Method for improving appearance of freeze-dried antithrombin III product

Abstract

The invention provides a method for improving the appearance of a freeze-dried antithrombin III product. The method comprises the following steps: carrying out pre-freezing treatment and sublimation drying treatment on a sample containing antithrombin III; the pre-freezing treatment comprises at least two cooling steps, and the temperature of each cooling step is-60 to-20 DEG C independently; the sublimation drying treatment comprises at least three heating steps, and the temperature of each heating step is-45 DEG C to 0 DEG C independently. The antithrombin III freeze-dried product obtained by the method disclosed by the invention meets the requirements of pharmacopoeia quality standards, and has the advantages of good molding, full appearance, no shrinkage, no collapse, no compact film layer at the top, high mechanical strength, good product stability and high market value, and is beneficial to storage and transportation.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biopharmaceuticals, in particular to a method for improving the appearance of lyophilized antithrombin III products. BACKGROUND

[0002] Antithrombin III (hereinafter referred to as "AT-III") can inhibit thrombin, IXa, Xa, XIa and other serine protease activities, and is the primary factor involved in physiological coagulation mechanism and control of blood coagulation, and is the main component of the blood anticoagulation system. Therefore, antithrombin III plays an important role in maintaining normal blood circulation and preventing thrombosis, and is valued by clinical and hemostasis research. In foreign countries, since the 1980s, AT-III lyophilized products isolated and purified from healthy human plasma and prepared by freeze-drying have been marketed. There is no such product on the market in China.

[0003] However, AT-III lyophilized products are not easy to shape, and are prone to shrinkage, collapse and other phenomena, and have low mechanical strength. During storage or transportation, the freeze-dried cake is prone to breakage and cracking, affecting product stability.

[0004] Therefore, the freeze-drying method for antithrombin still needs to be researched. SUMMARY

[0005] The present application aims to at least partially solve at least one of the technical problems existing in the prior art. To this end, the present application provides a method for improving the appearance of lyophilized antithrombin III products. The antithrombin III lyophilized products obtained by the method meet the quality standard requirements of the pharmacopoeia, and have good shaping, full appearance, no shrinkage, collapse, no dense film layer on the top, high mechanical strength, good storage and transportation, good product stability, and high market value.

[0006] In a first aspect of the present application, a method for improving the appearance of lyophilized antithrombin III products is provided. According to embodiments of the present application, the method comprises: pre-freezing and sublimation drying treatment of a sample containing antithrombin III; the pre-freezing treatment comprises at least two temperature reduction steps, and the temperature of each temperature reduction step is independently -60 to -20°C; the sublimation drying treatment comprises at least three temperature increase steps, and the temperature of each temperature increase step is independently -45 to 0°C. The antithrombin III lyophilized products obtained by the method according to the embodiments of the present application meet the quality standard requirements of the pharmacopoeia, and have good shaping, no shrinkage and collapse, high mechanical strength, good storage and transportation, good product stability, and high market value.

[0007] According to the embodiment of the present application, the temperature of each of the cooling steps is independently -57 to -23°C, preferably -55 to -25°C. Thereby, the appearance of the product is further improved and the mechanical strength is increased.

[0008] According to the embodiment of the present application, the cooling time of each of the cooling steps is independently 5 to 150 min, preferably 10 to 120 min, more preferably 15 to 120 min. Thereby, the lyophilized product with full appearance is obtained.

[0009] According to the embodiment of the present application, the maintaining time of each of the cooling steps is independently 10 to 700 min, preferably 15 to 700 min, more preferably 15 to 600 min. Thereby, the lyophilized product without atrophy, collapse and dense film layer on the top is obtained.

[0010] According to the embodiment of the present application, the vacuum pressure of each of the cooling steps is normal pressure. The inventors have determined through a large number of optimization experiments that under this condition, the obtained product meets the quality standard requirements of the pharmacopoeia and has a high pass rate.

[0011] According to the embodiment of the present application, the temperature of the first cooling step is -40 to -20°C, preferably -35 to -25°C. Thereby, the appearance of the product is further improved and the mechanical strength is increased.

[0012] According to the embodiment of the present application, the cooling time of the first cooling step is 10 to 120 min, preferably 15 min. Thereby, the appearance of the product is further improved and the lyophilized product with full appearance is obtained.

[0013] According to the embodiment of the present application, the maintaining time of the first cooling step is 10 to 200 min, preferably 180 min. Thereby, the appearance of the product is further improved and the lyophilized product without atrophy, collapse and dense film layer on the top is obtained.

[0014] According to the embodiment of the present application, the temperature of the second cooling step is -60 to -50°C, preferably -55°C. Thereby, the appearance of the product is further improved and the mechanical strength is increased.

[0015] According to the embodiment of the present application, the cooling time of the second cooling step is 10 to 20 min, preferably 15 min. Thereby, the appearance of the product is further improved and the lyophilized product with full appearance is obtained.

[0016] According to the embodiment of the present application, the maintaining time of the second cooling step is 300 to 700 min, preferably 360 min. Thereby, the appearance of the product is further improved and the lyophilized product without atrophy, collapse and dense film layer on the top is obtained.

[0017] According to the embodiment of the present application, the temperature of each of the temperature increasing steps is independently -43 to -0°C, preferably -40 to -0°C. Thus, the appearance of the product is further improved, and a freeze-dried product with no shrinkage, no collapse, high mechanical strength, and meeting the quality standard of the pharmacopoeia is obtained.

[0018] According to the embodiment of the present application, the temperature increasing time of each of the temperature increasing steps is independently 10 to 300 min, preferably 20 to 270 min, and more preferably 30 to 240 min. Thus, the appearance of the product is further improved.

[0019] According to the embodiment of the present application, the maintaining time of each of the temperature increasing steps is independently 280 to 4400 min, preferably 300 to 4250 min, and more preferably 300 to 4200 min. The inventors have determined through a large number of optimization experiments that, under this condition, the obtained product meets the quality standard of the pharmacopoeia, and the product pass rate is high.

[0020] According to the embodiment of the present application, the vacuum pressure of each of the temperature increasing steps is 0.05 to 0.5 mbar, preferably 0.1 to 0.3 mbar. The inventors have determined through a large number of optimization experiments that, under this condition, the qualified product meeting the quality standard of the pharmacopoeia can be obtained.

[0021] According to the embodiment of the present application, the temperature of the first temperature increasing step is -45 to -35°C, preferably -40°C. Thus, the mechanical strength of the product is further improved, and the appearance of the product is improved.

[0022] According to the embodiment of the present application, the temperature increasing time of the first temperature increasing step is 20 to 40 min, preferably 30 min. Thus, the appearance of the product is further improved.

[0023] According to the embodiment of the present application, the maintaining time of the first temperature increasing step is 360 to 960 min, preferably 480 min. The inventors have determined through a large number of optimization experiments that, under this condition, the obtained product meets the quality standard of the pharmacopoeia, and the product pass rate is high.

[0024] According to the embodiment of the present application, the vacuum pressure of the first temperature increasing step is 0.2 to 0.4 mbar, preferably 0.25 to 0.35 mbar, and more preferably 0.3 mbar. The inventors have determined through a large number of optimization experiments that, under this condition, the qualified product meeting the quality standard of the pharmacopoeia can be obtained.

[0025] According to the embodiment of the present application, the temperature of the second temperature increasing step is -40 to -20°C, preferably -30°C. Thus, the mechanical strength of the product is further improved, and the appearance of the product is improved.

[0026] According to an embodiment of the present application, the temperature of the second temperature increasing step is -10 to 0°C, preferably 0°C. The inventor has found through research that the third temperature increasing step of sublimation drying is essential to obtain the freeze-dried antithrombin III product meeting the pharmacopoeia quality standards.

[0027] According to an embodiment of the present application, the maintaining time of the second temperature increasing step is 2200 to 4400 min, preferably 4000 min. The inventor has determined through a large number of optimization experiments that under this condition, the product obtained meets the pharmacopoeia quality standard requirements and has a high pass rate.

[0028] According to an embodiment of the present application, the vacuum pressure of the second temperature increasing step is 0.1 to 0.3 mbar, preferably 0.15 to 0.25 mbar, and more preferably 0.2 mbar. The inventor has determined through a large number of optimization experiments that under this condition, the qualified product meeting the pharmacopoeia quality standard requirements can be obtained.

[0029] According to an embodiment of the present application, the temperature of the third temperature increasing step is -10 to 0°C, preferably 0°C. The inventor has found through research that the third temperature increasing step of sublimation drying is essential to obtain the freeze-dried antithrombin III product meeting the pharmacopoeia quality standards.

[0030] According to an embodiment of the present application, the temperature of the third temperature increasing step is -10 to 0°C, preferably 0°C. The inventor has found through research that the third temperature increasing step of sublimation drying is essential to obtain the freeze-dried antithrombin III product meeting the pharmacopoeia quality standards.

[0031] According to an embodiment of the present application, the maintaining time of the third temperature increasing step is 280 to 1400 min, preferably 300 min. The inventor has determined through a large number of optimization experiments that under this condition, the product obtained meets the pharmacopoeia quality standard requirements and has a high pass rate.

[0032] According to an embodiment of the present application, the vacuum pressure of the third temperature increasing step is 0.05 to 0.2 mbar, preferably 0.075 to 0.15 mbar, and more preferably 0.1 mbar. The inventor has determined through a large number of optimization experiments that under this condition, the product obtained meets the pharmacopoeia quality standard requirements.

[0033] According to an embodiment of the present application, the method further comprises: resolving drying treatment.

[0034] According to an embodiment of the present application, the temperature of the resolving drying is 25 to 35°C, preferably 30°C.

[0035] According to an embodiment of the present application, the temperature of the resolving drying is 25 to 35°C, preferably 30°C.

[0036] According to an embodiment of the present application, the maintaining time of the desiccation is not less than 300 minutes, preferably 600 minutes.

[0037] According to an embodiment of the present application, the vacuum pressure of the desiccation is 0.001-0.03 mbar. The inventor has determined through a large number of optimization experiments that under this condition, the product obtained meets the quality standard requirements of the pharmacopoeia.

[0038] In some specific embodiments of the present application, the sample contains one or more freeze-drying protective agents, buffers, surfactants.

[0039] In some more specific embodiments of the present application, the sample contains at least one selected from glycine or a salt thereof, lysine or a salt thereof, tyrosine or a salt thereof, aspartic acid or a salt thereof, arginine and a salt thereof, sodium citrate, mannitol, alanine, sucrose, sodium chloride, polysorbate, tris-hydroxymethyl aminomethane and sodium phosphate.

[0040] According to an embodiment of the present application, the sample contains: glycine, sodium citrate, sodium chloride.

[0041] According to an embodiment of the present application, the sample contains: 3% of the glycine, 0.2% sodium citrate, 0.8% sodium chloride, based on the total volume of the sample. In this way, the stability of the product is further improved.

[0042] According to an embodiment of the present application, the sample contains 3-9 g / L of the antithrombin III, preferably 6 g / L, based on the total volume of the sample. Exemplarily, the sample contains 3 g / L, 3.5 g / L, 4 g / L, 4.5 g / L, 5 g / L, 5.5 g / L, 6 g / L, 6.5 g / L, 7 g / L, 7.5 g / L, 8 g / L, 8.5 g / L or 9 g / L of the antithrombin III. In this way, the AT-III freeze-dried product meeting the pharmacopoeia requirements is obtained.

[0043] In the second aspect of the present application, a method for improving the appearance of a lyophilized antithrombin III product is provided. According to an embodiment of the present application, the method comprises: subjecting a sample containing antithrombin III to pre-freezing, sublimation drying and desorption drying; the pre-freezing comprises: (1) pre-cooling; (2) a first temperature decreasing step: decreasing the temperature to -35℃ within 15 minutes and maintaining for 180 minutes under normal pressure; (3) a second temperature decreasing step: decreasing the temperature to -55℃ within 15 minutes and maintaining for 360 minutes under normal pressure; the sublimation drying comprises: (4) a first temperature increasing step: increasing the temperature to -40℃ within 30 minutes and maintaining for 480 minutes under a vacuum pressure of 0.3 mbar; (5) a second temperature increasing step: increasing the temperature to -30℃ within 120 minutes and maintaining for 4000 minutes under a vacuum pressure of 0.2 mbar; (6) a third temperature increasing step: increasing the temperature to 0℃ within 60 minutes and maintaining for 300 minutes under a vacuum pressure of 0.1 mbar; and the desorption drying is: increasing the temperature to 30℃ within 240 minutes and maintaining for 600 minutes under a vacuum pressure of 0.01 mbar. The lyophilized antithrombin III product obtained by the method according to the embodiment of the present application meets the requirements of the pharmacopoeia quality standards, has good shape, no shrinkage or collapse, high mechanical strength, is beneficial for storage and transportation, has good product stability, and has high market value.

[0044] According to an embodiment of the present application, the pre-cooling step is performed at a temperature of 0-2℃. In this way, the uniformity of the lyophilized product is further improved.

[0045] According to an embodiment of the present application, the pre-cooling step is performed at a temperature of 1-2℃. The pre-cooling temperature is slightly higher than the freezing point, which makes the solute and solvent of the sample to be dried uniformly distributed but not directly crystallized, thereby avoiding the supercooling phenomenon and avoiding the phenomenon that some components of the sample are crystallized first and some components are not crystallized. In this way, the uniformity of the lyophilized product is further improved.

[0046] According to an embodiment of the present application, the sample contains 3% of the glycine, 0.2% of sodium citrate, and 0.8% of sodium chloride, based on the total volume of the sample.

[0047] According to an embodiment of the present application, the sample contains 3-9 g / L of the antithrombin III, based on the total volume of the sample.

[0048] According to an embodiment of the present application, the sample contains 6 g / L of the antithrombin III, preferably 6 g / L, based on the total volume of the sample.

[0049] The method of the present application can be used to prepare a lyophilized antithrombin product that meets the quality standard requirements, has good shape, full appearance, no shrinkage or collapse, no dense film layer on the top, high mechanical strength, is beneficial for storage and transportation, has good product stability, and has high market value.

[0050] It should be noted that the present application does not make strict limitation on the source of antithrombin III, which can be human antithrombin, bovine antithrombin, sheep antithrombin, pig antithrombin, etc.; and can be derived from mammalian plasma or from genetic recombination technology.

[0051] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0052] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description, including the appended drawings.

[0053] Figure 1 It is the appearance diagram of the freeze-dried product in Example 1 of the present application, wherein (A) is the side view of the product; (B) is the top view of the product; (C) is the bottom view of the product;

[0054] Figure 2 It is the appearance diagram of the freeze-dried product in Example 2 of the present application, wherein (A) is the side view of the product; (B) is the top view of the product;

[0055] Figure 3 It is the appearance of the freeze-dried product in Example 2 of the present application after slight shaking;

[0056] Figure 4 It is the appearance diagram of the freeze-dried product in Example 5 of the present application, wherein (A) is the side view of the product; (B) is the top view of the product;

[0057] Figure 5 It is the appearance diagram of the freeze-dried product in Example 5 of the present application, wherein (A) is the overall appearance diagram of the product; (B) is the bottom view of the product;

[0058] Figure 6 It is the appearance diagram of the freeze-dried product in Example 6 of the present application, wherein (A) is the overall appearance diagram of the product; (B) is the side view of the product;

[0059] Figure 7 It is the appearance diagram of the freeze-dried product in Example 6 of the present application, wherein (A) is the top view of the product; (B) is the bottom view of the product;

[0060] Figure 8 It is the overall appearance diagram of the freeze-dried product in Example 7 of the present application;

[0061] Figure 9 It is the side view appearance diagram of the freeze-dried product in Example 7 of the present application;

[0062] Figure 10 It is the overall appearance diagram of the freeze-dried product in Example 8 of the present application;

[0063] Figure 11 This is a side view of the freeze-dried product in Embodiment 8 of the present invention. Detailed Implementation

[0064] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0065] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0066] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0067] All quality inspection index data in the examples are average values.

[0068] Terms and Definitions

[0069] In this document, the term "cooling step" refers to the step in which the sample temperature is reduced from an initial temperature to a specific temperature, excluding the process of temperature increase. In this document, "cooling step" is used to describe the pre-freezing process in freeze-drying, the purpose of which is to rapidly freeze samples containing antithrombin III.

[0070] In this text, the term "sample" refers to any intermediate product containing the active ingredient that has not reached the lyophilization endpoint. In the context, it is equivalent to "lyophilized sample," "sample to be lyophilized," "human antithrombin III sample," "human antithrombin III lyophilized sample," "AT-III sample," and "AT-III lyophilized sample."

[0071] In this text, the term "product" refers to a finished product containing active ingredients that has reached the freeze-drying endpoint. In the context, it is equivalent to "freeze-dried product," "antithrombin III product," "antithrombin III freeze-dried product," "AT-III product," and "AT-III freeze-dried product."

[0072] In this document, the term "sublimation drying" is equivalent to "primary drying", which refers to a drying method that involves freezing the water-containing material below the freezing point to convert the liquid water into solid ice, and then converting the solid ice into gaseous vapor under vacuum conditions to remove the water.

[0073] In this document, the term "warming step" refers to a step in which the temperature of the sample is raised from a starting temperature to a specific temperature, and the process of temperature reduction is not included in this step. In this document, "warming step" is used to describe the sublimation drying process of freeze-drying, which aims to remove the water in the frozen antithrombin III sample.

[0074] In this document, the term "desorption drying" is equivalent to "secondary drying", which refers to a drying method that involves placing the material under negative pressure conditions and heating the material to its boiling point under negative pressure conditions to remove the water.

[0075] In this document, the term "maintenance time" is equivalent to "holding time", which refers to the time period after the sample reaches a specific state. In some cases, it is equivalent to "time" in the context.

[0076] In this document, the term "set time" refers to the time period required for the sample to reach a specific state. According to the embodiments of the present application, "set time" specifically refers to the time required to reach a specific "set temperature" to describe the rate of cooling / warming. In some cases, it is equivalent to "warming rate" or "cooling rate" or "warming time" or "cooling time" in the context.

[0077] In this document, the term "set temperature" refers to the temperature that the sample needs to reach in a specific step. In some cases, it is equivalent to "temperature" in the context.

[0078] The embodiments of the present application select human plasma-derived antithrombin III as the freeze-dried sample to explain and illustrate the thrombin freeze-drying method of the present application.

[0079] In the embodiments of the present application, the preparation method of human plasma-derived antithrombin III is as follows:

[0080] (1) Fresh frozen plasma is centrifuged to remove cryoprecipitate; (2) The cryoprecipitate-removed plasma is adsorbed by DEAE Sephdeax A50 gel to remove coagulation factors II, VII, IX, X, etc. in the plasma; (3) The plasma after A50 adsorption is clarified by filtration; (4) The filtrate is subjected to Unigel-65 Heparin affinity chromatography; (5) The eluate is subjected to ultrafiltration desalting and dilution of the protein; (6) The virus is inactivated by S / D reagent; (7) The S / D reagent and inactivated protein are removed by Unigel-65 Heparin affinity chromatography; (8) The ultrafiltration is replaced and the protein is concentrated; (9) The virus is removed by nanomembrane filtration, and the semi-finished product is prepared and subjected to sterilization filtration; and (10) Freeze-drying.

[0081] In the embodiments of the present application, the reconstitution time detection method of the antithrombin III lyophilized preparation is as follows: refer to the method in the Chinese Pharmacopoeia 2020 edition 3, each chapter: II treatment: human coagulation factor VIII 3.3.2.3.

[0082] In the embodiments of the present application, the titer detection method of the antithrombin III lyophilized preparation is as follows: refer to the method in the European Pharmacopoeia 10.0 2.7.17. "ASSAY OF HUMAN ANTITHROMBIN III".

[0083] In the embodiments of the present application, the molecular size distribution investigation method of the antithrombin III lyophilized preparation is as follows: refer to the "MOLECULAR WEIGHT DISTRIBUTION" under the "Antithrombin III Human" item in USP-NF.

[0084] In the embodiments of the present application, the appearance evaluation standard of the antithrombin III lyophilized preparation refers to the relevant requirements in the Chinese Pharmacopoeia.

[0085] In the embodiments of the present application, the antithrombin III lyophilized sample further comprises mannitol, amino acid, citrate and / or chloride salt.

[0086] Further, the human antithrombin III lyophilized preparation contains 3-10 g / L of antithrombin III, 1-3% of mannitol or glycine, 0.15-0.2% of sodium citrate, and 0.6-0.8% of sodium chloride.

[0087] The scheme of the present application will be explained below in combination with examples. Those skilled in the art will understand that the following examples are only used to illustrate the present application, and should not be regarded as limiting the scope of the present application. If the specific technology or condition is not specified in the examples, it is carried out according to the technology or condition described in the literature in the art or according to the product instruction. If the reagent or instrument is not specified by the manufacturer, it is a conventional product that can be obtained by purchase in the market.

[0088] Example 1

[0089] In this example, human plasma-derived antithrombin III was lyophilized according to the following procedure:

[0090] 1. A lyophilized sample of human plasma-derived antithrombin III was provided, containing 6 g / L antithrombin III, glycine 3%, sodium citrate 0.2%, sodium chloride 0.8%.

[0091] 2. The lyophilized sample was processed according to the following table to obtain a lyophilized preparation of antithrombin III.

[0092] Table 1 Lyophilization process parameters for Example 1

[0093]

[0094] Note: 1) "Set time" refers to the time required to reach the "set temperature" for the step, and is used to describe the rate of cooling / heating, which is controlled at 0.1-2.5°C / min; 2) "Controlled vacuum" refers to the vacuum condition that the sample is required to reach in the relatively closed space for the step. The same applies below; 3) " / " means that the vacuum is not controlled, and is under normal pressure.

[0095] Results and analysis:

[0096] 1) Reconstitution time: < 30 s (meets the requirement); appearance and visible foreign matter: does not meet the requirement, obvious visible foreign matter; potency: 70.5 IU / ml (meets the requirement); molecular size distribution: 2.08% multimers, 97.93% monomers (meets the requirement).

[0097] 2) The overall appearance is good, the side is smooth and flat, with a perfect rigid structure that will not crumble when hit, the bottom is smooth and flat, without concave, without holes, the top is loose, porous, without dense film Figure 1 ).

[0098] The above results show that the lyophilized preparation of antithrombin III obtained using the one-time cooling condition in the pre-freezing stage and the one-time heating condition in the primary drying stage of this example does not meet the quality standards of the pharmacopoeia.

[0099] Example 2

[0100] In this example, human plasma-derived antithrombin III was lyophilized according to the following procedure:

[0101] 1. A lyophilized sample of human plasma-derived antithrombin III was provided, specifically referring to Example 1.

[0102] 2. The lyophilized sample was processed according to the following table to obtain a lyophilized preparation of antithrombin III.

[0103] Table 2 Lyophilization process parameters for Example 2

[0104]

[0105] Note: 1) "Set time" refers to the time required to reach the "set temperature" of this step, which is used to describe the rate of temperature decrease / increase. The temperature decrease rate is controlled at 0.1-2.5°C / min; 2) "Controlled vacuum" refers to the vacuum condition required to be reached in the relatively closed space where the sample is located. The same applies to the following; 3) " / " means no control of the vacuum degree, which is under normal pressure.

[0106] 3. Test the potency, reconstitution time, molecular size distribution of the final lyophilized product, and observe whether the appearance of the product meets the requirements of the pharmacopoeia.

[0107] Results and analysis:

[0108] 1) Reconstitution time: <30S (meets the requirements); appearance and visible foreign matter: does not meet the requirements, with flocculent matter with a diameter >2mm; potency: 52IU / ml (meets the requirements); molecular size distribution: 0.78% multimers, 99.22% monomers (meets the requirements).

[0109] 2) The appearance is not ideal, with water vapor escape channels on the side of the lyophilized product, a relatively smooth bottom, a regular and flat bottom, and no dense film layer on the top, presenting a good appearance. However, the finished product is significantly shrunk, with insufficient mechanical strength and easy breakage under slight vibration. Figure 2 、 Figure 3 ).

[0110] The above results show that, on the basis of the lyophilization conditions of Example 1, the pre-freezing stage is increased by one temperature decrease step, and the temperature decrease holding time and the primary drying time are appropriately adjusted, resulting in an anti-thrombin III lyophilized product with poor mechanical strength, which does not meet the quality standards of the pharmacopoeia.

[0111] Example 3

[0112] In this example, human plasma-derived anti-thrombin III is freeze-dried according to the following method:

[0113] 1. Provide a human plasma-derived anti-thrombin III lyophilized sample, as described in Example 1.

[0114] 2. Process the lyophilized sample according to the conditions in the following table to obtain an anti-thrombin III lyophilized product.

[0115] Table 3 Lyophilization process parameters of Example 3

[0116]

[0117] Note: 1) "Set time" refers to the time required to reach the "set temperature" of this step, which is used to describe the rate of temperature decrease / increase. The temperature decrease rate is controlled at 0.1-2.5°C / min; 2) "Controlled vacuum" refers to the vacuum condition required to be reached in the relatively closed space where the sample is located. The same applies hereafter; 3) " / " means no vacuum control, which is under normal pressure.

[0118] Results and analysis:

[0119] 1) Reconstitution time: < 30 s (meets the requirement); appearance and visible foreign matter: does not meet the requirement, with flocculent material with a diameter > 2 mm.

[0120] 2) Appearance does not meet the requirement, with uneven channels on the side and a dense film on the top. Although the bottom is relatively flat, the overall appearance shows slight shrinkage, and the mechanical strength is not high enough, which can be easily broken by moderate shaking.

[0121] The above results show that, based on the freeze-drying conditions of Example 1, appropriate adjustment of the holding time of the freezing step and optimization of the primary drying stage step are made, and it is found that increasing three temperature increase steps cannot obtain antithrombin III freeze-dried products that meet the quality standards of the pharmacopoeia and have good mechanical strength.

[0122] Example 4

[0123] In this example, human plasma-derived antithrombin III was freeze-dried according to the following method:

[0124] 1. Provide a human plasma-derived antithrombin III freeze-dried sample, which is specifically referred to in Example 1.

[0125] 2. Process the freeze-dried sample according to the conditions in the following table to obtain antithrombin III freeze-dried products.

[0126] Table 4 Freeze-drying process parameters of Example 4

[0127]

[0128] Note: 1) "Set time" refers to the time required to reach the "set temperature" of this step, which is used to describe the rate of temperature decrease / increase. The temperature decrease rate is controlled at 0.1-2.5°C / min; 2) "Controlled vacuum" refers to the vacuum condition required to be reached in the relatively closed space where the sample is located. The same applies hereafter; 3) " / " means no vacuum control, which is under normal pressure.

[0129] 3. Detect the potency, reconstitution time, and molecular size distribution of the final freeze-dried products, and observe whether the appearance of the products meets the requirements of the pharmacopoeia.

[0130] Results and analysis: Freeze-drying failed, and the final product melted out of the cabinet immediately, without reaching the freeze-drying endpoint.

[0131] The above results show that the pre-freezing stage in this example uses a 2-step cooling procedure, and the drying stage uses a 2-step heating procedure, and the anti-thrombin III lyophilized product obtained still cannot meet the pharmacopoeia quality standards.

[0132] From the above four examples, it can be seen that the inventors cannot obtain anti-thrombin III lyophilized products meeting the pharmacopoeia quality standards by increasing the cooling step in the pre-freezing stage (Example 2), or by increasing the two-step heating procedure in the primary drying stage (Example 3), or by using two cooling steps in the pre-freezing stage and two heating steps in the primary drying stage (Example 4) based on the lyophilization conditions of Example 1. Therefore, in combination with Examples 1-4, the inventors further improve the lyophilization conditions.

[0133] Example 5

[0134] In this example, human plasma-derived anti-thrombin III is lyophilized according to the following method:

[0135] 1. A human plasma-derived anti-thrombin III lyophilized sample is provided, which contains 6 g / L anti-thrombin III, 3% glycine, 0.2% sodium citrate, and 0.8% sodium chloride.

[0136] 2. The lyophilized sample is treated according to the following table to obtain an anti-thrombin III lyophilized product.

[0137] Table 5 Lyophilization process parameters of Example 5

[0138]

[0139]

[0140] Note: 1) "Set time" refers to the time required to reach the "set temperature" of the step, which is used to describe the cooling / heating rate, and the cooling rate is controlled at 0.1-2.5°C / min; 2) "Controlled vacuum" refers to the vacuum condition required to be reached by the relatively closed space of the sample in this step. The same below; 3) " / " means not to control the vacuum degree, which is under normal pressure.

[0141] 3. The potency, reconstitution time, molecular size distribution of the final lyophilized product are detected, and whether the appearance of the product meets the pharmacopoeia requirements is observed.

[0142] Results and analysis:

[0143] 1) Reconstitution time: <30S (meets the requirements); appearance and visible foreign matter: meets the requirements, no visible foreign matter; potency: 54 IU / ml (meets the requirements); molecular size distribution: multimers 0.48%, monomers 99.52% (meets the requirements).

[0144] 2) The overall shape of the freeze-dried product is good, with a full appearance, no atrophy, no collapse, and no dense film layer on the top; it has a good rigid structure and will not break when shaken violently. Figure 4 , Figure 5 ).

[0145] The above results show that the pre-freezing stage in this example uses a 2-step cooling procedure, and the drying stage uses a 3-step heating procedure, and the antithrombin III freeze-dried product obtained can meet the pharmacopoeia quality standards.

[0146] Example 6

[0147] In this example, human plasma-derived antithrombin III was freeze-dried according to the following method:

[0148] 1. Provide a freeze-dried sample of human plasma-derived antithrombin III, which contains 6 g / L of antithrombin III, 3% mannitol, 0.2% sodium citrate, and 0.8% sodium chloride.

[0149] 2. Process the freeze-dried sample according to the following table to obtain an antithrombin III freeze-dried product.

[0150] Table 6 Freeze-drying process parameters of Example 6

[0151]

[0152]

[0153] Note: 1) "Set time" refers to the time required to reach the "set temperature" of this step, which describes the rate of cooling / heating, and the cooling rate is controlled at 0.1-2.5°C / min; 2) "Controlled vacuum" refers to the vacuum condition that the relatively closed space of the sample needs to reach in this step. The same below; 3) " / " means not to control the vacuum degree, which is under normal pressure.

[0154] 3. Detect the potency, reconstitution time, molecular size distribution of the final freeze-dried product, and observe whether the appearance of the product meets the requirements of the pharmacopoeia.

[0155] Results and analysis:

[0156] 1) Reconstitution time: <30S (meets the requirements); appearance and visible foreign matter: meets the requirements, no visible foreign matter; potency: 54 IU / ml (meets the requirements); molecular size distribution: multimers 0.54%, monomers 99.46% (meets the requirements).

[0157] 2) The overall shape of the freeze-dried product is good, with a full appearance, no atrophy, no collapse, and no dense film layer on the top; it has a good rigid structure and will not break when shaken violently. Figure 6 , Figure 7 ).

[0158] The above results show that, on the basis of the two-step cooling and three-step heating procedure of Example 5, the freeze-drying time of the pre-freezing stage and the primary drying stage was adjusted, and the freeze-dried antithrombin III product thus obtained met the pharmacopoeia quality standards.

[0159] Example 7

[0160] In this example, human plasma-derived antithrombin III was freeze-dried according to the following method:

[0161] 1. A freeze-dried sample of human plasma-derived antithrombin III was provided, which contained 6 g / L antithrombin III, 3% glycine, 0.2% sodium citrate, and 0.8% sodium chloride.

[0162] 2. The freeze-dried sample was processed according to the following table to obtain a freeze-dried antithrombin III product.

[0163] Table 7 Freeze-drying process parameters of Example 7

[0164]

[0165]

[0166] Note: 1) "Set time" refers to the time required to reach the "set temperature" of the step, which is used to describe the rate of cooling / heating, and the cooling rate is controlled at 0.1-2.5°C / min; 2) "Controlled vacuum" refers to the vacuum condition that the relatively closed space of the sample needs to reach in this step. The same applies below; 3) " / " means that the vacuum degree is not controlled, and it is under normal pressure.

[0167] 3. The potency, reconstitution time, molecular size distribution of the final freeze-dried product were detected, and the appearance of the product was observed to see if it met the pharmacopoeia requirements.

[0168] Results and analysis:

[0169] 1) Reconstitution time: <30 s (meets the requirements); appearance and visible foreign matter: meets the requirements, no visible foreign matter; potency: 52 IU / ml (meets the requirements); molecular size distribution: multimers 0.53%, monomers 99.47% (meets the requirements).

[0170] 2) The freeze-dried product was well shaped as a whole, with a full appearance, no atrophy, no collapse, and no dense film layer on the top; it had a good rigid structure and was not easily broken by violent shaking. Figure 8 、 Figure 9 ).

[0171] The above results show that, on the basis of the two-step cooling and three-step heating procedure, the freeze-drying time of the pre-freezing stage and the primary drying stage was adjusted, and the freeze-dried antithrombin III product thus obtained met the pharmacopoeia quality standards.

[0172] Example 8

[0173] In this example, human plasma-derived antithrombin III was lyophilized according to the following method:

[0174] 1. A lyophilized sample of human plasma-derived antithrombin III was provided, which contained 6 g / L antithrombin III, glycine 3%, sodium citrate 0.2%, sodium chloride 0.8%.

[0175] 2. The lyophilized sample was processed according to the following table to obtain a lyophilized antithrombin III product.

[0176] Table 8 Lyophilization process parameters of Example 8

[0177]

[0178] Note: 1) "Set time" refers to the time required to reach the "set temperature" of this step, which is used to describe the rate of temperature decrease / increase, and the temperature decrease rate is controlled at 0.1-2.5°C / min; 2) "Controlled vacuum" refers to the vacuum condition that the relatively closed space of the sample needs to reach in this step. The same applies below; 3) " / " means that the vacuum degree is not controlled, and it is under normal pressure.

[0179] 3. The potency, reconstitution time, molecular size distribution of the final lyophilized product were detected, and whether the appearance of the product met the requirements of the pharmacopoeia was observed.

[0180] Results and analysis:

[0181] 1) Reconstitution time: <30 s (meets the requirements); appearance and visible foreign matter: meets the requirements, no visible foreign matter; potency: 52 IU / ml (meets the requirements); molecular size distribution: multimers 0.43%, monomers 99.57% (meets the requirements).

[0182] 2) The overall shape of the lyophilized product was good, with a full appearance, no atrophy, no collapse, and no dense film layer on the top; it had a good rigid structure and was not easily broken by violent shaking Figure 10 、 Figure 11 ).

[0183] The above results show that the use of two-step gradient cooling in the pre-freezing stage and three-step gradient heating in the drying stage are essential for obtaining a lyophilized antithrombin III product that meets the quality standards of the pharmacopoeia.

[0184] Although the embodiments of the present application have been shown and described above, it should be understood that the above embodiments are exemplary and should not be construed as limiting the present application, and those of ordinary skill in the art can make changes, modifications, replacements and variations to the above embodiments within the scope of the present application.

Claims

1. A method of improving the appearance of a lyophilized antithrombin III preparation, characterized in that, The method comprises: subjecting a sample containing antithrombin III to a pre-freezing treatment and a sublimation drying treatment; the pre-freezing treatment comprises at least two temperature lowering steps, each of the temperature lowering steps independently has a temperature of -60 to -20°C; the sublimation drying treatment comprises at least three temperature raising steps, each of the temperature raising steps independently has a temperature of -45 to 0°C.

2. The method of claim 1, wherein, each of the temperature lowering steps independently has a temperature of -57 to -23°C, preferably -55 to -25°C; optionally, each of the temperature lowering steps independently has a temperature lowering time of 5 to 150 min, preferably 10 to 120 min, more preferably 15 to 120 min; optionally, each of the temperature lowering steps independently has a maintaining time of 10 to 700 min, preferably 15 to 700 min, more preferably 15 to 600 min; optionally, each of the temperature lowering steps independently has a vacuum pressure of normal pressure.

3. The method of claim 1, wherein, the first temperature lowering step has a temperature of -40 to -20°C, preferably -35 to -25°C; optionally, the first temperature lowering step has a temperature lowering time of 10 to 120 min, preferably 15 min; optionally, the first temperature lowering step has a maintaining time of 10 to 200 min, preferably 180 min.

4. The method of claim 1, wherein, the second temperature lowering step has a temperature of -60 to -50°C, preferably -55°C; optionally, the second temperature lowering step has a temperature lowering time of 10 to 20 min, preferably 15 min; optionally, the second temperature lowering step has a maintaining time of 300 to 700 min, preferably 360 min.

5. The method of claim 1, wherein, each of the temperature raising steps independently has a temperature of -43 to -0°C, preferably -40 to -0°C; optionally, each of the temperature raising steps independently has a temperature raising time of 10 to 300 min, preferably 20 to 270 min, more preferably 30 to 240 min; optionally, each of the temperature raising steps independently has a maintaining time of 280 to 4400 min, preferably 300 to 4250 min, more preferably 300 to 4200 min; optionally, each of the temperature raising steps independently has a vacuum pressure of 0.05 to 0.5 mbar, preferably 0.1 to 0.3 mbar.

6. The method of claim 1, wherein, the first temperature raising step has a temperature of -45 to -35°C, preferably -40°C; optionally, the first temperature raising step has a temperature raising time of 20 to 40 min, preferably 30 min; optionally, the first temperature raising step has a maintaining time of 360 to 960 min, preferably 480 min; optionally, the first temperature raising step has a vacuum pressure of 0.2 to 0.4 mbar, preferably 0.25 to 0.35 mbar, more preferably 0.3 mbar.

7. The method of claim 1, wherein, the second temperature raising step has a temperature of -40 to -20°C, preferably -30°C; optionally, the second temperature raising step has a temperature raising time of 100 to 300 min, preferably 120 min; optionally, the second temperature raising step has a maintaining time of 2200 to 4400 min, preferably 4000 min; Optionally, the vacuum pressure of the second temperature increasing step is 0.1-0.3 mbar, preferably 0.15-0.25 mbar, more preferably 0.2 mbar.

8. The method of claim 1, wherein, The temperature of the third temperature increasing step is -10-0℃, preferably 0℃; Optionally, the temperature increasing time of the third temperature increasing step is 40-120 min, preferably 60 min; Optionally, the maintaining time of the third temperature increasing step is 280-1400 min, preferably 300 min; Optionally, the vacuum pressure of the third temperature increasing step is 0.05-0.2 mbar, preferably 0.075-0.15 mbar, more preferably 0.1 mbar.

9. The method of claim 1, wherein, The method further comprises: resolving drying treatment; Optionally, the temperature of the resolving drying is 25-35℃, preferably 30℃; Optionally, the temperature increasing time of the resolving drying is 200-300 min, preferably 240 min; Optionally, the maintaining time of the resolving drying is no less than 300 min, preferably 600 min; Optionally, the vacuum pressure of the resolving drying is 0.001-0.03 mbar; Optionally, the sample comprises one or more of freeze-drying protective agents, buffers, surfactants; Optionally, the sample comprises at least one selected from glycine or its salt, lysine or its salt, tyrosine or its salt, aspartic acid or its salt, arginine and its salt, sodium citrate, mannitol, alanine, sucrose, sodium chloride, polysorbate, tris-hydroxymethyl aminomethane and sodium phosphate; Optionally, the sample comprises: glycine, sodium citrate, sodium chloride; Optionally, the sample comprises: 3% of the glycine, 0.2% sodium citrate, 0.8% sodium chloride, based on the total volume of the sample; Optionally, the sample contains 3-9 g / L of the antithrombin III, preferably 6 g / L, based on the total volume of the sample.

10. A method of improving the appearance of a lyophilized antithrombin III preparation, characterized in that, The method comprises: The sample containing antithrombin III is subjected to pre-freezing treatment, sublimation drying treatment and resolving drying treatment; The pre-freezing treatment comprises: (1) pre-cooling; (2) first temperature decreasing step: decreasing to -35℃ within 15 min, and maintaining for 180 min under normal pressure; (3) second temperature decreasing step: decreasing to -55℃ within 15 min, and maintaining for 360 min under normal pressure; The sublimation drying treatment comprises: (4) first temperature increasing step: increasing to -40℃ within 30 min, and maintaining for 480 min under vacuum pressure of 0.3 mbar; (5) second temperature increasing step: increasing to -30℃ within 120 min, and maintaining for 4000 min under vacuum pressure of 0.2 mbar; (6) third temperature increasing step: increasing to 0℃ within 60 min, and maintaining for 300 min under vacuum pressure of 0.1 mbar; The resolving drying treatment is: increasing to 30℃ within 240 min, and maintaining for 600 min under vacuum pressure of 0.01 mbar; Optionally, the temperature of the pre-cooling step is 0-2℃; preferably 1-2℃; Optionally, the sample contains 3% of the glycine, 0.2% sodium citrate, 0.8% sodium chloride, by total volume of the sample. Optionally, the sample contains 3-9 g / L of the antithrombin III, preferably 6 g / L, by total volume of the sample.

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