Stable vaccine formulations

Abstract

Provided is a stable vaccine comprising a protein antigen having a pI at or above about 7.0 or at least four consecutive positively charged amino acids, and a CpG oligonucleotide, and a buffer. Methods of stabilizing such subunit vaccines and assays determining whether the antigen adheres to container walls are also provided.

Description

STABLE VACCINE FORMULATIONSFIELD OF THE INVENTION

[0001] This invention is generally in the field of stable vaccine formulations containing protein antigens.SEQUENCE LISTING INFORMATION

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XMLfile format and is hereby incorporated by reference in its entirety. Said XML copy, created on June 6, 2024, is named ZP000517.xml and is 38,400 bytes in size, BACKGROUND

[0003] Subunit vaccines are prepared by extracting, purifying and / or recombinantly expressing some components of bacteria or viruses that can elicit an immunologic process. Compared to traditional attenuated vaccines, subunit vaccines are safer as they circumvent the stimulation of immune system thereby reducing the risk of subsequent, reversion, which can be caused by traditional attenuated microorganism vaccines.

[0004] Vaccine instability can be caused by light, heat, radiation, changes in the environment, or reactions with the container or with other components in the mixture, thus reducing the concentration of the antigen in the formulation.

[0005] Instability of vaccines in storage affects their efficiency and places additional burdens on storage, manufacturing, efficacy testing, and logistics of vaccine distribution. The development of storage-stable vaccines can greatly alleviate these problems.SUMMARY OF INVENTION

[0006] In the first aspect, the application discloses a vaccine formulation comprising: a protein antigen having a pl at or above about 7.0 or at least four consecutive positively charged amino acids, a CpG-containing immunostimulatory oligonucleotide, and a pharmaceutically acceptable carrier, wherein a) conductivity of said formulation is no greaterthan about 1.5 mS / cm; or b) saidvaccine formulation comprises an additional nonpolar or amphiphilic com pound and conductivity of said formulation is no greater than about 4.5 mS / cm.

[0007] In certain embodiments, the protein antigen comprises at least six, consecutive positively charged amino acids. In certain embodiments, conductivity of said formulation is no greaterthan about 1.0 mS / cm. In a set of embodiments applicable to the vaccine disclosed herein, the CpG-containing immunostimulatory oligonucleotide comprises a hydrophobic moiety that preferably comprises a 5' modification with iodo-modified uracil.

[0008] In a set of embodiments, the protein antigen in the vaccine formulation disclosed is selected from the group consisting of B burgdorferi OspC, a monoclonal antibody, IL-8, E canis TRP19, coronavirus spike protein, Chorionic gonadotropin (CG) or hemagglutinin (HA).

[0009] In a further set of embodiments, the vaccine formulation disclosed herein contains the additional nonpolar or amphiphilic compound, wherein said additional nonpolar or amphiphilic compound is an adjuvant or a part of an adjuvant combination. In certain embodiments, the vaccine formulation disclosed herein is an oil-in-water emulsion and wherein the conductivity of said formulation is no greaterthan about 4.5 mS / cm. In additional (or alternative) embodiments, the adjuvant combination is a combination of a triterpene saponin extracted from Quillaya saponaria and a sterol, and wherein the conductivity of said formulation is no greaterthan about 4.5 mS / cm. The vaccine accordingto any of the embodiments of thisfirst aspect of the invention is stable.

[0010] In a second aspect, the invention provides a kit for preparing a vaccine formulation according to the first aspect, comprising the protein antigen, the CpG-containing immunostimulatory oligonucleotide, optionally, a pharmaceutically acceptable diluent, wherein, upon combining the protein antigen and the CpG-containing immunostimulatory oligonucleotide in the pharmaceutically acceptable diluent, the conductivity of the resulting formulation is no greater than about 1.5 mS / cm.

[0011] In a third aspect, the invention provides a method of making the vaccine formulation disclosed herein or from the kit according to the second aspect, the method comprising combining the protein antigen, the CpG-containing immunostimulatory oligonucleotide in apharmaceutically acceptable diluent and, optionally, adjusting the conductivity of said vaccine formulation to no greater than about 4.5 mS / cm.

[0012] In a fourth aspect, the invention provides kit for preparing a vaccine formulation of according to certain embodiments of the first aspect, the kit comprising the protein antigen, the CpG-containing immunostimulatory oligonucleotide, optionally, a pharmaceutically acceptable diluent, wherein, upon combining the protein antigen, an additional nonpolar or amphiphilic compound and the CpG-containing immunostimulatory oligonucleotide in the pharmaceutically acceptable diluent, the conductivity of the resulting formulation is no greater than about 4.5 mS / cm. In certain embodiments, the kit according to this aspect further comprises the additional nonpolar or amphiphilic compound.

[0013] In a fifth aspect, the invention provides a method of making the vaccine formulation according to certain embodiments of the first aspect, and / or from the kit according to the fourth aspect, the method comprising combining the protein antigen, the CpG-containing immunostimulatory oligonucleotide and the additional nonpolar or amphiphilic compound in a pharmaceutically acceptable diluent and, optionally, adjusting the conductivity of said vaccine formulation to no greater than about 4.5 mS / cm.

[0014] In certain embodiments, of the second orthe fourth aspect of the invention, the adjuvant is a combination of the saponin extracted from Quillaya saponaria and a sterol. In additional or alternative embodiments, adjuvant is a combination an oil and a poloxamer, and wherein further the vaccine formulation is an oil-in-water emulsion.

[0015] In the sixth aspect, the invention provides an article of manufacturing comprising the vaccine formulation of the embodiments of the first aspect, wherein said vaccine formulation is within a glass or a plastic container.

[0016] In the seventh aspect, the invention provides a method of making the article of manufacturing according to the sixth aspect, the method comprising filling said glass or plastic container with the vaccine formulation.

[0017] In the eighth aspect, the invention provides a method of stabilizing a vaccine formulation comprising a protein antigen having a pl at or above about 7.0 or a His tag, a CpG-containingimmunostimulatory oligonucleotide, and a pharmaceutically acceptable carrier, the method comprising adjusting the conductivity of the vaccine formulation to 1.5 mS / cm or less.

[0018] In the ninth aspect, the invention provides a method of stabilizing a vaccine formulation comprising a protein antigen having a pl at or above about 7.0 or a His tag, a CpG-containing immunostimulatory oligonucleotide, an additional nonpolar or amphiphilic compound and a pharmaceutically acceptable carrier, the method comprising adjusting the conductivity of the vaccine formulation to 4.5 mS / cm or less.

[0019] In the tenth aspect, the invention provides a method of determining whether a protein antigen in a formulation adheres to walls of a vial, the method comprising: a) determining concentration of the protein antigen in the solution within the vial ("pre-incubation formulation"); b) incubating the formulation in the vial for time and at conditions sufficient for the protein subunit to adhere to walls of said vial; c) collecting the formulation after said incubation ("post-incubation formulation"); and at least one of: i) determining concentration of the protein antigen in the post-incubation formulation and subtracting the concentration of the protein antigen in the post-incubation formulation from the concentration of the protein antigen in the pre-incubation formulation; or ii) removing the protein antigen, if any, adhered to the walls of the vial, and determining the amount of the protein antigen removed from the walls of the vial.

[0020] In certain embodiments, the formulation used in said method contains a CpG oligonucleotide.

[0021] In certain embodiments, the concentration of the protein in said preincubation formulation and post-incubation formulation and the amount of the protein antigen removed from the walls of the vial is determined by optical density reading.

[0022] In other embodiments, the vial is washed after removal of the post-incubation formulation and prior to the step of removing the protein antigen.

[0023] Preferably, said step of removing the protein antigen comprises detergent wash. In particularly preferred embodiments the detergent comprises SDS (sodium dodecyl sulfate).BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is an illustration SDS-PAGE gel of protein adherence to vial walls in different adjuvant formulations. Panel A refers to a recombinant OspC antigen of B burgdopheri and Panel B refers to a recombinant Spike antigen of SARS-COVID-19.

[0025] Figure 2 is an illustration SDS-PAGE gel of protein adherence to polypropylene vial walls in formulations containing CpG in the presence, or absence of sodium chloride. Panel A refers to a recombinant OspC antigen of B burgdopheri and Panel B refers to a recombinant Spike antigen of SARS-COVID-19.

[0026] Figure 3 is an illustration SDS-PAGE gel analysis of protein adherence to glass vial walls in formulations containing CpG in the presence, or absence of sodium chloride.BRIEF DESCRIPTION OF SEQUENCES

[0027] SEQ ID NOs: 1-10 are exemplary sequences of CpG containing immunostimulatory oligonucleotides;

[0028] SEQ ID NOs: 11, 12, and 22 are exemplary amino acid sequences of OspC protein of B burgdoferi, as defined herein;

[0029] SEQ ID NO: 13 is an amino acid sequence of histidine tag (His-tag);

[0030] SEQ ID NO: 14 is an exemplary embodiment of OspA protein of B burgdoferi;

[0031] SEQ ID NO: 15 is recombinant Spike protein of SARS-COV-19;

[0032] SEQ ID NO: 16 is IL-8 protein;

[0033] SEQ ID NOs: 17 and 18 are subunits of equine Follicle-Stimulating Hormone heterodimer;

[0034] SEQ ID NO: 19 and 20 are subunits of equine Chorionic Gonadotropin heterodimer;

[0035] SEQ ID NO: 21 is BM86 antigen.DETAILED DESCRITION

[0036] The terms "about" and "approximately" are used interchangeably and refer to a range of values within ten percent of the reference value. Thus, for example and without limitations, "about 100" refers to a range from 90 to 110.

[0037] "Conductivity" of a solution refers to the ability of a solution to conduct electricity. Since electricity needs charged particles in order to flow, conductivity can be used as a measurementof ionic strength of the solution. Conductivity is measured in Siemens per unit of distance, most conveniently in milliSiemens per centimeter, or "mS / cm".

[0038] The term "Nonpolar compound" refers to compounds that have an even distribution of electric charge and no overall charge. Any charges are spread out evenly across the molecule. Nonpolar compounds typically interact by London dispersion forces. Nonpolar compounds are generally symmetrical and do not mix well with water. In certain embodiments, the term "nonpolar compounds" refers to organic molecules that dissolve in hydrophobic solvents better than in water.

[0039] The term "OspC" includes wild-type OspC proteins from different organisms, e.g., Borrelia species including without limitations B. burgdorferi, as well as recombinant constructs comprising fragments of different OspC phylotypes. More specifically and without limitations, the term “Borrelia burgdorferi OspC protein" encompasses loop and helix fragments from phylotypes I, H, N, C, M, D and a C-terminal tail from phylotype F. In the most preferred embodiments, the term “Borrelia burgdorferi OspC protein" encompasses SEQ ID NOs: 11 or 12.

[0040] The term "protein antigen" excludes whole cell extracts as well as bacterins and inactivated organisms. In certain preferred embodiments, the protein antigens are isolated from surrounding cellular or viral material. In more preferred embodiments, the protein antigens of the invention are produced in expression systems and purified therefrom.

[0041] The term "stable" refers to the degree of adherence of the protein antigen of the vaccine formulation to the wall of a container in which the vaccine formulation is stored. The vaccine formulation is "stable" (or "shelf-stable" or "storage-stable" or the like) if after 27 months of storage at 4⁰ C degrees Celsius the concentration of the protein antigen in an aliquot of the vaccine formulation is no less than 20% of the original concentration.

[0042] Applicants have discovered that in vaccine formulations comprising a combination of certain protein antigens and CpG oligonucleotides, the protein antigens tend to adhere to the walls of the container in which the vaccine formulations are stored. Applicants have further determined what features of the protein antigens make these proteins susceptible to adherence to the walls and how to prevent this side effect, thereby creating more stable vaccine formulations.

[0043] In vaccine formulations of the invention, the protein antigens have pl of 7.0 or above at pH of 7.0 or a positive surface charge created by a stretch of at least four, preferably, at least five, more preferably at least six consecutive positively charged amino acids, such as arginine, lysine, or histidine. One non-limiting example of said stretch is a His-tag created by six or more consecutive histidine amino acids (SEQ ID NO: 13, HHHHHH). As His-tag is a common tag used for purifying protein subunit antigens, knowledge that these protein antigens may adhere to the walls of the container when formulated in combination with CpG oligonucleotides is of particular importance for vaccines which is stable.

[0044] In suitable non-limiting examples, the protein antigen is selected from the group consisting of B burgdorferi OspC, a monoclonal antibody, IL-8, E canis TRP19, coronavirus spike protein, Chorionic gonadotropin (CG) or hemagglutinin (HA), each of which can further comprise a his-tag. In a particular example, the protein is B burgdorferi OspC which is optionally his-tagged.

[0045] The methods of making the protein antigens for use in the vaccine formulations described herein are well known. For example, in certain embodiments the protein antigens are purified from the extract or the supernatant of the pathogen. In other embodiments, nucleic acid sequences encoding the protein antigens described herein, and optionally, his-tag, may be subcloned into expression vectors, e.g., baculovirus, grown in suitable host cells, and harvested from extract or supernatants of said host cells. One of ordinary skill in the art is expected to be sufficiently skilled to be capable both of designing the sequence of the protein antigen and manufacturing said protein antigen in quantities sufficient for vaccine formulations.

[0046] The vaccine formulations disclosed herein also contain CpG containing immunostimulatory oligonucleotides, which are also referred to as "CpG oligonucleotides" or simply "CpGs". The effect of CpG containing oligonucleotides on the immune system has been known for over 20 years. Generally, the CpGs suitable for the invention are between 15 and 100 bases long, e.g., between 15 and 50 bases long, or between 18 and 40 bases long or between 20 and 30 bases long, or 20-24 bases long.

[0047] Several classes of CpGs have been described, including A-class CpGs, B-class CpGs, C-class CpGs, and P-class CpGs. In certain embodiments, the CpG containing immunostimulatory oligonucleotide is a P-class CpG. P-class CpGs are characterized by the presence of one or moreTLR-9 activating motif (s) and two palindromes or two complementarity areas. Preferably, the one or more TLR-9 activating motifs are at the 5' of the oligonucleotide and may be completely or partially be incorporated into the 5' palindrome or the 5' complementarity area. TLR-9 activating motifs are known and include, without limitations, TCG, TTCG, TTTCG, TYpR, TTYpR, TTTYpR, UCG, UUCG, UUUCG, TTT, or TTTT. The 5' palindrome or the 5' complementary area is at least 6 bases long. The 3' palindrome or the 3' complementary area is at least 8 bases long and is generally rich in C and G. These structural features of the P-class CpGs confer the ability to spontaneously self-assemble into concatamers either in vitro and / or in vivo.

[0048] In order to increase lipophilicity of the CpG oligonucleotides, at least one lipophilic substituted nucleotide analog may be included, preferably at the 5' end of the oligonucleotide. The P-class immunostimulatory oligonucleotides may be modified according to techniques known in the art. For example, J-modification refers to iodo-modified nucleotides. E-modification refers to ethyl-modified nucleotide(s). Thus, E-modified P-class immunostimulatory oligonucleotides are P-class immunostimulatory oligonucleotides, wherein at least one nucleotide (preferably 5' nucleotide) is ethylated. Additional modifications include attachment of 6-nitro-benzimidazol, O-Methylation, modification with proynyl-dll, inosine modification, 2-bromovinyl attachment (preferably to uridine).

[0049] The oligonucleotides modified by an addition of a lipophilic moiety are generally described in US 20100166780.

[0050] In certain embodiments, CpGs according to the invention comprise the modified backbone including, without limitations, phosphorothioate modifications, halogenations, alkylation (e.g., ethyl- or methyl-modifications), and phosphodiester modifications.

[0051] Suitable non-limiting examples of modified P-class immunostimulatory oligonucleotides are provided below ("*" refers to a phosphorothioate bond,refers to a phosphodiester bond, "JU" refers to 5'-lodo-2'-deoxyuridine and "EU" refers to 5-Ethyl-2'-deoxyuridine).SEQ ID NO: 1 5' T*C-G*T*C-G*A*C-G*A*T*C-G*G*C*G*C-G*C*G*C*C*G 3'SEQ ID NO: 2 5' T*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G 3' SEQ ID NO: 3 5' T*C*G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G*T 3' SEQ ID NO: 4 5' JU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G 3'SEQ ID NO: 5 5' JU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G*T 3' SEQ ID NO: 6 5' JU*C*G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C* G*T 3' SEQ ID NO: 7 5' EU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G 3' SEQ ID NO: 8 5' JU*C-G*T*C*G*A*C*G*A*T*C*G*G*C*G*G*C*C*G*C*C* G*T 3' SEQ ID NO: 9 5' JU*C*G*T*C*G*A*C*G*A*T*C*G*G*C*G*G*C*C*G*C*C* G*T 3' SEQ ID NO: 10 5' T*C-G*T*C-G*A*C-G*A*T*C-G*G*C*G*C-G*C*G*C*C*G 3'

[0052] In certain embodiments, the CpG oligonucleotide according to the invention comprises any one of SEQ ID NOs 1-10 or an oligonucleotide comprising at least 15 consecutive bases of any one of SEQ ID NOs 1-10. In the most preferred embodiment, the vaccine comprises an oligonucleotide comprising at least 15 consecutive bases of SEQ ID NO: 8 (e.g., at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23).

[0053] The CpG oligonucleotide may be present in the vaccine in the amount of 10-400 μg per dose of the vaccine, or 25-300 or 50-200 or 50-100 mg per dose.Optional additional nonpolar or amphiphilic compound

[0054] The vaccine may optionally contain an additional nonpolar or amphiphilic compound. Without wishing to be bound by theory, it is believed that the additional nonpolar or amphiphilic compound coats the protein antigen and prevents the adherence of the protein antigen to the walls of the container. In certain embodiments, the additional nonpolar or amphiphilic compounds have adjuvanting properties. Without limitations, such additional nonpolar or amphiphilic compounds include oils, sterols, poloxamers, and the like. In certain embodiments, the oil is the adjuvant, and the vaccine formulation is an emulsion such as, for example, a water-in-oil emulsion, an oil-in-water emulsion and the like. In other embodiments, the nonpolar or amphiphilic compound may form a part of an adjuvant. Suitable examples of such combination adjuvants include a combination of oil and poloxamer, and the sterol and a triterpenoid saponin extracted from Quillaya saponaria, e.g., Quil A ora fraction thereof, such as QS-21, QS-17, QS-18 or the like.

[0055] In certain embodiments, the additional nonpolar or amphiphilic compound is a mixture of a metabolizable oil, a poloxamer and, optionally, one or more surfactants.

[0056] In certain embodiments, in addition to the CpG-containing immunostimulatory oligonucleotide, the adjuvant comprises the metabolizable oil, the polyoxyethylenepolyoxypropylene block copolymer, and the surfactant.

[0057] Multiple metabolizable oils are known in the art including without limitation squalane, squalene, medium chain triglycerides, and long chain triglycerides. In one embodiment, the metabolizable oil is squalane. The metabolizable oil can be present in the vaccine in the amount of about 0.05% to about 35% v / v, e.g., about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, 1%, about 2%, about 3%, about 5%, about 8%, about 10%, about 15%, about 15%, about 20%, about 25%, or about 30% v / v. In certain preferred embodiments, the metabolizable oil is present in the amount of 0.1% to about 1% v / v, e.g., 0.3% to about 0.7%, or about 0.4% to about 0.5% v / v.

[0058] Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) triblock copolymers also known as poloxamers are also known in the art. They are composed of a central hydrophobic chain of polyoxypropylene (polypropylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (polyethylene oxide)). In certain embodiments, poloxamers are commercially available under the trade name PLURONIC®. The poloxamer can be present in the vaccine in the amount of amount of about 0.05% to about 0.8 % v / v.

[0059] The preferred Polyethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) triblock copolymer is poloxamer which is commercially available under the trade names PLURONIC® L-121 and ETHOX® L-121 and has the formula of (C3H6O·C2H4O)x.

[0060] Polyoxyethylene-polyoxypropylene block copolymers are known to have surface active properties. Nevertheless, in certain preferred embodiments, the adjuvant may further optionally comprise an additional optional surfactant (sometimes referred to as a second surfactant or the optional second surfactant) that is not a polyoxyethylene-polyoxypropylene block copolymer. Multiple suitable surfactants are known in the art. Preferably, second surfactant, is an amphipathic, nonionic surfactant that is derived from ethoxylated sorbitan. In certain embodiments, the second surfactant is polyoxyethylene sorbitan monooleate also known as TWEEN® 80 or polysorbate 80. The optional surfactant can be present in the vaccine in the amount of amount of about 0.005% to about 10% v / v, e.g., about 0.01%, or about 0.02%, orabout 0.03%, or about 0.04%, or about 0.05%, or about 0.06%, or about 0.07%, or about 0.08%, or about 0.09%, or about 0.1%, or about 0.15%, or about 0.20%, or about 0.5%, or about 0.8%, or about 1%, or about 5%, or about 8%, or about 9% v / v. In certain embodiments, the second surfactant us present in the amount from about 0.005% to about 0.1% v / v, or from about 0.01 to about 0.05% or from about 0.025% to about 0.04% v / v, or from about 0.1 % to about 0.5% v / v or from 0.25% to about 0.75% v / v.

[0061] A mixture of squalane, poloxamer 401, polyoxyethylene sorbitan monooleate and a buffered salt solution is known in the art and has been called "SP oil". In general, the SP oil emulsion contains about 1 to 3% vol / vol of polyoxyethylene-polyoxypropylene block copolymer, about 2 to 6% vol / vol of squalane, more particularly about 3 to 6% of squalane, and about 0.1 to 0.5% vol / vol of polyoxyethylene sorbitan monooleate, with the remainder being a buffered salt solution.

[0062] In certain embodiments, about 5% to about 20% SP oil is used in the vaccine. Thus, in certain embodiments, said metabolizable oil is present in the amount of about 0.2% to about 0.8% v / v of the vaccine; said polyoxyethylene-polyoxypropylene block copolymer is present in the amount of about 0.1% to about 0.4% v / v of the vaccine; and said polyoxyethylene sorbitan monooleate is present in the amount of about 0.016% to about 0.064% v / v of the vaccine.

[0063] In more specific embodiments, the metabolizable oil is present in the amount of about 0.4% v / v of the vaccine; said polyoxyethylene-polyoxypropylene block copolymer is present in the amount of about 0.2% v / v of the vaccine; and said polyoxyethylene sorbitan monooleate is present in the amount of about 0.032% v / v of the vaccine.

[0064] In addition to the CpG containing oligonucleotides, the protein antigens and optional additional nonpolar or amphiphilic compound(s), the vaccine formulations described herein also comprise a pharmaceutically acceptable carrier or diluent.

[0065] The inventors have surprisingly discovered that when ionic strength of the vaccine formulations described herein is lowered, the vaccine formulation is stable: the protein antigen does not adhere to the walls of the container where the vaccine formulation is stored. It was found that if the vaccine formulation contains the additional nonpolar or amphiphilic compound(s), then the conductivity of the vaccine, which is a measurement of the ionic strength,should preferably be no greater than about 4.5 mS / cm, e.g., no greater than about 4.0 mS / cm, or no greater than about 3.5 mS / cm, or no greater than about 3.0 mS / cm, or no greater than about 2.5 mS / cm, or no greater than about 2.0 mS / cm, or no greater than about no greater than about 1.5 mS / cm, or no greater than about 1.0 mS / cm, or no greater than about 0.5 mS / cm.

[0066] If the vaccine formulation does not contain the additional nonpolar or amphiphilic compound, then it is preferred that the conductivity of the solution should be no greater than about 2.0 mS / cm, or no greater than about 1.5 mS / cm, or no greater than about 1.0 mS / cm, or no greater than about 0.5 mS / cm.

[0067] Methods of determining the conductivity of the resulting vaccine formulation and devices for doing so are well known. Without limitations, such devices include Mettler Toledo FiveEasy Plus FP30, Mettler Toledo Seven Direct SD23, and Orion Star A212 conductivity meter.

[0068] If, after combining the ingredients of the vaccine composition, the conductivity is above the desired threshold, the conductivity can be lowered by adding a low-ionic-strength solution containing the protein antigen, the CpG containing oligonucleotide, and the optional additional nonpolar or amphiphilic compound in a low-ionic-strength solvent such as, for example, distilled water. One can add the low-ionic-strength solution to the vaccine formulation dropwise thus titrating the necessary amount to adjust the ionic strength of the final vaccine formulation.

[0069] The advantage of the vaccine formulations described herein is that said vaccine formulations are storage stable and can be stored at 4° C temperature for up to Tl months.

[0070] In another aspect, the invention provides an assay for determining whether the antigen in a given formulation is likely to adhere to vial wall. Conveniently, the assay can be conducted in a 96-well plate. The amount of protein can be quantified in the emptied wells. The amount of protein remaining in solution can also be quantified along with the original protein solution. With these three values, the amount of antigen adhered to the well walls can be determined. Thus, the optimal formulation condition can be determined for the antigen for prevention of vial wall adherence and the amount of adhered protein can be quantified.

[0071] Accordingly, in certain embodiments, the assay employs a method of determining whether a protein antigen in a formulation adheres to walls of a vial, the method comprising:a) determining concentration of the protein antigen in the solution within the vial ("pre-incubation formulation");b) incubating the formulation in the vial for time and at conditions sufficient for the protein subunit to adhere to walls of said vial;c) collecting the formulation after said incubation ("post-incubation formulation"); and at least one of:i) determining concentration of the protein antigen in the postincubation formulation and subtracting the concentration of the protein antigen in the post-incubation formulation from the concentration of the protein antigen in the pre-incubation formulation; orii) removing the protein antigen, if any, adhered to the walls of the vial, and determining the amount of the protein antigen removed from the walls of the vial.

[0072] In certain embodiments, the assay described herein may be used for the formulations comprising the protein antigen and the immunostimulatory oligonucleotide as described in any of the embodiment above.

[0073] The time and conditions sufficient for the protein subunit to adhere to walls of said vial depend on multiple criteria and need to be determined in course of the assay at different time points. Generally, time, temperature, the presence of surfactants, the sequence of the protein, the concentrations of the protein and the CpG are among the conditions that affect the occurrence and extent of the adherence of the antigen to the wall of the container. The inventors have discovered that as the temperature increases, the rate at which the protein adheres to the wall also increases. Accordingly, one can select higher temperature and thus emulate accelerated testing.

[0074] Conveniently, the concentration of the protein in said preincubation formulation and post-incubation formulation and the amount of the protein antigen removed from the walls of the vial is determined by optical density reading. Methods of determining the amount and / or concentration of a protein in a solution are well known. Kits for doing so may be obtained fromcommercial suppliers. In a set of particularly preferred embodiments, the concentration of the protein in said preincubation formulation and post-incubation formulation and the amount of the protein antigen removed from the walls of the vial is determined by use of BCA reagent (supplied in PIERCE™ BCA Protein Assay Kits, Thermofisher Scientific). This kit contains reagents for is a high-precision, detergent-compatible protein assay for determination of protein concentration and provides accurate determination of protein concentration with most sample types encountered in protein research. This assay can be used to assess yields in whole cell lysates, affinity-column fractions, purified proteins samples, as well as to monitor protein contamination in industrial applications.

[0075] Kits are available with or without Dilution-Free BSA Protein Standards, which are a set of seven pre-diluted BSA standards, packaged in a multichannel tubestrip. The tubestrip includes a single empty tube that enables users to add their own sample buffer for the purpose of blank subtraction.

[0076] Thus, after the Optical Density readings are obtained, the amounts and / or concentrations can be determined by comparing said Optical Density readings with the standard curve that is prepared using BSA standards.

[0077] If one wishes to directly determine the amount of the protein antigen that adheres to the walls of the vial, it is advantageous to wash the vial after removal of the post-incubation formulation and prior to the step of removing the protein antigen. In certain embodiments, the step of washing comprises addition of PBS or other suitable buffer to dilute and preferably remove the residual solution from the well. After the vial is washed, the protein antigen (if any) that remains adhered to the walls of the vial can be removed by detergent wash. In certain embodiments, the detergent wash entails the use of 0.1 to 5% (preferably 0.5 to 1.5, more preferably about 1% v / v) SDS solution for at least 2 hours, preferably 2 to about 7 hours, more preferably about 4 hours) at room temperature or 37C. After the detergent extraction, the solution containing the protein washed off is analyzed using SDS-PAGE gel.EXAMPLESMaterials and methods - in General

[0078] To determine the amount of the antigen bound to the walls of the vials, the contents of the vial were removed by aspiration. The vials were washed by adding an equal volume of DI water that was originally in the container and replace the cap and invert. This process was repeated for a total of three washes, lx Sodium dodecyl sulfate (SDS) solution was added at 1:5 the total volume of liquid that was originally in the container. The vials were incubated at 37° C, with rocking for a minimum of 2 hours and a maximum of 24 hours. 18 pL of the extracted solutions was transferred to individual tubes containing 6 pL of 4x SDS-PAGE loading dye and heat the samples at 90° C for 5 minutes. Gel tank was assembled and the samples were loaded into the gel wells along with the molecular weight standard and run the gel at 190 V for 45 minutes. The gel was stained with Coomassie blue and imaged with the BIORAD® imager.

[0079] To determine the amount of the antigen aggregated in solution, the entire contents of the vial was transferred to a centrifuge tube. The samples were centrifuged at 18,000 x g for 5 minutes at 4° C, and the liquid was removed by aspiration on the opposite side of the tube to where the pellet is. The pellet was washed by slow addition of 1 mL of PBS to the tube and aspiration of the PBS back out taking care to not disrupt the pellet. This procedure was repeated for a total of three washes. The pellet was solubilized in 6 M urea at 1:10 the original solution volume and vortexed for 30 sec. For small volumes, the urea was aspirated up and down to dissolve the pellet. 18 pL of the solubilized solutions was transferred to individual tubes containing 6 pL of 4x SDS-PAGE loading dye, and the samples were heated at 90° C for 5 minutes. Gel tank was assembled and the samples were loaded into the gel wells along with the molecular weight standard and run the gel at 190 V for 45 minutes. The gel was stained with Coomassie blue and imaged with the BIORAD® imager.

[0080] For the plate-based method of assaying vial wall adherence, a minimum of 1 mL for each formulation to be tested was prepared. BSA standard was prepared according to the manufacturer's instructions. 50 pL of each preparation in triplicate and BSA standard in duplicate were transferred to a flat-bottom 96-well polystyrene plate. BSA reagent was prepared according to the manufacturer's instructions and add 200 pL to each occupied well. The plate was covered with microplate adhesive film and the plate was incubated at 37° C for 30 minutes. The plate was read at a wavelength of 562 nm and the value was recorded as the "total" amountof protein. To a second plate, which was a polypropylene plate, 200 pL of each formulation in triplicate to a 96-well was added, and the plate was covered with microplate adhesive film and incubated overnight at 37° C. 50 pL of sample was transferred from each occupied well to a flatbottom, 96-well polystyrene plate and the BSA standard solution was added to unoccupied wells in duplicate. This plate is to be used to determine the "remaining" protein in solution ("remaining protein plate"). The residual sample in the 96-well polypropylene plate was removed, and the wells were washed the plate 3 times with PBS, and 50 pL of PBS were added to all wells that contained sample. The BSA standard was added to unoccupied wells in duplicate. This plate is to be used to determine the "adhered" protein to the well wall ("adhered protein plate"). The BCA reagent was prepared according to the manufacturer's instructions and added 200 pL to each occupied well in both plates (remaining protein plate and adhered protein plate). Each plate was covered with microplate adhesive film and incubated at 37° C for 30 minutes. Both plates at a wavelength of 562 nm and the values were record appropriately (i.e., the value for the adhered protein plate was recorded as adhered amount of protein, and the value for the remaining protein plate was recorded as the remaining amount of protein).

[0081] The data from each plate was analyzed independently, but in the same fashion. Average of the ODs for the BSA standard was determined and plotted the average OD vs. the concentration. BSA standard curve was fit to a line to determine the slope and Y-intercept. The equation of a line (Y - mX + b) was used to determine the concentration of the samples (average OD of triplicate samples). To estimate the amount of antigen that bound to the well walls the following formula was used: "total" protein concentration - "remaining" protein concentration = wall-adhered protein concentration. This value serves as an estimation to determine relative adherence.Example 1. SDS-PAGE analysis of protein adherence to vial walls in different adjuvant formulations.

[0082] In this example, it is demonstrated that at CpG may induce adherence of OspC and COVID protein antigens to vial walls.I. OspC

[0083] Seven formulations were investigated for this study. 1) CpG / SP-Oil / thimerosal / OspA / OspC. 2). Thimerosal / OspA / OspC. 3) OspA / OspC. 4) CpG / thimerosal / OspA / OspC. 5) SP Oil / thimerosal / OspA / OspC, 6) CpG / SP-Oil / thimerosal / OspC.7) CpG / SP-Oil / OspA / OspC.

[0084] For formulations containing CpG, a 20 mg / mL stock of CpG (SEQ ID NO: 8) was diluted to 100 pg / mL in 0.063% phosphate-buffered saline (PBS), pH = 7.4, 150 mM NaCI in the presence, or absence of other adjuvants, depending on the formulation.

[0085] Forformulations containing SP Oil (Squalane 4mL, Pluronic L1212mL, NaCI lg, KCI 0.025g, Sodium Phosphate Dibasic Heptahydrate 0.272g, Potassium Phosphate Monobasic Anhydrous 0.025g, Tween-800.032mL dissolved in lOOmL Purified water), a 100% stock solution was diluted to 10% in 0.063% phosphate-buffered saline (PBS), pH = 7.4, 150 mM NaCI in the presence, or absence of other adjuvants, depending on the formulation.

[0086] For formulations containing thimerosal, a 5% stock solution was diluted to 0.005% in 0.063% phosphate-buffered saline (PBS), pH = 7.4, 150 mM NaCI in the presence, or absence of other adjuvants, depending on the formulation. A stock concentration of OspC (SEQ ID NO: 11) was diluted to 30 pg / mL in each of the formulations. A stock concentration of OspA (SEQ ID NO: 14) was diluted to 20 pg / mL in formulations intended to contain the OspA antigen. Each formulation was prepared at 1 m L and added to a 3 mL polypropylene container and incubated, undisturbed, for 4 days at 4° C. after the incubation period, the formulation was removed from the container and the container was washed with PBS by adding 1 mL of PBS, inverting the container and removing the PBS by aspiration. The washing step was repeated three times to ensure removal of residual formulation liquid. A 1% SDS solution was prepared by diluting a 20x stock concentration of MES gel running buffer (NUPAGE™ MES SDS Running Buffer (20X), Invitrogen, Cat. No. NP0002 to a lx solution and 200 pL of the 1% SDS solution was added to the polypropylene container of each formulation and incubated, with rocking, for 12 hr at 37° C. the 1% SDS solution was then analyzed by Coomassie-stained, SDS-PAGE.II. SARS-CoV-2 spike protein (COVID)

[0087] Six formulations were investigated for this study. 1) CpG / QuilA / cholesterol / COVID (a recombinant Spike protein of SARS-COV-19, SEQ ID NO: 15). 2) QuilA / cholesterol / COVID. 3) QuilA / COVID. 4) cholesterol / COVID. 5). CpG / COVID. 6) CpG / QuilA / COVID.

[0088] For formulations containing CpG, a 20 mg / mL stock of CpG was diluted to 20 pg / mL in 0.063% phosphate-buffered saline (PBS), pH = 7.4, 150 mM NaCI in the presence, or absence of other adjuvants, depending on the formulation. For formulations containing QuilA, a 50 mg / mL stock solution was diluted to 0.02 mg / mL in 0.063% phosphate-buffered saline (PBS), pH = 7.4, 150 mM NaCI in the presence, or absence of other adjuvants, depending on the formulation. For formulations containing cholesterol, a 17 mg / mL stock solution was diluted to 0.02 mg / mL in 0.063% phosphate-buffered saline (PBS), pH = 7.4, 150 mM NaCI in the presence, or absence of other adjuvants, depending on the formulation.

[0089] A stock concentration of COVID was diluted to 20 pg / mL in each of the formulations. Each formulation was prepared at 1 mL and added to a 3 mL polypropylene container and incubated, undisturbed, for 4 days at 4° C. after the incubation period, the formulation was removed from the container and the container was washed with PBS by adding 1 mL of PBS, inverting the container, and removing the PBS by aspiration. The washing step was repeated three times to ensure removal of residual formulation liquid. A 1% SDS solution was prepared by diluting a 20x stock concentration of MES gel running buffer (NUPAGE™ MES SDS Running Buffer (20X), Invitrogen, Cat. No. NP0002 to a lx solution and 200 pL of the 1% SDS solution was added to the polypropylene container of each formulation and incubated, with rocking, for 12 hr at 37° C. the 1% SDS solution was then analyzed by Coomassie-stained, SDS-PAGE.

[0090] The results for OspC and SARS-COVID antigens are provided in panels A and B, respectively, of Fig. 1. For panel A, the lanes were loaded as follows: Lane 1. CpG / SP Oil / thimerosal / OspA / OspC (full formulation), lane 2. thimerosal / OspA / OspC, lane 3. OspA / OspC, lane 4. CpG / thimerosal / OspA / OspC, lane 5. SP Oil / thimerosal / OspA / OspC, lane 6. CpG / SP Oil / thimerosal / OspC, lane 7. CpG / SP Oil / OspA / OspC. Molecular weight marker (MW) is on the left.

[0091] For Panel B, the lanes were loaded as follows: QuilA / cholesterol / COVID, lane 3. QuilA / COVID, lane 4. cholesterol / COVID, lane 5. CpG / COVID, lane 6. CpG / QuilA / COVID. Molecular weight marker (MW) is on the left.

[0092] As one can readily see, antigen adherence to vial walls is particularly pronounced when the formulations contain CpG.Example 2. Research of the properties of proteins that are susceptible to adherence to vial walls.

[0093] The tested proteins are listed in Table 1 below. Three formulations were investigated for each tested proteins. 1) QuilA (0.02 mg / mL) in 0.063% phosphate-buffered saline (PBS), pH = 7.4, 150 mM NaCI and 20 pg / mL of each protein, separately. 2) CpG (100 pg / mL) in 0.063% phosphate-buffered saline (PBS), pH = 7.4, 150 mM NaCI and 20 pg / mL of each protein, separately. 3) QuilA (0.02 mg / mL) and CpG (100 pg / mL) in 0.063% phosphate-buffered saline (PBS), pH = 7.4, 150 mM NaCI and 30 pg / mL of each protein, separately. A 1 mL volume was made for each formulation and was added to 3 mL polypropylene containers and incubated at 4° C, undisturbed, for 4 days. After the incubation period, the formulation was removed from the container and the container was washed with PBS by adding 1 mLof PBS, inverting the container, and removing the PBS by aspiration. The washing step was repeated three times to ensure removal of residual formulation liquid. A 1% SDS solution was prepared by diluting a 20x stock concentration of MES gel running buffer (NUPAGE™ MES SDS Running Buffer (20X), Invitrogen, Cat. No. NP0002 to a lx solution and 200 pL of the 1% SDS solution was added to the polypropylene container of each formulation and incubated, with rocking, for 12 hr at 37° C. the 1% SDS solution was then analyzed by Coomassie-stained, SDS-PAGE.Table 1

[0094] As shown in Table 1, high pl (above 7.1) and / or the presence of a His tag were indicators of vial wall adherence.Example 3. Effect of salt concentration on vial wall adherence

[0095] Four formulations were investigated for each protein in this study. A formulation containing 0.063% PBS and 100 pg / mL CpG in the presence, or absence of 150 mM NaCI (saline) at a pH of either 7.4, or 6.4. A final concentration of 30 pg / mL of OspC and 50 pg / mL of COVID were added to each formulation, separately. A 1 mL volume was made for each formulation and was added to 3 mL polypropylene containers and incubated at 4° C, undisturbed, for 4 days. After the incubation period, the formulation was removed from the container and the container was washed with PBS by adding 1 mL of PBS, inverting the container, and removing the PBS by aspiration. The washing step was repeated a total of three times to ensure removal of residual formulation liquid. A 1% SDS solution was prepared by diluting a 20x stock concentration of MES gel running buffer (NuPAGE™ MES SDS Running Buffer (20X), Invitrogen, Cat. No. NP0002 to a lx solution and 200 pL of the 1% SDS solution was added to the polypropylene container of each formulation and incubated, with rocking, for 12 hr at 37° C. the 1% SDS solution was then analyzed by Coomassie-stained, SDS-PAGE.

[0096] Samples were incubated for 4 days at 4° C in the adjuvant formulations and the walls of the polypropylene container were treated with 1% SDS to remove any protein adhered to the container wall and examined by SDS-PAGE. SDS-PAGE analysis of the OspC and COVID antigens adherence to the container wall in various formulations. As shown in Fig. 2, the addition of salt (as indicated by the presence of saline) increases adherence of the antigen to vial walls.

[0097] Example 4

[0098] In this example, effect of different buffers on adherence to cell walls was investigated. Four proteins were selected for the experiment: BSA, OspA, OspC, and COVID antigen. BSA and OspA do not adhere to vial walls in the presence of CpG, whereas OspC and COVID antigen do. The results are provided in Tables 3A-3D. The buffers examined were Tris, phosphate, MES, and saline. Thus, sixteen different buffer-antigen formulations were tested.

[0099] All buffers were prepared at a concentration of 150 mM in deionized water. A 20 mg / mL stock of CpG was diluted to 100 pg / mL in each buffer and the volume was accounted for when producing the 150 mM starting buffer concentration. Separately, a 100 pg / mL CpG solution in deionized water was prepared to act as the diluent. Each buffer was diluted to concentrations of 150 mM, 75 mM, 37.5 mM, 21 mM, 10 mM, and 0 mM buffer and all dilutions contained 100 pg / mLCpG.

[0100] Each tested protein was then diluted to a concentration of 30 pg / mL in all dilutions of each buffer examined. The dilutions and the addition of protein was conducted in a 96 well, polypropylene plate and the plate was sealed with film and subsequently incubated at 37° C, with shaking for 12 hrs. Once the incubation was completed, the solution was removed, and the wells were washed with PBS by the addition of 200 pL PBS to each well and removal by aspiration. The wash step was repeated for a total of three washes. 50 pL of PBS was then added to each well and 200 pL of the BCA reagent was subsequently added and the plate was incubated at 37° C for thirty minutes according to the manufacturer's instructions. The plate was then read at 562 nm (OD562) using a Spectramax i 3x UV / VIS microplate reader.[OOlOlJThe results are summarized in Tables 2A-2D. As one can see, OD562 increased most significantly in proteins which are known to adhere to the walls of test vials, as the salt concentration increased.Table 2A. COVID antigen, OD562Table 2B. BSA, OD562Table 2C. OspA, OD562Table 2D. OspC, OD562*ND = not determinedExample 5. Conductivities of Tris, Phosphate, Saline, and MES

[0102] Buffer conductivities of A) Tris, B) Phosphate, C) Saline, and D) MES were determined. The results are provided in Table 3.Table 3. Buffer conductivity, mS / cm

[0103] The results suggest that is correlation between the conductivity of the buffers and the extent of antigen adherence to vial walls.Example 6 - formulation in a low-salt buffer reduces adherence of protein antigen to cell walls.

[0104] Based on the data above, it was hypothesized that decrease in salt content will decrease conductivity of the formulation and thus reduce the amount of the antigen that adheres to vial walls. Two formulations were investigated for each protein in this study. A formulation 0.063% PBS and 100 pg / mL CpG in the presence, or absence of 150 mM NaCI (saline) at a pH of 7.4. A final concentration of 20 pg / mL for each protein was added to each formulation, separately. A 1 mL volume was made for each formulation and was added to 3 mL glass (borosilicate) containers and incubated at 4° C, undisturbed, for 4 days. After the incubation period, the formulation was removed from the container and the container was washed with PBS by adding 1 mL of PBS, inverting the container, and removing the PBS by aspiration. The washing step was repeated a total of three times to ensure removal of residual formulation liquid. A 1% SDS solution was prepared by diluting a 20x stock concentration of MES gel running buffer (NUPAGE™ MES SDS Running Buffer (20X), Invitrogen, Cat. No. NP0002 to a lx solution and 200 pL of the 1% SDS solution was added to the polypropylene container of each formulation and incubated, with rocking, for 12 hr at 37° C. The 1% SDS solution was then analyzed by Coomassie-stained, SDS-PAGE. Samples were incubated for 4 days at 4° C in the adjuvant formulations and the walls of the glass container were treated with 1% SDS to remove any protein adhered to the container wall and examined by SDS-PAGE. The image is an SDS-PAGE of the OspA, OspC, and COVIDantigens extracted from the container wall by 1% SDS in the presence, or absence of 150 mM NaCI in lOOuM CpG-containing formulations. The control (C) protein remained at 4° C in an Eppendorf tube in the absence of CpG until analyzed by SDS-PAGE. the plus symbol (+) indicates formulations with 150 mM NaCI and the minus symbol (-) indicates formulations in the absence of salt. All formulations contained 0.063% PBS at pH 7.4.

[0105] The results are illustrated in Fig. 3 which shows that low-salt buffer did not affect OspA adherence to vial walls but decreased the adherence of COVID antigen and OspC to the vial walls.

Claims

CLAIMS1. A vaccine formulation comprising: a protein antigen having a pl at or above about 7.0 or at least four consecutive positively charged amino acids, a CpG-containing immunostimulatory oligonucleotide, and a pharmaceutically acceptable carrier, wherein a) conductivity of said formulation is no greater than about 1.5 mS / cm; orb) said vaccine formulation comprises an additional nonpolar or amphiphilic compound and conductivity of said formulation is no greater than about 4.5 mS / cm.

2. The vaccine formulation of claim 1 wherein the protein antigen comprises at least six consecutive positively charged amino acids.

3. The vaccine formulation of claim 1 wherein the protein antigen comprises His tag.

4. The vaccine formulation of any one of claims 1-3, wherein conductivity of said formulation is no greater than about 1.0 mS / cm.

5. The vaccine formulation of any one of claims 1-4, wherein the CpG containing immunostimulatory oligonucleotide comprises a hydrophobic moiety.

6. The vaccine formulation of claim 5, wherein the hydrophobic moiety comprises a 5' modification with iodo-modified uracil.

7. The vaccine formulation of any one of claims 1-6, wherein said protein is selected from the group consisting of B burgdorferi OspC, a monoclonal antibody, IL-8, E canis TRP19, coronavirus spike protein, Chorionic gonadotropin (CG) or hemagglutinin (HA).

8. The vaccine formulation of any one of claims 1-7 containing the additional nonpolar compound, wherein said additional nonpolar or amphiphilic compound is an adjuvant or a part of an adjuvant combination.

9. The vaccine formulation of claim 8 which is an oil-in-water emulsion and wherein the conductivity of said formulation is no greater than about 4.5 mS / cm.

10. The vaccine formulation of claim 8, wherein the adjuvant combination is a combination of a triterpene saponin extracted from Quillaya saponaria and a sterol, and wherein the conductivity of said formulation is no greater than about 4.5 mS / cm.

11. The vaccine formulation of any one of claims 1-10 which is stable.

12. A kit for preparing a vaccine formulation of any one of claims 1-7 or 11, comprising the protein antigen, the CpG-containing immunostimulatory oligonucleotide, optionally, a pharmaceutically acceptable diluent, wherein, upon combining the protein antigen and the CpG-containing immunostimulatory oligonucleotide in the pharmaceutically acceptable diluent, the conductivity of the resulting formulation is no greater than about 1.5 mS / cm.

13. A method of making the vaccine formulation according to anyone of claims 1-7 or 11, the method comprising combining the protein antigen, the CpG-containing immunostimulatory oligonucleotide in a pharmaceutically acceptable diluent and, optionally, adjusting the conductivity of said vaccine formulation to no greater than about 4.5 mS / cm.

14. A kit for preparing a vaccine formulation of any one of claims 1-11, comprising the protein antigen, the CpG-containing immunostimulatory oligonucleotide, optionally, a pharmaceutically acceptable diluent, wherein, upon combining the protein antigen, an additional nonpolar or amphiphilic compound and the CpG-containing immunostimulatory oligonucleotide in the pharmaceutically acceptable diluent, the conductivity of the resulting formulation is no greater than about 4.5 mS / cm.

15. The kit according to claim 14 further comprising the additional nonpolar or amphiphilic compound.

16. A method of making the vaccine formulation according to any one of claims 1-12, the method comprising combining the protein antigen, the CpG-containing immunostimulatory oligonucleotide and the additional nonpolar or amphiphiliccompound in a pharmaceutically acceptable diluent and, optionally, adjusting the conductivity of said vaccine formulation to no greater than about 4.5 mS / cm.

17. The kit according to claim 14 or 15, or the method according to claim 16, wherein the additional nonpolar or amphiphilic compound is an adjuvant or a part of an adjuvanting combination.

18. The kit according to claim 17, wherein the adjuvant is a combination of the saponin extracted from Quillaya saponaria and a sterol.

19. The kit according to claim 17, wherein the adjuvant is a combination an oil and a poloxamer, and wherein further the vaccine formulation is an oil-in-water emulsion.

20. An article of manufacturing comprising the vaccine formulation of any one of claims 1-11, wherein said vaccine formulation is within a glass or a plastic container.

21. A method of making the article of manufacturing according to claim 20, the method comprising filling said glass or plastic container with the vaccine formulation.

22. A method of stabilizing a vaccine formulation comprising a protein antigen having a pl at or above about 7.0 or a His tag, a CpG-containing immunostimulatory oligonucleotide, and a pharmaceutically acceptable carrier, the method comprising adjusting the conductivity of the vaccine formulation to 1.5 mS / cm or less.

23. A method of stabilizing a vaccine formulation comprising a protein antigen having a pl at or above about 7.0 or a His tag, a CpG-containing immunostimulatory oligonucleotide, an additional nonpolar or amphiphilic compound and a pharmaceutically acceptable carrier, the method comprising adjusting the conductivity of the vaccine formulation to 4.5 mS / cm or less.

24. A method of determining whether a protein antigen in a formulation adheres to walls of a vial, the method comprising:d) determining concentration of the protein antigen in the solution within the vial ("pre-incubation formulation");T1e) incubating the formulation in the vial for time and at conditions sufficient for the protein subunit to adhere to walls of said vial;f) collecting the formulation after said incubation ("post-incubation formulation"); and at least one of:iii) determining concentration of the protein antigen in the postincubation formulation and subtracting the concentration of the protein antigen in the post-incubation formulation from the concentration of the protein antigen in the pre-incubation formulation; oriv) removing the protein antigen, if any, adhered to the walls of the vial, and determining the amount of the protein antigen removed from the walls of the vial.

25. The method claim 24, wherein said formulation contains a CpG oligonucleotide.

26. The method of claim 24 or 25, wherein the concentration of the protein in said preincubation formulation and post-incubation formulation and the amount of the protein antigen removed from the walls of the vial is determined by optical density reading.

27. The method of any one of claims 24-26, wherein the vial is washed after removal of the post-incubation formulation and prior to the step of removing the protein antigen.

28. The method of claim 27, wherein said step of removing the protein antigen comprises detergent wash.

29. The method of claim 28, wherein the detergent comprises SDS (sodium dodecyl sulfate).

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