JP2012515752A - Stable vaccine composition and method of use - Google Patents

Abstract

  A stable lyophilized protein formulation comprising a recombinant protective antigen (rPA) is disclosed, which is reconstituted with a suitable diluent and used as a vaccine against anthrax infections in mammals, especially humans A highly potent protein concentration reconstituted formulation suitable for This formulation is prepared by lyophilizing rPA in the presence of a reducing sugar such as trehalose, and such lyophilization methods are also disclosed. The resulting lyophilized mixture or composition is then reconstituted to provide high immunogenicity without causing an apparent loss of stability of the rPA protein. A method of using the formulation in vaccination is also disclosed.

Description

  The present invention relates generally to the field of protein storage, and more particularly to the field of protein storage after industrial scale preparation, where the protein is part of a vaccine composition intended for administration to mammals, particularly humans. Form.

  Vaccines are usually formulated with adjuvants. A common type of adjuvant is a colloid mainly composed of aluminum, and is generally referred to as alum (alum). More specifically, these are usually aluminum hydroxide (aluminum oxyhydroxide) (also called alhydrogel) and aluminum phosphate (also called ajufos). For example, vaccines useful against life forms such as Bacillus anthracis are usually formulated with alhydrogels that bind anthrax antigens used in such vaccines (so-called subunit vaccines). The

  Recently, advances in vaccine therapy have enabled the production of various proteins for use in vaccines, including the use of recombinant protective antigen (rPA) from Bacillus anthracis using recombinant DNA technology, for example. Due to the relatively large size and complex nature of proteins, the formulation of such proteins in vaccines generally creates several unique problems. In order to maintain antigenic activity, the protein formulation must retain the conformational integrity of the protein that represents the basic antigenic structure in the vaccine. Protein degradation may be accompanied by chemical instability (eg, when bond formation or cleavage results in a new antigenic structure) or physical instability (eg, changes in protein conformation). Physical instability can result from, for example, denaturation or aggregation of proteins present in a vaccine, while chemical instability breaks the amino acid chain of a protein or one or more amino acid sides present in a protein. It can result from chemical reactions that contribute to the modification of chains (eg, groups essential for immunogenic activity).

  Drying at low temperatures (usually referred to as “lyophilization”) serves to remove water from the protein composition in question while preserving the protein. By this process, the material to be dried is first frozen and then the frozen solvent (ice in the case of water) is removed by sublimation under high vacuum. Additives may be included in the formulation prior to lyophilization to increase stability during the lyophilization process and / or improve the stability of the lyophilized product upon storage. Unfortunately, to date, such methods are unreliable for lyophilization and storage of certain antigenic proteins useful for vaccines, particularly anthrax vaccines, and the stability of lyophilized powders It has been demonstrated to result in a significant decrease in activity after reconstitution.

  The present invention addresses at least some of the conventional problems that have arisen in the lyophilization of protein formulations, particularly those comprising recombinant protected antigen bodies (rPA) of Bacillus anthracis, over a long period of time in a lyophilized state. Even storage, it is solved by providing a formulation that retains antigenic activity and therefore usefulness as a vaccine, so that the reconstituted vaccine composition is human resistant to anthrax infection. It is still very useful in protecting mammals such as

  In one aspect, the invention relates to a stable reconstituted formulation or vaccine composition, which formulation or composition may be isotonic and is from about 50 μg / ml to about 400 μg / ml. An antigen or immunogen used in the vaccine, preferably in an amount of about 200 μg / ml, preferably an anthrax antigen used in a vaccine against Bacillus anthracis, more preferably an anthrax protective antigen, most preferably A recombinant protective antigen (rPA) and a pharmaceutically acceptable carrier, the formulation is reconstituted from a lyophilized mixture or composition of Bacillus anthracis or other antigen. In one embodiment, the reconstituted formulation may include a component such as trehalose in an amount that provides lyophilization protection of the lyophilized mixture or composition.

  In one preferred embodiment of the reconstituted formulation, the formulation comprises a salt such as phosphate or NaCl or both, a detergent such as tween, and an adjuvant such as alhydrogel (alum), chitosan, MPL, or CpG. In a preferred embodiment, the adjuvant is CpG1018 or alhydrogel, particularly preferably both.

  The vaccine according to the invention may contain an adjuvant in addition to alum. Examples of such adjuvants are well known in the art and include oligonucleotides, especially so-called “CpG” oligonucleotides, phosphorothioate oligonucleotides, most preferably toll-like receptor 9 receptor. An oligonucleotide that targets the body. Other additional adjuvants that may be present include saponins, PCPP polymers, lipopolysaccharide derivatives, MPL [monophosphoryl lipid A], MDP muramyl dipeptide (MDP), t-MDP, flagellin protein, flagellin fusion protein , IC31, OM-174, Leishmania elongation factor, ISCOMS, chitosan, SB-AS2, AS02, SB-AS4, nonionic block copolymer, and SAF (Syntex adjuvant formulation).

  Where an additional adjuvant is used, it is preferably incorporated into the stabilized subunit vaccine, or alternatively, the additional adjuvant may be a separate adjuvant and / or diluted composition. May exist in the object.

  In certain embodiments, the stable subunit vaccine composition or formulation can further comprise an agent for reducing or preventing aggregation during storage, wherein the agent includes, for example, a small amount of nonionic surfactant. Agents, especially ethoxylated sorbitan esters, include, for example, polyoxyethylene sorbitan monolaurate typically containing about 20 ethyleneoxy units.

In another aspect, the present invention relates to a method for preparing a stable lyophilized vaccine formulation, comprising the following steps.
(A) forming a composition of anthrax antigen containing rPA and a lyophilized protective amount of reducing sugar, and (b) lyophilizing the composition to form a stable lyophilized vaccine composition.

  In a preferred embodiment, the lyophilized mixture or composition further comprises a buffer such as phosphate, a salt such as NaCl, a detergent such as tween, and an adjuvant such as alhydrogel (alum), chitosan, mpl, or CpG. CpG1018 and alhydrogel are particularly preferred.

  In one embodiment, the lyophilized composition further comprises a bulking agent added in an amount sufficient to impart mass to the lyophilized composition and contribute to the physical structure of the lyophilized bulk agent. .

  In a further aspect, the present invention uses a kit comprising a container holding a lyophilized mixture or composition of anthrax or other vaccine comprising rPA, trehalose, and a pharmaceutically acceptable carrier, and a diluent. A set of instructions for reconstituting the lyophilized composition to an rPA concentration of about 200 μg / ml In one embodiment, a diluent is provided in the kit. Preferably, the diluent includes an adjuvant such as alhydrogel.

  The present invention also relates to a method of treating infection in mammals that protects against microorganisms, especially bacteria, and to the therapeutic effect of the vaccine composition of the present invention on mammals that have developed or are likely to suffer from such infections. Administration of an effective amount. In further embodiments, the bacterial infection is a Bacillus anthracis infection and / or the mammal is a human.

The amino acid sequence of the recombinant protective antigen (rPA) from Bacillus anthracis (Anthrax).

-Definition-
As used herein, the term “stable” formulation refers to an antigenic protein such as rPA that retains its physical and chemical stability and integrity upon storage in the form of a lyophilized powder, solid, or cake. Say. Analytical techniques for measuring protein stability are known in the art and will not be described in detail herein. For example, Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991), and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). For example, a “stable” formulation can be one in which less than about 10%, preferably less than about 5% of the protein is present as an aggregate in the formulation. In another embodiment, any increase in aggregated formulation after lyophilization or storage of the lyophilized formulation can be measured. For example, when a “stable” lyophilized formulation is stored at 2-8 ° C. for at least about 16 months, preferably at least 9 months, more preferably 1 year or more, The increase in aggregation in the lyophilized formulation can be less than about 5%, preferably less than about 3%.

  As used herein, the term “about” means an amount that does not substantially reduce the effectiveness of the referenced ingredient by more than 10%. When the term “about” is used to refer to an amount or range, it should be understood that the invention specifically considers the same range or amount as if the term “about” was not used. That is, the range where “about” is added and the range where “about” is not added are selective embodiments of the method and the preparation of the present invention.

  The term “reconstituted” formulation in this application is a formulation prepared by dissolving a lyophilized protein formulation in a diluent and dispersing the protein in the reconstituted formulation. The reconstituted formulation is suitable for administration to a patient.

  The term “isotonic” in this application refers to a formulation or composition of the invention having substantially the same osmotic pressure as human blood. Isotonic formulations are generally considered to have an osmotic pressure from about 250 to 350 mOsm / Kg. Isotonicity can be measured using, for example, vapor pressure or an ice-making osmometer.

  The term “lioprotectant” in this application prevents chemical and / or physical instability of the protein when lyophilized and during subsequent storage when mixed with an immunogenic protein of the invention, such as rPA. By no means is meant a molecule that is substantially reduced and sufficient to maintain the immune properties of the protein, thereby ensuring continued utility as a vaccine. Examples of such lyoprotectants include non-reducing sugars such as saccharose and trehalose, amino acids such as monosodium glutamate and histidine, methylamines such as betaine, lyophilic salts such as magnesium sulfate, trivalent or higher sugars. Examples include polyols such as alcohols, such as glycerin, erythritol, glycerol, arabitol, xylitol, sorbitol, and mannitol, glycols such as propylene glycol and polyethylene glycol, and any combination thereof.

  As used herein, the term “lyophilized protective amount” substantially refers to the physical and chemical stability and integrity of an immunogen upon lyophilization and storage when added to an immunogen such as the rPA of the present invention. As a result of protection, when reconstituted, the immunogen retains its immune properties and refers to the amount of lyoprotectant such as trehalose that is well tolerated for its use in vaccines.

  The term “diluent” in this application means a carrier that is pharmaceutically acceptable (safe and non-toxic for administration to humans) and is useful in preparing reconstituted formulations. Typical diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffer (eg, phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solution.

  As used herein, the term “preservative” means a compound that can be added to a diluent to substantially reduce the bacterial action in a reconstituted formulation, thus facilitating, for example, the production of a versatile reconstituted formulation. can do. Examples include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl group is a long chain compound), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, 2-phenoxyethanol, thimerosal, benzethonium chloride, formaldehyde, butyl alcohol, benzyl alcohol, allyl parabens such as methyl paraben, propyl paraben, catechol, resorcinol, Examples include cyclohexanol, 3-pentanol, and m-cresol. The most preferred preservative in this application is 2-phenoxyethanol.

  The term “bulking agent” in this application refers to a compound that adds mass to the lyophilized mixture or composition and contributes to the physical structure of the lyophilized cake (eg, essentially maintaining an open pore structure). Promotes uniform lyophilized cake production). Exemplary bulking agents include mannitol, glycerin, dextran, polyethylene glycol, and xorbitol. “Treatment” refers to both therapeutic treatment and prophylactic or maintenance measures. Those in need of treatment include those already with the disease and those that should prevent the disease.

  As used herein, the term “effective amount” or “therapeutically effective amount” means a dose sufficient to treat, suppress, or reduce one or more symptoms of a bacterial infection, particularly anthrax infection. The exact dose will vary depending on factors such as the patient's age, immune system status, overall health, and environmental status, the nature and extent of the infection (or anticipated infection), and the availability of subsequent treatment / vaccination There are things to do.

  Antigens to be formulated are generally prepared using techniques well known in the art such as separation from organisms and synthesis by recombinant techniques or direct chemical synthesis of polypeptides used for antigens. The protective antigen used in the present invention is prepared by recombinant techniques and is referred to herein as recombinant protective antigen (rPA).

  According to the present invention, a stable reconstituted formulation is provided, which comprises, for example, a subunit vaccine antigen such as an antimicrobial antigen, an anthrax antigen comprising a recombinant protective antigen (rPA), etc. And a pharmaceutically acceptable carrier, prepared from a lyophilized composition of its anthrax antigen and a lyophilized protective amount of trehalose. In another preferred embodiment of such a formulation or composition intended for use as a vaccine, the rPA is in an amount of about 100-300 μg / ml, preferably about 150-250 μg / ml, most preferably about 200 μg / ml. In the reconstituted formulation.

  The reconstituted anthrax vaccine formulation of the present invention can also contain trehalose. In one embodiment, trehalose is in the reconstituted formulation in the range of about 3% to 7%, preferably in the range of about 4% to 6%, more preferably in the range of about 4.5% to 5.5%, most Preferably, it is present in an amount of about 5% (w / v).

  The stable reconstituted vaccine formulation of the present invention is maintained at a temperature of at least 5 ° C. prior to reconstitution for a period of at least 1 month, or at a temperature of at least 25 ° C. prior to reconstitution for a period of at least 1 month. Or a temperature of at least 30 ° C, 35 ° C, 40 ° C, 45 ° C, 50 ° C, 55 ° C, 60 ° C, 65 ° C, 70 ° C or more. The period is at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, or more, 1 year, 16 months, Or for at least two years.

  The reconstituted anthrax vaccine formulation of the present invention may further comprise a buffer. In that particular embodiment, the buffer (or salt) is a phosphate at a concentration ranging from 3 mM to 7 mM, preferably from 4 mM to 6 mM, more preferably from 4.5 mM to 5.5 mM, and from about 4 mM to A range of about 5.25 mM is particularly preferred. In another preferred embodiment, the reconstituted formulation comprises, for example, about 0.39% NaCl. The reconstituted formulation of the present invention can also include a cleaning agent such as Tween 20. In one non-limiting example, the tween 20 is present in an amount of about 0.02%.

  The reconstituted anthrax vaccine formulation of the present invention may further comprise an adjuvant, in particular one selected from alhydrogel (alum), chitosan, mpl, and CpG, wherein CpG and alhydrogel are preferable. In one embodiment, the adjuvant is CpG1018, eg, present at about 2 mg / ml. In another embodiment, the adjuvant is an alhydrogel, eg, present at about 0.26% (w / v).

  A complete but non-limiting example of such a formulation is 200 μg / ml rPA, 2 mg / ml CpG1018, 5% trehalose, 5.25 mM phosphate, 0.39% NaCl, 0.02% Tween 20, It contains 0.26% w / v alhydrogel (see Example 1). In one embodiment of the vaccine formulation of the present invention, the reconstituted formulation is isotonic.

The present invention also provides a method for preparing a stable reconstituted formulation, which comprises the following steps.
(A) lyophilizing a mixture or composition of a subunit vaccine antigen, for example an antimicrobial antigen such as anthrax antigen containing rPA, and a lyophilized protective amount of trehalose; and (b) a reconstituted formulation. The lyophilized mixture of step (a) in a diluent so as to have an rPA concentration of about 0.1 to about 10 times the rPA concentration in the mixture or composition prior to lyophilization Or reconstitute the composition.

  In another example of the method of the invention, the diluent used in step (b) for reconstituting the mixture of step (a) comprises an adjuvant, preferably Alhydrogel (alum), chitosan, An adjuvant selected from MPL or CpG, most preferably an alhydrogel. In a non-limiting example, the alhydrogel is present in the range of about 0.2% to about 0.3%, or is present in the range of about 0.25% to about 0.3%, or preferably about Present in an amount of 0.26%.

  In another such example, the diluent includes a salt such as phosphate and / or NaCl. In selected examples, the phosphate is present in the range of about 3-7 mM, or is present in the range of about 4-6 mM, or is present in the range of about 4.5-5.5 mM, or preferably, phosphate. Is present in an amount of about 5 mM. In another such example, NaCl is present in the range of about 0.3% to about 0.5%, or is present in the range of about 0.35% to about 0.45%, or preferably about 0. .39% exists. In a preferred embodiment, the diluent has a pH of about 7.4.

  In a preferred embodiment, the diluent comprises 0.26% alhydrogel and 0.39% NaCl in 5 mM phosphate and has a pH of 7.4.

  Subunit vaccines that can be used containing antimicrobial antigens are usually antigens based on recombinant proteins. Examples of such antimicrobial antigens include hepatitis B protective antigen, herpes simplex virus antigen, influenza antigen, congenital cytomegalovirus (CMV) antigen, Mycobacterium tuberculosis antigen, HIV antigen, diphtheria antigen, tetanus antigen, pertussis antigen, and Pestis protective antigens, as well as antigens containing one, two or more of the protective proteins disclosed in patent application WO 96/28551, incorporated by reference. Most preferably, the antigen is an anthrax protective antigen such as a recombinant protective antigen (rPA), especially one having the amino acid sequence of SEQ ID NO: 1.

The present invention further provides a method for preparing a stable lyophilized vaccine formulation comprising the following steps.
Forming a mixture or composition of a subunit vaccine antigen, for example an antimicrobial antigen such as anthrax antigen containing rPA and a lyophilized protective amount of reducing sugar, and (b) the mixture or composition. Lyophilize to form a stable lyophilized vaccine composition.

  In particular examples of the methods of the invention, the composition of step (a) is in the range of about 200-600 μg / ml, or about 300-500 μg / ml, or about 350-450 μg / ml, preferably about 400 μg / ml. Of rPA. In another example of the method of the present invention, the composition of step (a) is in the range of about 2-20%, alternatively about 4-18%, or about 6-16%, or about 8-14%, or Trehalose present in an amount in the range of about 8-12%, or about 9-12%, or preferably about 10% (w / v).

  In another embodiment of the method, the composition or mixture of step (a) comprises a salt, preferably phosphate and / or NaCl (or saline). In that example, the phosphate is in the range of about 0.3-0.7 mM, or in the range of about 0.35-0.65 mM, or in the range of about 0.4-0.6 mM, or about 0.45-0. It is present in the range of 55 mM, or preferably in an amount of about 0.5 mM. Further, in such examples, NaCl is present in the range of about 7-13 mM, or about 8-12 mM, or about 9-11 mM, or about 9.5-10.5 mM, or preferably in an amount of about 10 mM. Exists.

  In another example of the method of the present invention, the mixture or composition of step (a) comprises an adjuvant, preferably the adjuvant is selected from alhydrogel (alum), chitosan, MPL, or CpG. In its preferred embodiment, the adjuvant is CpG, such as CpG1018. In that example, CpG1018 is present in the range of about 2 mg / ml to about 6 mg / ml, or in the range of about 3 mg / ml to about 5 mg / ml, or in the range of about 3.5 mg / ml to about 4.5 mg / ml. Or preferably, CpG1018 is present in an amount of about 4 mg / ml.

  Examples of suitable oligonucleotides that may be employed as additional adjuvants include US Pat. Nos. 6,207,648, 6,589,940, 7,255,868, and 7,276,489, the disclosures of which are incorporated herein by reference. ).

  In certain embodiments, the stabilized vaccine composition can further comprise an agent that reduces or prevents aggregation during storage, including small amounts of nonionic surfactants, especially ethoxylated sorbitan esters. Illustratively, polyoxyethylene sorbitan monolaurate containing about 20 ethyleneoxy units is exemplified.

  In additional examples of the method, the mixture or composition of step (a) can further include a cleaning agent, such as, for example, Tween 20. In a non-limiting example, the tween 20 is present in the range of about 0.3 to about 0.5%, or is present in the range of about 0.35 to about 0.45%, or preferably about 0.04. % Present.

  In a particularly preferred embodiment of the method of the invention, the mixture or composition of step (a) comprises 400 μg / ml rPA, 4 mg / ml CpG1018, 10% trehalose, 0.5 mM phosphate, 10 mM NaCl, and 0 Contains 04% Tween 20.

  The present invention also provides a composition suitable for lyophilization, the composition comprising an anthrax antigen comprising a recombinant protective antigen (rPA) and a lyophilized protective amount of trehalose. In non-limiting embodiments of the composition, the rPA is present in the range of about 100 μg / ml to about 700 μg / ml, or present in the range of about 200-600 μg / ml, or in the range of about 250-550 μg / ml. Present, or present in the range of about 300-500 μg / ml, or present in the range of about 350-450 μg / ml, preferably present in an amount of about 400 μg / ml. In another example of the method of the present invention, the mixture or composition of step (a) is in the range of about 2-20%, or in the range of about 4-18%, or about 6-16%, or about 8-14. %, Or about 9-12%, or preferably, trehalose present in an amount of about 10% (w / v).

  In the present invention, suitably lyoprotectant is added to the formulation prior to lyophilization or the composition to be lyophilized. In a preferred embodiment, the additive is a non-reducing sugar, such as saccharose or trehalose, the latter being particularly preferred. In one embodiment, the amount of lyoprotectant added is such that upon reconstitution, the resulting formulation is isotonic. In addition, the amount such as is often generated during lyophilization of the protein, and must be sufficient to prevent undesired degradation and aggregation of an antigenic protein such as rPA.

  In a preferred embodiment of the present invention, it may be desirable to add a surfactant or detergent to the composition and / or the reconstituted formulation prior to lyophilization. Examples of useful surfactants include nonionic surfactant polysorbates such as polysorbate 20 (or Tween 20) or 80; Triton; sodium dodecyl sulfate (SDS); sodium laurel sulfate; octyl glycoside Sodium; lauryl-, mystyryl-, linoleyl-, or stearylsulfobetaine; lauryl-, mystyryl-, linoleyl-, or stearyl sarcosine; linoleyl-, myristyl-, or cetylbetaine; lauroamidopropyl-, cocamidopropyl-, Linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isosteramidopropyl-betaine (eg, lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isosteramidopropyl- Methylamine; methyl cocoyl sodium- or methyl oleyl-taurate disodium salt; Monaquato® series (Monaqua Industries, Patterson, NJ), polyethylene glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (For example, Pluronic, PF68, etc.). Tween 20 is a preferred surfactant or detergent. The amount added is sufficient to reduce aggregation of the reconstituted protein and minimize the generation of particles after reconstitution. For example, the surfactant or detergent may be present in the composition prior to lyophilization in an amount of about 0.001-0.5%, preferably about 0.005-0.05%.

  In certain embodiments of the invention, lyoprotectant (such as trehalose) can be used in combination with a bulking agent (eg, mannitol or glycerin) to prepare a pre-lyophilized formulation. The use of such extenders can facilitate the production of a more uniform lyophilized cake (ie, no excess pockets, etc.).

  In another embodiment, the composition comprises a salt, preferably phosphate and / or NaCl (or saline). In this example, the phosphate is in the range of about 0.3-0.7 mM, or in the range of about 0.35-0.65 mM, or in the range of about 0.4-0.6 mM, or about 0.45-0. It is present in the range of 55 mM, or preferably in an amount of about 0.5 mM. Further, in such examples, NaCl is present in the range of about 7-13 mM, or about 8-12 mM, or about 9-11 mM, or about 9.5-10.5 mM, or preferably in an amount of about 10 mM. Exists.

  In another example, the composition includes an adjuvant, preferably the adjuvant is selected from alhydrogel (alum), chitosan, MPL, or CpG. In its preferred embodiment, the adjuvant is CpG, such as CpG1018. In that example, CpG1018 is present in the range of about 2 mg / ml to about 6 mg / ml, or in the range of about 3 mg / ml to about 5 mg / ml, or in the range of about 3.5 mg / ml to about 4.5 mg / ml. Or preferably, CpG1018 is present in an amount of about 4 mg / ml.

  In additional examples, the composition further comprises a cleaning agent, such as, for example, Tween 20. In a non-limiting example, the tween 20 is present in the range of about 0.3 to about 0.5%, or is present in the range of about 0.35 to about 0.45%, or preferably about 0.04. % Present.

  In a particularly preferred embodiment, the present composition suitable for lyophilization comprises 400 μg / ml rPA, 4 mg / ml CpG1018, 10% trehalose, 0.5 mM phosphate, 10 mM NaCl, and 0.04% Tween 20. Including.

  Moreover, this composition provides the preparation method of such a composition, and this method fundamentally includes the procedure of the process (a) mentioned above about the method of this invention.

  The phosphate present in the composition to be lyophilized can be useful for buffering vaccine formulations produced after reconstitution at the same time as the composition. Here, the phosphate is not a limited buffer, and another buffer may be used in place of or in combination with the phosphate. Examples of such buffering agents include histidine, phosphate, tris, citrate, succinate, and other organic acids. The concentration of the buffer may be from about 1 mM to about 20 mM, or from about 3 mM to about 15 mM, depending on, for example, the buffer and the desired isotonicity of the vaccine formulation (eg, the reconstituted formulation). In addition to trehalose, such a buffer may provide a freeze-drying protective effect.

Moreover, this invention provides the kit containing the following.
(A) a container holding a lyophilized composition of anthrax vaccine comprising rPA, trehalose, and a pharmaceutically acceptable carrier, and (b) lyophilized to an rPA concentration of about 200 μg / ml using a diluent Instructions for reconstituting the composition.

  Such a kit can further include a container of a suitable diluent for reconstitution of the lyophilized composition. Specific embodiments of such diluents are described with reference to the diluent used in the method of the present invention.

  The kits of the present invention are typically lyophilized compositions in vials or other suitable containers such as bottles, vials (eg, dual chamber vials), syringes (eg, dual chamber syringes), and test tubes. To accommodate. The container can be formed from a variety of materials such as glass and plastic. The container holds the lyophilized formulation and the label thereon, or concomitantly, the container can provide instructions for reconstitution and / or use. For example, the label can indicate that the lyophilized formulation is reconstituted to a protein concentration as described above. In addition, the label can indicate that the formulation is intended or useful for subcutaneous administration. The container holding the formulation can also be a versatile vial that allows repeated administration of the reconstituted formulation. A kit that is an article of manufacture can further comprise a second container containing a suitable diluent of the type disclosed herein. When the diluent and lyophilized formulation are mixed, the final rPA concentration in the reconstituted formulation is generally in the above range, but is generally considered to be about 200 μg / ml. The kit can further include other materials desired from a commercial and user perspective, such as additional cushioning materials, diluents, filters, needles, syringes, and inserts containing instructions for use.

  In general, vaccines are prepared as injectables in the form of aqueous solutions or suspensions. Oil-based vaccines are well known for inhalation. It is also possible to formulate solids that are dissolved or suspended before use. Pharmaceutically acceptable carriers, diluents and excipients that are compatible with the active ingredient and suitable for pharmaceutical use are generally added.

  Pharmaceutical compositions and / or formulations useful in the present invention include a pharmaceutically acceptable carrier that includes any suitable diluent or excipient that is detrimental to the individual receiving the composition. Any pharmaceutical agent that does not cause production of the antibody itself and can be administered without undue toxicity is included. Pharmaceutically acceptable carriers include, but are not limited to, liquids such as water, saline, glycerol, and ethanol, as well as sprays for nasal and other airway delivery and delivery to the eye system Useful carriers are included. A detailed description of pharmaceutically acceptable carriers, diluents and excipients is given in REMINGTON'S PHARMACEUTICAL SCIENCES (Mac Publishing, NJ, current edition). Those described therein are particularly preferred for use in the formulations, compositions, methods, and kits of the present invention.

  The vaccine composition can further incorporate additional materials that stabilize the pH, function as adjuvants, wetting agents, or emulsifiers and help improve the effectiveness of the vaccine.

  Vaccines are generally formulated for parenteral administration and injected subcutaneously or intramuscularly. Such vaccines can also be formulated for use as suppositories or for oral administration or administration via the nasal or respiratory tract using methods known in the art.

  The amount of vaccine sufficient to confer immunity against pathogens, viruses, or other microorganisms can be determined by methods well known to those skilled in the art, after reviewing the description provided herein. This amount will be determined based on the vaccination characteristics and the required level of immunity (as described above). When the vaccine is administered by subcutaneous or intramuscular injection, purified protein in the range of 0.5-500 μg is given. Useful for the present invention provides about 1 μg, optionally 10 μg, even 50 μg, most often 100 μg, up to 500 μg of immunogenic protein, or immunogenic polypeptide, or an immunogenic active fragment thereof The dose is usually sufficient. Multiple of these substances may also be present in the vaccine. That is, a plurality of antigen structures can be used and can be used in the preparation of a vaccine or vaccine composition used in the method disclosed in the present application. This may include multiple individually immunogenic proteins or polypeptides, or proteins or polypeptides that exhibit immunogenic activity only when combined, and are present in the same or ratio quantitatively at each concentration. May be formulated as such, limited or non-limiting.

  The vaccine composition used in the process disclosed herein comprises one or more immunogenic proteins, one or more immunogenic polypeptides, and / or antigenic fragments of the immunogenic proteins or polypeptides. One or more immunogenic active immunogens can be included, and the fragments are present in any proportion selected by the use of the present invention. The exact components and respective amounts that make up the vaccines and vaccine compositions useful in the methods of the present invention determine, inter alia, the nature of the disease to be treated or prevented, the severity of symptoms already present, age, sex, and It is determined by the recipient's overall health and the personal and professional experience and intentions of researchers and / or physicians using these methods.

  With respect to the vaccine of the present invention, such as a subunit vaccine used against anthrax, as a specific non-limiting example of this vaccine composition, rPA is present at about 10-300 μg / ml, preferably 50-300 μg / ml Or rPA is present in an amount of about 100-300 μg / ml, more preferably about 150-250 μg / ml, and most preferably rPA is present in an amount of about 200 μg / ml.

  In another example. The dose of antigen administered, preferably rPA, is at least about 5 μg, or at least about 10 μg, or at least about 25 μg, or at least about 50 μg, or at least about 75 μg, or at least about 100 μg, or at least about 150 μg, or at least about 200 μg, the preferred dose is about 100 μg. A preferred dose volume is about 0.5 ml.

  The present invention further provides a purified antigen, preferably an anthrax antigen, more preferably an anthrax protective antigen (PA), most preferably a recombinant anthrax, bound to an adjuvant, preferably an adjuvant based on alum. It relates to a vaccine comprising a fungus protective antigen (rPA) (for example described in WO 2007/122373 published on Nov. 1, 2007, the disclosure of which is also incorporated herein by reference in its entirety). In a preferred embodiment, the present invention relates to a subunit vaccine comprising rPA conjugated to alum and at least about 2 mM phosphate salt, having a pH of about 7.1.

  Vaccine formulations used for in vivo administration are usually sterile, which can be achieved by filtration through a sterile filtration membrane before or after lyophilization and reconstitution. Sterilization of the entire composition is one non-limiting example, but can be achieved by autoclaving ingredients other than protein at about 120 ° C. for about 30 minutes or less.

  For the actual lyophilization procedure itself, various commercially available devices such as Hull 50 (trademark, Hull, USA), GT20 (trademark, Raybolt Heraois, Germany), BOC Edward, Christo GTS, FTS Royster freeze dryer are used. Is possible. Lyophilization is accomplished by freezing the formulation and then sublimating ice from the frozen material at a temperature suitable for primary drying. Under this condition, the product temperature is below the eutectic or disintegration temperature of the formulation. Typically, the primary drying shelf temperature is considered to be in the range of about -30 to 25 ° C at a suitable pressure typically in the range of about 50 to 250 millitorr (if the product remains frozen during primary drying). To do). The formulation, the size and type of the container holding the sample (eg, a glass vial), and the volume of the liquid are thought to primarily affect the time required for drying, which may be several hours to several days (eg, , 40-60 hours). The secondary drying stage can be performed at about 0-40 ° C. and depends mainly on the type and size of the container and the type of protein used. It has now been found that a secondary drying step can be unnecessary. For example, the shelf temperature throughout the entire water removal stage of lyophilization may be about 15-30 ° C. (eg, about 20 ° C.). The temperature and pressure required for secondary drying is believed to produce a suitable lyophilized cake and depends on, for example, temperature and other parameters. The secondary drying time is affected by the desired residual moisture level in the product and typically takes at least about 5 hours (eg, 10-15 hours). The pressure may be the same as that used in the primary drying process. Freeze-drying conditions may vary depending on the formulation and vial size. The resulting lyophilized powder will normally have a moisture content of less than about 5%, preferably less than about 3%.

  Examples of freeze-dry cycles useful in the method of the present invention are shown in Tables 1 and 2.

  In each cycle, the product is plugged before introducing air into the chamber. The aluminum seal is then applied to the vial and crimped when removed from the lyophilizer.

  The reconstituted formulation can be administered to a patient in need of treatment with the protein, preferably a human, by known methods, such as intravenous administration such as rapid administration or continuous infusion over time, and intramuscularly. , Intraperitoneal, intracerebral spinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical, or by inhalation route.

  Lyophilized vaccine compositions prepared by the methods of the invention are generally maintained in a lyophilized state by the time of use. When ready for administration to a patient, the lyophilized formulation is reconstituted with the diluents described herein.

  Reconstitution is usually performed at room temperature to ensure complete hydration, although other temperatures may be employed if desired. The time required for reconstitution will depend on, for example, the type of diluent, the amount of excipients, and a protein such as rPA. Examples of diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffer (eg, phosphate buffered saline is particularly preferred), sterile saline, Ringer's solution, or dextrose solution. The diluent optionally includes a preservative. Aromatic alcohols such as benzyl alcohol or phenol alcohol are preferred preservatives. The amount of preservative used is determined by assessing the concentration of various preservatives for protein compatibility and storage efficacy testing. For example, when the preservative is an aromatic alcohol (eg, benzyl alcohol), it is about 0.1-2.0%, preferably about 0.5-1.5%, most preferably about 1.0-1.2. % Can be present.

  The present invention further provides a method for protecting against or treating a microbial infection, preferably a bacterial infection, and administering to the mammal at risk of such infection a therapeutically effective amount of the stable pressure vaccine formulation described above. Including doing. The infecting organism may be any one or more of those described herein, and the formulation is reconstituted from the formulation described herein and / or a vaccine composition or mixture lyophilized by the method of the present invention. It is a formulation. In a preferred embodiment, the bacterial infection is an anthrax infection (ie caused by Bacillus anthracis). In one example, the animal to be protected is a mammal, especially a human patient. A preferred formulation for use in such treatments is 200 μg / ml rPA, 2 mg / ml CpG1018, 5% trehalose, 5.25 mM phosphate, 0.39% NaCl, 0.02% Tween 20, 0.02. A vaccine composition comprising 26% w / v alhydrogel.

  In practicing the procedure of the present invention, references to specific buffers, media, reagents, etc. are, of course, not intended to be limiting, and those skilled in the art will, in the specific context of the description, It should be understood to encompass all relevant materials that are of interest or value to be recognized. For example, replacing one buffer system with another often gives similar, if not identical, results. Those skilled in the art will be familiar with such systems and possible methodologies and, without undue experimentation, if the material is not essential to the invention, the methods and procedures disclosed herein. It is considered that an alternative that performs the purpose optimally can be performed by using.

  The invention is explained in more detail by the following non-limiting examples. These methods and examples are in no way intended to limit the invention to that embodiment, and alternative embodiments and applications will probably suggest themselves to those skilled in the art.

Example 1. Stability of Trehalose-Containing Lyophilized Vaccine 400 μg / ml rPA, 4 mg / ml CpG1018 in 10% trehalose, 0.5 mM phosphate, 10 mM NaCl, 0.04% Tween 20 was lyophilized. This formulation composition was twice the concentration of reconstituted material prior to lyophilization. The formulation was then reconstituted with 0.26% alhydrogel, 0.39% saline, pH 7.4 in 5 mM phosphate. The reconstituted formulation consisted of 200 μg / ml rPA, 2 mg / ml CpG1018, 5.25 mM phosphate, 0.39% NaCl, 0.02% Tween 20, 0.26% in 5% trehalose. Alhydrogel. All% values are w / v.

  At 70 ° C, rPA retained efficacy after as many as 8 months. Although 70 ° C is higher than Tg (glass transition temperature, the temperature at which trehalose sugar glass begins to soften and is no longer brittle), it was expected that high degradation would be seen, but such accelerated decomposition was observed. There wasn't. That is, the lyophilized formulation was unexpectedly stable in the presence of trehalose.

Example 2 Stability of lyophilized vaccine Female adult A / J mice were immunized by intramuscular injection (im) on day 0 using 0.1 ml of each sample shown in Table 3, where each sample Was formulated with a diluent prior to administration. All mice were exposed to S. anthracis STI strain spores for 21 days and survival was determined on day 11.

The results in Table 3 show that lyophilized vaccines show much higher stability than expected from previous experience with such vaccines (eg, compare storage results at 5 ° C and 70 ° C). . The diluent is 0.26% (w / w) alhydrogel (0.4% Al (OH) ₃ ) in 0.39% (w / v) sodium chloride buffered with 5 mM phosphate at pH 7.4. Made up of. Untreated animals were negative controls and received no treatment but received 21 days of exposure at the same spore dose as the test animals.

Claims (59)

  A stable reconstituted vaccine formulation comprising an anthrax antigen and a pharmaceutically acceptable carrier, prepared from the lyophilized composition of said anthrax antigen and a lyophilized protective amount of a non-reducing sugar Reconstituted vaccine formulation.   The stable reconstituted vaccine formulation of claim 1, wherein the anthrax antigen comprises a recombinant anthrax protective antigen (rPA).   The stable reconstituted vaccine formulation of claim 2, wherein the formulation retains substantially the same efficacy as the composition prior to the lyophilization.   The stable reconstituted vaccine formulation of claim 1, wherein the formulation retains at least 80% potency of the composition prior to lyophilization.   2. The stable reconstituted vaccine formulation of claim 1, wherein the formulation retains at least 85% efficacy of the composition prior to lyophilization.   The stable reconstituted vaccine formulation of claim 1, wherein the formulation retains at least 90% efficacy of the composition prior to lyophilization.   2. The stable reconstituted vaccine formulation of claim 1, wherein the formulation retains at least 95% efficacy of the composition prior to lyophilization.   4. The stable reconstituted vaccine formulation of claim 3, wherein the rPA is present in the reconstituted formulation in the range of about 100-300 [mu] g / ml.   4. The stable reconstituted vaccine formulation of claim 3, wherein the rPA is present in the reconstituted formulation in the range of about 150-250 [mu] g / ml.   4. The stable reconstituted vaccine formulation of claim 3, wherein the rPA is present in the reconstituted formulation in an amount of about 200 [mu] g / ml.   4. A stable reconstituted vaccine formulation according to claim 3, wherein the non-reducing sugar is trehalose.   12. The stable reconstituted vaccine formulation of claim 11, wherein the trehalose is present in the reconstituted formulation in the range of about 3% to 7% (w / v).   12. The stable reconstituted vaccine formulation of claim 11, wherein the trehalose is present in the range of about 4% to 6% (w / v).   12. The stable reconstituted vaccine formulation of claim 11, wherein the trehalose is present in the range of about 4.5% to 5.5% (w / v).   12. The stable reconstituted vaccine formulation of claim 11, wherein the trehalose is present in the reconstituted formulation in an amount of about 5% (w / v).   4. The stable reconstituted vaccine formulation of claim 3, wherein the lyophilized composition has been maintained at a temperature of at least 25 ° C. for a period of at least 3 months prior to reconstitution.   17. A stable reconstituted vaccine formulation according to claim 16, wherein the temperature is at least 40 ° C.   17. The stable reconstituted vaccine formulation of claim 16, wherein the temperature is at least 55 ° C.   17. The stable reconstituted vaccine formulation of claim 16, wherein the temperature is at least 70 ° C.   17. A stable reconstituted vaccine formulation according to claim 16, wherein the period is at least 3 months.   17. A stable reconstituted vaccine formulation according to claim 16, wherein the period is at least 7 months.   17. A stable reconstituted vaccine formulation according to claim 16, wherein the period is at least 8 months.   17. A stable reconstituted vaccine formulation according to claim 16, wherein the period is at least 16 months.   4. The stable reconstituted vaccine formulation of claim 3, wherein the stable formulation comprises an adjuvant.   25. The stable reconstituted vaccine formulation of claim 24, wherein the adjuvant is selected from alhydrogel (alum), chitosan, mpl, and CpG.   25. The stable reconstituted vaccine formulation of claim 24, wherein the adjuvant is alhydrogel (alum).   The reconstituted formulation was 200 μg / ml rPA, 2 mg / ml CpG1018, 5% trehalose, 5.25 mM phosphate, 0.39% NaCl, 0.02% Tween 20, and 0.26. 4. The stable reconstituted vaccine formulation of claim 3 comprising% w / v alhydrogel. The following steps:
(A) forming a composition of anthrax antigen containing rPA and a lyophilized protective amount of reducing sugar, and (b) lyophilizing said composition to form a stable lyophilized vaccine composition.
A process for the preparation of a stable lyophilized vaccine formulation.   30. The method of claim 28, wherein the composition of step (a) comprises rPA in the range of about 300-500 [mu] g / ml.   30. The method of claim 28, wherein the composition of step (a) comprises rPA in the range of about 350 to 450 [mu] g / ml.   30. The method of claim 28, wherein the composition of step (a) comprises rPA in an amount of about 400 [mu] g / ml.   29. The method of claim 28, wherein the reducing sugar in step (a) is trehalose.   30. The method of claim 28, wherein the trehalose is present in the range of about 8-12%.   30. The method of claim 28, wherein the trehalose is present in an amount of about 10% (w / v).   30. The method of claim 28, wherein the composition of step (a) comprises an adjuvant.   29. The method of claim 28, wherein the adjuvant is selected from the group consisting of alhydrogel (alum), chitosan, mpl, and CpG.   29. The method of claim 28, wherein the adjuvant is alhydrogel (alum).   30. The method of claim 28, wherein the composition of step (a) further comprises CpG.   40. The method of claim 38, wherein the CpG is present in an amount of about 4 mg / ml.   30. The method of claim 28, wherein the composition of step (a) further comprises a phosphate buffer.   41. The method of claim 40, wherein the phosphate is present in an amount of about 0.5 mM.   30. The method of claim 28, wherein the composition of step (a) further comprises a cleaning agent.   43. The method of claim 42, wherein the cleaning agent is Tween 20.   44. The method of claim 43, wherein the tween 20 is present in an amount of about 0.04%.   29. The composition of step (a) comprises 400 μg / ml rPA, 4 mg / ml CpG1018, 10% trehalose, 0.5 mM phosphate, 10 mM NaCl, and 0.04% Tween 20. The method described in 1.   A method for protection against microbial infection comprising administering to a mammal at risk of microbial infection a therapeutically effective amount of the stable vaccine formulation of claim 1.   48. The method of claim 46, wherein the microbial infection is an anthrax infection.   48. The method of claim 47, wherein the mammal is a human.   48. The method of claim 47, wherein the formulation is the stable vaccine formulation of claim 27.   29. A method of protection against microbial infection comprising administering to a mammal at risk of microbial infection a therapeutically effective amount of a stable vaccine formulation reconstituted from a lyophilized vaccine composition according to the method of claim 28.   51. The method of claim 50, wherein the microbial infection is an anthrax infection.   51. The method of claim 50, wherein the mammal is a human.   A method of treating a microbial infection in a mammal, comprising administering to the mammal infected with the microorganism a therapeutically effective amount of the stable vaccine formulation of claim 1.   54. The method of claim 53, wherein the microbial infection is an anthrax infection.   54. The method of claim 53, wherein the mammal is a human.   54. The method of claim 53, wherein the vaccine is the stable vaccine formulation of claim 27.   30. A method for treating microbial infection comprising administering to a microbially infected mammal a therapeutically effective amount of a stable vaccine formulation reconstituted from a lyophilized vaccine composition according to the method of claim 28.   58. The method of claim 57, wherein the microbial infection is an anthrax infection.   58. The method of claim 57, wherein the mammal is a human.

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