Snake venom hemocoagulase titer stabilizer as well as preparation method and application thereof
By using collagen peptide or a mixture of glycine as a titer stabilizer for venom hemagglutinin, the problem of titer decrease in venom hemagglutinin during liquid preparation, lyophilization and storage is solved, and the long-term stability and titer accuracy of the product are achieved.
Patent Information
- Application Number
- CN202510318592.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-18
- Publication Date
- 2025-06-27
AI Technical Summary
The titer of snake venom hemagglutinin is prone to decrease during liquid preparation, lyophilization and storage, and its long-term stability is poor, which affects its clinical application value.
A titer-stable preparation of venom hemagglutinin is prepared by an optimized lyophilization process using collagen peptide or a mixture of glycine as a titer stabilizer for venom hemagglutinin.
It significantly improves the titer stability of snake venom hemagglutinin under liquid mixing, lyophilization and storage conditions, avoids the expansion of snake venom hemagglutinin's activity, and ensures the long-term stability and titer accuracy of the product.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of pharmaceutical technology, and particularly relates to a snake venom hemocoagulase titer stabilizer, a preparation method thereof and an application thereof. Background Art
[0002] Snake venom serine proteinases (SVSPs) are an important class of proteolytic enzymes in snake venom, which are closely related to the coagulation system and have important clinical application values.
[0003] With the improvement of protein purification and separation technology and the in-depth research of biochemistry and pharmacology, the great value of snake venom-derived hemocoagulases in clinical hemostasis application has been determined. After decades of research and clinical application, it has been proved that snake venom hemocoagulase products have excellent hemostatic effects and are widely used in various hemorrhagic diseases. Snake venom hemocoagulase is a protease product with hemostatic effect, which is separated and extracted from snake venom by chromatography purification method and prepared by lyophilization. Without the protection of an enzyme activity stabilizer, the titer of snake venom hemocoagulase will decrease significantly during the liquid preparation and lyophilization processes, and there will also be a significant decrease in titer during long-term stability.
[0004] Collagen peptide is a polypeptide product prepared by directional enzymatic hydrolysis technology using bovine bone, cowhide or fish skin as the raw material of gelatin. At present, it has been widely used in oral health products and has the effects of locking water and whitening, repairing cells, balancing water and oil, delaying aging, etc. At present, some manufacturers have obtained registration numbers on the excipient registration and filing platform for medicinal use. However, there is no report on the application of pharmaceutical-grade collagen peptide and the mixture of collagen peptide and glycine as the titer stabilizer of snake venom hemocoagulase. Summary of the Invention
[0005] The purpose of the present invention is to provide a snake venom hemocoagulase titer stabilizer, a preparation method thereof and an application thereof.
[0006] The present invention firstly provides a snake venom hemocoagulase titer stabilizer, which is collagen peptide or a mixture of collagen peptide and glycine with a mass ratio of 1-3:1.
[0007] Specifically, in the mixture of collagen peptide and glycine, the mass ratio of collagen peptide to glycine is 1:1, 2:1 or 3:1.
[0008] The above-mentioned snake venom hemocoagulase titer stabilizer is used in the application of stabilizing the titer of snake venom hemocoagulase.
[0009] In the above application, the application is the titer stability during liquid preparation, lyophilization and / or storage.
[0010] Second, the present invention provides a hemocoagulase preparation from snake venom, which is prepared from the above-mentioned hemocoagulase titer stabilizer from snake venom and hemocoagulase from snake venom; In the hemocoagulase preparation from snake venom, the titer of the hemocoagulase from snake venom is 0.5 - 2 units per vial.
[0011] Third, the present invention provides a method for preparing a hemocoagulase preparation from snake venom, comprising the following steps: mixing a collagen peptide solution or a mixture solution of collagen peptide and glycine with hemocoagulase from snake venom to obtain the hemocoagulase preparation from snake venom.
[0012] In the above method, the mass percentage concentration of the collagen peptide solution is 2% - 10%, specifically 8%; In the mixture solution of collagen peptide and glycine, the mass percentage concentration of the collagen peptide is 2% - 6%, specifically 2%, 4% or 6%; the mass percentage concentration of glycine is 2% - 4%, specifically 2%; The solvent of the collagen peptide solution and the mixture solution of collagen peptide and glycine is water for injection; The relative molecular mass of the collagen peptide ≤ 2000 Da; The titer of the hemocoagulase from snake venom is not less than 400 units / mL; In the system after mixing the collagen peptide solution or the mixture solution of collagen peptide and glycine with hemocoagulase from snake venom, the titer of the hemocoagulase from snake venom is 0.5 - 2 units / mL; The pH of the system after mixing the collagen peptide solution or the mixture solution of collagen peptide and glycine with hemocoagulase from snake venom is 5.5 - 7.5; specifically, the pH value is adjusted with hydrochloric acid or sodium hydroxide solution.
[0013] The pH is specifically 6.6; The above method further comprises the step of freeze-drying the system after mixing the collagen peptide solution or the mixture solution of collagen peptide and glycine with hemocoagulase from snake venom; The lyophilization conditions are as follows: pre-freezing temperature is -40 ± 2°C, keep warm for 150 min; evacuate to a vacuum degree maintained at 30 - 100 Pa (specifically, it can be 35 Pa), start heating up, heat up to -35°C, keep warm for 60 min; heat up to -30°C, keep warm for 60 min; heat up to -25°C, keep warm for 60 min; heat up to -20°C, keep warm for 60 min; heat up to -15°C, keep warm for 60 min; heat up to -10°C, keep warm for 60 min; heat up to -5°C, keep warm for 60 min; heat up to 0°C, keep warm for 180 - 240 min; heat up to 5°C, keep warm for 60 min; heat up to 10°C, keep warm for 60 min; heat up to 15°C, keep warm for 60 min; heat up to 20°C, keep warm for 60 min; heat up to 25°C, keep warm for 60 min; heat up to 30°C, keep warm for 60 min; heat up to 35°C, keep warm for 60 min; heat up to 40°C, keep warm for 120 min; judge the end point of lyophilization when the pressure change within 1 minute is less than 30 Pa.
[0014] Fourth, the present invention provides a standard product of the hemocoagulase activity of snake venom, which is prepared from the above-mentioned hemocoagulase activity stabilizer of snake venom and hemocoagulase of snake venom; In the standard product of the hemocoagulase activity of snake venom, the hemocoagulase activity of the hemocoagulase of snake venom is 95 - 105 units per vial.
[0015] Fifth, the present invention provides a method for preparing a standard product of the hemocoagulase activity of snake venom, which includes the following steps: mixing a collagen peptide solution or a mixture solution of collagen peptide and glycine with hemocoagulase of snake venom to obtain the standard product of the hemocoagulase activity of snake venom.
[0016] In the above method, the mass percentage concentration of the collagen peptide solution is 2% - 10%, specifically it can be 8%; In the mixture solution of collagen peptide and glycine, the mass percentage concentration of the collagen peptide is 2% - 6%, specifically it can be 2%, 4% or 6%; the mass percentage concentration of glycine is 2% - 4%, specifically it can be 2%; The solvents of the collagen peptide solution and the mixture solution of collagen peptide and glycine are water for injection; The relative molecular mass of the collagen peptide ≤ 2000 Da; The hemocoagulase activity of snake venom is not less than 400 units / mL; In the system after mixing the collagen peptide solution or the mixture solution of collagen peptide and glycine with hemocoagulase of snake venom, the hemocoagulase activity of the hemocoagulase of snake venom is 95 - 105 units / mL; The pH of the system after mixing the collagen peptide solution or the mixture solution of collagen peptide and glycine with reptilase is 5.5 - 7.5, specifically 6.6. Specifically, the pH value is adjusted using hydrochloric acid or sodium hydroxide solution. The above method further includes a step of freeze - drying the system after mixing the collagen peptide solution or the mixture solution of collagen peptide and glycine with reptilase. The conditions for freeze - drying are as follows: pre - freezing temperature is - 40 ± 2°C, and keep warm for 150 min; evacuate to a vacuum degree maintained at 30 - 100 Pa (specifically 35 Pa), then start heating, heat up to - 35°C, and keep warm for 60 min; heat up to - 30°C, keep warm for 60 min; heat up to - 25°C, keep warm for 60 min; heat up to - 20°C, keep warm for 60 min; heat up to - 15°C, keep warm for 60 min; heat up to - 10°C, keep warm for 60 min; heat up to - 5°C, keep warm for 60 min; heat up to 0°C, keep warm for 180 - 240 min; heat up to 5°C, keep warm for 60 min; heat up to 10°C, keep warm for 60 min; heat up to 15°C, keep warm for 60 min; heat up to 20°C, keep warm for 60 min; heat up to 25°C, keep warm for 60 min; heat up to 30°C, keep warm for 60 min; heat up to 35°C, keep warm for 60 min; heat up to 40°C, keep warm for 120 min. Determine the freeze - drying end point when the pressure change within 1 minute is less than 30 Pa.
[0017] The reptilase described in the present invention is agkistrodon halys brevicaudus reptilase.
[0018] The present invention uses 2 - 10% pharmaceutical - grade collagen peptides with a specific relative molecular mass range (≤2000 Da) and a mixture of collagen peptide and glycine in a mass ratio of 1:1 - 3:1 as the potency stabilizer of agkistrodon halys brevicaudus reptilase, and obtains a potency - stable agkistrodon halys brevicaudus reptilase through an optimized freeze - drying process. These two types of stabilizers can be applied to snake - venom - type haemocoagulase preparations and snake - venom - type haemocoagulase potency reference standards.
[0019] The present invention has the following beneficial effects: (1) Compared with other macromolecular gelatin - type stabilizers, the stabilizer of the present invention does not show an expansion effect on the activity of agkistrodon halys brevicaudus reptilase with the increase of concentration; within the application concentration range, it not only ensures the stability of the product potency under the conditions of solution preparation, freeze - drying and storage, but also does not interfere with the potency detection due to the expansion effect of agkistrodon halys brevicaudus reptilase activity, resulting in deviation of potency accuracy.
[0020] (2)Compared with glycine, a commonly used freeze-drying excipient, the mixed stabilizer of collagen peptide and glycine has an extremely significant effect on the titer stability of hemocoagulase from Agkistrodon blomhoffii brevicaudus under formulation, freeze-drying and storage conditions. The titer is stable and controllable during the 10-day accelerated condition at 40 °C and the 36-month long-term stability study at -20 °C, and the coefficient of variation is less than 2%, meeting the requirements of the titer range of hemocoagulase preparations and the hemocoagulase from Agkistrodon blomhoffii brevicaudus titer reference substance.
[0021] (3)Compared with collagen peptide, the preparation obtained by freeze-drying the mixed stabilizer of collagen peptide and glycine has a smooth and flat appearance without shrinkage, and is more suitable for commercial product applications.
[0022] (4)The relative molecular masses of the stabilizers of the present invention are all less than 2000 Da, with an obvious difference from the molecular weight of hemocoagulase from Agkistrodon blomhoffii brevicaudus (36000 ± 5000 Da), and will not interfere with the purity detection of the working standard and the SDS-PAGE electrophoresis detection, meeting the inspection requirements for excipients of the working standard.
[0023] (5)The application of the hemocoagulase titer stabilizer of the present invention has a guiding effect on the preparation techniques of hemocoagulase preparations from Agkistrodon blomhoffii brevicaudus (0.5 unit - 2 units per vial) and the hemocoagulase from Agkistrodon blomhoffii brevicaudus titer reference substance (95 units - 105 units per vial). Specific Embodiments
[0024] The present invention will be further described in detail below in conjunction with specific embodiments. The provided examples are only for clarifying the present invention and not for limiting the scope of the present invention.
[0025] The experimental methods in the following examples are all conventional methods unless otherwise specified.
[0026] In the following examples, the quantitative tests are all set with four repeated experiments, and the results are averaged.
[0027] The materials, reagents, etc. used in the following examples can all be obtained from commercial channels unless otherwise specified.
[0028] Instruments and reagents used in the following examples: Mettler Toledo, MS205DU electronic balance; Mettler Toledo FE20-K pH meter; magnetic stirrer; lyo-0.2 freeze-dryer; TMS-5 transparent water bath; FB-40 four-channel semi-automatic blood coagulation analyzer.
[0029] Raw materials: Batroxobin concentrated solution, from Jinzhou Aohong Pharmaceutical Co., Ltd., batch number: 20231205, potency: 3000 units / mL. The definition of "unit" is as follows: Under the conditions of 37°C ± 0.5°C and shaking, the amount of enzyme in 0.2 mL of enzyme solution that causes 0.2 mL of coagulation quality control plasma suspension to form white flocs within 58 - 62 seconds, measured by the concentration of the enzyme solution, is defined as containing 1 unit of batroxobin in every 1 mL of enzyme solution.
[0030] Coagulation quality control plasma (freeze-dried powder, 1 vial reconstituted with 1 mL of water, 10 vials / box) was purchased from MDC Hemostasis; Collagen peptide (relative molecular mass 1700 - 1800 Da), pharmaceutical grade, was purchased from Baotou Dongbao Biotechnology Co., Ltd.; Glycine, pharmaceutical grade, was purchased from Hubei Bafeng Pharmaceutical Co., Ltd.; Other reagents were all of analytical grade.
[0031] Control group without enzyme activity stabilizer (1#) in Comparative Example 1 Add 100 mL of raw materials (batroxobin concentrated solution) to about 2000 mL of injection water, then adjust the pH to 6.5 with hydrochloric acid or sodium hydroxide solution, stir magnetically for 5 minutes, and make up the volume to 3000 mL (100 units / mL). After filtering through a 0.22 μm polyethersulfone sterilizing filter element, fill it into 2 mL middle borosilicate glass injection vials at a filling volume of 1.0 mL / vial, semi-plug, transfer to a freeze-dryer that has started the pre-freezing program for freeze-drying. The pre-freezing temperature is -40 ± 2°C, keep warm for 150 min; evacuate to a vacuum degree of 30 Pa and start heating up. Heat up to -35°C, keep warm for 60 min; heat up to -30°C, keep warm for 60 min; heat up to -25°C, keep warm for 60 min; heat up to -20°C, keep warm for 60 min; heat up to -15°C, keep warm for 60 min; heat up to -10°C, keep warm for 60 min; heat up to -5°C, keep warm for 60 min; heat up to 0°C, keep warm for 240 min; heat up to 5°C, keep warm for 60 min; heat up to 10°C, keep warm for 60 min; heat up to 15°C, keep warm for 60 min; heat up to 20°C, keep warm for 60 min; heat up to 25°C, keep warm for 60 min; heat up to 30°C, keep warm for 60 min; heat up to 35°C, keep warm for 60 min; heat up to 40°C, keep warm for 120 min. A 10 Pa change in pressure within 1 minute is used as the judgment criterion for the freeze-drying end point. After freeze-drying, fully plug and crimp the caps to obtain the batroxobin standard sample 1#.
[0032] Control group containing 2% - 6% glycine as enzyme activity stabilizer (2#) in Comparative Example 2 The preferred concentration of glycine is 2%: Weigh 60 g of glycine, dissolve it in an appropriate amount of water for injection, and dilute and make up the volume to 3000 mL to obtain a 2% glycine solution. Add 100 mL of the raw material (agkistrodon halys brevicaudus stejneger hemostatic enzyme concentrate) to about 2000 mL of the above glycine solution, then adjust the pH to 6.6 with hydrochloric acid or sodium hydroxide solution, and make up the volume to 3000 mL (100 units / mL). Stir magnetically for 5 minutes, filter through a 0.22 μm polyethersulfone sterilizing filter element, and fill into 2 mL middle borosilicate glass injection vials at a filling volume of 1.0 mL per vial, semi-plug, transfer to a freeze dryer that has started the pre-freezing program for freeze-drying. The pre-freezing temperature is -40 ± 2 °C, and keep warm for 150 min; evacuate to a vacuum degree of 35 Pa and start heating. Heat up to -35 °C and keep warm for 60 min; heat up to -30 °C and keep warm for 60 min; heat up to -25 °C and keep warm for 60 min; heat up to -20 °C and keep warm for 60 min; heat up to -15 °C and keep warm for 60 min; heat up to -10 °C and keep warm for 60 min; heat up to -5 °C and keep warm for 60 min; heat up to 0 °C and keep warm for 240 min; heat up to 5 °C and keep warm for 60 min; heat up to 10 °C and keep warm for 60 min; heat up to 15 °C and keep warm for 60 min; heat up to 20 °C and keep warm for 60 min; heat up to 25 °C and keep warm for 60 min; heat up to 30 °C and keep warm for 60 min; heat up to 35 °C and keep warm for 60 min; heat up to 40 °C and keep warm for 120 min. A 15 Pa change in pressure within 1 minute is used as the judgment for the freeze-drying end point. After freeze-drying, fully plug and crimp the caps to obtain the standard sample of agkistrodon halys brevicaudus stejneger hemostatic enzyme, sample 2#.
[0033] Example 1, the test group containing 3% - 10% collagen peptide as the enzyme activity stabilizer (3#) The preferred concentration of collagen peptide is 8%: Weigh 240 g of collagen peptide (relative molecular mass ≤ 2000 Da), dissolve it with an appropriate amount of water for injection, and dilute and make up the volume to 3000 mL to obtain an 8% collagen peptide solution. Add 100 mL of the raw material (agkistrodon halys brevicaudus stejneger hemostatic enzyme concentrate) to about 2000 mL of the above-mentioned collagen peptide solution, then adjust the pH to 6.6 with hydrochloric acid or sodium hydroxide solution, and make up the volume to 3000 mL (100 units / mL). Stir magnetically for 5 minutes, filter through a 0.22 μm polyethersulfone sterilizing filter element, and fill it into 2 mL medium borosilicate glass injection bottles at a filling volume of 1.0 mL per vial, semi-plug, transfer to a freeze dryer that has started the pre-freezing program for freeze-drying. The pre-freezing temperature is -40 ± 2 °C, and keep warm for 150 min; evacuate to a vacuum degree of 35 Pa and start heating. Heat up to -35 °C and keep warm for 60 min; heat up to -30 °C and keep warm for 60 min; heat up to -25 °C and keep warm for 60 min; heat up to -20 °C and keep warm for 60 min; heat up to -15 °C and keep warm for 60 min; heat up to -10 °C and keep warm for 60 min; heat up to -5 °C and keep warm for 60 min; heat up to 0 °C and keep warm for 240 min; heat up to 5 °C and keep warm for 60 min; heat up to 10 °C and keep warm for 60 min; heat up to 15 °C and keep warm for 60 min; heat up to 20 °C and keep warm for 60 min; heat up to 25 °C and keep warm for 60 min; heat up to 30 °C and keep warm for 60 min; heat up to 35 °C and keep warm for 60 min; heat up to 40 °C and keep warm for 120 min. A 15 Pa change in pressure within 1 minute is used as the judgment criterion for the freeze-drying end point. After freeze-drying, fully plug and crimp the caps to obtain the standard product of agkistrodon halys brevicaudus stejneger hemostatic enzyme 3#.
[0034] Example 2. Experimental group containing a mixed stabilizer of collagen peptide and glycine (4#) Weigh 60 g of glycine and 60 g of collagen peptide (relative molecular mass ≤ 2000 Da), dissolve them in an appropriate amount of water for injection, and dilute and make up the volume to 3000 mL to obtain a mixed solution of collagen peptide and glycine. Add 100 mL of raw material (agkistrodon halys brevicaudus stejneger hemostatic enzyme concentrate) to about 2000 mL of the above-mentioned mixed solution of collagen peptide and glycine, then adjust the pH to 6.6 with hydrochloric acid or sodium hydroxide solution, and make up the volume to 3000 mL (100 units / mL). Stir magnetically for 5 minutes, filter through a 0.22 μm polyethersulfone sterilizing filter element, and fill into 2 mL middle borosilicate glass injection vials at a filling volume of 1.0 mL per vial, semi-cork, and transfer to a freeze dryer that has started the pre-freezing program for freeze-drying. The pre-freezing temperature is -40 ± 2 °C, and keep warm for 150 min; evacuate to a vacuum degree of 35 Pa and start heating. Heat up to -35 °C and keep warm for 60 min; heat up to -30 °C and keep warm for 60 min; heat up to -25 °C and keep warm for 60 min; heat up to -20 °C and keep warm for 60 min; heat up to -15 °C and keep warm for 60 min; heat up to -10 °C and keep warm for 60 min; heat up to -5 °C and keep warm for 60 min; heat up to 0 °C and keep warm for 240 min; heat up to 5 °C and keep warm for 60 min; heat up to 10 °C and keep warm for 60 min; heat up to 15 °C and keep warm for 60 min; heat up to 20 °C and keep warm for 60 min; heat up to 25 °C and keep warm for 60 min; heat up to 30 °C and keep warm for 60 min; heat up to 35 °C and keep warm for 60 min; heat up to 40 °C and keep warm for 120 min. A 15 Pa change in pressure within 1 minute is used as the judgment criterion for the freeze-drying end point. After freeze-drying, fully cork and crimp to obtain the standard product of agkistrodon halys brevicaudus stejneger hemostatic enzyme 4#.
[0035] Example 3. Experimental group containing a mixed stabilizer of collagen peptide and glycine (5#) Weigh 60 g of glycine and 120 g of collagen peptide (relative molecular mass ≤ 2000 Da), dissolve them with an appropriate amount of water for injection, and dilute and make up the volume to 3000 mL to obtain a mixed solution of collagen peptide and glycine. Add 100 mL of the raw material (agkistrodon halys brevicaudus stejneger venom hemocoagulase concentrate) to about 2000 mL of the above-mentioned mixed solution of collagen peptide and glycine, then adjust the pH to 6.6 with hydrochloric acid or sodium hydroxide solution, and make up the volume to 3000 mL (100 units / mL). Stir magnetically for 5 minutes, filter through a 0.22 μm polyethersulfone sterilizing filter element, and fill into 2 mL middle borosilicate glass injection bottles at a filling volume of 1.0 mL per vial, semi-plug, transfer to a freeze-dryer that has started the pre-freezing program for freeze-drying. The pre-freezing temperature is -40 ± 2 °C, and keep warm for 150 min; evacuate to a vacuum degree of 35 Pa and start heating up. Heat up to -35 °C and keep warm for 60 min; heat up to -30 °C and keep warm for 60 min; heat up to -25 °C and keep warm for 60 min; heat up to -20 °C and keep warm for 60 min; heat up to -15 °C and keep warm for 60 min; heat up to -10 °C and keep warm for 60 min; heat up to -5 °C and keep warm for 60 min; heat up to 0 °C and keep warm for 240 min; heat up to 5 °C and keep warm for 60 min; heat up to 10 °C and keep warm for 60 min; heat up to 15 °C and keep warm for 60 min; heat up to 20 °C and keep warm for 60 min; heat up to 25 °C and keep warm for 60 min; heat up to 30 °C and keep warm for 60 min; heat up to 35 °C and keep warm for 60 min; heat up to 40 °C and keep warm for 120 min. A 15 Pa change in pressure within 1 minute is used as the judgment criterion for the freeze-drying end point. After freeze-drying, fully plug and crimp the caps to obtain the standard product of agkistrodon halys brevicaudus stejneger venom hemocoagulase 5#.
[0036] Example 4. Experimental group of agkistrodon halys brevicaudus stejneger venom hemocoagulase preparation containing a mixed stabilizer of collagen peptide and glycine (6#) Weigh 60 g of glycine and 180 g of collagen peptide (molecular weight ≤ 2000 Da), dissolve them with an appropriate amount of water for injection, and dilute and make up the volume to 3000 mL to obtain a mixed solution of collagen peptide and glycine. Add 2 mL of the raw material (Ancrod concentrated solution) to about 2000 mL of the above-mentioned mixed solution of collagen peptide and glycine, then adjust the pH to 6.6 with hydrochloric acid or sodium hydroxide solution, and make up the volume to 3000 mL (2 units / mL). Stir magnetically for 5 minutes, filter through a 0.22 μm polyethersulfone sterilizing filter element, and fill into 2 mL middle borosilicate glass injection vials at a filling volume of 0.25 mL per vial, semi-plug, transfer to a freeze-dryer that has started the pre-freezing program for freeze-drying. The pre-freezing temperature is -40 ± 2 °C, and keep warm for 150 min; evacuate to a vacuum degree of 35 Pa and start heating up. Heat up to -35 °C, keep warm for 60 min; heat up to -30 °C, keep warm for 60 min; heat up to -25 °C, keep warm for 60 min; heat up to -20 °C, keep warm for 60 min; heat up to -15 °C, keep warm for 60 min; heat up to -10 °C, keep warm for 60 min; heat up to -5 °C, keep warm for 60 min; heat up to 0 °C, keep warm for 240 min; heat up to 5 °C, keep warm for 60 min; heat up to 10 °C, keep warm for 60 min; heat up to 15 °C, keep warm for 60 min; heat up to 20 °C, keep warm for 60 min; heat up to 25 °C, keep warm for 60 min; heat up to 30 °C, keep warm for 60 min; heat up to 35 °C, keep warm for 60 min; heat up to 40 °C, keep warm for 120 min. A 15 Pa change in pressure within 1 minute is used as the judgment criterion for the freeze-drying end point. After freeze-drying, fully plug and crimp to obtain a sample 6# of Ancrod preparation (0.5 unit).
[0037] Example 5. Potency evaluation Potency evaluation method for the samples (sample 1# - 5#) for potency detection: Directly measure 1 mL of the prepared solution before preparation and before freeze-drying. After freeze-drying and for the samples in the accelerated stability study, first reconstitute 1 vial with 1 mL of water and then dilute with water to a solution containing approximately 1 unit per 1 mL as the test solution. Take a small test tube, add 0.2 mL of the coagulation quality control plasma suspension [Coagulation quality control plasma (MDCHemostasis) is placed at room temperature, 1.0 mL of water is added to each vial, and it is placed for 15 minutes, gently shaken intermittently during this period, and gently shaken well before each use], keep warm in a water bath at 37 °C ± 0.5 °C for 2 minutes, add 0.2 mL of the test solution, place it in a water bath at 37 °C ± 0.5 °C, immediately mix well and start timing, record the time when white flocs appear. Determine in parallel 4 times. If the visually observed time exceeds 60 ± 2 seconds for 1 time, re-determine; if the visually observed time exceeds 60 ± 2 seconds for 2 times, then re-adjust the concentration of the test solution and re-determine, record the concentration of the test solution, and calculate the enzyme activity. The amount of enzyme that can cause the coagulation quality control plasma to show white flocs within 60 seconds under the above conditions is defined as 1 unit. The potency calculation formula is as formula (1): (1) Method for evaluating the potency of the hemocoagulase preparation from Agkistrodon blomhoffii brevicaudus venom (Sample 6#): Standard solution: Take 1 vial of the hemocoagulase standard from Agkistrodon blomhoffii brevicaudus venom (for potency determination, Sample 5# in Example 3), accurately add 1.0 mL of water to dissolve it, and quantitatively dilute it with water for injection to prepare solutions containing approximately 0.3, 0.5, 1.0, 2.0, and 3.0 units per 1 mL respectively.
[0038] Test solution: Directly measure 1 mL of the prepared solution of Sample 6# before preparation and before freeze-drying. After freeze-drying and for the samples in the accelerated stability study, first reconstitute 1 vial with 1 mL of water as the test solution.
[0039] Determination method: Take 0.1 mL of the coagulation quality control plasma suspension [coagulation quality control plasma (MDC Hemostasis) is placed at room temperature, 1.0 mL of water is added to each vial, and it is left for 15 minutes, gently shaken intermittently during this period and shaken gently before each use], add it to 20 reaction cups respectively and place them in the FB-40 coagulation analyzer. Preheat at 37°C ± 0.5°C for 2 minutes. Accurately measure 0.2 mL of each standard solution with a series of concentrations and quickly add it to the above reaction cups, and record the coagulation time. Each concentration is determined 4 times, and the average value is calculated (the difference between the maximum and minimum values of the 4 determinations shall not exceed 10% of the average value, otherwise re-determine). Take the logarithm of the standard solution concentration as the abscissa and the logarithm of the coagulation time as the ordinate, calculate the linear regression equation, and the correlation coefficient should be greater than 0.99.
[0040] Take the test solution and determine it according to the above method. Obtain the concentration of the test solution from the regression equation and calculate to obtain the result.
[0041] Table 1 The calculation formula for the potency loss rate during the preparation process is as shown in formula (2): (2) The calculation formula for the potency loss rate under the accelerated condition of 40°C in Table 2 is as shown in formula (3): (3) Table 1 Summary of the potency loss of each group of samples during the process of preparation
[0042] Table 2 Summary of the potency loss of each group of samples after 10 days of acceleration under the conditions of 40°C and 75% RH
[0043] As can be seen from Table 1, for the sample of Comparative Example 1# without the addition of titer stabilizer, the titer loss rate reached 32% during the preparation of the sample solution and the freeze-drying process; for the sample of Comparative Example 2# with a single glycine solution added as the titer stabilizer, the titer loss rate reached 10% during the preparation of the sample solution and the freeze-drying process; while for Samples 3-6# using collagen peptide solution and using a mixed stabilizer of collagen peptide and glycine, the titer loss rates were both 0 during the solution preparation and freeze-drying processes, indicating that the mixed stabilizer of collagen peptide and glycine has a very significant titer protection effect on batroxobin during the solution preparation and freeze-drying processes, effectively avoiding the titer loss of batroxobin during the normal preparation process.
[0044] As can be seen from Table 2, for the sample of Comparative Example 1# without the addition of titer stabilizer, the titer loss rate was as high as 85.3% after being placed at 40°C under accelerated conditions for 10 days; for the sample of Comparative Example 2# with a single glycine solution added as the titer stabilizer, the titer loss rate reached 66.3% after being placed at 40°C under accelerated conditions for 10 days. Such a high titer loss makes the product unable to withstand relatively extreme short-term transportation conditions and room temperature storage, and cannot be commercialized. While for Samples 3-6# of the implementation using collagen peptide solution and using a mixed stabilizer of collagen peptide and glycine, the titer loss rates were both less than 5% after being placed at 40°C under accelerated conditions for 10 days, indicating that the mixed stabilizer of collagen peptide and glycine has a very significant titer protection effect on batroxobin during the storage period of the preparation, effectively avoiding the titer loss of batroxobin during the validity period.
[0045] Example 6. Stability study of the working reference standard for titer detection Preferably, Sample 5# in Example 3 was used as the working reference standard for batroxobin titer detection, and a long-term stability study was carried out under the condition of -20°C ± 2°C. The specific method is as follows: Place Sample 5# at -20°C ± 2°C for storage, sample at 0, 3, 6, 9, 12, 18, 24, and 36 months respectively, and determine the titer according to the titer evaluation method for the sample for titer detection in Example 5.
[0046] The calculation formula for the coefficient of variation is as shown in Formula (4): (4) The calculation formula for the 95% confidence interval range: sample mean ± (critical value × standard error) Table 3 Long-term stability test results of the working reference standard for batroxobin titer
[0047] As can be seen from Table 3, the working reference standard for batroxobin titer prepared with a mixed stabilizer of collagen peptide and glycine has stable titer after being stored at -20°C ± 2°C for 36 months, meeting the stability requirements for use as a working reference standard.
Claims
1. A snake venom hemocoagulase titer stabilizer, which is a collagen peptide or a mixture of collagen peptide and glycine in a mass ratio of 1 to 3:
1.
2. Use of the snake venom hemocoagulase titer stabilizer according to claim 1 in stabilizing the titer of snake venom hemocoagulase.
3. The use according to claim 2, characterized in that: The application is potency stabilization during formulation, lyophilization and / or storage.
4. A snake venom hemocoagulase preparation, which is prepared from the snake venom hemocoagulase titer stabilizer according to claim 1 and snake venom hemocoagulase; In the snake venom hemocoagulase preparation, the potency of the snake venom hemocoagulase is 0.5-2 units / vial.
5. A method for preparing a snake venom hemocoagulase preparation, comprising the following steps: mixing a collagen peptide solution or a mixture solution of collagen peptide and glycine with snake venom hemocoagulase to obtain the snake venom hemocoagulase preparation.
6. The method according to claim 5, characterized in that: The mass percentage concentration of the collagen peptide solution is 2% to 10%; In the mixture solution of the collagen peptide and glycine, the mass percentage concentration of the collagen peptide is 2% to 6%; the mass percentage concentration of the glycine is 2% to 4%; The solvent of the collagen peptide solution and the mixture solution of collagen peptide and glycine is water for injection; The relative molecular mass of the collagen peptide is ≤2000Da; The snake venom hemocoagulase titer is not less than 400 units / mL; In the system after the collagen peptide solution or the mixture solution of collagen peptide and glycine is mixed with snake venom hemocoagulase, the potency of the snake venom hemocoagulase is 0.5-2 units / mL; The pH value of the system after the collagen peptide solution or the mixture solution of collagen peptide and glycine and snake venom hemocoagulase are mixed is 5.5-7.5; specifically, the pH value is adjusted by using hydrochloric acid or sodium hydroxide solution.
7. The method according to claim 5 or 6, characterized in that: The method further comprises the step of freeze-drying the system after the collagen peptide solution or the mixture solution of the collagen peptide and glycine and the snake venom hemocoagulase are mixed; The freeze-drying conditions are as follows: pre-freezing temperature -40±2°C, keep warm for 150 minutes; evacuate to a vacuum degree of 30-100 Pa, start heating, heat to -35°C, keep warm for 60 minutes; heat to -30°C, keep warm for 60 minutes; heat to -25°C, keep warm for 60 minutes; heat to -20°C, keep warm for 60 minutes; heat to -15°C, keep warm for 60 minutes; heat to -10°C, keep warm for 60 minutes; heat to -5°C, keep warm for 60 minutes ; Heat to 0℃, keep warm for 180-240min; Heat to 5℃, keep warm for 60min; Heat to 10℃, keep warm for 60min; Heat to 15℃, keep warm for 60min; Heat to 20℃, keep warm for 60min; Heat to 25℃, keep warm for 60min; Heat to 30℃, keep warm for 60min; Heat to 35℃, keep warm for 60min; Heat to 40℃, keep warm for 120min; The freeze-drying endpoint is judged as the pressure change below 30Pa within 1 minute.
8. A snake venom hemocoagulase titer standard, which is prepared from the snake venom hemocoagulase titer stabilizer according to claim 1 and snake venom hemocoagulase; In the snake venom hemocoagulase titer standard product, the titer of the snake venom hemocoagulase is 95-105 units / vial.
9. A method for preparing a snake venom hemocoagulase titer standard product, comprising the following steps: mixing a collagen peptide solution or a mixture solution of collagen peptides and glycine with snake venom hemocoagulase to obtain the snake venom hemocoagulase titer standard product.
10. The method according to claim 9, characterized in that: The mass percentage concentration of the collagen peptide solution is 2% to 10%; In the mixture solution of the collagen peptide and glycine, the mass percentage concentration of the collagen peptide is 2% to 6%; the mass percentage concentration of the glycine is 2% to 4%; The solvent of the collagen peptide solution and the mixture solution of collagen peptide and glycine is water for injection; The relative molecular mass of the collagen peptide is ≤2000Da; The snake venom hemocoagulase titer is not less than 400 units / mL; In the system after the collagen peptide solution or the mixture solution of collagen peptide and glycine is mixed with snake venom hemocoagulase, the potency of the snake venom hemocoagulase is 95-105 units / mL; The pH value of the system after the collagen peptide solution or the mixture solution of collagen peptide and glycine and snake venom hemocoagulase are mixed is 5.5-7.5; specifically, the pH value is adjusted by using hydrochloric acid or sodium hydroxide solution; The method further comprises the step of freeze-drying the system after the collagen peptide solution or the mixture solution of the collagen peptide and glycine and the snake venom hemocoagulase are mixed; The freeze-drying conditions are as follows: pre-freezing temperature -40±2°C, keep warm for 150 minutes; evacuate to a vacuum degree of 30~100Pa, start heating, heat to -35°C, keep warm for 60 minutes; heat to -30°C, keep warm for 60 minutes; heat to -25°C, keep warm for 60 minutes; heat to -20°C, keep warm for 60 minutes; heat to -15°C, keep warm for 60 minutes; heat to -10°C, keep warm for 60 minutes; heat to -5°C, keep warm for 60 minutes ; Heat to 0℃, keep warm for 180~240min; Heat to 5℃, keep warm for 60min; Heat to 10℃, keep warm for 60min; Heat to 15℃, keep warm for 60min; Heat to 20℃, keep warm for 60min; Heat to 25℃, keep warm for 60min; Heat to 30℃, keep warm for 60min; Heat to 35℃, keep warm for 60min; Heat to 40℃, keep warm for 120min; The freeze-drying endpoint is judged as the pressure change below 30Pa within 1 minute.