Sterile glutamine and preparation method thereof

Abstract

The invention relates to sterile glutamine and a preparation method thereof, and belongs to the field of biological pharmacy, the sterile glutamine has the purity of more than or equal to 98.0% and the angle of repose of less than or equal to 40 degrees, the preparation method comprises the following steps: (1) mixing glutamine solid and water, (2) filtering, (3) crystallizing in a manner of adding a sterile organic solvent in two stages, (4) growing the crystal, and (5) carrying out solid-liquid separation, and (6) drying under reduced pressure to obtain the sterile glutamine. The invention also relates to the use of sterile glutamine for the preparation of a pharmaceutical formulation. The sterile glutamine disclosed by the invention has a sterile level of a medicinal grade, and the preparation method disclosed by the invention can be used for preparing the sterile glutamine with high purity and better flowability on an industrial scale and at high yield.

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceuticals. Specifically, the present invention relates to sterile glutamine, a preparation method thereof, and use of the sterile glutamine in preparing preparations. Background Art

[0002] Glutamine (molecular formula is C5H 10 N2O3, molecular weight of 146.15), is the amide form of glutamic acid and one of the 20 essential amino acids that constitute human protein. Glutamine, as an important amino acid analog, is widely used in the fields of medicine, nutritional supplements, sports health care, etc. Glutamine can be used as a gastric mucosal protectant to repair mucosal tissue and eliminate ulcer lesions. Glutamine is a substance necessary for lymphocyte secretion and proliferation and maintenance of its function, so it can enhance immune function. Glutamine can also pass through the blood-brain barrier, promote brain metabolism, improve brain function, and is an important nutrient for brain metabolism. In addition, glutamine also has the effect of promoting muscle growth and improving the body's antioxidant capacity. In particular, the latest research also shows that glutamine can also increase the sensitivity of drug-resistant bacteria to antibiotics, thereby reducing bacterial resistance. In view of the in-depth research, the health and therapeutic effects of glutamine are increasingly recognized, so the demand for glutamine that can be used to prepare pharmaceutical preparations in the field is increasing.

[0003] Currently, most commercially available glutamine is food grade (e.g., in accordance with GB29922), which does not require high sterility and cannot meet the requirements of raw / excipient materials for pharmaceutical preparations. In addition, it is a great challenge to prepare sterile glutamine on an industrial scale.

[0004] In view of the fact that there has long been a lack of sterile glutamine (especially sterile glutamine that meets the standards for injection preparations) products and preparation methods in the art, it is of great significance to research and develop such products and methods. Summary of the invention

[0005] The present invention is made in view of the above problems existing in the art.

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

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

[0008] (1) mixing solid glutamine with water and completely dissolving the glutamine to obtain a clear liquid;

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

[0010] (3) Adding selected from C to the filtrate in stages in the following manner 3-6 Ketone and C 1-6 One or more sterile organic solvents in alcohol to precipitate crystals:

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

[0012] The 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 the 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) performing solid-liquid separation on the solid-liquid mixture obtained after crystal growing to obtain crystals;

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

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

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

[0018] 1. The present invention provides a method for preparing sterile glutamine on an industrial scale. The method of the present invention adopts a two-stage addition of a sterile organic solvent for crystallization, and controls the flow rate and volume of the sterile organic solvent in each stage, so that sterile glutamine solid with good fluidity can be prepared efficiently with 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 fluidity, which is beneficial to quality control and improvement in the preparation production process.

[0020] 3. The method of the present invention avoids the use of high-temperature processes, which is beneficial to reducing energy consumption and improving product quality. DETAILED DESCRIPTION

[0021] The following is a detailed description of the purification and preparation methods of the compound of formula (I) of the present application. However, there may be cases where unnecessary detailed descriptions are omitted. For example, there are cases where the detailed description of well-known matters and the repeated description of the actual same structure are omitted. This is to avoid the following description from becoming unnecessarily lengthy and to facilitate the understanding of those skilled in the art. In addition, the following description is provided for those skilled in the art to fully understand the present application and is not intended to limit the subject matter described in the claims.

[0022] "Scope" disclosed in the present application is limited in the form of lower limit and upper limit, and a given range is limited by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundary of a special range. The scope limited in this way can be including end values ​​or not including end values, and can be arbitrarily combined, that is, any lower limit can be combined with any upper limit to form a scope. For example, if the scope of 60-120 and 80-110 is listed for a specific parameter, it is understood that the scope of 60-110 and 80-120 is also expected. In addition, if the minimum range values ​​1 and 2 are listed, and if the maximum range values ​​3, 4 and 5 are listed, the following scope can be all expected: 1-3, 1-4, 1-5, 2-3, 2-4 and 2-5. In the present application, unless otherwise specified, the numerical range "ab" represents the abbreviation of any real number combination between a and b, wherein a and b are real numbers. For example, the numerical range "0-5" means that all real numbers between "0-5" are listed in this document, and "0-5" is just an abbreviation of these numerical combinations. In addition, when a parameter is expressed as an integer ≥ 2, it is equivalent to disclosing that the parameter is, for example, an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0023] Unless otherwise specified, all embodiments and optional embodiments of the present application can be combined with each other to form a new technical solution.

[0024] If there is no special description, all technical features and optional technical features of the present application can be combined with each other to form a new technical solution. For example, features in the general range and features in the preferred range can be combined with each other.

[0025] If there is no special explanation, all steps of the present application can be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), which means that the method may include steps (a) and (b) performed sequentially, or may include steps (b) and (a) performed sequentially. For example, the method may further include step (c), which means that step (c) may be added to the method in any order. For example, the method may include steps (a), (b) and (c), or may include steps (a), (c) and (b), or may include steps (c), (a) and (b), etc.

[0026] As used herein, the term "comprising" and variations of the term, such as "containing," "including," and "having," are not intended to exclude other additives, components, or steps. They should be interpreted as open-ended rather than exclusive. Thus, disclosures of embodiments using the terms "containing," "including," and "comprising" include disclosures of embodiments "consisting essentially of" and "consisting of." The term "and / or" used in the context of "X and / or Y" should be interpreted as "X" or "Y" or "X and Y." Unless it is expressly stated that the alternatives are mutually exclusive, the use of the term "or" herein is intended to mean "and / or."

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

[0028] The sterile glutamine of the present invention complies with the "sterile" provisions of the Chinese Pharmacopoeia. Specifically, the term "sterile" means that the sterile glutamine prepared by the present invention is tested according to the test requirements of General Rule 1101 Sterility Test Method and General Rule 1143 Bacterial Endotoxin Test Method in the Chinese Pharmacopoeia (Part 4), and the test results meet the requirements.

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

[0030] The sterile glutamine of the present invention has good fluidity, which is beneficial to quality control in the pharmaceutical preparation process. The term "fluidity" used herein refers to the fluidity of glutamine solid (powder). Generally, the "angle of repose (θ)" is used to characterize the fluidity of solid powders, and the unit is "° (degree)". The term "angle of repose" used herein refers to the angle formed by the free inclined plane formed by the accumulation of powders and the horizontal plane in a static and balanced state. A variety of conventional methods can be used to determine the angle of repose, such as but not limited to the fixed cone method, the fixed funnel method, the tilting box method, the rotating cylinder method, and the fixed cone method is usually used for determination. It is generally believed that when θ≤40°, the fluidity requirements in the production process can be met (Han Li, ed., "Pharmaceutics Experiment", 2020 edition, page 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] The present application relates to a method for preparing sterile glutamine, which comprises the following steps:

[0034] (1) mixing solid glutamine with water and completely dissolving the glutamine to obtain a clear liquid;

[0035] (2) filtering the feed liquid to obtain a filtrate, wherein the filtration comprises filtering the feed liquid with a pore size of 0.22

[0036] Filtration by filter elements of μm and below;

[0037] (3) Adding selected from C to the filtrate in stages in the following manner 3-6 Ketone and C 1-6 One or more sterile organic solvents in alcohol to precipitate crystals:

[0038] The first stage: adding the sterile organic solvent to the filtrate at a flow rate of 1-2 liters / minute, and

[0039] The second stage: continue to add the sterile organic solvent at a flow rate of 2.5-4 liters / minute.

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

[0041] (4) crystal growth;

[0042] (5) performing solid-liquid separation on the solid-liquid mixture obtained after crystal growing to obtain crystals;

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

[0044] Without being bound by any theory, the method of the present invention can obtain sterile glutamine with high yield, high purity and good fluidity on an industrial scale and with high production efficiency. In particular, the sterile glutamine obtained by the method of the present invention meets the sterility standards of pharmaceutical preparations. In particular, the method of the present invention adopts a two-stage operation in the crystallization step (3), so that the sterile glutamine crystals are precipitated in a controlled manner, which is particularly helpful to improve the fluidity of the product.

[0045] In addition, the method of the present invention can efficiently prepare sterile glutamine at kilogram level to meet the raw / auxiliary material requirements in actual preparation production. At the same time, the method of the present invention also avoids high temperature processes (such as high temperature sterilization, high temperature drying, etc.), which not only reduces energy consumption and production costs, but also helps to reduce impurities that are not desired for pharmaceutical preparations, such as glutamic acid, pyroglutamic acid, etc.

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

[0047] In some embodiments, in step (1), the water may be purified water, preferably water for injection. Herein, the term "water for injection" refers to water that complies with the provisions of the Chinese Pharmacopoeia Water for Injection. Generally, it may be distilled water or deionized water obtained by distillation (also known as double distilled water). In some cases, water for injection may preferably be water that is effectively controlled for microbial contamination and that simultaneously controls the level of bacterial endotoxins.

[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. Under such temperature conditions, the dissolution rate of glutamine solid is fast and the dissolution is sufficient, which is conducive to improving production efficiency. Moreover, such a temperature will not cause thermal decomposition of the raw material glutamine, avoiding the generation of harmful impurities.

[0049] In some embodiments, in step (1), mixing glutamine solid with water (that is, throwing glutamine solid into water and mixing with it) can be carried out at a temperature of 15-30°C, preferably 18-30°C, and more preferably 20-28°C. In the present invention, the temperature may not be strictly controlled, and the feeding and mixing may be carried out at general room temperature. For example, glutamine solid may be thrown into water at a temperature of 18-30°C and mixed. After the glutamine solid is mixed with water, the system may be maintained at this temperature or appropriately heated (such as the dissolution temperature described above) to facilitate faster dissolution of the glutamine solid thrown into water and improve production efficiency.

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

[0051] In some embodiments, the dissolution in step (1) may be carried out with any operation that may accelerate the dissolution. Such operation can be routinely selected by those skilled in the art according to actual conditions. Preferably, it can be stirring. In the present invention, the stirring rate, stirring device and / or stirring method can be routinely 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 in sequence along the flow direction of the feed 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, and more preferably 0.45 μm, and the last stage filtration uses a filter element with a pore size of 0.22 μm and below, 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 in sequence along the flow direction of the feed liquid, wherein the feed liquid is sequentially subjected to a first stage filtration, a second stage filtration, and a third stage filtration, and the pore size used in the second stage filtration is 0.3 μm and below, preferably 0.25 μm and below, more preferably 0.22 μm and below, and more preferably 0.22 μm. The filter element.

[0053] As used herein, the term "filter element" or "pore size" or other similar terms refers 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 filter membrane. Preferably, the microporous filter membrane is a polyvinyl chloride microporous filter membrane, a polytetrafluoroethylene microporous filter membrane, a polyvinylidene fluoride microporous filter membrane (PVDF), or other organic solvent-resistant microporous filter membranes.

[0055] In some embodiments, during the filtration process of step (2), the temperature of the liquid to be filtered can be maintained at the dissolution temperature of step (1) described above (that is, step (2) can be carried out at the dissolution temperature of 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 one or more selected from methanol, ethanol, propanol, n-propanol and isopropanol; more preferably ethanol. 3-6 The ketone may be, for example, acetone. Although ketone solvents can achieve similar yields and purities as alcohol solvents, ethanol is more preferred, especially due to cost considerations.

[0057] As described above, in order to obtain a product with satisfactory yield, purity and fluidity, it is necessary to add a sterile organic solvent in two stages in the crystallization step of step (3) of the method of the present invention to precipitate sterile glutamine crystals, wherein the sterile organic solvent is added to the filtrate at a flow rate m1 of 1-2 L / min in the first stage, and the remaining sterile organic solvent is continued to be added at a flow rate m2 of 2.5-4 L / min in the second stage until the total volume to be added. In some embodiments, it is preferred that the sterile organic solvent is added at a flow rate m1 of 1-1.5 L / min in the first stage. In some embodiments, it is preferred that the remaining sterile organic solvent is added at a flow rate m2 of 2.5-3 L / min in the second stage. In the method of the present invention, the control of the flow rate m1 in the first stage is more important, which has a greater impact on the fluidity of the final product glutamine solid.

[0058] In some embodiments, 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 fluidity.

[0059] In the method of the present invention, the volume of the sterile organic solvent added in the first stage also has a great influence on the fluidity of the product. As described above, the ratio of the volume of the sterile organic solvent added in the first stage to the total sterile organic solvent volume can be in the range of 0.3-0.7:1, which can improve the production efficiency of the method and is also conducive to obtaining a product with good fluidity. In some embodiments, the ratio of the volume of the sterile organic solvent added in the first stage to the total sterile organic solvent volume added 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 more preferably 1:1. The ratio of the total volume of the organic solvent to the volume of the added water within an appropriate range is conducive to obtaining the product sterile glutamine with high yield and high purity; at the same time, the selection of the above range also takes into account the cost factor, because when the organic solvent is added in a larger amount, the purity and yield will not be substantially improved.

[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. Herein, this temperature may also be referred to as "crystallization temperature". In some embodiments, step (3) may preferably be performed at a temperature of 5-10°C. Using a lower temperature is more conducive to crystallization.

[0062] In the present invention, the temperature of the process of adding the sterile organic solvent in two stages in step (3) can be the same or different, as long as it is within the above-mentioned crystallization temperature range. In some embodiments, it is preferred that the temperature of the two stages can be the same.

[0063] In some embodiments, 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 interval) of 0.25-0.5 hours (h) between the first and second stages of step (3). Herein, the interval time period may also be referred to as an "intermittent stage". An interval time is inserted between the two solvent addition stages, especially an interval time within the range described above. During this time period, the sterile glutamine crystals produced after the addition of the solvent in the first stage are slightly nurtured, and then the solvent is added in the second stage. In this way, the sterile glutamine is crystallized in a controlled manner, which is beneficial to improve the fluidity of the product. Moreover, the selection of the interval time in the present invention also takes into account the production efficiency of the method; although it is beneficial to appropriately extend the interval time, too long a time will reduce the industrial production efficiency, and too short an interval time is not conducive to obtaining a product with good fluidity.

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

[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 and (optionally) the interval time between the two stages are controlled respectively, so that the crystallization process of sterile glutamine can be effectively controlled, so that the obtained sterile glutamine crystals have high yield, high purity and good fluidity.

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

[0068] Herein, the term "crystal cultivation" means the process of allowing the seed crystals produced in the crystallization step to naturally crystallize in the solution to produce crystals with ideal particle size distribution and good fluidity.

[0069] Preferably, in step (4), the crystal growing time can be 1-5 hours, preferably 1-3 hours. Controlling the crystal growing time is beneficial to improving the yield and purity of sterile glutamine while taking into account production efficiency.

[0070] Herein, the term "solid-liquid separation" refers to the process of separating the crystals from the liquid portion in the solid-liquid mixture (which contains the desired crystals and the liquid portion, such as a mixture of solvent and water) obtained after the crystal growing step to obtain the target crystals. In the present invention, the solid-liquid separation can be carried out by any conventional solid-liquid separation method known in the art, such as but not limited to filtration, centrifugation or sedimentation separation.

[0071] In some embodiments, in step (5), after solid-liquid separation, there may be a step of washing the crystals to further reduce the attachment of the mother liquor to the crystals, thereby improving the quality of the product. The step of washing the crystals may include one or more washings. The number of washings may be routinely determined by a person skilled in the art. In the present invention, there is no particular limitation on the washing liquid used in the washing step, and it may be routinely determined by a person 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 may be used. Preferably, the alcohol solvent may be, for example, but not limited to, methanol, ethanol, propanol, n-propanol or isopropanol; the ketone solvent may 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 may also be routinely determined by a person skilled in the art.

[0072] Herein, in step (6), the reduced pressure drying may be carried out by any conventional reduced pressure 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 the use of high temperatures is beneficial to reducing the decomposition of glutamine and the generation of impurities, which is beneficial to controlling product quality and is more suitable for the preparation of raw materials and auxiliary materials for medical use; and the energy consumption of drying at such a temperature is relatively low, which 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, in the sterile glutamine involved in the present invention or the sterile glutamine prepared by the method of the present invention, the glutamine is L-glutamine.

[0075] In the method of the present invention, at least steps (3)-(6) are performed under sterile conditions. Herein, "sterile 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 sterile conditions.

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

[0077] In a third aspect, the present application also relates to the use of sterile glutamine for preparing a pharmaceutical preparation. The pharmaceutical preparation described in the present invention is particularly an injectable preparation.

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

[0079] The method of the present invention will be described and illustrated in more detail below in conjunction with specific embodiments. It will be understood by those skilled in the art that the purpose of providing these embodiments is only for illustrative purposes and does not constitute any limitation to the scope of the present invention.

[0080] Example

[0081] I. Materials and Reagents

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

[0083] II. Evaluation and determination methods of physicochemical properties

[0084] 1. Sterility test

[0085] The determination was carried out using <1101 Sterility Test Method> and <1143 Bacterial Endotoxin Test Method> of Part IV of the Chinese Pharmacopoeia 2020 Edition.

[0086] a. Sterility test

[0087] The membrane filtration method described in the 2020 edition of the Chinese Pharmacopoeia, Part IV <1101 Sterility Test Method> is used for determination. Thioglycollate fluid culture medium and tryptic soy liquid culture medium used for sterility test should meet the requirements of sterility test and sensitivity test of 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 test, filter it with water for bacterial endotoxin test (water for bacterial endotoxin test should meet the standards for sterile water for injection) to wet the filter membrane, filter the test sample solution directly, and then rinse the filter membrane 3 times with water for bacterial endotoxin test. The rinsing volume and rinsing method used are the same as the method suitability test of <1101 Sterility Test Method> in Part IV of the 2020 edition of the Chinese Pharmacopoeia. Then, add 100 ml of thioglycollate fluid culture medium to one filter and 100 ml of tryptic soy bean liquid culture medium to one filter. The above filters are cultured for no less than 14 days at the temperature specified in each culture medium. If there is no sterile growth in the test sample tubes (specifically, all the test sample tubes are clear, or they appear turbid but are confirmed to have no sterile growth), the test sample is judged to meet the requirements; if any of the test sample tubes appears turbid and bacterial growth is confirmed, the test sample is judged to not meet the requirements, unless it can be fully proved that the test results are invalid, that is, the grown microorganisms are not contained in the test sample.

[0089] Positive control: Staphylococcus aureus is used as the control bacteria. The preparation of the bacterial solution for the positive control test is the same as the method suitability test of <1101 Sterility Test Method> in Part IV of the 2020 edition of the Chinese Pharmacopoeia. The amount of bacteria added is not more than 100 cfu, and the amount of the test sample is the same as the amount of sample inoculated in each culture medium during the sterility test of the above test sample. The positive control tube should be cultured for no more than 5 days and should grow well.

[0090] Negative control: Take the corresponding amount of water for bacterial endotoxin test and perform the sterility test for the test sample as a negative control. No bacteria should grow in the negative control.

[0091] b. Bacterial endotoxin test

[0092] The gel method described in the 2020 edition of the Chinese Pharmacopoeia, Part IV <1143 Bacterial Endotoxin Test> is used for determination, in which the test method adopts the gel limit test for determination. The test results should comply with the regulations.

[0093] 2. Moisture determination

[0094] The first method (Fischer method) in the volumetric titration method of the fourth part of the 2020 edition of the Chinese Pharmacopoeia <0832 Water Determination Method> is used for determination. The water content in the test sample is directly determined by using a moisture meter: accurately weigh an appropriate amount of the test sample, place it in a dry stoppered conical flask, add an appropriate amount of anhydrous methanol, and titrate with Fischer test solution under continuous shaking (or stirring) until the solution changes from light yellow to reddish brown. Perform a blank test and calculate according to the following formula:

[0095]

[0096] In the formula

[0097] A is the volume of Fischer's test solution consumed by the test sample, ml;

[0098] B is the volume of Fischer test solution consumed by the blank, ml;

[0099] F is the weight of water per ml of Fischer's test solution, mg;

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

[0101] 3. Particle size distribution determination

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

[0103] 4. Determination of the angle of repose

[0104] The fixed cone method recorded in the textbook "Pharmaceutics Experiment" (edited by Han Li, 2020 edition, page 177) was used for measurement.

[0105] Example 1

[0106] 6 kg of glutamine was added to 120 L of water for injection under stirring at room temperature (about 26 ° C), and the temperature was raised to 35-40 ° C, and the glutamine was stirred until the glutamine was dissolved to obtain a solution. The solution was filtered using a three-stage filter element (the filter element pore size was 0.45 μm, 0.22 μm, and 0.22 μm, respectively), and the filter element was washed with a small amount of water for injection. The filtrate and the washing liquid were combined, stirred, and cooled to 15-17 ° C. Under stirring (stirring rate was about 60-100 rpm), 40 L of anhydrous ethanol was added at a flow rate of 1 L / min m1, and the remaining anhydrous ethanol was continued to be added at a flow rate of 2.5 L / min m2 after an interval of 30 minutes until the total volume of anhydrous ethanol added was 120 L. Then, the system was cooled to 0-5 ° C and crystallized for 1 hour. Filter, vacuum dry the solid, and the drying temperature was 30-45 ° C. Dry to a product drying loss of ≤2% to obtain sterile glutamine. The sterility test and bacterial endotoxin test of the obtained sterile glutamine met the requirements. Other test results are summarized in Table 1.

[0107] Example 2

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

[0109] Example 3

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

[0111] Example 4

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

[0113] Example 5

[0114] This comparative example is substantially the same as Example 1, except that the volume of ethanol added in the first stage is 60 L. The sterility test and bacterial endotoxin test of the obtained sterile glutamine meet the requirements, and other test results are summarized in Table 1.

[0115] Example 6

[0116] This comparative example is substantially the same as Example 1, except that the volume of ethanol added in the first stage is 80 L. The sterility test and bacterial endotoxin test of the obtained sterile glutamine meet the requirements, and other test results are summarized in Table 1.

[0117] Comparative Example 1

[0118] This comparative example is substantially the same as Example 1, except that the sterile organic solvent ethanol is not added in stages, but all ethanol (total volume is 90 L) is added continuously at a flow rate of 1 L / min. The test results are summarized in Table 1.

[0119] Comparative Example 2

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

[0121] Comparative Example 3

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

[0123] Comparative Example 4

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

[0125] Comparative Example 5

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

[0127] As can be seen from Table 1 below, by adding the sterile organic solvent in stages and controlling the solvent flow rates and solvent volume ratios of the two stages, the method of the present invention can obtain sterile glutamine with improved fluidity at a higher yield and higher purity.

[0128]

Claims

1. A sterile glutamine having a purity of ≥98.0% and an angle of repose of ≤40°.

2. The sterile glutamine according to claim 1, having at least one of the following characteristics: a. a water content of less than 0.5%, preferably less than 0.2%; and b. The glutamine is L-glutamine.

3. A method for preparing sterile glutamine, comprising the following steps: (1) mixing solid glutamine with water and completely dissolving the glutamine to obtain a clear liquid; (2) filtering the feed liquid to obtain a filtrate, wherein the filtration includes filtering with a filter element having a pore size of 0.22 μm or less; (3) Adding a mixture selected from C 3-6 Ketone and C 1-6 One or more sterile organic solvents in alcohol to precipitate crystals: The first stage: adding the sterile organic solvent to the filtrate at a flow rate of 1-2 liters / minute, and The second stage: continue to add the sterile organic solvent at a flow rate of 2.5-4 liters / minute. The ratio of the volume of the sterile organic solvent added in the first stage to the total volume added in the two stages is 0.3-0.7:1; (4) crystal growth; (5) performing solid-liquid separation on the solid-liquid mixture obtained after crystal growing to obtain crystals; (6) Drying the crystals under reduced pressure to obtain the sterile glutamine.

4. The method according to claim 3, wherein step (3) has at least one of the following features: a. Flow rate m1 is 1-1.5 liters / minute; b. Flow rate m2 is 2.5-3 liters / minute; c. The ratio of m1 to m2 is 0.25-0.7:1, preferably 0.3-0.67:1, more preferably 0.3-0.4:1; d. The ratio of the volume of the sterile organic solvent added in the first stage to the total volume of the sterile organic solvent is 0.33-0.67:1, preferably 0.33-0.56:1; e. 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; f. an interval of 0.25-0.5 hours between the first and second stages; g. Step (3) is carried out at a temperature of 10-35 ° C, preferably 10-33 ° C, more preferably 12-32 ° C; h. Step (3) is carried out under stirring at a stirring rate of 60-100 rpm.

5. The method according to claim 3 or 4, wherein step (1) has at least one of the following features: a. The dissolution is carried out at a temperature of 20-60 ℃, preferably 30-50 ℃, more preferably 35-50 ℃; b. The mixing of solid glutamine with water is carried out at a temperature of 15-30°C, preferably 18-30°C, more preferably 20-28°C.

6. The method according to claim 3 or 4, wherein in step (2), the filtration comprises a first stage filtration and a last stage filtration arranged in sequence along the flow direction of the feed 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; Preferably, the filtering comprises two or more stages of filtering; Preferably, the filtration comprises three stages of filtration arranged in sequence along the flow direction of the feed liquid, wherein the feed liquid is sequentially subjected to first stage filtration, second stage filtration and third stage filtration, and the pore size of the filter element used in the second stage filtration is 0.3 μm or less, preferably 0.25 μm or less, more preferably 0.22 μm or less, more preferably 0.22 μm; Preferably, step (2) is carried out while maintaining the dissolution temperature of step (1).

7. The method according to claim 3 or 4, wherein step (4) has at least one of the following features: a. The crystal growing is carried out at a temperature of 3-15°C, preferably 5-10°C, more preferably 5-8°C; b. The crystal growing time is 1-5 hours, preferably 1-3 hours.

8. The method according to claim 3 or 4, wherein in step (6), the reduced pressure drying is carried out at a temperature of 30-80°C, preferably 30-50°C, more preferably 30-40°C.

9. The method according to claim 3 or 4, wherein the glutamine is L-glutamine.

10. Use of the sterile glutamine according to claims 1-2 or the sterile glutamine obtained by the method of any one of claims 3-9 for preparing a pharmaceutical preparation.

Images