Immobilized urease formulations with improved shelf life

Abstract

The present invention provides a urease formulation suitable for use in dialysis, the urease formulation comprising a urease, a non-reducing sugar, an antioxidant, a buffer, and a non-ionic surfactant. The urease preparation disclosed by the invention has excellent gamma-ray sterilization resistance stability and excellent shelf life.

Description

TECHNICAL FIELD

[0001] The present invention relates to a urease formulation having excellent stability to gamma sterilization and long-term storage. BACKGROUND

[0002] Kidney dialysis relies on the use of urease to remove urea from the patient. Urease is typically provided in an immobilized form attached to a solid support and is present in a sorbent through which the dialysis fluid can pass. In order to minimize the risk of infection associated with treatment, urease must be sterilized prior to treatment. However, urease formulations used in dialysis have poor stability to sterilization methods and also have poor storage stability. This creates logistical challenges for dialysis operators and patients, among others, as urease formulations cannot be purchased in bulk and cannot be easily stored at the treatment center or at the patient’s home.

[0003] Accordingly, existing urease formulations can require a cold chain, be stored at reduced temperatures, and be used for dialysis treatment within a few days or weeks of preparation. Non-compliance with these requirements can result in urea removal efficacy that is too low to be therapeutically effective. This low storage stability results in short expiration times and high levels of waste, increasing costs.

[0004] Accordingly, there is a need for improved urease formulations having long-term storage stability. That is, urease formulations that retain their full urea removal properties after storage for at least two months. SUMMARY

[0005] Surprisingly, the inventors have found that urease formulations as described herein can be stable at room temperature for a period of at least three months and, in certain more particular embodiments, stable to a gamma sterilization step followed by subsequent stability at room temperature for a period of at least three months.

[0006] Aspects and embodiments of the invention are mentioned in the numbered clauses below.

[0007] 1. A urease formulation suitable for use in dialysis, comprising:

[0008] urease;

[0009] non-reducing sugar;

[0010] antioxidant;

[0011] buffer; and

[0012] non-ionic surfactant.

[0013] 2. The urease formulation of clause 1, wherein the formulation further comprises:

[0014] activated carbon; and

[0015] hydrated zirconium oxide.

[0016] 3. The urease preparation of clause 1 or clause 2, wherein the urease is provided in the form of urease immobilised on a solid support.

[0017] 4. The urease preparation of any one of the preceding clauses, wherein the non-reducing sugar comprises one or more of the group consisting of trehalose and sucrose,

[0018] optionally wherein the non-reducing sugar comprises sucrose.

[0019] 5. The urease preparation of any one of the preceding clauses, wherein the antioxidant comprises one or more of the group consisting of glutathione, methionine and ascorbic acid,

[0020] optionally wherein the antioxidant comprises glutathione.

[0021] 6. The urease preparation of any one of the preceding clauses, wherein the buffer comprises one or more of histidine and phosphate buffer, and / or wherein the buffer has a pKa of 3 to 8;

[0022] optionally wherein the buffer comprises one or both of histidine and potassium phosphate,

[0023] more optionally wherein the buffer comprises histidine and potassium phosphate.

[0024] 7. The urease preparation of any one of the preceding clauses, wherein the non-ionic surfactant comprises one or both of polysorbate and poloxamer,

[0025] optionally wherein the non-ionic surfactant comprises polysorbate.

[0026] 8. The urease preparation of any one of the preceding clauses, wherein:

[0027] the non-reducing sugar comprises sucrose;

[0028] the antioxidant comprises glutathione;

[0029] the buffer comprises histidine; and

[0030] the non-ionic surfactant comprises polysorbate.

[0031] 9. The urease preparation of any one of clauses 1 to 7, wherein:

[0032] the non-reducing sugar comprises sucrose and trehalose;

[0033] the antioxidant comprises glutathione;

[0034] the buffer comprises potassium phosphate; and

[0035] The non-ionic surfactant comprises polysorbate.

[0036] 10. The urease formulation of any of the preceding clauses, wherein the antioxidant comprises one or more of the group consisting of reduced L-glutathione, reduced D-glutathione, oxidized L-glutathione, and oxidized D-glutathione,

[0037] Optionally, wherein the glutathione comprises reduced L-glutathione or reduced D-glutathione, more optionally, wherein the glutathione comprises reduced L-glutathione.

[0038] 11. The urease formulation of any of the preceding clauses, wherein the buffer comprises one or more of the group consisting of L-histidine and D-histidine,

[0039] Optionally wherein the buffer comprises L-histidine.

[0040] 12. The urease formulation of any of the preceding clauses, wherein the non-ionic surfactant comprises polysorbate-80.

[0041] 13. The urease formulation of any of the preceding clauses, wherein the formulation comprises, on a dry weight basis, about 0.5 wt.% to about 10 wt.% of non-reducing sugar (e.g., sucrose),

[0042] optionally about 1 wt.% to about 7 wt.% of non-reducing sugar,

[0043] more optionally about 1 wt.% to about 5 wt.% of non-reducing sugar.

[0044] 14. The urease formulation of any of the preceding clauses, wherein the formulation comprises, on a dry weight basis, about 0.001 wt.% to about 5 wt.% of antioxidant (e.g., glutathione),

[0045] optionally about 0.01 wt.% to about 2 wt.% of antioxidant,

[0046] more optionally about 0.05 wt.% to about 1 wt.% of antioxidant.

[0047] 15. The urease formulation of any of the preceding clauses, wherein the formulation comprises, on a dry weight basis, about 10 wt.% to about 25 wt.% of immobilized urease,

[0048] optionally about 14 wt.% to about 20 wt.% of immobilized urease.

[0049] 16. The urease formulation of any of the preceding clauses, wherein the non-ionic surfactant comprises polysorbate, and the formulation comprises about 0.005 wt.% to about 0.5 wt.% polysorbate by dry weight (e.g., about 0.01 wt.% to about 0.2 wt.% polysorbate).

[0050] 17. The urease formulation of any of the preceding clauses, wherein one or both of the following apply:

[0051] (a) the buffer comprises histidine, and the formulation comprises about 0.005 wt.% to about 1 wt.% histidine by dry weight (e.g., about 0.01 wt.% to about 0.3 wt.% histidine); and

[0052] (b) the buffer comprises potassium phosphate, and the formulation comprises about 0.1 wt.% to about 3 wt.% potassium phosphate by dry weight (e.g., about 0.3 wt.% to about 1.6 wt.% potassium phosphate).

[0053] 18. The urease formulation of clause 2 and any of clauses 3 to 17 dependent on clause 2, wherein the formulation comprises about 30 wt.% to about 45 wt.% activated carbon by dry weight, optionally about 34 wt.% to about 38 wt.% activated carbon.

[0054] 19. The urease formulation of clause 2 and any of clauses 3 to 18 dependent on clause 2, wherein the formulation comprises about 35 wt.% to about 48 wt.% zirconium oxide by dry weight, optionally about 39 wt.% to about 43 wt.% zirconium oxide.

[0055] 20. The urease formulation of any of the preceding clauses, wherein one or more of the following apply:

[0056] (a) the urease formulation further comprises one or more proteins, optionally wherein the one or more proteins comprise albumin, e.g., bovine serum albumin (BSA);

[0057] (b) the urease formulation further comprises one or more chelating agents, optionally wherein the one or more chelating agents comprise ethylenediaminetetraacetic acid (EDTA);

[0058] (c) the urease formulation provides greater than 99% urea clearance after sterilization using 25-40 Kgy of gamma radiation compared to urea clearance prior to sterilization using gamma radiation; and

[0059] (d) the urease formulation provides greater than 99% urea clearance after storage for three months at 30°C compared to urea clearance prior to storage.

[0060] 21. The urease formulation according to any one of Clauses 2 and 3 to 20 dependent on Clause 2, wherein the urease formulation provides greater than 99% urea clearance after:

[0061] (i) sterilisation using 25-40 Kgy of gamma radiation; and

[0062] (ii) subsequent storage at 30°C for three months, optionally wherein the formulation is a sterile urease formulation, compared to urea clearance and storage prior to sterilisation using gamma radiation.

[0063] 22. The urease formulation according to any one of the preceding Clauses, wherein the urease formulation is a lyophilised urease formulation. BRIEF DESCRIPTION OF DRAWINGS

[0064] Figure 1 An apparatus for testing the stability of the urease formulations disclosed herein is shown. DETAILED DESCRIPTION

[0065] As mentioned above, the inventors have disclosed urease formulations as described herein which are stable at room temperature for a period of at least three months, and in certain more particular embodiments, are stable to a gamma irradiation sterilisation step, and then subsequently stable at room temperature for a period of at least three months.

[0066] Thus, in a first aspect of the application, there is provided a urease formulation suitable for use in dialysis, comprising:

[0067] urease;

[0068] non-reducing sugar;

[0069] antioxidant;

[0070] buffer; and

[0071] non-ionic surfactant.

[0072] Without wishing to be bound by theory, it is believed that the formulation comprising the above components can maintain enzyme stability at ambient temperature for an extended period of time (e.g. three months).

[0073] In certain embodiments of the application which can be mentioned herein, the urease formulation further comprises activated charcoal and hydrated 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 application can maintain stability of the urease after a sterilisation step (e.g. with gamma radiation) at ambient temperature.

[0074] The word “comprising” as used herein can be interpreted as meaning that the mentioned features are present but does not exclude the presence of other features. Alternatively, the word “comprising” can also relate to situations where only the components / features listed are intended to be present (e.g. the word “comprising” can be replaced by the phrase “consisting of” or “consisting essentially of”). It is explicitly contemplated that both the broader and narrower interpretations can apply to all aspects and embodiments of the present application. In other words, the word “comprising” and its synonyms can be replaced by the phrase “consisting of’ or the phrase “consisting essentially of’ or vice versa.

[0075] The phrase “consisting essentially of’ and its permutations, as used herein, can be interpreted as meaning that the materials recited can be present in small amounts. For example, the materials can 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.

[0076] 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.

[0077] The term “urease” as used herein refers to an enzyme capable of reacting with urea as a substrate. Such an enzyme can be determined to have this function in vitro, for example, by reacting the enzyme with urea in solution and measuring a decrease in urea concentration. In healthy individuals, urea is typically removed from the body via urine. However, in certain individuals, urea is not removed from the body at a fast enough rate, resulting in uremic toxicity, a disease or condition characterized by an elevated level of at least one uremic toxin (e.g., urea) relative to physiologically normal levels of the uremic toxin. Non-limiting examples of conditions associated with uremic toxins include kidney disease or dysfunction, gout, and uremic toxicity in subjects receiving chemotherapy.

[0078] The term “predominantly” as used herein is intended to mean that a majority or a substantial amount of a condition or state occurs, while not excluding the possibility that a certain amount of another condition or state also occurs to a minimal extent. For example, it can be >80% or >90% or >95% or greater than 99%. For the avoidance of doubt, the term encompasses the possibility that only this condition or state occurs and does not include all other conditions or states.

[0079] The word “substantially” does not exclude “entirely” e.g. a composition “substantially free from” Y can be completely free from Y. The word “substantially” can be omitted from the definition of the application where necessary.

[0080] As used herein, the term "about," in the context of concentrations of components in a formulation, generally refers to ± 5% of the stated value, more usually + / - 4% of the stated value, more usually ± 3% of the stated value, more usually + / - 2% of the stated value, even more usually ± 1% of the stated value, and even more usually + / - 0.5% of the stated value.

[0081] Throughout this disclosure, certain embodiments can be disclosed in the range format. It is to be understood that the description in range format is merely for convenience and brevity and that one

[0082] In certain embodiments referred to herein, urease can be provided in the form of urease immobilized on a solid support.

[0083] The urease can be immobilized on any known support material, which can provide immobilization of the urease particles. Immobilization can be by physical means, for example by adsorption on alumina. In one embodiment, non-immobilized enzyme is used. Alternatively, other methods are used to convert urea to ammonia.

[0084] In one embodiment, the carrier material is a biocompatible substrate to which the urease is covalently bound. The biocompatible material can be a carbohydrate-based polymer, an organic polymer, a polyamide, a polyester, or an inorganic polymeric material. The biocompatible substrate can be a homogeneous substrate composed of one material or a composite substrate composed of at least two materials. The biocompatible substrate can be at least one of cellulose, Eupergit, silica (e.g., silica gel), zirconium phosphate, zirconium oxide, nylon, polycaprolactone, and chitosan.

[0085] In one embodiment, the urease is immobilized on the biocompatible substrate by an immobilization technique selected from glutaraldehyde activation, activation with an epoxy group, epichlorohydrin activation, bromoacetic acid activation, cyanogen bromide activation, thiol activation, and N-hydroxysuccinimide and diimide coupling. The immobilization technique used can also involve the use of a silane-based linker, such as (3-aminopropyl)triethoxysilane, (3-glycidyloxypropyl)trimethoxysilane, or (3- mercaptopropyl)trimethoxysilane. The surface of the biocompatible substrate can be further functionalized with reactive and / or stabilizing layers such as dextran or polyethylene glycol, 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 a crude extract, such as a urease extract from jack bean or other suitable urease source.

[0086] The urease particles can be capable of converting urea to ammonium carbonate. In one embodiment, the urease particles can have an average particle size of 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.

[0087] In one embodiment, 1000 units to 10000 units of urease are immobilized on the solid support. The total dry weight of the immobilized urease and substrate can range from about 0.5 g to about 100 g, such as about 0.5 g to about 50 g, or about 0.5 g to about 30 g.

[0088] As used herein, "non-reducing sugar" means a disaccharide, trisaccharide, or tetrasaccharide that does not contain a free aldehyde or a free ketone functional group and / or a carbohydrate that is not oxidized by a mild oxidizing agent (i.e., an oxidizing agent that can oxidize an aldehyde but not an alcohol, such as Tollen's reagent). Examples of non-reducing sugars include sugars formed from or comprising a cyclic moiety. Specific examples of non-reducing sugars that can be used in the present application include raffinose, stachyose, trehalose, sucrose, and lactose, such as sucrose.

[0089] The non-reducing sugar can be any suitable sugar material having the above- described properties. In particular embodiments of the application, the non-reducing sugar can comprise one or more of the group consisting of trehalose and sucrose. For example, the non-reducing sugar can be sucrose. Alternatively, for example, the non-reducing sugar can be a combination of sucrose and trehalose. In certain embodiments, wherein the non-reducing sugar is a combination of sucrose and trehalose, the weight ratio of sucrose to trehalose in the formulation is about 1 : 1 to about 1 :5, or about 1 : 1 to about 5: 1, for example, the weight ratio of sucrose to trehalose in the formulation can be about 1 : 1.

[0090] It will be appreciated that an antioxidant is a compound that inhibits oxidation. Any suitable antioxidant material can be used herein. For example, the antioxidant can comprise one or more of the group consisting of glutathione, methionine, and ascorbic acid. In particular embodiments of the application that can be referred to herein, the antioxidant can be glutathione. In yet more particular embodiments of the application that can be referred to herein, the antioxidant can comprise one or more of the group consisting of reduced L-glutathione, reduced D-glutathione, oxidized L-glutathione, and oxidized D-glutathione (e.g., the glutathione comprises reduced L-glutathione or reduced D-glutathione, such as the glutathione is reduced L-glutathione).

[0091] Any suitable buffer can be used herein. For example, the buffer can comprise one or more of histidine and phosphate buffer. Additionally or alternatively, the buffer can be a buffer having a pKa of 3 to 8. In certain embodiments that can be referred to herein, the buffer can comprise 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 can 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.

[0092] Any suitable non-ionic surfactant can be used in the formulations disclosed herein. In certain embodiments, the non-ionic surfactant can comprise one or both of polysorbate and poloxamer. For example, the non-ionic surfactant can be a polysorbate. More particularly, the polysorbate can comprise or can be polysorbate-80.

[0093] In certain embodiments, there is provided a urease formulation, wherein:

[0094] the non-reducing sugar comprises sucrose;

[0095] the antioxidant comprises glutathione;

[0096] the buffer comprises histidine; and

[0097] The non-ionic surfactant comprises a polysorbate.

[0098] Thus, the urease formulation can comprise urease, sucrose, glutathione, histidine, and a polysorbate (e.g., polysorbate-80).

[0099] In certain other embodiments, a urease formulation is provided, wherein:

[0100] The non-reducing sugar comprises sucrose and trehalose;

[0101] The antioxidant comprises glutathione;

[0102] The buffer comprises potassium phosphate; and

[0103] The non-ionic surfactant comprises a polysorbate.

[0104] Thus, the urease formulation can comprise urease, sucrose, glutathione, histidine, and a polysorbate (e.g., polysorbate-80).

[0105] Any suitable amount of the components listed above can be used in the urease formulation referred to herein. For example, the urease formulation can be a formulation comprising, on a dry weight basis, 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 can be a formulation comprising, on a dry weight basis, 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 can be a formulation comprising, on a dry weight basis, from about 10 wt.% to about 25 wt.% immobilized urease, such as from about 14 wt.% to about 20 wt.% immobilized urease. Additionally or alternatively, the urease formulation can be a formulation comprising, on a dry weight basis, from about 0.005 wt.% to about 0.5 wt.% polysorbate (e.g., from about 0.01 wt.% to about 0.2 wt.% polysorbate). Additionally or alternatively, the urease formulation can be a formulation comprising, on a dry weight basis, from about 0.005 wt.% to about 1 wt.% histidine (e.g., from about 0.01 wt.% to about 0.3 wt.% histidine). Additionally or alternatively, the urease formulation can be a formulation comprising, on a dry weight basis, from about 0.1 wt.% to about 3 wt.% potassium phosphate (e.g., from about 0.3 wt.% to about 1.6 wt.% potassium phosphate).

[0106] As noted above, in certain embodiments of the application, the urease formulation can further comprise activated carbon and zirconium oxide.

[0107] The activated carbon can be provided in any suitable form. In particular embodiments that can be mentioned herein, the activated carbon can be in the form of particles having an average particle size of less than 2,000 microns. In further embodiments of the application that can be mentioned herein, the activated carbon can have an average particle size of from 100 microns to 1,900 microns, for example from 500 microns to 1,500 microns. The activated carbon can be present in any suitable amount. For example, in embodiments in which activated carbon is present, it can be provided in an amount of from about 30 wt.% to about 45 wt.%, for example from about 34 wt.% to about 38 wt.%, based on the total weight of the formulation on a dry weight basis.

[0108] The zirconium oxide can be provided in any suitable form, for example zirconium oxide particles. When in the form of particles, the zirconium oxide particles can have an average particle size of from about 10 microns to about 1000 microns, from about 100 microns to about 900 microns, from about 200 microns to about 900 microns, from about 300 microns to about 800 microns, from about 400 microns to about 700 microns, from 500 microns to about 600 microns, from about 10 microns to about 200 microns, from about 10 microns to about 100 microns, from about 10 microns to about 30 microns, from about 10 microns to about 20 microns, from about 20 microns to about 50 microns, from about 25 microns to about 50 microns, from about 30 microns to about 50 microns, from about 40 microns to about 150 microns, from about 80 microns to about 120 microns, from about 160 microns to about 180 microns, from about 25 microns to about 250 microns, from about 250 microns to about 500 microns, or from about 250 microns to about 1000 microns. The zirconium oxide particles can 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 the physical compaction of the particles into a predetermined roll. In one embodiment, the immobilization of the zirconium oxide particles is achieved by sintering the 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 can be a carbohydrate-based polymer, an organic polymer, a polyamide, a polyester, a polyacrylate, a polyether, a polyolefin, or an inorganic polymer or ceramic material. The biocompatible substrate can be at least one of cellulose, Eupergit, silica, nylon, polycaprolactone, and chitosan.

[0109] The zirconium oxide can be a hydrous zirconium oxide. The hydrous zirconium oxide can be synthesized by conventional methods, for example, by reaction of an aqueous mixture of zirconium sodium carbonate and sodium hydroxide as described in US 4,256,718. After synthesis of the hydrous zirconium oxide, the product can be titrated to a pH of 12 to 13. This can be done by preparing an aqueous slurry of the hydrous zirconium oxide and titrating with 5M sodium hydroxide to a pH of 12-13 of the slurry. In some cases, the hydrous zirconium oxide can then be washed until the concentration of leachable in the filtrate is within acceptable levels, and then air dried. Alternatively, the hydrous zirconium oxide can be recovered directly from the slurry without washing prior to air drying.

[0110] The zirconium oxide can be present in any suitable amount. For example, in embodiments in which zirconium oxide is present, it can provide from about 35 wt.% to about 48 wt.%, for example, from about 39 wt.% to about 43 wt.%, based on the total weight of the formulation on a dry weight basis.

[0111] The urease formulations disclosed herein can further comprise one or more proteins. In certain embodiments, the one or more proteins can comprise albumin, for example, bovine serum albumin (BSA). Thus, in certain embodiments, the urease formulations of the present application can comprise albumin, for example, BSA.

[0112] The urease formulations disclosed herein can further comprise one or more chelating agents. In certain embodiments, the one or more chelating agents can comprise ethylenediaminetetraacetic acid (EDTA). Thus, in certain embodiments, the urease formulations of the present application can comprise a chelating agent, for example, EDTA.

[0113] Thus, in certain embodiments, the urease formulation comprises:

[0114] a urease;

[0115] a non-reducing sugar, including sucrose;

[0116] an antioxidant, including glutathione;

[0117] a buffer, including histidine;

[0118] a non-ionic surfactant, including polysorbate; and

[0119] optionally albumin (e.g., BSA), and optionally a chelating agent (e.g., EDTA).

[0120] For example, a urease formulation can include urease, sucrose, glutathione, histidine, polysorbate (e.g., polysorbate-80), optionally albumin (e.g., BSA), and optionally a chelator (e.g., EDTA). For example, a urease formulation can include urease, sucrose, glutathione, histidine, polysorbate (e.g., polysorbate-80), and albumin (e.g., BSA). Alternatively, for example, a urease formulation can include urease, sucrose, glutathione, histidine, polysorbate (e.g., polysorbate-80), and a chelator (e.g., EDTA). Alternatively, for example, a urease formulation can include urease, sucrose, glutathione, histidine, polysorbate (e.g., polysorbate-80), albumin (e.g., BSA), and a chelator (e.g., EDTA). As disclosed herein, a urease formulation can further include activated charcoal and zirconium oxide.

[0121] In certain other embodiments, a urease formulation includes:

[0122] urease;

[0123] non-reducing sugars, including sucrose and trehalose;

[0124] antioxidants, including glutathione;

[0125] buffering agents, including potassium phosphate;

[0126] non-ionic surfactants, including polysorbate; and

[0127] optionally albumin (e.g., BSA), and optionally a chelator (e.g., EDTA).

[0128] For example, a urease formulation can include urease, sucrose, glutathione, histidine, polysorbate (e.g., polysorbate-80), optionally albumin (e.g., BSA), and optionally a chelator (e.g., EDTA). For example, a urease formulation can include urease, sucrose, glutathione, histidine, polysorbate (e.g., polysorbate-80), and albumin (e.g., BSA). Alternatively, for example, a urease formulation can include urease, sucrose, glutathione, histidine, polysorbate (e.g., polysorbate-80), and a chelator (e.g., EDTA). Alternatively, for example, a urease formulation can include urease, sucrose, glutathione, histidine, polysorbate (e.g., polysorbate-80), albumin (e.g., BSA), and a chelator (e.g., EDTA). As disclosed herein, a urease formulation can further include activated charcoal and zirconium oxide.

[0129] For the avoidance of doubt, the urease formulations of the present application can be freeze-dried (i.e., lyophilized) formulations comprising the components described herein. For example, the urease formulations of the present application can be prepared by combining a base formulation solution with urease. The base formulation solution can comprise a non-reducing sugar, an antioxidant, a buffer, and a non-ionic surfactant, as described herein, as well as a solvent (e.g., water). The base formulation solution can also comprise albumin (e.g., BSA) and / or a chelating agent (e.g., EDTA), as described herein. The urease can be immobilized on a solid support described herein. Subsequent lyophilization of the base formulation solution and urease mixture provides a lyophilized urease formulation. Particular examples of urease formulations prepared in this manner are provided in the Examples section herein. When the urease is an immobilized urease, the lyophilized urease formulation can be referred to as a lyophilized immobilized urease formulation. Thus, in certain embodiments, the urease formulations of the present application are lyophilized urease formulations, e.g., lyophilized immobilized urease formulations.

[0130] The urease formulations disclosed herein can provide greater than 99% urea clearance after three months of storage at 30°C, as compared to urea clearance prior to storage.

[0131] The urease formulations disclosed herein that are sterilized using gamma radiation can provide greater than 99% urea clearance after sterilization using 25-40 Kgy of gamma radiation, as compared to urea clearance prior to sterilization using gamma radiation. In embodiments of the present application, the urease formulation, in the presence of activated carbon and zirconium oxide, can provide greater than 99% urea clearance after:

[0132] (i) sterilization using 25-40 Kgy of gamma radiation; and

[0133] (ii) subsequent storage for three months at 30°C, as compared to urea clearance prior to sterilization using gamma radiation and storage.

[0134] In embodiments of the present application, the formulation, in the presence of activated carbon and zirconium oxide, can be a sterile urease formulation. For example, the formulation can have been treated with gamma radiation.

[0135] Other aspects and embodiments of the present application will not be discussed by reference to the following non-limiting examples.

[0136] Examples

[0137] Materials

[0138]

[0139]

[0140] 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 the pH to 7.5 with a 300 g / L potassium hydroxide solution, diluting to 1000 mL with sterile water, and then filtering through a 0.2 micron filter.

[0141] General Method 1 : Preparation of base formulation solution

[0142] A sugar or polyol was dissolved in sterile water to make a 125 mL 25% sugar or 10% polyol solution, then the following excipients were added in order as required: histidine (buffer), antioxidants (cysteine, ascorbic acid, methionine, and glutathione), chelating agent (sodium EDTA), surfactant (polysorbate 80), amino acid (arginine), and protein (BSA). The solution was mixed on a shaker at 150 rpm for 10 minutes. The clear solution was stored at 4°C until further use. For formulations containing phosphate buffer, 0.2 M Phosphate Buffer Solution prepared as described above was used in place of sterile water.

[0143] General Method 2: Preparation of urease formulation

[0144] A sterile spatula was used to transfer 40 g of wet immobilized urease (proprietary blend prepared from JB urease purchased from Sigma Aldrich product number 94281-1G) into 125 mL of the base formulation solution from General Method 1 at 4°C. The resulting suspension was shaken at 150 rpm in an incubator at 4°C for 1 hour, the resulting solid was collected by filtration, and the filtered residue was placed in a sterile container and covered with a paper towel. The wet immobilized urease formulation was frozen at -20°C for 18 hours, and the material was lyophilized in a freeze-dryer with an operating vacuum of 0.1 mbar at 4°C for 7.0 hours to achieve a loss on drying (LOD) in the range of 15-25%. The loss on drying (LOD) was measured by heating the sample with a moisture analyzer (RADWAG MA200.3Y.WH) at 210°C until the weight was stable.

[0145] General Method 3: Gamma sterilization and storage method

[0146] A 5 g of the lyophilized immobilized urease formulation from General Method 2 was mixed with scavengers (8.5 g activated carbon and 7.5 g hydrous zirconium oxide, such that the weight-to-weight ratio of lyophilized immobilized urease formulation: activated carbon: hydrous zirconium oxide was 1:1.7:1.5, which ratio can be used to produce larger or smaller batches) and filled into a plastic urease cartridge, which was subsequently sealed with an airtight cap. The cartridge was sterilized by high gamma radiation dose (25-40 Kgy) at 4°C. The sterilized cartridge was stored at room temperature for the desired period of time.

[0147] General Method 4: Urease performance

[0148] 14 L of synthetic dialysate (a simulant of peritoneal dialysis waste) was prepared, having the composition shown in Table 1 below.

[0149] Table 1: 14 L Synthetic Dialysis Fluid

[0150]

[0151] The pH of the synthetic dialysate solution was adjusted to 7.5 (physiological pH). The concentrations of all components were verified using a Vitros 250 analyzer.

[0152] use Figure 1 The apparatus involved pumping the synthetic dialysate into the urease cartridge of General Method 3, followed by a flow rate of 50 mL / min through the adsorbent cartridge. The adsorbent cartridge contained anion and cation exchangers to absorb the ammonia produced by the urease module. This was necessary because the presence of ammonia could interfere with the measurement of urea concentration. The clearance efficiency of the immobilized urease was then monitored periodically by measuring the urea concentration after every 2 L of synthetic dialysate was used (measured using a Vitros 250 analyzer). Finally, all 14 L of synthetic dialysate was collected to measure the urea clearance rate. The total percentage of urea clearance was determined using cumulative and effluent data.

[0153] The amount of urea removed can be calculated as follows:

[0154] Total urea removed (mmol) = Total urea input (mmol) - Total urea in postdialysis fluid (mmol)

[0155] % Total urea removed = (Total urea removed) / (Total urea input) x 100

[0156] Example calculations are provided in Table 2 below.

[0157] Table 2: Example calculation of urea clearance rate

[0158]

[0159] Comparative Example 1 : Comparative formulation

[0160] Using the components listed in Table 3 below, prepare comparative formulations using general methods 1 and 2.

[0161] Table 3: Identification of Comparative Preparations

[0162]

[0163]

[0164] The concentrations indicated in this table relate to the concentration of each component in the base formulation solution used. The concentrations provided refer to the concentration of the component in the base formulation solution used (w / v).

[0165] The formulations listed in Table 3 were sterilized and stored according to the general method 3. After the appropriate storage time, the urease performance was tested according to the general method 4. The results are provided in Table 4. If the urea clearance rate dropped below 95%, the stability test of the formulation was stopped.

[0166] Table 4: Urea clearance rate of comparative formulations

[0167]

[0168] NT - not tested

[0169] Example 1 : Formulation of the invention

[0170] Using the components listed in the following Table 5, urease formulations were prepared using the general methods 1 and 2.

[0171] Table 5: Identification of urease formulations

[0172]

[0173] The concentrations indicated in this table relate to the concentration of each component in the base formulation solution used. The concentrations provided refer to the concentration of the component in the base formulation solution used (w / v).

[0174] The formulations listed in Table 5 were sterilized and stored according to the general method 3. After the appropriate storage time, the urease performance was tested according to the general method 4. The results are provided in Table 6.

[0175] Table 6: Urea clearance rate of urease formulations

[0176]

[0177] It is considered that the value of 1 month or 2 months can be wrong, since the urea clearance rate after two months should be equal or lower than the clearance rate of 1 month. NT = not tested

[0178] By comparing the results in Tables 4 and 6, it is clear that the formulations according to the present application are able to effectively stabilize urease and ensure its activity remains high during the gamma irradiation step and subsequent long-term storage at room temperature. In contrast, the comparative formulation, which does not include all non-reducing sugars, antioxidants, buffers, non-ionic surfactants, has a worse urea clearance rate after storage at room temperature, indicating a much shorter shelf-life.

[0179] Thus, the formulations according to the present application are highly advantageous and solve the problem of short shelf life of existing urease formulations for dialysis.

[0180] Example 2: Other formulations of the invention

[0181] Using the components listed in Table 7 below, urease formulations were prepared using General Methods 1 and 2.

[0182] Using the components listed in Table 5 below, urease formulations were prepared using General Methods 1 and 2.

[0183] Table 7: Identification of urease formulations

[0184]

[0185] The concentrations shown in this table relate to the concentration of each component in 100 mL of the base formulation solution used.

[0186] The formulations listed in Table 7 were sterilized and stored according to General Method 3. After the appropriate storage time, the urease performance was tested according to General Method 4. The results are provided in Table 8.

[0187] Table 8: Urea clearance of urease formulations

[0188]

[0189]

[0190] NT = not tested

[0191] By comparing the results in Tables 4 and 8, it is clear that the formulations according to the present application are able to effectively stabilize urease and ensure that its activity remains high during the gamma-irradiation sterilization step and subsequently during long-term storage at room temperature.

[0192] Thus, the formulations according to the present application are highly advantageous and solve the problem of short shelf life of existing urease formulations for dialysis.

Claims

1. 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 of claim 1, wherein the formulation further comprises: activated charcoal; and hydrated zirconium oxide.

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

4. The urease formulation of any one of the preceding claims, wherein the non- reducing 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 of 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 of any one of the preceding claims, wherein the buffer comprises one or more of the group consisting of histidine and phosphate buffer, and / or wherein the buffer has a pKa of 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 of any one of the preceding claims, wherein the non- ionic surfactant comprises one or both of polysorbate and poloxamer, optionally wherein the non-ionic surfactant comprises polysorbate.

8. The urease formulation of 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 of 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 of any one of the preceding claims, wherein the antioxidant comprises one or more of the group consisting of reduced L-glutathione, reduced D- glutathione, oxidized L-glutathione, and oxidized 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 of 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 of any one of the preceding claims, wherein the non- ionic surfactant comprises polysorbate-80.

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

14. The urease formulation of any one of the preceding claims, wherein, about 0.01 wt.% to about 2 wt.% of the antioxidant, more optionally about 0.05 wt.% to about 1 wt.% of the antioxidant.

15. The urease formulation of any one of the preceding claims, wherein, about 10 wt.% to about 25 wt.% immobilized urease by dry weight, optionally about 14 wt.% to about 20 wt.% immobilized urease.

16. The urease formulation of any one of the preceding claims, wherein the non-ionic surfactant comprises polysorbate, and the formulation comprises about 0.005 wt.% to about 0.5 wt.% polysorbate by dry weight (e.g., about 0.01 wt.% to about 0.2 wt.% polysorbate).

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

18. The urease preparation of claim 2 and any one of claims 3 to 17 as dependent on claim 2, wherein, about 30 wt.% to about 45 wt.% activated carbon by dry weight, optionally about 34 wt.% to about 38 wt.% activated carbon.

19. The urease preparation of claim 2 and any one of claims 3 to 18 as dependent on claim 2, wherein, about 35 wt.% to about 48 wt.% zirconium oxide by dry weight, optionally about 39 wt.% to about 43 wt.% zirconium oxide.

20. The urease formulation of 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, e.g., 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 sterilization using 25-40 Kgy of gamma radiation, as compared to urea clearance prior to sterilization using gamma radiation; and (d) the urease formulation provides greater than 99% urea clearance after three months of storage at 30 °C, as compared to urea clearance prior to storage.

21. The urease formulation of claim 2 and any one of claims 3-20 dependent on claim 2, wherein the urease formulation provides greater than 99% urea clearance after: (i) sterilization using 25-40 Kgy of gamma radiation; and (ii) subsequent storage for three months at 30 °C, as compared to urea clearance and storage prior to sterilization using gamma radiation, optionally wherein the formulation is a sterile urease formulation.

22. The urease formulation of any one of the preceding claims, wherein the urease formulation is a lyophilized urease formulation.

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