EA044571B1 - COMPOSITIONS OF GLYCOCONJUGATE VACCINE AGAINST EXPEC - Google Patents

Description

Field of technology to which the invention relates

This invention relates to compositions that induce an immune response against extraintestinal pathogenic Escherichia coli (ExPEC). In particular, embodiments of this invention relate to multivalent vaccines containing conjugates of E. Coli polysaccharide antigens covalently linked to a carrier protein that can withstand multiple environmental stresses.

BACKGROUND OF THE INVENTION

Extraintestinal pathogenic E. coli (ExPEC) is the most common Gram-negative pathogen in humans and can cause a variety of infections outside the gastrointestinal tract that lead to varied and serious morbidity resulting in significant morbidity and mortality. Increasing multidrug resistance among ExPEC strains is impeding treatment and leading to increasing numbers of hospitalizations and deaths and increasing healthcare costs associated with ExPEC infections.

In this regard, the need for a vaccine against ExPEC is urgent. O-antigen, a component of surface lipopolysaccharide, has been identified as a promising vaccine target, and is used as an antigen in a glycoconjugate vaccine currently under development (see, for example, Poolman and Wacker, 2016, J. Infect. Dis 213: 6-13).

Glycoconjugate ExPEC vaccines currently under development contain O-glycans belonging to different ExPEC serotypes, each of which is linked to a carrier protein such as Pseudomonas aeruginosa extracellular protein A (EPA) (see, for example, WO 2015/ 124769 and WO 2017/035181). Such vaccines containing O-glycans of E. coli serotypes 025b, O1A, O2 and O6A are, for example, in phase 2 studies (ClinicalTrials.gov Identifier: NCT02546960).

These inventors have found that although the existing formulation of ExPEC vaccines (25 mM Tris pH 7.4, 2.7 mM KCl, 137 mM NaCl) is suitable for short-term storage at 2-8°C and has an acceptable shelf life Once the package is opened, it is not freeze/thaw stable and does not withstand stirring stress well. Unintentional freezing, accidental heating, and shaking (eg, during storage or transportation) are detrimental to the product integrity of anti-ExPEC formulations. For pharmaceutical products intended for use in large populations, such as the ExPEC glycoconjugate vaccine, it is desirable that the formulation can be frozen in batches at low temperatures and, after thawing and before use, can be stored at approximately 2-8°C (i.e. e. when the drug substance is frozen for long-term storage in large volumes, but the drug product is stored at approximately 2-8°C during use, and thus the product necessarily undergoes at least one freeze/thaw cycle). It is further preferred that the formulation be compatible with a variety of materials so that the product (eg, glycoconjugate) can be stored in a variety of forms (eg, pouches, bottles, vials, prefilled syringes, and/or utensils).

There is a need in the art for ExPEC vaccine formulations that can withstand multiple environmental stresses (eg, freezing/thawing, shaking, elevated temperatures, exposure to light, exposure to oxidizing metals, etc.) and that would have longer stability and shelf life of the compositions, and would preferably be compatible with multiple materials (eg, container) when handling them. Any degradation resulting from environmental stress may lead to reduced biological activity, and may potentially result in the formation of by-products or derivatives of the formulation, thereby resulting in increased toxicity and/or altered immunogenicity of the ExPEC vaccine. Thus, a specialized approach is needed to find a stable formulation of glycoconjugate vaccines that ensures stability under a variety of conditions. This requires selecting the type of buffer, pH, and specific formulation excipients in a specific manner and methodically optimizing them to ensure the chemical, physical, and biological stability of the glycoconjugate vaccines. Given all these changing factors, finding the optimal formulation conditions for ExPEC glycoconjugate vaccines involves overcoming numerous obstacles, and a good formulation cannot be predicted.

Accordingly, there is a need in the art for ExPEC glycoconjugate vaccine formulations that allow the vaccine compositions to withstand multiple environmental stresses and have improved stability and longer shelf life. The purpose of the present invention is to obtain such compositions.

Brief description of the invention

Two new formulations of glycoconjugate vaccines against ExPEC have been proposed, which make it possible to obtain vaccine compositions with improved stabilizing effects during freezing/thawing, shaking stress, heat stress, metal-induced oxidative stress, and in addition, these formulations are compatible with various materials when handling them.

- 1 044571 lami (for example, a container). These improved formulations can be used during storage or transport of both the drug product and the drug substance, where freezing or violent shaking may (unintentionally) occur, which can be detrimental to the integrity of the product and thereby its effectiveness. In addition, the new formulations provide ExPEC glycoconjugate vaccines with improved stability under heat stress and for this reason may have broad commercial applications related to drug product and drug substance storage, handling and transportation, over a wide range of temperatures and a variety of conditions.

Provided herein are compositions comprising at least one E. coli O antigen polysaccharide, wherein the at least one O antigen polysaccharide is covalently linked to a Pseudomonas aeruginosa exotoxin A (EPA) carrier protein; 3% to 8% w/v sorbitol; from 5 to 15 mm methionine; 5 to 20 mM potassium/sodium phosphate buffer with pH 6.5 to 7.5; and 0.01% to 0.2% (w/v) surfactant. In preferred embodiments, the sorbitol concentration is between 4% and 6% (w/v). In preferred embodiments, the methionine concentration is between 8 and 12 mM. In preferred embodiments, the potassium/sodium phosphate buffer concentration is between 8 and 15 mM. Preferably, the composition is a multivalent immunogenic composition comprising an E. coli O25B antigen-polysaccharide, an E. coli O1A antigen-polysaccharide, an E. coli O2 antigen-polysaccharide, and an E. coli O6A antigen-polysaccharide, wherein each O antigen-polysaccharide independently covalently linked to Pseudomonas aeruginosa exotoxin A (EPA) carrier protein; 4% to 6% w/v sorbitol; from 8 to 12 mm methionine; 8 to 15 mM potassium/sodium phosphate buffer pH 6.5 to 7.5; and from 0.01% to 0.2% w/v surfactant.

Also provided herein are compositions comprising at least one E. Coli O antigen polysaccharide, wherein the at least one O antigen polysaccharide is covalently linked to a Pseudomonas aeruginosa exotoxin A (EPA) carrier protein; 3% to 12% w/v sucrose; 0.1 to 1.5 mM EDTA; 5 to 20 mM potassium/sodium phosphate buffer at pH 6.5 to 7.5; and from 0.01% to 0.2% w/v surfactant. In preferred embodiments, the sucrose concentration is from 3% to 10% w/v. In preferred embodiments, the potassium/sodium phosphate buffer concentration is between 8 and 15 mM. Preferably, the composition is a multivalent immunogenic composition comprising an E. coli O25B antigen polysaccharide, an E. coli O1A antigen polysaccharide, an E. coli O2 antigen polysaccharide, and an E. coli O6A antigen polysaccharide, wherein each antigen polysaccharide is independently covalently linked to Pseudomonas exotoxin A aeruginosa (EPA) carrier protein; 3% to 10% w/v sucrose; 0.1 to 1.5 mM EDTA; 8 to 15 mM potassium/sodium phosphate buffer at pH 6.5 to 7.5; and from 0.01% to 0.2% w/v surfactant.

In some embodiments, the E. coli O25B, O1A, O2, and O6A polysaccharide antigens are in a 1:1:1:1 or 2:1:1:1 weight ratio.

In certain embodiments, the sorbitol concentration is 5 w/v%.

In certain embodiments, the sucrose concentration is 8 w/v%.

In preferred embodiments, the methionine concentration is 10 mM.

In certain embodiments, the EDTA concentration is 1 mM.

In a certain embodiment, the potassium/sodium phosphate buffer concentration is 10 mM and the pH of the potassium/sodium phosphate buffer is 7.0.

In certain embodiments, the surfactant concentration is 0.02 w/v%. In preferred embodiments, the surfactant consists of a hydrophilic head (OH group) and a long hydrophobic tail (a carbon chain that may contain approximately 12, 14, 16, 18, 20, 30, 40, 50, 60, 80, 100, 120, 130 or more carbon atoms in the carbon chain). In preferred embodiments, the surfactant is a nonionic surfactant. In certain embodiments, the surfactant is selected from the group consisting of F-68, PS20, PS40, PS60 and PS80. In certain embodiments, the surfactant is PS80.

In certain embodiments, compositions are provided consisting essentially of an E. coli O25B antigen polysaccharide, an E. coli O1A antigen polysaccharide, an E. coli O2 antigen polysaccharide, and an E. coli O6A antigen polysaccharide, wherein each O antigen polysaccharide is covalently associated with Pseudomonas aeruginosa exotoxin A (EPA) carrier protein; 5 w/v% sorbitol; 10 mM methionine; 10 mM potassium/sodium phosphate buffer at pH 7.0; and 0.02% PS80. Also proposed are compositions consisting of E. coli O25B antigen-polysaccharide, E. coli O1A antigen-polysaccharide, E. coli O2 antigen-polysaccharide and E. coli O6A antigen-polysaccharide, where each O antigen-polysaccharide is covalently linked to Pseudomonas aeruginosa exotoxin A ( EPA) carrier protein; 5 w/v% sorbitol; 10 mM methionine; 10 mM potassium/sodium phosphate buffer at pH 7.0; and 0.02% PS80.

In certain embodiments, compositions are provided consisting essentially of an E. coli O25B polysaccharide antigen, an E. coli O1A polysaccharide antigen, an E. coli O2 polysaccharide antigen, an E. coli O6A polysaccharide antigen, and 1, 2, 3, 4 , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 other E. coli O polysaccharide antigens, where each O polysaccharide antigen is covalently linked to Pseudomonas aeruginosa exotoxin A (EPA) carrier protein; 5 w/v% sorbitol; 10 mM methionine; 10 mM potassium/sodium phosphate buffer at pH 7.0; and 0.02% PS80. Also proposed are compositions consisting of E. coli O25B antigen-polysaccharide, E. coli O1A antigen-polysaccharide, E. coli O2 antigen-polysaccharide, E. coli O6A antigen-polysaccharide and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 other E. coli O polysaccharide antigens, where each O polysaccharide antigen is covalently linked to Pseudomonas aeruginosa exotoxin A (EPA) carrier protein; 5 w/v% sorbitol; 10 mM methionine; 10 mM potassium/sodium phosphate buffer at pH 7.0; and 0.02% PS80.

In certain embodiments, compositions are provided consisting essentially of an E. coli O25B antigen polysaccharide, an E. coli O1A antigen polysaccharide, an E. coli O2 antigen polysaccharide, and an E. coli O6A antigen polysaccharide, wherein each O antigen polysaccharide is covalently associated with Pseudomonas aeruginosa exotoxin A (EPA) carrier protein; 8 w/v% sucrose; 1 mM EDTA; 10 mM potassium/sodium phosphate buffer at pH 7.0; and 0.02% PS80. Also proposed are compositions consisting of E. coli O25B antigen-polysaccharide, E. coli O1A antigen-polysaccharide, E. coli O2 antigen-polysaccharide and E. coli O6A antigen-polysaccharide, where each O antigen-polysaccharide is covalently linked to Pseudomonas exotoxin A aeruginosa (EPA) carrier protein; 8 w/v% sucrose; 1 mM EDTA; 10 mM potassium/sodium phosphate buffer at pH 7.0; and 0.02% PS80.

In certain embodiments, compositions are provided consisting essentially of an E. coli O25B polysaccharide antigen, an E. coli O1A polysaccharide antigen, an E. coli O2 polysaccharide antigen, an E. coli O6A polysaccharide antigen, and 1, 2, 3, 4 , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 other E. coli O polysaccharide antigens, where each O polysaccharide antigen is covalently linked to Pseudomonas aeruginosa exotoxin A (EPA) carrier protein; 8 w/v% sucrose; 1 mM EDTA; 10 mM potassium/sodium phosphate buffer at pH 7.0; and 0.02% PS80. Also proposed are compositions consisting of E. coli O25B antigen-polysaccharide, E. coli O1A antigen-polysaccharide, E. coli O2 antigen-polysaccharide, E. coli O6A antigen-polysaccharide and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 other E. coli O polysaccharide antigens, where each O polysaccharide antigen is covalently linked to Pseudomonas aeruginosa exotoxin A (EPA) carrier protein; 8 w/v% sucrose; 1 mM EDTA; 10 mM potassium/sodium phosphate buffer at pH 7.0; and 0.02% PS80.

Also provided herein are methods for preparing the compositions disclosed herein. In certain embodiments, methods for preparing compositions are provided that include adding at least one E. coli O antigen covalently bound to an EPA carrier, water, buffer salts (i.e., sodium (di)hydrogen phosphate and (di)hydrogen phosphate potassium [i.e. Na ₂ HPO ₄ and KH ₂ PO ₄ or NaH ₂ PO ₄ and K ₂ HPO ₄ ]), tonicity regulator (i.e. sorbitol or sucrose), antioxidant (i.e. methionine, if the regulator tonicity is sorbitol; EDTA if the tonicity regulator is sucrose) and surfactant (e.g. PS80) into a container, adjust pH to desired pH value (i.e. 6.5 to 7.5, e.g. 7.0) and stir these ingredients, and thus obtaining a liquid composition according to this invention. In a preferred embodiment, methods for preparing the compositions include the addition of E. coli O25B antigen polysaccharide, E. coli O1A antigen polysaccharide, E. coli O2 antigen polysaccharide and E. coli O6A antigen polysaccharide, wherein each antigen polysaccharide is covalently linked to an exotoxin A of Pseudomonas aeruginosa (EPA) carrier protein; water; potassium phosphate; sodium phosphate; sorbitol; methionine; and PS80 into a container, adjust the pH to 6.5-7.5 (e.g. 7.0), and mix each ingredient so that the final potassium/sodium phosphate buffer concentration is 5-20 mM (e.g. 10 mM) at pH 6.5-7.5 (e.g. 7.0), final sorbitol concentration 3-8% (e.g. 5%) (w/v), final methionine concentration 5-15 mM (e.g. 10 mM), and the final concentration of PS80 was 0.01-0.08% (eg 0.02%) (w/v). In a preferred embodiment, methods for preparing the compositions include the addition of E. coli O25B antigen polysaccharide, E. coli O1A antigen polysaccharide, E. coli O2 antigen polysaccharide and E. coli O6A antigen polysaccharide, wherein each antigen polysaccharide is covalently linked to an exotoxin A of Pseudomonas aeruginosa (EPA) carrier protein; water; potassium phosphate; sodium phosphate; sucrose; EDTA; and PS80 into a container, mixing each ingredient and adjusting the pH to 6.5-7.5 (e.g. 7.0) so that the final sucrose concentration is 3-12% (e.g. 8%) (w/v) , the final concentration of EDTA was 0.1-1.5 mM (eg 1 mM), the final concentration of potassium/sodium phosphate buffer was 5-20 mM (eg 10 mM) at a pH of 6.5-7.5 ( eg 7.0), and PS80 is 0.01-0.08% (eg 0.02%) (w/v).

In certain embodiments, these compositions are provided in the form of liquid compositions. By liquid composition is meant that it is in a liquid state at a temperature of 2-8°C and is preferably stored at 2-8°C.

In certain embodiments, these compositions can be stored and remain stable at 2-8°C, at 25°C, or at 40°C. In a preferred embodiment, the composition is stored and stable at 2-8°C. In certain embodiments, the composition is stable at 2-8°C for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or more months. In certain embodiments, the composition is stable at 25°C for at least about 1, 2, 3, 4, 5, 6 or more months. In certain embodiments, the composition is stable at 40°C for at least about 1, 2, 3, 4, 5, 6,

7, 8, 9, 10, 11, 12 or more weeks.

In certain embodiments, these compositions are provided as a frozen formulation. By frozen composition is meant a composition that is in a solid state when stored at a temperature of about -18°C or lower, for example, at about -20°C, -40°C, -60°C, -70°C, -80°C or any temperature in this range or below. In certain embodiments, these compositions can be stored and remain stable at -40°C or -60°C depending on the tonicity regulator present in the composition. In certain embodiments, the implementation of these compositions can be stored and remain stable at -70°C. In certain embodiments, the composition contains sucrose and is stable at -40°C for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 , 41, 42, 43, 44, 45, 46, 47, 48 or more months. In certain embodiments, the composition contains sorbitol and is stable at -60°C for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 , 41, 42, 43, 44, 45, 46, 47, 48 or more months. In certain embodiments, the composition contains sorbitol or sucrose and is stable at -70°C for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years.

In preferred embodiments, the compositions of the present invention do not contain sodium chloride.

In certain embodiments, the concentration of each O-antigen polysaccharide in the composition is from about 1 to 200 μg/ml, such as from about 2 to 100 μg/ml, such as from about 4 to 50 μg/ml. In certain embodiments, the polysaccharide:carrier protein ratio is from about 1:10 to about 1:2, such as from about 1:5 to 1:2, for each O-antigen polysaccharide in the composition.

Brief description of the figures

The foregoing brief description, as well as the following detailed description of preferred embodiments of the present invention, will be better understood when taken in conjunction with the accompanying drawings. It should be understood that the present invention is not limited to the specific embodiments shown in the drawings.

In graphic materials:

in fig. 1A-1C show that PS20, PS80 and F68, but not sorbitol, were able to prevent freeze/thaw induced aggregation of the ExPEC glycoconjugate under test conditions: PS20, PS80, F68 and sorbitol were added to the ExPEC glycoconjugate formulation and subjected to freeze/thaw stress. defrosting, in Fig. 1A shows a method for integrating a high-performance liquid chromatography (SEC-HPLC) chromatogram;

in fig. 1B is an SEC-HPLC chromatogram showing the experimental results before and after freezing/thawing; and in fig. 1C shows a diagram of the magnitude of the peak of impurity 1 as a percentage of the total integrated value of the peaks;

in fig. 2 shows a graph comparing the percentages of the EC-HPLC chromatographic peak of impurity 1 of formulations 26 and 28 with the old ExPEC glycoconjugate formulation, after prolonged temperature exposure at 40°C for a period of 12 weeks: both positions of the vial were examined, namely, vertical (4) and inverted (T), Formulations 26 and 28 demonstrated increased stability compared to the ExPEC glycoconjugate formulation when exposed to heat stress (40°C); and in fig. 3 shows graphs showing the behavior of impurity peak 1 of formulations 26 and 28 compared to the old ExPEC glycoconjugate formulation when exposed to three different concentrations of tungsten extract (HW, MW, LW) and stored at 40°C for 4 weeks.

in fig. 4 shows EC-HPLC chromatograms showing the experimental results before and after shaking stress (T0 - control sample, i.e. without shaking stress) for formulations 26 and 26 in comparison with the old formulation in contact with PETG and plastic materials PC.

Detailed Description of the Invention

Various publications, articles and patents are cited or described in the Background section and throughout the specification; each of these references being incorporated herein by reference in its entirety. The discussion of documents, acts, materials, devices, products, and the like that have been included herein is intended to provide context for the present invention. Such discussion is not an admission that any or all of this material is part of the prior art with respect to any disclosed or claimed inventions.

- 4 044571

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention relates. Otherwise, certain terms used herein have the meanings set forth in the specification.

It should be noted that as used herein and in the accompanying claims, the singular number includes a reference to the plural unless the context clearly indicates otherwise.

Unless otherwise indicated, any numerical values, such as concentration or concentration range limits, described herein are in all cases to be understood as modified by the term approximately. Thus, the numerical value usually includes ±10% of the specified value. For example, a concentration of 1 mg/ml includes all values from 0.9 mg/ml to 1.1 mg/ml. Likewise, the concentration range from 1% to 10% w/v includes values from 0.9% w/v to 11% w/v. As used herein, the use of a numeric interval clearly includes all possible subintervals, all numeric values within that interval, including integers within such intervals, and fractional values, unless the context explicitly states otherwise.

As used herein, the terms contains, contains, includes, including, has, having, or any other form thereof are intended to mean that they include the integer or group of integers indicated, but do not exclude any other integer or group of integers, and are intended be non-exclusive or amendable. For example, a composition, mixture, process, method, article or device containing a list of elements is not necessarily limited to only those elements, but may include other elements not expressly listed or integral to such composition, mixture, process method, article or device. Also, unless explicitly stated otherwise, or refers to an inclusive or rather than an exclusive or. For example, the condition A or B is satisfied by any of the following: A is true (or present) and B is false (or absent), A is false (or absent) and B is true (or present), and both A and B are true (or present). availability).

As used herein, the term consists of, or variations such as consist of or consisting of, throughout the specification or claims, indicate the inclusion of any of the specified elements or group of elements, but additional elements or group of elements may not be added to the specified method , structure or composition.

As used herein, the term essentially consists of, or variations such as essentially consist of or essentially consisting of, throughout the specification or claims, indicate the inclusion of any of the recited elements or group of elements and the optional inclusion of any of the recited elements or group elements that do not significantly change the basis of the new features of the specified method, structure or composition.

The terms approximately, approximately, generally, generally and the like, when used in this context in relation to the size or property of a component of a preferred invention, should be taken to indicate that the size/property described does not constitute a strict limitation or parameter and does not exclude small deviations from it that are functionally the same or similar, as would be understood by one of ordinary skill in the art. At a minimum, references to such numerical parameters will include variations that, when using mathematical and industrial approaches accepted in the art (for example, rounding, measurement errors or other systematic errors, manufacturing tolerances, etc.), will not change the latter significant figure.

As used herein, the terms O polysaccharide, O antigen, O antigen, O antigen polysaccharide, O polysaccharide antigen, and the abbreviation OPS all refer to the O antigen of gram-negative bacteria, which is a component of lipopolysaccharide (LPS) and is specific to each serotype or sero( sub)type of gram-negative bacteria. O antigen typically contains repeating units (RUs) of two to seven sugar residues. In this context, the PU is taken to be equal to the biological repeat unit (BRU). BEP describes the O-antigen PE as it is synthesized in vivo.

As used herein, the terms conjugate and glycoconjugate all refer to a conjugation product containing E. coli O antigen covalently linked to a carrier protein (eg, Pseudomonas aeruginosa exotoxin A (EPA)). The conjugate may be a bioconjugate, which is a conjugation product produced in a host cell, where the host cell machinery produces the O antigen and the carrier protein and binds the O antigen to the carrier protein, for example, via N-bonds. The conjugate can also be produced by other methods, for example by chemically linking the protein and the sugar residue of the antigen.

E. coli O antigens in embodiments of the present invention include O1A, O2, O6A and 025b disclosed in WO 2015/124769 and WO 2017/035181. Each of these references is incorporated herein by reference in its entirety. Other E. coli O antigens may also be used, for example, in addition to specific O1A, O2, O6A and/or O25B antigens, and may, for example, include, without limitation, O antigens from their E serotypes or subserotypes coli O1, O2, O4, O6, O7, O8, O15,

O16, O18, O21, O25, O73, O75 and O153.

In this context, the term drug substance refers to the bulk finished product of a single glycoconjugate (E. coli O antigen-polysaccharide covalently linked to the EPA of a carrier protein, e.g., E. coli O25B O antigen covalently linked to EPA), the concentration of which is higher than at the final product that will be administered to the subject. The drug substance can be obtained after completion of the downstream process after purification of the glycoconjugate. The drug substance can, for example, be stored in a more concentrated form in the buffer of the composition of the present invention, for example, in a frozen state, for example at -70°C.

As used herein, the term drug product refers to the composition of the glycoconjugates in their final form intended for administration to a subject. The drug product contains all of the glycoconjugates (E. coli O polysaccharide antigens bound to the carrier protein EPA in the case of a multivalent vaccine, e.g. 025b, O1A, O2 and O6A each individually bound to the carrier protein EPA in the case of a quadrivalent vaccine, and , optionally, more O E. coli antigens bound to the EPA carrier protein if the valency of the vaccine is increased). The drug product can usually be prepared by mixing the drug substances of the appropriate glycoconjugates and diluting with the formulation buffer, if necessary, to obtain the target dose of the vaccine. The drug product of this invention can be stored at 2-8°C and remain stable at this temperature for at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16. 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or more months.

The term approximately, when used in reference to a number, refers to any number with a value of ±1, ±5, or ±10% of the specified number.

If the O antigen is covalently linked to a carrier protein, the effective amount or dosage of the O antigen is determined based only on the O antigen-polysaccharide fragment in the conjugate.

Illnesses associated with ExPEC or ExPEC infections include, but are not limited to, urinary tract infections, postoperative infections, bacteremia, abdominal or pelvic infection, pneumonia, hospital-acquired pneumonia, osteomyelitis, cellulitis, pyelonephritis, wound infection, meningitis, neonatal meningitis, peritonitis, inflammation of the biliary tract, soft tissue infections, intermuscular abscess, purulent arthritis and sepsis.

As used herein, the term combination when referring to the administration of two or more therapies to a subject refers to the administration of more than one therapy. Use of the term in combination does not impose a limitation on the order in which these therapies are administered to a subject. For example, a first therapy (eg, a composition described herein) may be administered to a subject before, concurrently with, or after the administration of a second therapy (eg, a composition described herein or another therapy, such as an antibiotic treatment).

As used herein, the term subject refers to an animal, preferably a mammal, and may include non-primate animals (eg, pig, horse, goat, sheep, cat, dog, rabbit, rat or mouse) and primates (eg, monkey, chimpanzee or person). In certain embodiments, the subject is a non-human animal. In another embodiment, the subject is a human. The terms subject and patient may be used interchangeably. The compositions of this invention are suitable for administration to a subject and can be used to generate an immune response to ExPEC O antigens contained in the composition.

As used herein, an immunological response or immune response to an antigen or composition refers to the development in a subject of a humoral and/or cellular immune response to the antigen or to an antigen present in the composition.

Compositions containing E. coli O polysaccharide antigens

In one general aspect, the invention provides a multivalent vaccine containing O-antigen serotypes that are found primarily among clinical isolates of E. coli that can be used to provide active immunization to prevent disease caused by ExPEC having the O-antigen serotype contained in vaccine. In one embodiment, the invention provides a composition comprising at least one E. coli O antigen polysaccharide. In some embodiments, the composition may comprise an E. coli O25B antigen polysaccharide. In other embodiments, the composition contains E. coli O1A, O2 and/or O6A antigen polysaccharide. In preferred embodiments, the composition contains E. coli O1A, O2, O6A and 025B polysaccharide antigens disclosed in WO 2015/124769 and WO 2017/035181. Each of these references is incorporated herein by reference in its entirety. In some embodiments, other or additional E. coli O polysaccharide antigens are present in the composition. Such E. coli O polysaccharide antigens may include, without limitation, O antigens from E. coli (sub)serotypes 01, 02, 04, O6, 07, 08, 015, 016, 018, 021, 025, 073, 075, and 0153. Depending on the need, the compositions may include more than one additional E. coli O antigen, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more additional E. coli O antigens to provide protection

- 6 044571 from numerous serotypes of E. coli.

In some embodiments, the compositions and methods may relate to the E. coli O25B antigen and one or more additional E. coli O antigens. Examples of E. coli O antigens may include, but are not limited to, E. coli O25B, O1A, O2 and O6A antigens.

In this context, E. coli O25B antigen refers to an O antigen specific for the E. coli O25B serotype. In one embodiment, the E. coli O25B antigen includes a structure with formula 025B':

D-GIc

4 ⁶ ₽ [ * а а 1 —D-GIc---► L-Rha2Ac —D-GIcNAc -4—*-

-TG 113 1.з U

L-Rha where n in the formula O25B' is an integer from 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 10 to 30, 15 to 30, 20 to 30 , 25 to 30, 5 to 25, 10 to 25, 15 to 25, 20 to 25, 10 to 20, or 15 to 20. In one embodiment of the present invention, n in formula 025B' is an integer from 10 to 20.

In this context, E. coli O1A antigen refers to the O antigen specific for the E. coli O1A serotype. In one embodiment, the E. coli O1A antigen includes a structure with the formula O1A':

where n in formula O1A' is an integer from 1 to 30, 1 to 20, from 1 to 15, from 1 to 10, from 1 to 5, from 10 to 30, from 15 to 30, from 20 to 30, from 25 to 30, 5 to 25, 10 to 25, 15 to 25, 20 to 25, 10 to 20, or 15 to 20. In one embodiment, n in formula O1A' is an integer from 7 to 15.

In this context, E. coli O2 antigen refers to the O antigen specific for the E. coli O2 serotype. In one embodiment, the E. coli O2 antigen includes a structure with the formula O2':

a a r

L-Rha----► L-Rha , >·L-Rha —— i V 1.3 1.4

D-GIcNAc n

^Ε Ί ²

D-Fuc3NAc f

where n in formula O2' is an integer from 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 25, 10 to 25, 15 to 25, 20 to 25, 10 to 20, or 15 to 20. In one embodiment, n in formula O2' is an integer from 8 to 16.

In this context, E. coli O6 antigen refers to the O antigen specific for the E. coli O6 serotype. In one embodiment, the E. coli O6 antigen includes E. coli O6A.

In this context, E. coli O6A antigen, also referred to as E. coli O6K2 antigen or E. coli O6Glc antigen, refers to the O antigen specific for the E. coli O6A serotype. In one embodiment, the E. coli O6A antigen includes a structure with the formula O6A':

where the β 1, 2 bond is also called a p2 bond, n in the formula O6A' is an integer from 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 25, 10 to 25, 15 to 25, 20 to 25, 10 to 20, or 15 to 20. In one embodiment, n in the formula O6A' is an integer from 8 to 18.

The E. coli O polysaccharide antigens in the compositions of the present invention are covalently linked (sometimes referred to as conjugated) to a carrier protein, the carrier protein of the invention being the P. aeruginosa exotoxin A, preferably an inactivated version thereof (EPA; see, for example, Ihssen, et al (2010) Microbial cell factories 9, 61; WO 2015/124769). The combination of a carrier protein and an O antigen-polysaccharide is referred to as a glycoconjugate. Typically, the O antigen from each E. coli serotype in the composition is covalently linked to a separate carrier protein, i.e. if the composition contains four O polysaccharide antigens, then each of them is separately and independently associated with the EPA of the carrier protein, and thus the composition contains four different glycoconjugates. One way to make glycoconjugates is bioconjugation, in which the host cell machinery produces O antigen and carrier protein and binds O antigen to the carrier protein, e.g.

- 7 044571 with N-bonds (preparation of bioconjugates of E. coli O antigen 025B, O1A, O2 and O6A polysaccharides with EPA carrier protein, see, for example, WO 2015/124769). For EPA, various detoxified protein variants have been described in the literature and can be used as carrier proteins. Inactivated (or non-toxic) EPA refers to any Pseudomonas aeruginosa exotoxin A that does not have ADP-ribosylation activity. The ribosylation activity of wild-type EPA is located approximately between amino acids 400 and 613 in the EPA sequence, and, for example, removing the glutamic acid amino acid at position 553 from wild-type EPA or replacing the histidine at position 426 with a tyrosine in wild-type EPA renders the EPA molecule inactive. Other amino acids among amino acids 400-613 in wild-type EPA can be modified, for example by deletion, insertion or substitution of amino acid residues, to remove ADP-ribosylation activity and thereby toxicity, as known to one of ordinary skill in the art. In preferred embodiments of the present invention, the carrier protein EPA is a detoxified version. In some non-limiting and illustrative embodiments, the carrier protein EPA may comprise an amino acid sequence as set forth in SEQ ID NO: 1 (representing an embodiment of a deactivated EPA), or an amino acid sequence that is at least 80%, 81%, 82% , 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % identical to SEQ ID NO: 1, or different by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids from SEQ ID NO: 1, any of which optionally additionally contain 1, 2, 3 , 4, 5, 6, 7, 8 or more recombinantly inserted consensus glycosylation sequences, as described below. In specific embodiments, the introduction of glycosylation sites is accomplished by inserting consensus glycosylation sequences (e.g., Asn-X-Ser(Thr), where X can be any amino acid other than Pro; or, preferably, Asp(Glu)-X-Asn-Z-Ser (Thr), where X and Z are independently selected from natural amino acids, excluding Pro (SEQ ID NO: 2) (see, for example, WO 2006/119987 and WO 2015/124769)) anywhere in the primary structure of the EPA protein. In preferred embodiments, the carrier protein EPA comprises one or more recombinantly inserted N-linked consensus glycosylation sites having the amino acid sequence Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from natural amino acids except Pro (SEQ ID NO: 2). In some embodiments, the carrier protein EPA comprises one, two, three, four, five, six, seven, eight, or more such inserted consensus sequences. In other embodiments, EPA does not contain such recombinantly inserted N-linked consensus glycosylation sites, for example, if the glycoconjugate is prepared using classical conjugation chemistry (see, for example, US 5,370,872; Cryz et al., 1990, Infection and Immunity 58: 373- 377).

In some embodiments, the E. coli O antigens are covalently linked to a carrier protein in a polysaccharide to protein weight ratio of about 1:20 to 20:1, preferably 1:10 to 10:1, such as 1: 10 to 3:1. In some non-limiting embodiments of the bioconjugates, the polysaccharide/protein ratio is in the range of about 0.1 to 0.5 (i.e., polysaccharide:protein is about 1:10 to 1:2, e.g., about 1:2, 1: 3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10 or any intermediate value) for each bioconjugate depending on the 0-antigen serotype.

In some non-limiting embodiments, the concentration of E. coli O antigen polysaccharides in the composition can be from about 1 to 200 μg/ml for each O antigen polysaccharide, for example, from about 2 to 100 μg/ml, for example, from about 4 to 50 μg/ ml, for example from about 4 to 48 µg/ml, for example from about 8 to 48 µg/ml, for example from about 4 to 32 µg/ml, for example from about 8 to 32 µg/ml, for example 8, 16, 20 or 32 μg/ml or equal to any other value in this range Preferably, in a composition of at least two E. coli O antigen polysaccharides, each O antigen polysaccharide is present in weight ratios of 1:1 to 1:2 or any intermediate value for each combination of O antigens-polysaccharides in the composition.

In some non-limiting embodiments, the O-antigen polysaccharides are present in the composition at a total O-antigen polysaccharide concentration of from about 4 to 1000 μg/ml, for example, from about 10 to 500 μg/ml, for example, from about 20 to 250 μg/ml, for example, from about 24 to 120 μg/ml.

In some non-limiting embodiments, the O-antigen polysaccharides are present in the composition at a total O-antigen polysaccharide concentration of from about 40 to 2000 μg/ml, such as from about 100 to 1500 μg/ml, such as from about 200 to 1200 μg/ml, for example from about 250 to 600 μg/ml.

In some embodiments, the composition contains 025b, O1A, O2 and O6A antigen polysaccharides in a weight ratio of 1:1:1:1, with each O antigen polysaccharide covalently linked to the EPA of a carrier protein. In another embodiment, the composition contains E. coli O25B, O1A, O2 and O6A polysaccharide antigens in a weight ratio of 2:1:1:1, with each O polysaccharide antigen covalently linked to the EPA of a carrier protein. In each such embodiment, the concentration of 025B polysaccharide antigen may, by way of non-limiting example, be 8, 16, or 32 μg/ml.

The compositions described herein are useful in treating and preventing infections in subjects (eg, human subjects) with ExPEC. In some embodiments, in addition to the polysaccharide antigens contained in the E. coli O antigens covalently bound to a carrier protein EPA, the compositions described herein contain a pharmaceutically acceptable carrier. As used herein, pharmaceutically acceptable means approved by a regulatory agency of the federal or state government, or listed in the U.S. Pharmacopeia or other generally accepted pharmacopoeia for use in animals or, more specifically, in humans. The term carrier, as used in the context of a pharmaceutically acceptable carrier, refers to the diluent, excipient, excipient or carrier with which the pharmaceutical composition is administered. The water in the composition of the present invention is preferably water for injection.

The composition proposed in this document contains a surfactant. The surfactants used herein are organic amphiphilic compounds, which means that they contain both hydrophobic groups (their tail parts) and hydrophilic groups (their head parts). In preferred embodiments, the surfactant consists of a hydrophilic head (containing OH groups) and a long hydrophobic tail (a carbon chain that may contain approximately 12, 14, 16, 18, 20, 30, 40, 50, 60, 80, 100 , 120, 130 or more carbon atoms in the carbon chain). Preferred surfactants according to this invention are nonionic surfactants. Examples of surfactants suitable for the compositions of this invention include, but are not limited to, polysorbate 20 (PS20), polysorbate 40 (PS40), polysorbate 60 (PS60), polysorbate 80 (PS80) and Pluronic® F-68 (F-68). In a preferred embodiment, the surfactant is PS80. I In some embodiments, the concentration of surfactant in the composition is from 0.01% (w/v (w/v)) to 0.2% w/v. In some embodiments, the concentration of surfactant in the composition is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0 .09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0 .19%, 0.2% or any value in between. In embodiments where the surfactant is F-68, the concentration is preferably from about 0.05% to about 0.2%. In embodiments where the surfactant is PS20, PS40, PS60, or PS80, the concentration is preferably from about 0.01% to about 0.08%, such as about 0.01%, 0.02%, 0.03% , 0.04%, 0.05%, 0.06%, 0.07%, 0.08% or any value in between.

The compositions provided herein additionally contain a buffer with a specific pH. According to the present invention, the composition contains a potassium/sodium phosphate buffer. Concentration ranges for such a buffer may be, for example, from about 5 mM to about 20 mM, from about 8 mM to about 15 mM, from about 8 mM to about 13 mM, from about 9 mM to about 11 mM. In some embodiments, the buffer concentration is 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, or any value in between. In some embodiments, the buffer concentration is from about 8 mM to about 15 mM. In a preferred embodiment, the concentration of the buffer in the composition is 10 mM. The pH ranges for such buffers may be from about pH 6.5 to about pH 7.5, from about pH 6.6 to about pH 7.4, from about pH 6.7 to about pH 7.3, from about pH 6 .8 to about pH 7.2, from about pH 6.9 to about pH 7.1. In certain embodiments, the pH may be 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7. 5 or any other intermediate value. In a preferred embodiment, the pH of the buffer is 7.0. In a preferred embodiment, the composition contains potassium/sodium phosphate buffer at a concentration of 10 mM and at a pH of 7.0.

Potassium/sodium phosphate buffer solutions may, for example, contain potassium from K2HPO4 (dibasic) or KH2P4 (monobasic) and sodium from Na ₂ HPO ₄ (dibasic) or NaH ₂ PO ₄ (monobasic). In some embodiments, the potassium/sodium phosphate buffer contains monobasic potassium phosphate (KH2P4) and dibasic sodium phosphate (Na ₂ HPO ₄ ). In some embodiments, the potassium/sodium phosphate buffer contains dibasic potassium phosphate (K ₂ HPO ₄ ) and monobasic sodium phosphate (NaH ₂ PO ₄ ). The molar ratio of monobasic phosphate particles and dibasic phosphate particles ([H ₂ PO ₄ ]/[HPO ₄ ] ^2- ), regardless of the counterion, varies from 5.13 to 0.52.

The composition provided herein may further comprise a tonicity regulator (sometimes called a stabilizer). The tonicity regulators used in the composition of this invention are either sucrose or sorbitol, depending on other excipients as defined herein. In certain embodiments, the composition contains sucrose. In some embodiments, the composition contains sucrose at a concentration ranging from about 3% to about 12% w/v, from about 3% to about 11% w/v, from about 3% to about 10% w/v. %, from about 4% to about 10% w/v, from about 5% to about 9% w/v, from about

- 9 044571

6% to approximately 9 w/v%. In certain embodiments, the sucrose concentration may be 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, or any other value in between. In a preferred embodiment, the composition contains sucrose, the concentration of which is from about 3% to about 10%. In a preferred embodiment, the composition contains sucrose, the concentration of which is 8%. In other preferred embodiments, the composition contains sorbitol. In a preferred embodiment, the composition contains sorbitol at a concentration of from about 3% to about 8% w/v, from about 3% to about 7% w/v, from about 4% to about 6% w/v. In preferred embodiments, the composition contains sorbitol at a concentration of from about 4% to about 6% w/v. In certain embodiments, the composition contains sorbitol at a concentration of 3%, 4%, 5%, 6%, 3%, 4%, 5%, 6%, 7%, 8%, or any other value in between. In a preferred embodiment, the composition contains sorbitol at a concentration of 5%.

The composition provided herein further contains an antioxidant. The antioxidant used in the compositions of this invention is EDTA or methionine depending on other excipients as described herein. In certain embodiments, the composition contains EDTA. In some embodiments, the composition contains EDTA at a concentration of from about 0.1 mM to about 1.5 mM, from about 0.2 mM to about 1.4 mM, from about 0.5 mM to about 1.3 mM, from about 0.7 mm to about 1.2 mm, from about 0.8 mm to about 1.2 mm, from about 0.9 mm to about 1.1 mm. In some embodiments, the concentration of EDTA in the composition is 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0 .9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM or any value in between. In a preferred embodiment, the composition contains EDTA, the concentration of which is 1.0 mM. The composition of the present invention containing EDTA also contains sucrose. In other embodiments, the composition contains methionine. In some embodiments, the composition contains methionine at a concentration of from about 5 mM to about 15 mM, from about 6 mM to about 14 mM, from about 7 mM to about 13 mM, from about 8 mM to about 12 mM, from about 9 mM to approximately 11 mM. In a preferred embodiment, the composition contains methionine, the concentration of which is from about 8% to about 12%. In some embodiments, the methionine concentration is 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, or any value in between. In a preferred embodiment, the composition contains methionine, the concentration of which is 10 mM. The composition of the present invention containing methionine also contains sorbitol.

Compositions are proposed containing E. coli O25B antigen-polysaccharide, E. coli O1A antigen-polysaccharide, E. coli O2 antigen-polysaccharide and E. coli O6A antigen-polysaccharide, where each O antigen-polysaccharide is covalently linked to Pseudomonas aeruginosa exotoxin A (EPA) protein- carrier; 5 w/v% sorbitol; 10 mM methionine; 10 mM potassium/sodium phosphate buffer at pH 7.0; and 0.02% PS80. Also provided are compositions consisting primarily of E. coli O25B antigen-polysaccharide, E. coli O1A antigen-polysaccharide, E. coli O2 antigen-polysaccharide and E. coli O6A antigen-polysaccharide, where each O antigen-polysaccharide is covalently linked to Pseudomonas exotoxin A aeruginosa (EPA) carrier protein; 5 w/v% sorbitol; 10 mM methionine; 10 mM potassium/sodium phosphate buffer at pH 7.0; and 0.02% PS80. Also proposed are compositions consisting of E. coli O25B antigen-polysaccharide, E. coli O1A antigen-polysaccharide, E. coli O2 antigen-polysaccharide and E. coli O6A antigen-polysaccharide, where each O antigen-polysaccharide is covalently linked to Pseudomonas exotoxin A aeruginosa (EPA) carrier protein; 5 w/v% sorbitol; 10 mM methionine; 10 mM potassium/sodium phosphate buffer at pH 7.0; and 0.02% PS80. Also proposed are compositions containing E. coli O25B antigen-polysaccharide, E. coli O1A antigen-polysaccharide, E. coli O2 antigen-polysaccharide, E. coli O6A antigen-polysaccharide and 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 other E. coli O polysaccharide antigens, where each O polysaccharide antigen is covalently linked to Pseudomonas aeruginosa exotoxin A (EPA ) carrier protein; 5 w/v% sorbitol; 10 mM methionine; 10 mM potassium/sodium phosphate buffer at pH 7.0; and 0.02% PS80. Also provided are compositions consisting primarily of E. coli O25B polysaccharide antigen, E. coli O1A polysaccharide antigen, E. coli O2 polysaccharide antigen, E. coli O6A polysaccharide antigen and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 other E. coli O polysaccharide antigens, where each O polysaccharide antigen is covalently linked to Pseudomonas exotoxin A aeruginosa (EPA) carrier protein; 5 w/v% sorbitol; 10 mM methionine; 10 mM potassium/sodium phosphate buffer at pH 7.0; and 0.02% PS80. Also proposed are compositions consisting of E. coli O25B antigen-polysaccharide, E. coli O1A antigen-polysaccharide, E. coli O2 antigen-polysaccharide, E. coli O6A antigen-polysaccharide and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 other E. coli O polysaccharide antigens, where each O polysaccharide antigen is covalently linked to Pseudomonas aeruginosa exotoxin A ( EPA) carrier protein; 5 w/v% sorbitol;

- 10044571 mM methionine; 10 mM potassium/sodium phosphate buffer at pH 7.0; and 0.02% PS80.

Compositions are proposed containing E. coli O25B antigen-polysaccharide, E. coli O1A antigen-polysaccharide, E. coli O2 antigen-polysaccharide and E. coli O6A antigen-polysaccharide, where each O antigen-polysaccharide is covalently linked to Pseudomonas aeruginosa exotoxin A (EPA) protein- carrier; 8 w/v% sucrose; 1 mM EDTA; 10 mM potassium/sodium phosphate buffer at pH 7.0; and 0.02% PS80. Also provided are compositions consisting primarily of E. coli O25B antigen-polysaccharide, E. coli O1A antigen-polysaccharide, E. coli O2 antigen-polysaccharide and E. coli O6A antigen-polysaccharide, where each O antigen-polysaccharide is covalently linked to an exotoxin A Pseudomonas aeruginosa (EPA) carrier protein; 8 w/v% sucrose; 1 mM EDTA; 10 mM potassium/sodium phosphate buffer at pH 7.0; and 0.02% PS80. Also proposed are compositions consisting of E. coli O25B antigen-polysaccharide, E. coli O1A antigen-polysaccharide, E. coli O2 antigen-polysaccharide and E. coli O6A antigen-polysaccharide, where each O antigen-polysaccharide is covalently linked to Pseudomonas exotoxin A aeruginosa (EPA) carrier protein; 8 w/v% sucrose; 1 mM EDTA; 10 mM potassium/sodium phosphate buffer at pH 7.0; and 0.02% PS80. Also proposed are compositions containing E. coli O25B antigen-polysaccharide, E. coli O1A antigen-polysaccharide, E. coli O2 antigen-polysaccharide, E. coli O6A antigen-polysaccharide and 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 other E. coli O polysaccharide antigens, where each O polysaccharide antigen is covalently linked to Pseudomonas aeruginosa exotoxin A (EPA ) carrier protein; 8 w/v% sucrose; 1 mM EDTA; 10 mM potassium/sodium phosphate buffer at pH 7.0; and 0.02% PS80. Also provided are compositions primarily consisting of E. coli O25B polysaccharide antigen, E. coli O1A polysaccharide antigen, E. coli O2 polysaccharide antigen, E. coli O6A polysaccharide antigen and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 other E. coli O polysaccharide antigens, where each O polysaccharide antigen is covalently linked to Pseudomonas exotoxin A aeruginosa (EPA) carrier protein; 8 w/v% sucrose; 1 mM EDTA; 10 mM potassium/sodium phosphate buffer at pH 7.0; and 0.02% PS8 0. Also proposed are compositions consisting of E. coli O25B antigen-polysaccharide, E. coli O1A antigen-polysaccharide, E. coli O2 antigen-polysaccharide, E. coli O6A antigen-polysaccharide and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 other E. coli O polysaccharide antigens, where each O antigen is a polysaccharide covalently linked to Pseudomonas aeruginosa exotoxin A (EPA) carrier protein; 8 w/v% sucrose; 1 mM EDTA; 10 mM potassium/sodium phosphate buffer at pH 7.0; and 0.02% PS80.

The compositions described herein may optionally contain a preservative such as the mercury derivative thimerosal, phenoxyethanol or parabens. In a specific embodiment, the pharmaceutical compositions described in this invention contain from 0.001% to 0.01% thiomersal. In other embodiments, the pharmaceutical compositions described in this invention do not contain a preservative.

In certain embodiments, the compositions described herein (eg, immunogenic compositions) contain or are administered in combination with an adjuvant. The administration adjuvant in combination with the composition described herein can be administered before, simultaneously with, or after administration of the composition. In some embodiments, the term adjuvant or adjuvant refers to a compound that, when administered in combination with or as part of a composition described herein, increases, enhances and/or promotes the immune response to the bioconjugate, but when administered alone, the immune response is not formed on the bioconjugate. Adjuvant agents enhance the immune response through several mechanisms, including, for example, lymphocyte recruitment, stimulation of B and/or T cells, and stimulation of macrophages. In certain embodiments, the compositions described herein do not contain an adjuvant other than bioconjugates and excipients, and/or it is not administered in combination with an adjuvant other than bioconjugates and excipients (in the case where the bioconjugates and excipients have inherent adjuvant properties, this will not be taken into account and additional foreign aid will not be added in these embodiments).

Specific examples of adjuvants include, but are not limited to, aluminum salts (alum) (such as aluminum hydroxide, aluminum phosphate and aluminum sulfate), 3 de-Oacylated monophosphoryl lipid A (MPL) (see UK patent GB2220211), MF59 (Novartis ), AS03 (GlaxoSmithKline), AS04 (GlaxoSmithKline), imidazopyridine compounds (see WO 2007/109812), imidazoquinoxaline compounds (see WO 2007/109813) and saponins such as QS21 (see Kensil et al., in Vaccine Design : The Subunit and Adjuvant Approach (eds. Powell & Newman, Plenum Press, NY, 1995); US Patent No. 5057540). Other excipients are oil-in-water emulsions (such as squalene or peanut oil), optionally in combination with immunostimulants such as monophosphoryl lipid A (see Stoute et al., 1997, N. Engl. J. Med. 336 , 86-91). Another adjuvant is CpG (Bioworld Today, Nov. 15, 1998).

In some embodiments, the compositions described herein are formulated to be suitable for a particular route of administration to a subject. For example, the formulations described herein can be formulated, for example, for intramuscular, subcutaneous, steam

- 11 044571 teral, oral, intranasal, intradermal, transdermal, colorectal, intraperitoneal, intratracheal, topical, rectal or pulmonary administration. In certain embodiments, the compositions described herein are useful for administration by intramuscular injection. In other embodiments, the compositions described herein can be administered intradermally. In other embodiments, the compositions described herein can be administered through the skin.

In another aspect, this document also provides drug substance compositions containing at least one of the four conjugates described in detail herein (i.e., an O-antigen from E. coli serotypes 025b, O1A, O2, or O6A) , in the formulations described herein. Such compositions containing only one or a set of less than four conjugates may be useful, for example, in the case of storing a bulk batch of antigens, for example, in the form of a drug substance, before mixing into a final drug product, and have the same beneficial stability properties that are described for drug product compositions containing all four conjugates.

Methods/applications

The compositions of the present invention can be used, for example, in a method of inducing an immune response against ExPEC in a subject in need thereof. Preferably, the immune response is effective in preventing or treating disease associated with ExPEC in a subject in need thereof. The method includes administering to a subject a composition according to this invention.

In some embodiments, the compositions provided herein can be stored in containers. Suitable containers may include, but are not limited to, bags, vials, syringes, bottles and tubes. In some embodiments, the syringeable stopper vials contain any of the compositions described herein. The containers provided herein can be made from a variety of materials, such as glass (eg, borosilicate glass), metal or plastic (eg, polycarbonates). In some embodiments, the container is a Type I borosilicate glass vial. In other embodiments, the container is a glass syringe with either a luer lock or a permanent needle. In other embodiments, the container is a syringe made of a plastic material, such as polycarbonate (PC) or polyethylene terephthalate glycol (PET), which material has been demonstrated to be compatible with the drug substance of the present invention. The vials may optionally contain rubber stoppers, for example, (chloro/bromo)butyl rubber coated with a fluoropolymer film (eg, Flurotec [ethylene tetrafluoroethylene (EFTE)] or Teflon [fluorinated ethylene propylene (FEP)]). Other materials and types of containers may be used, and compatibility with the compositions of the present invention can be determined by one skilled in the art based on the present invention.

In certain embodiments, the drug substance is stored in polycarbonate containers, such as bottles. In certain embodiments, the drug substance is stored in polyethylene terephthalate glycol containers, such as bottles. In certain embodiments, the drug product is stored in glass containers, such as vials.

The compositions proposed herein improve the stability of glycoconjugates in the compositions. By stability is generally meant that the composition retains its physical stability and/or chemical stability and/or biological activity upon storage. Preferably, the composition substantially retains its physical and chemical stability and its biological activity upon storage. Those skilled in the art can determine physical stability, chemical stability, and biological activity using the methods disclosed herein and methods known in the art. In this invention, a composition is considered stable (and the corresponding formulation is considered stabilizing) if a change in the impurity peak of 5% or less is observed on HPLC size exclusion chromatography (indicative of glycoconjugate aggregation) compared to the zero time point, as described in the examples herein. For example, the existing ExPEC vaccine formulation (in 25 mM Tris pH 7.4, 2.7 mM KCl, 137 mM NaCl) has a greater than 5% increase in peak impurity after 8 weeks at 40°C and is therefore unstable at 8 week at this temperature, while the compositions of the present invention exhibit an increase in peak impurity of less than 5% after 12 weeks at 40°C and are therefore stable for at least 12 weeks at this temperature. The compositions of this invention are stored in a glass container at 2-8°C for at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21. 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or more months. The compositions of this invention are stored in plastic containers for at least 7 days at 25°C, allowing the ExPEC glycoconjugate product to be handled under these conditions.

In certain embodiments, these compositions are provided in the form of liquid compositions. By liquid composition is meant that it is in a liquid state at a temperature of 2-8°C and is preferably stored at 2-8°C. Alternatively, the liquid composition is stored at 25°C or 40°C, for example, for the purpose of accelerated stability testing under thermal conditions.

- 12 044571 th stress.

In certain embodiments, these compositions can be stored and remain stable at 2-8°C, at 25°C, or at 40°C. In a preferred embodiment, the composition is stored and stable at 2-8°C. In certain embodiments, the composition is stable at 2-8°C for at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or more months. In certain embodiments, the composition remains stable at 25°C for 1, 2, 3, 4, 5, 6 or more months. In certain embodiments, the composition is stable at 40°C for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more weeks.

In certain embodiments, these compositions are provided in the form of a frozen composition. By frozen composition is meant a composition that is in a solid state when stored at a temperature of about -18°C or lower, for example, at about -20°C, -40°C, -60°C, -70°C, -80°C or any temperature in this range or below. In certain embodiments, these compositions can be stored and remain stable at -40°C or -60°C depending on the tonicity regulator present in the composition. In certain embodiments, the implementation of these compositions can be stored and remain stable at -70°C. In certain embodiments, the composition contains sucrose and is stable at -40°C for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 , 41, 42, 43, 44, 45, 46, 47, 48 or more months. In certain embodiments, the composition contains sorbitol and is stable at -60°C for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 , 41, 42, 43, 44, 45, 46, 47, 48 or more months. In certain embodiments, the composition contains sorbitol or sucrose and is stable at -70°C for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years. The compositions of the present invention are designed to be more stable than the older compositions described previously. The stability of the composition can be determined by methods described herein and by methods known in the art. Using these methods, the compositions of the present invention will be more stable for a specified period of time at a given temperature compared to the compositions of the old formulation for the same period of time and at the same temperature.

Also provided herein are methods for preparing the compositions disclosed herein. In certain embodiments, methods for preparing compositions are provided that include adding at least one E. coli O antigen covalently bound to an EPA carrier, water, buffer salts (i.e., sodium (di)hydrogen phosphate and (di)hydrogen phosphate potassium [i.e. Na ₂ HPO ₄ and KH2P4 or NaH ₂ PO ₄ and K2HPO4]), tonicity regulator (i.e. sorbitol or sucrose), antioxidant (i.e. methionine if sorbitol is the tonicity regulator; EDTA, if the tonicity regulator is sucrose) and a surfactant (e.g. PS80) into a container, adjust the pH to the required pH value (i.e. 6.5 to 7.5, e.g. 7.0) and mix these ingredients, and thus obtaining a liquid composition according to this invention. In a preferred embodiment, methods for preparing the compositions include the addition of E. coli O25B antigen polysaccharide, E. coli O1A antigen polysaccharide, E. coli O2 antigen polysaccharide and E. coli O6A antigen polysaccharide, wherein each antigen polysaccharide is covalently linked to an exotoxin A of Pseudomonas aeruginosa (EPA) carrier protein; water; potassium phosphate; sodium phosphate; sorbitol; methionine; and PS80 into a container, adjust the pH to 7.0, and mix each ingredient so that the final potassium/sodium phosphate buffer concentration is 10 mM at pH 7.0, the final sorbitol concentration is 3-8% (e.g., 5 %) (w/v), sorbitol was 5% w/v, methionine was 10 mM, and PS80 was 0.02% w/v. One non-limiting, illustrative way to prepare the composition according to this invention is as follows: to approximately 3.5 liters of water add: 3.3696 g KH ₂ P ₄ (Mw=136.09 g/mol), 2.1635 g Na ₂ HPO ₄ ( Mw=141.96 g/mol), 200 g sorbitol, 5.9684 g methionine (Mw=149.21 g/mol), 8 ml 10% (w/v) PS80 stock solution, giving a pH of approx. 6.73, which is then adjusted to the target pH of 7.0 using 500 μL of 10 N NaOH and the volume adjusted to 4 L by adding water. A formulation containing sucrose and EDTA in place of sorbitol and methionine can be prepared in a similar manner as would be readily apparent to one of ordinary skill in the art and the information provided herein. One possible way to prepare a composition according to this invention is by buffer substitution, for example, using along-flow filtration, filtration, dialysis, size exclusion chromatography, etc., to replace the composition prepared as described above with any buffer in which ExPECt glycoconjugate, for example, during or after the ExPEC glycoconjugate purification step. This buffer exchange step is routine for one skilled in the art when using the information provided herein.

The following examples of the invention further illustrate the nature of the present invention. It should be understood that the following examples do not limit the present invention, the scope of which is defined by the appended claims.

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Embodiments

Embodiment 1 is an immunogenic composition comprising E. coli O25B antigen polysaccharide, E. coli O1A antigen polysaccharide, E. coli O2 antigen polysaccharide, and E. coli O6A antigen polysaccharide, preferably all four E. coli O25B , O1A, O2 and O6A polysaccharide antigen, wherein each polysaccharide antigen is independently covalently linked to a Pseudomonas aeruginosa exotoxin A (EPA) carrier protein; 3% to 8% (preferably 4% to 6%) (w/v) sorbitol; 5 to 15 mM (preferably 8 to 12 mM) methionine; 5 to 20 mM (preferably 8 to 15 mM) potassium/sodium phosphate buffer pH 6.5 to 7.5; and from 0.01% to 0.2% w/v surfactant.

Embodiment 2 is the immunogenic composition of embodiment 1, wherein the E. coli 025B, O1A, O2 and O6A polysaccharide antigens are in a weight ratio of 1:1:1:1 or 2:1:1:1.

Embodiment 3 is the immunogenic composition of embodiment 1 or 2 where the sorbitol concentration is 5 w/v%.

Embodiment 4 is the immunogenic composition of embodiments 1-3, where the concentration of methionine is 10 mM.

Embodiment 5 is an immunogenic composition of any of embodiments 1-4, wherein the potassium/sodium phosphate buffer concentration is 10 mM and the potassium/sodium phosphate buffer pH is 7.0.

Embodiment 5 is an immunogenic composition of any of embodiments 1-5, wherein the surfactant comprises a hydrophilic head and a hydrophobic tail, wherein the surfactant is preferably selected from the group consisting of F-68, PS20 and PS80.

Embodiment 7 is the immunogenic composition of Embodiment 6, wherein the surfactant is PS80.

Embodiment 8 is the immunogenic composition of embodiment 7, wherein the surfactant concentration is 0.02 w/v%.

Embodiment 7 is an immunogenic composition comprising:

E. coli O25B antigen-polysaccharide, E. coli O1A antigen-polysaccharide, E. coli 02 antigen-polysaccharide, and E. coli O6A antigen-polysaccharide, wherein each antigen-polysaccharide is covalently linked to a Pseudomonas aeruginosa exotoxin A (EPA) carrier protein;

w/v% sorbitol;

mM methionine;

6.19 mM KH2P4 and 3.81 mM Na ₂ HPO ₄ buffers with a pH of 7.0; And

0.02 w/v% PS80.

Embodiment 10 is an immunogenic composition comprising E. coli O25B antigen polysaccharide, E. coli O1A antigen polysaccharide, E. coli O2 antigen polysaccharide, and E. coli O6A antigen polysaccharide, preferably all four E. coli O25B , O1A, 02 and O6A polysaccharide antigen, wherein each polysaccharide antigen is independently covalently linked to a Pseudomonas aeruginosa exotoxin A (EPA) carrier protein; 3% to 12% (preferably 3% to 10%) (w/v) sucrose; 0.1 to 1.5 mM EDTA; 5 to 20 mM (preferably 8 to 15 mM) potassium/sodium phosphate buffer pH 6.5 to 7.5; and from 0.01% to 0.2% w/v surfactant.

Embodiment 11 is the immunogenic composition of embodiment 10, wherein the E. coli O25B, O1A, O2 and O6A polysaccharide antigens are in a weight ratio of 1:1:1:1 or 2:1:1:1.

Embodiment 12 is the immunogenic composition of embodiment 10 or 11 where the sucrose concentration is 8 w/v%.

Embodiment 13 is the immunogenic composition of embodiments 10-12, where the concentration of EDTA is 1 mM.

Embodiment 14 is an immunogenic composition of any of embodiments 10-13, wherein the potassium/sodium phosphate buffer concentration is 10 mM and the potassium/sodium phosphate buffer pH is 7.0.

Embodiment 15 is an immunogenic composition of any of embodiments 10-14, wherein the surfactant comprises a hydrophilic head and a hydrophobic tail, wherein the surfactant is preferably selected from the group consisting of F-68, PS20 and PS80.

Embodiment 16 is the immunogenic composition of embodiment 15, wherein the surfactant is PS80.

Embodiment 17 is the immunogenic composition of embodiment 16, wherein the surfactant concentration is 0.02 w/v%.

Embodiment 18 is an immunogenic composition comprising:

E. coli O25B antigen-polysaccharide, E. coli O1A antigen-polysaccharide, E. coli O2 antigen-polysaccharide, E. coli O6A antigen-polysaccharide, wherein each antigen-polysaccharide is covalently linked to a Pseudomonas aeruginosa exotoxin A (EPA) carrier protein;

w/v% sucrose;

- 14 044571 mM EDTA;

mM potassium/sodium phosphate (eg, 6.19 mM KH2P ₄ and 3.81 mM Na ₂ HPO ₄ ) buffer with a pH of 7.0; And

0.02 w/v% PS80.

Embodiment 19 is an immunogenic composition of any one of embodiments 1-18, wherein the concentration of each O-antigen polysaccharide is in the range of from about 1 to 200 μg/ml, preferably in the range of 1 to 100 μg/ml, more preferably in ranging from 2 to 50 μg/ml, for example, ranging from about 4 μg/ml to 32 μg/ml.

Embodiment 20 is an immunogenic composition of any one of embodiments 1-19, wherein the ratio of polysaccharide:carrier protein (wt:wt) is from about 1:10 to about 1:2, such as from about 1:5 to about 1 :2 for each O-antigen polysaccharide.

Embodiment 21 is an immunogenic composition of any one of embodiments 1-20 in liquid form suitable for administration by injection or infusion.

Embodiment 22 is an immunogenic composition of any one of embodiments 1-21 that elicits an immune response in a subject in need thereof.

Embodiment 23 is the use of an immunogenic composition of any one of embodiments 1-21 to produce a medicament that induces an immune response in a subject in need thereof.

Embodiment 24 is a composition of any one of embodiments 1-21 in a glass container.

Embodiment 25 is a composition of any one of embodiments 1-21 in a polycarbonate container.

Embodiment 26 is a composition of any one of embodiments 1-21 in a polyethylene terephthalate glycol container.

Embodiment 27 is a composition of any one of embodiments 1-21 in a vial.

Embodiment 28 is a composition of any one of embodiments 1-21 in a syringe.

Embodiment 29 is a composition of any one of embodiments 1-21 or 24-28 that is stable for no less than 6 months, preferably no less than 12 months, more preferably no less than 18 months, more preferably no less than 24 months, more preferably no less than 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or more months of storage at a temperature of 2-8°C.

Embodiment 30 is a method of maintaining a stable state of a liquid immunogenic composition containing E. coli O antigen covalently linked to a carrier protein EPA, comprising storing the composition of any one of embodiments 1-21 or 24-29 at a temperature of 2-8 °C for at least 6 months, preferably at least 12 months, more preferably at least 18 months, more preferably at least 24 months, more preferably at least 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or more months.

Examples

Example 1 Surfactant Addition Prevents Freeze/Thaw Induced Aggregation of ExPEC Glycoconjugate

To determine which combination of excipients can be added to the E. coli 025b, O1A, O2, O6A saccharide antigens, each independently covalently linked to a separate (i.e. four separate glycoconjugates in total) Pseudomonas aeruginosa exotoxin A (EPA), here and hereinafter referred to as ExPEC glycoconjugates, to promote stabilization during freezing/thawing, shaking, heat stress, metal-induced oxidative stress, various excipients were added to the original ExPEC glycoconjugate composition (ExPEC glycoconjugate, 25 mM Tris, pH 7.4, 137 mM NaCl, 2 .7 mM KCl) (hereinafter referred to as the legacy formulation, which is the ExPEC glycoconjugate formulation currently used in phase 2 clinical trials, ClinicalTrials.gov Identifier: NCT02546960), and the resulting formulation was tested by appropriate methods for increased stabilization.

In the old formulation (sometimes referred to here as control) ExPEC glycoconjugate forms aggregates under freeze/thaw stress. Aggregation of the ExPEC glycoconjugate is observed by the impurity peak on the size exclusion chromatography-high performance liquid chromatography (SEC-HPLC) curve (see, for example, FIGS. 1A and 1B), in which impurity peak 1 is an indicator of instability under stress.

First, a cryoprotectant (eg, sorbitol) was added to the ExPEC glycoconjugate formulation and the resulting formulation was examined to determine whether it could improve stability when exposed to freeze/thaw stress. It was found that the introduction of sorbitol does not protect the ExPEC glycoconjugate from aggregation during freezing/thawing according to EH-15 044571

HPLC (Fig. 1B).

Surprisingly, however, the addition of a surfactant (eg, F-68 [also known as Poloxamer 188 or Plutonic F-68], PS20, or PS80) to the ExPEC glycoconjugate formulation increased the stability of the ExPEC glycoconjugate when exposed to freeze/thaw stress. Specifically, the surfactant prevented aggregation of the ExPEC glycoconjugate when exposed to freeze/thaw stress. Addition of 0.01% PS-80, 0.01% PS 20, or 0.1% F-68 (all w/v) to the ExPEC glycoconjugate prevented freeze/thaw-induced aggregation as measured by SEC-HPLC ( Fig. 1B and Fig. 1C). Each surfactant tested (all surfactants were nonionic) consisted of a hydrophilic head part (containing OH groups) and a long hydrophobic tail part (a carbon chain that can contain approximately 12, 14, 16, 18, 20, 30, 40, 50, 60, 80, 100, 120, 130 or more carbon atoms in the carbon chain). Without resorting to theorizing, it was hypothesized that these physical properties allow the studied surfactants to prevent freeze/thaw-induced aggregation of the ExPEC glycoconjugate. In subsequent development, PS80 was used as a forming agent to prevent freeze/thaw-induced aggregation, as this surfactant has the added benefit of being easy to use in later stages of ExPEC glycoconjugate development.

Example 2: Evaluation of formulation with respect to buffer, pH value and tonicity regulator

After it was shown that surfactants could prevent freeze/thaw-induced aggregation of the ExPEC glycoconjugate, a suitable buffer, pH, and tonicity regulator for this formulation was sought. In a subsequent experiment, several formulations were tested with different buffer-pH combinations in the presence of NaCl (150 mM) or 5% sorbitol (w/v) as a tonicity adjuster, all containing PS80 (0.01 w/v%). The old formulation (25 mM Tris, pH 7.4, 137 mM NaCl, 2.7 mM KCl) was used as a control.Several concentrations of ExPEC glycoconjugate were tested (e.g., 16 or 8 μg/ml for each polysaccharide), but there was no effect on stability We did not observe any effects on the studied compositions from the use of different concentrations (data not shown).

These formulations were subjected to several stresses, including freezing/thawing, shaking, light exposure, and heat stress at various temperatures (eg, 2-8°C, 25°C, and 40°C) (Table 1).

Table 1: Stress conditions to which the newly formulated ExPEC glycoconjugates were subjected.

Stress Conditions Time points Temperature 5°С 0, 4, 8, 12 weeks 2 5°С 2, 4, 8 weeks 4 0°C 1, 2, 4 weeks Agitation Vortex mixer (1000 rpm at ambient temperature) 4 hours Freezing/thawing -70°C to ambient temperature 5 consecutive cycles Photosensitivity Light exposure (ICH Q1B option 2) 2 00 W/ ^m2 UV-A light (~6 hours) + 1200 Klux-hour visible light (~40 hours)

Table 2: Assay Kit for Reformulated ExPEC Glycoconjugates

Method of analysis Target Visual inspection Appearance and Transparency pH Acidity and basicity samples Osmolality Osmolality SEC-HPLC Cleanliness, units, products splitting Reversed chromatography phase (RP-HPLC) Purity, chemical transformation Isoelectric focusing (IEF) Purity, chemical transformation Dynamic light scattering (DRS) Invisible particles FlowCAM Invisible particles

Using a set of analytical methods described in table. 2, it was shown that formulations containing histidine at pH 7 or potassium phosphate at pH 7 were more stable compared to other buffer-pH combinations (data not shown). Also, at extreme pH values (i.e., pH 5.0 and

- 16 044571 pH 8.0) a higher level of aggregation of the ExPEC glycoconjugate is observed. In addition, it was noted that formulations containing sorbitol as a tonicity regulator were surprisingly more stabilizing than formulations containing NaCl as a tonicity regulator. Indeed, it was noted that formulations containing NaCl were unable to stabilize the ExPEC glycoconjugate against aggregation during storage at 40°C. When sorbitol was used as a tonicity regulator, aggregation of the ExPEC glycoconjugate did not occur, indicating an unexpected stabilizing effect of sorbitol. In addition, aggregation at extreme pH values (ie, pH 5.0 and pH 8.0) is reduced in sorbitol-containing formulations compared to NaCl-containing formulations. This shows that sorbitol makes the formulations more stable and is able to provide the ExPEC glycoconjugate with a stabilizing effect even if pH changes occur during production.

Example 3: Evaluation of formulation stability

Additional formulations were developed to identify the best candidate formulations with respect to ExPEC glycoconjugate stability. For buffers and pH, histidine was assessed at pH 6.5 and 7.0, and instead of potassium phosphate alone, potassium/sodium phosphate buffer (KH2PO ₄ /Na2HPO ₄ ) was tested at pH 6.5 and 7.0. The combination of potassium and sodium was chosen based on initial results and works better locally to buffer the pH of the freeze/thaw tank. When preparing the buffer system, an excess of potassium over sodium was used (for example, for 10 mM phosphate buffer with pH 7.0, we used 6.19 mM KH2P4 and 3.81 mM Na ₂ HPO ₄ ). Sucrose (8 wt/vol%) and sorbitol (5 wt/vol%) were studied as tonicity regulators. In addition, the effect of administering the antioxidant EDTA (1 mM) or methionine (10 mM) was assessed. It was also examined whether the absence of NaCl or the presence of sorbitol had the protective effect observed in Example 2 by incorporating a formulation that contained a combination of the two (each at half the concentration compared to when these components were used separately as in Example 2). All formulations contained PS80 at a concentration of 0.02 w/v%.

The different formulations were subjected to the same stress conditions as in Example 2. Based on the combination of stability data, a selection step was carried out where the effectiveness of each formulation was assessed and excluded based on the following criteria: (a) the appearance of an additional impurity peak in the RP-HPLC chromatogram according to at least two time points out of three for each temperature; (b) a peak for impurity 1 was visible/increased in the SEC-HPLC chromatogram at at least two of the three time points for each temperature; (c) the appearance of an additional impurity peak in the SEC-HPLC chromatogram after exposure to stress (eg, freezing/thawing, shaking, exposure to light); and (d) the peak of impurity 1 was visible/increased in the SEC-HPLC chromatogram after exposure to stress (e.g., freezing/thawing, shaking, exposure to light). It was found that two specific formulations, referred to herein as formulations 26 and 28, were able to allow the ExPEC glycoconjugate to withstand each of the stress conditions tested.

Formulation 26 contained 10 mM Na/K phosphate buffer pH 7.0, 5% w/v sorbitol, 10 mM methionine, 0.02% w/v PS-80, and ExPEC glycoconjugate.

Formulation 28 contained 10 mM Na/K phosphate buffer pH 7.0, 8% sucrose, 1 mM EDTA, 0.02% PS-80, and ExPEC glycoconjugate.

Surprisingly, certain combinations of tonicity regulator and antioxidant made a difference, as formulations containing either (i) sorbitol with methionine or (ii) sucrose with EDTA were significantly more effective than (a) formulations in which sorbitol was combined with EDTA, and as well as (b) formulations in which sucrose was combined with methionine, and (c) formulations without the antioxidant. It has also surprisingly been found that ExPEC glycoconjugate formulations preferably do not contain sodium chloride. The compositions of 26 and 28 can be varied within certain limits and still stabilize the ExPEC glycoconjugate. In table Table 3 shows the applicable limits for pH and excipient concentrations for formulations 26 and 28.

Table 3: pH and excipient concentration limits for ExPEC formulations. glycoconjugates

Formative Limits pH 6, 5 7.5 K/Na phosphate (mM) 5 20 Sorbitol (%) (w/v) 3 8 Sucrose (%) (w/v) 3 12 Methionine (mM) 5 15 EDTA (mM) 0, 1 1.5 Surfactant (%): F-68 0.05 0.2 0.01 0.08 0.01 0.08

- 17 044571

PS20

PS80

Formulations 26 and 28 were further tested in a conformation study for comparison with the old ExPEC glycoconjugate formulation (ExPEC glycoconjugate, 25 mM Tris, pH 7.4, 137 mM NaCl, 2.7 mM KCl). Both upright and inverted vial positions were examined to determine the effect of contact with the stopper of the final container. Formulations 26 and 28 consist of excipients that do not pose any hazard at the proposed concentrations, are used in licensed vaccines and/or are on the list of approved excipients for parenteral administration. The combination of these excipients for formulations 26 and 28 and the pH of the buffer solution contribute to the improved stabilizing effect of the ExPEC glycoconjugate compared to the older ExPEC glycoconjugate formulation. The proposed combination of these excipients for formulations 26 and 28 turned out to be capable of preserving the drug substances (DS) and drug product (DP) of glycoconjugate vaccines against ExPEC during freezing/thawing and heat stress (for example, Fig. 2), while meeting the requirements for the expected stability at storage at 2-8°C and 25°C (data not shown). The polysaccharide concentration tested for LP was 20 μg/ml for each strain (total 80 μg/ml), and the total protein concentration was 300 μg/ml. In the case of drugs of serotypes 025b (i.e. containing E. coli O25B antigen-polysaccharide covalently linked to the exotoxin EPA carrier protein), the polysaccharide concentration tested was 200 μg/ml, and the protein concentration tested was 830 μg/ml. Such compositions were stable over a period of time.

Thus, the drug product in formulations 26 and 28 has demonstrated stability for at least 6 months at 2-8°C, at least six months at 25°C, and stability studies are ongoing.

Example 4: Evaluation of Formulations under Metal-Induced Oxidative Stress Conditions

Formulations 26 and 28 were further examined for their stabilizing effect on the ExPEC glycoconjugate in the presence of tungsten as a model of metal-induced oxidation of the product. Residual amounts of tungsten are typically present at the tip of glass prefilled syringes (GPS) as a result of the tip shaping process using a tungsten needle. The deposition of residual amounts of tungsten in PZH depends on the manufacturing process and can vary from manufacturer to manufacturer in the range of 250-1250 nanograms/package. Tungsten has been linked to protein aggregation in PZS (Jiang et al., J. Pharmaceutical Sci. 98(12):4695-710 (2009); Seidl et al., Pharmaceutical Res. 29:1454-67 (2012)). Thus, the stability of formulations 26 and 28 and the old formulation was examined when exposed to tungsten at various concentrations, while observing the oxidative stress and aggregation tendency of the ExPEC glycoconjugate. This study tested three tungsten levels (high (HW), medium (MW) and low (LW)) across a range of tungsten residue levels in currently commercially marketed tungsten. The stress conditions applied to the formulations are summarized in Table. 4.

Table 4: Stress Conditions Applicable to Tungsten Compounds____

Stress Conditions Time points Temperature 4 0°C 0, 1, 2 and 4 weeks Agitation Vortex mixer (200 rpm at ambient temperature) 24 hours

Formulations 26 and 28 were shown to contain the least amount of ExPEC glycoconjugate aggregates over time as determined by SEC-HPLC analysis (stability impurity peak 1). Impurity peak values at time point 4 weeks reached 3.1% compared to 1.0% at time point T-0, even when exposed to the highest levels of tungsten (average 0.5% increase per week, Fig. 3) .

Thus, the invention provides two different improved liquid formulations for ExPEC glycoconjugate vaccine. The first, most preferred composition contains ExPEC glycoconjugate, sorbitol, methionine, K/Na-phosphate buffer with pH 7 and a surfactant. A second preferred composition contains ExPEC glycoconjugate, sucrose, EDTA, K/Na-phosphate buffer pH 7 and a surfactant. The advantage of these compositions over the previously described composition is that when stored at 2-8°C, the ExPEC glycoconjugate vaccine exhibits an improved stability profile in the new formulations of the present invention, in which aggregation and formation of degradation products are prevented, as evidenced by assays characterizing stability (EC-HPLC and RP-HPLC). The stabilizing effect of both inventive formulations is even more apparent when exposed to stresses such as freeze/thaw, heat (40°C) and oxidizing metals when analyzed using the above analytical techniques. These features allow the use of many options for storage and transportation of both drugs and drugs of the glycoconjugate vaccine against ExPEC. Additionally, improved oxidative stress tolerance properties

-

Claims (14)

metals also apply to long-term storage of ExPEC vaccine in alternative primary packaging systems (eg, prefilled syringe and/or application devices). Example 5: Study of the compatibility of the composition with plastic materials. Formulations 26 and 28 were further examined for their stabilizing effect on the drug substance ExPEC glycoconjugate (025b conjugate, polysaccharide concentrations tested were 227-242 µg/ml and protein 952-1048 µg/ml) when in contact with various plastic materials such as polycarbonate (PC) and polyethylene terephthalate glycol (PET). Plastic materials are associated with protein degradation, such as when the plastic material is sterilized by irradiation. The formulations were examined for stability under stress conditions caused by intense stirring (24 h at 200 rpm at room temperature) in PET and PC containers. The old formulations completely decomposed under these conditions in PET containers. In contrast, formulations 26 and 28 were shown to remain stable under the same conditions in the PET and PC containers tested, as shown by SEC-HPLC analysis (see Figure 4, using impurity peak 1 as an indicator of instability). In addition, the stability of the drug substance in these formulations was established during storage for seven days in PC and PET containers at 25°C (data not shown). CLAIM 1. Immunogenic composition containing: at least one E. coli O antigen polysaccharide, wherein the at least one O antigen polysaccharide is covalently linked to a Pseudomonas aeruginosa exotoxin A (EPA) carrier protein; 3% to 8% w/v sorbitol; from 5 to 15 mm methionine; 5 to 20 mM potassium/sodium phosphate buffer with pH 6.5 to 7.5; and from 0.01% to 0.2% w/v surfactant. 2. An immunogenic composition according to claim 1, containing E. coli O25B antigen-polysaccharide, E. coli O1A antigen-polysaccharide, E. coli O2 antigen-polysaccharide and E. coli O6A antigen-polysaccharide, where each antigen-polysaccharide is covalently linked to Pseudomonas aeruginosa exotoxin A (EPA) carrier protein. 3. Immunogenic composition according to claim 1 or 2, where the concentration of sorbitol is 5 wt/vol.%. 4. Immunogenic composition according to any one of claims 1-3, where the concentration of methionine is 10 mM. 5. An immunogenic composition according to any one of claims 1 to 4, wherein the concentration of the potassium/sodium phosphate buffer is 10 mM and the pH of the potassium/sodium phosphate buffer is 7.0. 6. An immunogenic composition according to any one of claims 1 to 5, where the surfactant is a non-ionic surfactant, where the surfactant is preferably selected from the group consisting of F-68, PS20 and PS80. 7. An immunogenic composition according to claim 6, wherein the surfactant is PS80, wherein the concentration of PS80 is preferably 0.02 w/v%. 8. Immunogenic composition according to any one of claims 1-7, containing: E. coli O25B antigen-polysaccharide, E. coli O1A antigen-polysaccharide, E. coli O2 antigen-polysaccharide, and E. coli O6A antigen-polysaccharide, wherein each antigen-polysaccharide is covalently linked to a Pseudomonas aeruginosa exotoxin A (EPA) carrier protein; 5 w/v% sorbitol; 10 mM methionine; 10 mM KH ₂ PO4/Na ₂ HPO4 buffer with pH equal to 7.0; 0.02 w/v% PS80; and water. 9. Immunogenic composition containing: at least one E. coli O antigen polysaccharide, wherein the at least one O antigen polysaccharide is covalently linked to a Pseudomonas aeruginosa exotoxin A (EPA) carrier protein; 3% to 12% w/v sucrose; 0.1 to 1.5 mM EDTA; 5 to 20 mM potassium/sodium phosphate buffer with pH 6.5 to 7.5; and from 0.01% to 0.2% w/v surfactant. 10. An immunogenic composition according to claim 9, containing E. coli O25B antigen-polysaccharide, E. coli O1A antigen-polysaccharide, E. coli O2 antigen-polysaccharide and E. coli O6A antigen-polysaccharide, where each antigen-polysaccharide is covalently linked to Pseudomonas aeruginosa exotoxin A (EPA) carrier protein. 11. Immunogenic composition according to claim 9 or 10, where the concentration of sucrose is 8 wt/vol.%. 12. Immunogenic composition according to any one of claims 9-11, where the concentration of EDTA is 1 mM. 13. An immunogenic composition according to any one of claims 9 to 12, wherein the concentration of the potassium/sodium phosphate buffer is 10 mM and the pH of the potassium/sodium phosphate buffer is 7.0. 14. Immunogenic composition according to any one of claims 9-13, where the surfactant is nonionic -