detergent to protein in the immunogenic composition is from about 1 to about 5. In some embodiments, the molar ratio of detergent to protein in the immunogenic composition is from about 1.4 to about 4.2. In some embodiments, the molar ratio of detergent to protein in the immunogenic composition is about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 5.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 5.0, about 5, about 5.6, about 5, about 5.0, about 5, about 5.0, about 5.8, about 5, about 5.0, about 5, about 5.8, about 5, about 5.0, about 5. In some embodiments, the detergent is a nonionic detergent. In some embodiments, the detergent is a polysorbate detergent. In some embodiments, the detergent is polysorbate 80.

In some embodiments, the immunogenic composition further comprises a multivalent cation. In some embodiments, the multivalent cation is calcium or aluminum. In some embodiments, the aluminum is AlPO₄、Al(OH)₃、Al₂(SO₄)₃And alum. In some embodiments, the immunogenic composition comprises from about 0.1mg/mL to about 1mg/mL, from about 0.25mg/mL to about 0.75mg/mL, or from about 0.4mg/mL to about 0.6mg/mL of aluminum. In some embodiments, the immunogenic composition comprises about 0.1mg/mL, about 0.15mg/mL, about 0.2mg/mL, about 0.25mg/mL, about 0.3mg/mL, about 0.35mg/mL, about 0.4mg/mL, about 0.45mg/mL, about 0.5mg/mL, about 0.55mg/mL, about 0.6mg/mL, about 0.65mg/mL, about 0.7mg/mL, about 0.75mg/mL, about 0.8mg/mL, about 0.85mg/mL, about 0.9mg/mL, about 0.95mg/mL, or about 1mg/mL of aluminum. In some embodiments, the aluminum is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% bound to the protein.

In some embodiments, the immunogenic composition further comprises a buffer comprising histidine. In some embodiments, the concentration of histidine is about 2mM to about 20mM, about 5mM to about 15mM, or about 8mM to 12 mM. In some embodiments, the concentration of histidine is about 2mM, about 3mM, about 4mM, about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 11mM, about 12mM, about 13mM, about 14mM, about 15mM, about 16mM, about 17mM, about 18mM, about 19mM, or about 20 mM.

In some embodiments, the immunogenic composition further comprises a buffer comprising succinate. In some embodiments, the concentration of succinate is from about 2mM to about 20mM, from about 2mM to about 10mM, or from about 3mM to 7 mM. In some embodiments, the concentration of succinate is about 2mM, about 3mM, about 4mM, about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 11mM, about 12mM, about 13mM, about 14mM, about 15mM, about 16mM, about 17mM, about 18mM, about 19mM, or about 20 mM.

In some embodiments, the pH of the immunogenic composition is from about 5.0 to about 8.0, from about 5.5 to about 7.0, or from about 5.8 to about 6.0. In some embodiments, the pH of the immunogenic composition is about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, or about 6.5.

In some embodiments, the formulation of the MnB rLP2086 subfamily B protein antigen immunogenic composition is 10mM histidine buffered saline ph6.0 containing 0.5mg/mL aluminum in the form of aluminum phosphate and polysorbate 80 at a molar ratio to protein of 2.8.

In some embodiments, the formulation of the MnB rLP2086 subfamily B protein antigen immunogenic composition is 5mM ph6.0 succinate buffered saline containing 0.5mg/mL aluminum in the form of aluminum phosphate and polysorbate 80 at a molar ratio to protein of 2.8.

In some embodiments, the invention provides a method of stabilizing an rLP2086 subfamily B antigen over time, comprising storing the antigen in a buffer having a low detergent to protein molar ratio.

In some embodiments, the molar ratio of detergent to protein in the buffer is from about 0.5 to about 10. In some embodiments, the molar ratio of detergent to protein in the buffer is from about 1 to about 5. In some embodiments, the molar ratio of detergent to protein in the buffer is from about 1.4 to about 4.2. In some embodiments, the molar ratio of detergent to protein in the buffer is about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.5, about 4.6, about 4.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4.5, about 4.5, about 4.0, about 5.5, about 5, about 5.0, about 5, about 5.5, about 5, about 5.0, about 5, about 5.8, about 5.9, about 5, about 5.0, about 5, about 5.8. In some embodiments, the detergent is a nonionic detergent. In some embodiments, the detergent is a polysorbate detergent. In some embodiments, the detergent is polysorbate-80.

In some embodiments, the buffer further comprises a multivalent cation. At one endIn some embodiments, the multivalent cation is calcium or aluminum. In some embodiments, the aluminum is AlPO₄、Al(OH)₃、Al₂(SO₄)₃And alum. In some embodiments, the stabilizer in the buffer is from about 0.1mg/mL to about 1mg/mL, from about 0.25mg/mL to about 0.75mg/mL, or from about 0.4mg/mL to about 0.6mg/mL of aluminum. In some embodiments, the stabilizer in the buffer is about 0.1mg/mL, about 0.15mg/mL, about 0.2mg/mL, about 0.25mg/mL, about 0.3mg/mL, about 0.35mg/mL, about 0.4mg/mL, about 0.45mg/mL, about 0.5mg/mL, about 0.55mg/mL, about 0.6mg/mL, about 0.65mg/mL, about 0.7mg/mL, about 0.75mg/mL, about 0.8mg/mL, about 0.85mg/mL, 0.9mg/mL, about 0.95mg/mL, or about 1mg/mL of aluminum. In some embodiments, the aluminum is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% bound to the protein.

In some embodiments, the buffer further comprises histidine. In some embodiments, the concentration of histidine is about 2mM to about 20mM, about 5mM to about 15mM, or about 8mM to 12 mM. In some embodiments, the concentration of histidine is about 2mM, about 3mM, about 4mM, about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 11mM, about 12mM, about 13mM, about 14mM, about 15mM, about 16mM, about 17mM, about 18mM, about 19mM, or about 20 mM.

In some embodiments, the buffer further comprises succinate. In some embodiments, the concentration of succinate is from about 2mM to about 20mM, from about 2mM to about 10mM, or from about 3mM to 7 mM. In some embodiments, the concentration of succinate is about 2mM, about 3mM, about 4mM, about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 11mM, about 12mM, about 13mM, about 14mM, about 15mM, about 16mM, about 17mM, about 18mM, about 19mM, or about 20 mM.

In some embodiments, the pH of the buffer is from about 5.0 to about 8.0, from about 5.5 to about 7.0, or from about 5.8 to about 6.0. In some embodiments, the pH of the buffer is about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, or about 6.5.

In some embodiments, the buffer in which the MnB rLP2086 subfamily B protein antigen is stored is 10mM histidine buffered saline ph6.0 containing 0.5mg/mL aluminum in the form of aluminum phosphate and polysorbate 80 at a molar ratio to protein of 2.8.

In some embodiments, the buffer in which the MnB rLP2086 subfamily B protein antigen is stored is 5mM succinate buffered saline at ph6.0 containing 0.5mg/mL aluminum in the form of aluminum phosphate and polysorbate 80 at a molar ratio to protein of 2.8.

Stability of potency of rLP2086 subfamily A and B antigens

In some embodiments, the present invention provides an immunogenic composition for stabilizing rLP2086 subfamily a and/or rLP2086 subfamily B antigens over time, the composition comprising a buffer with a high stabilizer concentration and a low detergent to protein molar ratio.

In some embodiments, the immunogenic composition further comprises a multivalent cation. In some embodiments, the multivalent cation is calcium or aluminum. In some embodiments, the aluminum is AlPO₄、Al(OH)₃、Al₂(SO₄)₃And alum. In some embodiments, the immunogenic composition comprises from about 0.1mg/mL to about 1mg/mL, from about 0.25mg/mL to about 0.75mg/mL, or from about 0.4mg/mL to about 0.6mg/mL of aluminum. In some embodiments, the immunogenic composition comprises about 0.1mg/mL, about 0.15mg/mL, about 0.2mg/mL, about 0.25mg/mL, about 0.3mg/mL, about 0.35mg/mL, about 0.4mg/mL, about 0.45mg/mL, about 0.5mg/mL, about 0.55mg/mL, about 0.6mg/mL, about 0.65mg/mL, about 0.7mg/mL, about 0.75mg/mL, about 0.8mg/mL, about 0.85mg/mL, 0.9mg/mL, about 0.95mg/mL, or about 1mg/mL of aluminum. In some embodiments, the aluminum is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% bound to the protein.

In some embodiments, the formulation of MnB rLP2086 subfamily a and B protein antigens is 10mM histidine buffered saline, ph6.0, containing 0.5mg/mL aluminum in the form of aluminum phosphate and polysorbate 80 at a molar ratio to protein of 2.8.

In some embodiments, the present invention provides a method of stabilizing an rLP2086 subfamily a and/or rLP2086 subfamily B antigen over time, comprising storing the antigen in a buffer having a high stabilizer concentration and a low detergent to protein molar ratio.

In some embodiments, the molar ratio of detergent to protein is less than 10: 1. In some embodiments, the molar ratio of detergent to protein in the buffer is from about 0.5 to about 10. In some embodiments, the molar ratio of detergent to protein in the buffer is from about 1 to about 5. In some embodiments, the molar ratio of detergent to protein in the buffer is from about 1.4 to about 4.2. In some embodiments, the molar ratio of detergent to protein in the buffer is about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.5, about 4.6, about 4.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4.5, about 4.5, about 4.0, about 5.5, about 5, about 5.0, about 5, about 5.5, about 5, about 5.0, about 5, about 5.8, about 5.9, about 5, about 5.0, about 5, about 5.8. In some embodiments, the detergent is a nonionic detergent. In some embodiments, the detergent is a polysorbate detergent. In some embodiments, the detergent is polysorbate 80.

In some embodiments, the stabilizing agent in the buffer is a multivalent cation. In some embodiments, the multivalent cation is calcium or aluminum. In some embodiments, the aluminum is AlPO₄、Al(OH)₃、Al₂(SO₄)₃And alum. In some embodiments, the stabilizer in the buffer is from about 0.1mg/mL to about 1mg/mL, from about 0.25mg/mL to about 0.75mg/mL, or from about 0.4mg/mL to about 0.6mg/mL of aluminum. In some embodiments, the stabilizer in the buffer is about 0.1mg/mL, about 0.15mg/mL, about 0.2mg/mL, about 0.25mg/mL, about 0.3mg/mL, about 0.35mg/mL, about 0.4mg/mL, about 0.45mg/mL, about 0.5mg/mL, about 0.55mg/mL, about 0.6mg/mL, about 0.65mg/mL, about 0.7mg/mL, about 0.75mg/mL, about 0.8mg/mL, about 0.85mg/mL, 0.9mg/mL, about 0.95mg/mL, or about 1mg/mL of aluminum. In some embodiments, the aluminum is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% bound to the protein.

In some embodiments, the buffer in which MnB rLP2086 subfamily a and B protein antigens are stored is 10mM histidine buffered saline ph6.0 containing 0.5mg/mL aluminum in the form of aluminum phosphate and polysorbate 80 at a molar ratio to protein of 2.8.

In some embodiments, the buffer in which MnB rLP2086 subfamily a and B protein antigens are stored is 5mM ph6.0 succinate buffered saline containing 0.5mg/mL aluminum in the form of aluminum phosphate and polysorbate 80 at a 2.8 molar ratio to the protein.

In order that the invention may be better understood, the following examples are set forth. These examples are for illustrative purposes only and should not be construed as limiting the scope of the invention.

All references cited herein are incorporated herein by reference.

Examples

Example 1: experimental procedures

Determination of aluminum bonding

Centrifuging the composition comprising aluminum and at least one protein antigen, thereby precipitating the aluminum (pelleted). Dissociation of aluminium-adsorbing proteins is known in the artAnd (4) a heart. See for example, Egan et al,vaccinevol.27(24):3175-3180 (2009). Precipitation of aluminum-binding proteins also occurred, but non-aluminum-binding proteins remained in the supernatant. Total protein in the supernatant and pellet was determined by Lowry test. The percentage of bound protein was calculated by dividing the total protein in the supernatant by the total protein added to the composition and multiplying by 100%. Likewise, the percentage of unbound protein was calculated by dividing the total protein in the supernatant by the total protein added to the composition and multiplying by 100%.

For compositions comprising both subfamily a and subfamily B antigens, the respective subfamily a and B protein concentrations in the supernatant were determined by ion exchange chromatography. Subfamily a and B proteins were separated and eluted using a strong anion column and an eluent of high salt concentration. Both subfamily a and B proteins were detected and quantified using fluorescence detectors set at excitation =280run and emission =310 run. Subfamily a and subfamily B proteins eluted at different retention times and were quantified using standard curves generated for the rLP2086 protein reference material. The percentage of unbound protein was calculated by dividing the total protein in the supernatant by the total protein added to the composition and multiplying by 100%. The percentage of bound protein was calculated by subtracting the percentage of unbound protein from 100%.

In vitro potency assay

The rLP2086 potency assay is a homogeneous capture assay or sandwich assay that relies on two functional monoclonal antibodies that recognize conformational and non-overlapping epitopes on a single protein molecule of the rLP2086 drug. A purified monoclonal antibody is used as a capture antibody (mAb) chemically conjugated to carboxylated polystyrene beads with unique color-coded identifiers. The second antibody was biotinylated and used as a detection antibody that was subsequently bound by streptavidin (SA-PE) conjugated to the fluorophore R-phycoerythrin. The fluidics of the Bio-Plex detector quantified individual microspheres and their associated SA-PE signals. The fluorescence signal from R-phycoerythrin associated with the microspheres is detected only by the formation of a ternary complex between the antibody conjugated to the bead, the antigen and the detection antibody, and is proportional to the number of functional epitopes in the rLP2086 sample. Changes in one or both epitopes result in a loss of fluorescence relative to the reference standard, indicating a loss of potency.

Reagent

Monoclonal antibodies conjugated to microspheres (conjugated to Luminex MicroPlex Microsphere bead #12 or to bead # 66).

Biotinylated monoclonal antibodies.

rLP2086 reference substance, subfamilies A and B, 2 mg/ml. Stored at-70 ℃.

rLP2086 subfamily A and B bivalent controls

Lyophilized streptavidin conjugated with R-phycoerythrin

Buffer solution

10mM histidine, 150mM NaCl, pH6.0

5% w/v Polysorbate 80(PS-80) in 0.85% w/v saline.

Matrix buffer (10mM histidine, 0.02% polysorbate 80, 150mM NaCl, ph 6.0).

Test buffer (PBS, ph7.4, containing 0.1% BSA, 0.02% polysorbate 80, 0.1% azide).

100 × streptavidin conjugated with R-phycoerythrin (SA-PE) — open the vial containing lyophilized streptavidin, R-phycoerythrin and add 1mL distilled water. Vortex until completely dissolved.

Operation of

mu.L of the subfamily A protein and 200. mu.L of the subfamily B protein were added to 600. mu.L of the matrix buffer so that the concentration of each subfamily was 400. mu.g/ml. Eight concentration (3333-1.5ng/mL) standard curves were generated by diluting the stock solution in test buffer.

200 μ L of the bivalent control was added to 800 μ L of matrix buffer to give a concentration of 400 μ g/mL per subfamily. The 400. mu.g/mL stock solutions used to formulate the working concentrations of 100ng/mL, 50ng/mL and 12.5ng/mL were diluted in test buffer. 100ng/mL and 12.5ng/mL represent high (CH) and low (CH) controls, respectively.

The test sample was diluted in matrix buffer to a concentration of 400. mu.g/mL. From this 400. mu.g/mL stock solution, working solutions of 100ng/mL, 50ng/mL and 12.5ng/mL were prepared.

Using 2x10⁵A homogeneous assay mixture was prepared in assay buffer with a concentration of conjugate beads per mL and a concentration of detection antibody of 30. mu.g/mL. Sample plates were prepared by adding 0.4mL of standard, control, sample or blank to 2mL of 96-well deep-well plates. Pre-wetting of 96-well MultiScreen by adding 100. mu.L of test buffer_HTS-a filter of BV filter plates, then extracted by vacuum suction. 25 μ L of the prepared homogeneous assay mixture was added to a 96-well plate. 25 μ L each of the standard, control, sample or blank solution was added to each well of the 96-well filter plate. The plates were incubated at room temperature for 1 hour with shaking.

After antigen-antibody incubation, the buffer was removed by vacuum aspiration through the filter. The filter of each well was washed three times with 100. mu.L of test buffer and then subjected to vacuum aspiration. After the last wash, 50. mu.L of 1xSA-PE was added to each well. The plate was incubated for 10 minutes at room temperature with shaking on a titer plate shaker (titer) protected from light.

After SA-PE incubation, 75. mu.L of test buffer was added to each well of the plate to a total volume of 125. mu.L. Plates were read immediately on the Bio-Plex200 system.

Serum sterilization test

New Zealand female white rabbits (2.5-3.0 kg) obtained from Charles River Canada (St. constant, QC, Canada) were pre-screened by whole cell ELISA to identify pairs of different meningesThose rabbits with low reactivity of the immunoglobulin strain (one per P2086 subfamily). Generally, rabbits have a very low background, and those with the lowest value are selected for use. Rabbits were vaccinated intramuscularly at weeks 0, 4 and 9 using either a monovalent rLP2086-a05, monovalent rLP2086-B01, or divalent rLP2086-a05 + B01 vaccine. Each dose of the monovalent vaccine contains 100. mu.g of protein, and each dose of the bivalent vaccine contains 100. mu.g of each protein, formulated in 10mM histidine buffer pH6.0, 150mM NaCl, 0.02% polysorbate 80 and 250. mu.g AlPO₄In (1). The vaccine was injected intramuscularly to the right hind limb (0.5 ml/dose). As a control, one group of rabbits was inoculated with formulation buffer only. Pre-immune (week 0) and immune (week 10) serum samples were obtained for analysis. All animal procedures were in compliance with the guidelines of the institutional animal care and use committee.

SBA and human complement were used to determine serum bactericidal antibodies in rabbits immunized with rLP2086 vaccine. Rabbit immune blood is heat inactivated to remove intrinsic complement activity, followed by serial 1:2 dilutions in 96-well microtiter plates containing Ca²⁺And Mg²⁺In Dulbecco's PBS (D-PBS) to test serum bactericidal activity against N.meningitidis strains. The bacteria used in this test were grown in GC medium (GCK) supplemented with Kellogg supplement and monitored by optical density at 650 nm. Collecting the final OD₆₅₀Bacteria from 0.50 to 0.55 were used in this assay, diluted in D-PBS and 1000-.

Human serum with no detectable bactericidal activity was used as the exogenous source of complement. Testing the applicability of the complement source to each detection strain. The complement source was used only if the number of bacteria surviving in the control without added immune serum was > 75%. Ten unique complement sources were added to perform the SBA described in this study.

At 37 ℃ with 5% CO₂After 30 min incubation, D-PBS was added to the reaction mixture and aliquots were transferred to a micro-filter plate containing 50% GCK medium. Filtering the micro-scale filtrateFilter plate, which is treated at 37 ℃ with 5% CO₂Incubate overnight, stain and quantify the microcolonies. Serum bactericidal titer was defined as: CFU was reduced by 50% of the reciprocal interpolated serum dilution (interpolated reciprocal serum dilution) compared to CFU in control wells without immune serum. SBA titers were defined as: reciprocal of interpolated dilution of test serum causing 50% reduction of bacterial count after 30 min incubation at 37 ℃. Susceptibility to killing by P2086 immune sera is determined if the SBA titer of the P2086 immune sera is 4-fold or more than 4-fold increased relative to the corresponding preimmune sera. The limit of detection of rabbit serum was a titer of 8. The sera that had no effect on the test strain at the initial dilution were assigned a titer of half the limit of detection tested (i.e., 4 for rabbits).

Flow cytometry

Growth of MnB cells to OD₆₅₀From 0.45 to 0.55, which is subsequently fixed in 1% (v/v) paraformaldehyde in 1 XPBS for 10 minutes. One hundred microliters/well of bacteria were inoculated into a 96-well U-bottom polystyrene plate, centrifuged, and washed once with 1% (w/v) BSA in 1 × PBS. The anti-LP 2086 monoclonal antibody was added to the bacterial pellet, resuspended and incubated on ice for 30 minutes. After two washes in 1% BSA/PBS, biotinylated goat anti-mouse IgG (subclass 1+2a +2b +3) (jackson immunoresearch) was added to the cell pellet, resuspended and incubated on ice for 30 minutes. Cells were washed twice, resuspended in streptavidin-pe (bd biosciences), and incubated on ice for 30 minutes. After washing twice in 1% BSA/PBS, the cell pellet was resuspended in 1% paraformaldehyde. Mouse IgG was introduced as a negative control. Twenty thousand (20,000) events/well were obtained on a BD LSR II flow cytometer and analyzed using FlowJo v7 software (Treestar, Ashland, Oregon). The Mean Fluorescence Intensity (MFI) of the PE channels for each sample was determined after gating (gate) the bacterial cells in dot plots against the counter FSC versus SSC. MFI is considered positive if it is three times that of control mouse IgG.

Example 2:binding of polysorbate 80 to rLP2086 protein

To understand the stability of polysorbate 80 binding to each of rLP2086 proteins a and B, one rLP2086 sample was formulated with 200 μ g/mL subfamily a and aluminum (Al) and another rLP2086 sample was formulated with 200 μ g/mL subfamily B, both samples were stored at 2-8 ℃ and 25 ℃, and both samples were tested for protein and polysorbate 80 content after five months. Placebo (buffer + Al, no protein) was also analyzed. The distribution of polysorbate 80 in placebo is shown in figure 14, while the distribution of polysorbate 80 for subfamily a and B proteins is shown in figure 15 and figure 16, respectively. The relative potency (%) of subgroup B was compared to the binding mole ratio as shown in fig. 17.

Results

As shown in figure 14, the total polysorbate 80 percentage and the percentage of polysorbate 80 in the supernatant were the same (0.017%), indicating that polysorbate 80 did not bind to aluminum or become trapped in the pellet. Additionally, polysorbate 80 is stable after five months at 2-8 ℃ and 25 ℃.

The distribution of polysorbate 80 in bound (pellet), unbound (supernatant) and total rLP2086 subfamily a and subfamily B samples is shown in fig. 15 and 16, respectively. For subfamily a, the percentage of polysorbate 80 in the supernatant and pellet at the five month time point, 2-8 ℃ and 25 ℃ did not change. However, for the subfamily B sample, more polysorbate 80 was observed in the precipitate at 25 ℃ at the five month time point. Although the polysorbate 80 concentrations in the supernatant and the pellet differ at 2-8 ℃ and 25 ℃, an accurate mass balance is achieved for both subfamilies. Since rLP2086 protein binds one hundred percent to aluminum phosphate in the matrix, polysorbate 80, which is associated with precipitation, is most likely to bind to the protein molecule.

Although both proteins A and B bind to polysorbate 80, the binding of protein A is the same for samples stored at 2-8 ℃ and 25 ℃, whereas the binding of protein B is almost two times that of samples stored at 25 ℃And (4) doubling. At T₀And five month time points at 2-8 ℃ and 25 ℃ to determine the relative potency of subgroup B, and it was found that the relative potency exhibited opposite to the binding mole ratio, as shown in FIG. 17. Percent efficacy from T₀120 at 5M/25 ℃ to 16%, while the binding mole ratio increased from 5.3 to 13.9 over the same period of time.

Example 3: critical molar ratio study

To determine the critical concentration of polysorbate 80 required for rLP2086 stability, forty (40) rLP2086 formulations were prepared containing only subfamily a, only subfamily B, and both subfamilies a and B (200 μ g/mL and 400ug/mL), with varying polysorbate 80 concentrations, as shown in table 1. Total and bound proteins were determined for each sample, and zero (T) at time point₀) Polysorbate 80 percentage in whole samples, supernatants and pellets at 2-8 ℃ and 25 ℃, 14 days and 1 month. The results of this study are shown in fig. 18 to 24.

TABLE 1

Results

Polysorbate 80 was measured in the supernatant, pellet, and whole samples of all 40 rLP2086 formulation samples containing aluminum phosphate. The total and binding mole ratios of both subfamilies a and B were determined and appeared to be similar for the two subfamilies of 200 μ g/mL containing 0.005% (mole ratio of 5.4) or less polysorbate 80, as shown in figures 18 and 19, respectively. However, for samples containing 0.0065% (mole ratio of 7.0) or more polysorbate 80, the total mole ratio of subfamily B is much higher than the binding mole ratio. The data for the total and bound mole ratios of subfamily a, subfamily B, and subfamily a + B at 400 μ g/mL each are also very similar for formulations containing 0.008% (at a molar ratio of 8.6) or less polysorbate 80, but the total mole ratio is much higher than the bound mole ratio for formulations containing 0.017% (at a molar ratio of 18.4) polysorbate 80, as shown in figure 20.

Example 4Polysorbate 80 binding over time

At T₀Determination of polysorbate 80 in AlPO-containing solution at 14 days/25 ℃, 1 month/4 ℃, and 1 month/25 ℃₄In the supernatant and in the pellet of the subfamily a and B preparation samples (%). The percentage of polysorbate 80 in the supernatants of both sub-family a and B formulation samples was relatively the same for the samples stored at 2-8 ℃. However, for samples stored at 25 ℃, the percentage of polysorbate 80 in the supernatant decreased dramatically even after only 14 days. At T₀The percentage of polysorbate 80 in the precipitation of both subfamilies a and B was relatively similar at/5 ℃ and 1 month/5 ℃. However, for samples stored at 25 ℃, particularly for subfamily B containing 0.008% (molar ratio of 8.6) or more polysorbate 80, the percentage of polysorbate 80 in the supernatant was significantly increased. Also determined at T₀Polysorbate 80 in AlPO at 14 days/25 ℃, 1 month/4 ℃, and 1 month/25 ℃₄Percent in supernatant and pellet of rLP2086 subfamily a and B preparations. As shown in fig. 21, the concentration of polysorbate 80 was approximately the same for the sample containing 0.008% at all four time points. However, for the sample containing 0.017% polysorbate 80 and stored at 25 ℃, the concentration of polysorbate 80 was elevated. In the supernatant of the sample containing 0.008% or less of polysorbate 80, no polysorbate 80 was found. As shown in fig. 22, the binding mole ratio was stable for the sample containing 0.008% or less polysorbate 80 at all four time points. However, for the sample containing 0.017% polysorbate 80 and stored at 25 ℃, the binding mole ratio increased.

Determination of AlPO content₄Of subfamily A and B of (1) at T₀And efficacy at 14 days/25 ℃ (see fig. 23 and fig. 25, respectively). As shown in FIG. 23, the efficacy of different total molar ratios of subgroup A at 5 ℃ and 25 ℃Are 91-102. Although the binding mole ratio results are relatively identical at either temperature, a slight increase in potency was observed as the total mole ratio/binding mole ratio increased.

For samples of subfamily B at 5 ℃ with a total molar ratio of up to 9.0, the efficacy is about 95%. However, subfamily B efficacy decreased to 79% as the total molar ratio increased to 18.1. In addition, the sample with an overall molar ratio of 18.1 had a higher binding molar ratio than the other samples. At 25 ℃, the potency of subgroup B showed a significant drop from 83% to 5% with increasing total molar ratio from 5.3 to 18.1. The bound mole ratio of the 25 ℃ sample increased from 5.3 to 13.8 as the total mole ratio increased. Thus, the potency of subfamily B is inversely proportional to the binding mole ratio.

Both subfamily a and subfamily B proteins bind to polysorbate 80. For samples stored at 2-8 ℃ and 25 ℃, the binding of subfamily a was the same, but for samples stored at 25 ℃, the binding of subfamily B was almost doubled. In addition, critical molar ratio studies indicate that 200 μ g/mL of a formulation sample is stable when 0.008% or less polysorbate 80 is present (equivalent to a total molar ratio of 4.2 or less).

Example 5:detergent concentration and rLP2086 subfamily B antigen potency

Another stability study with different concentrations of polysorbate 80 demonstrated a critical state of polysorbate 80 to protein molar ratio for maintaining efficacy. In one experiment, a 200 μ g dose of immunogenic composition (ph6.3, total protein concentration 400 μ g/mL) was prepared with 10mM Histidine Buffered Saline (HBS) containing 0.5mg/mL aluminium (as aluminium phosphate) and incorporated with 0.01%, 0.02%, 0.05% or 0.1% polysorbate 80 (polysorbate 80 to rLP2086 protein at respective molar ratios of 5.3, 10.7, 26.7 and 53.4). The formulated samples were incubated at 25 ℃ and control samples were stored at 2-8 ℃. At time "0", polysorbate 80 concentrations up to 0.1%, there was no significant change in efficacy. However, a decline in efficacy as a function of temperature and polysorbate 80 concentration was observed at 2-8 ℃ and 25 ℃ for longer periods of time. As the concentration of polysorbate 80 in the immunogenic composition increased from 0.01% to 0.1%, the three month stability point demonstrated that the potency of the subfamily B protein decreased to less than 10% and 25% at 25 ℃ and 2-8 ℃, respectively (fig. 1).

Another stability study was performed (figure 2) evaluating the subfamily B protein, which was present in HBS at a concentration of 4mg/mL and incorporated polysorbate 80 at final concentrations of 0.06%, 0.5% and 1% (corresponding molar ratios of 3.3, 26.7 and 53.4). The control contained 0.09% polysorbate 80. Subfamily B protein was stable in 0.06% polysorbate 80 (3.3 molar ratio). The same samples containing polysorbate 80 at concentrations increased to 0.5% and 1% (molar ratios 26.7 and 53.4, respectively) were unstable. For the 400 μ g/mL immunogenic composition formulation, instability of the subfamily B protein was observed in all formulations containing polysorbate 80 concentrations of 0.01% (molar ratio of 5.3) or higher. However, at 4mg/mL protein and 0.06% polysorbate 80 concentration, there was no decrease in potency due to the lower polysorbate 80 to protein ratio (3.3) compared to the molar ratio at 400 μ g/mL protein +0.01% polysorbate 80 concentration (5.3). Thus, the decrease in potency of the subgroup B protein by polysorbate 80 is related to the molar ratio of polysorbate 80 detergent to protein, and not to the absolute concentration of polysorbate 80 in the matrix.

Therefore, the concentration of polysorbate 80 in the immunogenic composition must be reduced to maintain the stability of the subfamily B protein in the vaccine and during subsequent storage at 2-8 ℃. A 28-day accelerated stability study was designed for immunogenic compositions containing polysorbate 80(0, 1.1, 2.7 and 5.3) at doses of 20 μ g and 200 μ g at different molar ratios (fig. 3 and 4). Bivalent (subfamily a and subfamily B) formulations were prepared in 10mM ph6.0 histidine buffered saline, 0.5mg/mL aluminum in the form of aluminum phosphate with different polysorbate 80 concentrations. The samples were incubated with a 2-8 ℃ control at 25 ℃. Samples were analyzed for efficacy on days 0, 7, 14 and 28. Both subfamily a (data not shown) and B proteins were stable for all groups containing polysorbate 80 at a molar ratio to protein of less than 5.3. Potency values greater than 80% are considered to be within the test variability. At a molar ratio of 5.3, a tendency was observed to decrease the potency of the subgroup B protein in the 25 ℃ samples.

A full study evaluated all possible clinical doses (20 μ g, 60 μ g, 120 μ g and 200 μ g doses) formulated at different polysorbate 80 to protein molar ratios under accelerated storage stability conditions to study the effect of polysorbate 80 to protein molar ratio on the stability of MnB rLP2086 protein. A divalent MnB rLP2086 immunogenic composition formulated with a polysorbate 80 to protein molar ratio of about 1.4 to 10.7 was used. To generate immunogenic compositions formulated with increased polysorbate 80 to protein molar ratios (1.4, 2.4, 3.4, 3.9, 4.3, 4.7, and 10.7), antigens were adjusted to variable molar ratios by adding polysorbate 80 so that no additional polysorbate 80 was added during formulation of the immunogenic composition. Two series of antigen batches were used in this study. One series of sub-group a and B batches with a polysorbate 80 to protein molar ratio of 1.4 was generated, while the other series was 2.4. A protein series with a molar ratio of 2.4 was used to adjust the molar ratio to 3.4, 3.9, 4.3 and 10.7 by incorporating additional polysorbate 80. The final matrix of the immunogenic composition was 10mM histidine, 150mM NaCl, ph6.0, 0.5mg/mL aluminum phosphate, and had the polysorbate 80 to protein molar ratio described above. After storage at 2-8 ℃ or 25 ℃ for a specific time interval, gently mixed with a shaker for 24 hours, and then tested. Supernatant fractions were tested for total protein (by IEX-HPLC), potency, appearance, optical density at 320nm, and pH.

The efficacy results for the 200 μ g and 20 μ g doses are shown in figures 5 and 6, respectively. The efficacy test is more sensitive than the other tests used in this study. In summary, no significant decrease in potency of both subfamily a or B antigens was observed compared to the initial time point for all doses at a molar ratio of 4.3 and lower. Since the potency of the subfamily B protein stored at 25 ℃ is slightly reduced, the formulation with a molar ratio of 4.7 is considered to be a critical formulation. For samples stored at 25 ℃, the potency results for the subgroup B antigen of the formulations at a molar ratio of 10.7 were significantly lower than those stored at 2-8 ℃.

Example 6:aluminum concentration and potency of rLP2086 subfamily A and B antigens

Numerous experiments were performed to determine the optimum aluminum phosphate level to ensure greater than 95% binding of both subfamily a and B proteins. Initial studies focused on formulations optimized for ph 6.5. Formulations with a target dose of 200 μ g/mL of each protein were prepared from subfamily a and B proteins in 10mM histidine buffer (ph6.5) containing 0.02% polysorbate 80 and 0.25mg/mL or 0.5mg/mL aluminum (as aluminum phosphate). Subfamily B proteins bind to aluminum phosphate to a lesser extent than subfamily a proteins (fig. 7). Increasing the aluminum content from 0.25mg/mL to 0.5mg/mL increased the binding of subgroup B proteins to > 80%. Since the binding mechanism between proteins and aluminum suspensions is mainly ionic interactions, the pH of the suspension is one factor that affects binding.

Formulation pH was optimized to ensure over 90-95% binding of subfamily B protein. Multiple formulations of 200 μ g/mL each of protein a and B, ph5.6 to 6.5, and with different batches of immunogenic composition were examined (fig. 8). Over 90-95% binding of both proteins occurred in the formulations at pH5.6 to 6.4. As the pH of the formulation increased to 6.5 and above, the binding of subgroup B proteins decreased significantly. The recommended target pH is 6.0 to ensure that the subfamily a and B proteins are more than 90% bound.

The stability of the formulation (robustness) at formulation variables and/or limits was also assessed by varying pH, buffer, protein and polysorbate 80 concentrations (fig. 9). Although the binding of subfamily A proteins was consistently higher (> 95%) at total protein concentrations as high as 500. mu.g/mL (250. mu.g/mL per protein), the binding of subfamily B proteins was more sensitive to protein concentration and pH. The results of this experiment further support the recommended formulation at pH6 and containing 0.5mg/mL aluminum phosphate, as a 200 μ g dose of the commercial formulation was used.

Formulations with and without aluminum phosphate were evaluated to investigate the feasibility of providing stable formulations that did not contain aluminum phosphate and that had polysorbate 80 concentrations low enough to maintain the stability of the subfamily B protein. The immunogenic compositions were formulated at doses of 20 μ g and 200 μ g in histidine buffered saline buffer with polysorbate 80 concentration at 0 to 5.3 molar ratio. Half of the samples were stirred for 24 hours with a digital multi-tube vortex set at 500rpm in pulsed mode (2 seconds on, 1 second off) and then examined. This condition was used to simulate the ISTA test (international association for safe transport) typically performed at the final shipping package stage of the immunogenic composition to simulate extreme vibration during shipping conditions.

Under agitation, the formulation without aluminum phosphate precipitates, eventually resulting in loss of potency of both subfamily a and B antigens. Appearance tests (figure 10) and absorbance measurements at λ =320nm (figure 11) showed that aggregates and/or precipitates were formed when the formulation without aluminium phosphate was stirred. Potency assays on these samples (fig. 12 and 13) showed a significant loss of potency for both subfamily a and B proteins at all time points tested. The loss of efficacy was most pronounced in formulations containing low amounts of polysorbate 80. Since a small amount of polysorbate 80 is necessary to maintain subfamily B protein stability, inclusion of aluminum phosphate in the formulation is required to maintain stability. rLP2086 immunogenic compositions can be formulated with aluminum phosphate, which will serve to enhance potency stability as measured by an in vitro potency test.

Example 7:succinate and histidine as buffers

A series of formulations were prepared to compare the binding of rLP2086 subfamily A and B proteins in succinate and histidine, as well as pH, polysorbate 80, and MgCl₂Effect on binding (table 2). Stability of the formulations at formulation variables and/or limits was evaluated by varying pH, buffer, protein and polysorbate 80 concentrations (figures 25 and 26). The binding of aluminum to both subfamily a and subfamily B proteins is similar regardless of the buffer (histidine or succinate) used.

TABLE 2 evaluation of histidine and succinate buffers, MgCl₂Polysorbate 80 and pH5.6-6.0 on rLP2086 and AlPO₄ ¹Of

¹All formulations shown in Table 2 contained 0.5mg Al/mL.

The effect of buffer salts and mixing time on aluminum binding was evaluated with three commonly used buffer salts, which were chosen because their pKa was within the physiological range and because these salts are generally considered safe. The rLP2086 subfamily a and B proteins were formulated with one of the following three buffer salts at a pH appropriate for the pKa of each salt: 5mM succinate, 10mM histidine or 10mM phosphate to determine the extent of binding at each condition. The time required to complete binding was assessed by mixing the sample for 5 minutes or 120 minutes before measuring the amount of bound protein.

As shown in figure 27, subfamily B protein in phosphate buffer showed reduced binding at ph6.8, whereas subfamily a protein was not significantly affected under the same conditions. The amount of protein bound to aluminum was similar in samples formulated with histidine or succinate. Therefore, these two buffer salts were selected for further evaluation. While not wishing to be bound by theory, the reduction in binding in phosphate buffer may be due to competition for AlPO with added phosphate ions₄Due to the binding site of (a).

In proteins and AlPO₄At these conditions and concentrations, the binding was completed after five minutes of mixing at room temperature, which is similar to the results obtained after 2 hours of mixing.

To further examine whether the reduced binding of subgroup B proteins in phosphate buffer at pH6.8 was due to differences between pH or buffering salts, the binding in histidine or succinate buffered formulations was measured in the pH range of 5.3 to 7.0. A bivalent preparation containing 0.2mg/mL of each subfamily protein (total protein 0.4mg/mL), 0.02% PS80, 0.5mg Al/mL, and 150mM NaCl was prepared. Samples were prepared in 10mM histidine or 5mM succinate to compare the effect of buffer salts. After formulation, the pH of each sample was checked individually.

The binding properties of subfamily A protein at pH5.3 to 7.0 are shown in FIG. 28, while those of subfamily B protein are shown in FIG. 29. The amount of bound protein of subfamily a protein was hardly changed and binding was retained over 95% in the tested pH range. The formulation containing histidine and having a target pH of 7 gave a pH of 6.8. The pH is not adjusted to 7.0 (e.g., by addition of base) to avoid treatment of the protein or AlPO₄Possible effects, and therefore the result of this data point is not known.

The binding properties of the subfamily B proteins (shown in figure 29) show a tendency to pH dependence. However, regardless of whether the binding was performed in histidine or succinate buffered formulations, the amount of protein bound to aluminum was similar. Binding is dependent on the pH of the formulation, not the buffer salt. Binding remained at 95% until pH6.5 (94% in histidine, 95% in succinate), but binding decreased when pH was greater than 6.5. At pH 7.0, binding dropped to about 82%, with a slight difference between the buffer salts.

To obtain subfamily B protein and AlPO at these concentrations₄Preferably a pH of 6.5 or less.

Example 8:safety, tolerability and immunogenicity studies

A study was conducted to evaluate the safety, tolerability and immunogenicity of the administered rLP2086 vaccine in healthy young populations, according to a schedule of 0 and 2 months; 0.2 and 6 months; 0 and 2 months, followed by 12 months booster doses.

The immunogenic composition is the rLP2086 vaccine (recombinant and lipidated). What is needed isThe immunogenic composition comprises a recombinant ORF2086 protein of neisseria meningitidis serogroup B expressed in Escherichia coli (Escherichia coli) and formulated as a bivalent vaccine comprising one subfamily a strain and one subfamily B strain of rLP 2086. Specifically, the dose of the immunogenic composition was 0.5mL, formulated to contain 60 μ g, 120 μ g, or 200 μ g of each purified subfamily a and B rLP2086 protein, polysorbate 80 at a molar ratio of 2.8, and 0.25mg of Al³⁺(AlPO₄Form), 10mM histidine buffered saline (pH 6.0). The control composition contained a dose of 0.5mL of physiological saline solution (0.9% sodium chloride).

Individuals were randomly assigned to 5 groups. See table 3. Individuals were divided into two age groups: from 11 years old to <14 years old; and from ≧ 14 to <19 years of age.

Saline was used as a placebo because there was no proven safe, immunogenic, and effective vaccine against MnB that could serve as an active control.

According to table 3, subjects received one dose of rLP2086 vaccine or saline at each vaccination visit (e.g., visits 1, 2, 4, and 6). The vaccine was not injected into the blood vessels following standard vaccination practice. The rLP2086 vaccine was administered intramuscularly by injecting 0.5mL into the upper deltoid muscle. Saline is administered intramuscularly to the upper deltoid muscle.

A. Visit 1

At visit 1, day 1, at inoculation 1, the individuals were first bled and then inoculated. The 1 st visit blood draw and the 1 st inoculation occurred on the same day. Blood samples (approximately 20mL) were collected from the individuals prior to vaccination. For individuals randomized to groups 1, 2, 3, and 4, a single 0.5mL intramuscular injection of rLP2086 vaccine was administered to the upper deltoid muscle. For individuals in group 5, a single 0.5mL intramuscular injection of saline was administered to the upper deltoid muscle.

B. Visit 2 (42-70 days after visit 1), inoculation 2

For groups 1, 2 and 3, a single intramuscular injection of 0.5mLr LP2086 vaccine was administered to the upper deltoid muscle. For groups 4 and 5, a single 0.5mL intramuscular injection of saline was administered to the upper deltoid muscle.

C. Visit 3 (28-42 days after visit 2), draw blood after vaccination 2

Blood samples (approximately 20mL) were collected from the individuals.

D. Visit 4 (105-126 days after visit 2), and inoculation 3

For groups 2, 4 and 5, a single 0.5mL intramuscular injection of rLP2086 vaccine was administered to the upper deltoid muscle. For groups 1 and 3, a single 0.5mL intramuscular injection of saline was administered to the upper deltoid muscle.

E. Visit 5 (28-42 days after visit 4), draw blood after vaccination 3

Blood samples (approximately 20mL) were collected from the individuals.

F. Visit 6 (161-175 days after visit 4), and inoculation 4

At visit 6, the individual was first bled and then vaccinated. The 6 th visit blood draw and the 4 th vaccination occurred on the same day. Blood samples (approximately 20mL) were collected from the individuals prior to vaccination. For groups 3 and 5, a single 0.5mL intramuscular injection of rLP2086 vaccine was administered to the upper deltoid muscle. For individuals in groups 1, 2 and 4, a single 0.5mL saline intramuscular injection was administered to the upper deltoid muscle.

G. 7 th visit (28-42 days after 6 th visit), and blood draw after 4 th inoculation

Blood samples (approximately 20mL) were collected from the individuals.

Immunogenic results

The main objective of this study was to evaluate the immunogenicity of 60 μ g, 120 μ g and 200 μ g rLP2086 vaccines as measured by SBA with MnB strains expressing LP2086 subfamily A and B proteins.

The second objective of this study was to evaluate the immunogenicity of 60 μ g, 120 μ g and 200 μ g rLP2086 vaccines as determined by quantifying the amount of Ig binding to the rLP2086 vaccine subfamily a and B proteins.

SBA activity was evaluated using 3 subfamily a strains and 3 subfamily B strains, as shown in table 4.

TABLE 4 analysis of individuals with SBA titers increasing 4-fold or more before the 1 st dose (mITT population) (study 6108A1-2001-WW/B1971005)

Abbreviations CI = confidence interval, SBA = serum bactericidal test.

Note: the test assay supports a lower limit of quantitation (LLQQ) for subfamily a strain 1=9 and subfamily B strain 1= 10. SBA titers above LLQQ were considered accurate and their quantitative values were reported. Values below or indicated below LLQQ were set to 0.5 × LLOQ for analysis.

The proportion of individuals whose titer reached the specified level is given in table 5. For both subfamilies, the proportion of individuals reaching the prescribed SBA titer level after the 3 rd dose is higher than after the 2 nd dose.

Immunogenicity data indicates that the vaccine can produce antibodies with significant SBA activity against subgroup a and subgroup B strains of MnB. For subfamily a strain 2, SBA response rates were 88.9% to 90.9% after the 2 nd dose and 90.0% to 97.4% after the 3 rd dose. For the subfamily a strain 1 variant, 100.0% of individuals responded to SBA at both the 60 μ g and 120 μ g dose levels of this variant after the 2 nd and 3 rd doses. At the 200 μ g dose level, 96.5% and 99.0% of the individuals had SBA responses after the 2 nd and 3 rd dose, respectively. For the subgroup a strain 1 variants, the SBA response rate was 85.0% to 96.3% after the 2 nd dose and 95.2% to 97.4% after the 3 rd dose.

For the subgroup B strain 1 variant, the SBA response rate was 76.2% to 81.0% after the 2 nd dose and 89.5% to 92.0% after the 3 rd dose. For subgroup B strain 2 variants after the 2 nd dose, the percentage of individuals with SBA response rate was 21.1% to 33.3%. However, 53.3%, 75.6% and 67.9% of the individuals had SBA responses at dose levels of 60 μ g, 120 μ g and 200 μ g, respectively, after the 3 rd dose. For the subgroup B strain 3 variants, the SBA response rate was 61.9% to 70.8% after the 2 nd dose and 76.2% to 88.7% after the 3 rd dose.

In summary, a higher proportion of individuals responded with SBA titers of ≧ LLOQ, regardless of whether subfamily A or B strains were tested. The hSBA data indicate strong immune responses at doses of 60 μ g to 200 μ g, but there is no clear dose-response relationship. Regardless of the assay tested, the frequency of response was numerically highest in the 120 μ g group. The 200 μ g group did not have an increased immune response above the 120 μ g dose level.

Claims

1. An immunogenic composition comprising a LP2086(fHBP) subfamily B polypeptide formulated such that the potency of the LP2086(fHBP) subfamily B polypeptide remains stable over time.

2. An immunogenic composition comprising a detergent and a LP2086(fHBP) subfamily B polypeptide in a detergent to protein molar ratio of less than 10: 1.

3. The immunogenic composition according to claim 1 or 2, wherein at least 50% of the LP2086(fHBP) subfamily B polypeptide potency is maintained for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, 12 months, 18 months, 24 months, 30 months, 36 months, 42 months, 48 months, 54 months, or 60 months.

4. The immunogenic composition of any one of claims 1-3, further comprising a LP2086(fHBP) subfamily A polypeptide.

5. The immunogenic composition of claim 1, further comprising a detergent.

6. The immunogenic composition of any one of claims 1-5, wherein the detergent to protein molar ratio is from about 0.5:1 to about 10: 1.

7. The immunogenic composition of claim 6, wherein the detergent to protein molar ratio is from about 1:1 to about 5: 1.

8. The immunogenic composition of claim 7, wherein the detergent to protein molar ratio is about 1.4:1 to about 4.2: 1.

9. The immunogenic composition of claim 8, wherein the detergent to protein molar ratio is about 2.8: 1.

10. The immunogenic composition of any one of claims 1-9, wherein the amount of detergent is sufficient to reduce binding of the polypeptide to silicon in the container.

11. The immunogenic composition of claim 10, wherein the container is a syringe or a vial.

12. The immunogenic composition of any one of claims 1-11, wherein the detergent is a non-ionic detergent.

13. The immunogenic composition of claim 12, wherein the detergent is a polysorbate detergent.

14. The immunogenic composition of claim 13, wherein the detergent is polysorbate 80.

15. The immunogenic composition of any one of claims 1-14, further comprising a multivalent cation.

16. The immunogenic composition of claim 15, wherein the multivalent cation is calcium or aluminum.

17. The immunogenic composition of claim 16, wherein the immunogenic composition comprises (Ca)₃(PO₄)₂。

18. The immunogenic composition of claim 16, wherein the concentration of aluminum is from about 0.1mg/ml to about 1 mg/ml.

19. The immunogenic composition of claim 18, wherein the concentration of aluminum is about 0.5 mg/ml.

20. The immunogenic composition of any one of claims 16-19, wherein the composition comprises AlPO₄、Al(OH)₃、Al₂(SO₄)₃And alum.

21. The immunogenic composition of any one of claims 1-20, further comprising histidine.

22. The immunogenic composition according to claim 21, wherein the concentration of histidine is between about 2mM and about 20 mM.

23. The immunogenic composition according to claim 22, wherein the concentration of histidine is between about 5mM and about 15 mM.

24. The immunogenic composition of any one of claims 1-20, further comprising a succinate salt.

25. The immunogenic composition of claim 24, wherein the concentration of succinate is from about 2mM to about 10 mM.

26. The immunogenic composition of claim 25, wherein the concentration of succinate is from about 3mM to about 7 mM.

27. The immunogenic composition of claim 25, wherein the concentration of succinate is about 5 mM.

28. The immunogenic composition of any one of claims 1-27, wherein the pH is from about 5.0 to about 8.0.

29. The immunogenic composition of claim 28, wherein the pH is about 5.8 to 6.0.

30. The immunogenic composition according to claim 29, wherein histidine is at a concentration of about 10mM and pH 6.0.

31. The immunogenic composition of any one of claims 1-30, wherein the composition is lyophilized.

32. The immunogenic composition of claim 31, wherein the lyophilized composition is resuspended in a buffer comprising aluminum.

33. The immunogenic composition of claim 32, wherein the buffer comprises AlPO₄、Al(OH)₃、Al₂(SO₄)₃Or alum.

34. The immunogenic composition of claim 1 or 2, wherein the composition comprises polysorbate 80 at a molar ratio of about 2.8:1 to protein, AlPO of 0.5mg/mL₄Form aluminum, 10mM histidine pH6.0, and 150mM NaCl.

35. The immunogenic composition of claim 1 or 2, wherein the composition consists essentially of 200ug/mL LP2086(fHBP) subfamily B polypeptide, polysorbate 80 at a molar ratio to protein of about 2.8:1, 0.5mg/mL AlPO₄Form aluminum, 10mM histidine pH6.0, and 150mM NaCl.

36. The immunogenic composition of claim 1 or 2, wherein the composition consists essentially of 200ug/mL of rLP2086(fHBP) subfamily a polypeptide, 200ug/mL of LP2086(fHBP) subfamilyGroup B polypeptides, polysorbate 80 at a molar ratio to protein of about 2.8:1, AlPO 0.5mg/mL₄Form aluminum, 10mM histidine pH6.0 and 150mM NaCl.

37. The immunogenic composition of claim 1 or 2, wherein the composition comprises polysorbate 80 at a molar ratio of about 2.8:1 to protein, AlPO of 0.5mg/mL₄Form aluminum, 5mM succinate pH6.0 and 150mM NaCl.

38. The immunogenic composition of claim 1 or 2, wherein the composition consists essentially of 200ug/mL LP2086(fHBP) subfamily B polypeptide, polysorbate 80 at a molar ratio to protein of about 2.8:1, 0.5mg/mL AlPO₄Form of aluminum, 5mM succinate pH6.0, and 150mM NaCl.

39. The immunogenic composition of claim 1 or 2, wherein the composition consists essentially of 200ug/mL of rLP2086(fHBP) subfamily a polypeptide, 200ug/mL of LP2086(fHBP) subfamily B polypeptide, polysorbate 80 at a molar ratio to protein of about 2.8:1, 0.5mg/mL of AlPO₄Form of aluminum, 5mM succinate pH6.0 and 150mM NaCl.

40. A method of stabilizing the potency of a LP2086(fHBP) subfamily B polypeptide in an immunogenic composition, the method comprising step (a): formulating the LP2086(fHBP) subfamily B polypeptide into a composition having a detergent to protein molar ratio of about 0.5:1 to about 10: 1.

41. The method of claim 40, wherein the molar ratio of detergent to protein is from about 1:1 to about 5: 1.

42. The method of claim 41, wherein the molar ratio of detergent to protein is from about 1.4:1 to about 4.2: 1.

43. The method of claim 42, wherein the molar ratio of detergent to protein is about 2.8: 1.

44. The method of any of claims 40-43, wherein the amount of detergent is sufficient to reduce binding of the polypeptide to silicon in the container.

45. The method of claim 44, wherein the container is a syringe or a vial.

46. The method of any one of claims 40-45, wherein the detergent is a non-ionic detergent.

47. The method of claim 46, wherein the detergent is a polysorbate detergent.

48. The method of claim 47, wherein the detergent is polysorbate 80.

49. The method of any one of claims 40-48, wherein the composition further comprises a multivalent cation.

50. The method of claim 49, wherein the multivalent cation is calcium or aluminum.

51. The method of claim 50, wherein the composition comprises (Ca)₃(PO₄)₂。

52. The method of claim 50, wherein the concentration of aluminum is from about 0.1mg/ml to about 1 mg/ml.

53. The method of claim 52, wherein the aluminum concentration is about 0.5 mg/ml.

54. The method of any one of claims 49-53, wherein the composition comprises AlPO₄、Al(OH)₃、Al₂(SO₄)₃Or alum.

55. The method of any one of claims 40-54, further comprising histidine.

56. The method of claim 55, wherein the concentration of histidine is between about 2mM and about 20 mM.

57. The method of claim 56, wherein the concentration of histidine is between about 5mM and about 15 mM.

58. The method of any one of claims 40-54, further comprising a succinate salt.

59. The method of claim 58, wherein the concentration of succinate is from about 2mM to about 10 mM.

60. The method of claim 59, wherein the concentration of succinate is from about 3mM to about 7 mM.

61. The method of any one of claims 40-60, wherein the pH is from about 5.0 to about 8.0.

62. The method of claim 61, wherein the pH is about 5.8 to 6.0.

63. The method of claim 62, wherein histidine is at a concentration of about 10mM and pH 6.0.

64. The method of claim 62, wherein the concentration of succinate is about 5mM and the pH is 6.0.

65. The method of any one of claims 40-64, further comprising step (b): lyophilizing the immunogenic composition.

66. The method of claim 65, further comprising step (c): resuspending the lyophilized composition in a buffer comprising aluminum.

67. The method of claim 66, wherein the buffer comprises AlPO₄、Al(OH)₃、Al₂(SO₄)₃Or alum.

68. The method of claim 40, wherein said subfamily B protein is formulated to consist essentially of polysorbate 80, 0.5mg/mL AlPO at a molar ratio of about 2.8:1 to protein₄Aluminum in the form of 10mM histidine pH6.0 and 150mM NaCl.

69. The method of claim 40, wherein said subfamily B protein is formulated to consist essentially of polysorbate 80, 0.5mg/mL AlPO at a molar ratio of about 2.8:1 to protein₄Form of aluminum, 5mM succinate pH6.0 and 150mM NaCl.

70. A method of stabilizing the potency of a LP2086(fHBP) subfamily B polypeptide in an immunogenic composition, the method comprising step (a): the LP2086(fHBP) subfamily B polypeptide is formulated into a composition containing about 0.1mg/ml to about 1mg/ml aluminum and a detergent to protein molar ratio of about 0.5:1 to about 10: 1.

71. The method of claim 70, wherein the molar ratio of detergent to protein is from about 1:1 to about 5: 1.

72. The method of claim 71, wherein the molar ratio of detergent to protein is from about 1.4:1 to about 4.2: 1.

73. The method of claim 72, wherein the molar ratio of detergent to protein is about 2.8: 1.

74. The method of any of claims 70-73, wherein the amount of detergent is sufficient to reduce binding of the polypeptide to silicon in the container.

75. The method of claim 74, wherein the container is a syringe or a vial.

76. The method of any one of claims 70-75, wherein the detergent is a non-ionic detergent.

77. The method of claim 76, wherein the detergent is a polysorbate detergent.

78. The method of claim 77, wherein said detergent is polysorbate 80.

79. The method of any one of claims 70-78, wherein the concentration of aluminum is about 0.5 mg/ml.

80. The method of any of claims 70-79, wherein the composition comprises AlPO₄、Al(OH)₃、Al₂(SO₄)₃Or alum.

81. The method of any one of claims 70-80, further comprising histidine.

82. The method of claim 81, wherein histidine is at a concentration of about 2mM to about 20 mM.

83. The method of claim 82, wherein the concentration of histidine is between about 5mM and about 15 mM.

84. The method of any one of claims 70-80, further comprising a succinate salt.

85. The method of claim 84, wherein the concentration of succinate is from about 2mM to about 10 mM.

86. The method of claim 85, wherein the concentration of succinate is from about 3mM to about 7 mM.

87. The method of any one of claims 70-86, wherein the pH is about 5.0 to about 8.0.

88. The method of claim 87, wherein the pH is about 5.8 to 6.0.

89. The method of claim 88, wherein histidine is at a concentration of about 10mM and pH 6.0.

90. The method of any one of claims 70-89, further comprising step (b): lyophilizing the immunogenic composition.

91. The method of claim 90, further comprising step (c): resuspending the lyophilized composition in a buffer comprising aluminum.

92. The method of claim 91, wherein the buffer comprises AlPO₄、Al(OH)₃、Al₂(SO₄)₃Or alum.

93. The method of claim 70, wherein the subfamily B protein is formulated to consist essentially of polysorbate 80, 0.5mg/mL AlPO at a molar ratio of about 2.8:1 to protein₄Aluminum in the form of 10mM histidine pH6.0 and 150mM NaCl.

94. The method of claim 70, wherein the subfamily B protein is formulated to consist essentially of polysorbate 80, 0.5mg/mL AlPO at a molar ratio of about 2.8:1 to protein₄Form of aluminum, 5mM succinate pH6.0 and 150mM NaCl.

95. A method of determining the efficacy of a 2086(fHBP) polypeptide in an immunogenic composition, the method comprising the steps of: (1) incubating a first monoclonal Ab and a second mAb with an immunogenic composition comprising a 2086 protein, wherein the first mAb is conjugated to a first label for capturing the mAb and the second mAb is conjugated to a second label that is detectable, and wherein the first and second mabs are directed to different conformational epitopes on a 2086 reference protein; (2) capturing the first mAb-bound 2086 protein using the first label; and (3) detecting and quantifying the amount of captured second mAb-bound 2086 protein using the second label.

96. The method of claim 95, wherein the first tag is biotin, GST, 6xHis tag, or a bead.

97. The method of claim 96, wherein the beads are carboxylated polystyrene beads or paramagnetic beads.

98. The method of claim 96, wherein the first label is captured with streptavidin beads, streptavidin columns, glutathione beads, glutathione columns, nickel beads, nickel columns, by centrifugation, or with a magnetic field.

99. The method of any one of claims 95-98, wherein the second label is biotin, HRP, a fluorophore, or a radiolabel.

100. The method of claim 99, wherein the second label is detected with streptavidin conjugated to a fluorophore or HRP, by electrochemiluminescence, fluorescence detection, or radioactivity detection.

101. The method according to any one of claims 95-100, where in the 2086 polypeptide is a subfamily a polypeptide.

102. The method according to any one of claims 95-100, where in the 2086 polypeptide is a subfamily B polypeptide.

103. The method according to any one of claims 95-102, where in the 2086 polypeptide is lipidated.

104. The method according to any one of claims 95-102, where in the 2086 polypeptide is non-lipidated.

105. The method according to any one of claims 95-104, where in the 2086 polypeptide is recombinant.

106. The method of any one of claims 95-105, wherein polypeptides exhibiting epitopes recognized by both antibodies can be quantified.

107. The method of any one of claims 95-106, wherein the potency of the sample is compared to the potency of a reference substance.