Immobilized urease preparations with improved shelf life

Abstract

The present invention provides a urease preparation suitable for use in dialysis, which comprises urease, a non-reducing sugar, an antioxidant, a buffer, and a nonionic surfactant. The urease preparation of the present invention has excellent stability against gamma ray sterilization and an excellent shelf life.

Description

Technical Field

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

Background Art

[0002] Hemodialysis relies on the use of urease enzyme to remove urea from patients. Urease is typically provided in an immobilized form attached to a solid support and present in an adsorbent through which the dialysate can pass. To minimize the risk of infection associated with the treatment, it is necessary to sterilize urease before treatment. However, urease preparations used in dialysis have low stability against sterilization methods and also low storage stability. This causes logistical problems for both dialysis technicians and patients alike. Because urease preparations cannot be purchased in bulk and cannot be easily stored at the treatment center or the patient's home.

[0003] Therefore, existing urease preparations may require a cold distribution system, storage at low temperature, and use within a few days to a few weeks of preparation for dialysis procedures. If these requirements are not met, it can result in urea removal efficacy that is too low to be therapeutically effective. This low storage stability results in a short expiration date and a high level of loss, increasing costs.

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

Summary of the Invention

[0005] Surprisingly, the inventors have discovered that urease preparations as described herein can be stable at room temperature for at least a three-month period and, in more specific particular embodiments, are stable against a gamma-ray sterilization process and can then be stable at room temperature for at least a three-month period.

[0006] Aspects and embodiments of the present invention are referred to in the numbered sections below. [Item 1] Urease; Non-reducing sugars; Antioxidants; buffer; and Nonionic surfactants, A urease preparation suitable for use in dialysis, containing [specific ingredient / method]. [Item 2] Activated carbon; and Zirconium hydrated oxide, A urease preparation that further includes items according to item 1. [Item 3] The urease is provided in the form of urease immobilized on a solid support. A urease preparation conforming to item 1 or item 2. [Item 4] The non-reducing sugar includes one or more types from the group consisting of trehalose and sucrose; Optionally, non-reducing sugars include sucrose. A urease preparation that conforms to any one of the items listed above. [Item 5] The antioxidant includes one or more of the following: glutathione, methionine, and ascorbic acid; Optionally, antioxidants, including glutathione, A urease preparation that conforms to any one of the items listed above. [Item 6] The buffer solution comprises one or more types from the group consisting of histidine and phosphate buffer; and / or buffer, 3-8 pK a Having; The buffer optionally contains either histidine or potassium phosphate, or both; More optionally, the buffer contains histidine and potassium phosphate. A urease preparation that conforms to any one of the items listed above. [Item 7] The nonionic surfactant comprises either or both polysorbate and poloxamer; Optionally, a nonionic surfactant, including polysorbate, A urease preparation that conforms to any one of the items listed above. [Item 8] Non-reducing sugars include sucrose; The antioxidant contains glutathione; The buffer contains histidine; and Nonionic surfactants include polysorbate. A urease preparation that conforms to any one of the items listed above. [Item 9] Non-reducing sugars include sucrose and trehalose; The antioxidant contains glutathione; The buffer solution contains potassium phosphate; and Nonionic surfactants include polysorbate. A urease preparation that conforms to one of items 1 through 7. [Item 10] The antioxidant comprises one or more of the following: reduced L-glutathione, reduced D-glutathione, oxidized L-glutathione, and oxidized D-glutathione; Optionally, glutathione may include reduced L-glutathione or reduced D-glutathione; More optionally, glutathione, including reduced L-glutathione, A urease preparation that conforms to any one of the items listed above. [Item 11] The buffer solution contains one or more of the group consisting of L-histidine and D-histidine; The buffer may optionally contain L-histidine. A urease preparation that conforms to any one of the items listed above. [Item 12] The nonionic surfactant contains polysorbate-80. A urease preparation that conforms to any one of the items listed above. [Item 13]. Based on the dry weight, the formulation contains from about 0.5 wt% to about 10 wt% of a non-reducing sugar (e.g., sucrose); Optionally from about 1 wt% to about 7 wt% of a non-reducing sugar, More optionally from about 1 wt% to about 5 wt% of a non-reducing sugar, The urease formulation according to any one of the above items. [Item 14] Based on the dry weight, the formulation contains from about 0.001 wt% to about 5 wt% of an antioxidant (e.g., glutathione); Optionally from about 0.01 wt% to about 2 wt% of an antioxidant, More optionally from about 0.05 wt% to about 1 wt% of an antioxidant, The urease formulation according to any one of the above items. [Item 15] Based on the dry weight, the formulation contains from about 10 wt% to about 25 wt% of immobilized urease, Optionally from about 14 wt% to about 20 wt% of immobilized urease, The urease formulation according to any one of the above items. [Item 16] The non-ionic surfactant contains polysorbate; Based on the dry weight, the formulation contains from about 0.005 wt% to about 0.5 wt% of polysorbate (e.g., from about 0.01 wt% to about 0.2 wt% of polysorbate), The urease formulation according to any one of the above items. [Item 17] The following: (a) the buffer contains histidine and based on the dry weight, the formulation contains from about 0.005 wt% to about 1 wt% of histidine (e.g., from about 0.01 wt% to about 0.3 wt% of histidine); And (b) the buffer contains potassium phosphate and based on the dry weight, the formulation contains from about 0.1 wt% to about 3 wt% of potassium phosphate (e.g., from about 0.3 wt% to about 1.6 wt% of potassium phosphate), The urease formulation according to any one of the above items that applies to either one or both of the above. [Item 18] Based on dry weight, the formulation contains approximately 30 wt% to 45 wt% activated carbon; Optionally, approximately 34 wt% to 38 wt% activated carbon. A urease preparation that includes item 2 and any one of items 3-17 that are dependent on item 2. [Item 19] Based on dry weight, the formulation contains approximately 35 wt% to 48 wt% zirconium oxide; Optionally, approximately 39 wt% to 43 wt% of zirconium oxide, A urease preparation that includes item 2 and any one of items 3-18 that are dependent on item 2. [Item 20] below: (a) The urease preparation further contains one or more proteins, optionally including albumin, for example, bovine serum albumin (BSA); (b) The urease preparation further contains one or more chelating agents, and optionally one or more chelating agents contain ethylenediaminetetraacetic acid (EDTA); (c) The urease preparation provides urea clearance of over 99% after sterilization using gamma radiation at 25-40 kGy, compared to urea clearance before sterilization using gamma radiation; and (d) The urease preparation provides a urea clearance of more than 99% after storage at 30°C for 3 months compared to the urea clearance before storage; A urease preparation that meets one or more of the above criteria and conforms to any one of the above items. [Item 21] Urease preparations, Compared to urea clearance before sterilization and preservation using gamma radiation, (i) Sterilization using gamma radiation at 25-40 kGy; and (ii) Subsequent storage at 30°C for 3 months. It then provides over 99% urea clearance; The preparation is optionally a sterile urease preparation. A urease preparation that conforms to item 2 and any one of items 3-20 that are dependent on item 2. [Item 22] The urease preparation is a freeze-dried urease preparation. A urease preparation that conforms to any one of the above items. [Brief explanation of the drawing]

[0007] [Figure 1] The apparatus used to test the stability of the urease preparations disclosed herein is shown. [Modes for carrying out the invention]

[0008] As described above, the present inventors have found a urease formulation, as described herein, that is stable at room temperature for a period of at least three months, and in a more specific embodiment, stable against a gamma ray sterilization process, and subsequently stable at room temperature for a period of at least three months.

[0009] Therefore, in the first aspect of the present invention, Urease; Non-reducing sugars; Antioxidants; buffer; and Nonionic surfactants, The present invention provides a urease preparation suitable for use in dialysis, which includes [specific ingredient / method].

[0010] While not limited by theory, formulations containing the above components are thought to be able to maintain enzyme stability for extended periods (e.g., 3 months) at ambient temperatures.

[0011] In certain embodiments of the present invention that may be referenced herein, the urease preparation further comprises activated carbon and hydrated zirconium oxide. Although not limited by theory, the addition of these two components to the preparations described in the first aspect of the present invention is thought to maintain the stability of the urease enzyme at ambient temperature after a sterilization process (e.g., by gamma rays).

[0012] The term “comprising” as used herein may be interpreted as requiring the features mentioned, but not as limiting the presence of other features. Alternatively, the term “comprising” may also relate to a situation in which only the listed components / features are intended to exist (for example, the term “comprising” may be replaced by the expression “consisting of” or “essentially consisting of”). It is expressly intended that both broader and more restrictive interpretations may apply to all aspects and embodiments of the invention. In other words, the term “comprising” and its synonyms may be replaced by the expression “consisting of” or “essentially consisting of” or its synonyms, and vice versa.

[0013] The expression "essentially consisting of" and its alternative names may be interpreted in this specification as referring to materials that may contain small amounts of impurities. For example, a material may be 90% or more pure, such as 95% or more pure, 97% or more pure, 99% or more pure, 99.9% or more pure, 99.99% or more pure, 99.999% or more pure, 100% pure, etc.

[0014] As used herein, the singular forms “a,” “an,” and “the” refer to multiple objects unless the context explicitly indicates otherwise. For example, “a composition” includes mixtures of two or more such compositions; “a non-reducing sugar” includes mixtures of two or more such non-reducing sugars, and so on.

[0015] As used herein, the term "urease" refers to an enzyme capable of reacting with urea as a substrate. Such an enzyme may be determined to be functional in vitro, for example, by reacting the enzyme with urea in solution and measuring the decrease in urea concentration.

[0016] In healthy individuals, urea is normally excreted from the body through urine. However, in certain individuals, urea is not removed from the body at a sufficiently rapid rate, leading to uremic toxicity, a disease or condition characterized by elevated levels of at least one uremic toxin (e.g., urea) relative to physiologically normal levels of uremic toxins. Non-limiting examples of disorders associated with uremic toxins include renal disease or renal failure, gout, and uremic toxicity in patients undergoing chemotherapy.

[0017] As used herein, the term “predominantly” is intended to describe a situation or condition that occurs most of the time, or most often, but does not exclude the possibility that some other situation or condition may also occur less frequently. For example, it could be over 80%, over 90%, over 95%, or over 99%. To avoid misunderstanding, this term covers the possibility that only that situation or condition occurs, excluding all others.

[0018] The term "substantially" does not exclude "completely"; for example, a composition that "substantially does not contain" Y may contain Y at all. Where necessary, the term "substantially" may be omitted from the definition of this invention.

[0019] As used herein, the term “about” typically means ±5% of the value mentioned, in relation to the concentration of the components of the formulation, more typically ±4% of the value mentioned, more typically ±3% of the value mentioned, more typically ±2% of the value mentioned, even more typically ±1% of the value mentioned, and even more typically ±0.5% of the value mentioned.

[0020] Throughout this disclosure, certain embodiments may be disclosed in the form of a scope. It should be understood that such scope descriptions are merely for convenience and brevity and should not be considered inflexible limitations on the scope of the disclosure. Therefore, a scope description should be considered to include all possible sub-scopes specifically disclosed, as well as the individual numbers within those scopes. For example, a scope description such as 1–6 should be considered to have specifically disclosed sub-scopes such as 1–3, 1–4, 1–5, 2–4, 2–6, 3–6, as well as the individual numbers within those scopes, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the scope.

[0021] In certain embodiments referred to herein, the urease may be provided in the form of urease immobilized on a solid support.

[0022] Urease can be immobilized on any known support material that can provide immobilization of urease enzyme particles. Immobilization may be by physical means, such as adsorption onto alumina. In one embodiment, an unimmobilized enzyme is used. Alternatively, other methods are used to convert urea to ammonia.

[0023] In one embodiment, the support material is a biocompatible substrate to which a urease enzyme is covalently bonded. The biocompatible material may be a carbohydrate-based polymer, an organic polymer, a polyamide, a polyester, or an inorganic polymer material. The biocompatible substrate may be a homogeneous substrate composed of one type of material, or a composite substrate composed of at least two types of 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.

[0024] In one embodiment, immobilization of urease enzymes on a biocompatible substrate is carried out by an immobilization technique selected from the group consisting of glutaraldehyde activation, epoxy activation, epichlorohydrin activation, bromoacetic acid activation, cyanide bromide activation, thiol activation, and coupling with N-hydroxysuccinimide and diimidoamide. The immobilization technique used may also include 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 may be further functionalized with a reactive layer and / or stabilizing layer such as dextran or polyethylene glycol, and with suitable linker molecules and / or stabilizing molecules such as ethylenediamine, 1,6-diaminohexane, thioglycerol, mercaptoethanol, and trehalose. Urease may be used in a purified form or in the form of a crude extract, such as a urease extract from canavalia cordata or other suitable urease sources.

[0025] Urease particles may be capable of converting urea to ammonium carbonate. In one embodiment, urease particles may have an average particle size in the range of approximately 10 to 1000 microns, approximately 100 to 900 microns, approximately 200 to 900 microns, approximately 300 to 800 microns, approximately 400 to 700 microns, 500 to 600 microns, approximately 25 to 250 microns, approximately 25 to 100 microns, approximately 250 to 500 microns, approximately 250 to 1000 microns, approximately 125 to 200 microns, approximately 150 to 200 microns, approximately 100 to 175 microns, or approximately 100 to 150 microns.

[0026] In one embodiment, 1,000 to 10,000 units of urease are immobilized on a solid support. The total dry weight of the immobilized urease and its substrate may be in the range of about 0.5 g to about 100 g, for example, about 0.5 g to about 50 g, or about 0.5 g to about 30 g.

[0027] As used herein, “non-reducing sugars” refers to disaccharides, trisaccharides, or tetrasaccharides that do not contain free aldehydes or free ketone functional groups and / or are not oxidized by weak oxidizing agents (i.e., oxidizing agents that can oxidize aldehydes but not alcohols, such as Torrens’ reagent). Examples of non-reducing sugars include sugars that are formed from or contain cyclic moieties. Specific examples of non-reducing sugars that may be useful in the present invention include raffinose, stachyose, trehalose, sucrose, and lactose, for example, sucrose.

[0028] The non-reducing sugar may be any suitable sugar material having the properties described herein. In certain embodiments of the present invention, the non-reducing sugar may include one or more from the group consisting of trehalose and sucrose. For example, the non-reducing sugar may be sucrose. Alternatively, for example, the non-reducing sugar may be a combination of sucrose and trehalose. In certain embodiments, when the non-reducing sugar is a combination of sucrose and trehalose, the weight ratio of sucrose to trehalose in the formulation may be 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 may be about 1:1.

[0029] As is understood, antioxidants are compounds that inhibit oxidation. Any suitable antioxidant material can be used herein. For example, the antioxidant may include one or more from the group consisting of glutathione, methionine, and ascorbic acid. In certain embodiments of the invention that may be referenced herein, the antioxidant may be glutathione. In more detailed embodiments of the invention that may be referenced herein, the antioxidant may include one or more from the group consisting of reduced L-glutathione, reduced D-glutathione, oxidized L-glutathione, and oxidized D-glutathione (for example, glutathione may include reduced L-glutathione or reduced D-glutathione, and glutathione may be reduced L-glutathione, etc.).

[0030] Any suitable buffer can be used herein. For example, the buffer may include one or more of the group consisting of histidine and phosphate buffers. Additionally or alternatively, the buffer may have a pK of 3 to 8. a The buffer may have the following properties. In certain embodiments that may be referenced herein, the buffer may contain one or both histidine and potassium phosphate (for example, the buffer may contain both histidine and potassium phosphate). In more specific embodiments, the buffer may contain one or more of the group consisting of L-histidine and D-histidine (for example, the buffer may contain L-histidine). In certain embodiments, if the buffer contains potassium phosphate, the non-reducing sugars may include sucrose and trehalose.

[0031] Any suitable nonionic surfactant can be used in the formulations disclosed herein. In certain embodiments, the nonionic surfactant may comprise one or both of polysorbate and poloxamer. For example, the nonionic surfactant may be polysorbate. More specifically, the polysorbate may comprise polysorbate-80, or the polysorbate may be polysorbate-80.

[0032] In a particular embodiment, a urease preparation is provided. Non-reducing sugars include sucrose; Antioxidants include glutathione; The buffer contains histidine; and Nonionic surfactants include polysorbates.

[0033] Therefore, urease preparations may contain urease, sucrose, glutathione, histidine, and polysorbate (e.g., polysorbate-80).

[0034] In other specific embodiments, a urease preparation is provided. Non-reducing sugars include sucrose and trehalose; Antioxidants include glutathione; The buffer solution contains potassium phosphate; and Nonionic surfactants include polysorbates.

[0035] Therefore, urease preparations may contain urease, sucrose, trehalose, glutathione, potassium phosphate, and polysorbate (e.g., polysorbate-80).

[0036] Any suitable amount of the components listed above may be used in the urease preparations referred to herein. For example, a urease preparation may contain, on dry weight, about 0.5 wt% to about 10 wt% of non-reducing sugars (e.g., sucrose or trehalose, or both sucrose and trehalose), such as about 1 wt% to about 7 wt% of non-reducing sugars, such as about 1 wt% to about 5. Additionally or alternatively, a urease preparation may contain, on dry weight, about 0.01 wt% to about 2 wt% of antioxidants, such as about 0.05 wt% to about 1 wt% of antioxidants, such as about 0.001 wt% to about 5 wt% of antioxidants (e.g., glutathione), such as about 0.01 wt% to about 2 wt% of antioxidants, such as about 0.05 wt% to about 1 wt% of antioxidants, such as about 5 wt% of antioxidants, such as about 5 wt% of antioxidants, such as glutathione. Additionally or alternatively, the urease preparation may contain approximately 10 wt% to 25 wt% of immobilized urease, such as approximately 14 to 20 wt% of immobilized urease, based on dry weight. Additionally or alternatively, the urease preparation may contain approximately 0.005 wt% to 0.5 wt% of polysorbate (e.g., approximately 0.01 wt% to 0.2 wt% of polysorbate) based on dry weight. Additionally or alternatively, the urease preparation may contain approximately 0.005 wt% to 1 wt% of histidine (e.g., approximately 0.01 wt% to 0.3 wt% of histidine) based on dry weight. Additionally or alternatively, the urease preparation may contain approximately 0.1 wt% to approximately 3 wt% of potassium phosphate (e.g., approximately 0.3 wt% to approximately 1.6 wt% of potassium phosphate) based on dry weight.

[0037] As described above, in certain embodiments of the present invention, the urease preparation may further contain activated carbon and zirconium oxide.

[0038] Activated carbon may be provided in any preferred form. In certain embodiments that may be referenced herein, the activated carbon may be in the form of particles having an average particle size of less than 2,000 microns. In further embodiments of the invention that may be referenced herein, the activated carbon may have an average particle size of 100 to 1,900 microns, such as 500 to 1,500 microns. Activated carbon may be present in any preferred amount. For example, in embodiments in which activated carbon is present, the activated carbon may be provided in an amount of about 30 wt% to about 45 wt% (e.g., about 34 wt% to about 38 wt%) of the total weight of the formulation, based on dry weight.

[0039] Zirconium oxide may be provided in any preferred form, such as zirconium oxide particles. When in particulate form, zirconium oxide particles may have an average particle size in the range of approximately 10 microns to 1000 microns, approximately 100 microns to 900 microns, approximately 200 microns to 900 microns, approximately 300 microns to 800 microns, approximately 400 microns to 700 microns, 500 microns to 600 microns, approximately 10 microns to 200 microns, approximately 10 microns to 100 microns, approximately 10 microns to 30 microns, approximately 10 microns to 20 microns, approximately 20 microns to 50 microns, approximately 25 microns to 50 microns, approximately 30 microns to 50 microns, approximately 40 microns to 150 microns, approximately 80 microns to 120 microns, approximately 160 microns to 180 microns, approximately 25 microns to 250 microns, approximately 250 microns to 500 microns, or approximately 250 microns to 1000 microns. Zirconium oxide particles may be immobilized on any known support material capable of providing immobilization of zirconium oxide particles. In one embodiment, immobilization of zirconium phosphate particles is the physical compression of particles to a predetermined volume. In one embodiment, immobilization of zirconium phosphate 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 polyacrylic acid, a polyether, a polyolefin, or an inorganic polymer or a ceramic material. The biocompatible substrate may be at least one of cellulose, eupergit, silicon dioxide, nylon, polycaprolactone, and chitosan.

[0040] Zirconium oxide may be hydrated zirconium oxide. Hydrated zirconium oxide may be synthesized by conventional methods, for example, by the reaction of an aqueous mixture of sodium zirconium carbonate and sodium hydroxide, as described in U.S. Patent Application No. 4,256,718. After the synthesis of hydrated zirconium oxide, the product may be titrated to a pH of 12-13. This may be done by preparing an aqueous slurry of hydrated zirconium oxide and titrating it with 5 M sodium hydroxide until the slurry reaches a pH of 12-13. In some examples, the hydrated zirconium oxide may then be washed until the concentration of leaches in the filtrate is within an acceptable level, and then air-dried. Alternatively, the hydrated zirconium oxide may be recovered directly from the slurry and not washed before air-drying.

[0041] Zirconium oxide may be present in any suitable amount. For example, in embodiments in which zirconium oxide is present, it may be provided in an amount of approximately 35 wt% to approximately 48 wt% (approximately 39 wt% to approximately 43 wt%, etc.) of the total weight of the formulation, based on dry weight.

[0042] The urease preparations disclosed herein may further contain one or more proteins. In certain embodiments, the one or more proteins may include albumin, for example, bovine serum albumin (BSA). Therefore, in certain embodiments, the urease preparations of the present invention may contain albumin, for example, BSA.

[0043] The urease formulations disclosed herein may further contain one or more chelating agents. In certain embodiments, the one or more chelating agents may include ethylenediaminetetraacetic acid (EDTA). Therefore, in certain embodiments, the urease formulations of the present invention may contain a chelating agent, such as EDTA.

[0044] Therefore, in a particular embodiment, the urease preparation is Urease; Non-reducing sugars, including sucrose; Antioxidants containing glutathione; Histidine-containing buffer; Nonionic surfactants containing polysorbate; and Optionally, albumin (e.g., BSA); and Optionally, a chelating agent (e.g., EDTA) Includes.

[0045] For example, a urease preparation may contain urease, sucrose, glutathione, histidine, polysorbate (e.g., polysorbate-80), optionally albumin (e.g., BSA), and optionally a chelating agent (e.g., EDTA). Alternatively, a urease preparation may contain, for example, urease, sucrose, glutathione, histidine, polysorbate (e.g., polysorbate-80), and albumin (e.g., BSA). Alternatively, for example, a urease preparation may comprise urease, sucrose, glutathione, histidine, polysorbate (e.g., polysorbate-80), albumin (e.g., BSA), and a chelating agent (e.g., EDTA). As disclosed herein, the urease preparation may further comprise activated carbon and zirconium oxide.

[0046] In certain other embodiments, the urease preparation is Urease; Non-reducing sugars, including sucrose and trehalose; Antioxidants containing glutathione; Buffer solution containing potassium phosphate; Nonionic surfactants containing polysorbate; and Optionally, albumin (e.g., BSA); and Optional chelating agent (e.g., EDTA), Includes.

[0047] For example, a urease preparation may contain urease, sucrose, trehalose, glutathione, potassium phosphate, polysorbate (e.g., polysorbate-80), optionally albumin (e.g., BSA), and optionally a chelating agent (e.g., EDTA). Alternatively, for example, a urease preparation may contain urease, sucrose, trehalose, glutathione, potassium phosphate, polysorbate (e.g., polysorbate-80), and albumin (e.g., BSA). Alternatively, for example, a urease preparation may comprise urease, sucrose, trehalose, glutathione, potassium phosphate, polysorbate (e.g., polysorbate-80), albumin (e.g., BSA), and a chelating agent (e.g., EDTA). As disclosed herein, the urease preparation may further comprise activated carbon and zirconium oxide.

[0048] To avoid misunderstanding, the urease formulations of the present invention may be freeze-dried (i.e., lyophilized) formulations containing the components described herein. For example, the urease formulations of the present invention may be prepared by combining a basic formulation solution with urease. The basic formulation solution may include non-reducing sugars, antioxidants, buffers, and nonionic surfactants, as well as a solvent (e.g., water), as described herein. The basic formulation solution may also include albumin (e.g., BSA) and / or a chelating agent (e.g., EDTA), as described herein. The urease may be immobilized on a solid support as described herein. Subsequent lyophilization of the basic formulation solution and urease mixture yields a lyophilized urease formulation. Specific examples of urease formulations prepared in this manner are shown in the Examples section herein. If the urease is immobilized urease, the lyophilized urease formulation may be called a lyophilized and immobilized urease formulation. Therefore, in certain embodiments, the urease preparation of the present invention is a lyophilized urease preparation, for example, a lyophilized and immobilized urease preparation.

[0049] The urease preparations disclosed herein may provide more than 99% urea clearance after 3 months of storage at 30°C compared to urea clearance before storage.

[0050] The urease preparations disclosed herein, subjected to sterilization using gamma radiation, can provide urea clearance of over 99% after sterilization using gamma radiation at 25-40 kGy compared to urea clearance before sterilization using gamma radiation. In embodiments of the present invention, in the presence of activated carbon and zirconium oxide, the urease preparation provides urea clearance compared to urea clearance before sterilization using gamma radiation and storage. (i) Sterilization using gamma radiation at 25-40 kGy; (ii) Subsequent storage at 30°C for 3 months, It can provide over 99% urea clearance afterward.

[0051] In embodiments of the present invention, when activated carbon and zirconium oxide are present, the formulation may be a sterile urease formulation. For example, the formulation may be treated with gamma radiation.

[0052] Further aspects and embodiments of the present invention will be described by reference to the following non-limiting embodiments.

[0053] Examples material

[0054] [Table 1]

[0055] 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 300 g / L of potassium hydroxide solution, diluting to 1000 mL with sterile water, and then filtering through a 0.2 micron filter.

[0056] General method 1: Preparation of basic formulation solution Prepare 125 mL of a 25% sugar or 10% polyol solution by dissolving the sugar or polyol in sterile water. Then, add the following excipients sequentially as needed: histidine (buffer), antioxidants (cysteine, ascorbic acid, methionine, and glutathione), chelating agent (EDTA sodium salt), surfactant (polysorbate 80), amino acid (arginine), and protein (BSA). Stir this solution in a shaker at 150 rpm for 10 minutes. Store the clear solution at 4°C until further use. For formulations containing phosphate buffer, replace the sterile water with the 0.2 M phosphate buffer prepared as described above.

[0057] General method 2: Preparation of urease preparations 40 g of wet-immobilized urease (a proprietary mixture prepared from JB urease obtained from Sigma Aldrich product number 94281-1G) is transferred to 125 mL of the basic formulation solution from General Method 1 at 4°C using a sterile spatula. The resulting suspension is shaken at 150 rpm for 1 hour at 4°C in an incubator. The resulting solid is collected by filtration, and the filtration residue is placed in a sterile container and covered with a paper towel. The wet-immobilized urease formulation is frozen at -20°C for 18 hours, and this material is freeze-dried in a freeze-dryer at 4°C for 7.0 hours under an operating vacuum of 0.1 mmbar until the loss on drying (LOD) reaches a range of 15-25%. The loss on drying (LOD) is measured by heating the sample at 210°C until the weight stabilizes using a hygrometer (RADWAG MA20 0.3Y.WH).

[0058] General Method 3: Gamma Ray Sterilization and Preservation Method 5 g of lyophilized and immobilized urease preparation from General Method 2 is mixed with a scavenger (8.5 g of activated carbon and 7.5 g of hydrated zirconium oxide, so that the weight-to-weight ratio of lyophilized and immobilized urease preparation:activated carbon:hydrated zirconium oxide is 1:1.7:1.5, and the above ratio can be used to create larger or smaller batches), filled into a plastic urease cartridge, and then sealed with an airtight lid. The cartridge is sterilized by high-dose gamma irradiation (25-40 kGy) at 4°C. The sterilized cartridge is stored at room temperature for the desired period.

[0059] General method 4: Urease performance Prepare 14 L of synthetic dialysate (a mimic of peritoneal dialysis waste fluid) having the composition shown in Table 1 below.

[0060] [Table 2]

[0061] The pH of the synthetic dialysate was adjusted to 7.5 (physiological pH). The concentrations of all components were confirmed using a Vitros 250 Analyzer.

[0062] Using the apparatus shown in Figure 1, synthetic dialysate was pumped into a urease cartridge from General Method 3 and then passed through an adsorption cartridge at a flow rate of 50 mL / min. The adsorption cartridge contained anion and cation exchangers to absorb ammonia produced from the 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 regularly monitored by measuring the urea concentration every 2 L of synthetic dialysate used (measurements were performed using a Vitros250 analyzer). Finally, all 14 L of synthetic dialysate was collected to measure urea clearance. The total percentage of urea clearance was determined using cumulative and discharge data.

[0063] The amount of urea removed can be calculated as follows: Total urea removed (mmol) = Total urea added (mmol) - Total urea in the solution after dialysis (mmol) % Total urea removed = (Total urea removed) / (Total urea added) x 100

[0064] An example calculation is shown in Table 2 below.

[0065] [Table 3]

[0066] Comparative Example 1: Comparison Formulation Comparison formulations were prepared using the components listed in Table 3 below, employing general methods 1 and 2.

[0067] [Table 4]

[0068] The formulations shown in Table 3 were sterilized and stored according to General Method 3. After an appropriate storage period, the urease performance was tested according to General Method 4. The results are shown in Table 4. If the urea clearance fell below 95%, the stability test of the formulation was discontinued.

[0069] [Table 5]

[0070] Example 1: Formulation of the present invention Urease preparations were prepared using the components listed in Table 5 below, employing general methods 1 and 2.

[0071] [Table 6]

[0072] The formulations shown in Table 5 were sterilized and stored according to General Method 3. After an appropriate storage period, the urease performance was tested according to General Method 4. The results are shown in Table 6.

[0073] [Table 7]

[0074] By comparing the results in Tables 4 and 6, it is clear that formulations according to the present invention can effectively stabilize urease and ensure that its activity remains high during gamma ray sterilization at room temperature and subsequent long-term storage. In contrast, a comparison formulation that does not contain any non-reducing sugars, antioxidants, buffers, or nonionic surfactants exhibits inferior urea clearance after storage at room temperature, suggesting a much shorter shelf life.

[0075] Therefore, formulations according to the present invention are highly advantageous and solve the problem of the short shelf life of existing urease formulations for dialysis.

[0076] Example 2: Further formulations of the present invention Urease preparations were prepared using the components listed in Table 7 below, employing general methods 1 and 2. Urease preparations were prepared using the components listed in Table 5 below, employing general methods 1 and 2.

[0077] [Table 8]

[0078] The formulations shown in Table 7 were sterilized and stored according to General Method 3. After an appropriate storage period, the urease performance was tested according to General Method 4. The results are shown in Table 8.

[0079] [Table 9]

[0080] By comparing the results in Tables 4 and 8, it is clear that formulations according to the present invention can effectively stabilize urease and ensure that its activity level remains high during gamma ray sterilization at room temperature and subsequent long-term storage.

[0081] Therefore, formulations according to the present invention are highly advantageous and solve the problem of the short shelf life of existing urease formulations for dialysis.

Claims

1. Urease; Non-reducing sugars; Antioxidants; buffer; and Nonionic surfactants, A urease preparation suitable for use in dialysis, including the above.

2. The formulation, Activated carbon; and Zirconium hydrated oxide A urease preparation according to claim 1, further comprising:

3. The urease is provided in the form of urease immobilized on a solid support. A urease preparation according to claim 1 or claim 2.

4. The non-reducing sugar includes one or more types from the group consisting of trehalose and sucrose. Optionally, non-reducing sugars include sucrose. A urease preparation according to any one of the claims described above.

5. The antioxidant contains one or more of the following: glutathione, methionine, and ascorbic acid. Optionally, antioxidants, including glutathione, A urease preparation according to any one of the claims described above.

6. The buffer contains one or more types from the group consisting of histidine and phosphate buffer, and / or the buffer has a pK of 3 to 8. ａ Having; The buffer optionally contains either histidine and potassium phosphate, or both. More optionally, the buffer contains histidine and potassium phosphate. A urease preparation according to any one of the claims described above.

7. The nonionic surfactant includes one or both of polysorbate and poloxamer. Optionally, a nonionic surfactant, including polysorbate, A urease preparation according to any one of the claims described above.

8. Non-reducing sugars include sucrose; The antioxidant contains glutathione; The buffer solution contains histidine; and Nonionic surfactants include polysorbate. A urease preparation according to any one of the claims described above.

9. Non-reducing sugars include sucrose and trehalose; The antioxidant contains glutathione; The buffer solution contains potassium phosphate; and Nonionic surfactants include polysorbate. A urease preparation according to any one of claims 1 to 7.

10. The antioxidant comprises one or more types from the group consisting of reduced L-glutathione, reduced D-glutathione, oxidized L-glutathione, and oxidized D-glutathione. Optionally, the glutathione may include reduced L-glutathione or reduced D-glutathione. More optionally, glutathione, including reduced L-glutathione, A urease preparation according to any one of the claims described above.

11. The buffer solution contains one or more types from the group consisting of L-histidine and D-histidine. Optionally, the buffer may contain L-histidine. A urease preparation according to any one of the claims described above.

12. The nonionic surfactant contains polysorbate-80. A urease preparation according to any one of the claims described above.

13. Based on dry weight, the formulation contains approximately 0.5 wt% to approximately 10 wt% of non-reducing sugars (e.g., sucrose). Optionally, approximately 1 wt% to 7 wt% of non-reducing sugars, More optionally, approximately 1 wt% to 5 wt% of non-reducing sugars, A urease preparation according to any one of the claims, comprising:

14. Based on dry weight, the formulation contains approximately 0.001 wt% to approximately 5 wt% of an antioxidant (e.g., glutathione). Optionally, approximately 0.01 wt% to approximately 2 wt% of antioxidants, More optionally, approximately 0.05 wt% to approximately 1 wt% of antioxidants, A urease preparation according to any one of the claims, comprising:

15. Based on dry weight, the formulation contains approximately 10 wt% to 25 wt% immobilized urease. Optionally, approximately 14 wt% to approximately 20 wt% of immobilized urease, A urease preparation according to any one of the claims, comprising:

16. A urease preparation according to any one of the claims, wherein the nonionic surfactant comprises polysorbate and, based on dry weight, the preparation contains about 0.005 wt% to about 0.5 wt% of polysorbate (e.g., about 0.01 wt% to about 0.2 wt% of polysorbate).

17. The following conditions apply: (a) The buffer contains histidine, and based on dry weight, the formulation contains approximately 0.005 wt% to approximately 1 wt% histidine (e.g., approximately 0.01 wt% to approximately 0.3 wt% histidine); and (b) The buffer contains potassium phosphate and, based on dry weight, the formulation contains about 0.1 wt% to about 3 wt% potassium phosphate (for example, about 0.3 wt% to about 1.6 wt% potassium phosphate), A urease preparation according to any one of the claims described above, which is one or both of the above.

18. Based on dry weight, the formulation contains approximately 30 wt% to 45 wt% activated carbon. Optionally, approximately 34 wt% to approximately 38 wt% activated carbon. A urease preparation according to claim 2 and claims 3 to 17 dependent on claim 2, comprising the above.

19. Based on dry weight, the formulation contains approximately 35 wt% to 48 wt% zirconium oxide. Optionally, approximately 39 wt% to approximately 43 wt% of zirconium oxide, A urease preparation according to any one of claims 2 and any one of claims 3 to 18 dependent on claim 2, including the above.

20. The following: (a) The urease preparation further contains one or more proteins, optionally including albumin, such as bovine serum albumin (BSA); (b) The urease preparation further contains one or more chelating agents, and optionally one or more chelating agents contain ethylenediaminetetraacetic acid (EDTA); (c) The urease preparation provides urea clearance of more than 99% after sterilization using gamma radiation at 25-40 kGy, compared to urea clearance before sterilization using gamma radiation; and (d) The urease preparation provides a urea clearance of more than 99% after storage at 30°C for 3 months compared to the urea clearance before storage; A urease preparation according to any one of the above claims, which satisfies one or more of the above.

21. (i) Sterilization using gamma radiation at 25-40 kGy; and (ii) Subsequent storage at 30°C for 3 months. After that, It provides over 99% urea clearance compared to urea clearance before sterilization and preservation using gamma radiation. The preparation is optionally a sterile urease preparation. A urease preparation according to claim 2 and any one of claims 3 to 20 dependent on claim 2.

22. It is a freeze-dried urease preparation. A urease preparation according to any one of the above claims.

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