HK1254329B - Method for stabilizing the potency of an lp2086 (fhbp) subfamily b polypeptide in an immunogenic composition - Google Patents

Description

Method for stabilizing the potency of LP2086 (fHBP) subfamily B polypeptides in immunogenic compositions

The present application is a divisional application of Chinese patent application No. 201180050859.8 filed on August 22, 2011, entitled “Stable Formulation of Neisseria meningitidis rLP2086 Antigen”.

Technical Field

The present invention relates to the preparation of Neisseria meningitidis rLP2086 subgroup B antigens in the form of immunogenic compositions as described herein. The present invention also relates to methods for preserving the conformation of Neisseria meningitidis rLP2086 antigens and methods for determining the potency of Neisseria meningitidis rLP2086 antigens.

Background of the Invention

rLP2086 is a 28 kDa recombinant lipoprotein that elicits cross-reactive bacterial antibodies against multiple strains of Neisseria meningitidis. Based on putative amino acid sequence homology, two distinct subfamilies of rLP2086 were identified: A and B. These subfamilies were used to formulate MnB-rLP2086 vaccine samples with varying levels of polysorbate 80 (PS-80) at 20 μg/mL, 60 μg/mL, 120 μg/mL, and 200 μg/mL of each subfamilies in 10 mM histidine (pH 6.0), 150 mM NaCl, and 0.5 mg/mL aluminum. Polysorbate 80, also known as TWEEN 80, 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 shipping, reduce filter absorption, and reduce tube absorption.

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 an 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 from 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 nonionic detergent such as a polysorbate detergent. In some embodiments, the detergent is polysorbate-80.

In some embodiments, the immunogenic composition further comprises a polyvalent cation. In some embodiments, the polyvalent 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 0.1 mg/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 2 mM to about 20 mM, or from about 5 mM to about 15 mM. In some embodiments, the concentration of histidine is from 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, the concentration of histidine is 10 mM and the pH is 6.0.

In some embodiments, the immunogenic composition further comprises succinate. In some embodiments, the concentration of succinate is about 2 mM to about 10 mM, or about 3 mM to about 7 mM. In some embodiments, the concentration of succinate is about 5 mM. In some embodiments, the pH of succinate is about 5.0 to about 8.0, or about 5.8 to about 6.0. In some embodiments, the concentration of succinate is 5 mM and the pH is 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 in the form of 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, 0.5 mg/mL aluminum in the form of AlPO ₄ , 10 mM histidine at pH 6.0, and 150 mM NaCl. In some embodiments, the immunogenic composition consists essentially of 200 ug/mL LP2086 (fHBP) subfamily B polypeptide, polysorbate 80 at a molar ratio of about 2.8:1 to protein, 0.5 mg/mL aluminum in the form of AlPO ₄ , 10 mM histidine at pH 6.0, and 150 mM NaCl. In some embodiments, the immunogenic composition consists essentially of 200 ug/mL rLP2086 (fHBP) subfamily A polypeptide, 200 ug/mL LP2086 (fHBP) subfamily B polypeptide, polysorbate 80 at a molar ratio of about 2.8:1 to protein, 0.5 mg/mL aluminum in the form of AlPO ₄ , 10 mM histidine at pH 6.0, and 150 mM NaCl.

In some embodiments, the immunogenic composition comprises polysorbate 80 at a molar ratio of about 2.8:1 to protein, 0.5 mg/mL aluminum in the form of AlPO ₄ , 5 mM succinate at pH 6.0, and 150 mM NaCl. In some embodiments, the immunogenic composition consists essentially of 200 ug/mL LP2086 (fHBP) subfamily B polypeptide, polysorbate 80 at a molar ratio of about 2.8:1 to protein, 0.5 mg/mL aluminum in the form of AlPO ₄ , 5 mM succinate at pH 6.0, and 150 mM NaCl. In some embodiments, the immunogenic composition consists essentially of 200 ug/mL rLP2086 (fHBP) subfamily A polypeptide, 200 ug/mL LP2086 (fHBP) subfamily B polypeptide, polysorbate 80 at a molar ratio to protein of about 2.8:1, 0.5 mg/mL aluminum in the form of AlPO ₄ , 5 mM succinate at pH 6.0, and 150 mM NaCl.

In another aspect, the present invention provides a method for stabilizing the potency of an LP2086 subfamily B polypeptide in an immunogenic composition by storing the LP2086 subfamily B polypeptide 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 detergent-to-protein molar ratio 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 nonionic detergent, such as a polysorbate detergent. In some embodiments, the detergent is polysorbate 80.

In some embodiments, the buffer further comprises a polyvalent cation. In some embodiments, the polyvalent 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 0.1 mg/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 about 2 mM to about 20 mM or about 5 mM to about 15 mM. In some embodiments, the concentration of histidine is about 10 mM. In some embodiments, the pH of histidine is about 5.0 to about 8.0 or about 5.8 to about 6.0. In some embodiments, the concentration of histidine is 10 mM and the pH is 6.0.

In some embodiments, the buffer further comprises succinate. In some embodiments, the concentration of succinate is about 2 mM to about 10 mM or about 3 mM to about 7 mM. In some embodiments, the concentration of succinate is about 5 mM. In some embodiments, the pH of succinate is about 5.0 to about 8.0 or about 5.8 to about 6.0. In some embodiments, the concentration of succinate is 10 mM and the pH is 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 in the form of aluminum phosphate, aluminum hydroxide, aluminum sulfate, or alum.

In some embodiments, the buffer consists essentially of polysorbate 80 at a molar ratio of about 2.8:1 to protein, 0.5 mg/mL aluminum in the form of AlPO ₄ , 10 mM histidine at pH 6.0, and 150 mM NaCl. In some embodiments, the immunogenic composition consists essentially of 200 ug/mL LP2086 (fHBP) subfamily B polypeptide, polysorbate 80 at a molar ratio of about 2.8:1 to protein, 0.5 mg/mL aluminum in the form of AlPO ₄ , 10 mM histidine at pH 6.0, and 150 mM NaCl.

In some embodiments, the buffer consists essentially of polysorbate 80 at a molar ratio of about 2.8:1 to protein, 0.5 mg/mL aluminum in the form of AlPO ₄ , 5 mM succinate at pH 6.0, and 150 mM NaCl. In some embodiments, the immunogenic composition consists essentially of 200 ug/mL LP2086 (fHBP) subfamily B polypeptide, polysorbate 80 at a molar ratio of about 2.8:1 to protein, 0.5 mg/mL aluminum in the form of AlPO ₄ , 5 mM succinate at pH 6.0, and 150 mM NaCl.

In another aspect, the present invention provides a method for stabilizing the potency of LP2086 subfamily A polypeptides and LP2086 subfamily B polypeptides in an immunogenic composition by storing the LP2086 subfamily A polypeptides and the LP2086 subfamily B polypeptides in a buffer containing about 0.1 mg/mL to about 10 mg/mL of aluminum and a detergent-to-protein molar ratio of about 0.5:1 to 10:1. In some embodiments, the detergent-to-protein molar ratio is about 1:1 to about 5:1, or about 1.4:1 to about 4.2:1. In some embodiments, the detergent-to-protein molar ratio 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 aluminum is present in the form of 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 further comprises histidine. In some embodiments, the concentration of histidine is about 2 mM to about 20 mM or about 5 mM to about 15 mM. In some embodiments, the concentration of histidine is about 10 mM. In some embodiments, the pH of histidine is about 5.0 to about 8.0 or about 5.8 to about 6.0. In some embodiments, the concentration of histidine is 10 mM and the pH is 6.0.

In some embodiments, the buffer further comprises succinate. In some embodiments, the concentration of succinate is about 2 mM to about 10 mM or about 3 mM to about 7 mM. In some embodiments, the concentration of succinate is about 5 mM. In some embodiments, the pH of succinate is about 5.0 to about 8.0 or about 5.8 to about 6.0. In some embodiments, the concentration of succinate is 10 mM and the pH is 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 in the form of aluminum phosphate, aluminum hydroxide, aluminum sulfate, or alum.

In some embodiments, the buffer consists essentially of polysorbate 80 at a molar ratio of about 2.8:1 to protein, 0.5 mg/mL aluminum in the form of AlPO ₄ , 10 mM histidine at pH 6.0, and 150 mM NaCl. In some embodiments, the immunogenic composition consists essentially of 200 ug/mL LP2086 (fHBP) subfamily A polypeptide, 200 ug/mL LP2086 (fHBP) subfamily B polypeptide, polysorbate 80 at a molar ratio of about 2.8:1 to protein, 0.5 mg/mL aluminum in the form of AlPO ₄ , 10 mM histidine at pH 6.0, and 150 mM NaCl.

In some embodiments, the buffer consists essentially of polysorbate 80 at a molar ratio of about 2.8:1 to protein, 0.5 mg/mL aluminum in the form of AlPO ₄ , 5 mM succinate at pH 6.0, and 150 mM NaCl. In some embodiments, the immunogenic composition consists essentially of 200 ug/mL LP2086 (fHBP) subfamily A polypeptide, 200 ug/mL LP2086 (fHBP) subfamily B polypeptide, polysorbate 80 at a molar ratio of about 2.8:1 to protein, 0.5 mg/mL aluminum in the form of AlPO ₄ , 5 mM succinate at pH 6.0, and 150 mM NaCl.

In another aspect, the present invention provides a method for determining the potency of rLP2086 subfamily A polypeptides and/or rLP2086 subfamily B polypeptides, the method comprising the steps of: (a) binding a first and a second functional monoclonal antibody that recognizes a conformational epitope on each subfamily protein to the immunogenic composition, and (b) quantifying the antibodies bound to the polypeptides. In some embodiments, the quantification is performed by electrochemiluminescence. In some embodiments, the polypeptides presenting 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 separated by a compound bound to a conjugated label, such as streptavidin beads or a streptavidin column. In some embodiments, the second antibody is bound 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 THE DRAWINGS

Figure 1: Stability of subfamily B in formulations with different polysorbate 80 concentrations.

Figure 2: Accelerated stability of subfamily B with different polysorbate 80 concentrations.

Figure 3: Efficacy of subfamily B at 200 μg/mL over 28 days.

Figure 4: Efficacy of subfamily B at 20 μg/mL over 28 days.

Figure 5: Potency results of 200 μg/mL with different molar ratios.

Figure 6: Potency results of 20 μg/mL with different molar ratios.

Figure 7: Protein binding to aluminum phosphate at pH 6.5.

Figure 8: Binding of MnB rLP2086 subfamilies A and B as a function of pH.

Figure 9: Effect of pH, buffer and protein concentration on the binding of rLP2086 subfamily A and B.

Figure 10: Appearance of rLP2086 formulations without aluminum phosphate.

Figure 11: OD measurements of appearance samples at 2-8°C.

Figure 12: Efficacy results for subfamily A of formulations with and without _AlPO4 .

Figure 13: Efficacy results for subfamily B of formulations with and without _AlPO4 .

Figure 14: Polysorbate 80 results in rLP2086 placebo containing 0.5 mg/mL aluminum.

Figure 15: Polysorbate 80 results for subfamily A.

Figure 16: Polysorbate 80 results for subfamily B.

Figure 17: Correlation between the potency of subfamily B and the bound molar ratio.

Figure 18: Molar ratio results for subfamily A.

Figure 19: Molar ratio results for subfamily B.

Figure 20: Molar ratio results for 400 μg/mL rLP2086 formulation.

Figure 21: Polysorbate 80 results for rLP2086 drug product at different time points.

FIG22 : The binding molar ratio results of rLP2086 drug at different time points.

Figure 23: Potency and binding molar ratio results for subfamily A.

Figure 24: Subfamily B potency and binding molar ratio results.

Figure 25: Binding of subfamily A to _AlPO4 in succinate and histidine buffers.

Figure 26: Binding of subfamily B to _AlPO4 in succinate and histidine buffers.

Figure 27: Comparison of binding in succinate, histidine and phosphate buffers.

Figure 28: pH dependent binding of subfamily A to _AlPO4 .

Figure 29: pH dependent binding of subfamily B to _AlPO4 .

Detailed Description of the Invention

Unless otherwise specified, all scientific and technical terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention pertains. Although methods and materials similar or equivalent to those described herein can be used to implement or test the present invention, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patents, and other documents mentioned herein are incorporated herein by reference in their entirety.

Throughout the 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

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 methods and/or steps of the type described herein and/or which will become apparent to those of ordinary skill in the art upon reading this disclosure.

Unless the context clearly indicates otherwise, plural forms used herein include the singular referent. Thus, for example, reference to "the method" includes one or more methods and/or steps of the type described herein and/or that would become apparent to one of ordinary skill in the art upon reading this disclosure.

As used herein, "about" means within a statistically significant range of values, such as a referenced concentration range, time frame, molecular weight, temperature, or pH. Such a range can 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 encompassed by the term "about" will depend on the specific system under study and will be readily understood by one of ordinary skill in the art. Whenever a range is mentioned in this application, each integer within that range is also considered an embodiment of the invention.

The term "adjuvant" refers to a compound or mixture that enhances the immune response 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 the 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 adjuvant 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, and Avridine lipid-amine adjuvant.

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

As used herein, the term "effective immunogenic amount" refers to an amount of a polypeptide or composition comprising a polypeptide that is effective in eliciting an immune response in a vertebrate host. For example, an effective immunogenic amount of the rLP2086 protein of the present invention is an amount that is effective in eliciting 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, as well as the method of administration. Appropriate dosages are readily determined by those skilled in the art.

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

For example, a composition comprising 0.01% polysorbate 80 and 200 μg of protein has a molar ratio of detergent to protein of 10.8:1 [(0.01/0.2) x 216]. A ratio of 3 moles of polysorbate 80 to 2 moles of protein can be expressed as a molar ratio of PS80 to protein of 3:2. 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. As used herein, the terms "detergent to protein" and "polysorbate 80 to protein" generally refer to the molar ratio of detergent (or polysorbate 80) to protein antigen (particularly P2086 antigen). Based on the teachings disclosed herein, one skilled in the art will be able to determine how to calculate the molar ratios of other detergents and the optimal molar ratios for formulations containing other detergents. As used herein, a "low" molar ratio generally refers to a molar ratio of detergent to protein antigen in an immunogenic composition that is lower than a "high" molar ratio. A "high" molar ratio generally refers to a molar ratio of detergent to protein antigen in an 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 greater than 10:1. In some embodiments, a "low molar ratio" of detergent to protein refers to a molar ratio of 0.5:1 to 10:1.

The term "ORF2086" as used herein refers to the open-label region 2086 derived from Neisseria bacteria. Neisseria ORF2086, proteins encoded thereby, fragments of those proteins, and immunogenic compositions comprising these proteins are known in the art and are described in, for example, U.S. Patent Application Publications US 20060257413 and US 20090202593 (each of which is incorporated herein by reference in its entirety). The term "P2086" generally refers to a protein encoded by ORF2086. The P2086 protein of the present invention may be lipidated or non-lipidated. Typically, "LP2086" and "P2086" refer to lipidated and non-lipidated forms of the 2086 protein, respectively. The P2086 protein of the present invention may be recombinant. Typically, "rLP2086" and "rP2086" refer to recombinant 2086 proteins in lipidated and non-lipidated forms, 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 agents, and absorption delaying agents that are compatible with administration to humans or other vertebrate hosts. Typically, a pharmaceutically acceptable carrier is one that is licensed by a regulatory agency of the federal government, a state government, or other regulatory agency, or is listed in the U.S. Pharmacopoeia or other generally recognized pharmacopeia for use in animals (including humans and non-human mammals). The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle that is administered together with the pharmaceutical composition. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, plant, or synthetic origin. Water, saline solutions, aqueous dextrose solutions, and glycerol solutions can be used as liquid carriers, particularly liquid carriers for solutions for injection. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. If necessary, the composition may also include a small amount of wetting agent, filler, emulsifier, or pH buffer. These compositions may be in the form of solutions, suspensions, emulsions, sustained-release formulations, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W.Martin. The preparation should be suitable for 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 an epitope to bind to an antibody. Potency may be lost or reduced over time due to loss of antigen or epitope integrity, or changes in antigen or epitope conformation. Potency may be lost or reduced due to factors including, but not limited to, light, temperature, freeze-thaw cycles, agitation, and pH. Potency can be measured by methods disclosed herein and tests known in the art. Such potency determination tests include, but are not limited to, animal vaccination models, serum bactericidal assays (SBAs), flow cytometry, and in vitro potency tests. Preferred methods for determining potency are SBAs and in vitro potency tests. More preferred methods for determining potency are SBAs. In some embodiments, potency can be determined using at least one monoclonal antibody directed against at least one epitope involved in the immune response. In some embodiments, the potency of a test sample is compared to that of a reference standard. In some embodiments, the reference standard is a test sample at T _0. In some embodiments, the reference standard is an immunogenic composition without detergent. In some embodiments, the reference standard is an immunogenic composition having a detergent to protein molar ratio greater than 10:1.

A "protective" immune response refers to the ability of an immunogenic composition to elicit an immune response (including humoral and cell-mediated immune responses) that acts to protect an individual from infection. If there is a statistically significant improvement compared to a control group of individuals (e.g., infected animals that have not been 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 alleviating the severity or speed of onset of symptoms of infection. Typically, a "protective immune response" includes an increase in the level of antibodies specific for a particular antigen (including some measurable functional antibody response levels for each antigen) elicited in at least 50% of individuals. In specific cases, a "protective immune response" may include a two-fold or four-fold increase in the level of antibodies specific for a particular antigen (including some measurable functional antibody response levels for each antigen) elicited in at least 50% of individuals. In certain embodiments, opsonizing antibodies are associated with a protective immune response. Thus, a protective immune response can be tested by measuring the percentage decrease in bacterial counts in opsonophagocytosis assays (e.g., those described below). In some embodiments, the bacterial count is reduced by at least 10%, 25%, 50%, 65%, 75%, 80%, 85%, 90%, 95% or more compared to the bacterial count in the absence of the immunogenic composition.

The terms "protein," "polypeptide," and "peptide" refer to polymers of amino acid residues, and there is no restriction on the minimum length of such products. Thus, 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 to the native sequence such as deletions, additions, and substitutions (usually conservative in nature, but also non-conservative), preferably such that the protein retains the ability to elicit an immune response in the animal to which it is administered. Post-expression modifications such as glycosylation, acetylation, lipidation, phosphorylation, and the like are also included.

As used herein, the term "recombinant" refers to any protein, polypeptide, or cell expressing a gene of interest produced by genetic engineering methods. With respect to proteins or polypeptides, the term "recombinant" refers to polypeptides produced by expressing recombinant polynucleotides. Proteins of the present invention can be isolated from natural sources or produced by genetic engineering methods. As used herein, "recombinant" also describes nucleic acid molecules that, due to their origin or manipulation, are not related to all or part of the polynucleotides with which they are associated in nature. With respect to host cells, the term "recombinant" refers to host cells that contain recombinant polynucleotides.

The terms "stable" and "stability" refer to the ability of an antigen to maintain immunogenicity over a period of time. The stability of potency 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 a protein composition may be caused by chemical degradation or aggregation of the protein molecules to form higher polymers; dissociation of heterodimers into monomers; deglycosylation; glycosylation modification; or any other structural modification that reduces at least one biological activity of a protein composition encompassed by the present 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 (by differential scanning calorimetry (DSC)) of a sample. Other methods for assessing stability are also known in the art and can also be used in accordance with the present invention.

In some embodiments, the antigen in the stable formulation of the present invention can maintain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% efficacy over 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 compared to reference standard. In some embodiments, the antigen in the stable formulation of the present invention can maintain at least 50% efficacy over at least 1 year, 2 years, 3 years, 4 years or 5 years compared to 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, compared to a reference standard, the antigen in the stable formulation of the present invention can 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. In some embodiments, stability is measured according to environmental conditions. Non-limiting examples of environmental conditions include light, temperature, freeze-thaw cycles, stirring and pH. Those skilled in the art can determine the presence of antigenic epitopes or immunoreactivity using methods disclosed herein or other methods known in the art. See McNeil et al., Vaccine , 27:3417-3421 (2009). In some embodiments, stability is measured from the day of formulation of the antigen. In some embodiments, the stability of the antigen is measured on the day the storage conditions are changed. Non-limiting examples of changes in storage conditions include changing from frozen to refrigerated, from frozen to room temperature, from refrigerated to room temperature, from refrigerated to frozen, from room temperature to frozen, from room temperature to refrigerated, from light to dark, or introducing agitation.

The term "stabilizer" refers to a compound that binds to an antigen and maintains the 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 "individual" refers to a mammal, bird, fish, reptile, or any other animal. The term "individual" 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 "individual" also includes domestic animals. Non-limiting examples of domestic animals include alpacas, bison, camels, cattle, deer, pigs, horses, llamas, mules, donkeys, sheep, goats, rabbits, reindeer, yaks, chickens, geese, and turkeys.

The terms "vaccine" or "vaccine composition" are used interchangeably to 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 based on the novel discovery that rLP2086 subgroup B antigens, but not rLP2086 subgroup A antigens, lose potency over time in bivalent vaccine formulations and are therefore unstable. By varying the components in the bivalent formulations, it was determined that a high molar ratio of detergent to protein in the bivalent vaccine formulations resulted in instability specific to the rLP2086 subgroup B antigens. Reducing the molar ratio of detergent to protein in both the bivalent and monovalent formulations resulted in increased stability of the rLP2086 subgroup B antigens (measured by maintenance of potency over time) without affecting the stability of the rLP2086 subgroup A antigens. This result is surprising because lipoproteins are typically purified and preserved using high detergent concentrations in order to prevent aggregation of their hydrophobic lipid portions. Thus, in some embodiments, the present invention provides an immunogenic composition comprising an rLP2086 subgroup B antigen and a low molar ratio of detergent to protein. In some embodiments, the present invention provides a method for maintaining the stability of rLP2086 subgroup B antigen in an immunogenic composition, the method comprising the step of storing the rLP2086 subgroup B antigen in a buffer comprising a low molar ratio of detergent to protein.

Further studies have shown that low molar ratio formulations result in aggregation of rLP2086 subgroup A and B antigens when the low molar ratio immunogenic composition is stirred. However, increasing the aluminum concentration in the low molar ratio composition prevents aggregation of rLP2086 subgroup A and B antigens, even when stirred. In addition, in the absence of aluminum, rLP2086 subgroup A antigens are more sensitive to the effects of low detergent molar ratios. Therefore, in some embodiments, the present invention provides an immunogenic composition comprising rLP2086 subgroup A antigen, rLP2086 subgroup B antigen, a high concentration of aluminum, and a low molar ratio of detergent to protein. In some embodiments, the present invention provides a method for maintaining the stability of rLP2086 subgroup A antigen and rLP2086 subgroup B antigen in an immunogenic composition, the method comprising the steps of storing the rLP2086 subgroup A antigen and rLP2086 subgroup B antigen in a buffer comprising a high concentration of aluminum and a low molar ratio of detergent to protein.

Immunogenic composition

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 US 20060257413 and US20090202593, both of which are incorporated herein by reference in their entirety. Such immunogenic compositions described therein comprise a protein exhibiting bactericidal activity, referred to as an ORF2086 protein, an immunogenic portion thereof, and/or a biological equivalent thereof. The ORF2086 protein refers to a protein encoded by open-label region 2086 of a Neisseria species.

The protein can be a recombinant protein or a protein isolated from a natural Neisseria species. For example, the Neisseria ORF2086 protein can 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, and Neisseria lactamica, as well as immunogenic portions and/or biological equivalents of the protein.

ORF2086 proteins include 2086 subfamily A proteins and subfamily B proteins, immunogenic portions thereof, and/or biological equivalents thereof. ORF2086 proteins or their equivalents may be lipidated or non-lipidated. Preferably, the Neisseria 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 Neisseria ORF2086.

In one embodiment, the immunogenic composition comprises an isolated protein having at least 95% amino acid sequence identity to a subgroup A protein encoded by a nucleotide sequence from Neisseria ORF2086. Preferably, the immunogenic composition comprises an isolated subgroup A protein encoded by a nucleotide sequence from Neisseria ORF2086.

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

In another embodiment, the immunogenic composition comprises an isolated protein having at least 95% amino acid sequence identity to a subgroup A protein encoded by a nucleotide sequence from Neisseria ORF2086, and an isolated protein having at least 95% amino acid sequence identity to a subgroup B protein encoded by a nucleotide sequence from Neisseria ORF2086. Preferably, the immunogenic composition comprises a subgroup A protein encoded by a nucleotide sequence from Neisseria ORF2086 and a subgroup B protein encoded by a nucleotide sequence from Neisseria ORF2086.

In one embodiment, the immunogenic composition comprises subgroup A proteins to subgroup B proteins in a 1:1 ratio.

The immunogenic composition may comprise a protein encoded by a nucleotide sequence from Neisseria ORF2086, a polynucleotide, or its equivalent, as the sole active immunogen in the immunogenic composition. Alternatively, the immunogenic composition may further comprise active immunogens, including immunogenic polypeptides from other Neisseria species or immunologically active proteins or capsular polysaccharides from one or more other microbial pathogens (such as, but not limited to, viruses, prions, bacteria, or fungi). The composition may comprise one or more desired proteins, fragments, or pharmaceutical compounds, as needed for the selected indication.

The present invention includes any multi-antigen or multivalent immunogenic composition. For example, the immunogenic composition can include a combination of two or more ORF2086 proteins, a combination of ORF2086 protein and one or more Por A proteins, a combination of ORF2086 protein and meningococcal serogroups A, C, Y and W135 polysaccharides and/or polysaccharide conjugates, a combination of ORF2086 protein and meningococcal and pneumococcal combinations, or a combination of any of the foregoing combinations in a form suitable for desired administration (e.g., for mucosal delivery). Those skilled in the art can readily formulate such multi-antigen or multivalent immunogenic compositions.

The present invention also includes multiple immunization protocols in which any composition for combating pathogens can be combined therein or in combination with the compositions of the present invention. For example, but not limitation, an immunogenic composition of the present invention and another immunological composition for immunizing against human papillomavirus (HPV) (e.g., an HPV vaccine) can be administered to a patient as part of a multiple immunization protocol. One skilled in the art can readily select immunogenic compositions for use in combination with the immunogenic compositions of the present invention to develop and implement multiple immunization protocols.

ORF2086 polypeptides, fragments, and equivalents can be used as part of a conjugated immunogenic composition, in which one or more proteins or polypeptides are conjugated to a carrier to generate a composition immunogenic against multiple serotypes and/or multiple 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 composition of the present invention preferably comprises 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.

Pharmaceutically acceptable carriers may also contain small amounts of auxiliary substances, such as wetting agents, emulsifiers, preservatives, or buffers, which may extend the shelf life or effectiveness of the antibody. The preparation and use of pharmaceutically acceptable carriers are well known in the art. Unless any conventional media or agents are incompatible with the active ingredient, they are contemplated for use in the immunogenic compositions of the present invention.

The immunogenic composition can be administered parenterally (e.g., by injection (subcutaneous or intramuscular)) as well as orally or intranasally. Methods for intramuscular immunization are described in Wolff et al. Biotechniques ; 11(4):474-85, (1991) and Sedegah et al. PNAS Vol.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 commonly used excipients as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc., but are not limited thereto. Preferably, the immunogenic composition is administered intramuscularly.

The immunogenic compositions of the present invention may include one or more adjuvants. Exemplary adjuvants include, but are not limited to, aluminum hydroxide; aluminum phosphate; STIMULON ^™ QS-21 (Aquila Biopharmaceuticals, Inc., Framingham, Mass.); MPL ^™ (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 (theronyl)-D-isoglutamine (thr-MDP); N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP); 11637, referred to as n-MDP); N-acetylmuramoyl-L-alanyl-D-isoglutamyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy-ethylamine) (CGP 19835A, referred to as MTP-PE); and cholera toxin. In certain preferred embodiments, the adjuvant is QS-21.

Other exemplary adjuvants include non-toxic derivatives of cholera toxin, including its A subunit, and/or conjugates or genetically engineered fusions of Neisseria meningitidis polypeptides with cholera toxin or its B subunit ("CTB"), procholeragenoids, fungal polysaccharides (including schizophyllans), muramyl dipeptides, muramyl dipeptide ("MDP") derivatives, phorbol esters, heat-labile toxin of Escherichia coli (E. coli), block polymers, or saponins.

Aluminum phosphate has been used as an adjuvant in phase 1 clinical trials at a concentration of 0.125 mg/dose, well below the limit of 0.85 mg/dose specified in the US Code of Federal Regulations [610.15(a)]. Aluminum-containing adjuvants are widely used in humans to enhance the immune response to antigens when administered intramuscularly or subcutaneously.

In certain preferred embodiments, the proteins of the present invention are used in immunogenic compositions comprising a mucosal adjuvant for oral administration and for treating or preventing Neisseria meningitidis infection in a human host. The mucosal adjuvant may be cholera toxin, but preferably, other mucosal adjuvants other 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 can be particularly used to prepare the immunogenic compositions of the present invention, see the mutant cholera holotoxin E29H disclosed in published international application WO 00/18434 (incorporated herein by reference in its entirety). These substances can be added to or conjugated to the polypeptides of the present invention. The same technology can be applied to other molecules with mucosal adjuvant or delivery properties, such as Escherichia coli heat-labile toxin (LT). Other compounds with mucosal adjuvant or delivery activity can 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 the mucosal surface. Other compounds with mucosal activity include derivatives of microbial structures, such as MDP, acridine, and cimetidine. As described above, STIMULON ^™ QS-21, MPL, and IL-12 can also be used.

The immunogenic compositions of the present invention can be delivered in the form of ISCOMS (immune stimulating complexes), ISCOMS containing CTB, liposomes, or encapsulated in compounds such as acrylates or poly (DL-lactide-glycolide) to form microspheres of a size suitable for absorption. The proteins of the present invention can also be incorporated into oily emulsions.

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

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

In vitro potency testing

Potency is determined by quantifying functional epitopes in subgroup A and subgroup B proteins in the immunogenic composition using conformationally specific monoclonal antibodies directed against an rLP2086 reference material. Potency is determined by quantitatively measuring functional epitopes in subgroup A or subgroup Br LP2086 proteins that elicit an immune response in vivo, thereby producing bactericidal antibodies. Quantitative techniques are employed for potency testing using selected monoclonal antibodies (mAbs). For each subgroup rLP2086 protein in the immunogenic composition, two functional monoclonal antibodies are selected that are conformationally and non-overlapping. In each of the two purified monoclonal antibodies, the first antibody is conjugated to a first label, wherein the first label is used to capture rLP2086 protein molecules. In some embodiments, the first label is biotin, glutathione-S transferase (GST), a 6×His tag, or beads (e.g., carboxylated polystyrene beads or paramagnetic beads). In some embodiments, the first label is captured using streptavidin beads, a streptavidin column, nickel beads, a nickel column, by centrifugation, or using 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 by electrochemiluminescence, fluorescence detection, or radioactive detection using streptavidin conjugated to a fluorophore or HRP. Only proteins that exhibit two epitopes recognized by the two monoclonal antibodies in each immunogenic composition are measured. This reflects changes in either or both epitopes of the protein. The effectiveness of the sample is reported relative to the effectiveness of the reference substance.

In some embodiments, the present invention includes a method for determining the potency of a 2086 protein. In some embodiments, the method comprises the following steps: (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, the second mAb is conjugated to a detectable second label, and wherein the first and second mAbs are directed against different conformational epitopes on a 2086 reference protein; (2) using the first label to capture the 2086 protein bound to the first mAb; and (3) using the second label to detect and quantify the amount of captured 2086 protein bound to the second mAb. 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 label is biotin, 6xHis label or beads (such as carboxylated polystyrene beads or paramagnetic beads). In some embodiments, the first label is captured by centrifugation or magnetic field with streptavidin beads, streptavidin columns, glutathione columns, nickel beads, nickel columns. In some embodiments, the second label is biotin, HRP, fluorophore or radioactive label. In some embodiments, the second label is detected by electrochemiluminescence, fluorescence detection or radioactive detection with streptavidin conjugated to fluorophore or HRP. In some embodiments, the immunogenic composition comprises a plurality of 2086 protein variants.

Stability of rLP2086 subgroup B antigen potency

In some embodiments, the present invention provides immunogenic compositions for stabilizing rLP2086 subgroup B antigens over time, the compositions comprising a buffer having 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. 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 polyvalent cation. In some embodiments, the polyvalent cation is calcium or aluminum. In some embodiments, the aluminum is present in the form of one or more of AlPO ₄ , Al(OH) ₃ , Al ₂ (SO ₄ ) ₃ , and alum. In some embodiments, the immunogenic composition comprises aluminum in an amount of about 0.1 mg/mL to about 1 mg/mL, about 0.25 mg/mL to about 0.75 mg/mL, or about 0.4 mg/mL to about 0.6 mg/mL. In some embodiments, the immunogenic composition comprises about 0.1 mg/mL, about 0.15 mg/mL, about 0.2 mg/mL, about 0.25 mg/mL, about 0.3 mg/mL, about 0.35 mg/mL, about 0.4 mg/mL, about 0.45 mg/mL, about 0.5 mg/mL, about 0.55 mg/mL, about 0.6 mg/mL, about 0.65 mg/mL, about 0.7 mg/mL, about 0.75 mg/mL, about 0.8 mg/mL, about 0.85 mg/mL, about 0.9 mg/mL, about 0.95 mg/mL, or about 1 mg/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 containing histidine. In some embodiments, the concentration of histidine is about 2mM to about 20mM, about 5mM to about 15mM, or about 8mM to 12mM. 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 20mM.

In some embodiments, the immunogenic composition further comprises a buffer containing succinate. In some embodiments, the concentration of succinate is about 2mM to about 20mM, about 2mM to about 10mM or about 3mM to 7mM. 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 20mM.

In some embodiments, the pH of the immunogenic composition is about 5.0 to about 8.0, about 5.5 to about 7.0, or 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 10 mM histidine-buffered saline, pH 6.0, containing 0.5 mg/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 5 mM succinate buffered saline, pH 6.0, containing 0.5 mg/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 for stabilizing an rLP2086 subgroup 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 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 polyvalent cation. In some embodiments, the polyvalent cation is calcium or aluminum. In some embodiments, the aluminum is present in the form of one or more of AlPO ₄ , Al(OH) ₃ , Al ₂ (SO ₄ ) ₃ , and alum. In some embodiments, the stabilizer in the buffer is aluminum at about 0.1 mg/mL to about 1 mg/mL, about 0.25 mg/mL to about 0.75 mg/mL, or about 0.4 mg/mL to about 0.6 mg/mL. In some embodiments, the stabilizer in the buffer is about 0.1 mg/mL, about 0.15 mg/mL, about 0.2 mg/mL, about 0.25 mg/mL, about 0.3 mg/mL, about 0.35 mg/mL, about 0.4 mg/mL, about 0.45 mg/mL, about 0.5 mg/mL, about 0.55 mg/mL, about 0.6 mg/mL, about 0.65 mg/mL, about 0.7 mg/mL, about 0.75 mg/mL, about 0.8 mg/mL, about 0.85 mg/mL, 0.9 mg/mL, about 0.95 mg/mL, or about 1 mg/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 12mM. 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 20mM.

In some embodiments, buffer also comprises succinate.In some embodiments, the concentration of succinate is about 2mM to about 20mM, about 2mM to about 10mM or about 3mM to 7mM.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 20mM.

In some embodiments, the pH of the buffer is about 5.0 to about 8.0, about 5.5 to about 7.0, or 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 10 mM histidine-buffered saline, pH 6.0, containing 0.5 mg/mL aluminum in the form of aluminum phosphate and polysorbate 80 at a molar ratio of 2.8 to protein.

In some embodiments, the buffer in which the MnB rLP2086 subfamily B protein antigen is stored is 5 mM succinate buffered saline, pH 6.0, containing 0.5 mg/mL aluminum in the form of aluminum phosphate and polysorbate 80 at a molar ratio to protein of 2.8.

Stability of rLP2086 subgroup A and B antigen potency

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 having a high stabilizer concentration and 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. 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 polyvalent cation. In some embodiments, the polyvalent cation is calcium or aluminum. In some embodiments, the aluminum is present in the form of one or more of AlPO ₄ , Al(OH) ₃ , Al ₂ (SO ₄ ) ₃ , and alum. In some embodiments, the immunogenic composition comprises aluminum in an amount of about 0.1 mg/mL to about 1 mg/mL, about 0.25 mg/mL to about 0.75 mg/mL, or about 0.4 mg/mL to about 0.6 mg/mL. In some embodiments, the immunogenic composition comprises about 0.1 mg/mL, about 0.15 mg/mL, about 0.2 mg/mL, about 0.25 mg/mL, about 0.3 mg/mL, about 0.35 mg/mL, about 0.4 mg/mL, about 0.45 mg/mL, about 0.5 mg/mL, about 0.55 mg/mL, about 0.6 mg/mL, about 0.65 mg/mL, about 0.7 mg/mL, about 0.75 mg/mL, about 0.8 mg/mL, about 0.85 mg/mL, 0.9 mg/mL, about 0.95 mg/mL, or about 1 mg/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 containing histidine. In some embodiments, the concentration of histidine is about 2mM to about 20mM, about 5mM to about 15mM, or about 8mM to 12mM. 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 20mM.

In some embodiments, the immunogenic composition further comprises a buffer containing succinate. In some embodiments, the concentration of succinate is about 2mM to about 20mM, about 2mM to about 10mM or about 3mM to 7mM. 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 20mM.

In some embodiments, the pH of the immunogenic composition is about 5.0 to about 8.0, about 5.5 to about 7.0, or 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 MnB rLP2086 subfamily A and B protein antigens is 10 mM histidine buffered saline, pH 6.0, containing 0.5 mg/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 5 mM succinate buffered saline, pH 6.0, containing 0.5 mg/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 for stabilizing rLP2086 subfamily A and/or rLP2086 subfamily B antigens over time, comprising storing the antigens 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 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 stabilizer in the buffer is a multivalent cation. In some embodiments, the multivalent cation is calcium or aluminum. In some embodiments, the aluminum is present in the form of one or more of AlPO ₄ , Al(OH) ₃ , Al ₂ (SO ₄ ) ₃ , and alum. In some embodiments, the stabilizer in the buffer is aluminum at about 0.1 mg/mL to about 1 mg/mL, about 0.25 mg/mL to about 0.75 mg/mL, or about 0.4 mg/mL to about 0.6 mg/mL. In some embodiments, the stabilizer in the buffer is about 0.1 mg/mL, about 0.15 mg/mL, about 0.2 mg/mL, about 0.25 mg/mL, about 0.3 mg/mL, about 0.35 mg/mL, about 0.4 mg/mL, about 0.45 mg/mL, about 0.5 mg/mL, about 0.55 mg/mL, about 0.6 mg/mL, about 0.65 mg/mL, about 0.7 mg/mL, about 0.75 mg/mL, about 0.8 mg/mL, about 0.85 mg/mL, 0.9 mg/mL, about 0.95 mg/mL, or about 1 mg/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 12mM. 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 20mM.

In some embodiments, buffer also comprises succinate.In some embodiments, the concentration of succinate is about 2mM to about 20mM, about 2mM to about 10mM or about 3mM to 7mM.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 20mM.

In some embodiments, the pH of the buffer is about 5.0 to about 8.0, about 5.5 to about 7.0, or 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 A and B protein antigens are stored is 10 mM histidine-buffered saline, pH 6.0, containing 0.5 mg/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 A and B protein antigens are stored is 5 mM succinate buffered saline, pH 6.0, containing 0.5 mg/mL aluminum in the form of aluminum phosphate and polysorbate 80 at a molar ratio to protein of 2.8.

In order to better understand the present invention, the following examples are given. These examples are only for illustrative purposes and should not be construed as limiting the scope of the present invention.

All references cited herein are hereby incorporated by reference.

Example

Example 1 : Experimental operation

Determination of aluminum binding

The composition comprising aluminum and at least one protein antigen is centrifuged so that the aluminum is pelleted. Centrifugation of proteins that absorb aluminum is known in the art. See, for example, Egan et al., Vaccine , Vol. 27(24): 3175-3180 (2009). Aluminum-bound proteins also precipitate, but non-aluminum-bound proteins remain in the supernatant. The total protein in the supernatant and the pellet is determined by Lowry's test. The percentage of bound protein is calculated by dividing the total protein in the supernatant by the total protein added to the composition and multiplying by 100%. Similarly, the percentage of unbound protein is calculated by dividing the total protein in the supernatant by the total protein added to the composition and multiplying by 100%.

For compositions containing both subgroup A and subgroup B antigens, the concentration of each subgroup A and B protein in the supernatant was determined by ion exchange chromatography. A strong anion column and a high salt concentration eluent were used to separate and elute the subgroup A and B proteins. A fluorescence detector set to excitation = 280 run and emission = 310 run was used to detect and quantify both subgroup A and B proteins. The subgroup A and subgroup B proteins eluted at different retention times and were quantified using a standard curve 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 testing

The rLP2086 potency test is a homogeneous capture test or sandwich test that relies on two functional monoclonal antibodies that recognize conformational epitopes and non-overlapping epitopes on a single protein molecule of the rLP2086 drug. A purified monoclonal antibody is used as a capture antibody (mAb) that is chemically conjugated to carboxylated polystyrene beads with a unique color-coded identifier. The second antibody is biotinylated and used as a detection antibody that is subsequently bound to streptavidin (SA-PE) conjugated to the fluorophore R-phycoerythrin. The flow control technology of the Bio-Plex detector quantifies each microsphere and its associated SA-PE signal. The fluorescent signal from the R-phycoerythrin associated with the microsphere is detected only by the formation of a ternary complex between the antibody conjugated to the beads, the antigen, and the detection antibody, and is proportional to the number of functional epitopes in the rLP2086 sample. Alterations in one or both epitopes result in a loss of fluorescence relative to the reference standard, which indicates a loss of potency.

Reagents

• Monoclonal antibodies conjugated to microspheres (either to Luminex MicroPlex Microsphere beads region #12 or to beads region #66).

Biotinylated monoclonal antibodies.

rLP2086 reference material, subgroups A and B, 2 mg/ml. Store at -70°C.

rLP2086 subfamily A and B bivalent control

Lyophilized streptavidin conjugated to R-phycoerythrin

buffer

10 mM histidine, 150 mM NaCl, pH 6.0

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

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

• Assay buffer (PBS, pH 7.4, containing 0.1% BSA, 0.02% polysorbate 80, 0.1% azide).

100× Streptavidin conjugated to R-phycoerythrin (SA-PE) – Open the vial containing lyophilized streptavidin and R-phycoerythrin and add 1 mL of distilled water. Vortex until completely dissolved.

operate

200 μL of subgroup A protein and 200 μL of subgroup B protein were added to 600 μL of matrix buffer to give a concentration of 400 μg/ml for each subgroup. A standard curve of eight concentrations (3333-1.5 ng/mL) was generated by diluting this stock solution in assay buffer.

Add 200 μL of bivalent control to 800 μL of matrix buffer to a concentration of 400 μg/mL for each subfamily. This 400 μg/mL stock solution, used to prepare working concentrations of 100 ng/mL, 50 ng/mL, and 12.5 ng/mL, was diluted in assay buffer. 100 ng/mL and 12.5 ng/mL represent high (CH) and low (CH), controls, respectively.

The test sample was diluted in matrix buffer to a concentration of 400 μg/mL. Working solutions of 100 ng/mL, 50 ng/mL, and 12.5 ng/mL were prepared from the 400 μg/mL stock solution.

A homogeneous test mixture was prepared in assay buffer using a conjugated bead concentration of 2 x 10 ⁵ beads/mL and a detection antibody concentration of 30 μg/mL. A sample plate was prepared by adding 0.4 mL of standard, control, sample, or blank to a 2 mL volume of a 96-well deep-well plate. The filter of a 96-well MultiScreen _HTS -BV filter plate was pre-wetted by adding 100 μL of assay buffer and then removed by vacuum aspiration. 25 μL of the prepared homogeneous test mixture was added to the 96-well plate. 25 μL of each standard, control, sample, or blank solution was added to each well of the 96-well filter plate. The plate was incubated with shaking at room temperature for 1 hour.

After the antigen-antibody incubation, the buffer solution was removed by vacuum suction through the filter. The filter in each well was washed three times with 100 μL of test buffer solution and then vacuum suction was performed. After the final wash, 50 μL of 1x SA-PE was added to each well. The plate was incubated for 10 minutes in a titer plate shaker at room temperature in the dark.

Following the SA-PE incubation, 75 μL of assay buffer was added to each well of the plate, bringing the total volume to 125 μL. The plate was immediately read on a Bio-Plex 200 system.

Serum bactericidal test

Female New Zealand white rabbits (2.5–3.0 kg) obtained from Charles River Canada (St. Constant, QC, Canada) were prescreened by whole-cell ELISA to identify those with low reactivity to two different meningococcal strains (one from each P2086 subfamily). Rabbits generally have very low background, and those with the lowest values were selected for use. Rabbits were vaccinated intramuscularly at weeks 0, 4, and 9 with either monovalent rLP2086-A05, monovalent rLP2086-B01, or bivalent rLP2086-A05+B01 vaccines. Each dose of the monovalent vaccine contained 100 μg of protein, and each dose of the bivalent vaccine contained 100 μg of each protein, formulated in 10 mM histidine buffer, pH 6.0, 150 mM NaCl, 0.02% polysorbate 80, and 250 μg AlPO ₄ . The vaccine was injected intramuscularly into the right hind limb (0.5 ml/dose). As a control, one group of rabbits was vaccinated with formulation buffer alone. Pre-immune (week 0) and immune (week 10) serum samples were obtained for analysis. All animal manipulations were performed in accordance with the established Laboratory Animal Care and Use Committee guidelines.

Serum bactericidal antibodies in rabbits immunized with the rLP2086 vaccine were determined using SBA and human complement. Rabbit immune serum was heat-inactivated to remove inherent complement activity and subsequently serially diluted in 96-well microtiter plates at 1:2 in Dulbecco's PBS (D-PBS) containing Ca ²⁺ and ^{Mg 2+} to detect serum bactericidal activity against Neisseria meningitidis strains. The bacteria used in this test were grown in GC culture medium (GCK) supplemented with Kellogg's supplements and monitored by optical density at 650nm. Bacteria with a final OD ₆₅₀ of 0.50-0.55 were collected for use in this test, diluted in D-PBS, and 1000-3000 CFU were added to a test mixture containing 20% human complement.

Human serum without detectable bactericidal activity was used as an exogenous complement source. The complement source was tested for suitability with each test strain. A complement source was used only if the number of bacteria surviving the control without added immune serum was >75%. Ten unique complement sources were required to perform the SBA described in this study.

After incubation at 37°C with 5% _CO2 for 30 minutes, D-PBS was added to the reaction mixture and aliquots were transferred to microfiltration plates filled with 50% GCK medium. The microfiltration plates were filtered, incubated overnight at 37°C with 5% _CO2 , and the microcolonies were stained and quantified. The serum bactericidal titer was defined as the interpolated reciprocal serum dilution that reduced the CFU by 50% compared to the CFU in the control wells without immune serum. The SBA titer was defined as the interpolated reciprocal serum dilution that caused a 50% reduction in bacterial counts after incubation at 37°C for 30 minutes. Sensitivity to killing by the P2086 immune serum was determined if the SBA titer of the P2086 immune serum had a 4-fold or greater increase relative to the corresponding pre-immune serum. The detection limit for rabbit serum was a titer of 8. Sera that were inactive against the test strain at the initial dilution were assigned a titer of half the detection limit of the test (ie, 4 for rabbit).

Flow cytometry

MnB cells were grown to an _OD650 of 0.45-0.55 and then fixed in 1% (v/v) paraformaldehyde in 1× PBS for 10 minutes. One hundred microliters of bacteria per well were inoculated into a 96-well U-bottom polystyrene plate, centrifuged, and washed once with 1% (w/v) BSA in 1× PBS. Anti-LP2086 monoclonal antibody was added to the bacterial pellet, resuspended, and incubated on ice for 30 minutes. After washing twice in 1% BSA/PBS, biotinylated goat anti-mouse IgG (subclasses 1+2a+2b+3) (Jackson Immunoresearch) was added to the cell pellet, resuspended, and incubated on ice for 30 minutes. The 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 included as a negative control. Twenty thousand (20,000) events/well were acquired on a BD LSRII flow cytometer and analyzed using FlowJo v7 software (Treestar, Ashland, Oregon). After gating bacterial cells in a dot plot of log FSC vs. SSC, the mean fluorescence intensity (MFI) of the PE channel was determined for each sample. An MFI was considered positive if it was three times the MFI of control mouse IgG.

Example 2: Binding of Polysorbate 80 to rLP2086 Protein

To understand the stability of polysorbate 80 binding to each of the rLP2086 proteins A and B, one rLP2086 sample was formulated with 200 μg/mL subgroup A and aluminum (Al), and another rLP2086 sample was formulated with 200 μg/mL subgroup B. Both samples were stored at 2-8°C and 25°C, and the protein and polysorbate 80 content of both samples were measured after five months. A placebo (buffer + Al, no protein) was also analyzed. The distribution of polysorbate 80 in the placebo is shown in Figure 14, while the distribution of polysorbate 80 for subgroup A and B proteins is shown in Figures 15 and 16, respectively. The relative potency (%) of subgroup B is compared to the binding molar ratio in Figure 17.

result

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 was not bound to aluminum or trapped in the precipitate. In addition, polysorbate 80 was stable at 2-8°C and 25°C after five months.

The distribution of polysorbate 80 in bound (precipitate), unbound (supernatant), and total rLP2086 subfamily A and subfamily B samples is shown in Figures 15 and 16, respectively. For subfamily A, the percentage of polysorbate 80 in the supernatant and precipitate did not change at the five-month time point at 2-8°C and 25°C. However, for subfamily B samples, more polysorbate 80 was observed in the precipitate at 25°C at the five-month time point. Despite the different concentrations of polysorbate 80 in the supernatant and precipitate at 2-8°C and 25°C, both subfamilies achieved a precise mass balance. Since the rLP2086 protein is 100% bound to the aluminum phosphate in this matrix, the polysorbate 80 associated with the precipitate is most likely bound to the protein molecule.

Although both proteins A and B bound to polysorbate 80, protein A binding was identical for samples stored at 2-8°C and 25°C, whereas protein B binding was nearly double that of samples stored at 25°C. The relative potency of subfamily B was measured at _T0 and five months at 2-8°C and 25°C, and the relative potency behavior was inversely proportional to the molar ratio of binding, as shown in Figure 17. The percent potency decreased from 120 at _T0 to 16% at 5M/25°C, while the molar ratio of binding increased from 5.3 to 13.9 over the same time period.

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 subfamily A only, subfamily B only, and both subfamily A and B (200 μg/mL and 400 μg/mL) with varying polysorbate 80 concentrations, as shown in Table 1. Total protein and bound protein were determined for each sample, as well as the percentage of polysorbate 80 in the whole sample, supernatant, and pellet at time points zero (T ₀ ), 14 days, and 1 month at 2-8° C. and 25° C. The results of this study are shown in Figures 18 to 24 .

Table 1

result

The concentrations of polysorbate 80 in the supernatant, pellet, and whole sample of all 40 rLP2086 formulations containing aluminum phosphate were determined. The total and bound molar ratios for both subfamilies A and B were determined. For 200 μg/mL of polysorbate 80 containing 0.005% (molar ratio of 5.4) or less, the molar ratios appeared similar for both subfamilies, as shown in Figures 18 and 19, respectively. However, for samples containing 0.0065% (molar ratio of 7.0) or more of polysorbate 80, the total molar ratio of subfamilies B was much higher than the bound molar ratio. For formulations containing 0.008% (molar ratio 8.6) or less of polysorbate 80, the data for the total molar ratio and the combined molar ratio of subfamily A, subfamily B, and subfamily A+B at 400 μg/mL each were also very similar. However, for formulations containing 0.017% (molar ratio 18.4) of polysorbate 80, the total molar ratio was much higher than the combined molar ratio, as shown in Figure 20.

Example 4 : Polysorbate 80 Binding over Time

The percentage (%) of polysorbate 80 in the supernatant and precipitate of samples of subfamily A and B formulations containing AlPO ₄ was measured at T ₀ , 14 days/25°C, 1 month/4°C, and 1 month/25°C. For samples stored at 2-8°C, the percentage of polysorbate 80 in the supernatant was relatively the same for both subfamily A and B formulations. However, for samples stored at 25°C, the percentage of polysorbate 80 in the supernatant decreased dramatically even after only 14 days. At T ₀ /5°C and 1 month/5°C, the percentage of polysorbate 80 in the precipitate was relatively similar for both subfamily A and B. However, for samples stored at 25°C, particularly for subfamily B containing 0.008% (8.6 molar ratio) or more of polysorbate 80, the percentage of polysorbate 80 in the supernatant increased significantly. The percentage of polysorbate 80 in the supernatant and pellet of rLP2086 subfamily A and B formulations containing _AlPO4 was also measured at _T0 , 14 days at 25°C, 1 month at 4°C, and 1 month at 25°C. As shown in Figure 21, for samples containing 0.008% polysorbate 80, the concentration of polysorbate 80 was approximately the same at all four time points. However, for samples containing 0.017% polysorbate 80 and stored at 25°C, the concentration of polysorbate 80 increased. No polysorbate 80 was found in the supernatant of samples containing 0.008% or less polysorbate 80. As shown in Figure 22, the bound molar ratio was stable at all four time points for samples containing 0.008% or less polysorbate 80. However, the bound molar ratio increased for samples containing 0.017% polysorbate 80 and stored at 25°C.

The efficacy of the formulation samples of subfamily A and B containing AlPO ₄ was determined at T ₀ and 14 days/25°C (see Figures 23 and 25, respectively). As shown in Figure 23, the efficacy of subfamily A at different total molar ratios at 5°C and 25°C ranged from 91 to 102. Although the combined molar ratio results were relatively the same at either temperature, a slight increase in efficacy was observed as the total molar ratio/combined molar ratio increased.

For 5°C samples of Subfamily B with a total molar ratio as high as 9.0, the potency was approximately 95%. However, as the total molar ratio increased to 18.1, the potency of Subfamily B decreased to 79%. Additionally, the sample with a total molar ratio of 18.1 had a higher bound molar ratio than the other samples. At 25°C, the potency of Subfamily B showed a significant decrease from 83% to 5% as the total molar ratio increased from 5.3 to 18.1. As the total molar ratio increased, the bound molar ratio values for the 25°C samples increased from 5.3 to 13.8. Therefore, the potency of Subfamily B is inversely proportional to the bound molar ratio.

Both subfamily A and subfamily B proteins bind to polysorbate 80. Subfamily A binding is the same for samples stored at 2-8°C and 25°C, but subfamily B binding is almost twice as high for samples stored at 25°C. In addition, critical molar ratio studies indicate that 200 μg/mL formulation samples are stable when containing 0.008% or less polysorbate 80 (equivalent to a total molar ratio of 4.2 or less).

Example 5: Detergent Concentration and rLP2086 Subgroup B Antigen Potency

Another stability study using varying concentrations of polysorbate 80 demonstrated the criticality of the polysorbate 80 to protein molar ratio for maintaining potency. In one experiment, a 200 μg dose of immunogenic composition was prepared in 10 mM histidine-buffered saline (HBS) containing 0.5 mg/mL aluminum (as aluminum phosphate) at pH 6.3, with a total protein concentration of 400 μg/mL, and spiked with 0.01%, 0.02%, 0.05%, or 0.1% polysorbate 80 (corresponding molar ratios of polysorbate 80 to rLP2086 protein of 5.3, 10.7, 26.7, and 53.4). The formulated samples were incubated at 25°C, and control samples were stored at 2-8°C. At time "0," there was no significant change in potency at polysorbate 80 concentrations up to 0.1%. However, at 2-8°C and 25°C for extended periods, a decrease in potency was observed as a function of temperature and concentration of polysorbate 80. As the concentration of polysorbate 80 in the immunogenic composition was increased from 0.01% to 0.1%, the three-month plateau demonstrated that the potency of the subgroup B proteins decreased to less than 10% and 25% at 25°C and 2-8°C, respectively ( FIG1 ).

Another stability study ( FIG2 ) was conducted to evaluate the stability of subgroup B proteins in HBS at a concentration of 4 mg/mL and spiked with polysorbate 80 at final concentrations of 0.06%, 0.5%, and 1% (respectively, molar ratios of 3.3, 26.7, and 53.4). The control contained 0.09% polysorbate 80. Subgroup B proteins were stable in 0.06% polysorbate 80 (molar ratio of 3.3). The same samples containing polysorbate 80 at concentrations increasing to 0.5% and 1% (molar ratios of 26.7 and 53.4, respectively) were unstable. For the 400 μg/mL immunogenic composition formulation, instability of subgroup B proteins was observed in all formulations containing polysorbate 80 concentrations of 0.01% (molar ratio of 5.3) or higher. However, at a concentration of 4 mg/mL protein and 0.06% polysorbate 80, there was no decrease in potency because the molar ratio of polysorbate 80 to protein (3.3) was lower than the molar ratio (5.3) at a concentration of 400 μg/mL protein + 0.01% polysorbate 80. Therefore, the decrease in potency of subgroup B proteins caused by polysorbate 80 was related to the molar ratio of polysorbate 80 detergent to protein, rather than 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 subfamily B protein in the vaccine and during subsequent storage at 2-8°C. A 28-day accelerated stability study (Fig. 3 and Fig. 4) was designed for immunogenic compositions containing polysorbate 80 (0, 1.1, 2.7, and 5.3) at a dose of 20 μg and 200 μg. Bivalent (subfamily A and subfamily B) formulations were prepared in histidine buffered saline with 10 mM pH 6.0, 0.5 mg/mL aluminum in the form of aluminum phosphate with different polysorbate 80 concentrations. The samples were incubated at 25°C together with the 2-8°C control group. The efficacy of the samples was analyzed on day 0, 7, 14, and 28. For all groups containing polysorbate 80 with a molar ratio to protein less than 5.3, both subfamily A (data not shown) and B proteins were stable. Efficacy values greater than 80% were considered to be within the test variability range. At a molar ratio of 5.3, a trend toward decreased potency of the subgroup B proteins was observed for the 25°C samples.

A comprehensive study evaluated all possible clinical doses (20 μg, 60 μg, 120 μg, and 200 μg doses) formulated at different polysorbate 80 to protein molar ratios under accelerated storage stability conditions to investigate the effect of the polysorbate 80 to protein molar ratio on the stability of the MnB rLP2086 protein. A bivalent MnB rLP2086 immunogenic composition formulated at polysorbate 80 to protein molar ratios of approximately 1.4 to 10.7 was used. To generate immunogenic compositions formulated at increasing polysorbate 80 to protein molar ratios (1.4, 2.4, 3.4, 3.9, 4.3, 4.7, and 10.7), the antigen was adjusted to a variable molar ratio by adding polysorbate 80, eliminating the need for additional polysorbate 80 during immunogenic composition formulation. Two series of antigen batches were used in this study. The subfamily A and B batches of a series with a polysorbate 80 to protein molar ratio of 1.4 are generated, while another series is 2.4. The protein series with a molar ratio of 2.4 is used to adjust the molar ratio to 3.4, 3.9, 4.3 and 10.7 by incorporating other polysorbate 80. The final matrix of the immunogenic composition is 10mM histidine, 150mM NaCl, pH 6.0, 0.5mg/mL aluminum phosphate, and has the molar ratio of the polysorbate 80 and protein mentioned above. After preserving the specific time interval at 2-8 ℃ or 25 ℃, gently mix with a shaker for 24 hours, then test. The total protein (by IEX-HPLC), efficacy, appearance, optical density at 320nm and pH of the test supernatant fraction are measured.

The potency results for the 200 μg and 20 μg doses are shown in Figures 5 and 6, respectively. This potency test is more sensitive than the other assays used in this study. Overall, no significant decrease in potency was observed for either subgroup A or B antigens compared to the initial time point for all doses with a molar ratio of 4.3 and lower. The formulation with a molar ratio of 4.7 was considered borderline due to a slight decrease in potency for the subgroup B proteins stored at 25°C. For samples stored at 25°C, the potency results for the subgroup B antigens for the formulation with a molar ratio of 10.7 were significantly lower than those for samples stored at 2-8°C.

Example 6: Aluminum Concentration and rLP2086 Subgroup A and B Antigen Potency

Numerous experiments were conducted to determine the optimal aluminum phosphate level to ensure greater than 95% binding of both subgroup A and B proteins. Initial studies focused on optimizing formulations at pH 6.5. Formulations with a target dose of 200 μg/mL of each protein were prepared from subgroup A and B proteins in 10 mM histidine buffer (pH 6.5) containing 0.02% polysorbate 80 and either 0.25 mg/mL or 0.5 mg/mL aluminum (as aluminum phosphate). Subgroup B proteins bound to aluminum phosphate to a lesser extent than subgroup A proteins ( FIG7 ). Increasing the aluminum content from 0.25 mg/mL to 0.5 mg/mL increased the binding of subgroup B proteins to >80%. Because the binding mechanism between proteins and aluminum suspensions is primarily ionic, the pH of the suspension is a factor influencing binding.

The formulation pH was optimized to ensure greater than 90-95% binding of the subfamily B proteins. Multiple formulations were examined at 200 μg/mL each of the A and B proteins at pH 5.6 to 6.5, and with different batches of immunogenic composition ( FIG8 ). Greater than 90-95% binding of both proteins occurred in formulations at pH 5.6 to 6.4. Binding of the subfamily B proteins decreased significantly as the pH of the formulation was increased to 6.5 and above. The recommended target pH is 6.0 to ensure greater than 90% binding of the subfamily A and B proteins.

The robustness of the formulation under formulation variables and/or limits was also assessed by varying pH, buffer, protein, and polysorbate 80 concentrations ( FIG. 9 ). Although binding of subfamily A proteins was consistently high (≥95%) up to a total protein concentration of 500 μg/mL (250 μg/mL of each protein), binding of subfamily B proteins was more sensitive to protein concentration and pH. Since a commercial formulation at a dose of 200 μg was used, the results of this experiment further support the recommended formulation of pH 6 containing 0.5 mg/mL aluminum phosphate.

Formulations containing and not containing aluminum phosphate were evaluated to investigate the feasibility of providing such stable formulations, which do not contain aluminum phosphate and at concentrations of polysorbate 80 low enough to maintain the stability of subgroup B proteins. 20 μg and 200 μg of immunogenic compositions were prepared in histidine-buffered saline buffer with a polysorbate 80 concentration ranging from 0 to 5.3 molar ratios. Half of the samples were stirred for 24 hours using a digital multi-tube vortexer set to 500 rpm in pulse mode (on for 2 seconds, off for 1 second) and then tested. This condition was used to simulate the ISTA test (International Safe Transit Association) typically performed at the final transport packaging stage of the immunogenic composition to simulate extreme vibrations during transport conditions.

Upon stirring, the formulations without aluminum phosphate precipitated, ultimately resulting in a loss of potency for both subgroup A and B antigens. Appearance testing ( FIG. 10 ) and absorbance measurements at λ=320 nm ( FIG. 11 ) indicated the formation of aggregates and/or precipitates when the formulations without aluminum phosphate were stirred. Potency testing of these samples ( FIG. 12 and FIG. 13 ) indicated a significant loss of potency for both subgroup A and B proteins at all time points tested. The loss of potency was most pronounced in formulations containing low amounts of polysorbate 80. Because low amounts of polysorbate 80 are necessary to maintain the stability of the subgroup B proteins, aluminum phosphate is required in the formulation to maintain stability. The rLP2086 immunogenic compositions can be formulated with aluminum phosphate, which will serve to enhance potency stability, as measured by in vitro potency testing.

Example 7: Succinate and Histidine as Buffer

A series of formulations were prepared to compare the binding of rLP2086 subfamily A and B proteins in succinate and histidine, as well as the effects of pH, polysorbate 80, and _MgCl2 on binding (Table 2). The stability of the formulations under formulation variables and/or limits was assessed by varying the pH, buffer, protein, and polysorbate 80 concentrations (Figures 25 and 26). Aluminum binding to subfamily A and subfamily B proteins was similar regardless of the buffer used (histidine or succinate).

Table 2: Formulations used to evaluate the effects of histidine and succinate buffers, MgCl ₂ , polysorbate 80, and pH 5.6-6.0 on the binding of rLP2086 to AlPO ₄ ¹

Table 2: Formulations used to evaluate the effects of histidine and succinate buffers, MgCl ₂ , polysorbate 80, and pH 5.6-6.0 on the binding of rLP2086 to AlPO ₄ ¹

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

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

As shown in Figure 27, at pH 6.8, subgroup B proteins exhibited reduced binding in phosphate buffer, while subgroup A proteins were not significantly affected under the same conditions. The amount of protein bound to aluminum was similar in samples formulated with either histidine or succinate. Therefore, these two buffer salts were selected for further evaluation. While not wishing to be bound by theory, the reduced binding in phosphate buffer may be due to competition with the added phosphate ions for binding sites on _AlPO4 .

Under these conditions and concentrations of protein and AlPO ₄ , binding was complete after five minutes of mixing at room temperature, which was 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 pH 6.8 was due to differences in pH or buffer salts, binding was measured in histidine- or succinate-buffered formulations over a pH range of 5.3 to 7.0. Bivalent formulations containing 0.2 mg/mL of each subgroup protein (0.4 mg/mL total protein), 0.02% PS80, 0.5 mg Al/mL, and 150 mM NaCl were prepared. Samples were prepared in either 10 mM histidine or 5 mM succinate to compare the effects of buffer salts. Following preparation, the pH of each sample was individually checked.

The binding properties of subfamily A proteins from pH 5.3 to 7.0 are shown in Figure 28, while those of subfamily B proteins are shown in Figure 29. The amount of bound protein for subfamily A proteins was almost unchanged, and binding was retained over 95% across the pH range tested. Formulations containing histidine and targeting a pH of 7 resulted in a pH of 6.8. This pH was not adjusted to 7.0 (e.g., by adding base) to avoid possible effects on the protein or AlPO ₄ , so the results for this data point are not available.

The binding properties of subgroup B proteins (shown in Figure 29) showed a pH-dependent trend. However, the amount of protein bound to aluminum was similar regardless of whether binding was performed in histidine or succinate buffered formulations. Binding was dependent on the pH of the formulation, not the buffer salt. Binding remained at 95% up to pH 6.5 (94% in histidine and 95% in succinate), but decreased above pH 6.5. At pH 7.0, binding decreased to approximately 82%, with slight differences between the buffer salts.

To obtain strong binding of subgroup B proteins to _AlPO4 at these concentrations, a pH of 6.5 or lower is preferred.

Example 8: Safety, Tolerability and Immunogenicity Studies

The study was conducted to evaluate the safety, tolerability, and immunogenicity of the rLP2086 vaccine in a healthy young adult population according to a schedule of 0 and 2 months; 0, 2, and 6 months; and 0 and 2 months, followed by a booster dose at 12 months.

The immunogenic composition is a rLP2086 vaccine (recombinant and lipidated). The immunogenic composition comprises a recombinant ORF2086 protein of Neisseria meningitidis serogroup B, expressed in Escherichia coli and formulated as a bivalent vaccine comprising a subgroup A strain and a subgroup B strain of rLP2086. Specifically, the dose of the immunogenic composition is 0.5 mL, which is formulated to contain 60 μg, 120 μg, or 200 μg of each purified subgroup A and purified subgroup B rLP2086 protein, polysorbate 80 at a molar ratio of 2.8, and 0.25 mg of Al ³⁺ (in the form of AlPO ₄ ), 10 mM histidine buffered saline (pH 6.0). The control composition comprises a 0.5 mL dose of physiological saline solution (0.9% sodium chloride).

The subjects were randomly assigned to 5 groups. See Table 3. The subjects were divided into two age groups: ≥11 years to <14 years; and ≥14 years to <19 years.

Saline was used as a placebo because there is no proven safe, immunogenic, and efficacious 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). Following standard vaccination practices, the vaccine was not injected into a blood vessel. The rLP2086 vaccine was administered by intramuscular injection of 0.5 mL into the superior deltoid muscle. Saline was administered intramuscularly into the superior deltoid muscle.

A. First visit

At Visit 1, Day 1, Vaccination 1, blood was drawn from the individual prior to vaccination. The Visit 1 blood draw and vaccination 1 occurred on the same day. Prior to vaccination, a blood sample (approximately 20 mL) was collected from the individual. For individuals randomized to Groups 1, 2, 3, and 4, a single 0.5 mL intramuscular injection of rLP2086 vaccine was administered into the superior deltoid muscle. For individuals in Group 5, a single 0.5 mL intramuscular injection of saline was administered into the superior deltoid muscle.

B. Second visit (42 to 70 days after the first visit), second vaccination

A single 0.5 mL intramuscular injection of rLP2086 vaccine was administered to the superior deltoid muscle for Groups 1, 2, and 3. A single 0.5 mL intramuscular injection of saline was administered to the superior deltoid muscle for Groups 4 and 5.

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

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

D. Visit 4 (105 to 126 days after visit 2), 3rd vaccination

A single 0.5 mL intramuscular injection of rLP2086 vaccine was administered to the superior deltoid muscle for Groups 2, 4, and 5. A single 0.5 mL intramuscular injection of saline was administered to the superior deltoid muscle for Groups 1 and 3.

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

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

F. Visit 6 (161 to 175 days after visit 4), 4th vaccination

At visit 6, blood was drawn from the individual and then the vaccination was performed. The blood draw for visit 6 and the fourth vaccination occurred on the same day. Before vaccination, a blood sample (approximately 20 mL) was collected from the individual. For groups 3 and 5, a single 0.5 mL intramuscular injection of rLP2086 vaccine was administered to the superior deltoid muscle. For groups 1, 2, and 4, a single 0.5 mL intramuscular injection of saline was administered to the superior deltoid muscle.

G. Visit 7 (28 to 42 days after Visit 6), blood draw after 4th vaccination

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

Immunogenicity results

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

A secondary objective of this study was to evaluate the immunogenicity of 60 μg, 120 μg, and 200 μg rLP2086 vaccine by quantifying Ig bound to rLP2086 vaccine subfamily A and B proteins.

SBA activity was assessed using three subfamily A strains and three subfamily B strains, as shown in Table 4. Table 4: Analysis of individuals with a ≥ 4-fold increase in SBA titer prior to the first dose (mITT population) (Study 6108A1-2001-WW/B1971005)

Table 4: Analysis of Subjects with a ≥ 4-fold Rise in SBA Titer Before Dose 1 (mITT Population) (Study 6108A1-2001-WW/B1971005)

Abbreviations: CI = confidence interval; SBA = serum bactericidal assay.

Note: The assay supports a lower limit of quantification (LLQQ) of 1 = 9 for subgroup A strains and 1 = 10 for subgroup B strains. SBA titers above the LLQQ were considered accurate and reported as such. Values below the LLQQ or indicated as below the LLQQ were set to 0.5*LLOQ for analysis.

The proportion of subjects reaching the prescribed titer level is given in Table 5. For both subpopulations, the proportion of subjects reaching the prescribed SBA titer level was higher after the third dose than after the second dose.

Immunogenicity data demonstrated that the vaccine elicited antibodies with significant SBA activity against both subgroup A and subgroup B strains of MnB. For subgroup A strain 2, SBA response rates ranged from 88.9% to 90.9% after the second dose and from 90.0% to 97.4% after the third dose. For the subgroup A strain 1 variant, 100.0% of individuals had an SBA response to this variant at both the 60 μg and 120 μg dose levels after the second and third doses. At the 200 μg dose level, 96.5% and 99.0% of individuals had an SBA response after the second and third doses, respectively. For the subgroup A strain 1 variant, SBA response rates ranged from 85.0% to 96.3% after the second dose and from 95.2% to 97.4% after the third dose.

For subgroup B strain 1 variants, SBA response rates ranged from 76.2% to 81.0% after the second dose and from 89.5% to 92.0% after the third dose. For subgroup B strain 2 variants, the percentage of individuals with an SBA response rate after the second dose ranged from 21.1% to 33.3%. However, after the third dose, 53.3%, 75.6%, and 67.9% of individuals had an SBA response at the 60 μg, 120 μg, and 200 μg dose levels, respectively. For subgroup B strain 3 variants, SBA response rates ranged from 61.9% to 70.8% after the second dose and from 76.2% to 88.7% after the third dose.

Overall, a high proportion of individuals responded with SBA titers ≥ LLOQ, regardless of whether subgroup A or subgroup B strains were tested. hSBA data demonstrated robust immune responses at doses ranging from 60 μg to 200 μg, but without a clear dose-response relationship. Regardless of the assay tested, the frequency of responses was numerically highest in the 120 μg group. The 200 μg group did not exhibit an enhanced immune response above the 120 μg dose level.

Claims (7)

1. A method for stabilizing the potency of a Neisseria meningitidis serogroup BLP2086 subgroup B peptide in an immunogenic composition, the method comprising step (a): formulating the LP2086 subgroup B peptide with a Neisseria meningitidis serogroup B LP2086 subgroup A peptide, polysorbate 80, aluminum in the form of _AlPO4 , and histidine to form a composition, wherein the composition comprises polysorbate 80 in a molar ratio of 2.8:1 to protein, 0.5 mg/mL aluminum in the form of AlPO4, ₁₀ mM histidine at pH 6.0, and 150 mM NaCl; more than 95% of the subgroup B peptide is bound to the aluminum; and the composition is not lyophilized. 2. A method for stabilizing the potency of a Neisseria meningitidis serogroup BLP2086 subgroup B peptide in an immunogenic composition, the method comprising step (a): formulating the LP2086 subgroup B peptide with a Neisseria meningitidis serogroup B LP2086 subgroup A peptide, polysorbate 80, aluminum in the form of _AlPO4 , and histidine to form a composition, wherein the composition comprises primarily 200 μg/mL of the LP2086 subgroup B peptide, polysorbate 80 in a molar ratio of 2.8:1 to protein, 0.5 mg/mL of aluminum in the form of _AlPO4 , 10 mM histidine at pH 6.0, and 150 mM NaCl; more than 95% of the subgroup B peptide is bound to the aluminum; and the composition is not lyophilized. 3. A method for stabilizing the potency of a Neisseria meningitidis serogroup BLP2086 subgroup B peptide in an immunogenic composition, the method comprising step (a): formulating the LP2086 subgroup B peptide with a Neisseria meningitidis serogroup B LP2086 subgroup A peptide, polysorbate 80, aluminum in the form of _AlPO4 , and histidine to form a composition, wherein the composition comprises primarily 200 μg/mL rLP2086 subgroup A peptide, 200 μg/mL LP2086 subgroup B peptide, polysorbate 80 in a molar ratio of 2.8:1 to protein, 0.5 mg/mL aluminum in the form of _AlPO4 , 10 mM histidine at pH 6.0, and 150 mM NaCl; more than 95% of the subgroup B peptide is bound to the aluminum; and the composition is not lyophilized. 4. A method for stabilizing the potency of a Neisseria meningitidis serogroup BLP2086 subgroup B peptide in an immunogenic composition, the method comprising step (a): formulating the LP2086 subgroup B peptide with a Neisseria meningitidis serogroup B LP2086 subgroup A peptide, polysorbate 80, aluminum in the form of _AlPO4 , and succinate to form a composition, wherein the composition comprises polysorbate 80 in a molar ratio of 2.8:1 to protein, 0.5 mg/mL aluminum in the form of _AlPO4 , 5 mM succinate at pH 6.0, and 150 mM NaCl; more than 95% of the subgroup B peptide is bound to the aluminum; and the composition is not lyophilized. 5. A method for stabilizing the potency of a Neisseria meningitidis serogroup BLP2086 subgroup B peptide in an immunogenic composition, the method comprising step (a): formulating the LP2086 subgroup B peptide with a Neisseria meningitidis serogroup B LP2086 subgroup A peptide, polysorbate 80, aluminum in the form of _AlPO4 , and succinate to form a composition, wherein the composition comprises primarily 200 μg/mL of the LP2086 subgroup B peptide, polysorbate 80 in a molar ratio of 2.8:1 to protein, 0.5 mg/mL of aluminum in the form of _AlPO4 , 5 mM succinate at pH 6.0, and 150 mM NaCl; more than 95% of the subgroup B peptide is bound to the aluminum; and the composition is not lyophilized. 6. A method for stabilizing the potency of a Neisseria meningitidis serogroup BLP2086 subgroup B peptide in an immunogenic composition, the method comprising step (a): formulating the LP2086 subgroup B peptide with a Neisseria meningitidis serogroup B LP2086 subgroup A peptide, polysorbate 80, aluminum in the form of _AlPO4 , and succinate to form a composition, wherein the composition comprises primarily 200 μg/mL rLP2086 subgroup A peptide, 200 μg/mL LP2086 subgroup B peptide, polysorbate 80 in a molar ratio of 2.8:1 to protein, 0.5 mg/mL aluminum in the form of _AlPO4 , 5 mM succinate at pH 6.0, and 150 mM NaCl; more than 95% of the subgroup B peptide is bound to the aluminum; and the composition is not lyophilized. 7. The method of any one of claims 1-6, wherein at least 50% of the potency of the LP2086 subfamily B peptide 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.