Sterile glutamine and its preparation method

Abstract

This invention relates to sterile glutamine and its preparation method, belonging to the field of biopharmaceuticals. The sterile glutamine has a purity of ≥98.0% and an angle of repose of ≤40°. The preparation method includes the following steps: (1) mixing solid glutamine with water; (2) filtering; (3) crystallizing by adding a sterile organic solvent in two stages; (4) crystal growth; (5) solid-liquid separation; and (6) vacuum drying to obtain the sterile glutamine. This invention also relates to the use of sterile glutamine in the preparation of pharmaceutical formulations. The sterile glutamine of this invention has a pharmaceutical-grade sterility level, and the preparation method of this invention can prepare sterile glutamine with high purity and good flowability on an industrial scale and in high yield.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceuticals. Specifically, this invention relates to a sterile glutamine, a method for preparing the same, and the use of the sterile glutamine in the preparation of formulations. Background Technology

[0002] Glutamine (molecular formula C5H) 10 N₂O₃ (molecular weight 146.15) is the amide form of glutamic acid and one of the 20 essential amino acids for the formation of human proteins. As an important amino acid analog, glutamine has wide applications in medicine, nutritional supplementation, and sports health. Glutamine can act as a gastric mucosal protectant, repairing mucosal tissue and eliminating ulcer lesions. Glutamine is essential for the secretion, proliferation, and functional maintenance of lymphocytes, thus enhancing immune function. Glutamine can also cross the blood-brain barrier, promoting brain metabolism and improving brain function, making it an important nutrient for brain metabolism. Furthermore, glutamine promotes muscle growth and enhances the body's antioxidant capacity. In particular, recent studies have shown that glutamine can also increase the sensitivity of drug-resistant bacteria to antibiotics, thereby reducing bacterial resistance. Given the deepening research, the health and therapeutic effects of glutamine are increasingly recognized, leading to a growing demand for glutamine that can be used in pharmaceutical preparations.

[0003] Currently, most commercially available glutamine is food-grade (e.g., conforming to GB29922), which does not have high sterility requirements and cannot meet the requirements for pharmaceutical raw materials / excipients. Furthermore, preparing sterile glutamine on an industrial scale is a significant challenge.

[0004] Given the long-standing lack of sterile glutamine products (especially sterile glutamine that meets the standards for injectable formulations) and their preparation methods in this field, researching and developing such products and methods is of great significance. Summary of the Invention

[0005] This invention is made to address the aforementioned problems existing in the field.

[0006] In a first aspect, this application relates to a sterile glutamine having a purity of ≥98.0% and an angle of repose of ≤40°.

[0007] In a second aspect, this application relates to a method for preparing sterile glutamine, comprising the following steps:

[0008] (1) Mix solid glutamine with water and dissolve it completely to obtain a clear liquid;

[0009] (2) Filter the liquid to obtain filtrate, wherein the filtration includes filtration using a filter element with a pore size of 0.22 μm or less;

[0010] (3) Add the following ingredients selected from C to the filtrate in stages as follows: 3-6 Ketones and C 1-6 One or more sterile organic solvents in alcohols cause crystals to precipitate:

[0011] First stage: Add sterile organic solvent to the filtrate at a flow rate of 1-2 liters per minute (L / min) m1, and

[0012] Second stage: Continue to add sterile organic solvent at a flow rate of 2.5-4 liters / minute m2, wherein the ratio of the volume of sterile organic solvent added in the first stage to the total volume added in the two stages is 0.3-0.7:1;

[0013] (4) Crystal growth;

[0014] (5) Separate the solid-liquid mixture obtained after crystal growth to obtain crystals;

[0015] (6) The crystals are dried under reduced pressure to obtain the sterile glutamine.

[0016] In a third aspect, this application relates to the use of the sterile glutamine of the present invention or the sterile glutamine obtained by the method of the present invention in the preparation of pharmaceutical formulations.

[0017] Compared with the prior art, the method of the present invention achieves the following beneficial effects:

[0018] 1. This invention provides a method suitable for the industrial-scale preparation of sterile glutamine. The method employs a two-stage addition of sterile organic solvent for crystallization, controlling the flow rate and volume of the sterile organic solvent in each stage, thereby efficiently preparing sterile glutamine solid with good flowability in high yield and high purity.

[0019] 2. The glutamine solid prepared by the method of the present invention can meet the sterility standards of drug raw materials / excipients and has good flowability, which is beneficial to quality control and improvement in the formulation production process.

[0020] 3. The method of the present invention avoids the use of high-temperature processes, which helps to reduce energy consumption and improve product quality. Detailed Implementation

[0021] The following details the embodiments of the purification and preparation methods of the compound of formula (I) of this application. However, unnecessary details may be omitted. For example, detailed descriptions of well-known matters and repetitive descriptions of actually identical structures may be omitted. This is to avoid making the following description unnecessarily lengthy and to facilitate understanding by those skilled in the art. Furthermore, the following description is provided to enable those skilled in the art to fully understand this application and is not intended to limit the subject matter of the claims.

[0022] The "range" disclosed in this application is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60-120 and 80-110 are listed for a specific parameter, it is expected that ranges of 60-110 and 80-120 are also included. Furthermore, if minimum range values ​​of 1 and 2 are listed, and if maximum range values ​​of 3, 4, and 5 are listed, then the following ranges are all expected: 1-3, 1-4, 1-5, 2-3, 2-4, and 2-5. In this application, unless otherwise stated, the numerical range "ab" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0-5" indicates that all real numbers between "0-5" have been listed in this article; "0-5" is simply a shortened representation of these numerical combinations. Furthermore, when a parameter is stated as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0023] Unless otherwise specified, all embodiments and optional embodiments of this application can be combined to form new technical solutions.

[0024] Unless otherwise specified, all technical features and optional technical features of this application can be combined to form new technical solutions. For example, features of the general scope and features of the preferred scope can be combined with each other.

[0025] Unless otherwise specified, all steps in this application may be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the mention that the method may also include step (c) indicates that step (c) may be added to the method in any order. For example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0026] The term “comprising” and its variations, such as “containing,” “including,” and “having,” as used herein are not intended to exclude other additives, components, or steps. They should be interpreted as open-ended rather than exclusive. Therefore, disclosures of embodiments using the terms “containing,” “including,” and “comprising” include disclosures of embodiments “consisting substantially of” and “consisting of.” The term “and / or” as used in the context of “X and / or Y” should be interpreted as “X” or “Y” or “X and Y.” Unless explicitly stated that alternatives are mutually exclusive, the term “or” as used herein refers to “and / or.”

[0027] The present invention relates in a first aspect to a sterile glutamine having a purity of ≥98.0% and an angle of repose of ≤40°.

[0028] The sterile glutamine of this invention meets the definition of "sterile" in the Chinese Pharmacopoeia. Specifically, the term "sterile" means that the sterile glutamine prepared according to this invention is tested according to the requirements of the sterility test method in General Chapter 1101 and the bacterial endotoxin test method in General Chapter 1143 of the Chinese Pharmacopoeia (Part IV), and the test results meet the requirements.

[0029] The sterile glutamine of this invention has high purity. In this document, "purity" refers to the percentage of the target substance glutamine based on the total weight of the dried product. In this document, "dried product" means a product dried to a weight loss of ≤2%.

[0030] The sterile glutamine of this invention exhibits good flowability, which is beneficial for quality control in pharmaceutical formulation processes. The term "flowability" as used herein refers to the flowability of solid glutamine (powder). Generally, the flowability of solid powder is characterized by the "angle of repose (θ)," measured in degrees (°). The term "angle of repose" as used herein refers to the angle formed between a free inclined plane formed by powder accumulation in a static, equilibrium state and a horizontal plane. The angle of repose can be determined using various conventional methods, such as, but not limited to, the fixed cone method, the fixed funnel method, the tilting box method, and the rotating cylinder method; the fixed cone method is commonly used. It is generally considered that θ ≤ 40° is sufficient to meet the flowability requirements in the production process (Han Li, ed., *Pharmaceutics Experiments*, 2020 edition, p. 176).

[0031] In some embodiments, the water content of the sterile glutamine is less than 0.5%, preferably less than 0.2%, based on the weight of the sterile glutamine.

[0032] In some embodiments, the sterile glutamine of the present invention is sterile L-glutamine.

[0033] This application relates to a method for preparing sterile glutamine, which includes the following steps:

[0034] (1) Mix solid glutamine with water and dissolve it completely to obtain a clear liquid;

[0035] (2) Filtering the liquid to obtain a filtrate, wherein the filtration includes using a filter with a pore size of 0.22 mm.

[0036] Filtration is performed using filter cartridges with a filter size of μm and below;

[0037] (3) Add the following ingredients selected from C to the filtrate in stages as follows: 3-6 Ketones and C 1-6 One or more sterile organic solvents in alcohols cause crystals to precipitate:

[0038] First stage: Add the sterile organic solvent to the filtrate at a flow rate of 1-2 liters / minute (m1), and...

[0039] Second stage: Continue adding the sterile organic solvent at a flow rate of 2.5-4 liters / minute (m2).

[0040] The ratio of the volume of sterile organic solvent added in the first stage to the total volume added in both stages is 0.3-0.7:1;

[0041] (4) Crystal growth;

[0042] (5) Separate the solid-liquid mixture obtained after crystal growth to obtain crystals;

[0043] (6) The crystals are dried under reduced pressure to obtain the sterile glutamine.

[0044] Unrestricted by any particular theory, the method of this invention can produce sterile glutamine with high yield, high purity, and good flowability on an industrial scale with high production efficiency. Specifically, the sterile glutamine obtained by the method of this invention meets the sterility standards for pharmaceutical preparations. In particular, the method of this invention, due to the two-stage operation in the crystallization step (3), allows for the controlled precipitation of sterile glutamine crystals, which particularly helps to improve the flowability of the product.

[0045] Furthermore, the method of this invention can efficiently prepare sterile glutamine in kilogram-scale quantities to meet the raw / excipient requirements in actual pharmaceutical production. Simultaneously, the method of this invention avoids high-temperature processes (e.g., high-temperature sterilization, high-temperature drying), which not only reduces energy consumption and production costs but also helps reduce undesirable impurities for pharmaceutical formulations, such as glutamic acid and pyroglutamic acid.

[0046] In this invention, the glutamine solid used to prepare sterile glutamine can be glutamine solid from any source; for example, it can be commercially available glutamine solid (e.g., but not limited to commercially available food-grade glutamine solid), or glutamine solid prepared by processes such as fermentation.

[0047] In some embodiments, in step (1), the water may be purified water, preferably water for injection. Hereinafter, the term "water for injection" refers to water that conforms to the specifications for water for injection in the Chinese Pharmacopoeia. It can generally be water obtained by distillation of distilled or deionized water (also known as double-distilled water). In some cases, the water for injection is preferably water in which microbial contamination is effectively controlled and the level of bacterial endotoxins is also controlled.

[0048] In some embodiments, in step (1), the dissolution is carried out at a temperature of 20-60°C, preferably 30-50°C, and more preferably 35-50°C. Dissolving solid glutamine at such temperatures results in a rapid and complete dissolution, which is beneficial for improving production efficiency. Furthermore, such temperatures prevent the thermal decomposition of the raw material glutamine, avoiding the generation of harmful impurities.

[0049] In some embodiments, in step (1), mixing the glutamine solid with water (i.e., adding the glutamine solid to water and mixing it) can be carried out at a temperature of 15-30°C, preferably 18-30°C, and more preferably 20-28°C. In this invention, this temperature is not strictly controlled; the addition and mixing can be carried out under normal room temperature conditions. For example, the glutamine solid can be added to water and mixed at a temperature of 18-30°C. After mixing the glutamine solid with water, the system can be maintained at this temperature or appropriately heated (as described above at the dissolution temperature) to facilitate faster dissolution of the added glutamine solid in the water and improve production efficiency.

[0050] In some embodiments, in step (1), the weight ratio of the glutamine solid to the volume of the water, in kg / L, is 1:5-50, preferably 1:10-35, and more preferably 1:15-30. Mixing the glutamine solid with water in an appropriate ratio not only ensures the complete dissolution of the glutamine solid but also facilitates the crystallization process in step (3) and improves production efficiency.

[0051] In some embodiments, the dissolution in step (1) can be performed in conjunction with any operation that may accelerate dissolution. Such an operation is conventionally selected by those skilled in the art based on the actual situation. Stirring is preferred. In this invention, the stirring rate, stirring equipment, and / or stirring method are conventionally selected by those skilled in the art.

[0052] In some embodiments, in step (2), the filtration includes a first-stage filtration and a last-stage filtration arranged sequentially along the flow direction of the liquid, wherein the first-stage filtration uses a filter element with a pore size of 0.22-0.8 μm, preferably 0.3-0.6 μm, more preferably 0.45 μm, and the last-stage filtration uses a filter element with a pore size of 0.22 μm or less, preferably 0.22 μm. In some embodiments, the filtration includes two or more stages of filtration. In some embodiments, the filtration includes three stages of filtration arranged sequentially along the flow direction of the liquid, wherein the liquid undergoes first-stage filtration, second-stage filtration, and third-stage filtration sequentially, and the second-stage filtration uses a filter element with a pore size of 0.3 μm or less, preferably 0.25 μm or less, more preferably 0.22 μm or less, more preferably 0.22 μm.

[0053] In this article, the terms "filter element" or "pore size" or other similar terms refer to the filter's ability to retain microorganisms, rather than the distribution coefficient of the average pore size.

[0054] In some embodiments, in step (2), the filtration uses a microporous membrane. Preferably, the microporous membrane is a polyvinyl chloride microporous membrane, a polytetrafluoroethylene microporous membrane, a polyvinylidene fluoride microporous membrane (PVDF), or other microporous membranes resistant to organic solvents.

[0055] In some implementations, during the filtration process in step (2), the temperature of the liquid to be filtered can be maintained at the dissolution temperature in step (1) described above (i.e., step (2) can be performed at the dissolution temperature in step (1)).

[0056] As the sterile organic solvent in step (3) of the method of the present invention, C 1-6 The alcohol is preferably selected from one or more of methanol, ethanol, propanol, n-propanol, and isopropanol; more preferably, ethanol. 3-6 Ketones can be, for example, acetone. Although ketone solvents can achieve similar yields and purities as alcohol solvents, ethanol is preferred, especially for cost reasons.

[0057] As described above, to obtain a product with satisfactory yield, purity, and flowability, sterile organic solvent needs to be added in two stages in step (3) of the method of the present invention—the crystallization step—to precipitate sterile glutamine crystals. In the first stage, sterile organic solvent is added to the filtrate at a flow rate m1 of 1-2 L / min, while in the second stage, the remaining sterile organic solvent is added at a flow rate m2 of 2.5-4 L / min until the desired total volume is reached. In some embodiments, it is preferred that the sterile organic solvent is added in the first stage at a flow rate m1 of 1-1.5 L / min. In some embodiments, it is preferred that the remaining sterile organic solvent is added in the second stage at a flow rate m2 of 2.5-3 L / min. In the method of the present invention, the control of the flow rate m1 in the first stage is crucial, as it significantly affects the flowability of the final product, solid glutamine.

[0058] In some implementations, the ratio of the flow rate m1 in the first stage to the flow rate m2 in the second stage is 0.25-0.7:1, preferably 0.3-0.67:1. Most preferably, the ratio of the flow rate m1 in the first stage to the flow rate m2 in the second stage is 0.3-0.4:1. The inventors have found that this is more conducive to obtaining products with better yield, purity, and flowability.

[0059] In the method of this invention, the volume of the sterile organic solvent added in the first stage also has a significant impact on the flowability of the product. As mentioned above, the ratio of the volume of the sterile organic solvent added in the first stage to the total volume of sterile organic solvent can be in the range of 0.3-0.7:1, which can improve the production efficiency of the method and is also beneficial to obtaining a product with good flowability. In some embodiments, the ratio of the volume of the sterile organic solvent added in the first stage to the total volume of added sterile organic solvent is preferably 0.33-0.67:1, more preferably 0.33-0.56:1.

[0060] In some embodiments, the ratio of the total volume of the sterile organic solvent added in step (3) to the volume of water in step (1) is 0.7-1.5:1, preferably 0.8-1.3:1, more preferably 0.9-1.2:1, and even more preferably 1:1. A suitable ratio of the total volume of the organic solvent to the volume of water is beneficial for obtaining sterile glutamine with high yield and high purity; at the same time, the selection of the above range also takes into account cost factors, because adding a larger amount of organic solvent does not substantially improve purity and yield.

[0061] In some embodiments, step (3) is performed at a temperature of 10-35°C, preferably 10-33°C, and more preferably 12-32°C. This temperature may also be referred to herein as the "crystallization temperature." In some embodiments, step (3) is preferably performed at a temperature of 5-10°C. Lower temperatures are more conducive to crystallization.

[0062] In this invention, the temperatures of the two-stage addition of sterile organic solvent in step (3) can be the same or different, as long as they are within the crystallization temperature range described above. In some embodiments, it is preferred that the temperatures of the two stages be the same.

[0063] In some implementations, step (3) may be performed under stirring. The stirring rate may be, for example, 60-100 revolutions per minute (rpm).

[0064] In some embodiments, there may be an interval (or period) of 0.25-0.5 hours between the first and second stages in step (3). This interval period may also be referred to herein as the "interval phase". Inserting an interval period, particularly within the range described above, between the two solvent addition stages allows for slight crystal growth of the sterile glutamine crystals formed after the solvent addition in the first stage, before the second stage solvent addition. This allows for controlled crystallization of the sterile glutamine, thereby improving the flowability of the product. Furthermore, the selection of the interval period in this invention also considers the production efficiency of the method; while a suitable extension of the interval period is advantageous, an excessively long interval reduces industrial production efficiency, while an excessively short interval is detrimental to obtaining a product with good flowability.

[0065] During the intermittent phase, the system temperature does not need to be specially controlled, as long as it is within the crystallization temperature range described above; for example, the intermittent phase can be carried out at the same temperature as the first phase.

[0066] As described above, in step (3) of the present invention, the sterile organic solvent is added in two stages and the flow rate and volume of the sterile organic solvent in the two stages are controlled respectively, as well as the interval time between the two stages. This can effectively control the crystallization process of sterile glutamine, resulting in a high yield, high purity, and good fluidity of the obtained sterile glutamine crystals.

[0067] Preferably, in step (4), the crystal growth is carried out at a temperature of 3-15°C, more preferably 5-10°C, and even more preferably 5-8°C. After the crystallization step in step (3) is completed, the system temperature can be lowered to carry out crystal growth, which is beneficial to obtaining uniform crystals with high yield and high purity, and helps to improve the flowability of the obtained product.

[0068] In this article, the term "crystallization" refers to the process of allowing the seed crystals produced in the crystallization step to naturally crystallize in the solution in order to produce crystals with ideal particle size distribution and good fluidity.

[0069] Preferably, in step (4), the crystal growth time can be 1-5 hours, more preferably 1-3 hours. Controlling the crystal growth time helps improve the yield and purity of sterile glutamine while also taking into account production efficiency.

[0070] In this document, the term "solid-liquid separation" refers to the process of separating the crystals from the liquid portion of a solid-liquid mixture (containing the desired crystals and a liquid portion, such as a mixture of solvent and water) obtained after a crystal growth step to obtain the target crystals. In this invention, the solid-liquid separation can employ any conventional solid-liquid separation method known in the art, such as, but not limited to, filtration, centrifugation, or sedimentation.

[0071] In some embodiments, after solid-liquid separation, step (5) may include a crystal washing step to further reduce the adhesion of mother liquor to the crystals, thereby improving product quality. The crystal washing step may include one or more washing cycles. The number of washing cycles can be conventionally determined by those skilled in the art. In this invention, there are no particular limitations on the washing liquid used in the washing step, and it can be conventionally determined by those skilled in the art. For example, an alcohol solvent or a ketone solvent, or a solution of one or more alcohols and / or ketones mixed in any proportion, can be used. Preferably, the alcohol solvent can be, for example, but not limited to, methanol, ethanol, propanol, n-propanol, or isopropanol; the ketone solvent can be, for example, but not limited to, acetone. In some embodiments, preferably, the washing liquid is ethanol or acetone. The volume of the washing liquid used is also conventionally determined by those skilled in the art.

[0072] In this document, in step (6), the vacuum drying can be performed using any conventional vacuum drying method and equipment known in the art.

[0073] In some embodiments, the sterile glutamine crystals obtained in step (5) can be dried under reduced pressure at a temperature of 30-80°C, preferably 30-50°C, and more preferably 30-40°C. Avoiding high temperatures helps reduce the decomposition of glutamine and the generation of impurities, which is beneficial for controlling product quality and is more suitable for the preparation of raw materials and excipients for pharmaceutical applications; moreover, drying at such temperatures consumes less energy and is more suitable for industrial production.

[0074] In some embodiments, the glutamine solid can be any commercially available glutamine solid, preferably food-grade glutamine solid. In some embodiments, the glutamine in the sterile glutamine of the present invention or the sterile glutamine prepared by the method of the present invention is L-glutamine.

[0075] In the method of the present invention, at least steps (3)-(6) are performed under aseptic conditions. Herein, "aseptic conditions" means a state in which no pathogenic microorganisms or other pathogens are present in the environment. In some embodiments, steps (1)-(6) of the method of the present invention can all be performed under aseptic conditions.

[0076] The present invention also relates to a sterile glutamine obtained by the method of the present invention.

[0077] In a third aspect, this application also relates to the use of sterile glutamine in the preparation of pharmaceutical formulations. The pharmaceutical formulations described in this invention are particularly injectable formulations.

[0078] In addition, this application also relates to a pharmaceutical preparation comprising the sterile glutamine of the present invention or sterile glutamine obtained by the method of the present invention.

[0079] The method of the present invention will now be described and illustrated in more detail with reference to specific embodiments. Those skilled in the art will understand that these embodiments are provided for illustrative purposes only and do not constitute any limitation on the scope of the present invention.

[0080] Example

[0081] I. Raw Materials and Reagents

[0082] In the following embodiments, all raw materials and reagents used were commercially available, and all equipment used was conventional equipment in the art.

[0083] II. Evaluation and Determination Methods for Physicochemical Properties

[0084] 1. Sterility test

[0085] The assay was performed using the methods described in Part IV of the 2020 edition of the Chinese Pharmacopoeia: <1101 Sterility Test Method> and <1143 Bacterial Endotoxin Test Method>.

[0086] a. Aseptic testing method

[0087] The membrane filtration method described in Part IV, 1101 Sterility Test, of the 2020 edition of the Chinese Pharmacopoeia was used for the determination. Thioglycolate fluid culture medium and tryptic soy peptone liquid culture medium used for sterility testing should meet the requirements for sterility and sensitivity testing of the culture medium.

[0088] Sterility test of the test sample: Take a specified amount of the test sample (e.g., sterile glutamine), dissolve it in water for bacterial endotoxin testing, filter it with water for bacterial endotoxin testing (the water for bacterial endotoxin testing should meet the standards for sterile water for injection) to wet the filter membrane, then filter the test sample solution directly. Next, rinse the filter membrane three times with water for bacterial endotoxin testing, using the same rinsing volume and method as the suitability test method in Part IV <1101 Sterility Test Method> of the 2020 edition of the Chinese Pharmacopoeia. Then, add 100 ml of thioglycolate fluid medium to one filter and 100 ml of tryptic soy peptone liquid medium to another filter. Incubate the above filters separately at the temperatures specified for each medium for no less than 14 days. If no sterile growth is observed in any of the test tubes (specifically, all test tubes are clear, or although they appear turbid, sterile growth is confirmed), the test sample is deemed to comply with the regulations; if any of the test tubes appears turbid and bacterial growth is confirmed, the test sample is deemed to fail to comply with the regulations, unless it can be sufficiently proven that the test results are invalid, i.e., the growing microorganisms are not contained in the test sample.

[0089] Positive control: Staphylococcus aureus was used as the control bacterium. The preparation of the bacterial suspension for the positive control test was the same as the suitability test method in Part IV, <1101 Sterility Test Method> of the 2020 edition of the Chinese Pharmacopoeia. The bacterial load should not exceed 100 CFU, and the amount of test sample used should be the same as the sample amount inoculated per culture medium during the sterility test of the test sample mentioned above. The positive control tubes should be incubated for no more than 5 days and should show good growth.

[0090] Negative control: Take an appropriate amount of water for bacterial endotoxin testing and perform aseptic testing as required for the test sample. No bacterial growth should be observed in the negative control.

[0091] b. Bacterial endotoxin test

[0092] The determination was performed using the gel method described in Section 1143, Test Method for Bacterial Endotoxins, of the 2020 edition of the Chinese Pharmacopoeia, Volume IV, with the test method employing the gel limit test. The test results should meet the specified requirements.

[0093] 2. Moisture content determination

[0094] The water content was determined using the first method (Fischer method) described in the Chinese Pharmacopoeia 2020 edition, Part IV, <0832 Determination of Moisture>. A moisture content was directly determined using a moisture analyzer: an appropriate amount of the sample was accurately weighed and placed in a dry, stoppered conical flask. An appropriate amount of anhydrous methanol was added, and the solution was titrated with Fischer reagent while continuously shaking (or stirring) until the solution changed from pale yellow to reddish-brown. A blank test was also performed, and the moisture content was calculated using the following formula:

[0095]

[0096] In the formula

[0097] A represents the volume of Fischer solution consumed by the test sample, in ml;

[0098] B represents the volume of Fischer solution consumed in the blank, in ml;

[0099] F represents the weight of water equivalent to 1 ml of Fischer solution, in mg.

[0100] W represents the weight of the test sample, in mg.

[0101] 3. Particle size distribution determination

[0102] The determination was performed using the dry method described in the third method (light scattering method) of the Chinese Pharmacopoeia 2020 edition, Part IV, <0982 Determination of Particle Size and Particle Size Distribution>.

[0103] 4. Angle of repose measurement

[0104] The determination was performed using the fixed cone method described in the textbook "Pharmaceutics Experiments" (edited by Han Li, 2020 edition, page 177).

[0105] Example 1

[0106] Add 6 kg of glutamine to 120 L of water for injection while stirring at room temperature (approximately 26°C). Heat to 35-40°C and stir until the glutamine dissolves, obtaining a solution. Filter this solution through a three-stage filter (pore sizes of 0.45 μm, 0.22 μm, and 0.22 μm respectively), and wash the filter with a small amount of water for injection. Combine the filtrate and washings, stir, and cool to 15-17°C. While stirring (at a rate of approximately 60-100 rpm), add 40 L of anhydrous ethanol at a flow rate (m1) of 1 L / min. After a 30-minute interval, continue adding the remaining anhydrous ethanol at a flow rate (m2) of 2.5 L / min until the total volume of anhydrous ethanol added is 120 L. Then, cool the system to 0-5°C and allow crystals to grow for 1 hour. Filter, and vacuum dry the solid at 30-45°C. Dry until the product's weight loss on drying is ≤2%, obtaining sterile glutamine. The sterility and bacterial endotoxin tests of the obtained sterile glutamine met the requirements. Other test results are summarized in Table 1.

[0107] Example 2

[0108] Add 6 kg of glutamine to 140 L of water for injection at room temperature (approximately 26°C) with stirring, and stir until the glutamine dissolves. Filter the solution using a three-stage filter (filter pore sizes of 0.45 μm, 0.22 μm, and 0.22 μm, respectively), and wash the filter with a small amount of water for injection. Combine the filtrate and washings, stir, and cool to 25-30°C. While stirring (at a stirring speed of approximately 60-100 rpm), first add 100 L of anhydrous acetone at a flow rate of 2 L / min (m1), pause for 15 minutes, and then continue adding the remaining anhydrous acetone at a flow rate of 4 L / min (m2) until the total volume of anhydrous acetone added is 180 L. Then, cool the system to 10-15°C and allow crystals to grow for 2 hours. Filter, and dry the solid under vacuum at a temperature of 45-55°C. The product was dried until the loss on drying was ≤2%. The sterility and bacterial endotoxin tests of the obtained sterile glutamine met the requirements. Other test results are summarized in Table 1.

[0109] Example 3

[0110] Add 6 kg of glutamine to 100 L of water for injection at room temperature (approximately 26°C) with stirring. Heat to 40-50°C and stir until the glutamine dissolves. Filter the solution using a three-stage filter (pore sizes of 0.45 μm, 0.22 μm, and 0.22 μm), and wash the filter with a small amount of water for injection. Combine the filtrate and washings, stir, and cool to 20-25°C. While stirring (at a rate of approximately 60-100 rpm), first add 50 L of anhydrous ethanol at a flow rate of 2 L / min, pause for 15 minutes, then continue adding the remaining anhydrous ethanol at a flow rate of 3 L / min until the total volume of anhydrous ethanol added is 80 L. Then, cool the system to 5-10°C and allow crystals to grow for 3 hours. Filter, and vacuum dry the solid at 45-50°C. Dry until the product's loss on drying is ≤2%, obtaining sterile glutamine. The sterility and bacterial endotoxin tests of the obtained sterile glutamine met the requirements. Other test results are summarized in Table 1.

[0111] Example 4

[0112] The preparation method in this embodiment is basically the same as that in Example 1, except that the interval between the two ethanol addition stages is 0.25 hours. The sterility and bacterial endotoxin tests of the obtained sterile glutamine both met the requirements. Other test results are summarized in Table 1.

[0113] Example 5

[0114] The comparative example was essentially the same as Example 1, except that the volume of ethanol added in the first stage was 60 L. The sterility and bacterial endotoxin tests of the obtained sterile glutamine met the requirements. Other test results are summarized in Table 1.

[0115] Example 6

[0116] The comparative example was essentially the same as Example 1, except that the volume of ethanol added in the first stage was 80 L. The sterility and bacterial endotoxin tests of the obtained sterile glutamine both met the requirements. Other test results are summarized in Table 1.

[0117] Comparative Example 1

[0118] This comparative example is essentially the same as Example 1, except that: instead of adding sterile organic solvent ethanol in stages, all ethanol (total volume 90L) is continuously added at a flow rate of 1L / min. The test results are summarized in Table 1.

[0119] Comparative Example 2

[0120] The preparation method in this embodiment is basically the same as that in Example 1, except that the interval between the two ethanol addition stages is 0.1 hours. The sterility and bacterial endotoxin tests of the obtained sterile glutamine both met the requirements. Other test results are summarized in Table 1.

[0121] Comparative Example 3

[0122] The comparative example is basically the same as Example 1, except that the flow rate m1 in the first stage is 3 L / min and the flow rate m2 in the second stage is 2 L / min (that is, m1 and m2 are not within the scope of this invention, and m1>m2). The test results are summarized in Table 1.

[0123] Comparative Example 4

[0124] The comparative example is basically the same as Example 1, except that the volume of ethanol added in the first stage is 30L. The test results are summarized in Table 1.

[0125] Comparative Example 5

[0126] The comparative example is basically the same as Example 1, except that the volume of ethanol added in the first stage is 96L. The test results are summarized in Table 1.

[0127] As shown in Table 1 below, by adding sterile organic solvent in stages and controlling the solvent flow rate and solvent volume ratio in the two stages, the method of the present invention can obtain sterile glutamine with improved flowability in high yield and high purity.

[0128]

Claims

1. A process for preparing sterile glutamine, comprising the steps of: (1) mixing glutamine solids with water and dissolving completely to obtain a clear feed solution; (2) filtering the feed solution to obtain a filtrate, wherein the filtering comprises filtering with a filter having a pore size of 0.22 μm or less; (3) at 10-35°C, under stirring, the filtrate is added, in stages, with one or more sterile organic solvents selected from C 3-6 ketones and C 1-6 alcohols, in such a way that the crystals precipitate: a first stage of adding the sterile organic solvent to the filtrate at a flow rate ml of 1-1.5 L / min, and a second stage of continuing to add the sterile organic solvent at a flow rate m2 of 2.5-3 L / min, wherein the ratio of the volume of sterile organic solvent added in the first stage to the total volume added in both stages is 0.33-0.67:1, and wherein the ratio of the total volume of sterile organic solvent added to the volume of water in step (1) is 0.7-1.5:1, wherein the ratio of ml to m2 is 0.25-0.7:1, wherein there is an intermittent period of 0.25-0.5 hours between the first and second stages; (4) seeding at 3-15 °C for 1-5 hours; (5) subjecting the solid-liquid mixture obtained after seeding to solid-liquid separation to obtain crystals; (6) drying the crystals under reduced pressure to obtain the sterile glutamine.

2. The process according to claim 1, wherein step (3) has at least one of the following characteristics: a. the ratio of ml to m2 is 0.3-0.67:1; b. the ratio of the volume of sterile organic solvent added in the first stage to the total volume of sterile organic solvent is 0.33-0.56:1; c. the ratio of the total volume of sterile organic solvent added in step (3) to the volume of water in step (1) is 0.8-1.3:1; d. step (3) is carried out at a temperature of 10-33 °C; e. the stirring rate in step (3) is 60-100 rpm.

3. The method of claim 2, wherein, the ratio of ml to m2 is 0.3-0.4:

1.

4. The method of claim 2, wherein, the ratio of the total volume of sterile organic solvent added in step (3) to the volume of water in step (1) is 0.9-1.2:

1.

5. The method of claim 2, wherein, step (3) is carried out at a temperature of 12-32 °C.

6. The process according to claim 1 or 2, wherein step (1) has at least one of the following characteristics: a. the dissolving is carried out at a temperature of 20-60 °C; b. the mixing of glutamine solids with water is carried out at a temperature of 15-30 °C.

7. The method of claim 6, wherein, the dissolving is carried out at a temperature of 30-50 °C.

8. The method of claim 7, wherein, the dissolving is carried out at a temperature of 35-50 °C.

9. The method of claim 6, wherein, the mixing of glutamine solids with water is carried out at a temperature of 18-30 °C.

10. The method of claim 9, wherein, the mixing of glutamine solids with water is carried out at a temperature of 20-28 °C.

11. The process according to claim 1 or 2, wherein in step (2), the filtering comprises a first stage of filtering and a last stage of filtering arranged in sequence along the flow direction of the feed solution, wherein the first stage of filtering employs a filter having a pore size of 0.22-0.8 μm, and the last stage of filtering employs a filter having a pore size of 0.22 μm or less.

12. The process according to claim 11, wherein the first stage of filtering employs a filter having a pore size of 0.3-0.6 μm.

13. The method according to claim 12, wherein the first stage filtration employs a filter having a pore size of 0.45 μιη.

14. The method according to claim 11, wherein the last stage filtration employs a filter having a pore size of 0.22 μιη.

15. The method according to claim 11, wherein the filtration comprises two or more stages of filtration.

16. The method according to claim 15, wherein the filtration comprises three stages of filtration arranged in sequence along the flow direction of the feed liquid, wherein the feed liquid is subjected to the first stage filtration, the second stage filtration and the third stage filtration in sequence, and the second stage filtration employs a filter having a pore size of 0.3 μιη or less.

17. The method according to claim 16, wherein the second stage filtration employs a filter having a pore size of 0.25 μιη or less.

18. The method according to claim 17, wherein the second stage filtration employs a filter having a pore size of 0.22 μιη or less.

19. The method according to claim 18, wherein the second stage filtration employs a filter having a pore size of 0.22 μιη.

20. The method according to claim 11, wherein step (2) is performed at a temperature maintained at the dissolving temperature of step (1).

21. The method according to claim 1 or 2, wherein step (4) has at least one of the following features: a. the seeding is performed at a temperature of 5-10 °C; b. the seeding time is 1-3 hours.

22. The method of claim 21, wherein, the seeding is performed at a temperature of 5-8 °C.

23. The method according to claim 1 or 2, wherein in step (6) the reduced pressure drying is performed at a temperature of 30-80 °C.

24. The method according to claim 23, wherein the reduced pressure drying is performed at a temperature of 30-50 °C.

25. The method according to claim 24, wherein the reduced pressure drying is performed at a temperature of 30-40 °C.

26. The method according to claim 1 or 2, wherein the glutamine is L-glutamine.

27. Use of the sterile glutamine obtained by the method of any one of claims 1-26 for the preparation of a formulation for injection; wherein the sterile glutamine has a purity of > 98.0% and a static angle of < 40°.

28. The use according to claim 27, wherein the sterile glutamine has at least one of the following features: a. a water content of less than 0.5%; and b. the glutamine is L-glutamine.

29. The use of claim 28, wherein, a water content of less than 0.2%.

Images