Thrombin freeze-drying method

Abstract

The invention provides a thrombin freeze-drying method. The thrombin freeze-drying method comprises the following steps: carrying out pre-freezing, sublimation drying and desorption drying on a sample containing thrombin, wherein the sublimation drying stage comprises at least three heating steps; the temperature of each heating step is less than or equal to 0 DEG C; the pre-freezing stage comprises a primary freezing step, and the temperature of the freezing step is less than or equal to-45 DEG C. According to the method, the titer loss of the thrombin freeze-dried sample in the freeze-drying process can be effectively reduced, the process is stable, the product percent of pass is high, and the thrombin freeze-dried product obtained through the method has the advantages of being good in forming, high in titer recovery rate, high in uniformity and the like and is high in application value.

Description

Technical Field

[0001] The invention relates to the field of biopharmaceuticals, and in particular to a thrombin freeze-drying method. Background Art

[0002] Thrombin is a proteolytic enzyme that is converted from prothrombin (coagulation factor II) during the coagulation process. It directly acts on the last step of the blood coagulation process, prompting the soluble fibrinogen in the plasma to be converted into insoluble fibrin, thereby achieving the purpose of rapid hemostasis. Thrombin also participates in the activation of coagulation factor V and coagulation factor VIII, plays a role in stabilizing fibrin, and can also promote the mitosis of epithelial cells and accelerate wound healing. Therefore, thrombin is a fast-acting local hemostatic drug, suitable for ligation of small blood vessels, capillaries, bleeding of solid organs and other various bleeding that are difficult to stop bleeding, and has important clinical value.

[0003] Human thrombin freeze-dried products are mainly products obtained by lyophilizing human thrombin through plasma separation and purification and with auxiliary materials (such as stabilizers, crystallization excipients, etc.), and are widely produced and used at present. Specifically, human thrombin is extracted from healthy human plasma, and through processing steps such as DEAE A50 gel adsorption elution, calcium chloride activation, S / D virus inactivation, nano-membrane filtration and freeze drying, human thrombin freeze-dried products are finally obtained. Wherein, human thrombin is unstable after activation, which affects the titer recovery rate of subsequent process steps, especially the freeze drying step, and the activity loss is serious, and the titer recovery rate is low. Meanwhile, human thrombin freeze-dried products also often appear problems such as shrinkage in appearance, irregular shape, poor molding, etc., which affects the product qualification rate.

[0004] Therefore, the freeze-drying method of thrombin still needs to be studied. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present invention provides a thrombin freeze-drying method, which can effectively reduce the potency loss of thrombin freeze-dried samples during the freeze-drying process, has a stable process and a high product qualification rate, and the thrombin freeze-dried products obtained by the method have the advantages of good molding, high potency recovery rate and high uniformity, and have high application value.

[0006] It should be noted that the present invention is completed based on the following work of the inventors:

[0007] In view of the problem that human thrombin is unstable after activation and its activity is easily affected during freeze drying, the existing marketed external use frozen human thrombin freeze-dried products optimize the formulation formula and select human albumin, sodium chloride and calcium chloride as freeze-drying protective agents to improve the potency recovery rate of human thrombin freeze drying process. During the process development, the inventors rationally designed the freeze drying process and found that the condition control in the sublimation drying stage is of great significance for reducing freeze drying damage of thrombin and improving the quality of freeze-dried drugs.

[0008] The inventors further optimized the freeze-drying process and obtained a thrombin freeze-drying method in which the sublimation drying stage includes at least three temperature-raising steps. The method can effectively reduce the potency loss of thrombin freeze-dried samples during the freeze-drying process. The thrombin freeze-dried products obtained by the method have the advantages of good molding, low potency loss rate and high uniformity, and have high application value.

[0009] Therefore, in one aspect of the present invention, the present invention provides a thrombin freeze-drying method. The method comprises:

[0010] The sample containing thrombin was processed in three stages: pre-freezing, sublimation drying and analytical drying;

[0011] Wherein, the sublimation drying stage includes at least three heating steps; the temperature of each heating step is ≤0°C;

[0012] The pre-freezing stage includes a freezing step, and the temperature of the freezing step is ≤ -5°C.

[0013] It should be noted that, in this article, the temperatures of the "at least three heating steps" in the sublimation drying stage are increased sequentially.

[0014] The inventors found during the process of thrombin process development that introducing at least three heating steps in the sublimation drying stage can effectively reduce the potency loss of thrombin freeze-dried samples during the freeze-drying process, thereby obtaining a thrombin product that meets the requirements of the pharmacopoeia.

[0015] According to an embodiment of the present invention, the temperature of each heating step is independently -53 to 0°C.

[0016] According to an embodiment of the present invention, the heating time of each heating step is independently 20 to 420 minutes.

[0017] According to an embodiment of the present invention, the maintenance time of each temperature increasing step is independently 30 to 1260 minutes.

[0018] Therefore, within the above temperature range, the potency loss of the thrombin freeze-dried sample during the freeze-drying process is further reduced.

[0019] It should be noted that, in the present invention, the temperature reduction / heating rate is limited by limiting the time taken to reach a specific temperature.

[0020] It should be noted that, in the present invention, the duration of controlling the specific temperature is limited by limiting the maintenance time after reaching the specific temperature.

[0021] After extensive process exploration, the inventors obtained the temperature range, heating time and temperature maintenance time of the heating step in the above-mentioned sublimation drying stage, thereby further reducing the potency loss during the freeze-drying process and obtaining a thrombin freeze-dried product with high activity and qualified appearance.

[0022] According to an embodiment of the present invention, when the temperature of the heating step is -53 to -20°C, the heating time of the heating step is independently 20 to 420 minutes, preferably 30 to 390 minutes; more preferably 210 to 390 minutes. The inventor unexpectedly found in the experiment that the heating rate under the temperature condition of -53 to -20°C is important for reducing the potency loss of thrombin freeze-drying process. At this heating rate, the potency recovery rate of the freeze-dried product is significantly improved.

[0023] According to an embodiment of the present invention, when the temperature of the heating step is -53 to -20°C, the maintenance time of the heating step is independently 540 to 1260 minutes, 570 to 1200 minutes, and more preferably 600 to 810 minutes. Thus, the potency recovery rate of the freeze-dried product can be further improved.

[0024] According to an embodiment of the present invention, when the temperature of the heating step is -15 to 0°C, the heating time of the heating step is independently 30 to 150 minutes. Thus, the potency recovery rate of the freeze-dried product can be further improved.

[0025] According to an embodiment of the present invention, when the temperature of the temperature-raising step is -15 to 0°C, the maintenance time of the temperature-raising step is independently 30 to 810 minutes. Thus, the potency recovery rate of the freeze-dried product can be further improved.

[0026] According to an embodiment of the present invention, the absolute value of the temperature difference between two adjacent temperature increasing steps is ≤30° C., preferably ±10 to ±25° C. Thus, the potency recovery rate of the freeze-dried product can be further improved, the product appearance can be improved, and the product qualification rate can be increased.

[0027] According to an embodiment of the present invention, the sublimation drying stage includes at least four heating steps, and the temperatures of the four heating steps are respectively -53 to -50°C, -30 to -25°C, -10 to -5°C, and -5 to 0°C. The inventors have conducted in-depth research and found that if the temperature change of the heating step in the sublimation drying stage is controlled within the above range, the appearance of the freeze-dried product can be further improved, the potency recovery rate can be increased, and the consistency of the freeze-dried product can be improved.

[0028] According to an embodiment of the present invention, the heating time of the four heating steps is 30 to 270 minutes, 60 to 390 minutes, 40 to 90 minutes, and 90 to 150 minutes, respectively. When the heating rate is within the above range, the potency loss is low, the freeze-dried product has a good appearance, and the product qualification rate is high.

[0029] According to an embodiment of the present invention, the maintenance time of the four temperature increasing steps is 570-1260 minutes, 750-950 minutes, 30-90 minutes, and 750-810 minutes, respectively. By controlling the temperature maintenance time within the above range, a freeze-dried product with high potency recovery rate can be obtained with less energy consumption and short process time.

[0030] According to an embodiment of the present invention, the vacuum pressure is 0.05-0.3 mbar.

[0031] In some embodiments of the present invention, the vacuum pressure can be 0.05 mbar, 0.1 mbar, 0.15 mbar, 0.2 mbar, 0.25 mbar or 0.3 mbar. In combination with the actual situation of the freeze-drying equipment, the vacuum pressure is controlled between 0.05 and 0.3 mbar. The lower the pressure, the better the sublimation effect.

[0032] The inventors creatively obtained the parameter conditions of the temperature rising steps in the above four sublimation drying stages in a pilot-scale production test, thereby obtaining a thrombin freeze-drying process with stable process and high product qualification rate while ensuring a high potency recovery rate.

[0033] According to an embodiment of the present invention, the analytical drying includes at least two heating steps. After the sublimation drying stage, there is still about 10% moisture in the product, and this part of moisture is adsorbed on the capillary walls and polar groups of the dry substance. When they reach a certain content, they provide conditions for the growth and reproduction of microorganisms and certain chemical reactions. Therefore, the analytical drying stage further removes residual moisture, thereby improving the storage stability of the product and extending its shelf life. The inventors have found that adding a heating step in the analytical drying treatment stage can not only reduce the moisture content of the freeze-dried product, but also increase the potency recovery rate of the freeze-dried product, thereby further improving the quality and yield of the thrombin freeze-dried product.

[0034] According to an embodiment of the present invention, the temperature of each heating step is independently 5 to 50°C.

[0035] According to an embodiment of the present invention, the heating time of each heating step is independently 30 to 150 minutes, and the maintaining time of each heating step is independently 30 to 750 minutes.

[0036] According to an embodiment of the present invention, when the temperature of the heating step is 5 to 15° C., the heating time of the heating step is independently 30 to 90 minutes, and the holding time of the heating step is independently 10 to 90 minutes. Thus, the potency recovery rate of the freeze-dried product can be further improved and the moisture content of the freeze-dried product can be reduced.

[0037] According to an embodiment of the present invention, when the temperature of the temperature-raising step is 20-30° C., the temperature-raising time of the temperature-raising step is independently 90-150 minutes, and the holding time of the temperature-raising step is independently 510-750 minutes. Thus, the potency recovery rate of the freeze-dried product can be further improved and the moisture content of the freeze-dried product can be reduced.

[0038] According to an embodiment of the present invention, the temperatures of the two heating steps are 5-15° C. and 20-30° C. respectively.

[0039] According to an embodiment of the present invention, the heating time of the two heating steps is 30 to 90 minutes and 90 to 150 minutes, respectively, preferably 45 to 75 minutes and 105 to 135 minutes.

[0040] According to an embodiment of the present invention, the maintenance time of the two heating steps is 10 to 90 minutes and 510 to 750 minutes, respectively, preferably 45 to 75 minutes and 690 to 750 minutes.

[0041] According to an embodiment of the present invention, the vacuum pressure is 0-0.1 mbar.

[0042] In some embodiments of the present invention, the vacuum pressure can be exemplarily 0.01mbar, 0.02mbar, 0.03mbar, 0.04mbar, 0.05mbar, 0.06mbar, 0.07mbar, 0.08mbar, 0.09mbar, 0.01mbar, 0.03mbar, 0.05mbar, 0.07mbar, 0.09mbar or 0.1mbar. In combination with the actual situation of the freeze-drying equipment, the vacuum pressure is controlled between 0 and 0.1mbar, and the lower the pressure, the better the analytical drying effect.

[0043] After analyzing a large amount of pilot test data, the inventors obtained the process parameters such as the number of heating steps, temperature range, heating time and holding time in the above-mentioned analytical drying stage, thereby further improving the potency recovery rate of the freeze-dried product, reducing the moisture content of the freeze-dried product, and improving the potency recovery rate of the freeze-dried product.

[0044] According to an embodiment of the present invention, the temperature of the freezing step is -55 to -45° C. Thus, the sample is frozen to a low temperature and maintained until the sample is completely frozen and crystallized.

[0045] According to an embodiment of the present invention, the cooling time of the freezing step is 5 to 90 minutes, and the holding time is 20 to 360 minutes; further, the cooling time of the freezing step is 15 to 75 minutes, and the holding time is 30 to 300 minutes. Thus, the obtained freeze-dried product has a good appearance and a short reconstitution time.

[0046] By controlling the pre-freezing rate and pre-freezing time within the above conditions, the sample can be frozen solid with less energy consumption and in a short process time. As a result, the thrombin activity in the freeze-dried sample is retained to the maximum extent, and the probability of product quality defects such as unqualified moisture content and appearance that does not meet the pharmacopoeia requirements of the freeze-dried product is effectively reduced. As a result, a thrombin freeze-drying process suitable for production levels is developed.

[0047] Therefore, by using the method of the present invention to treat a sample containing thrombin, the obtained lyophilized thrombin product (such as a lyophilized human thrombin product) has the advantages of high specific activity, good molding, high uniformity, etc., and has high clinical application value.

[0048] According to an embodiment of the present invention, the pre-freezing stage includes: a. maintaining 0°C for 20 to 360 minutes; b. cooling to -75 to -50°C within 5 to 90 minutes and maintaining for 150 to 360 minutes;

[0049] The sublimation drying stage includes: at least four heating steps, the temperatures of the four heating steps are -53 to -50°C, -30 to -25°C, -10 to -5°C and -5 to 0°C, the heating time of the four heating steps is 30 to 270 minutes, 60 to 390 minutes, 40 to 90 minutes and 90 to 150 minutes, the holding time of the four heating steps is 570 to 1260 minutes, 750 to 950 minutes, 30 to 90 minutes and 750 to 810 minutes, and the vacuum pressure is controlled to be 0.05 to 0.3 mbar;

[0050] The analytical drying stage includes: at least two heating steps; the temperatures of the two heating steps are 5-15°C and 20-30°C respectively; the heating time of the two heating steps is 30-90 minutes and 90-150 minutes respectively, the holding time of the two heating steps is 10-90 minutes and 510-750 minutes respectively, and the vacuum pressure is controlled to be 0-0.1 mbar.

[0051] In another aspect of the present invention, a method for freeze-drying thrombin is provided. The method comprises:

[0052] The sample containing thrombin was processed in three stages: pre-freezing, sublimation drying and analytical drying;

[0053] The pre-freezing stage includes: a. maintaining 0°C for 30 minutes; b. cooling to -55°C within 15 minutes and maintaining for 180 minutes;

[0054] The sublimation drying stage includes: at least four heating steps, the temperatures of the four heating steps are -50°C, -28°C, -10°C and 0°C, the heating time of the four heating steps is 240 minutes, 360 minutes, 60 minutes and 120 minutes, the holding time of the four heating steps is 600 minutes, 780 minutes, 60 minutes and 780 minutes, and the vacuum pressure is controlled to be 0.1 mbar;

[0055] The analytical drying stage includes: at least two heating steps, the temperatures of the two heating steps are 10°C and 30°C respectively, the heating times of the two heating steps are 60 minutes and 120 minutes respectively, the holding times of the two heating steps are 60 minutes and 720 minutes respectively, and the vacuum pressure is controlled to be 0 mbar.

[0056] Therefore, the thrombin freeze-drying method according to the embodiment of the present invention has the advantages of high titer recovery rate, stable process, high product qualification rate, etc., and can be applied to the mass production of thrombin freeze-dried products. The thrombin freeze-dried products prepared thereby have high specific activity, good molding, high uniformity, stable product quality, and high clinical application value.

[0057] According to an embodiment of the present invention, the sample contains one or more of 8-20 g / L of glycine, 3-12 g / L of sodium chloride, 30-70 μg / mL of polysorbate, and 2-5 g / L of amino acid hydrochloride.

[0058] It should be noted that the present invention does not impose strict restrictions on the source of thrombin, which may be human thrombin, bovine thrombin, sheep thrombin, porcine thrombin, etc.; it may be derived from mammalian plasma or from genetic recombination technology.

[0059] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0061] Figure 1 The following are appearance pictures of freeze-dried products in the examples and comparative examples of the present invention, wherein, from left to right, they are appearance pictures of freeze-dried thrombin products prepared in Examples 1 to 5 and Comparative Examples 1 to 6, respectively. DETAILED DESCRIPTION

[0062] 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 understood as limiting the present invention.

[0063] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0064] The endpoints and any values ​​of the ranges disclosed in this article 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 each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0065] Terms and Definitions

[0066] In the text, the term "freezing step" refers to the step in which the sample is cooled from the initial temperature of the pre-freezing stage or the temperature of the previous annealing step, and this step does not include a temperature increase step. The term "annealing step" refers to the step in which the sample is heated up after the freezing step, and this step does not include a temperature decrease step. The purpose of the freezing step is to quickly freeze the sample containing thrombin, and the purpose of the annealing step is to further promote the freezing and crystallization of the sample containing thrombin, and to avoid the phenomenon that the eutectic point resistance curve of the sample decreases during the temperature increase stage of sublimation drying.

[0067] In the text, the term "sample" refers to any intermediate product that contains active ingredients and has not reached the freeze-drying endpoint. In combination with the context, it is equivalent to "lyophilized sample", "sample to be freeze-dried", "human thrombin sample", and "human thrombin freeze-dried sample".

[0068] In the text, the term "product" refers to the finished product that contains active ingredients and reaches the freeze-dried end point. Combined with the context, it is equivalent to "lyophilized product", "human thrombin product", and "human thrombin lyophilized product".

[0069] In this article, the term "sublimation drying" is equivalent to "primary drying", which refers to a drying method in which a water-containing material is frozen below the freezing point, the water is converted into ice, and then the ice is converted into vapor under a relatively high vacuum and removed. The material can be frozen in a freezing device before being dried. However, it can also be directly frozen in a drying chamber by rapidly evacuating the chamber. The water vapor generated by sublimation is removed by a condenser. The vaporization heat required in the sublimation process is generally supplied by thermal radiation.

[0070] In this article, the term "analytical drying" is equivalent to "secondary drying", which refers to a drying method in which the material is placed under negative pressure conditions and appropriately heated to reach the boiling point under negative pressure or cooled to solidify the material and then dried.

[0071] In this article, the term "maintenance time" is equivalent to "holding time", which refers to the length of time that a sample continues to maintain after reaching a specific state. In combination with the context, it is equivalent to "time" in some cases.

[0072] In this article, the term "set time" refers to the length of time required for a sample to reach a specific state. According to an embodiment of the present invention, "set time" specifically refers to the time required to reach a specific step "set temperature", which is used to describe the rate of cooling / heating. In combination with the context, in some cases it is equivalent to "heating rate" or "cooling rate" or "heating time" or "cooling time".

[0073] In this article, the term "set temperature" refers to the temperature that the sample is to reach in a specific step. In combination with the context, it is equivalent to "temperature" in some cases.

[0074] In this article, the term "moisture content" is equivalent to "residual moisture content" and refers to the percentage of moisture in the product or finished product to the total mass. The moisture content of a freeze-dried product is usually related to its storage stability.

[0075] The present invention provides a thrombin freeze-drying method, which comprises: subjecting a sample containing thrombin to three-stage processing of pre-freezing, sublimation drying and analytical drying; wherein the sublimation drying stage comprises at least three heating steps.

[0076] According to an embodiment of the present invention, the freeze-drying method of the present invention is suitable for freeze-drying a thrombin freeze-dried sample with a titer of 700 to 5000 IU / mL, including but not limited to: human thrombin, bovine thrombin, sheep thrombin, porcine thrombin, and the like.

[0077] According to an embodiment of the present invention, the thrombin is human thrombin. The source of the human thrombin can be human plasma or obtained by gene recombination.

[0078] Unless otherwise specified, the method for preparing thrombin from human plasma in the embodiments of the present invention is as follows:

[0079] (1) The plasma is combined and centrifuged to remove the cryoprecipitate. The plasma supernatant after the cryoprecipitate is removed (hereinafter referred to as "cryoprecipitate-free plasma").

[0080] (2) The temperature of the cryoprecipitated plasma is raised to 8-22° C. DEAE-Sephadex A50 (DEAE-A50) gel pre-equilibrated with equilibration solution A is added at 0.5-3% of the total weight of the plasma, stirred, and adsorbed for more than 30 minutes.

[0081] Formula of balanced solution A: Taking the preparation of 1kg solution as an example, add 5.88g sodium citrate, 4.68g sodium chloride, 5-15g glycine, add WFI (water for injection) to 1kg, stir until completely dissolved. Add 1mol / L hydrochloric acid solution to adjust the pH to 7.0±0.3.

[0082] (3) Collect the DEAE-A50 gel, wash it with equilibration solution A, discard the supernatant, and remove the impurities; finally, elute it with elution solution A, collect the eluted protein solution, concentrate it by ultrafiltration, desalt it by dialysis, and re-concentrate it to collect the ultrafiltration concentrate (the weight of the concentrate is controlled at 10 to 40 kg / ton of plasma), and obtain the DEAE eluate.

[0083] Formula of balancing solution A: same as in step (2) above.

[0084] Formula of eluent A: Taking the preparation of 1kg solution as an example, add 6g sodium citrate, 100g sodium chloride, 5-15g glycine, add WFI (water for injection) to 1kg, stir until completely dissolved. Add 1mol / L hydrochloric acid solution to adjust the pH to 7.0±0.3.

[0085] (4) Preparation of crude thrombin: The pH of the DEAE-A50 eluate was adjusted to 6.5±0.5 and the potency of coagulation factor II to 5-15 IU / mL, and 1 mol / L calcium chloride solution was added to make the final calcium ion concentration 20-50 mmol / L. After activation treatment, a crude thrombin solution with a human thrombin potency greater than 200 IU / mL was obtained.

[0086] (5) S / D reagent inactivates the virus.

[0087] (6) UniGel 50SP gel cation exchange chromatography to remove S / D reagent and inactivated protein; the buffer formula used in the cation exchange chromatography process is as follows:

[0088] Balance solution: Taking the preparation of 1kg solution as an example, add 2.92g sodium chloride, 15g glycine, 1.2g lysine hydrochloride, add 950g WFI, stir until completely dissolved, adjust the pH to 6.5±0.5, and then add WFI to 1kg.

[0089] Washing liquid: Taking the preparation of 1kg solution as an example, add 7.01g sodium chloride, 15g glycine, 1.2g lysine hydrochloride, add 950g WFI, stir until completely dissolved, adjust the pH to 6.5±0.5, and then add WFI to 1kg.

[0090] Eluent: Taking the preparation of 1kg solution as an example, add 23.4g sodium chloride, 15g glycine, 1.2g lysine hydrochloride, add 950g WFI, stir until completely dissolved, adjust the pH to 6.5±0.5, and then add WFI to 1kg.

[0091] (7) Nano-membrane filtration to remove viruses and prepare semi-finished products. The semi-finished product has a human thrombin titer of 700 IU / mL and a formulation of: glycine 5-25 g / L, sodium chloride 5-15 g / L, and polysorbate 80 20-70 μg / mL.

[0092] (8) Freeze drying.

[0093] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0094] Unless otherwise specified, the embodiments of the present invention use freeze-dried thrombin samples derived from human plasma to illustrate the freeze-drying method of thrombin of the present invention.

[0095] Unless otherwise specified, the quality inspection index data in the embodiments and comparative examples of the present invention are all average values.

[0096] Example 1

[0097] In this example, thrombin derived from human plasma was freeze-dried according to the following method:

[0098] 1. Provide a lyophilized sample of thrombin derived from human plasma, which contains human thrombin with a titer of 650 IU / mL, glycine 15 g / L, sodium chloride 7 g / L, and polysorbate 80 50 μg / mL.

[0099] 2. Treat the freeze-dried sample according to the conditions in the table below to obtain a freeze-dried thrombin product.

[0100] Table 1 Example 1 Freeze-drying process parameters

[0101]

[0102]

[0103] Notes: 1) "Set time" refers to the time required to reach the "set temperature" of this step, which is used to describe the rate of cooling / heating; 2) "Controlled vacuum" refers to the vacuum condition that needs to be achieved in the relatively closed space where the sample is located in this step; 3) " / " means that the vacuum degree is not controlled and the pressure is normal. The same below.

[0104] 3. Test or calculate the potency, potency recovery rate, pH value (after reconstitution), reconstitution time, moisture content of the final freeze-dried product, and observe whether the product appearance meets the requirements of the pharmacopoeia. The specific methods are as follows:

[0105] Human thrombin titer detection: refer to the "Human Thrombin Titer" detection method in the three monographs of the 2020 edition of the "Chinese Pharmacopoeia": Ⅱ Therapeutic Human Thrombin.

[0106] The titer recovery rate (%) was determined as follows: titer after lyophilization / titer before lyophilization × 100.

[0107] pH value: Refer to Part III of the 2020 edition of the Chinese Pharmacopoeia.

[0108] Reconstitution time: Refer to the detection method in Part III of the 2020 edition of the Chinese Pharmacopoeia: II Therapeutic Human Thrombin.

[0109] Moisture content: refer to the method in the first method of Part III General Chapter 0832 of the 2020 edition of the Chinese Pharmacopoeia.

[0110] Appearance inspection standard for freeze-dried products: refer to the detection method for therapeutic human thrombin in Part III II of the 2020 edition of the Chinese Pharmacopoeia.

[0111] Example 2

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

[0113] 1. Provide a freeze-dried sample of thrombin derived from human plasma, with specific reference to Example 1.

[0114] 2. Treat the freeze-dried sample according to the conditions in the table below to obtain a freeze-dried thrombin product.

[0115] Table 2 Example 2 Freeze-drying process parameters

[0116]

[0117]

[0118] 3. Detect or calculate the potency, potency recovery rate, pH value (after reconstitution), reconstitution time, and moisture content of the final freeze-dried product, and observe whether the appearance of the product meets the requirements of the pharmacopoeia. Specific method: Same as Example 1.

[0119] Example 3

[0120] In this example, thrombin derived from human plasma was freeze-dried according to the following method:

[0121] 1. Provide a freeze-dried sample of thrombin derived from human plasma, with specific reference to Example 1.

[0122] 2. Treat the freeze-dried sample according to the conditions in the table below to obtain a freeze-dried thrombin product.

[0123] Table 3 Freeze-drying process parameters of Example 3

[0124]

[0125] 3. Detect or calculate the potency, potency recovery rate, pH value (after reconstitution), reconstitution time, and moisture content of the final freeze-dried product, and observe whether the appearance of the product meets the requirements of the pharmacopoeia. Specific method: Same as Example 1.

[0126] Example 4

[0127] In this example, thrombin derived from human plasma was freeze-dried according to the following method:

[0128] 1. Provide a freeze-dried sample of thrombin derived from human plasma, with specific reference to Example 1.

[0129] 2. Treat the freeze-dried sample according to the conditions in the table below to obtain a freeze-dried thrombin product.

[0130] Table 4 Example 4 Freeze-drying process parameters

[0131]

[0132] 3. Detect or calculate the potency, potency recovery rate, pH value (after reconstitution), reconstitution time, and moisture content of the final freeze-dried product, and observe whether the appearance of the product meets the requirements of the pharmacopoeia. The specific method is the same as Example 1.

[0133] Example 5

[0134] In this example, thrombin derived from human plasma was freeze-dried according to the following method:

[0135] 1. Provide a lyophilized sample of thrombin derived from human plasma, which contains human thrombin with a titer of 650 IU / mL, 15 g / L of glycine, 7 g / L of sodium chloride, 3.5 g / L of proline hydrochloride, and 50 μg / mL of polysorbate 80.

[0136] 2. Treat the freeze-dried sample according to the freeze-drying conditions of Example 1 to obtain a freeze-dried thrombin product.

[0137] Example 6

[0138] Further, the inventors, with reference to the freeze-drying process of Example 1, trial-produced 9 batches of 3 specifications (1000 IU / bottle, 2500 IU / bottle, 5000 IU / bottle) of human plasma-derived human thrombin on a production scale, all of which were prepared at 650 IU / mL before freeze-drying. The key quality indicators of the finished product, such as potency, potency recovery rate, pH value, reconstitution time, and moisture content, were observed and tested. The results are shown in Table 5.

[0139] The results showed that the overall shape of the human thrombin freeze-dried product was full and the skeleton was loose. No shrinkage, collapse, cracks or melting were observed. The potency recovery rate was greater than 80%, the residual moisture content (≤1.5%) was low (the acceptable moisture range for human thrombin specified in the Chinese Pharmacopoeia: the moisture content does not exceed 3%), the pH value was stable, the differences between products were small, and the qualified rate of freeze-dried products was 100%.

[0140] Table 5 Key quality indexes of human thrombin finished product obtained by freeze-drying process production scale in Example 1

[0141]

[0142]

[0143] Comparative Example 1

[0144] In this example, thrombin derived from human plasma was freeze-dried according to the following method:

[0145] 1. Provide a freeze-dried sample of thrombin derived from human plasma, with specific reference to Example 1.

[0146] 2. Treat the freeze-dried sample according to the conditions in the table below to obtain a freeze-dried thrombin product.

[0147] Table 6 Freeze-drying process parameters of Comparative Example 1

[0148]

[0149] 3. Detect or calculate the potency, potency recovery rate, pH value (after reconstitution), reconstitution time, and moisture content of the final freeze-dried product, and observe whether the appearance of the product meets the requirements of the pharmacopoeia. Specific method: Same as Example 1.

[0150] Comparative Example 2

[0151] In this example, thrombin derived from human plasma was freeze-dried according to the following method:

[0152] 1. Provide a freeze-dried sample of thrombin derived from human plasma, with specific reference to Example 1.

[0153] 2. Treat the freeze-dried sample according to the conditions in the table below to obtain a freeze-dried thrombin product.

[0154] Table 7 Comparative Example 2 Freeze-drying Process Parameters

[0155]

[0156] 3. Detect or calculate the potency, potency recovery rate, pH value (after reconstitution), reconstitution time, and moisture content of the final freeze-dried product, and observe whether the appearance of the product meets the requirements of the pharmacopoeia. The specific method is the same as Example 1.

[0157] Comparative Example 3

[0158] In this comparative example, thrombin derived from human plasma was freeze-dried according to the following method:

[0159] 1. Provide a freeze-dried sample of thrombin derived from human plasma, with specific reference to Example 1.

[0160] 2. Referring to the freeze-drying process disclosed in CN201810585957.5, the freeze-dried sample was treated according to the conditions in the following table to obtain a freeze-dried thrombin product.

[0161] Table 8 Comparative Example 3 Freeze-drying Process Parameters

[0162]

[0163]

[0164] 3. Detect or calculate the potency, potency recovery rate, pH value (after reconstitution), reconstitution time, and moisture content of the final freeze-dried product, and observe whether the appearance of the product meets the requirements of the pharmacopoeia. The specific method is the same as Example 1.

[0165] Comparative Example 4

[0166] In this comparative example, thrombin derived from human plasma was freeze-dried according to the following method:

[0167] 1. Provide a freeze-dried sample of thrombin derived from human plasma, with specific reference to Example 1.

[0168] 2. Refer to the freeze-drying process disclosed in CN201810585957.5, optimize the analytical drying parameters, treat the freeze-dried sample according to the conditions in the table below, and obtain a thrombin freeze-dried product.

[0169] Table 9 Comparative Example 4 Freeze-drying Process Parameters

[0170]

[0171] 3. Detect or calculate the potency, potency recovery rate, pH value (after reconstitution), reconstitution time, and moisture content of the final freeze-dried product, and observe whether the appearance of the product meets the requirements of the pharmacopoeia. The specific method is the same as Example 1.

[0172] Comparative Example 5

[0173] In this comparative example, thrombin derived from human plasma was freeze-dried according to the following method:

[0174] 1. Provide a freeze-dried sample of thrombin derived from human plasma, with specific reference to Example 1.

[0175] 2. Referring to the freeze-drying process disclosed in CN201810585957.5, the sublimation drying parameters were optimized, and the freeze-dried samples were treated according to the conditions in the following table to obtain a thrombin freeze-dried product.

[0176] Table 10 Comparative Example 5 Freeze-drying Process Parameters

[0177]

[0178]

[0179] 3. Detect or calculate the potency, potency recovery rate, pH value (after reconstitution), reconstitution time, and moisture content of the final freeze-dried product, and observe whether the appearance of the product meets the requirements of the pharmacopoeia. The specific method is the same as Example 1.

[0180] Comparative Example 6

[0181] In this comparative example, thrombin derived from human plasma was freeze-dried according to the following method:

[0182] 1. Provide a freeze-dried sample of thrombin derived from human plasma, with specific reference to Example 1.

[0183] 2. Treat the freeze-dried sample according to the conditions in the table below to obtain a freeze-dried thrombin product.

[0184] Table 11 Freeze-drying process parameters of Comparative Example 6

[0185]

[0186] 3. Detect or calculate the potency, potency recovery rate, pH value (after reconstitution), reconstitution time, and moisture content of the final freeze-dried product, and observe whether the appearance of the product meets the requirements of the pharmacopoeia. The specific method is the same as Example 1.

[0187] Result analysis:

[0188] The results of the investigation of the potency, potency recovery rate, pH value (after reconstitution), reconstitution time, moisture content, and product appearance of the lyophilized thrombin products derived from human plasma of Examples 1 to 5 and Comparative Examples 1 to 6 are shown in Table 12.

[0189] The results showed that increasing the number of annealing steps in the pre-freezing stage or optimizing the temperature conditions of analytical drying or sublimation drying according to conventional ideas could not effectively improve the potency recovery rate; optimizing the temperature conditions of analytical drying or sublimation drying according to conventional ideas caused the products to shrink, not form a cake shape, and the appearance did not meet the requirements and had poor uniformity.

[0190] Table 12 Comparison of the quality of the freeze-dried products of Examples 1 to 5 and Comparative Examples 1 to 6

[0191]

[0192]

[0193] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, unless they are contradictory.

[0194] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A method for freeze-drying thrombin, It is characterized in that The method comprises: The sample containing thrombin was processed in three stages: pre-freezing, sublimation drying and analytical drying; Wherein, the sublimation drying stage includes at least three heating steps; the temperature of each heating step is ≤0°C; The pre-freezing stage includes a freezing step, and the temperature of the freezing step is ≤ -45°C.

2. The method according to claim 1, It is characterized in that The temperature of each heating step is independently -53 to 0°C; Optionally, the heating time of each heating step is independently 20 to 420 minutes; Optionally, the maintenance time of each of the heating steps is independently 30 to 1260 minutes; Optionally, when the temperature of the heating step is -53 to -20°C, the heating time of the heating step is independently 20 to 420 minutes, preferably 30 to 390 minutes; more preferably 210 to 390 minutes; Optionally, when the temperature of the heating step is -53 to -20°C, the maintenance time of the heating step is independently 540 to 1260 minutes, preferably 570 to 1200 minutes; more preferably 600 to 810 minutes; Optionally, when the temperature of the heating step is -15 to 0°C, the heating time of the heating step is independently 30 to 150 minutes; Optionally, when the temperature of the heating step is -15 to 0°C, the maintenance time of the heating step is independently 30 to 810 minutes.

3. The method according to claim 1 or 2, It is characterized in that The absolute value of the temperature difference between two adjacent heating steps is ≤30°C, preferably 10-25°C.

4. The method according to claim 1 or 2, It is characterized in that The sublimation drying stage includes at least four heating steps, and the temperatures of the four heating steps are -53 to -50°C, -30 to -25°C, -10 to -5°C, and -5 to 0°C, respectively; Optionally, the heating time of the four heating steps is 30 to 270 minutes, 60 to 390 minutes, 40 to 90 minutes, and 90 to 150 minutes, respectively; Optionally, the maintenance time of the four heating steps is 570 to 1260 minutes, 750 to 950 minutes, 30 to 90 minutes, and 750 to 810 minutes, respectively; Optionally, the vacuum pressure is 0.05-0.3 mbar.

5. The method according to claim 1, It is characterized in that The analytical drying comprises at least two heating steps; Optionally, the temperature of each heating step is independently 5 to 50° C.; Optionally, the heating time of each heating step is independently 30 to 150 minutes, and the maintaining time of each heating step is independently 30 to 750 minutes.

6. The method according to claim 5, It is characterized in that When the temperature of the heating step is 5 to 15°C, the heating time of the heating step is independently 30 to 90 minutes, and the holding time of the heating step is independently 0 to 90 minutes; Optionally, when the temperature of the heating step is 20 to 30° C., the heating time of the heating step is independently 90 to 150 minutes, and the holding time of the heating step is independently 510 to 750 minutes.

7. The method according to claim 6, It is characterized in that The temperatures of the two heating steps are 5-15°C and 20-30°C respectively; Optionally, the heating time of the two heating steps is 30 to 90 minutes, 90 to 150 minutes, preferably 45 to 75 minutes, 105 to 135 minutes; Optionally, the maintenance time of the two heating steps is 10 to 90 minutes, 510 to 750 minutes, preferably 45 to 75 minutes, 690 to 750 minutes; Optionally, the vacuum pressure is 0 to 0.1 mbar.

8. The method according to claim 1, It is characterized in that The temperature of the freezing step is -55 to -45°C; Optionally, the cooling time of the freezing step is 5 to 90 minutes, and the holding time is 20 to 360 minutes; further, the cooling time of the freezing step is 15 to 75 minutes, and the holding time is 30 to 300 minutes.

9. The method according to claim 1, It is characterized in that The pre-freezing stage includes: a. maintaining 0°C for 20 to 360 minutes; b. cooling to -75 to -50°C within 5 to 90 minutes and maintaining for 150 to 360 minutes; The sublimation drying stage includes: at least four heating steps, the temperatures of the four heating steps are -53 to -50°C, -30 to -25°C, -10 to -5°C and -5 to 0°C, the heating time of the four heating steps is 30 to 270 minutes, 60 to 390 minutes, 40 to 90 minutes and 90 to 150 minutes, the holding time of the four heating steps is 570 to 1260 minutes, 750 to 950 minutes, 30 to 90 minutes and 750 to 810 minutes, and the vacuum pressure is controlled to be 0.05 to 0.3 mbar; The analytical drying stage includes: at least two heating steps; the temperatures of the two heating steps are 5-15°C and 20-30°C respectively; the heating time of the two heating steps is 30-90 minutes and 90-150 minutes respectively, the holding time of the two heating steps is 10-90 minutes and 510-750 minutes respectively, and the vacuum pressure is controlled to be 0-0.1 mbar.

10. A method for freeze-drying thrombin, It is characterized in that The method comprises: subjecting a sample containing thrombin to three-stage processing: pre-freezing, sublimation drying and analytical drying; The pre-freezing stage includes: a. maintaining 0°C for 30 minutes; b. cooling to -55°C within 15 minutes and maintaining for 180 minutes; The sublimation drying stage includes: at least four heating steps, the temperatures of the four heating steps are -50°C, -28°C, -10°C and 0°C, the heating time of the four heating steps is 240 minutes, 360 minutes, 60 minutes and 120 minutes, the holding time of the four heating steps is 600 minutes, 780 minutes, 60 minutes and 780 minutes, and the vacuum pressure is controlled to be 0.1 mbar; The analytical drying stage includes: at least two heating steps, the temperatures of the two heating steps are 10°C and 30°C respectively, the heating times of the two heating steps are 60 minutes and 120 minutes respectively, the holding times of the two heating steps are 60 minutes and 720 minutes respectively, and the vacuum pressure is controlled to be 0 mbar.

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