Composition for stabilizing human prothrombin complex and application thereof

Abstract

The invention relates to a composition for stabilizing a human prothrombin complex and application of the composition. The composition comprises an anticoagulant and a protective agent, the anticoagulant is heparin and / or a pharmaceutically acceptable salt thereof; the protective agent is selected from arginine and / or histidine and one or more of their respective pharmaceutically acceptable salts, and the ratio of the anticoagulant to the protective agent is (4.5-9.5) IU: (0.02-0.04) g. The activity of FII, FVII, FIX and FX of the prothrombin complex preparation prepared by adopting the composition for stabilizing the human prothrombin complex provided by the invention is not obviously reduced even under the condition of high temperature (40 + / -2 DEG C), and the activity of the FII, the FVII, the FIX and the FX is not obviously reduced after the prothrombin complex preparation is stored at the high temperature for at least 24 months. The biological activity index of the prothrombin complex preparation still meets the clinical use standard.

Description

Technical Field

[0001] This invention belongs to the field of plasma product technology. Specifically, this invention relates to a composition for stabilizing human prothrombin complex and its use. Background Technology

[0002] Human prothrombin complex concentrates (PCC) are obtained by separating and extracting from fresh plasma of healthy individuals. They primarily contain four vitamin K-dependent clotting factors—factor II (FII), factor VII (FVII), factor IX (FIX), and factor X (FX)—and a small amount of anticoagulant proteins. This complex exhibits significant coagulation activity and is clinically used primarily for the prevention and treatment of bleeding caused by deficiencies in FII, FVII, FIX, and FX, such as bleeding due to hemophilia B, severe liver diseases (e.g., acute severe hepatitis, cirrhosis), disseminated intravascular coagulation (DIC), and surgery.

[0003] However, once FII, FVII, FIX, and FX are removed from the body's natural environment, they are easily abnormally activated, accompanied by hydrolysis, decreased activity, denaturation, and changes in appearance. Preparing PCCs into lyophilized formulations not only significantly improves thermal stability and ensures no change in quality, but also simplifies transportation, storage, and clinical administration procedures, combining high efficiency and safety. Before use, they can be rapidly reconstituted by injecting an appropriate amount of sterile water for injection, 5% glucose, or sodium chloride injection according to specifications, making intravenous infusion convenient. However, the individual proteins in PCCs are still easily inactivated during lyophilization and subsequent processing and storage; therefore, maintaining the biological activity of FII, FVII, FIX, and FX is crucial.

[0004] Currently, existing PCC formulations are typically designed for storage at low temperatures of 2-8°C, which fails to meet the stability requirements of PCC formulations during prolonged high-temperature storage and transportation. In practical applications, PCC formulations often face complex storage and transportation environments, especially in areas with high summer temperatures or lacking adequate cold chain facilities, where frequent high-temperature exposures can occur within short periods. For example, temporary malfunctions in the cold chain system during transportation, or difficulties in precisely controlling the ambient temperature during temporary storage in medical institutions, can all lead to damage to the quality of the formulation, thereby affecting the treatment efficacy and safety for patients. Furthermore, in emergency treatment scenarios, such as field medical rescue or sudden large-scale bleeding events, ensuring the long-term stability of PCC formulations under high-temperature environments is crucial for protecting patients' lives and health.

[0005] Therefore, developing a PCC formulation that can remain stable at high temperatures for extended periods is not only a significant breakthrough for existing technologies, but also crucial for meeting actual clinical needs and ensuring the treatment efficacy and safety for patients. Summary of the Invention

[0006] To address the above problems, the object of this invention is to provide a composition for stabilizing human prothrombin complex and its use. The inventors have discovered that using one or more positively charged (basic) amino acids, such as arginine, histidine, and their respective pharmaceutically acceptable salts, as a protective agent is particularly advantageous for the stability of prothrombin complex formulations.

[0007] During the preparation process, heparin and / or its salts prevent premature coagulation of blood components, ensuring the effective extraction and purification of prothrombin complexes. The inventors further discovered that combining heparin with positively charged (basic) amino acids in a specific mass ratio can significantly improve the high-temperature stability of the formulation.

[0008] Even under high temperature (40±2℃) conditions, the activities of FII, FVII, FIX, and FX in PCC formulations prepared using the composition provided by this invention did not show a significant decrease. Moreover, after 24 months of high-temperature storage, the bioactivity indicators of the PCC formulations of this invention still meet the standards for clinical use. Therefore, this invention provides the above-mentioned composition for stabilizing human prothrombin complex and its uses.

[0009] The above-mentioned objective of the present invention is achieved by providing the following technical solution:

[0010] In a first aspect, the present invention provides a composition for stabilizing human prothrombin complex, comprising an anticoagulant and a protectant;

[0011] The anticoagulant is heparin and / or a pharmaceutically acceptable salt thereof;

[0012] The protective agent is a positively charged basic amino acid and / or its pharmaceutically acceptable salt, wherein the positively charged basic amino acid and / or its pharmaceutically acceptable salt is selected from one or more of the following:

[0013] (i) Arginine,

[0014] (ii) Histidine,

[0015] (iii) Pharmaceutically acceptable salts of arginine,

[0016] (iv) Pharmaceutically acceptable salts of histidine;

[0017] The ratio of the anticoagulant to the protective agent is (4.5-9.5) IU:(0.02-0.04) g.

[0018] Preferably, the ratio of the anticoagulant to the protective agent is (7-9) IU:0.025g, and more preferably 7 IU:0.025g.

[0019] According to some embodiments of the present invention, the concentration of the anticoagulant in the composition is 5-9 IU / mL;

[0020] According to some embodiments of the present invention, the concentration of the protective agent is 0.02-0.04 g / mL;

[0021] According to some embodiments of the present invention, the pharmaceutically acceptable salt of heparin is heparin sodium.

[0022] According to some embodiments of the present invention, the composition further comprises a buffer sodium citrate and / or an osmotic pressure regulator sodium chloride.

[0023] In this invention, an osmotic pressure regulator is added to the formulation system to adjust the osmotic pressure and maintain the electrolyte balance in the lyophilized formulation.

[0024] The osmotic pressure regulator and buffer described in this invention do not interact with the drug formulation, such as not causing adverse reactions such as oxidation, hydrolysis, or complexation, and do not decompose or affect the activity of the drug formulation during the freeze-drying process; wherein, the osmotic pressure regulator can adjust the osmotic pressure of the freeze-dried drug solution to make it similar to the osmotic pressure of body fluids, thereby preventing cell rupture or dehydration caused by osmotic pressure differences and ensuring the stability of the active ingredients of the drug.

[0025] According to some embodiments of the present invention, the weight ratio of sodium citrate to protective agent is (0.001-0.006):(0.02-0.04)g, preferably (0.001-0.003):0.025.

[0026] According to some embodiments of the present invention, the weight ratio of sodium chloride to protective agent is (0.004-0.01):(0.02-0.04)g, preferably (0.004-0.009):0.025.

[0027] The sodium chloride described in this invention has high solubility in water, does not absorb moisture or decompose during freeze-drying, and sodium ions are the main cations in body fluids, thus having less interference with protein stability.

[0028] According to some embodiments of the present invention, the concentration of sodium citrate in the composition is 0.001-0.006 g / mL, preferably 0.001-0.003 g / mL.

[0029] According to some embodiments of the present invention, the concentration of sodium chloride in the composition is 0.004-0.01 g / mL, preferably 0.004-0.009 g / mL.

[0030] According to various embodiments of the invention, the composition does not contain glycine and / or lysine, or their respective pharmaceutically acceptable salts.

[0031] In a second aspect, the present invention provides a pharmaceutical composition comprising a human prothrombin complex and the composition described in the first aspect of the present invention.

[0032] According to some embodiments of the present invention, the human prothrombin complex comprises human coagulation factors II, VII, IX and X.

[0033] According to some embodiments of the present invention, the activity of human coagulation factor II in the pharmaceutical composition is not less than 8 IU / mL.

[0034] According to some embodiments of the present invention, the activity of human coagulation factor VII in the pharmaceutical composition is not less than 4 IU / mL.

[0035] According to some embodiments of the present invention, the activity of human coagulation factor IX in the pharmaceutical composition is not less than 8 IU / mL.

[0036] According to some embodiments of the present invention, the activity of human coagulation factor X in the pharmaceutical composition is not less than 8 IU / mL.

[0037] According to some embodiments of the present invention, the pharmaceutical composition is prepared by a method comprising the following steps:

[0038] (1) Take plasma from healthy individuals, and sequentially separate, purify, S / D inactivate, and refine it to obtain a human prothrombin complex solution;

[0039] (2) Sodium citrate, sodium chloride, heparin or a pharmaceutically acceptable salt thereof, and arginine or a pharmaceutically acceptable salt thereof are added to the human prothrombin complex solution at the final concentration of each component in the composition according to the first aspect of the present invention, and mixed evenly.

[0040] Thirdly, the present invention provides the use of the composition according to the first aspect of the invention or the pharmaceutical composition according to the second aspect of the invention in the preparation of human prothrombin complex formulations.

[0041] Fourthly, the present invention provides a human prothrombin complex formulation, which is prepared from the pharmaceutical composition described in the second aspect of the present invention.

[0042] Fifthly, the present invention provides a method for preparing a human prothrombin complex formulation according to the fourth aspect of the present invention, comprising: sterilizing, lyophilizing and dry heat treating the pharmaceutical composition according to the second aspect of the present invention.

[0043] According to some embodiments of the present invention, the sterilization is carried out by filtration through a filter membrane with a diameter of 0.1-0.3 micrometers, preferably 0.2 micrometers.

[0044] According to some embodiments of the present invention, the freeze-drying includes: holding at a pressure of less than 0.2 mbar at -40°C for 0.5-5 hours, then holding at 1-10°C for 15-30 hours, and finally holding at a pressure of less than 0.1 mbar at 25-40°C for 6 hours or more.

[0045] According to some embodiments of the present invention, the dry heat treatment includes: first treating at 75-85°C for 2-6 hours, and then treating at 95-105°C for 10-50 minutes.

[0046] In a sixth aspect, the present invention provides a kit comprising a composition according to the first aspect of the invention, a pharmaceutical composition according to the second aspect of the invention, a human prothrombin complex preparation according to the fourth aspect of the invention, or a human prothrombin complex preparation prepared by the method according to the fifth aspect of the invention.

[0047] The present invention has at least the following beneficial effects:

[0048] The PCC formulation prepared using the composition for stabilizing human prothrombin complex provided by this invention can remain stable for a long time not only at room temperature (25±2℃) but also at high temperature (40±2℃), representing a significant improvement over existing technologies, as detailed below:

[0049] (1) The present invention's prothrombin complex formulation successfully solves the problem of insufficient stability of existing PCC formulations under high temperature conditions. Existing PCC formulations can only be stored and transported at low temperatures of 2-8℃, while the PCC formulation of the present invention, under high temperature conditions of 40±2℃, not only maintains the integrity of the protein structure, but also the activity of key coagulation factors (such as FII, FVII, FIX, FX) does not show a significant decrease. After being stored at high temperature (40±2℃) for at least 24 months, the bioactivity indicators of the PCC formulation of the present invention still meet the standards for clinical use. Whether it is a brief exposure to high temperature during transportation or temperature fluctuations that may occur in the clinical environment, the PCC formulation of the present invention can effectively maintain its coagulation activity and quality, ensuring that the drug can be safely and effectively used in clinical treatment under various complex environments.

[0050] (2) The PCC formulation of this invention exhibits significant high-temperature stability, reducing the strict reliance on cold chain transportation and storage. During transportation and storage, even in areas with inadequate cold chain facilities or in emergency situations, the quality of the formulation can be guaranteed to remain unaffected. This not only reduces the risk of losses due to cold chain failures but also significantly saves on the investment and operating costs of cold chain equipment. Attached Figure Description

[0051] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0052] Figure 1 This shows the trend of the percentage of FII potency over time in accelerated stability testing;

[0053] Figure 2 This shows the trend of the percentage of potency of FVII over time in accelerated stability testing;

[0054] Figure 3 This shows the trend of FIX potency percentage over time in accelerated stability testing;

[0055] Figure 4 This shows the trend of the percentage of FX potency over time in accelerated stability testing;

[0056] Figure 5 This shows the trend of the percentage of FII potency over time in the forced stability test;

[0057] Figure 6 This shows the trend of the percentage of potency of FVII over time in the forced stability test;

[0058] Figure 7 This shows the trend of the percentage of FIX potency over time in the forced stability test;

[0059] Figure 8 This shows the trend of the percentage of FX valence over time in the forced stability test. Detailed Implementation

[0060] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0061] Unless otherwise specified, the methods or conditions used in the following examples were performed according to conventional methods disclosed in the art. Unless otherwise stated, all reagents or instruments used in the following examples are commercially available products. The preparation method of the human prothrombin complex solution used in the following examples is as follows:

[0062] I. Separation and purification

[0063] Plasma from healthy individuals was collected and adsorbed onto a DEAE-A50 gel. The DEAE-A50 gel after plasma adsorption was collected and washed with 5 times the volume of washing buffer (composed of 0.2M NaCl and 0.02M sodium citrate, pH 6.5). Then, it was eluted with 5 times the volume of elution buffer (composed of 0.02M sodium citrate and 2M sodium chloride, pH 6.5). The eluted protein solution was collected and ultrafiltered to obtain a protein concentrate.

[0064] II. S / D Inactivation

[0065] Slowly add 11% (v / v) S / D concentrate (composition: 11% (v / v) Tween-80 and 3.3% (v / v) tributyl phosphate) to the protein concentrate while stirring, until the final concentration of Tween-80 is 0.8% (v / v) and the final concentration of tributyl phosphate is 0.36% (v / v), and the pH value is 6.5. After the addition is complete, keep it at 24°C for no less than 6 hours.

[0066] III. Refined

[0067] (1) Re-adsorption of the product

[0068] Add 0.5% (v / v) of the S / D inactivated protein solution to the pre-equilibrated DEAE-A50 gel. After stirring and adsorption for 30 minutes, collect the DEAE-A50 gel and wash it with 5 times the volume of washing solution (composed of 0.2M NaCl and 0.02M sodium citrate, pH 6.5).

[0069] (2) Purification ultrafiltration

[0070] Then, elute with 5 times the amount of gel eluent (composed of 0.02M sodium citrate and 1M sodium chloride, pH 6.5), collect the eluent protein solution, and perform ultrafiltration and concentration to obtain the human prothrombin complex solution.

[0071] The results showed that the activity of FII in the obtained human prothrombin complex solution was not less than 10 IU / mL, the activity of FVII was not less than 5 IU / mL, the activity of FIX was 30±5 IU / mL, and the activity of FX was not less than 10 IU / mL.

[0072] Example 1

[0073] Take the human prothrombin complex solution prepared by the above method, and add the following components to the following final concentrations: sodium citrate 3 mg / mL, sodium chloride 7 mg / mL, heparin sodium 7 IU / mL and arginine hydrochloride 25 mg / mL. The pH of the solution after addition is 7. After thorough mixing, sterilize, dispense, freeze dry and dry heat treat in sequence to obtain the human prothrombin complex preparation.

[0074] The sterilization process involves filtration through a 0.2-micron filter membrane. The freeze-drying steps are as follows: vacuum is applied to below 0.2 mbar, and the heat transfer oil temperature is controlled below -40°C for 2 hours; then the heat transfer oil temperature is set to 4°C and maintained for 22 hours to allow the product to dry gradually; subsequently, under a vacuum of less than 0.1 mbar, the heat transfer oil temperature is set to 32°C and maintained for at least 6 hours to complete the freeze-drying process. The dry heat treatment steps are as follows: first, treatment at 80°C for 4 hours, followed by treatment at 100°C for 30 minutes.

[0075] Experimental Example 1

[0076] (1) Screening of protective agents:

[0077] In this invention, amino acids or their hydrochlorides, as shown in Table 1, are added to the stock solution as protective agents. Arginine, lysine, and histidine are positively charged basic amino acids; glutamic acid is a negatively charged acidic amino acid; glycine is a polar (neutral) amino acid; and leucine is a nonpolar (hydrophobic) amino acid. Other steps are the same as in Example 1. After dry heat inactivation, the solution is reconstituted with sterile water for injection. The appearance after reconstitution is observed, and the reconstitution time, coagulation factor IX titer, and its recovery rate are measured. The test results are shown in the table below.

[0078]

[0079] According to the results shown in Table 1, when glycine or a mixture of glycine and arginine was used as a protective agent (formulas ① and ②), the appearance after reconstitution was unacceptable, with fine flocculent matter appearing, and the recovery rate of coagulation factor IX was low, only about 78%. However, when arginine, histidine, or their hydrochloride salts were used (formulas ③, ④, ⑥, and ⑦), the recovery rate of coagulation factor IX was significantly improved, all reaching over 97%, and the appearance after lyophilization and reconstitution met the requirements. Formulations with added glycine, lysine, or their hydrochloride salts (⑤, ⑧, and ⑨) performed poorly, with recovery rates all below 80%, generally prolonged reconstitution times, and some exhibiting slight shrinkage or flocculent matter in the lyophilized appearance. Furthermore, formulations ⑩ (glutamic acid) and ⑪ (leucine) were the least effective, with recovery rates of only 61.6% and 62.4%, respectively. They also had long reconstitution times, severe shrinkage and adhesion to the freeze-dried surface, and flocculent matter after reconstitution, failing to effectively protect the formulation during preparation.

[0080] (2) Screening of osmotic pressure regulators

[0081] An osmotic pressure regulator was added to the stock solution, and the other steps were the same as in Example 1. After dry heat inactivation, the solution was reconstituted with sterile water for injection. The appearance after reconstitution was observed, and the reconstitution time, coagulation factor IX titer, and its titer recovery rate were measured. The test results are shown in the table below.

[0082]

[0083] It can be seen that the appearance and reconstituted products of lyophilized preparations made with potassium chloride and calcium chloride as protective agents at different concentrations were all unqualified. Among them, the reconstituted products made with sodium chloride as protective agent were qualified in appearance, with no visible foreign matter, and the recovery rate of coagulation factor IX was relatively higher than that of potassium chloride and calcium chloride.

[0084] (3) Screening of buffers

[0085] A buffer was added to the stock solution, and the other steps were the same as in Example 1. After dry heat inactivation, the solution was reconstituted with sterile water for injection. The appearance after reconstitution was observed, and the reconstitution time, coagulation factor IX titer, and its titer recovery rate were measured. The test results are shown in the table below.

[0086]

[0087] Table 3 shows that the formulations using sodium citrate, sodium histidine, and sodium succinate as buffers all met the requirements for reconstitution time. However, sodium histidine at concentrations of 0.002-0.004 g / mL showed fine protein flocculent precipitation after reconstitution, while sodium succinate not only exhibited flocculent precipitation after reconstitution but also showed severe shrinkage and collapse in its lyophilized appearance, indicating that these two buffers have certain defects in the physical stability of the formulations.

[0088] In contrast, sodium citrate exhibited good formulation stability at all concentrations, with no shrinkage or collapse in the lyophilized appearance, and the appearance after reconstitution met the requirements. The reconstitution time was less than 10 minutes, and the recovery rate of coagulation factor IX remained stable between 78.6% and 78.8%, significantly higher than that of sodium histidine (70.8%-71.6%) and sodium succinate (60.9%-61.4%). Therefore, sodium citrate is superior to sodium histidine and sodium succinate in terms of reconstitution efficiency, appearance quality, and protein activity retention, making it more suitable as a buffer component in prothrombin complex formulations.

[0089] Based on the screening of protective agents, osmolarity regulators, and buffers, 0.025 g / mL arginine hydrochloride was selected as a protective agent, 0.007 g / mL sodium chloride as an osmolarity regulator, and 0.003 g / mL sodium citrate as a buffer. An anticoagulant was further added to prepare a prothrombin complex formulation. The appearance after reconstitution was observed, and the reconstitution time, coagulation factor IX titer, and its recovery rate were measured, among other indicators, as detailed below:

[0090] (4) Screening of anticoagulants

[0091]

[0092] As shown in Table 4, heparin, heparin sodium, and heparin lithium at concentrations of 5-9 IU / mL can all shorten the reconstitution time of lyophilized formulations. Among them, the lyophilized formulations prepared with heparin and heparin sodium have acceptable appearances and the reconstituted products have acceptable appearances and can stabilize the recovery rate of coagulation factor IX. However, heparin lithium has no effect on improving the appearance of lyophilized formulations or the appearance after reconstitution.

[0093] In summary, by adding arginine hydrochloride as a protective agent, sodium chloride as an osmotic pressure regulator, and sodium citrate as an anticoagulant to the original prothrombin complex solution, the prepared prothrombin complex formulation has qualified lyophilized appearance and reconstituted appearance, and the dissolution time can be less than 1 min. The recovery rate of coagulation factor IX titer is stable and can reach more than 99%.

[0094] Investigation of formulations in Examples 1-12 and Comparative Examples 1-12

[0095] The human prothrombin complex formulation was prepared according to the method of Example 1. The differences between other examples and comparative examples and Example 1 are that the amount of some components added is different or the added components are different, so as to investigate the effect of different formulations on the quality of the finished product, as shown in the table below.

[0096]

[0097] Test case

[0098] 1. Performance Testing

[0099] The human prothrombin complex formulations prepared in the above examples and comparative examples were subjected to the following stability tests:

[0100] Long-term stability test: 42 months, under the conditions of 5±3℃ and protection from light;

[0101] Accelerated stability test: 42 months, under conditions of 25±2℃, relative humidity of 60±5%, protected from light;

[0102] Forced stability test: 24 months, under conditions of 40±2℃, relative humidity of 75±5%, protected from light.

[0103] Key testing items include: appearance, vacuum degree, reconstitution time, visible foreign matter, moisture, pH value, FIX titer, FIX specific activity, FII titer, FVII titer, FX titer, human thrombin activity, and activated coagulation factor activity.

[0104] 2. Results of long-term stability study

[0105] Upon examination, the formulations of Examples 1-12, Comparative Examples 1-4, and 11, after being stored for 42 months, met the standards of the Chinese Pharmacopoeia (2020 edition, Part III) in terms of appearance, vacuum degree, reconstitution time, visible foreign matter, moisture content, pH value, FII potency (300 IU / vial), FIX specific activity, FVII potency (150 IU / vial), FIX potency (300 IU / vial), FX potency (300 IU / vial), human thrombin activity, and activated coagulation factor activity. Each vial contains 10 mL.

[0106] The reconstitution times of formulations in Comparative Examples 5 and 12 at 0 months were 15 minutes 21 seconds and 16 minutes 03 seconds, respectively, which did not meet the pharmacopoeia requirements, and therefore accelerated stability studies were not conducted.

[0107] The formulations of Comparative Examples 7 and 10 showed visible foreign matter after reconstitution at 0 months; the formulations of Comparative Examples 8 and 9 collapsed after freeze-drying and shrank after dry heat, which did not meet the pharmacopoeia standards and were not subject to accelerated stability testing.

[0108] When the formulation of Comparative Example 6 was investigated for 36 months, it began to show a yellow powder appearance and visible foreign matter appeared in the reconstituted product. Other test indicators met the pharmacopoeia standards during the investigation period (before 36 months).

[0109] Based on this, accelerated stability tests were further conducted on the formulations prepared in Examples 1-12 and Comparative Examples 1-4, 6, and 11.

[0110] 3. Accelerated stability test results

[0111]

[0112]

[0113]

[0114]

[0115]

[0116] The accelerated stability test results show that, after being placed at a temperature of 25±2℃ and a relative humidity of 60±5% for 42 months, the activities of FII, FVII, FIX, and FX did not show a significant decrease, and the potency fluctuations were within the normal range with a stable trend (see [link to relevant documentation]). Figure 1-4In particular, the percentage fluctuation of coagulation factor titers in the formulation of Example 1 was relatively small. After dry heat treatment at 80°C and 100°C, the protective effect of the formulations of Examples 4-12 on the titers of active factors was slightly weaker than that of the formulations of Examples 1-3. However, at room temperature, the percentage of titers of each active factor remained relatively stable. Among them, the activities of FII, FVII, FIX, and FX in Example 11 (a combination of arginine hydrochloride and arginine) and Example 12 (the amino acid is histidine hydrochloride) were lower than those in other examples. The formulation of Example 12 had the lowest protective strength for the activities of FII, FVII, FIX, and FX, but the trend of titer change remained stable. In addition, the appearance and visible foreign matter of the formulations of each example met the pharmacopoeia requirements.

[0117] The compositions of Comparative Examples 1, 2-4, 6, and 11 are not within the scope of protection of this invention. The preparations obtained showed visible foreign matter after 12 months of reconstitution, which does not meet the pharmacopoeia standards, indicating that their stability is poor.

[0118] The formulations of Comparative Example 1 (heparin sodium to arginine hydrochloride ratio of 4 IU: 0.025 g) and Comparative Example 2 (10 IU: 0.025 g) showed FIX potency lower than the pharmacopoeia standard at 1 month and 9 month, respectively. Comparative Example 3 (6 IU: 0.015 g), Comparative Example 4 (9.5 IU: 0.045 g), and Comparative Example 6 were unstable in the early stages due to improper amino acid types, and Comparative Example 11 was unstable due to incorrect heparin sodium dosage and amino acid combination.

[0119] Based on the long-term stability study results, it can be seen that for the amino acids used in the formulation preparation process, Comparative Examples 7-9, which combined arginine hydrochloride with glycine or lysine hydrochloride, showed extremely poor appearance stability compared to Examples 1-12, which used arginine hydrochloride and / or arginine, or histidine alone. The appearance of the formulations after lyophilization following high-temperature dry heat treatment and the presence of visible foreign matter upon reconstitution were already unacceptable. Comparative Examples 5 and 10, which used only one amino acid (glycine or lysine hydrochloride), also failed to meet pharmacopoeia standards in appearance at month 0. Comparative Example 12, which used glycine, showed that the appearance of the formulation failed to meet pharmacopoeia requirements in terms of reconstitution time after dry heat treatment.

[0120] Based on this, during the accelerated stability assessment phase, Examples 1-12 and Comparative Examples 2 and 11, which showed slightly better performance, were selected for subsequent forced stability assessment.

[0121] 4. Results of forced stability tests

[0122]

[0123]

[0124]

[0125]

[0126]

[0127] The experimental results of forced stability show that after undergoing dry heat treatment at 80°C and 100°C, the formulations prepared in Examples 1-12 of this invention exhibit good stability under initial (0 months) and long-term high temperature (40±2°C) storage conditions, but there are significant differences between different formulations.

[0128] At 0 months, although the protective effect of the formulations of Examples 7-11 of the present invention on the potency of active factors was weaker than that of the formulations of Examples 1-3, the potency percentage of each active factor remained relatively stable after being placed at 40±2℃ for 24 months. Among them, the protective effect of Example 1 (containing only arginine hydrochloride) was the best, and the potency percentage of each coagulation factor (FII, FVII, FIX, FX) remained at a high level with the smallest fluctuation.

[0129] Example 7 (containing only arginine) also showed good stability, although slightly inferior to Example 1, but still significantly better than most comparative examples. In contrast, Example 11 (containing both arginine hydrochloride and arginine) showed lower percentage potency of FII, FVII, and FX in accelerated and forced stability studies than Examples 1 and 7, indicating that the coexistence of two salt forms in the formulation system of this invention is unexpectedly detrimental to protein conformation stability, thereby weakening potency protection.

[0130] In Example 12, the potency percentage of FIX fell below 80% after 12 months. The potency percentages of FII, FVII, and FX also gradually decreased after 12 months of formulation storage, indicating that this component could not provide long-term effective protection under high-temperature conditions. The longest stable period of the formulation did not exceed 12 months, and its effectiveness was far inferior to that of Examples 1-11, which were mainly composed of arginine hydrochloride and / or arginine. Nevertheless, all the formulations in these examples consistently met pharmacopoeia requirements in terms of appearance and visible foreign matter.

[0131] In contrast, the formulations of Comparative Examples 2 (heparin to arginine hydrochloride in a ratio of 10 IU: 0.025 g) and 11 (arginine hydrochloride and glycine combination) showed problems such as visible foreign matter and a severe decrease in potency after reconstitution in the early stage of the investigation (0-1 month), which did not meet the pharmacopoeia standards. Therefore, they were not included in the subsequent mandatory stability test, further highlighting the significant advantage of the formulation protected by this invention in maintaining the stability of the formulation.

[0132] The above descriptions are merely several exemplary embodiments of the present invention and are not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any equivalent or similar implementation schemes obtained by those skilled in the art by making some modifications or alterations to the above-disclosed technical content without departing from the scope of the present invention are within the scope of the present invention.

Claims

1. A composition for stabilizing human prothrombin complex, comprising an anticoagulant and a protective agent; The anticoagulant is heparin and / or a pharmaceutically acceptable salt thereof; The protective agent is a positively charged basic amino acid and / or its pharmaceutically acceptable salt, wherein the positively charged basic amino acid and / or its pharmaceutically acceptable salt is selected from one or more of the following: (i) Arginine, (ii) Histidine, (iii) Pharmaceutically acceptable salts of arginine, (iv) Pharmaceutically acceptable salts of histidine; in, The ratio of the anticoagulant to the protective agent is (4.5-9.5) IU: (0.02-0.04) g.

2. The composition according to claim 1, wherein, The ratio of the anticoagulant to the protective agent is (7-9) IU:0.025g, preferably 7 IU:0.025g; Preferably, the concentration of the anticoagulant in the composition is 5-9 IU / mL; Preferably, the concentration of the protective agent is 0.02-0.04 g / mL; Preferably, the pharmaceutically acceptable salt of heparin is heparin sodium.

3. The composition according to claim 1 or 2, wherein, The composition also includes an osmotic pressure regulator and a buffer; Furthermore, the osmotic pressure regulator is sodium chloride; Furthermore, the buffer is sodium citrate; Preferably, the weight ratio of sodium citrate to the protective agent is (0.001-0.006):(0.02-0.04), more preferably (0.001-0.003):0.025; Preferably, the weight ratio of sodium chloride to the protective agent is (0.004-0.01):(0.02-0.04), and more preferably (0.004-0.009):0.

025.

4. The composition according to claim 3, wherein, The concentration of sodium citrate in the composition is 0.001-0.006 g / mL, preferably 0.001-0.003 g / mL; Preferably, the concentration of sodium chloride in the composition is 0.004-0.01 g / mL, more preferably 0.004-0.009 g / mL.

5. A pharmaceutical composition comprising a human prothrombin complex, comprising a human prothrombin complex and the composition according to any one of claims 1 to 4.

6. The pharmaceutical composition according to claim 5, wherein, The human prothrombin complex comprises human coagulation factors II, VII, IX and X; Preferably, the activity of human coagulation factor II in the pharmaceutical composition is not less than 8 IU / mL; Preferably, the activity of human coagulation factor VII in the pharmaceutical composition is not less than 4 IU / mL; Preferably, the activity of human coagulation factor IX in the pharmaceutical composition is not less than 8 IU / mL; Preferably, the activity of human coagulation factor X in the pharmaceutical composition is not less than 8 IU / mL.

7. Use of the composition of any one of claims 1 to 4 or the pharmaceutical composition of claim 5 or 6 in the preparation of human prothrombin complex formulations.

8. A human prothrombin complex formulation, prepared from the pharmaceutical composition of claim 5 or 6.

9. A method for preparing the human prothrombin complex formulation according to claim 8, comprising: The pharmaceutical composition according to claim 5 or 6 is subjected to sterilization, freeze-drying, and dry heat treatment.

10. A kit comprising the composition according to any one of claims 1 to 4, the pharmaceutical composition according to claim 5 or 6, the human prothrombin complex preparation according to claim 8, or the human prothrombin complex preparation prepared according to the method of claim 9.

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