HK1185001A - Vaccine formulations - Google Patents

Description

Vaccine formulations

Background

Pneumococcal disease caused by the bacterium Streptococcus pneumoniae (also known as pneumococcus) is one of the more important bacterial pathogens worldwide. The burden of the disease is high in children under 5 years of age in developing countries without vaccines. Pneumococcal disease is a complex disease group, including invasive infections such as bacteremia/sepsis, meningitis, pneumonia and otitis media, which affect both children and adults. Prevnar 13 (also referred to as "Prevenar 13", and herein as "Prev (e) nar 13") is from 13 pneumococcal serotypes (1,3,4,5,6A,6B,7F, 9V, 14,18C,19A,19F, and 23F), respectively, and CRM₁₉(cross-reactive material from Corynebacterium diphtheriae (Corynebacterium diphtheria) mutants). Prev (e) nar13 is recommended for active immunization of infants and young children to provide broad serotype coverage for any pneumococcal conjugate vaccine. In particular, serotype 19A in prev (e) nar13 is prevalent in many parts of the world and is often associated with antibiotic resistance. See, e.g., WO2006/110381, WO2008/079653, WO2008/079732, WO2008/143709, and references cited therein.

Thimerosal (also known as sodium Thimerosal, merthiolate) is an ethylmercuric-containing preservative that has been added to many multi-dose injectable formulations and topical solutions since the early 20 th 30 s to protect them from potential contamination during the exposure period and when multiple individuals are administered. Thimerosal continues to be administered as part of forced immunization as well as in other pharmaceutical products in the united states and in other countries of the world. It is claimed to be an effective preservative for eliminating potential contaminating bacteria during multiple uses of the product in the vaccine field, with minimal interaction with the antigenic structure and properties of the vaccine. Due to increasing debate about the potential safety issues and side effects of ethyl mercury on brain development in infants and young adults, several organizations have begun to suggest identifying alternative preservatives with lower or negligible safety risks. In 1999, review by the U.S. food and drug administration mandated by the U.S. congress found that some infants may receive more mercury from vaccines than what is considered acceptable under some national guidelines. The American Academy of Pediatrics (AAP) and the U.S. public health agency (usps) have signed a joint statement regarding thimerosal in a vaccine, and then the AAP issues a provisional report to clinicians suggesting that thimerosal be removed from the vaccine as soon as possible while maintaining efforts to ensure that high levels of vaccination continue to be performed globally without affecting safety.

The need to add preservatives to the vaccine can be reduced or eliminated by preparing and using only a single dose of the vaccine formulation. However, the use of a single dose, preservative-free formulation increases the overall cost of vaccination and compromises the effectiveness of the immunization program in developing countries. Furthermore, complete removal of preservatives from multi-dose vials is not considered a priority, particularly in countries with limited refrigeration conditions and undesirable Health care standards (Drain et al, Bull World Health organization 81(10): 726-,The Hazards of Immunizationathlone Press, London. pp.75-78 (1967). Thus, while multi-dose vials appear to be most suitable for producing a less expensive vaccine, it is desirable to formulate a multi-dose vaccine with at least one preservative in order to protect an individual from infection by microorganisms that inadvertently introduce the vaccine during multiple uses or after one or more non-sterile events. However, the efficacy of a preservative in combating bacterial and other microbial contamination must be balanced against the effect of the particular preservative on the immunogenicity of the selected immunogenic composition and on the long-term stability of each different antigenic determinant. Compatibility of the prev (e) nar13 formulations with preservatives has not been previously discussed. It would be desirable to prepare an optimized formulation comprising at least one preservative that protects and/or stabilizes antigenic determinants of pneumococcal antigen serotypes present in prev (e) nar 13.

Summary of The Invention

In a first aspect, the present invention provides a multivalent immunogenic composition comprising a plurality of capsular polysaccharides from streptococcus pneumoniae serotypes and 2-phenoxyethanol (2-PE). In certain embodiments, the capsular polysaccharide is from one or more streptococcus pneumoniae serotypes selected from 1,3,4,5,6A,6B,7F, 9V, 14,18C,19A,19F and 23F. In certain embodiments, the capsular polysaccharides are from seven or more streptococcus pneumoniae serotypes selected from 1,3,4,5,6A,6B,7F, 9V, 14,18C,19A,19F and 23F. In certain embodiments, the capsular polysaccharide is from each of streptococcus pneumoniae serotypes 1,3,4,5,6A,6B,7F, 9V, 14,18C,19A,19F, and 23F.

In certain embodiments of the invention, the composition comprises 2-PE at a concentration of 7mg/mL to 15mg/mL, about 10mg/mL, not less than 7mg/mL, not less than 10mg/mL, or not less than 15 mg/mL.

In certain embodiments, the immunogenic compositions of the invention may further comprise one or more of adjuvants, buffers, cryoprotectants, salts, divalent cations, nonionic detergents, and inhibitors of free radical oxidation. In certain embodiments, the adjuvant is aluminum phosphate.

Preferred multivalent immunogenic compositions of the invention are conjugated to CRM separately₁₉Of (a) from serotypes 1,3,4,5,6A,6B,7F, 9V, 14,18C,19A,19F and 23F, wherein the multivalent immunogenic composition is formulated in a sterile liquid to comprise: about 4.4 μ g/mL of each polysaccharide, except about 8.8 μ g/mL of 6B; about 58. mu.g/mL CRM₁₉₇A carrier protein; about 0.25mg/mL of elemental aluminum in the form of aluminum phosphate; about 0.85% sodium chloride; about 0.02% polysorbate 80; about 5mM sodium succinate buffer at pH 5.8; and about 10mg/mL of 2-phenoxyethanol.

In certain embodiments of the invention, the antigenicity of the immunogenic composition is stable at a temperature of 2-8 ℃, 20-25 ℃ or 37 ℃ for not less than 1 year, 1.5 years, 2 years or 2.5 years.

In certain embodiments of the invention, the concentration of one or more microorganisms decreases over time after the immunogenic composition is inoculated with the microorganisms. In certain embodiments, the composition exhibits a log reduction of at least 1.0 from the initial microbial count at 24 hours (log reduction), at 7 days at least 3.0 from the previously measured value, and after 28 days, exhibits a log increase of no more than 0.5 from the previously measured value (log increate) after inoculation with the one or more bacterial strains in certain embodiments, the composition exhibits a log reduction of at least 2.0 from the initial calculated count at 6 hours, at 24 hours at least 3.0 from the previously measured value, and no recovery at 28 days after inoculation with the one or more bacterial strains. The microbial strain includes one or more strains selected from the group consisting of pseudomonas aeruginosa (p.aeruginosa), staphylococcus aureus (s.aureus), escherichia coli (e.coli), and bacillus subtilis (b.subtilis).

In certain embodiments, the immunogenic composition is inoculated multiple times. In certain embodiments, the second inoculation occurs 6 hours after the first inoculation, the third inoculation occurs 24 hours after the first inoculation, the third inoculation occurs 7 days after the first inoculation, and the fourth inoculation occurs 14 days after the first inoculation.

In a second aspect, the invention also provides a vial containing a multivalent immunogenic composition of the invention. The vial may contain a single dose or multiple doses of the immunogenic composition. The present invention also provides a pre-filled vaccine delivery device comprising a multivalent immunogenic composition of the invention. In certain embodiments, the pre-filled vaccine delivery device is or comprises a syringe. The vaccine delivery device of the present invention may comprise a dual or multi-chamber syringe or vial or a combination thereof. In certain embodiments, the prefilled vaccine delivery device comprises a multivalent immunogenic composition formulated for intramuscular or subcutaneous injection.

In a third aspect, the invention also provides a kit for preparing an immunogenic composition of the invention, wherein the kit comprises (i) a plurality of capsular polysaccharides in a composition in lyophilized form, and (ii) an aqueous material for reconstitution of component (i) so as to provide an aqueous composition.

In a fourth aspect, the invention provides a multi-dose vaccine comprising four doses of the vaccine in vials, each dose comprising 4 to 20mg/mL, preferably 10mg/mL, of 2-phenoxyethanol, wherein one dose is 0.5mL of the vaccine.

In a fifth aspect, the invention also provides a method of measuring the efficacy of a vaccine formulation comprising one or more selected preservatives in the presence of some or all of the immunogenic and non-immunogenic components of the vaccine composition, wherein the test comprises at least the following two steps: inoculating a test composition with a selected population of microorganisms, and comparing the log reduction of the inoculated microorganism or microorganisms over time and under particular environmental conditions (e.g., temperature) to the log reduction in a control composition lacking the test preservative.

Brief Description of Drawings

Figure 1-efficacy of thimerosal as a vaccine preservative in various formulations.

Figure 2-2-phenoxyethanol (2-PE) efficacy and stability as a vaccine preservative in various formulations and at different concentrations.

Figure 3-time course of microbial colony counts reduction in preservative-free prev (e) nar13 vaccine formulations at 20-25 ℃ after a single microbial challenge (expressed as mean log of time to challenge at t =0, 6 hours, 24 hours, 7 days, 14 days, and 28 days₁₀Change).

FIG. 4-time course of microbial colony counts reduction in Prev (e) nar13 vaccine formulations containing 0.01% thimerosal at 20-25 deg.C after a single microbial challenge (expressed as mean log of time to challenge at t =0, 6 hours, 24 hours, 7 days, 14 days, and 28 days₁₀Change).

FIG. 5-Single passThe time course of microbial colony counts reduction in the 0.02% thimerosal-containing prev (e) nar13 vaccine formulations at 20-25 ℃ after microbial challenge (expressed as the mean log of time to challenge at t =0, 6 hours, 24 hours, 7 days, 14 days and 28 days compared to time of challenge₁₀Change).

FIG. 6-time course of microbial colony counts decrease in saline containing 0.02% thimerosal at 20-25 deg.C after a single microbial challenge (expressed as mean log of time to challenge at t =0, 6 hours, 24 hours, 7 days, 14 days, and 28 days)₁₀Change).

FIG. 7-time course of microbial colony counts reduction in Prev (e) nar13 vaccine formulations containing 5mg/0.5mL 2-phenoxyethanol at 20-25 deg.C after a single microbial challenge (expressed as mean log of time to challenge at t =0, 6 hours, 24 hours, 7 days, 14 days, and 28 days₁₀Change).

Figure 8-time course of microbial colony count reduction in preservative-free prev (e) nar13 vaccine formulations at t =0, 6 hours, 24 hours, 7 days, 14 days and 28 days at (a) 22-24 ℃ or at (B) 2-8 ℃ after multiple microbial challenge (expressed as mean log of time to challenge at t =0, 6 hours, 24 hours, 7 days, 14 days and 28 days₁₀Change).

FIG. 9-time course of microbial colony count reduction in Prev (e) nar13 vaccine formulations containing 0.01% thimerosal at t =0, 6 hours, 24 hours, 7 days, 14 days and 28 days at (A) 22-24 ℃ or at (B) 2-8 ℃ after multiple microbial challenge (expressed as mean log of time to challenge at t =0, 6 hours, 24 hours, 7 days, 14 days and 28 days₁₀Change).

FIG. 10-time course of microbial colony count reduction in Prev (e) nar13 vaccine formulations containing 0.02% thimerosal at t =0, 6 hours, 24 hours, 7 days and 14 days at (A) 22-24 ℃ or at (B) 2-8 ℃ after multiple microbial challenge (expressed as mean log of time to challenge at t =0, 6 hours, 24 hours, 7 days, 14 days and 28 days₁₀Change).

FIG. 11-time course of microbial colony counts decrease in saline containing 0.02% thimerosal at t =0, 6 hours, 24 hours, 7 days and 14 days at (A) 22-24 deg.C or at (B) 2-8 deg.C after multiple microbial challenge (expressed as mean log of time of challenge versus time of challenge at t =0, 6 hours, 24 hours, 7 days, 14 days and 28 days₁₀Change).

FIG. 12-non-linear regression analysis of Staphylococcus aureus depletion in various challenge studies.

FIG. 13-2-comparison of PE and Thimerosal as vaccine preservatives against single or multiple microbial challenge: pass or fail EP 5.1.3 standard B.

Figure 14-antigenicity of streptococcus pneumoniae polysaccharide preparations from each serotype long term stability in prev (e) nar13 formulated with 5mg 2-PE.

FIG. 15-2-PE Long term stability in Prev (e) nar13 vaccine formulations.

Detailed Description

Percent concentration as used herein is a mass to volume ratio (w/v) or a mass to mass ratio (w/w).

Unless otherwise specified, "dose" refers to a vaccine dose of 0.5 mL.

The term "multiple dose" refers to a composition comprising more than one dose of a vaccine, which may be administered to an individual or multiple individuals in different administration steps or over time.

The present invention provides multivalent immunogenic compositions comprising a plurality of capsular polysaccharides from streptococcus pneumoniae (also known as pneumococcus) serotypes and a preservative. The composition may also be referred to as a vaccine and is used to induce an immune response against pneumococci and to protect an individual, e.g. a human individual, preferably a human child or infant, from infection.

Any of a variety of streptococcus pneumoniae capsular polysaccharides are suitable for use in the compositions of the invention. In certain embodiments of the invention, the multivalent immunogenic composition comprises capsular polysaccharides prepared from streptococcus pneumoniae serotypes 4,6B,9V, 14,18C,19F and 23F. In certain embodiments, the capsular polysaccharide is prepared from serotypes 4,6B,9V, 14,18C,19F, 23F and at least one additional streptococcus pneumoniae serotype. In certain embodiments, the capsular polysaccharides are prepared from at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 serotypes selected from streptococcus pneumoniae serotypes 1,4,5,6B,7F, 9V, 14,18C,19F, and 23F. In certain embodiments, the capsular polysaccharides are prepared from streptococcus pneumoniae serotypes 1,3,4,5,6A,6B,7F, 9V, 14,18C,19A,19F, and 23F. The capsular polysaccharides of the invention are prepared from streptococcus pneumoniae serotypes using known techniques. See, e.g., international patent applications WO2006/110381, WO2008/079653, WO2008/079732, and WO2008/143709, each of which is incorporated herein by reference.

In certain embodiments of the invention, the capsular polysaccharide is conjugated to a carrier protein. These pneumococcal conjugates can be prepared separately. For example, in one embodiment, each pneumococcal polysaccharide serotype is grown in soy medium. The individual polysaccharides are then purified by centrifugation, precipitation, ultrafiltration and column chromatography. The purified polysaccharide is chemically activated so that the saccharide can react with the selected carrier protein to form a pneumococcal conjugate.

Once activated, each capsular polysaccharide is conjugated to a carrier protein to form a glycoconjugate, respectively. In certain embodiments, each different capsular polysaccharide is conjugated to the same carrier protein. In such embodiments, conjugation can be achieved by, for example, reductive amination.

Chemical activation of the polysaccharide and subsequent conjugation to the carrier protein is accomplished by conventional methods. See, for example, U.S. Pat. nos. 4,673,574 and 4,902,506, which are incorporated herein by reference.

The carrier protein is preferably a protein that is non-toxic and non-reactogenic and that can be obtained in sufficient quantity and purity. The carrier protein should be amenable to standard conjugation procedures. In certain embodiments of the invention, CRM₁₉₇Used as a carrier protein.

CRM₁₉₇(Pfizer, Sanford, NC) is a strain derived from Corynebacterium diphtheriae (Corynebacterium diphtheria) strain C7 (CRM) grown in casamino acid and yeast extract medium₁₉₇) A non-toxic variant of diphtheria toxin (i.e. toxoid) isolated from the culture. CRM₁₉₇Purification by ultrafiltration, ammonium sulfate precipitation and ion exchange chromatography. Alternatively, CRM is recombinantly produced, e.g., according to U.S. Pat. No. 5,614,382, which is incorporated herein by reference₁₉₇. Other diphtheria toxoids are also suitable as carrier proteins.

Other suitable carrier proteins include inactivated bacterial toxins such as tetanus toxoid, pertussis toxoid, cholera toxoid (e.g., as described in international patent application WO 2004/083251), escherichia coli LT, escherichia coli ST, and exotoxin a from pseudomonas aeruginosa. Bacterial outer membrane proteins such as outer membrane complex C (ompc), porins, transferrin binding proteins, pneumolysin, pneumococcal surface protein a (pspa), pneumococcal adhesion protein (PsaA), C5a peptidase from group a or group B streptococci, or haemophilus influenzae (haemophilus fluuenzae) protein D may also be utilized. Other proteins such as ovalbumin, Keyhole Limpet Hemocyanin (KLH), Bovine Serum Albumin (BSA), or purified protein derivatives of tuberculin (PPD) may also be used as carrier proteins.

After conjugation of the capsular polysaccharide to the carrier protein, the polysaccharide-protein conjugate is purified (i.e., enriched in the amount of polysaccharide-protein conjugate) by a variety of techniques. These techniques include concentration/diafiltration operations, precipitation/elution, column chromatography and depth filtration.

As discussed in more detail below, the immunogenic compositions of the invention comprise at least one preservative for the preparation of multi-dose vaccine formulations, which have advantageous properties in terms of long-term stability of one or more antigenic determinants of the multivalent pneumococcal capsular polysaccharide-protein conjugate, and which advantageously protect the composition from contamination by conferring resistance to one or more microorganisms prior to administration to an individual in need thereof.

Additional formulations of the immunogenic compositions of the invention comprising preservatives may be obtained using art-recognized methods. For example, thirteen individual pneumococcal conjugates can be formulated with a physiologically acceptable carrier to prepare a composition. Examples of such carriers include, but are not limited to, water, buffer salts, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and dextrose solutions, as described in more detail below.

The immunogenic compositions of the invention comprise one or more preservatives in addition to the various pneumococcal capsular polysaccharide-protein conjugates. The FDA requires that multi-dose (multi-dose) vials of bioproducts contain preservatives with few exceptions. Preservative-containing vaccine products include vaccines comprising: benzethonium chloride (anthrax vaccine), 2-phenoxyethanol (acellular diphtheria vaccine (DTaP), hepatitis a vaccine, lyme disease vaccine, polio vaccine (parenteral)), phenol (pneumonia vaccine, typhoid vaccine (parenteral), vaccinia vaccine), and thimerosal (acellular diphtheria vaccine, diphtheria vaccine (DT), tetanus-diphtheria vaccine (Td), hepatitis b vaccine, haemophilus influenzae type b vaccine (Hib), influenza vaccine, encephalitis b vaccine (JE), epidemic encephalitis vaccine, pneumonia vaccine, rabies vaccine). Preservatives approved for use in injectable pharmaceuticals include, for example, chlorobutanol, m-cresol, methyl paraben, propyl paraben, 2-phenoxyethanol, benzethonium chloride, benzalkonium chloride, benzoic acid, benzyl alcohol, phenol, thimerosal, and phenylmercuric nitrate.

Having tested a variety of potentially suitable formulations comprising preservatives for enhancing the efficacy and stability of prev (e) nar13 immunogenic compositions, the invention disclosed herein provides such pneumococcal immunogenic compositions comprising 2-phenoxyethanol (2-PE) at a concentration of about 2.5-10 mg/dose (0.5-2%). In certain embodiments, the concentration of 2-PE is about 3.5-7.5 mg/dose (0.7-1.5%). In certain embodiments, the concentration of 2-PE is about 5 mg/dose (1%). In certain embodiments, the concentration of 2-PE is no less than 3.5 mg/dose (0.7%), no less than 4.0 mg/dose (0.8%), no less than 4.5 mg/dose (0.9%), no less than 5.0 mg/dose (1%), no less than 5.5 mg/dose (1.1%), no less than 6.0 mg/dose (1.2%), no less than 6.5 mg/dose (1.3%), no less than 7.0 mg/dose, no less than 7.5 mg/dose (1.5%), no less than 8.0 mg/dose (1.6%), no less than 9.0 mg/dose (1.8%), or no less than 10 mg/dose (2%).

In certain embodiments of the invention, the pneumococcal immunogenic composition comprises one or more additional preservatives, including but not limited to thimerosal and formalin.

In certain embodiments, the immunogenic composition may comprise one or more adjuvants. As defined herein, an "adjuvant" is a substance used to enhance the immunogenicity of the immunogenic composition of the invention. Thus, adjuvants are often used to boost the immune response and are well known to those skilled in the art. Suitable adjuvants to enhance the efficacy of the composition include, but are not limited to:

(1) aluminum salts (alum) such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, and the like;

(2) oil-in-water emulsion formulations (with or without other specific immunostimulants such as muramyl peptides (as defined below) or bacterial cell wall components), such as for example,

(a) MF59(PCT application WO 90/14837) comprising 5% squalene, 0.5% tween 80 and 0.5% Span (Span)85 (optionally containing different amounts of MTP-PE (see below, although not required)) formulated in submicron particles using a microfluidizer such as model 110Y microfluidizer (Microfluidics, Newton, MA),

(b) a SAF comprising 10% squalene, 0.4% Tween 80, 5% pluronic block copolymer L121 and thr-MDP (see below), microfluidized in a submicron particle emulsion or vortexed to produce a larger particle size emulsion, and

(c) a Ribi Adjuvant System (RAS), (Corixa, Hamilton, MT) comprising 2% squalene, 0.2% tween 80 and one or more bacterial cell wall components from the group consisting of 3-O-deacylated monophosphoryl lipid a (MPL), (Corixa), Trehalose Dimycolate (TDM) and Cell Wall Skeleton (CWS) described in U.S. Pat. No. 4,912,094, preferably MPL + CWS (detox);

(d) polysorbate 80 (tween 80);

(3) saponin adjuvants, such as Quil a or STIMULON QS-21 (antibiotics, Framingham, MA) (U.S. patent No. 5,057,540), may utilize or produce particles therefrom, such as ISCOMs (immune stimulating complexes);

(4) bacterial lipopolysaccharides; synthetic lipid a analogs such as aminoalkyl glucosamine phosphate compound (AGP), or derivatives or analogs thereof, which are commercially available from Corixa and described in U.S. patent No. 6,113,918, one such AGP is 2- [ (R) -3-tetradecanoyloxytetradecanoylamino ] ethyl 2-deoxy-4-O-phosphoryl-3-O- [ (R) -3-tetradecanoyloxytetradecanoyl ] -2- [ (R) -3-tetradecanoyloxytetradecanoylamino ] -b-D-glucopyranoside, which is also known as 529 (formerly RC529), formulated as an aqueous form or as a stable emulsion; synthetic polynucleotides such as oligonucleotides containing CpG motifs (U.S. Pat. No. 6,207,646);

(5) cytokines such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, IL-18, etc.), interferons (e.g., gamma interferon), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), Tumor Necrosis Factor (TNF), co-stimulatory molecules B7-1 and B7-2, etc.;

(6) detoxified mutants of bacterial ADP-ribosylating toxins, such as the wild-type or mutant forms of Cholera Toxin (CT), for example according to published international patent application No. WO 00/18434 (see also WO 02/098368 and WO 02/098369) wherein the glutamic acid at amino acid position 29 is substituted with another amino acid, preferably histidine; pertussis Toxin (PT); or E.coli heat-Labile Toxins (LT), in particular LT-K63, LT-R72, CT-S109, PT-K9/G129 (see, for example, WO 93/13302 and WO 92/19265); and

(7) other substances that enhance the efficacy of the compositions AS immunostimulants, such AS calcium salts, iron, zinc, acylated tyrosine suspensions, acylated sugars, derivatized sugars/saccharides, polyphosphazenes, biodegradable microspheres, monophosphoryl lipid a (MPL), lipid a derivatives (e.g., with reduced toxicity), 3-O-deacylated MPL, quil a, saponin, QS21, tocol, freund's incomplete adjuvant (Difco Laboratories, destroit, MI), Merck adjuvant 65(Merck and Company, inc., Rahway, NJ), klith-2 (Smith-ine Beecham, philiadelphia, PA), CpG oligonucleotides (preferably unmethylated), bioadhesives and mucoadhesives, microparticles, liposomes, polyoxyethylene ether formulations, polyoxyethylene ester formulations, and muramyl or imidazoquinolone compounds. Muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-orthopolymuramyl-L-alanine-2- (1'-2' dipalmitoyl-sn-glycero-3-hydroxyphosphonoxy) -ethylamine (MTP-PE), and the like.

In certain embodiments, the adjuvant composition is one that facilitates induction of THl-type cytokines (e.g., IFN- γ, TNF α, IL-2, and IL-12) to a greater extent than H2-type cytokines, which may facilitate induction of a cell-mediated immune response against the administered antigen. Specific adjuvant systems that predominantly promote the THl reaction include, but are not limited to, lipid a derivatives, such as monophosphoryl lipid a (MPL) or derivatives thereof, for example 3-de-O-acylated MPL (3D-MPL); combinations of MPL and/or 3D-MPL with aluminium salts and/or saponin derivatives (e.g. QS21 in combination with 3D-MPL as disclosed in WO 94/00153, or QS21 and cholesterol as disclosed in WO 96/33739); triterpenoids and oil-in-water emulsions such as tocopherol-containing emulsions (as disclosed in WO 95/17210).

Adjuvants may optionally be adsorbed by or combined with one or more immunogenic components of the preservative vaccine formulations of the present invention. As used herein, the term "adsorbed antigen" refers to a mixture in which greater than 20%,30%,40%,50%,60%,70%,80%, or 90% of the antigen is adsorbed to an adjuvant. In certain embodiments, the adjuvant is adsorbed aluminum phosphate (Al +) or aluminum hydroxyphosphate. Typically, the total aluminum content is 200-. Alternatively, the Al + content may come from a mixture of aluminum hydroxide and aluminum phosphate in various ratios, for example aluminum phosphate to aluminum hydroxide is 1:8-8:1,1:4-4:1,3:8-8:3,1:2-2:1 or 1: 1. Although most of the aluminum is provided by the pre-adsorbed antigen prior to mixing to form the combination vaccine, some of the aluminum may be added in free form during formulation of the combination vaccine of the invention, e.g., prior to the pH adjustment step described herein. In general, the free aluminum content per 0.5mL dose can be 0-300. mu.g, 50-250. mu.g, 75-200. mu.g, 100-150. mu.g or about 120. mu.g of Al3 +. The free Al3+ may be all Al (oh)3 or all AlPO4, or a mixture of Al (oh)3 and A1PO4 in various proportions.

The vaccine antigen components may be separately pre-adsorbed onto the aluminium salt prior to mixing. In another embodiment, the antigen mixture may be pre-adsorbed prior to mixing with additional adjuvants. Alternatively, certain components of the vaccine of the invention may be formulated for unintended adsorption to the adjuvant.

The formulations of the present invention may also comprise one or more of a buffer, a salt, a divalent cation, a non-ionic detergent, a cryoprotectant such as a sugar, and an antioxidant such as a free radical scavenger or a chelating agent, or any combination of more of these. The selection of any one component, such as a chelating agent, can determine whether another component (e.g., a scavenger) is required. The final composition formulated for administration should be sterile and/or pyrogen-free. One skilled in the art can empirically determine which combinations of these and other components are optimal for inclusion in the preservative containing vaccine compositions of the present invention, depending on a variety of factors such as the particular storage and administration conditions desired.

In certain embodiments, formulations of the invention compatible with parenteral administration comprise one or more physiologically acceptable buffers selected from, but not limited to, tris (hydroxymethyl) aminomethane (trimethylamine), phosphate, acetate, borate, citrate, glycine, histidine and succinate. In certain embodiments, the formulation is buffered to a pH range of about 6.0 to about 9.0, preferably from about 6.4 to about 7.4.

In certain embodiments, it is desirable to adjust the pH of the immunogenic compositions or formulations of the invention. The pH of the formulations of the present invention can be adjusted using techniques standard in the art. The pH of the formulation may be adjusted to 3.0-8.0. In certain embodiments, the pH of the formulation may be or may be adjusted to 3.0-6.0, 4.0-6.0, or 5.0-8.0. In other embodiments, the pH of the formulation may be or may be adjusted to be about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 5.8, about 6.0, about 6.5, about 7.0, about 7.5, or about 8.0. In certain embodiments, the pH may be or may be adjusted to be in the range of 4.5 to 7.5, or 4.5 to 6.5, 5.0 to 5.4, 5.4 to 5.5, 5.5 to 5.6, 5.6 to 5.7, 5.7 to 5.8, 5.8 to 5.9, 5.9 to 6.0, 6.0 to 6.1, 6.1 to 6.2, 6.2 to 6.3, 6.3 to 6.5, 6.5 to 7.0, 7.0 to 7.5, or 7.5 to 8.0. In a particular embodiment, the pH of the formulation is about 5.8.

In certain embodiments, formulations of the invention compatible with parenteral administration comprise one or more divalent cations, including but not limited to MgCl, at a concentration of from about 0.1mM to about 10mM, preferably up to about 5mM₂、CaCl₂And MnCl₂。

In certain embodiments, formulations of the invention that are compatible with parenteral administration comprise one or more salts, including but not limited to sodium chloride, potassium chloride, sodium sulfate, and potassium sulfate, present at an ionic strength that is physiologically acceptable to the subject upon parenteral administration, and included at a final concentration to produce a selected ionic strength or osmolarity in the final formulation. The final ionic strength or osmolality of the formulation is determined by the various components (e.g., ions from buffering compounds and other non-buffering salts). The preferred salt NaCl is present in a range up to about 250mM, with the salt concentration being selected to complement other components (e.g., sugars) so that the final total osmolarity of the formulation is compatible with parenteral administration (e.g., intramuscular or subcutaneous injection), and will promote long-term stability of the immunogenic components of the vaccine formulation over a variety of temperature ranges. A salt-free formulation would allow for an increased range of one or more selected cryoprotectants to maintain the desired final osmolarity level.

In certain embodiments, formulations of the invention compatible with parenteral administration comprise one or more cryoprotectants selected from, but not limited to, disaccharides (e.g., lactose, maltose, sucrose, or trehalose) and polyhydric hydrocarbons (e.g., galactitol, glycerol, mannitol, and sorbitol).

In certain embodiments, the osmolality of the formulation ranges from about 200mOs/L to about 800mOs/L, with a preferred range of about 250mOs/L to about 500mOs/L, or about 300mOs/L to about 400 mOs/L. The salt-free formulation may comprise, for example, from about 5% to about 25% sucrose, preferably from about 7% to about 15%, or from about 10% to about 12% sucrose. Alternatively, the salt-free formulation may comprise, for example, from about 3% to about 12% sorbitol, preferably from about 4% to 7%, or from about 5% to about 6% sorbitol. If a salt such as sodium chloride is added, the effective range of sucrose or sorbitol is relatively reduced. These and other such osmolality and osmolality considerations are well within the skill of the art.

In certain embodiments, formulations of the present invention that are compatible with parenteral administration comprise one or more free radical oxidation inhibitors and/or chelating agents. A variety of free radical scavengers and chelating agents are known in the art and are suitable for use in the formulations and methods described herein. Examples include, but are not limited to, ethanol, EDTA/ethanol combinations, triethanolamine, mannitol, histidine, glycerol, sodium citrate, phytate, tripolyphosphate, ascorbic acid/ascorbate, succinic acid/succinate, malic acid/maleate, deferoxamine mesylate, EDDHA and DTPA, and various combinations of two or more of the foregoing. In certain embodiments, at least one non-reducing free radical scavenger may be added at a concentration effective to enhance long-term stability of the formulation. One or more free radical oxidation inhibitors/chelators may also be added in various combinations, such as scavengers and divalent cations. The choice of chelating agent will determine whether or not the addition of a scavenger is required.

In certain embodiments, formulations of the invention compatible with parenteral administration comprise one or more non-ionic surfactants including, but not limited to, polyoxyethylene sorbitan fatty acid ester polysorbate-80 (tween 80), polysorbate-60 (tween 60), polysorbate-40 (tween 40), and polysorbate-20 (tween 20), polyoxyethylene alkyl ethers including, but not limited to, Brij58, Brij35, and others such as Triton (Triton) X-100; triton X-114, NP40, span 85, and the pluronic series of nonionic surfactants (e.g., pluronic 121), wherein the preferred component is polysorbate-80 at a concentration of about 0.001% to about 2% (up to about 0.25% being preferred), or polysorbate-40 at a concentration of about 0.001% to 1% (as high as about 0.5% being preferred).

In certain embodiments, the formulations of the invention comprise one or more additional stabilizing agents suitable for parenteral administration, such as a reducing agent comprising at least one thiol (-SH) group (e.g., cysteine, N-acetylcysteine, reduced glutathione, sodium thioglycolate, thiosulfate, thioglycerol, or mixtures thereof). Alternatively or optionally, the preservative-containing vaccine formulations of the present invention can be further stabilized by removing oxygen from the storage container, protecting the formulation from light (e.g., by using an amber glass container).

The preservative containing vaccine formulations of the present invention may contain one or more pharmaceutically acceptable carriers or excipients, including any excipient that does not itself induce an immune response. Suitable excipients include, but are not limited to, macromolecules such as proteins, sugars, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, sucrose (Paoletti et al,2001, Vaccine,19:2118), trehalose, lactose, and lipid polymers such as oil droplets or liposomes. Such vectors are well known to those skilled in the art. Pharmaceutically acceptable excipients are described, for example, in Gennaro,2000, Remington: The Science and Practice of pharmacy, 20^thedition, ISBN: 0683306472.

The compositions of the invention may be lyophilized or in aqueous form, such as a solution or suspension. Liquid formulations can conveniently be administered directly from their packaged form and are therefore ideal for injection without the need for re-dissolution in aqueous media as is otherwise required with the lyophilised compositions of the invention.

In a particular embodiment of the invention, the vaccine is a multivalent immunogenic composition comprising one or more pneumococcal capsular polysaccharides selected from serotypes 1,3,4,5,6A,6B,7F, 9V, 14,18C,19A,19F and 23F, respectively, and CRM₁₉₇And (6) conjugation. The vaccine is formulated to comprise: 1 to 5 μ g, preferably about 4.4 μ g/mL of each polysaccharide, but preferably about 8.8 μ g/mL of 6B; CRM of 20-100. mu.g/mL, preferably about 58. mu.g/mL₁₉₇A carrier protein; 0.02-2mg/mL, preferably 0.25mg/mL, of elemental aluminium in the form of aluminium phosphate; 0.5-1.25%, preferably about 0.85% sodium chloride; 0.002-0.2%, preferably about 0.02% polysorbate 80; 1-10mM, preferably about 5mM, sodium succinate buffer, pH 4-7, preferably pH 5.8; and 4-20mg/mL, preferably about 10mg/mL, of 2-phenoxyethanol.

In certain preferred embodiments of the invention, the vaccine is a multivalent immunogenic composition comprising pneumococcal capsular polysaccharides selected from serotypes 1,3,4,5,6A,6B,7F, 9V, 14,18C,19A,19F and 23F, respectively, and CRM₁₉₇And (6) conjugation. The vaccine is formulated to comprise: about 4.4 μ g/mL of each saccharide, except that 6B is about 8.8 μ g/mL; CRM at about 58. mu.g/mL₁₉₇A carrier protein; about 0.25mg/mL of elemental aluminum in the form of aluminum phosphate; about 0.85% sodium chloride; about 0.02% polysorbate 80; about 5mM sodium succinate buffer at pH 5.8; and about 10mg/mL of 2-phenoxyethanol.

The amount of many substances that the compositions of the present invention may comprise may be expressed as weight/dose, weight/volume or percent concentration (e.g., weight/volume or weight/weight). All these values can be converted to each other. The volume of the dose is specified for conversion to or from a weight/dose unit. For example, given a dose of 0.5mL, 5.0 mg/dose of 2-PE corresponds to a concentration of 10mg/mL or 1.0% (g/100 mL).

The vaccine formulation may also be expressed as a ratio of polysaccharide to 2-PE. For example, a preferred formulation having a dose of 0.5mL with 4.4. mu.g/mL of each polysaccharide (except for 6B of 8.8. mu.g/mL) and 10mg/mL of 2-PE would have 30.8. mu.g of polysaccharide (2.2. mu.g of serotype 12 + 4. mu.g of serotype 6B) and 5000. mu.g of 2-PE. Thus, the weight ratio of polysaccharide to 2-PE was 30.8: 5000.

In certain embodiments of the invention, the vaccine has a polysaccharide to 2-PE weight ratio of 5:5000 to 100: 5000. In a preferred embodiment of the invention, the polysaccharide to 2-PE weight ratio is about 30.8 to 5000.

Delivery of vaccine formulations

Also provided are methods of using the disclosed pharmaceutical compositions and formulations comprising at least one preservative to induce an immune response against pneumococci in a mammalian subject, such as a human subject, preferably a child or infant, and thereby protect against infection. The vaccine formulations of the present invention may be used to protect human individuals from pneumococcal infection by administering the vaccine via the systemic or mucosal route. Such administration may include, for example, parenteral or mucosal administration to the oral/digestive, respiratory or genitourinary tracts.

Direct delivery of the vaccine formulation of the present invention to an individual may be achieved by parenteral administration (intramuscular, intraperitoneal, intradermal, subcutaneous, intravenous or to the interstitial space), or by rectal, buccal, vaginal, topical, transdermal, intranasal, ocular, ear, lung or other mucosal administration. In a preferred embodiment, parenteral administration is by intramuscular injection, e.g., to the thigh or upper arm of the individual. The injection may be via a needle (e.g., a hypodermic needle), but alternatively, a needle-free injection may be utilized. The usual intramuscular dose is 0.5 mL. The compositions of the present invention may be prepared in a variety of forms, such as liquid solutions or suspensions for injection. In certain embodiments, the compositions may be prepared as a powder or spray for administration to the respiratory tract, for example in an inhaler. In other embodiments, the composition may be prepared as a suppository or pessary, or for nasal, aural, or ocular administration, for example as a spray, drop, gel, or powder.

In one embodiment, intranasal administration may be used to prevent pneumonia or otitis media (as nasopharyngeal transport of pneumococci can be more effectively prevented, thereby reducing infection in its early stages).

The amount of conjugate per vaccine dose is selected to induce an immune protective response without significant side effects. This amount may vary depending on the pneumococcal serotype. Typically, each dose will contain 0.1-100. mu.g of polysaccharide, particularly 0.1-10. mu.g, and more particularly 1-5. mu.g.

The optimal amounts of the components of a particular vaccine can be determined by standard studies involving observation of appropriate immune responses in individuals. Following the initial immunization, the individual may receive one or several booster immunizations at sufficient intervals.

The conventional schedule for infants and young children against invasive diseases caused by streptococcus pneumoniae because of the serotypes included in the prev (e) nar13 vaccine is 2,4,6 and 12-15 months of age. The compositions of the present invention are also suitable for use in older children, adolescents and adults, where the same or different conventional schedules may be employed, depending on the discretion of the skilled practitioner.

Packaging and dosage form

The vaccines of the present invention may be packaged in unit-dose or multi-dose form (e.g., 2 doses, 4 doses, or more). For multi-dose forms, vials are generally, but not necessarily, preferred to pre-filled syringes. Suitable multi-dose profiles include, but are not limited to: 2-10 doses per container, 0.1-2mL per dose. In certain embodiments, the dose is a 0.5mL dose. See, for example, international patent application WO2007/127668, which is incorporated herein by reference. The composition may be present in a vial or other suitable storage container, or may be present in a pre-filled delivery device, such as a single or multi-component syringe, which may or may not be equipped with a needle. Syringes are typically, but not necessarily, required to contain a single dose of the preservative containing vaccine composition of the present invention, although multi-dose, pre-filled syringes are also contemplated. Likewise, the vial may comprise a single dose, but may alternatively comprise multiple doses.

Effective dosage volumes can be routinely established, but typical doses of compositions for injection have a volume of 0.5 mL. In certain embodiments, the dosage is formulated for administration to a human subject. In certain embodiments, the dosage is formulated for administration to human subjects in adults, adolescents, toddlers, or infants (i.e., no more than 1 year of age), and in preferred embodiments, may be administered by injection.

The liquid vaccines of the present invention are also suitable for reconstitution of other vaccines in lyophilized form. If the vaccine is to be used for such immediate reconstitution, the present invention provides the following kits: having two or more vials, two or more ready-to-fill syringes, or one or more of each, wherein the contents of the syringes are used to reconstitute the contents of the vials prior to injection, or vice versa.

Alternatively, the vaccine compositions of the invention may be lyophilized and reconstituted, for example using one of a number of methods well known in the art for lyophilization, to form dry, regularly shaped (e.g. spherical) particles, such as micropellets or microspheres, having particle characteristics, such as average diameter size, which can be selected and controlled by varying the exact method used to prepare the particles. The vaccine composition may also comprise an adjuvant, optionally prepared from or contained within individual dry, regularly shaped (e.g. spherical) particles such as micropellets or microspheres. In such embodiments, the invention also provides a vaccine kit comprising a first component comprising the stable dry vaccine composition, optionally further comprising one or more preservatives of the invention, and a second component comprising a sterile aqueous solution for reconstituting the first component. In certain embodiments, the aqueous solution comprises one or more preservatives, and may optionally comprise at least one adjuvant (see, e.g., WO2009/109550 (which is incorporated herein by reference)).

In yet another embodiment, the multi-dose format of the container is selected from one or more of, but not limited to, the following: general laboratory glassware, flasks, beakers, graduated cylinders, fermentors, bioreactors, tubes, tubing, bags, jars, vials, vial closures (e.g., rubber stoppers, screw caps), ampoules, syringes, dual or multi-chamber syringes, syringe stoppers, syringe plungers, rubber closures, plastic closures, glass closures, sleeves, disposable pens, and the like. The container of the present invention is not limited by the materials of processing, including materials such as glass, metals (e.g., steel, stainless steel, aluminum, etc.), and polymers (e.g., thermoplastics, elastomers, thermoplastic elastomers). In a particular embodiment, this style of container is a 5mL Schott type 1 glass bottle with a butyl plug. The skilled artisan will recognize that the above-described formats are by no means exhaustive and merely serve as guides for the skilled artisan in regard to the variety of formats that may be utilized with the present invention. Additional models contemplated for use in the present invention can be found in published catalogues from laboratory equipment suppliers and manufacturers such as United States Plastic Corp. (Lima, OH), vwr.

Methods for evaluating preservative efficacy in vaccine compositions

The present invention also provides novel methods for measuring the efficacy of vaccine formulations comprising one or more selected preservatives in the presence of some or all of the immunogenic and non-immunogenic components of the vaccine composition. The WHO protocol for preservative efficacy utilizes USP and EP tests and includes Open bottle Policy (Open visual Policy) conditions when certain tests are performed. A typical preservative efficacy test is a single challenge test in which the tested composition is inoculated once with a selected microbial population and the log reduction of the inoculated microorganism over time and under specific environmental conditions (e.g., temperature) is compared to the log reduction of the inoculated microorganism in a control composition lacking the test preservative. See, e.g., examples 2 and 3 below. However, no additional tests have been required to address the efficacy of preservatives on multiple contaminations, such as vials and stoppers by multiple inoculations of the same vial.

Accordingly, the present invention provides a multiple challenge test for assessing the efficacy of one or more preservatives in an immunogenic composition, wherein the test comprises at least the following two steps: the test compositions are inoculated with a selected population of microorganisms and the reduction in the inoculated microorganisms over time and under particular environmental conditions (e.g., temperature) is compared to the reduction in a control composition lacking the test preservative. See, examples 4 and 5 below.

Preservative efficacy

The preservative containing vaccine formulations of the present invention are suitable for filling in multi-dose vaccine vials or containers, e.g., compatible with parenteral administration, and remain stable at 2-8 ℃, room temperature, or 37 ℃ for extended periods of time, with reduced or negligible impairment of activity when compared to the same formulation lacking the preservative.

The amount of preservative in the formulation is selected to meet the safety requirements of the vaccine as defined in the united states (u.s.), european or World Health Organization (WHO) pharmacopoeia, or combinations thereof.

To determine the level of preservative according to the United states and European pharmacopoeias (USP and EP, respectively), about 10 was used at time 0⁵-10⁶CFU/ml (CFU = colony forming unit) the following bacteria were inoculated once with the vaccine formulation:

1. staphylococcus aureus (bacterium; ATCC #6538; "SA")

2. Pseudomonas aeruginosa (bacteria;;;;; "ATCC #9027;" PA ")

3. Candida albicans (Yeast; ATCC #10231; "CA")

4. Aspergillus niger (mold; ATCC #16404; "AN")

To represent the worst reasonable scenario of contamination that may occur during repeated use of multiple dose formulations in practice, the WHO requires the use of the bacterial strains pseudomonas aeruginosa ("PA"), staphylococcus aureus ("SA"), escherichia coli ("EC"), and bacillus subtilis ("BA"), the peace of intentional exposure to multiple contamination eventsAnd (5) testing the completeness. 5x10 was added to the formulation at times 0, 6 hours, 24 hours, 7 days and 14 days after the initial challenge³CFU/ml of each organism and stored at 2-8 ℃ or 22-24 ℃ to simulate potential storage conditions in practice.

USP 29NF 24 supplemented version 2(USP) requires at least a 1.0 log reduction from the initial calculated count (i.e. time of inoculation) at 7 days, at least a 3.0 log reduction from the previously measured value at 14 days, and no increase from the previously measured value at 28 days after inoculation of the bacterial microorganism. See table 1. For yeast and fungi, USP requires no increase from inoculation time at 7, 14 and 28 days.

The requirements of EP are more stringent. EP 5 th edition 5.6(5.1.3) has two parts for the requirements of parenteral and ophthalmic formulations: category a and category B. Category A (EP-A) requires, for bacteriｃA, ｃA log reduction of at least 2.0 from the initial calculated count at 6 hours after inoculation; there was at least a 3.0 log reduction from the previously measured value at 24 hours and no recovery at 28 days. Category B (EP-B) requires, for bacteria, at 24 hours there is a log reduction of at least 1.0 from the initial calculated count, at 7 days there is a log reduction of at least 3.0 from the previously measured value, and at 28 days there is no more than a 0.5log increase (i.e. no increase) from the previously measured value. See table 1. For yeast and fungi, category a requires at least a 2.0 log reduction in counts from initial calculations at 7 days, and no increase from previously measured values at 28 days; and category B requires at least a 1.0 log reduction in counts from the initial calculation at 14 days, and no increase from the previously measured values at 28 days.

TABLE 1 acceptance criteria for efficacy testing of preservatives between United states, European and Japanese pharmacopoeias

The a criteria express the recommended efficacy to be achieved. In reasonable circumstances, the B criterion must be met if the a criterion cannot be met.

NI no increase: it is defined as not more than 0.5log greater than the previously measured value₁₀Is increased.

NR-No recovery

In certain embodiments of the invention, the preservatives of the present invention are effective in reducing the concentration of microorganisms in an immunogenic formulation. In certain embodiments of the invention, a vaccine formulation comprising at least one preservative reduces the concentration of one or more microorganisms following vaccination with said microorganisms compared to a vaccine formulation not comprising the one or more preservatives. In a particular embodiment of the invention, the formulation exhibits at least a 1.0 log reduction in initial microbial count at 24 hours, at least a 3.0 log reduction from a previously measured value at 7 days, and no more than a 0.5log increase from a previously measured value at 28 days. In another particular embodiment of the invention, the formulation exhibits a log reduction of at least 2.0 from the initial calculated count at 6 hours, a log reduction of at least 3.0 from the previously measured value at 24 hours, and no recovery from the initial microbial count at 28 days. In another embodiment of the invention, the formulation meets the requirements of the European PharmacopoeiｃA (EP) for parenteral and ophthalmic formulations, in particular the requirements of category ｃA (EP- ｃA) and/or category B of EP 5 th edition 5.6 (5.1.3). In another embodiment of the invention, the formulation meets the requirements of the United States Pharmacopeia (USP)29NF 24 supplement 2 for parenteral formulations.

In certain embodiments of the invention, the at least one preservative of the invention is effective in reducing the concentration of microorganisms in the formulation when challenged with a microorganism, as compared to a formulation lacking the one or more preservatives. Without limitation, the microorganism may be one or more of the following: pseudomonas aeruginosa, staphylococcus aureus, escherichia coli, bacillus subtilis, candida albicans, aspergillus niger and others.

In certain embodiments of the invention, the microorganisms may be introduced or inoculated in the vaccine one or more times at different intervals. Vaccination may occur in the case of planned experimental vaccination or in the case of a contaminated hypodermic needle into a container of multiple doses of vaccine formulation. The interval between inoculations may be between 1 minute and 1 month. In particular embodiments, multiple inoculations occur 6 hours after the initial inoculation, 24 hours after the initial inoculation, 7 days after the initial inoculation, and 14 days after the initial inoculation.

Parameters for vaccine and preservative stability

In certain embodiments of the invention, the antigenicity of at least one antigenic determinant (i.e., a polysaccharide preparation from a streptococcus pneumoniae serotype) in a vaccine formulation is stable over storage time and temperature. Antigenicity can be measured by methods known in the art. For example, total antigenicity can be determined by using type-specific antisera, as described in example 3.

In certain embodiments of the invention, at least one epitope in the vaccine formulation is antigenically stable for not less than 4 weeks, not less than 6 weeks, not less than 8 weeks, not less than 10 weeks, not less than 12 weeks, not less than 18 weeks, not less than 24 weeks, not less than 48 weeks, not less than 1 year, not less than 1.25 years, not less than 1.5 years, not less than 1.75 years, not less than 2 years, not less than 2.25 years, or not less than 2.5 years. Preferably, the antigenicity of most epitopes of the vaccine in the formulation is stable for not less than 4 weeks, not less than 6 weeks, not less than 8 weeks, not less than 10 weeks, not less than 12 weeks, not less than 18 weeks, not less than 24 weeks, not less than 48 weeks, not less than 1 year, not less than 1.25 years, not less than 1.5 years, not less than 1.75 years, not less than 2 years, not less than 2.25 years or not less than 2.5 years, for example, at least 50%, 75%, 80%, 85%, 90%, 95% or more of the epitopes.

In certain embodiments of the invention, the antigenicity of at least one epitope in a vaccine formulation is stable when stored at about-25 ℃ to about 37 ℃, or-20 to-10 ℃, or 2 to 8 ℃, or about room temperature, or 22 ℃ to 28 ℃, or about 37 ℃. In a particular embodiment of the invention, the antigenicity of at least one antigenic determinant in the vaccine formulation is stable for not less than 2.5 years stored at a temperature of 2 to 8 ℃.

In certain embodiments of the invention, the concentration of the preservative of the invention is stable after storage of the vaccine at the time periods and storage temperatures described above. In a particular embodiment of the invention, the concentration of preservative in the vaccine formulation is stable for not less than 2.5 years of storage of the vaccine at a temperature of 2 to 8 ℃. The concentration of the preservative can be measured by methods known in the art. For example, thimerosal can be measured using Cold Vapor Atomic Absorption Spectrometry (CVAAS), as described in example 3. The concentration of 2-EP can be measured by reverse phase HPLC assay, as also described in example 3. Reverse phase HPLC assays can be performed in the following manner: the samples were vortexed and diluted 1:10 in 5mM succinate buffer in saline, then the samples were centrifuged and again diluted 1:10 in 5mM succinate buffer in saline (final dilution of test sample 1: 100). The samples were then analyzed using an Agilent Eclipse XDB-C18HPLC column and a linear gradient of water and acetonitrile containing trifluoroacetic acid. The amount of preservative is then compared to a standard curve. See also, Sharma et al, Biologicals36(1):61-63 (2008).

The foregoing disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples. These examples are described for illustrative purposes only and are not intended to limit the scope of the present invention.

Examples

Example 1 preliminary preservative screening study

Formulation development of multi-dose prev (e) nar13 vaccines began with a preliminary screen of preservatives including phenol (0.25%), 2-phenoxyethanol (5mg/mL), m-cresol (0.3%), methyl paraben and propyl paraben (0.18% and 0.12%, respectively) in the prev (e) nar13 formulation.

To test the efficacy of the preservative, aliquots of the vaccine were inoculated with the following organisms:

1. golden yellow grape ball (bacterium; ATCC #6538)

2. Pseudomonas aeruginosa (bacterium; ATCC #9027)

3. Candida albicans (Yeast; ATCC #10231)

4. Aspergillus niger (mold; ATCC #16404)

30 milliliters (ml) of each vaccine formulation or saline containing 0.02% thimerosal, with or without the indicated concentrations of thimerosal or 2-PE, were inoculated in triplicate into each suspension of organisms tested so that the inoculum density reached about 10 at time 0⁵To 10⁶CFU/ml (CFU = colony forming unit). The volume of each inoculum during each intentional challenge did not exceed 1% of the product volume. The samples were mixed to ensure a uniform distribution of the attacking organisms. Triplicate additional 30ml of vaccine (with and without preservatives) were used as negative controls and medium alone was added to evaluate the inherent contamination that may be present in the sample or medium. Each of the three series of vaccines, as well as positive and negative controls, were then incubated at 20-25 ℃ respectively. Aliquots (1ml) of challenged sample and control (or their appropriate 10-fold serial dilutions) were counted in duplicate by plate counting at time 0 and at intervals of 6 hours, 24 hours, 7 days, 14 days and 28 days post inoculation.

USP 29NF 24 supplement 2(USP) requires at least a 1.0 log reduction from the initial calculated count (i.e. time of inoculation) at 7 days, at least a 3.0 log reduction from the previously measured value at 14 days, and no increase from the previously measured value at 28 days after inoculation of the bacterial microorganism. See table 1. For yeast and fungi, the requirements of USP are no increase from inoculation time to 7, 14 and 28 days.

The requirements of EP are more stringent. EP 5 th edition 5.6(5.1.3) has two parts for the requirements of parenteral and ophthalmic formulations: category a and category B. Category A (EP-A) requires, for the bacteriｃA, ｃA log reduction of at least 2.0 from the initial calculated count at 6 hours after inoculation, ｃA log reduction of at least 3.0 from the previously measured value at 24 hours and no recovery at 28 days. Category B (EP-B) requires, for bacteria, at 24 hours there is a logarithmic decrease of at least 1.0 from the initial calculated count, at 7 days there is a logarithmic decrease of at least 3.0 from the previously measured value, and at 28 days there is a logarithmic increase (i.e. no increase) of no more than 0.5 from the previously measured value. See table 1. For yeast and fungi, category a requires at least a 2.0 log reduction at 7 days, and no increase from the previously measured values at 28 days; and category B requires at least a 1.0 log reduction at 14 days and no increase from the previously measured value at 28 days.

In counting, for bacteria, the method comprises<300CFU plates, or for yeasts or molds, using plates containing<100CFU plate. For single challenge studies, the arithmetic mean counts of all viable microorganisms (6 values per time point) plus their diluted samples in triplicate and on duplicate plates were measured and normalized to CFU/ml. Results are expressed as mean log₁₀CFU/ml decreased (compared to time 0). In this case, the viable microorganism count at time 0 was evaluated as baseline and compared to incubation times of 6 hours, 24 hours, 7 days, 14 days, and 28 days.

The preservatives tested showed no significant effect on the stability of prev (e) nar13, except for the parabens (methyl and propyl parabens) which were shown to reduce the antigenicity of the prev (e) nar13 binding. In addition, phenol, m-cresol, methyl paraben and propyl paraben interfere with the modified Lowry protein assay (protein concentration of the vaccine is determined by the commercial modified Lowry protein assay).

Preservative Efficacy Test (PET) results show that all preservatives tested meet the USP requirements but do not meet the EP standards (EP-A or EP-B). See table 2. 2-PE is the only candidate preservative known to be safe at higher doses. Thus, preservative efficacy was further tested with higher doses of 2-PE.

TABLE 2 potential preservatives after a single microbial challenge meet USP and EP^*Efficacy in terms of vaccine safety requirements

^*EP-B

Example 2-preservative efficacy testing by single challenge method: 2-PE and Thimerosal

Thimerosal is commonly used at a concentration of 0.01% in most vaccines licensed in the united states. The efficacy of thimerosal as a preservative was tested using the same single challenge method described in example 1 above. Prev (e) nar13 vaccine formulations containing 0.01% thimerosal (equivalent to 25 μ g mercury per 0.5mL dose) did not meet the European acceptance criteriｃA EP-A or EP-B established by the preservative antimicrobial efficacy method of EP. However, it passes the acceptance range established by the U.S. or japanese pharmacopoeias because the acceptance range established by these legal methods is less strict than that established in EP. See fig. 1.

Thimerosal at 0.02% equivalent to 2-fold the recommended concentration of thimerosal in some us licensed vaccines (containing 50 μ g of mercury per dose), or thimerosal at 0.04% equivalent to 4-fold the recommended concentration of thimerosal in some us licensed vaccines (containing 100 μ g of mercury per dose) met EP acceptance criteria B but not the more stringent a acceptance criteria (with a single microbial challenge). See fig. 1.

2-PE is more effective as a preservative than thimerosal. Although 2-PE at 2.5 mg/dose does not meet EP acceptance criteria A and B, 2-PE at concentrations of 3.5 to 5.5 mg/dose meets EP acceptance criteria B. At concentrations above 6.0 mg/dose, 2-PE met the EP-A and EP-B antimicrobial efficacy acceptance criteriｃA (FIG. 2).

Example 3-single challenge method with 2-PE and thimerosal: variation of contaminant level

At 20-25 ℃, the absence of preservative in the prev (e) nar13 vaccine formulation resulted in slow growth of pseudomonas aeruginosa, no alteration of levels of candida albicans and aspergillus niger, and a slow reduction of staphylococcus aureus colony forming units during the 28 day challenge period (fig. 3).

The presence of 0.01% thimerosal (25 μ g mercury per dose) reduced the contamination level of all four inoculated microorganisms. However, inhibition of staphylococcus aureus and candida albicans was less than inhibition of pseudomonas aeruginosa and aspergillus niger (fig. 4). A dose-response relationship of the rate of thiomersal antimicrobial action in prev (e) nar13 vaccine formulations, particularly against candida albicans, was observed, with the reduction in contamination levels being more pronounced with 0.02% thiomersal (figure 5). The absence of prev (e) nar13 in the saline formulation containing 0.02% thimerosal mildly increased the growth inhibitory potency of thimerosal against staphylococcus aureus and candida albicans (fig. 6).

2-PE is more effective as a preservative than thimerosal. For example, antimicrobial efficacy of 5.0 mg/dose of 2-PE resulted in a drop to baseline in s.aureus over 24 hours post-inoculation, compared to slow s.aureus depletion by thimerosal (fig. 7). Although 2-PE was less effective as a preservative against Aspergillus niger (FIG. 7) and the rate of contamination reduction of Aspergillus niger was slower compared to Thimerosal (FIGS. 4 and 5), the superior efficacy of 2-PE for other strains was such that it met the preservative acceptance criteria EP-B (FIG. 2) at concentrations of 3.5 and 5 mg/dose, whereas 0.01% Thimerosal was not met (FIG. 1).

Preservative efficacy of 3.5 to 5.0 mg/dose of 2-PE persists when the formulation is stored at 37 ℃ for one month or two and a half years at 2-8 ℃ (figure 2). The concentration of 2-PE in the formulation was also stable (FIG. 15). Prev (e) the immunological activity (total antigenicity) of each of the 13 serotypes present in the nar13 preparation was also stable under these storage conditions (figure 14).

For each serotype, the total antigenicity comes from both conjugated and unconjugated polysaccharides present in the vaccine. Type-specific antigenicity was determined by using type-specific antisera. Prior to assay, 13-valent vaccine formulated with aluminum phosphate was first dissolved. The solution is then neutralized to avoid base-induced degradation. This assay measures the rate of change of light scattering intensity from antibody-antigen complex formation using a turbidimeter. The antigenicity of the test samples was determined by linear regression using a standard curve measured immediately before or after sample analysis.

To clarify the thimerosal content of prev (e) nar13 vaccine and salt formulations, the mercury concentration in some formulations was determined by a Cold Vapor Atomic Absorption Spectrometry (CVAAS) method. The measured mercury concentration was very close to its expected value, indicating that the thimerosal concentration in these formulations was correct and was not underestimated. The measured concentration of 2-PE was also very close to its expected value and did not change over time when the Prev (e) nar13 formulation was stored at 2-8 ℃ or 37 ℃. The 2-PE concentration was measured using a reverse phase HPLC assay. Samples were vortexed and diluted 1:10 in 5mM succinate buffer in saline, then centrifuged and again diluted 1:10 in 5mM succinate buffer in saline. The final dilution of the test sample was 1: 100. The assay utilizes an Agilent Eclipse XDB-C18HPLC column and a linear gradient of water and acetonitrile containing trifluoroacetic acid. 2-PE in the 13vPnC multi-dose vaccine samples was quantified against a 2-PE standard curve. See also, Sharma et al, Biologicals36(1):61-63 (2008).

Example 4-preservative efficacy testing by multiple challenge method: thimerosal

To evaluate the suitability of the WHO multi-dose vaccine decap policy for multiple immunization sessions up to 4 weeks, the experimental design provided by WHO was applied. In this study, the efficacy of thimerosal was evaluated at the concentration (0.01%) present in most U.S. licensed vaccines, as well as at concentrations of 0.02% higher. To represent a practical multi-dose systemWorst case rationale for contamination that may occur during repeated use of the agent, and to test WHO requirements, WHO recommended bacterial strains were utilized: pseudomonas aeruginosa, staphylococcus aureus, escherichia coli and bacillus subtilis, a prev (e) nar13 vaccine formulation containing 0.01 or 0.02% thimerosal or containing 5.0 mg/dose of 2-PE was intentionally exposed to multiple contamination events. At time 0, 6 hours, 24 hours, 7 days and 14 days after the initial challenge, the formulations were added at 5x10³CFU/ml of each organism and stored at 2-8 ℃ or 22-24 ℃ to simulate potential storage conditions in practice. Saline formulations containing 0.02% thimerosal were also used as controls to evaluate the potential effect of prev (e) nar13 on the antimicrobial efficacy of thimerosal in the formulations.

Prev (e) multiple intentional contamination of nar13 vaccine formulations levels of pseudomonas aeruginosa and escherichia coli organisms increased with the progress of the study in the absence of preservatives, particularly when stored at 22-24 ℃ (fig. 8A and 8B). The levels of staphylococcus aureus in the formulations stored at 22-24 ℃ were slowly reduced, similar to that observed in the single challenge study (fig. 8A, compare fig. 3). The reduction in viability of bacillus subtilis was more pronounced (fig. 8A and 8B). These results indicate that bacillus subtilis is not a robust organism in this formulation to be used as a model for this challenge study in preservative efficacy testing, although it is recommended by WHO.

In prev (e) nar13 vaccine formulations, 0.01% thimerosal antibacterial efficacy was highest against bacillus subtilis, followed by pseudomonas aeruginosa. However, the reduction of staphylococcus aureus and escherichia coli was slow, especially when the formulations were stored at 2-8 ℃ (fig. 9A and 9B).

As shown by the non-linear regression analysis of the viability depletion of Staphylococcus aureus summarized in FIG. 12, the protein was compared with the protein stored at 22-24 deg.C (-1.39 log/day)₁₀By 50% in 6.2 days), the reduction rate of staphylococcus aureus was much slower when the formulation was stored at 2-8 ℃ (5.98 log per day)₁₀50% in 30.28 days (fig. 12). These results indicate that the effect is in the middle of multiple dose delivery0.01% thimerosal in a prev (e) nar13 vaccine formulation contaminated with ground and further stored at refrigeration temperatures is ineffective in reducing bacterial contamination.

The efficacy of thimerosal was both concentration dependent and temperature dependent (fig. 9 and 10). Thimerosal was the more effective preservative at higher 0.02% concentrations. It is also a more effective preservative at higher storage temperatures of 22-24 ℃. However, as discussed above, thimerosal did not meet the EP requirements of EP-A or EP-B even when stored at ｃA concentration of 0.02% at 22-24 ℃, when this criterion was applied in ｃA multi-challenge study (FIG. 1).

To investigate whether the vaccine itself affected the preservative effects of thimerosal, the efficacy of 0.02% thimerosal at multiple challenge times was compared between saline and prev (e) nar13 vaccine formulations. The reduction rate of both s.aureus and e.coli in the presence of 0.02% thimerosal was more pronounced in the saline formulation than in the vaccine formulation (fig. 11, compare fig. 10 and 12), confirming the efficacy of the presence of the vaccine to some extent inhibiting thimerosal as a preservative. However, even in the saline control formulation without vaccine, 0.02% thimerosal still failed to meet the acceptance criteriｃA of EP-A or EP-B at multiple challenge times (FIG. 1).

Example 5-preservative efficacy testing by multiple challenge method: 2-PE

The prev (e) nar13 vaccine formulation containing 2-PE as a preservative at 5 mg/dose inactivated staphylococcus aureus viability more strongly than the lack of efficacy of thimerosal as a preservative, especially when inoculated multiple times or stored at 2-8 °, regardless of challenge method (i.e. single or multiple times) or storage temperature (fig. 12).

In fact, 5 mg/dose of 2-PE as preservative is superior to 0.01% thimerosal, regardless of storage temperature, when using the multi-challenge method, and with all tested organisms (Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli and Bacillus subtilis). In the non-linear regression analysis of Staphylococcus aureus depletion in different challenge studies, at 50% depletion and the average of the depletionSlope (log)₁₀Depletion/day), vaccine formulations containing 2-PE have a faster rate of microbial contamination depletion than vaccine formulations containing thimerosal. See fig. 12. Furthermore, 2-PE at 5 mg/dose satisfied the EP-B criteria under multiple challenge, whereas thimerosal in any form was not satisfied under the same conditions (FIG. 13).

Thimerosal is an ineffective preservative in protecting prev (e) nar13 in multi-dose formulations from potential contamination that may be introduced during dispensing. This is even more evident when contamination is introduced multiple times during administration to an individual in a multi-dose formulation. Thimerosal has a slow inactivation rate, particularly against staphylococcus aureus and escherichia coli, with a delayed immediate effect in clearing potentially contaminating organisms that might be generated by a general practitioner when removing a vaccine from a multi-dose vial under harsh sanitary conditions. However, 2-PE at 3.5 to 5 mg/dose is stable, has a much higher rate of antimicrobial efficacy compared to thimerosal, and thus protects the product from inadvertent contamination when administered to an individual.

Example 6 immunization with Prevenar 13 vaccine with or without 2-phenoxyethanol as preservative agent immune response elicited in non-human primates

Prevenar 13 and Prevenar 13 containing 2-phenoxyethanol were evaluated for their ability to induce an immune response in cynomolgus monkeys (cynomolgus macaques).

A total of 20 cynomolgus monkeys were divided into two immunization groups of 10 macaques each for the study detailed in table 3.

TABLE 3

Group of	Kiwi fruit/group	Vaccine	Final volume	Delivery of
					1	10	13vPnC	0.5mL	IM
2	10	13vPnC+5mg 2PE	0.5mL	IM

Pre-screened animals were randomly grouped based on their body weight and baseline titer.

Macaques are administered a clinical dose of 13vPnC containing 0 or 5mg of 2-phenoxyethanol as a preservative. The vaccine was administered intramuscularly in a single site in the quadriceps of each monkey. The final volume delivered was 0.5 mL.

All macaques received three doses and were vaccinated at weeks 2,4 and 8.

Blood sampling schedule: peripheral blood was taken at weeks 0, 6, 8, 10, 12 and 16 in order to monitor induction of immune response to the vaccine.

The immune response elicited by vaccination was monitored by performing the following assays on sera collected during the study:

in vitro binding and functional antibodies

o serotype-specific IgG by ELISA (see, e.g., Fernsten P, et al, Hum vaccine.2011Jan 1;7:75-84)

Serotype-specific opsonophagocytosis assay (OPA) (see, e.g., Fernsten P, et al, Hum vaccine.2011 Jan 1;7:75-84)

In vivo protection in a young mouse challenge model (see, e.g., Fernsten P, et al, HumVacin.2011Jan 1;7: 75-84):

o evaluation of serotype-specific protection of the collected cynomolgus monkey sera.

Claims (29)

1. A multivalent immunogenic composition comprising a plurality of capsular polysaccharides from Streptococcus pneumoniae (Streptococcus pneumoniae) serotypes 1,3,4,5,6A,6B,7F, 9V, 14,18C,19A,19F and 23F conjugated to a carrier protein, and further comprising 2-phenoxyethanol (2-PE).

2. The multivalent immunogenic composition of claim 1, wherein the composition comprises seven or more capsular polysaccharides from Streptococcus pneumoniae serotypes 1,3,4,5,6A,6B,7F, 9V, 14,18C,19A,19F, and 23F.

3. The multivalent immunogenic composition of any one of claims 1-2, wherein the composition comprises 2-PE at a concentration of 7mg/mL to 15 mg/mL.

4. The multivalent immunogenic composition of claim 3, wherein the composition comprises 2-PE at a concentration of about 10 mg/mL.

5. The multivalent immunogenic composition of any one of claims 1-4, wherein the composition comprises not less than 7mg/mL of 2-PE.

6. The multivalent immunogenic composition of any one of claims 1-4, wherein the composition comprises not less than 10mg/mL of 2-PE.

7. The multivalent immunogenic composition of any one of claims 1-4, wherein the composition comprises not less than 15mg/mL of 2-PE.

8. The multivalent immunogenic composition of any one of claims 1-7, wherein the composition further comprises an adjuvant, and wherein the adjuvant is aluminum phosphate.

9. The multivalent immunogenic composition of any one of claims 1-8, wherein the antigenicity of the immunogenic composition is stable for no less than 1 year, 1.5 years, 2 years, or 2.5 years.

10. The multivalent immunogenic composition of any one of claims 1-9, wherein the concentration of the one or more microorganisms decreases over time after inoculation with the microorganisms.

11. The multivalent immunogenic composition of claim 10, wherein upon inoculation with one or more bacterial strains, the composition exhibits a log reduction of at least 1.0 from the initial microbial count at 24 hours, a log reduction of at least 3.0 from a previously measured value at 7 days, and a log increase of no more than 0.5 from a previously measured value at 28 days.

12. The multivalent immunogenic composition of claim 10, wherein upon inoculation with the one or more bacterial strains, the composition exhibits at least a 2.0 log reduction from the initial calculated count at 6 hours post inoculation, at least a 3.0 log reduction from the previously measured value at 24 hours, and no recovery at 28 days.

13. The multivalent immunogenic composition of any one of claims 10-12, wherein the microorganism strain is one or more strains selected from the group consisting of pseudomonas aeruginosa (p. aeruginosa), staphylococcus aureus (s. aureus), escherichia coli (e.coli), and bacillus subtilis (b.subtilis).

14. The multivalent immunogenic composition of any one of claims 10-13, wherein the composition is inoculated multiple times.

15. The multivalent immunogenic composition of claim 13 or 14, wherein the second vaccination occurs 6 hours after the initial vaccination, the third vaccination occurs 24 hours after the initial vaccination, the third vaccination occurs 7 days after the initial vaccination, and the fourth vaccination occurs 14 days after the initial vaccination.

16. The multivalent immunogenic composition of any one of claims 1-15, wherein the composition further comprises one or more of a buffer, a cryoprotectant, a salt, a divalent cation, a nonionic detergent, and an inhibitor of free radical oxidation.

17. Multivalent immunogenic composition formulations of pneumococcal capsular polysaccharides from serotypes 1,3,4,5,6A,6B,7F, 9V, 14,18C,19A,19F and 23F conjugated to CRM, respectively₁₉₇Wherein the multivalent immunogenic composition is formulated in a sterile liquid to comprise: about 4.4 μ g/mL of each polysaccharide, except that 6B is about 8.8 μ g/mL; CRM at about 58. mu.g/mL₁₉₇A carrier protein; about 0.25mg/mL of elemental aluminum in the form of aluminum phosphate; about 0.85% sodium chloride; about 0.02% polysorbate 80; about 5mM sodium succinate buffer at pH 5.8; and about 10mg/mL of 2-phenoxyethanol.

18. A vial comprising the multivalent immunogenic composition of any one of claims 1-17.

19. The vial of claim 18, wherein the vial comprises more than one dose of the immunogenic composition.

20. A pre-filled vaccine delivery device comprising the multivalent immunogenic composition of any one of claims 1-19.

21. The pre-filled vaccine delivery device of claim 20, wherein the device is or comprises a syringe.

22. The pre-filled vaccine delivery device of claim 19, wherein the device is or comprises a dual or multi-chamber syringe or vial or a combination thereof.

23. The prefilled vaccine of claims 20-22, wherein said multivalent immunogenic composition is formulated for intramuscular or subcutaneous injection.

24. A kit for the preparation of a multivalent immunogenic composition according to any one of claims 1-17, wherein the kit comprises (i) the plurality of capsular polysaccharides in frozen form of a composition according to any one of the preceding claims, and (ii) an aqueous material for reconstitution of component (i) so as to provide an aqueous composition.

25. A multi-dose vaccine comprising 4 doses of the vaccine in a vial, each dose comprising 4-20mg/mL, preferably 10mg/mL, of 2-phenoxyethanol, wherein the dose is 0.5mL of the vaccine.

26. A container comprising two doses or more of the multivalent immunogenic composition of any one of claims 1-17 at 0.1-2mL per dose.

27. The container of claim 26, wherein the dose is a 0.5mL dose.

28. The container of claims 26-27, comprising 2-10 doses.

29. A method of measuring the efficacy of a vaccine formulation comprising one or more selected preservatives in the presence of some or all of the immunogenic and non-immunogenic components of the vaccine composition, wherein the test comprises at least the following two steps: inoculating a test composition with a selected population of microorganisms, and comparing the log reduction of the inoculated microorganism or microorganisms over time and under particular environmental conditions (e.g., temperature) to the log reduction in a control composition lacking the test preservative.