Immobilised urease formulations with improved shelf life

Abstract

The present invention provides urease formulations suitable for use in dialysis, the urease formulations comprise urease, a non-reducing sugar, an antioxidant, a buffer, and a non-ionic surfactant. The urease formulations of the invention have excellent stability against gamma ray sterilisation and excellent shelf life.

Description

[0001] IMMOBILISED UREASE FORMULATIONS WITH IMPROVED SHELF LIFE

[0002] FIELD OF THE INVENTION

[0003] The invention relates to urease formulations with excellent stability against gamma ray sterilisation and long term storage.

[0004] BACKGROUND TO THE INVENTION

[0005] Kidney dialysis relies on the use of urease enzymes to remove urea from a patient. Urease is typically provided in immobilised form attached to a solid support, and present in a sorbent through which dialysis fluid may be passed. In order to minimise the infection risk associated with treatment, the urease must be sterilised before a treatment. However, the urease formulations used in dialysis have poor stability against sterilisation methods, and also have poor storage stability. This causes logistical challenges for dialysis operators and patients alike, since urease formulations cannot be purchased in bulk and easily stored at a treatment centre or patient’s home.

[0006] Thus, existing urease formulations may require a cold-chain, storage at reduced temperature, and use in a dialysis treatment within days or weeks of preparation. Failure to adhere to these requirements may result in urea removal efficacy that is too low to be therapeutically effective. This low storage stability results in short expiry and high levels of wastage, increasing costs.

[0007] There is therefore a need for improved urease formulations that have long term storage stability. That is, for urease formulations that maintain their full urea removal properties after storage for at least two months.

[0008] SUMMARY OF THE INVENTION

[0009] Surprisingly, the inventors have found that a urease formulation as described herein may be stable at room temperature for a period of at least three months and, in certain more particular embodiments, stable to a gamma ray sterilisation step and then subsequently stable at room temperature for a period of at least three months.

[0010] Aspects and embodiments of the current invention are referred to in the below numbered clauses.

[0011] 1 . A urease formulation suitable for use in dialysis, comprising: urease; a non-reducing sugar; an antioxidant; a buffer; and a non-ionic surfactant.

[0012] 2. The urease formulation according to Clause 1 , wherein the formulation further comprises: activated carbon; and hydrous zirconium oxide.

[0013] 3. The urease formulation according to Clause 1 or Clause 2, wherein the urease is provided in the form of urease immobilized on a solid support.

[0014] 4. The urease formulation according to any one of the preceding clauses, wherein the nonreducing sugar comprises one or more of the group consisting of trehalose and sucrose, optionally wherein the non-reducing sugar comprises sucrose.

[0015] 5. The urease formulation according to any one of the preceding clauses, wherein the antioxidant comprises one or more of the group consisting of glutathione, methionine and ascorbic acid, optionally wherein the antioxidant comprises glutathione.

[0016] 6. The urease formulation according to any one of the preceding clauses, wherein the buffer comprises one or more of the group consisting of histidine and a phosphate buffer, and / or wherein the buffer has a pKa of from 3 to 8; optionally wherein the buffer comprises one or both of histidine and potassium phosphate, more optionally wherein the buffer comprises histidine and potassium phosphate.

[0017] 7. The urease formulation according to any one of the preceding clauses, wherein the nonionic surfactant comprises one or both of a polysorbate and a poloxamer, optionally wherein the non-ionic surfactant comprises a polysorbate.

[0018] 8. The urease formulation according to any one of the preceding clauses, wherein: the non-reducing sugar comprises sucrose; the antioxidant comprises glutathione; the buffer comprises histidine; and the non-ionic surfactant comprises polysorbate. 9. The urease formulation according to any one of clauses 1 to 7, wherein: the non-reducing sugar comprises sucrose and trehalose; the antioxidant comprises glutathione; the buffer comprises potassium phosphate; and the non-ionic surfactant comprises polysorbate.

[0019] 10. The urease formulation according to any one of the preceding clauses, wherein the antioxidant comprises one or more of the group consisting of reduced L-glutathione, reduced D- glutathione, oxidised L-glutathione, and oxidised D-glutathione, optionally wherein the glutathione comprises reduced L-glutathione or reduced D- glutathione, more optionally wherein the glutathione comprises reduced L-glutathione.

[0020] 11 . The urease formulation according to any one of the preceding clauses, wherein the buffer comprises one or more of the group consisting of L-histidine and D-histidine, optionally wherein the buffer comprises L-histidine.

[0021] 12. The urease formulation according to any one of the preceding clauses, wherein the nonionic surfactant comprises polysorbate-80.

[0022] 13. The urease formulation according to any one of the preceding clauses, wherein, on a dry weight basis, the formulation comprises from about 0.5 wt. % to about 10 wt. % non-reducing sugar (e.g. sucrose), optionally from about 1 wt. % to about 7 wt. % non-reducing sugar, more optionally from about 1 wt. % to about 5 wt. % non-reducing sugar.

[0023] 14. The urease formulation according to any one of the preceding clauses, wherein, on a dry weight basis, the formulation comprises from about 0.001 wt. % to about 5 wt. % antioxidant (e.g. glutathione), optionally from about 0.01 wt. % to about 2 wt. % antioxidant, more optionally from about 0.05 wt. % to about 1 wt. % antioxidant.

[0024] 15. The urease formulation according to any one of the preceding clauses, wherein, on a dry weight basis, the formulation comprises from about 10 wt. % to about 25 wt. % immobilised urease, optionally from about 14 wt. % to about 20 wt. % immobilised urease.

[0025] 16. The urease formulation according to any one of the preceding clauses, wherein the nonionic surfactant comprises polysorbate and on a dry weight basis, the formulation comprises from about 0.005 wt. % to about 0.5 wt. % polysorbate (e g. about 0.01 wt. % to about 0.2 wt. % polysorbate).

[0026] 17. The urease formulation according to any one of the preceding clauses, wherein one or both of the following apply:

[0027] (a) the buffer comprises histidine and on a dry weight basis, the formulation comprises from about 0.005 wt. % to about 1 wt. % histidine (e.g. about 0.01 wt. % to about 0.3 wt. % histidine); and

[0028] (b) the buffer comprises potassium phosphate and on a dry weight basis, the formulation comprises from about 0.1 wt. % to about 3 wt. % potassium phosphate (e.g. about 0.3 wt. % to about 1.6 wt. % potassium phosphate).

[0029] 18. The urease formulation according to any one of Clause 2 and Clauses 3 to 17 as dependent upon Clause 2, wherein, on a dry weight basis, the formulation comprises from about 30 wt. % to about 45 wt. % activated carbon, optionally from about 34 wt. % to about 38 wt. % activated carbon.

[0030] 19. The urease formulation according to any one of Clause 2, and Clauses 3 to 18 as dependent upon Clause 2, wherein, on a dry weight basis, the formulation comprises from about 35 wt. % to about 48 wt. % zirconium oxide, optionally from about 39 wt. % to about 43 wt. % zirconium oxide.

[0031] 20. The urease formulation according to any one of the preceding clauses, wherein one or more of the following apply:

[0032] (a) the urease formulation further comprises one or more proteins, optionally wherein the one or more proteins comprise albumin, for example bovine serum albumin (BSA);

[0033] (b) the urease formulation further comprises one or more chelating agents, optionally wherein the one or more chelating agents comprise ethylenediaminetetraacetic acid (EDTA); (c) the urease formulation provides greater than 99% urea clearance after sterilisation using gamma radiation at 25-40 Kgy, as compared to the urea clearance prior to sterilisation using gamma radiation; and

[0034] (d) the urease formulation provides greater than 99% urea clearance after storage at 30°C for three months, as compared to the urea clearance prior to storage.

[0035] 21. The urease formulation according to any one of Clause 2, and Clauses 3 to 20 as dependent upon Clause 2, wherein the urease formulation provides greater than 99% urea clearance after:

[0036] (i) sterilisation using gamma radiation at 25-40 Kgy; and

[0037] (ii) subsequent storage at 30 °C for three months, as compared to the urea clearance prior to sterilisation using gamma radiation and storage, optionally wherein the formulation is a sterile urease formulation.

[0038] 22. The urease formulation according to any one of the preceding clauses, wherein the urease formulation is a lyophilised urease formulation.

[0039] BRIEF DESCRIPTION OF THE FIGURES

[0040] FIG. 1 shows the apparatus used to test the stability of urease formulations disclosed herein.

[0041] DETAILED DESCRIPTION OF THE INVENTION

[0042] As noted above, the inventors have uncovered urease formulations as described herein that are stable at room temperature for a period of at least three months and, in certain more particular embodiments, stable to a gamma ray sterilisation step and then subsequently stable at room temperature for a period of at least three months.

[0043] Thus, in a first aspect of the invention, there is provided a urease formulation suitable for use in dialysis, comprising: urease; a non-reducing sugar; an antioxidant; a buffer; and a non-ionic surfactant. Without wishing to be bound by theory, it is believed that a formulation comprising the components above can maintain enzyme stability at ambient temperature for an extended period of time (e.g. three months).

[0044] In certain embodiments of the invention that may be mentioned herein, the urease formulation further comprises activated carbon and hydrous zirconium oxide. Without wishing to be bound by theory, it is believed that the addition of these two components to the formulation described in the first aspect of the invention can maintain the urease enzyme’s stability at ambient temperature following a sterilisation step (e g. with gamma rays).

[0045] The word “comprising” as used herein may be interpreted as requiring the features mentioned, but not limiting the presence of other features. Alternatively, the word “comprising” may also relate to the situation where only the components / features listed are intended to be present (e.g. the word “comprising” may be replaced by the phrases “consists of’ or “consists essentially of”). It is explicitly contemplated that both the broader and narrower interpretations can be applied to all aspects and embodiments of the present invention. In other words, the word “comprising” and synonyms thereof may be replaced by the phrase “consisting of” or the phrase “consists essentially of” or synonyms thereof and vice versa.

[0046] The phrase, “consists essentially of’ and its pseudonyms may be interpreted herein to refer to a material where minor impurities may be present. For example, the material may be greater than or equal to 90% pure, such as greater than 95% pure, such as greater than 97% pure, such as greater than 99% pure, such as greater than 99.9% pure, such as greater than 99.99% pure, such as greater than 99.999% pure, such as 100% pure.

[0047] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” includes mixtures of two or more such compositions, reference to “a non-reducing sugar” includes mixtures of two or more such non-reducing sugars, and the like.

[0048] When used herein, the term “urease” refers to an enzyme able to react with urea as a substrate. Such enzymes can be determined to have this function in vitro, for example, by allowing the enzyme to react with a urea in solution and measuring a decrease in the concentration of the urea. In healthy individuals, urea is usually excreted from the body through the urine. However, in certain individuals, urea is not removed from the body at a sufficiently fast rate, leading to uremic toxicity, i.e. a disease or condition characterized by elevated levels of at least one uremic toxin (e g. urea) with respect to physiologically normal levels of the uremic toxin. Non-limiting examples of disorders associated with uremic toxins include renal disease or dysfunction, gout, and uremic toxicity in subjects receiving chemotherapy.

[0049] The term “predominantly” as used herein is intended to represent a situation or state which occurs for the most part or principally, while not excluding the possibility that some amount of another situation or state also occurs to a minimal extent. For example, it may be >80% or >90% or >95% or greater than 99%. For the avoidance of doubt, the possibility that only that situation or state occurs, to the exclusion of all others, is covered by the term.

[0050] The word "substantially" does not exclude "completely" e.g. a composition which is "substantially free" from Y may be completely free from Y. Where necessary, the word "substantially" may be omitted from the definition of the invention.

[0051] As used herein, the term "about", in the context of concentrations of components of the formulations, typically means ± 5% of the stated value, more typically + / - 4% of the stated value, more typically ± 3% of the stated value, more typically, + / - 2% of the stated value, even more typically ± 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0052] Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0053] In certain embodiments mentioned herein, the urease may be provided in the form of urease immobilized on a solid support. The urease may be immobilized on any known support material, which can provide immobilization for the urease enzyme particles. Immobilization may be by physical means such as by adsorption on alumina. In an embodiment, non-immobilised enzyme is used. Alternatively, other methods are used to convert urea to ammonia.

[0054] In one embodiment, the support material is a biocompatible substrate to which the urease enzyme is covalently bound. The biocompatible material may be a carbohydrate-based polymer, an organic polymer, a polyamide, a polyester, or an inorganic polymeric material. The biocompatible substrate may be a homogeneous substrate made up of one material or a composite substrate made up of at least two materials. The biocompatible substrate may be at least one of cellulose, Eupergit, silicon dioxide (e.g. silica gel), zirconium phosphate, zirconium oxide, nylon, polycaprolactone and chitosan.

[0055] In one embodiment, the immobilization of the urease enzyme on the biocompatible substrate is carried out by immobilization techniques selected from the group consisting of glutaric aldehyde activation, activation with epoxy groups, epichlorohydrin activation, bromoacetic acid activation, cyanogen bromide activation, thiol activation, and N-hydroxysuccinimide and diimide amide coupling. The immobilization techniques used may also involve the use of silane-based linkers such as (3-ami nopropyl) triethoxysilane, (3-glycidyloxypropyl) trimethoxysilane or (3- mercaptopropyl) trimethoxysilane. The surface of the biocompatible substrate may be further functionalized with a reactive and / or stabilizing layer such as dextran or polyethyleneglycol, and with suitable linker- and / or stabilizer molecules such as ethylenediamine, 1 ,6-diaminohexane, thioglycerol, mercaptoethanol and trehalose. The urease can be used in purified form, or in the form of crude extract such as extract of urease from Jack Bean or other suitable urease sources.

[0056] The urease particles may be capable of converting urea to ammonium carbonate. In one embodiment the urease particles may have an average particle size in the range of from about 10 microns to about 1000 microns, about 100 microns to about 900 microns, about 200 microns to about 900 microns, about 300 microns to about 800 microns, about 400 microns to about 700, 500 microns to about 600 microns, about 25 microns to about 250 microns, about 25 microns to about 100 microns, about 250 microns to about 500 microns, about 250 microns to about 1000 microns, about 125 microns to about 200 microns, about 150 microns to about 200 microns, about 100 microns to about 175 microns, or about 100 microns to about 150 microns. In one embodiment, 1000 units to 10000 units of urease are immobilized on said solid support. The overall dry weight of immobilized urease and the substrate may range from about 0.5 g to about 100 g, for example from about 0.5 g to about 50 g, or from about 0.5 g to about 30 g.

[0057] As used herein, a “non-reducing sugar” refers to a di-, tri- or tetra-saccharide that does not comprise a free aldehyde or free ketone functional group and / or is a carbohydrate that is not oxidised by a weak oxidising agent (i.e. an oxidising agent that can oxidise an aldehyde but not an alcohol, such as Tollen’s reagent). Examples of non-reducing sugars include sugars that are formed form, or comprise, cyclic moieties. Specific examples of non-reducing sugars that may be useful in the invention include raffinose, stachyose, trehalose, sucrose and lactose, for example sucrose.

[0058] The non-reducing sugar may be any suitable sugar material that has the properties mentioned hereinbefore. In particular embodiments of the invention, the non-reducing sugar may comprise one or more of the group consisting of trehalose, and sucrose. For example, the non-reducing sugar may be sucrose. Or, for example, the non-reducing sugar may be a combination of sucrose and trehalose. In certain embodiments, where the non-reducing sugar is a combination of sucrose and trehalose, the weight ratio of sucrose to trehalose in the formulation is from about 1 : 1 to about 1 :5, or from about 1 :1 to about 5:1, for example, the weight ratio of sucrose to trehalose in the formulation may be about 1 :1.

[0059] As will be appreciated, antioxidants are compounds that inhibit oxidation. Any suitable antioxidant material may be used herein. For example, the antioxidant may comprises one or more of the group consisting of glutathione, methionine, and ascorbic acid. In particular embodiments of the invention that may be mentioned herein the antioxidant may be glutathione. In yet more particular embodiments of the invention that may be mentioned herein, the antioxidant may comprise one or more of the group consisting of reduced L-glutathione, reduced D-glutathione, oxidised L- glutathione, and oxidised D-glutathione (e.g. the glutathione comprises reduced L-glutathione or reduced D-glutathione, such as the glutathione is reduced L-glutathione).

[0060] Any suitable buffer may be used herein. For example, the buffer may comprise one or more of the group consisting of histidine and a phosphate buffer. Additionally or alternatively, the buffer may be one that has a pKa of from 3 to 8. In certain embodiments that may be mentioned herein, the buffer may comprises one or both of histidine and potassium phosphate (e.g. the buffer comprises both histidine and potassium phosphate). In certain more particular embodiments, the buffer may comprise one or more of the group consisting of L-histidine and D-histidine (e.g. the buffer comprises L-histidine). In certain embodiments, when the buffer comprises potassium phosphate, the non-reducing sugar comprises sucrose and trehalose.

[0061] Any suitable non-ionic surfactant may be used in the formulation disclosed herein. In certain embodiments, the non-ionic surfactant may comprise one or both of a polysorbate and a poloxamer. For example, the non-ionic surfactant may be a polysorbate. Yet more particularly, the polysorbate may comprise or may be polysorbate-80.

[0062] In certain embodiments, there is provided a urease formulation, where: the non-reducing sugar comprises sucrose; the antioxidant comprises glutathione; the buffer comprises histidine; and the non-ionic surfactant comprises polysorbate.

[0063] Thus, the urease formulation may comprise urease, sucrose, glutathione, histidine, and a polysorbate (e.g. polysorbate-80).

[0064] In certain other embodiments, there is provided a urease formulation, where: the non-reducing sugar comprises sucrose and trehalose; the antioxidant comprises glutathione; the buffer comprises potassium phosphate; and the non-ionic surfactant comprises polysorbate.

[0065] Thus, the urease formulation may comprise urease, sucrose, trehalose, glutathione, potassium phosphate, and a polysorbate (e.g. polysorbate-80).

[0066] Any suitable amount of the components listed above may be used in the urease formulations mentioned herein. For example, the urease formulation may, on a dry weight basis, be one that comprises from about 0.5 wt. % to about 10 wt. % non-reducing sugar (e.g. sucrose or trehalose, or both sucrose and trehalose), such as from about 1 wt. % to about 7 wt. % non-reducing sugar, such as from about 1 wt. % to about 5 wt. % non-reducing sugar. Additionally or alternatively, the urease formulation may, on a dry weight basis, may be one that comprises from about 0.001 wt. % to about 5 wt. % antioxidant (e.g. glutathione), such as from about 0.01 wt. % to about 2 wt. % antioxidant, such as from about 0.05 wt. % to about 1 wt. % antioxidant. Additionally or alternatively, the urease formulation may, on a dry weight basis, be one that comprises from about 10 wt. % to about 25 wt. % immobilised urease, such as from about 14 to about 20 wt. % immobilised urease. Additionally or alternatively, the urease formulation may, on a dry weight basis, be one that comprises from about 0.005 wt. % to about 0.5 wt. % polysorbate (e.g. about 0.01 wt. % to about 0.2 wt. % polysorbate). Additionally or alternatively, the urease formulation may, on a dry weight basis, be one that comprises from about 0.005 wt. % to about 1 wt. % histidine (e.g. about 0.01 wt. % to about 0.3 wt. % histidine). Additionally or alternatively, the urease formulation may, on a dry weight basis, be one that comprises from about 0.1 wt. % to about 3 wt. % potassium phosphate (e.g. about 0.3 wt. % to about 1.6 wt. % potassium phosphate).

[0067] As noted above, in certain embodiments of the invention, the urease formulation may further comprise activated carbon and zirconium oxide.

[0068] The activated carbon may be provided in any suitable form. In particular embodiments that may be mentioned herein, the activated carbon may be in the form of particles with an average particle size that is less than 2,000 microns. In further embodiments of the invention that may be mentioned herein, the activated carbon may have an average particle size of from 100 microns to 1 ,900 microns, such as from 500 microns to 1 ,500 microns. The activated carbon may be present in any suitable amount. For example, in embodiments where the activated carbon is present, then it may provide, on a dry weight basis, from about 30 wt. % to about 45 wt. %, such as from about 34 wt. % to about 38 wt. % of the total weight of the formulation.

[0069] The zirconium oxide may be provided in any suitable form, such as zirconium oxide particles. When in the form of particles, the zirconium oxide particles may have an average particle size in the range of from about 10 microns to about 1000 microns, about 100 microns to about 900 microns, about 200 microns to about 900 microns, about 300 microns to about 800 microns, about 400 microns to about 700 microns, 500 microns to about 600 microns, about 10 microns to about 200 microns, about 10 microns to about 100 micron, about 10 microns to about 30 microns, about 10 microns to about 20 microns, about 20 microns to about 50 microns, about 25 microns to about 50 microns, about 30 microns to about 50 microns, about 40 microns to about 150 microns, about 80 microns to about 120 microns, about 160 microns to about 180, about 25 microns to about 250 microns, about 250 microns to about 500 microns, or about 250 microns to about 1000 microns. The zirconium oxide particles may be immobilized on any known support material which can provide immobilization for the zirconium oxide particles. In one embodiment, the immobilization of the zirconium phosphate particles is a physical compaction of the particles into a predetermined volume. In one embodiment, the immobilization of the zirconium oxide particles is achieved by sintering zirconium oxide, or a mixture of zirconium oxide and a suitable ceramic material. In one embodiment, the support material is a biocompatible substrate. The biocompatible material may be a carbohydrate-based polymer, an organic polymer, a polyamide, a polyester, a polyacrylate, a polyether, a polyolefin or an inorganic polymeric or ceramic material. The biocompatible substrate may be at least one of cellulose, Eupergit, silicon dioxide, nylon, polycaprolactone and chitosan.

[0070] The zirconium oxide may be a hydrous zirconium oxide. Hydrous zirconium oxide may be synthesised by conventional methods, for example by reaction of an aqueous mixture of sodium zirconium carbonate and sodium hydroxide as described in US Pat No 4,256,718. After synthesis of hydrous zirconium oxide, the product may be titrated to a pH of from 12 to 13. This can be done by making an aqueous slurry of the hydrous zirconium oxide and titrating it with 5M sodium hydroxide until the slurry is at a pH of 12 to 13. In some instances, the hydrous zirconium oxide may then be washed until the concentration of leachables in the filtrate is within acceptable levels, and then air dried. Alternatively, the hydrous zirconium oxide may recovered directly from the slurry and not washed before being air dried.

[0071] The zirconium oxide may be present in any suitable amount. For example, in embodiments where the zirconium oxide is present, then it may provide, on a dry weight basis, from about 35 wt. % to about 48 wt. %, such as from about 39 wt. % to about 43 wt. % of the total weight of the formulation.

[0072] The urease formulations disclosed herein may further comprise one or more proteins. In certain embodiments, the one or more proteins may comprise albumin, for example bovine serum albumin (BSA). Thus, in certain embodiments, the urease formulations of the present invention may comprise albumin, for example BSA.

[0073] The urease formulations disclosed herein may further comprise one or more chelating agents. In certain embodiments, the one or more chelating agents may comprise ethylenediaminetetraacetic acid (EDTA). Thus, in certain embodiments, the urease formulations of the present invention may comprise a chelating agent, for example EDTA.

[0074] Accordingly, in certain embodiments, the urease formulation comprises: urease; a non-reducing sugar comprising sucrose; an antioxidant comprising glutathione; a buffer comprising histidine; a non-ionic surfactant comprising polysorbate; and optionally albumin (e g. BSA), and optionally a chelating agent (e g. EDTA).

[0075] For example, the urease formulation may comprise urease, sucrose, glutathione, histidine, a polysorbate (e g. polysorbate-80), optionally albumin (e.g. BSA), and optionally a chelating agent (e.g. EDTA). For example, the urease formulation may comprise urease, sucrose, glutathione, histidine, a polysorbate (e.g. polysorbate-80), and albumin (e.g. BSA). Or, for example, the urease formulation may comprise urease, sucrose, glutathione, histidine, a polysorbate (e.g. polysorbate- 80), and a chelating agent (e.g. EDTA). Or, for example, the urease formulation may comprise urease, sucrose, glutathione, histidine, a polysorbate (e.g. polysorbate-80), albumin (e.g. BSA), and a chelating agent (e.g. EDTA). As disclosed herein, the urease formulation may further comprise activated carbon and zirconium oxide.

[0076] In certain other embodiments, the urease formulation comprises: urease; a non-reducing sugar comprising sucrose and trehalose; an antioxidant comprising glutathione; a buffer comprising potassium phosphate; a non-ionic surfactant comprising polysorbate; and optionally albumin (e.g. BSA), and optionally a chelating agent (e.g. EDTA).

[0077] For example, the urease formulation may comprise urease, sucrose, trehalose, glutathione, potassium phosphate, a polysorbate (e.g. polysorbate-80), optionally albumin (e.g. BSA), and optionally a chelating agent (e.g. EDTA). For example, the urease formulation may comprise urease, sucrose, trehalose, glutathione, potassium phosphate, a polysorbate (e.g. polysorbate- 80), and albumin (e.g. BSA). Or, for example, the urease formulation may comprise urease, sucrose, trehalose, glutathione, potassium phosphate, a polysorbate (e.g. polysorbate-80), and a chelating agent (e.g. EDTA). Or, for example, the urease formulation may comprise urease, sucrose, trehalose, glutathione, potassium phosphate, a polysorbate (e.g. polysorbate-80), albumin (e.g. BSA), and a chelating agent (e.g. EDTA). As disclosed herein, the urease formulation may further comprise activated carbon and zirconium oxide. For the avoidance of doubt, the urease formulation of the present invention may be a freeze-dried (i.e. lyophilised) formulation comprising the components described herein. For example, the urease formulation of the invention may be prepared by combining a base formulation solution with urease. The base formulation solution may comprise a non-reducing sugar, an antioxidant, a buffer, and a non-ionic surfactant, as described herein, and a solvent (e.g. water). The base formulation solution may also comprise albumin (e.g. BSA) and / or a chelating agent (e.g. EDTA), as described herein. The urease may be immobilised on a solid support as described herein. Subsequent lyophilisation of the base formulation solution and urease mixture provides a lyophilised urease formulation. Specific examples of urease formulations prepared in this manner are provided in the Examples section herein. When the urease is an immobilised urease, the lyophilised urease formulation may be referred to as a lyophilised immobilised urease formulation. Thus, in certain embodiments, the urease formulation of the invention is a lyophilised urease formulation, for example a lyophilised immobilised urease formulation.

[0078] The urease formulations disclosed herein may provide greater than 99% urea clearance after storage at 30°C for three months, as compared to the urea clearance prior to storage.

[0079] The urease formulations disclosed herein that are subjected to sterilisation using gamma radiation may provide greater than 99% urea clearance after sterilisation using gamma radiation at 25-40 Kgy, as compared to the urea clearance prior to sterilisation using gamma radiation. In embodiments of the invention, where activated carbon and zirconium oxide are present, the urease formulation may provide greater than 99% urea clearance after:

[0080] (i) sterilisation using gamma radiation at 25-40 Kgy; and

[0081] (ii) subsequent storage at 30°C for three months, as compared to the urea clearance prior to sterilisation using gamma radiation and storage.

[0082] In embodiments of the invention, where activated carbon and zirconium oxide are present, the formulation may be a sterile urease formulation. For example, the formulation may have been treated with gamma radiation.

[0083] Further aspects and embodiments of the invention will not be discussed by reference to the following non-limiting examples. EXAMPLES

[0084] Materials

[0085] A 0.2 M phosphate buffer solution (PBS) was prepared by dissolving 27.22 g of potassium dihydrogen phosphate in 930 mL of sterile water, adjusting to pH 7.5 with a 300 g / L solution of potassium hydroxide and diluting to 1000 mL with sterile water, followed by filtering through a 0.2 micron filter.

[0086] General Method 1: Preparation of base formulation solution

[0087] Dissolve sugar or polyols in sterile water to make 125 mL of 25% sugar or 10% of polyol solution, Then sequentially add the following excipients as required: histidine (buffer), antioxidants (cysteine, ascorbic acid, methionine, and glutathione), chelating agents (EDTA sodium salt), surfactant (Polysorbate 80), amino acid (arginine), and protein (BSA). Mix the solution on the shaker for 10 min at 150 rpm. Store the clear solution at 4 °C until further use. For formulations comprising a phosphate buffer, the sterile water was substituted with the 0.2 M phosphate buffer prepared as described above.

[0088] General Method 2: Preparation of urease formulation

[0089] Transfer 40 g of wet Immobilized Urease (proprietary blend prepared from prepared from JB Urease procured from Sigma Aldrich Product No. 94281-1G) into 125 mL of base formulation solution from General Method 1 at 4°C using a sterile spatula. Shake the resulting suspension at 150 rpm for 1 hour at 4°C in an incubator Collect the resulting solid by filtration, and place the filtered residue in a sterile container and cover with paper towel. Freeze the wet immobilized urease formulation at -20°C for 18 hours and lyophilize the material in a freeze dryer with an operating vacuum of 0.1 mbar for 7.0 hours at 4°C to achieve the loss on drying (LOD) in the range of 15-25%. Loss on Drying (LOD) is measured by heating the sample at 210 °C until the weight gets stabilized with a moisture analyzer (RADWAG MA200.3Y.WH).

[0090] General Method 3: Gamma ray sterilisation and storage method

[0091] Mix 5 g of freeze-dried immobilized urease formulation from General Method 2 with scavengers (8.5 g activated carbon and 7.5 g hydrous zirconium oxide, such that the weight to weight ratio of freeze-dried immobilized urease formulation:activated carbon:hydrous zirconium oxide is 1 : 1 .7: 1 .5, said ratio may be used to create larger or smaller batches) and fill into a plastic urease cartridge, followed by sealing with an airtight lid. Sterilize the cartridge by high gamma irradiation dosage (25-40 Kgy) at 4 °C. Store the sterilized cartridge at room temperature for a desired time period.

[0092] General Method 4: Urease performance

[0093] Prepare 14 L of synthetic dialysate (mimic of peritoneal dialysis fluid waste), having the composition shown in Table 1 below.

[0094] Table 1 : 14 L of synthetic dialysate The pH of the synthetic dialysate solution was adjusted to 7.5 (physiological pH). The concentration of all components was verified using a Vitros 250 Analyzer.

[0095] Using the apparatus of FIG. 1 , the synthetic dialysate was pumped into the urease cartridge from General Method 3, and subsequently through a sorbent cartridge at a flow rate of 50 mL / min. The sorbent cartridge contained anion and cation exchangers to absorb ammonia generated from urease module. This is necessary because the presence of ammonia may interfere with the measurement of urea concentration. The clearance efficiency of the Immobilized urease was then monitored regularly by measuring urea concentration after every 2 L of synthetic dialysate was used (measurement conducted using a Vitros 250 Analyzer). Finally, collect the entire 14 L of synthetic dialysate to measure urea clearance. Determine the total percentage of urea clearance using cumulative and drain data.

[0096] The amount of urea cleared may be calculated as follows:

[0097] Total Urea Cleared (mmol) = Total Input Urea (mmol) - Total urea in post-dialysis solution (mmol)

[0098] % total urea cleared = (Total urea cleared) / (total input urea) x 100

[0099] An example calculation is provided in Table 2 below.

[0100] Table 2: Example calculation for urea clearance

[0101] Comparative Example 1 : Comparative Formulations

[0102] Comparative formulations were prepared using General Methods 1 and 2, using the components listed in Table 3 below.

[0103] Table 3: Identity of comparative formulations

[0104] *The concentrations shown in this table relate to the concentration of each component in the base formulation solution used. The % concentrations provided refer to the % (w / v) of a component in the base formulation solution used.

[0105] The formulations set out in Table 3 were sterilised and stored according to General Method 3. After the appropriate storage time, the urease performance was tested according to General Method 4. Results are provided in Table 4. Stability testing for formulations was stopped if urea clearance dropped below 95%.

[0106] Table 4: Urea clearance of comparative formulations.

[0107] NT - not tested

[0108] Example 1 : Formulations of the invention

[0109] Urease formulations were prepared using General Methods 1 and 2, using the components listed in Table 5 below.

[0110] Table 5: Identity of urease formulations

[0111] *The concentrations shown in this table relate to the concentration of each component in the base formulation solution used. The % concentrations provided refer to the % (w / v) of a component in the base formulation solution used.

[0112] The formulations set out in Table 5 were sterilised and stored according to General Method 3. After the appropriate storage time, the urease performance was tested according to General Method 4. Results are provided in Table 6.

[0113] Table 6: Urea clearance of urease formulations

[0114] *lt is believed that the value for 1 month or 2 months may be erroneous, since the urea clearance after two months should be equal to, or lower, than that for 1 month. NT = not tested

[0115] By comparing the results in Tables 4 and 6, it is clear that formulations according to the invention are able to effectively stabilise urease and ensure its activity remains high during a gamma ray sterilisation step and subsequent long term storage at room temperature. In contrast, the comparative formulations that do not include all of a non-reducing sugar, an antioxidant, a buffer, a non-ionic surfactant, had worse urea clearance after storage at room temperature, indicating a much shorter shelf life.

[0116] Thus, the formulations according to the invention are highly advantageous and solve the problem of short shelf life of existing urease formulations for dialysis.

[0117] Example 2: Further formulations of the invention

[0118] Urease formulations were prepared using General Methods 1 and 2, using the components listed in Table 7 below.

[0119] Urease formulations were prepared using General Methods 1 and 2, using the components listed in Table 5 below.

[0120] Table 7: Identity of urease formulations

[0121] *The concentrations shown in this table relate to the concentration of each component in 100 mL of the base formulation solution used.

[0122] The formulations set out in Table 7 were sterilised and stored according to General Method 3. After the appropriate storage time, the urease performance was tested according to General Method 4. Results are provided in Table 8. Table 8: Urea clearance of urease formulations

[0123] NT = not tested

[0124] By comparing the results in Tables 4 and 8, it is clear that formulations according to the invention are able to effectively stabilise urease and ensure its activity remains high during a gamma ray sterilisation step and subsequent long term storage at room temperature.

[0125] Thus, the formulations according to the invention are highly advantageous and solve the problem of short shelf life of existing urease formulations for dialysis.

Claims

CLAIMS1 . A urease formulation suitable for use in dialysis, comprising: urease; a non-reducing sugar; an antioxidant; a buffer; and a non-ionic surfactant.

2. The urease formulation according to Claim 1 , wherein the formulation further comprises: activated carbon; and hydrous zirconium oxide.

3. The urease formulation according to Claim 1 or Claim 2, wherein the urease is provided in the form of urease immobilized on a solid support.

4. The urease formulation according to any one of the preceding claims, wherein the nonreducing sugar comprises one or more of the group consisting of trehalose and sucrose, optionally wherein the non-reducing sugar comprises sucrose.

5. The urease formulation according to any one of the preceding claims, wherein the antioxidant comprises one or more of the group consisting of glutathione, methionine and ascorbic acid, optionally wherein the antioxidant comprises glutathione.

6. The urease formulation according to any one of the preceding claims, wherein the buffer comprises one or more of the group consisting of histidine and a phosphate buffer, and / or wherein the buffer has a pKa of from 3 to 8; optionally wherein the buffer comprises one or both of histidine and potassium phosphate, more optionally wherein the buffer comprises histidine and potassium phosphate.

7. The urease formulation according to any one of the preceding claims, wherein the nonionic surfactant comprises one or both of a polysorbate and a poloxamer, optionally wherein the non-ionic surfactant comprises a polysorbate.

8. The urease formulation according to any one of the preceding claims, wherein: the non-reducing sugar comprises sucrose; the antioxidant comprises glutathione; the buffer comprises histidine; and the non-ionic surfactant comprises polysorbate.

9. The urease formulation according to any one of claims 1 to 7, wherein: the non-reducing sugar comprises sucrose and trehalose; the antioxidant comprises glutathione; the buffer comprises potassium phosphate; and the non-ionic surfactant comprises polysorbate.

10. The urease formulation according to any one of the preceding claims, wherein the antioxidant comprises one or more of the group consisting of reduced L-glutathione, reduced D- glutathione, oxidised L-glutathione, and oxidised D-glutathione, optionally wherein the glutathione comprises reduced L-glutathione or reduced D- glutathione, more optionally wherein the glutathione comprises reduced L-glutathione.

11. The urease formulation according to any one of the preceding claims, wherein the buffer comprises one or more of the group consisting of L-histidine and D-histidine, optionally wherein the buffer comprises L-histidine.

12. The urease formulation according to any one of the preceding claims, wherein the non- ionic surfactant comprises polysorbate-80.

13. The urease formulation according to any one of the preceding claims, wherein, on a dry weight basis, the formulation comprises from about 0.5 wt. % to about 10 wt. % non-reducing sugar (e g. sucrose), optionally from about 1 wt. % to about 7 wt. % non-reducing sugar, more optionally from about 1 wt. % to about 5 wt. % non-reducing sugar.

14. The urease formulation according to any one of the preceding claims, wherein, on a dry weight basis, the formulation comprises from about 0.001 wt. % to about 5 wt. % antioxidant (e.g. glutathione),optionally from about 0.01 wt. % to about 2 wt. % antioxidant, more optionally from about 0.05 wt. % to about 1 wt. % antioxidant.

15. The urease formulation according to any one of the preceding claims, wherein, on a dry weight basis, the formulation comprises from about 10 wt. % to about 25 wt. % immobilised urease, optionally from about 14 wt. % to about 20 wt. % immobilised urease.

16. The urease formulation according to any one of the preceding claims, wherein the nonionic surfactant comprises polysorbate and on a dry weight basis, the formulation comprises from about 0.005 wt. % to about 0.5 wt. % polysorbate (e.g. about 0.01 wt. % to about 0.2 wt. % polysorbate).

17. The urease formulation according to any one of the preceding claims, wherein one or both of the following apply:(a) the buffer comprises histidine and on a dry weight basis, the formulation comprises from about 0.005 wt. % to about 1 wt. % histidine (e.g. about 0.01 wt. % to about 0.3 wt. % histidine); and(b) the buffer comprises potassium phosphate and on a dry weight basis, the formulation comprises from about 0.1 wt. % to about 3 wt. % potassium phosphate (e.g. about 0.3 wt. % to about 1.6 wt. % potassium phosphate).

18. The urease formulation according to any one of Claim 2 and Claims 3 to 17 as dependent upon Claim 2, wherein, on a dry weight basis, the formulation comprises from about 30 wt. % to about 45 wt. % activated carbon, optionally from about 34 wt. % to about 38 wt. % activated carbon.

19. The urease formulation according to any one of Claim 2, and Claims 3 to 18 as dependent upon Claim 2, wherein, on a dry weight basis, the formulation comprises from about 35 wt. % to about 48 wt. % zirconium oxide, optionally from about 39 wt. % to about 43 wt. % zirconium oxide.

20. The urease formulation according to any one of the preceding claims, wherein one or more of the following apply:(a) the urease formulation further comprises one or more proteins, optionally wherein the one or more proteins comprise albumin, for example bovine serum albumin (BSA);(b) the urease formulation further comprises one or more chelating agents, optionally wherein the one or more chelating agents comprise ethylenediaminetetraacetic acid (EDTA);(c) the urease formulation provides greater than 99% urea clearance after sterilisation using gamma radiation at 25-40 Kgy, as compared to the urea clearance prior to sterilisation using gamma radiation; and(d) the urease formulation provides greater than 99% urea clearance after storage at 30°C for three months, as compared to the urea clearance prior to storage.21 . The urease formulation according to any one of Claim 2, and Claims 3 to 20 as dependent upon Claim 2, wherein the urease formulation provides greater than 99% urea clearance after:(i) sterilisation using gamma radiation at 25-40 Kgy; and(ii) subsequent storage at 30 °C for three months, as compared to the urea clearance prior to sterilisation using gamma radiation and storage, optionally wherein the formulation is a sterile urease formulation.

22. The urease formulation according to any one of the preceding claims, wherein the urease formulation is a lyophilised urease formulation.

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