Thrombin freeze-drying protective agent and thrombin freeze-drying preparation

Abstract

The invention provides a thrombin freeze-drying protective agent and a thrombin freeze-drying preparation. The thrombin freeze-drying protective agent comprises glycine, hydrochloric acid amino acid, sodium chloride and polysorbate. The thrombin freeze-drying protective agent can protect freeze-dried thrombin, avoid loss of biological activity of the freeze-dried thrombin, improve stability of the freeze-dried thrombin and facilitate storage and transportation of a thrombin freeze-drying preparation. In addition, the prepared human thrombin freeze-drying preparation is good in resolubility, high in clarity and high in application value.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a thrombin lyophilization protectant and a thrombin lyophilized preparation. Background Art

[0002] Thrombin is a proteolytic enzyme, which is converted from prothrombin (coagulation factor II) during the coagulation process. Thrombin 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. At the same time, thrombin also participates in the activation of coagulation factor V and coagulation factor VIII, thereby stabilizing fibrin, and can also promote the mitosis of epithelial cells and accelerate wound healing. It is a rapid-acting local hemostatic drug, suitable for bleeding of small blood vessels, capillaries, solid organs and other bleeding that are difficult to ligate.

[0003] As an enzyme protein, thrombin, like most biologically active proteins, has relatively poor stability and is easily inactivated and denatured. Therefore, thrombin preparations used as hemostatic drugs are generally made into freeze-dried preparations, which are white or off-white sterile freeze-dried blocks or powders and are required to be stored below 10°C. Freeze-drying is a complex phase change process. Freeze-dried products are subject to various stresses throughout the process, including low-temperature stress, freezing stress, drying stress, etc., which are often factors that directly or indirectly cause protein denaturation in the product, so protective agents need to be used during the freeze-drying process.

[0004] However, lyoprotectants for thrombin remain to be studied. Summary of the invention

[0005] It should be noted that the present invention is completed based on the following findings of the inventors:

[0006] The most commonly used protective agents for blood products include amino acids, albumin, sugars, and polyols, among which most protective agents contain glycine. The inventors initially tried to select glycine, human albumin, sodium chloride, and calcium chloride as protective agent compositions, which can protect thrombin during the freeze-drying process. Among them, human albumin mainly plays a role in improving stability, but its production cost is high, which restricts its large-scale use.

[0007] In view of this, the inventor attempts to select other substances to replace human albumin to play a role. After a large number of theoretical research analysis and experimental screening verification, the inventor creatively found that human albumin is replaced with hydrochloric acid amino acids, and glycine and hydrochloric acid amino acids are compounded, which can effectively extend the storage period of thrombin and reduce production costs. At the same time, adding polysorbate can increase protein surface activity, and the re-dissolution time is short, improve product quality, and facilitate clinical use. Adding sodium chloride can shorten the re-dissolution time and maintain protein activity. Finally, it is determined that the composition of the thrombin lyoprotectant includes glycine, hydrochloric acid amino acids, sodium chloride and polysorbate, and the thrombin lyoprotectant of the present invention can protect freeze-dried thrombin, avoid its biological activity loss, improve its stability, and facilitate thrombin lyophilized preparation storage and transportation. In addition, the prepared thrombin lyoprotectant has good solubility, high clarity, and high application value.

[0008] To this end, in one aspect of the present invention, the present invention provides a thrombin lyophilization protectant. According to an embodiment of the present invention, the thrombin lyophilization protectant comprises: glycine, amino acid hydrochloride, sodium chloride and polysorbate.

[0009] In the thrombin lyophilization protective agent according to the embodiment of the present invention, glycine has a good excipient effect, and has a significant effect in both the lyophilization and dry heat processes, and the clarity of the preparation after reconstitution is good. In addition, glycine also has the function of a protein protective agent, and the amino and carboxyl groups contained in it can make the lyophilized preparation have strong buffering properties.

[0010] The inventors found that glycine and hydrochloric acid amino acid compounding can effectively prolong the storage period of thrombin and reduce production costs. At the same time, adding polysorbate can increase protein surface activity, shorten the reconstitution time, improve product quality, and facilitate clinical use. Sodium chloride can make the lyophilized preparation present a loose mesh shape, significantly shortening the reconstitution time. Neutral sodium chloride can also increase the hydration degree of protein, protect the protein space chain from being directly exposed to the environment, thereby maintaining protein activity.

[0011] Therefore, the thrombin lyoprotectant according to the embodiment of the present invention can protect the lyophilized thrombin, avoid the loss of its biological activity, improve its stability, and facilitate the storage and transportation of the lyophilized thrombin preparation.

[0012] According to an embodiment of the present invention, the above-mentioned thrombin lyophilization protectant may also have the following additional technical features:

[0013] According to an embodiment of the present invention, the hydrochloric acid amino acid is selected from at least one of the following: proline hydrochloride, arginine hydrochloride and lysine hydrochloride. The above hydrochloric acid amino acids, especially proline hydrochloride, are used to synergistically improve the stability of thrombin lyophilized powder with glycine.

[0014] According to an embodiment of the present invention, the polysorbate is selected from at least one of the following: polysorbate 80, polysorbate 20, and polysorbate 60. The use of the above polysorbates, especially polysorbate 80, can further increase the surface activity of the protein, shorten the re-dissolution time, and improve the product quality.

[0015] According to an embodiment of the present invention, the content ratio of glycine to amino acid hydrochloride is (5-15): (2-5), preferably (9-11): (3-4). Thus, the above compounding ratio can further protect the freeze-dried thrombin, avoid the loss of its biological activity, and improve its stability.

[0016] In some embodiments, the content ratio of glycine to the amino acid hydrochloride is any value in (5-15): (2-5) or a range value between any two values, preferably any value in (9-11): (3-4) or a range value between any two values, for example 5:2, 5:3, 5:4, 5:5, 5:5, 6:2, 6:3, 6:4, 6:5, 7:2, 7:3, 7:4, 7:5, 7:5, 8:2, 8:3, 8:4, 8:5, 8:5, 8:2, 8:3, 8:4, 8:5, 9:2, 9:3, 9:4, 9:5, 10:2, 10:3, 10:4, 10:5, 10:5, 11:2, 11:3, 11:4, 11:5, 12:2, 12:3, 12:4, 12:5, 13:2, 13:3, 13:4, 13:5, 13:5, 14:2, 14:3, 14:4, 14:5, 15:2, 15:3, 15:4, 15:5, etc.

[0017] According to an embodiment of the present invention, based on the total volume of the thrombin lyoprotectant, the thrombin lyoprotectant includes: 1-20 g / L of glycine; 1-10 g / L of amino acid hydrochloride; 1-15 g / L of sodium chloride; and 20-90 μg / mL of polysorbate.

[0018] In some embodiments, based on the total volume of the thrombin lyoprotectant, the thrombin lyoprotectant includes: 8-12 g / L of glycine; 1-5 g / L of proline hydrochloride; 5-10 g / L of sodium chloride; and 40-60 μg / mL of polysorbate 80.

[0019] In some embodiments, the concentration of glycine is any value between 1 and 20 g / L or a range between any two values, preferably any value between 8 and 12 g / L or a range between any two values, for example, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L, etc.

[0020] In some embodiments, the concentration of the amino acid hydrochloride is any value in the range of 1 to 10 g / L or a range value between any two values, preferably any value in the range of 1 to 5 g / L or a range value between any two values, for example, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 4.5 g / L, 5 g / L, 5.5 g / L, 6 g / L, 7 g / L, 7.5 g / L, 8 g / L, 8.5 g / L, 9 g / L, 9.5 g / L, 10 g / L, etc.

[0021] In some embodiments, the concentration of sodium chloride is any value in the range of 1 to 15 g / L or a range between any two values, preferably any value in the range of 5 to 10 g / L or a range between any two values, for example, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, etc.

[0022] In some embodiments, the concentration of the polysorbate is any value in the range of 20 to 90 μg / mL or a range between any two values, preferably any value in the range of 40 to 60 μg / mL or a range between any two values, for example 20 μg / mL, 21 μg / mL, 22 μg / mL, 23 μg / mL, 24 μg / mL, 25 μg / mL, 30 μg / mL, 35 μg / mL, 40 μg / mL, 45 μg / mL, 50μg / mL, 55μg / mL, 60μg / mL, 65μg / mL, 70μg / mL, 72μg / mL, 75μg / mL, 77μg / mL, 79μg / mL, 80μg / mL, 81μg / mL, 82μg / mL, 83μg / mL, 84μg / mL, 85μg / mL, 86μg / mL, 87μg / mL, 88μg / mL, 89μg / mL, 90μg / mL, etc.

[0023] The inventors obtained the above-mentioned optimal ratio through a large number of experiments, thereby further protecting the freeze-dried thrombin, avoiding the loss of its biological activity and improving its stability.

[0024] According to an embodiment of the present invention, the thrombin is selected from thrombin of mammalian origin. In some embodiments, the mammal includes human, cattle, horse, pig or sheep.

[0025] In addition, the present invention proposes another human thrombin lyophilization protective agent. According to an embodiment of the present invention, based on the total volume of the human thrombin lyophilization protective agent, the human thrombin lyophilization protective agent includes: 10g / L of glycine, 3.5g / L of proline hydrochloride, 7g / L of sodium chloride, 50μg / mL of polysorbate 80 and water. The inventor obtained the above-mentioned preferred human thrombin lyophilization protective agent composition through a large number of experiments, which can be used to protect freeze-dried human thrombin, avoid the loss of its biological activity, improve its stability, and facilitate the storage and transportation of human thrombin lyophilized preparations.

[0026] In another aspect of the present invention, the present invention provides a thrombin freeze-dried preparation. According to an embodiment of the present invention, the thrombin freeze-dried preparation includes: thrombin; the thrombin freeze-dried protective agent described above or the human thrombin freeze-dried protective agent described above. As described above, the thrombin freeze-dried protective agent of the present invention can protect the freeze-dried thrombin, avoid the loss of its biological activity, and improve its stability. Therefore, the thrombin freeze-dried preparation of the present invention has strong stability, high biological activity, convenient storage and transportation, and high application value.

[0027] It should be noted that the present invention does not impose strict restrictions on the method for obtaining thrombin, and thrombin can be prepared by conventional techniques in the art, or obtained through commercial purchase or donation.

[0028] According to an embodiment of the present invention, based on the total volume of the thrombin lyoprotectant or the human thrombin lyoprotectant, the content of thrombin is 400-700 IU / mL. Thus, thrombin can further efficiently maintain stable biological activity in the thrombin lyoprotectant.

[0029] In another aspect of the present invention, the present invention proposes the use of the above-mentioned thrombin lyoprotectant or the human thrombin lyoprotectant in the preparation of lyophilized thrombin preparations. As mentioned above, the thrombin lyoprotectant of the present invention can protect lyophilized thrombin, avoid the loss of its biological activity, improve its stability, facilitate the storage and transportation of lyophilized thrombin preparations, and has high application value.

[0030] 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. DETAILED DESCRIPTION

[0031] 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.

[0032] Example 1

[0033] 1. Obtaining human thrombin

[0034] (1) Human plasma is combined, centrifuged at low temperature to remove cold precipitates, and the plasma supernatant after centrifugation to remove cold precipitates is collected, i.e., cryoprecipitate-free plasma.

[0035] (2) adding 3% of the total weight of the cryoprecipitated plasma to the pre-equilibrated DEAE-Sephadex A50 gel for adsorption, collecting the DEAE-A50 gel, and then washing and eluting with water to obtain a DEAE eluate;

[0036] Formula of washing solution: 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, and adjust the pH to 7.0±0.3;

[0037] Eluent formula: 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, and adjust the pH to 7.0±0.3.

[0038] (3) The pH of the DEAE eluate was adjusted to 6.5±0.5 and the potency of coagulation factor II was adjusted to 5 IU / mL, and a 1 mol / L calcium chloride solution was added to make the final calcium ion concentration 20 mmol / L. After activation treatment, a crude thrombin solution with a human thrombin potency greater than 200 IU / mL was obtained.

[0039] (3) The crude thrombin solution was subjected to S / D virus inactivation using an S / D virus inactivation solution containing 0.5% glycine, 0.2% TNBP, 0.7% polysorbate 80, and a pH of 6.5. The S / D reagent was removed by UniGel 50SP gel cation exchange chromatography, and purified human thrombin was obtained by filtration using a 0.1 μm nanomembrane filter.

[0040] 2. Human thrombin lyophilized preparation

[0041] (1) Glycine, proline hydrochloride, sodium chloride, and polysorbate 80 with a final concentration of 10 g / L, 3.5 g / L, and 7 g / L, respectively, were mixed with water to prepare a human thrombin lyophilization protective agent.

[0042] (2) Add the purified human thrombin to the human thrombin lyophilized protective agent at 600 IU / mL, mix well, divide into packages, freeze at -55°C for 3 h, keep at -28°C for 15 h, keep at -10°C for 1 h, keep at 0°C for 13 h, keep at 10°C for 1 h, and keep at 30°C for 12 h to obtain a human thrombin lyophilized preparation.

[0043] Example 2

[0044] 1. Obtaining human thrombin

[0045] Same as Example 1.

[0046] 2. Human thrombin lyophilized preparation

[0047] (1) Glycine, arginine hydrochloride, sodium chloride, and polysorbate 80 with a final concentration of 10 g / L, 3.5 g / L, and 50 μg / mL, were mixed with water to prepare a human thrombin lyophilized protective agent.

[0048] (2) The purified human thrombin was added to the human thrombin lyophilization protective agent at 600 IU / mL, mixed, divided and freeze-dried under the same conditions as in Example 1 to obtain a human thrombin lyophilized preparation.

[0049] Example 3

[0050] 1. Obtaining human thrombin

[0051] Same as Example 1.

[0052] 2. Human thrombin lyophilized preparation

[0053] (1) Glycine, lysine hydrochloride, sodium chloride, and polysorbate 80 with a final concentration of 10 g / L were mixed with water to prepare a human thrombin lyophilization protective agent.

[0054] (2) The purified human thrombin was added to the human thrombin lyophilization protective agent at 600 IU / mL, mixed, divided and freeze-dried under the same conditions as in Example 1 to obtain a human thrombin lyophilized preparation.

[0055] Example 4

[0056] 1. Obtaining human thrombin

[0057] Same as Example 1.

[0058] 2. Human thrombin lyophilized preparation

[0059] (1) Glycine, arginine hydrochloride, sodium chloride, and polysorbate 80 with a final concentration of 20 g / L were mixed with water to prepare a human thrombin lyophilized protective agent.

[0060] (2) The purified human thrombin was added to the human thrombin lyophilization protective agent at 600 IU / mL, mixed, divided and freeze-dried under the same conditions as in Example 1 to obtain a human thrombin lyophilized preparation.

[0061] Example 5

[0062] 1. Obtaining human thrombin

[0063] Same as Example 1.

[0064] 2. Human thrombin lyophilized preparation

[0065] (1) Mix 8 g / L glycine, 1 g / L lysine hydrochloride, 5 g / L sodium chloride and 40 μg / mL polysorbate 20 with water to obtain a human thrombin lyophilized protective agent.

[0066] (2) The purified human thrombin was added to the human thrombin lyophilization protective agent at 600 IU / mL, mixed, divided and freeze-dried under the same conditions as in Example 1 to obtain a human thrombin lyophilized preparation.

[0067] Example 6

[0068] 1. Obtaining human thrombin

[0069] Same as Example 1.

[0070] 2. Human thrombin lyophilized preparation

[0071] (1) Glycine, lysine hydrochloride, sodium chloride, and polysorbate 60 with a final concentration of 11 g / L were mixed with water to prepare a human thrombin lyophilized protective agent.

[0072] (2) The purified human thrombin was added to the human thrombin lyophilization protective agent at 600 IU / mL, mixed, divided and freeze-dried under the same conditions as in Example 1 to obtain a human thrombin lyophilized preparation.

[0073] Example 7

[0074] 1. Obtaining human thrombin

[0075] Same as Example 1.

[0076] 2. Human thrombin lyophilized preparation

[0077] (1) Mix 9 g / L glycine, 3 g / L lysine hydrochloride, 7 g / L sodium chloride and 20 μg / mL polysorbate 80 with water to obtain a human thrombin lyophilization protective agent.

[0078] (2) The purified human thrombin was added to the human thrombin lyophilization protective agent at 600 IU / mL, mixed, divided and freeze-dried under the same conditions as in Example 1 to obtain a human thrombin lyophilized preparation.

[0079] Comparative Example 1

[0080] 1. Obtaining human thrombin

[0081] Same as Example 1.

[0082] 2. Human thrombin lyophilized preparation

[0083] (1) Glycine, sodium chloride and polysorbate 80 with a final concentration of 10 g / L were mixed with water to prepare a human thrombin lyophilization protective agent.

[0084] (2) The purified human thrombin was added to the human thrombin lyophilization protective agent at 600 IU / mL, mixed, divided and freeze-dried under the same conditions as in Example 1 to obtain a human thrombin lyophilized preparation.

[0085] Comparative Example 2

[0086] 1. Obtaining human thrombin

[0087] Same as Example 1.

[0088] 2. Human thrombin lyophilized preparation

[0089] (1) Glycine, 0.02 mol / L Tris, 7 g / L sodium chloride and 50 μg / mL polysorbate 80 with final concentrations of 10 g / L were mixed with water to prepare a human thrombin lyophilization protective agent.

[0090] (2) The purified human thrombin was added to the human thrombin lyophilization protective agent at 600 IU / mL, mixed, divided and freeze-dried under the same conditions as in Example 1 to obtain a human thrombin lyophilized preparation.

[0091] Comparative Example 3

[0092] 1. Obtaining human thrombin

[0093] Same as Example 1.

[0094] 2. Human thrombin lyophilized preparation

[0095] (1) Glycine, 0.02 mol / L sodium citrate, 7 g / L sodium chloride and 50 μg / mL polysorbate 80 with final concentrations of 10 g / L were mixed with water to prepare a human thrombin lyophilization protective agent.

[0096] (2) The purified human thrombin was added to the human thrombin lyophilization protective agent at 600 IU / mL, mixed, divided and freeze-dried under the same conditions as in Example 1 to obtain a human thrombin lyophilized preparation.

[0097] Comparative Example 4

[0098] 1. Obtaining human thrombin

[0099] Same as Example 1.

[0100] 2. Human thrombin lyophilized preparation

[0101] (1) Glycine, 0.5% human albumin solution, 7 g / L sodium chloride and 50 μg / mL polysorbate 80 with a final concentration of 10 g / L were mixed with water to prepare a human thrombin lyophilization protective agent.

[0102] (2) The purified human thrombin was added to the human thrombin lyophilization protective agent at 600 IU / mL, mixed, divided and freeze-dried under the same conditions as in Example 1 to obtain a human thrombin lyophilized preparation.

[0103] Comparative Example 5

[0104] 1. Obtaining human thrombin

[0105] Same as Example 1.

[0106] 2. Human thrombin lyophilized preparation

[0107] (1) Glycine, 0.25% human albumin solution, 7 g / L sodium chloride and 50 μg / mL polysorbate 80 with a final concentration of 10 g / L were mixed with water to prepare a human thrombin lyophilization protective agent.

[0108] (2) The purified human thrombin was added to the human thrombin lyophilization protective agent at 600 IU / mL, mixed, divided and freeze-dried under the same conditions as in Example 1 to obtain a human thrombin lyophilized preparation.

[0109] Comparative Example 6

[0110] 1. Obtaining human thrombin

[0111] Same as Example 1.

[0112] 2. Human thrombin lyophilized preparation

[0113] (1) Glycine, sodium proline, sodium chloride, and polysorbate 80 with a final concentration of 10 g / L, 3.5 g / L, 7 g / L, and 50 μg / mL, were mixed with water to prepare a human thrombin lyophilization protective agent.

[0114] (2) The purified human thrombin was added to the human thrombin lyophilization protective agent at 600 IU / mL, mixed, divided and freeze-dried under the same conditions as in Example 1 to obtain a human thrombin lyophilized preparation.

[0115] Comparative Example 7

[0116] 1. Obtaining human thrombin

[0117] Same as Example 1.

[0118] 2. Human thrombin lyophilized preparation

[0119] (1) Glycine, proline, sodium chloride, and polysorbate 80 with a final concentration of 10 g / L, 3.5 g / L, and 7 g / L, respectively, were mixed with water to prepare a human thrombin lyophilized protective agent.

[0120] (2) The purified human thrombin was added to the human thrombin lyophilization protective agent at 600 IU / mL, mixed, divided and freeze-dried under the same conditions as in Example 1 to obtain a human thrombin lyophilized preparation.

[0121] Comparative Example 8

[0122] 1. Obtaining human thrombin

[0123] Same as Example 1.

[0124] 2. Human thrombin lyophilized preparation

[0125] (1) Glycine, arginine hydrochloride, and sodium chloride with final concentrations of 10 g / L, 3.5 g / L, and 7 g / L, were mixed with water to prepare a human thrombin lyophilization protective agent.

[0126] (2) The purified human thrombin was added to the human thrombin lyophilization protective agent at 600 IU / mL, mixed, divided and freeze-dried under the same conditions as in Example 1 to obtain a human thrombin lyophilized preparation.

[0127] Comparative Example 9

[0128] 1. Obtaining human thrombin

[0129] Same as Example 1.

[0130] 2. Human thrombin lyophilized preparation

[0131] (1) Glycine, lysine hydrochloride, sodium chloride, sodium citrate, calcium chloride, and water with final concentrations of 10 g / L, 3.5 g / L, 7 g / L, and 0.02 mol / L, respectively, were mixed to prepare a human thrombin lyophilization protective agent.

[0132] (2) The purified human thrombin was added to the human thrombin lyophilization protective agent at 600 IU / mL, mixed, divided and freeze-dried under the same conditions as in Example 1 to obtain a human thrombin lyophilized preparation.

[0133] Comparative Example 10

[0134] 1. Obtaining human thrombin

[0135] Same as Example 1.

[0136] 2. Human thrombin lyophilized preparation

[0137] (1) Mannitol, 3.5 g / L arginine hydrochloride, 3.5 g / L lysine hydrochloride, 7 g / L sodium chloride, and 0.02 mol / L sodium citrate with final concentrations of 10 g / L were mixed with water to prepare a human thrombin lyophilization protective agent.

[0138] (2) The purified human thrombin was added to the human thrombin lyophilization protective agent at 600 IU / mL, mixed, divided and freeze-dried under the same conditions as in Example 1 to obtain a human thrombin lyophilized preparation.

[0139] Effect example

[0140] The human thrombin lyophilized preparations prepared in Examples 1 to 7 and Comparative Examples 1 to 10 were tested. For specific detection methods, please refer to Part III of the 2020 edition of the Chinese Pharmacopoeia. The results are shown in Table 1. It can be seen that the overall effect of Examples 1 to 5 is better than that of Comparative Examples 1 to 9, with a short reconstitution time, stable titer of human thrombin before and after lyophilization, high recovery rate, good stability in accelerated experiments, high protein content of lyophilized preparations, and high specific activity. Among them, Example 1 has the best effect.

[0141] Table 1 Test results of Examples 1 to 7 and Comparative Examples 1 to 3

[0142]

[0143]

[0144] Table 1: Test results of comparative examples 4 to 10

[0145]

[0146] 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, without contradiction.

[0147] 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 thrombin freeze-drying protective agent, It is characterized in that include: Glycine, amino acid hydrochloride, sodium chloride and polysorbate.

2. The thrombin lyophilization protective agent according to claim 1, It is characterized in that The hydrochloric acid amino acid is selected from at least one of the following: proline hydrochloride, arginine hydrochloride and lysine hydrochloride, preferably proline hydrochloride.

3. The thrombin lyophilization protective agent according to claim 1, It is characterized in that The polysorbate is selected from at least one of the following: polysorbate 80, polysorbate 20, polysorbate 60, preferably polysorbate 80.

4. The thrombin lyophilization protective agent according to claim 1, It is characterized in that The content ratio between the glycine and the hydrochloric acid amino acid is (5-15):(2-5), preferably (9-11):(3-4).

5. The thrombin lyophilization protectant according to claim 1, It is characterized in that Based on the total volume of the thrombin lyophilized protective agent, the thrombin lyophilized protective agent comprises: 1-20 g / L glycine; 1~10g / L amino acid hydrochloride; 1-15 g / L sodium chloride; 20-90 μg / mL polysorbate; Optionally, based on the total volume of the thrombin lyoprotectant, the thrombin lyoprotectant comprises: 8-12 g / L of glycine; 1-5 g / L proline hydrochloride; 5-10 g / L sodium chloride; 40-60 μg / mL polysorbate 80.

6. The thrombin lyophilization protectant according to claim 1, It is characterized in that The thrombin is selected from thrombin of mammalian origin; Optionally, the mammal comprises a human, a cow, a horse, a pig or a sheep.

7. A human thrombin lyophilization protective agent, It is characterized in that Based on the total volume of the human thrombin lyophilized protective agent, the human thrombin lyophilized protective agent includes: 10 g / L of glycine, 3.5 g / L of proline hydrochloride, 7 g / L of sodium chloride, 50 μg / mL of polysorbate 80 and water.

8. A lyophilized preparation of thrombin, It is characterized in that include: Thrombin; The thrombin lyoprotectant according to any one of claims 1 to 6 or the human thrombin lyoprotectant according to claim 7.

9. The lyophilized thrombin preparation according to claim 8, It is characterized in that Based on the total volume of the thrombin lyoprotectant or the human thrombin lyoprotectant, the content of thrombin is 400 to 700 IU / mL.

10. Use of the thrombin lyoprotectant according to any one of claims 1 to 6 or the human thrombin lyoprotectant according to claim 7 in the preparation of a lyophilized thrombin preparation.

Images