Human blood coagulation factor VIII and preparation method thereof
By employing a dual precipitation method combining acid precipitation and polyethylene glycol precipitation, along with the application of carboxylated iron tetroxide nanoparticles, the challenges of achieving high recovery rate and high purity in the preparation of plasma-derived human coagulation factor VIII in existing technologies have been solved, resulting in efficient impurity removal and improved product stability.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SINOPHARM GRP SHANGHAI BLOOD PROD CO LTD
- Filing Date
- 2026-02-27
- Publication Date
- 2026-05-12
AI Technical Summary
Existing plasma-derived human coagulation factor VIII preparation processes struggle to balance high recovery rates with high purity, especially when avoiding aluminum ion residues, and high-speed centrifugation may lead to a decrease in purity.
A dual precipitation method combining acid precipitation and polyethylene glycol precipitation was employed, and carboxylated iron oxide nanoparticles were introduced to form a shear-resistant magnetic floc network through a bridging flocculation mechanism. Combined with appropriate centrifugation and incubation conditions, impurities were removed and the activity of human coagulation factor VIII was protected.
It significantly improved the specific activity and purity of human coagulation factor VIII, avoided damage to the product caused by high-speed shearing, and ensured high activity recovery rate and product stability.
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Abstract
Description
Technical Field
[0001] This application relates to the field of biopharmaceutical technology, and in particular to a human coagulation factor VIII and its preparation method. Background Technology
[0002] Plasma-derived human coagulation factor VIII (FVIII) is a key blood product isolated and purified from healthy human plasma. It is primarily used to prevent and treat bleeding events in patients with hemophilia A and is one of the most important alternative therapies for this disease. This product is included in my country's medical insurance catalog, and patients typically require lifelong medication; therefore, its safety, efficacy, and accessibility are of great importance. Statistics show that there are approximately 50,000 to 100,000 hemophilia A patients in my country, requiring about 800,000 vials of plasma-derived FVIII annually (at 200 IU / vial), but the market supply still has a shortage of about 400,000 vials. Although recombinant FVIII products reduce the risk of viral transmission to some extent, multiple clinical studies have shown that they pose a higher risk of inducing inhibitor (antibody) formation than plasma-derived products; therefore, the latter still holds an irreplaceable position in clinical practice.
[0003] Currently, the industrial preparation process of plasma-derived human coagulation factor VIII mainly includes two major steps: pre-purification and post-purification. Pre-purification often employs precipitation methods, such as polyethylene glycol precipitation, glycine precipitation, acid precipitation, or aluminum hydroxide gel adsorption, aiming to remove impurities such as fibrinogen and fibronectin. Post-purification often utilizes chromatographic techniques such as ion exchange chromatography, affinity chromatography, or immunoaffinity chromatography to further improve purity. Among these methods, aluminum hydroxide gel adsorption is widely used because it can effectively remove impurities such as prothrombin complexes, but this method easily leads to residual aluminum ions in the final product. Existing processes still face challenges in balancing "high recovery rate" and "high purity": for example, acid precipitation has a high recovery rate but insufficient purity; polyethylene glycol precipitation has high purity but a low recovery rate; and centrifugation, as a key pretreatment step, has relatively limited research on the optimization of its process parameters (such as centrifugal force and temperature), and high-speed centrifugation can cause acid and polyethylene glycol precipitates to be sheared and dispersed, leading to a decrease in purity.
[0004] Therefore, there is an urgent need for a process method that can achieve high activity recovery rate and high product purity in the current preparation technology of plasma-derived human coagulation factor VIII. Summary of the Invention
[0005] To improve the recovery activity and purity of human coagulation factor VIII, this application provides a human coagulation factor VIII and its preparation method.
[0006] This application provides a method for preparing human coagulation factor VIII, which adopts the following technical solution: A method for preparing human coagulation factor VIII includes the following steps: S1. Centrifuge the cryoprecipitate at room temperature to remove the supernatant and obtain the extract; S2. Mix the extract and heparin sodium solution and stir until completely dissolved to obtain a solution; S3. Adjust the pH of the solution to 6.2-6.4 using hydrochloric acid, stir well, and control the temperature at 20-30℃ to obtain an acid precipitate; S4. Mix the acid precipitate and polyethylene glycol, stir until homogeneous, and obtain polyethylene glycol precipitate; S5. Add carboxylated iron oxide nanoparticles to the polyethylene glycol precipitate, incubate, and magnetically separate to obtain the first separation liquid; S6. Centrifuge the first separation liquid to obtain the second separation liquid; S7. Clarify and filter the second separated liquid, and adjust the pH value to 6.4-7.4 to obtain a clear liquid; S8. Add the stabilizer and inactivator to the clarified liquid, stir evenly, and keep warm in a water bath to obtain human coagulation factor VIII stock solution.
[0007] By adopting the above technical solution, this application first constructs a highly efficient primary purification step through a dual precipitation method combining acid precipitation and polyethylene glycol (PEG) precipitation, which can effectively remove major impurities without using aluminum gel adsorption. Furthermore, the introduction of carboxylated iron oxide nanoparticles allows for the adsorption of protein aggregates using their large specific surface area and surface functional groups. The bridging flocculation mechanism strengthens the internal structure of the PEG-precipitated protein aggregates, forming a shear-resistant rigid magnetic floc network. This magnetically separates residual trace proteins (such as immunoglobulins, prothrombin complexes, complement system proteins, etc.), lipids, and fine particulate matter for adsorption and removal, avoiding the shear dispersion effect of subsequent high-speed shear centrifugation on the PEG precipitation. This synergistic process significantly improves the specific activity and purity of the final product while ensuring high activity recovery.
[0008] Optionally, in step S5, the oscillation speed during incubation is 1000-2000 rpm.
[0009] By adopting the above technical solution, this application uses an oscillation speed of 1000-2000 rpm for incubation, which ensures that the carboxylated iron oxide nanoparticles are uniformly dispersed in the polyethylene glycol precipitation system and maintain sufficient and gentle contact with impurities, proteins, lipids, and microparticles in the solution, thereby maximizing the adsorption efficiency on their surface. At the same time, this medium-to-high speed oscillation avoids the problems of uneven mixing and incomplete adsorption that may be caused by low-speed stirring, and also avoids the damage to human coagulation factor VIII caused by high shear force.
[0010] Optionally, in step S6, the centrifugal force is 10000-20000g and the centrifugal temperature is 15-18℃.
[0011] By adopting the above technical solution, this application uses centrifugation at 10000-20000g and 15-18℃, which can help to remove extremely small amounts of nanoparticles, nanoparticle-protein complexes and other submicron-sized insoluble impurities that are not completely captured by magnetism, thus avoiding protein denaturation and inactivation due to local overheating.
[0012] Optionally, in step S1, the temperature of the refrigerated centrifugation is 0-4℃, and the centrifugation speed is 4500-6500 r / min.
[0013] By adopting the above technical solution, the centrifugation temperature is strictly controlled within a low temperature range of 0-4℃ in the initial step of protein separation, and a suitable rotation speed of 4500-6500 r / min is set. This can maximally inhibit the thermal denaturation and enzymatic degradation of human coagulation factor VIII protein during centrifugation, while ensuring that the target protein in the cryoprecipitate is fully and gently separated and collected. This provides a high-activity, high-recovery starting material for subsequent purification steps, ensuring the high specific activity and stability of the final product from the source.
[0014] Optionally, in step S2, the concentration of heparin sodium in the heparin sodium solution is 70-90 IU / ml, and the volume ratio of the heparin sodium solution to the cryoprecipitate is 1:(1.5-2.5).
[0015] By adopting the above technical solution, the concentration of heparin sodium can be controlled within the range of 70-90 IU / mL, which can provide a binding and protective environment for human coagulation factor VIII protein, help stabilize its spatial conformation, and inhibit its spontaneous activation or degradation during the dissolution process. This maximizes the maintenance of the protein's biological activity in the initial dissolution stage, laying a highly active material basis for the subsequent acid precipitation step.
[0016] Optionally, in step S3, the concentration of hydrochloric acid is 0.05-0.15 mol / L.
[0017] By adopting the above technical solution, this application uses 0.05-0.15 mol / L hydrochloric acid, which can not only quickly and accurately adjust the pH value of the solution to the target range, achieving strict control over precipitation conditions and the stability of the final product, but also, as an inorganic strong acid, the chloride ions introduced by hydrochloric acid are easy to remove or compatible in subsequent processes, without introducing additional organic impurities or metal ions, thus avoiding the potential interference of complex residues on the activity of human coagulation factor VIII protein and product safety.
[0018] Furthermore, in step S4, the polyethylene glycol is a 30% polyethylene glycol solution, and the volume ratio of the acid precipitate to the 30% polyethylene glycol solution is 1:0.13.
[0019] Optionally, in step S7, the clarification filtration involves first using a 2μm filter membrane for initial filtration, and then using a 0.45-0.55μm filter membrane for further filtration.
[0020] By adopting the above technical solution and employing a graded clarification filtration strategy (first 2μm, then 0.45-0.55μm), the larger particles and aggregates remaining in the centrifuged liquid are first efficiently removed through a larger pore size filter membrane, effectively protecting the subsequent precision filter membrane and reducing its clogging pressure; then, a submicron-sized filter membrane is used for final fine filtration, which can thoroughly remove fine particles, potential microorganisms and some large molecular aggregates, significantly improving the clarity and physical stability of the final product.
[0021] Optionally, in step S8, the stabilizer is a mixture of sucrose and trisodium citrate, and the inactivator is a mixture of tributyl phosphate and polysorbate-80.
[0022] By adopting the above technical solution, using sucrose and trisodium citrate as stabilizers, an effective osmotic pressure protection and buffering environment can be provided for human coagulation factor VIII protein during the S / D virus inactivation process, maintaining its spatial conformation and biological activity; at the same time, the inactivating agent composed of tributyl phosphate and polysorbate-80 can efficiently destroy the lipid envelope virus structure, achieving reliable viral safety.
[0023] Furthermore, in step S8, the weight ratio of sucrose to trisodium citrate is 2:1, and the weight ratio of tributyl phosphate to polysorbate-80 is 3:10.
[0024] Optionally, in step S8, the temperature of the water bath heating is 24-26℃.
[0025] By adopting the above technical solution, the water bath heating temperature is strictly controlled within the range of 24-26℃, which can provide a stable and suitable thermal environment for the system during the S / D virus inactivation process. This can effectively activate the inactivating agent to fully destroy the lipid envelope virus and ensure the safety of the virus, while avoiding denaturation or loss of activity of human coagulation factor VIII protein due to excessive temperature. Thus, while ensuring the safety of the virus in the product, its biological activity and structural integrity are maintained to the greatest extent.
[0026] Secondly, this application provides a method for preparing human coagulation factor VIII to obtain human coagulation factor VIII.
[0027] In summary, this application includes at least one of the following beneficial technical effects: 1. This application first employs a dual precipitation method combining acid precipitation and polyethylene glycol (PEG) precipitation, forming a highly efficient primary purification step that effectively removes major impurities without the need for aluminum gel adsorption. Furthermore, carboxylated iron oxide nanoparticles are introduced, utilizing their large specific surface area and surface functional groups to adsorb protein aggregates. The bridging flocculation mechanism strengthens the internal structure of the PEG-precipitated protein aggregates, forming a shear-resistant, rigid magnetic floc network. This magnetically separates residual trace proteins (such as immunoglobulins, prothrombin complexes, complement system proteins, etc.), lipids, and fine particulate matter, avoiding the shear dispersion effect of subsequent high-speed shear centrifugation on the PEG precipitate. This synergistic process significantly improves the specific activity and purity of the final product while ensuring high activity recovery. 2. This application employs an oscillation speed of 1000-2000 rpm for incubation, which ensures uniform dispersion of carboxylated iron oxide nanoparticles in the polyethylene glycol precipitation system and maintains sufficient and gentle contact with impurities, proteins, lipids, and microparticles in the solution, thereby maximizing the adsorption efficiency on their surface. Simultaneously, this medium-to-high-speed oscillation avoids the problems of uneven mixing and incomplete adsorption that may occur with low-speed stirring, and also avoids damage to human coagulation factor VIII caused by high shear forces. 3. This application uses centrifugation at 10,000-20,000g and 15-18℃, which helps to remove trace amounts of nanoparticles, nanoparticle-protein complexes, and other submicron-sized insoluble impurities that are not completely captured by magnetism, thus avoiding protein denaturation and inactivation due to local overheating. Attached Figure Description
[0028] Figure 1 This is a process flow diagram of Embodiment 1 of this application. Detailed Implementation
[0029] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.
[0030] This application discloses a method for preparing human coagulation factor VIII, comprising the following steps: S1. Centrifuge the cold precipitate at 0-4℃ and 4500-6500r / min for 10-20min, remove the supernatant, and obtain the extract; S2. Mix the extract and 70-90 IU / ml heparin sodium solution at a volume ratio of 1:(1.5-2.5), and stir at 400-600 rpm for 2-4 hours to obtain a solution; S3. Adjust the pH of the solution to 6.2-6.4 using 0.05-0.15 mol / L hydrochloric acid, stir at 400-600 rpm for 5-10 min, and control the temperature at 20-30℃ to obtain acid precipitate; S4. Mix the acid precipitate and the polyethylene glycol solution, and stir at 400-600 rpm for 20-40 min to obtain polyethylene glycol precipitate; S5. Add carboxylated iron oxide nanoparticles to the polyethylene glycol precipitate, shake and incubate at 1000-2000 rpm for 1-2 hours, and then magnetically separate to obtain the first separation liquid. S6. Centrifuge the first separation liquid at 5000-20000g for 10-20 minutes at a centrifugation temperature of 15-30℃ to obtain the second separation liquid; S7. The second separated liquid is first filtered using a 2μm filter membrane, and then clarified using a 0.45-0.5μm filter membrane. The pH value is adjusted to 6.4-7.4 to obtain a clear liquid. S8. Add the stabilizer and inactivator to the clarified solution, stir at 400-600 rpm for 20-40 min, and keep warm in a water bath at 24-26℃ for 5-7 h to obtain human coagulation factor VIII stock solution.
[0031] All raw materials used in the embodiments of this application are commercially available, wherein: Cryoprecipitate, Sinopharm Shanghai Blood Products Co., Ltd. Heparin sodium, Shanghai Pharmaceutical First Biochemical Pharmaceutical Co., Ltd.; Polyethylene glycol-4000, Sinopharm Chemical Reagent Co., Ltd.; Hydrochloric acid, 0.1 mol / L, Sinopharm Chemical Reagent Co., Ltd. Sugar, Hunan Ercon Pharmaceutical Co., Ltd.; Trisodium citrate, Hunan Ercon Pharmaceutical Co., Ltd.; Polysorbate 80, Merck GmbH, Germany Tributyl phosphate, Sinopharm Chemical Reagent Co., Ltd.; Carboxylated iron tetroxide nanoparticles, 100nm in diameter, Xi'an Qiyue Biotechnology Co., Ltd. Example 1
[0032] A method for preparing human coagulation factor VIII includes the following steps: S1. Centrifuge 1 kg of cryoprecipitate at 2℃ and 5500 r / min for 10 min, remove the supernatant, and obtain the extract; S2. Mix the extract with 2L of 80IU / ml heparin sodium solution and stir at 500rpm for 3h to obtain the solution; S3. Adjust the pH of the solution to 6.3 using 0.1 mol / L hydrochloric acid, stir at 500 rpm for 5 min, and control the temperature at 25℃ to obtain acid precipitate; S4. Mix the acid precipitate with 1000 mL of 30% polyethylene glycol-4000 solution (mix 316.8 g of polyethylene glycol-4000 with 700 mL of water and bring the volume to 1 L), stir at 500 rpm for 30 min to obtain polyethylene glycol precipitate; S5. Add 2g of carboxylated iron oxide nanoparticles to the polyethylene glycol precipitate, shake and incubate at 1500rpm for 2h, and then magnetically separate to obtain the first separation liquid. S6. Centrifuge the first separation liquid at 5000g for 10min at a centrifugation temperature of 17℃ to obtain the second separation liquid; S7. First, filter the second separated liquid through a 2μm filter membrane, then filter it through a 0.5μm filter membrane for clarification. Adjust the pH value to 6.8 to obtain a clear liquid. S8. Add 10g sucrose, 5g trisodium citrate, 10g polysorbate 80 and 3g tributyl phosphate to the clarified solution, stir at 500rpm for 30min, and keep warm in a water bath at 25℃ for 6h to obtain human coagulation factor VIII stock solution.
[0033] Examples 2-9 Based on Example 1, the difference is that in step S6, the centrifugal force and / or centrifugal temperature are different, while the other steps remain the same as in Example 1. Example 2
[0034] The difference between this embodiment and Embodiment 1 is that the centrifugation temperature is 20-23℃. Example 3
[0035] The difference between this embodiment and Embodiment 1 is that the centrifugation temperature is 25-30℃. Example 4
[0036] The difference between this embodiment and Embodiment 1 is that the centrifugal force in this embodiment is 10000g and the centrifugal temperature is 15-18℃. Example 5
[0037] The difference between this embodiment and Embodiment 1 is that the centrifugal force in this embodiment is 10000g and the centrifugal temperature is 20-23℃. Example 6
[0038] The difference between this embodiment and Embodiment 1 is that the centrifugal force in this embodiment is 10000g and the centrifugal temperature is 25-30℃. Example 7
[0039] The difference between this embodiment and Embodiment 1 is that the centrifugal force in this embodiment is 20000g and the centrifugal temperature is 15-18℃. Example 8
[0040] The difference between this embodiment and Embodiment 1 is that the centrifugal force in this embodiment is 20000g and the centrifugal temperature is 20-23℃. Example 9
[0041] The difference between this embodiment and Embodiment 1 is that the centrifugal force in this embodiment is 20000g and the centrifugal temperature is 25-30℃.
[0042] Comparative Example 1 The difference between this comparative example and Example 1 is that this comparative example does not include step S5. Specifically, it includes the following steps: Centrifuge 1 kg of cryoprecipitate at 2℃ and 5500 r / min for 10 min, remove the supernatant to obtain the extract; mix the extract with 2 L of 80 IU / ml heparin sodium solution, stir at 500 rpm for 3 h to obtain the solution; adjust the pH of the solution to 6.3 with 0.1 mol / L hydrochloric acid, stir at 500 rpm for 5 min, and control the temperature at 25℃ to obtain the acid precipitate; mix the acid precipitate with 1000 mL of 30% polyethylene glycol-4000 solution (316.8 g... Polyethylene glycol-4000 and 700 mL of water were mixed and brought to a final volume of 1 L. The mixture was stirred at 500 rpm for 30 min to obtain a polyethylene glycol precipitate. The polyethylene glycol precipitate was centrifuged at 5000 g for 10 min at 17 °C to obtain a second separation liquid. The second separation liquid was first filtered through a 2 μm filter membrane and then clarified through a 0.5 μm filter membrane. The pH was adjusted to 6.8 to obtain a clear liquid. 10 g of sucrose, 5 g of trisodium citrate, 10 g of polysorbate 80 and 3 g of tributyl phosphate were added to the clear liquid. The mixture was stirred at 500 rpm for 30 min and incubated in a water bath at 25 °C for 6 h to obtain human coagulation factor VIII stock solution.
[0043] Comparative Examples 2-3 The difference between Comparative Examples 2-3 and Example 1 is that the oscillation speed during incubation is different in step S5, while the other steps are the same as in Example 1.
[0044] Comparative Example 2 The difference between this comparative example and Example 1 is that the oscillation speed during incubation in this comparative example is 900 rpm.
[0045] Comparative Example 3 The difference between this comparative example and Example 1 is that the oscillation speed during incubation in this comparative example is 2100 rpm.
[0046] Performance Test 1 The human coagulation factor VIII prepared in Examples 1-9 and Comparative Examples 1-3 were subjected to potency, protein content, and specific activity determination. Protein content and specific activity were determined according to the protein content determination method in General Chapter 0731 of the current edition of the Chinese Pharmacopoeia. Potency was determined according to the human coagulation factor VIII potency determination method in General Chapter 3521 of the current edition of the Chinese Pharmacopoeia. Three batches of samples were selected for each example for repeated testing, and the measurement results of each batch were recorded. The average value of the three batches was calculated.
[0047] Table 1 Performance of human coagulation factor VIII in Examples 1-9 and Comparative Examples 1-3
[0048] Table 2. Protein analysis results in three batches of human coagulation factor VIII stock solution from Example 7.
[0049] As shown in Example 1, Comparative Example 1, and Table 1, the human coagulation factor VIII stock solution prepared in Example 1 of this application has a potency of 92.80 IU / mL, a protein content of 0.209 mg / mL, and a specific activity of 44.52 IU / mg, which is significantly better than that of Comparative Example 1. This indicates that the introduction of a carboxylated iron oxide nanoparticle adsorption step after the polyethylene glycol precipitation step in this application can effectively adsorb hydrophobic impurities (such as immunoglobulins, prothrombin complexes, complement system proteins, etc.), thereby reducing the protein content and increasing the specific activity. However, direct centrifugation after polyethylene glycol precipitation can lead to the dispersion of protein aggregates, resulting in incomplete separation of small proteins.
[0050] As shown in Example 1, Comparative Examples 2-3, and Table 1, the human coagulation factor VIII prepared in Example 1 of this application has a potency of 92.80 IU / mL, a protein content of 0.209 mg / mL, and a specific activity of 44.52 IU / mg, which is significantly better than that of Comparative Examples 2-3. This indicates that the oscillation speed of 1000-2000 rpm used in this application ensures that the carboxylated iron oxide nanoparticles are uniformly dispersed in the polyethylene glycol precipitation system and maintain sufficient and gentle contact with impurities, lipids, and microparticles in the solution, thereby maximizing the adsorption efficiency of its surface. At the same time, the 1000-2000 rpm oscillation incubation avoids the problems of uneven mixing and incomplete adsorption that may be caused by low-speed stirring, and also avoids the damage to human coagulation factor VIII caused by high-speed shear.
[0051] As shown in Examples 1-9 and Table 1, the human coagulation factor VIII stock solution prepared in Example 7 of this application has a potency of 104.7 IU / mL, a protein content of 0.184 mg / mL, and a specific activity of 57.05 IU / mg, which is significantly better than that of Examples 1-6 and Examples 8-9. This indicates that the centrifugation force of 10000-20000g and centrifugation at 15-18℃ used in this application can help to remove trace amounts of nanoparticles, nanoparticle-protein complexes, and other submicron-sized insoluble impurities that are not completely captured by magnetism, thus avoiding protein denaturation and inactivation due to local overheating, thereby improving the purity and specific activity of human coagulation factor VIII.
[0052] The above are all preferred embodiments of this application, and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made in accordance with the structure, shape and principle of this application should be covered within the scope of protection of this application.
Claims
1. A method for preparing human coagulation factor VIII, characterized in that, Includes the following steps: S1. Centrifuge the cryoprecipitate at room temperature to remove the supernatant and obtain the extract; S2. Mix the extract and heparin sodium solution and stir until completely dissolved to obtain a solution; S3. Adjust the pH of the solution to 6.2-6.4 using hydrochloric acid, stir well, and control the temperature at 20-30℃ to obtain an acid precipitate; S4. Mix the acid precipitate and polyethylene glycol, stir until homogeneous, and obtain polyethylene glycol precipitate; S5. Add carboxylated iron oxide nanoparticles to the polyethylene glycol precipitate, incubate, and magnetically separate to obtain the first separation liquid; S6. Centrifuge the first separation liquid to obtain the second separation liquid; S7. Clarify and filter the second separated liquid, and adjust the pH value to 6.4-7.4 to obtain a clear liquid; S8. Add the stabilizer and inactivator to the clarified liquid, stir evenly, and keep warm in a water bath to obtain human coagulation factor VIII stock solution.
2. The method for preparing human coagulation factor VIII according to claim 1, characterized in that, In step S5, the oscillation speed during incubation is 1000-2000 rpm.
3. The method for preparing human coagulation factor VIII according to claim 1, characterized in that, In step S6, the centrifugal force is 10000-20000g and the centrifugal temperature is 15-18℃.
4. The method for preparing human coagulation factor VIII according to claim 1, characterized in that, In step S1, the temperature of the refrigerated centrifuge is 0-4℃, and the centrifugation speed is 4500-6500 r / min.
5. The method for preparing human coagulation factor VIII according to claim 1, characterized in that, In step S2, the concentration of heparin sodium in the heparin sodium solution is 70-90 IU / ml, and the volume ratio of the heparin sodium solution to the cryoprecipitate is 1:(1.5-2.5).
6. The method for preparing human coagulation factor VIII according to claim 1, characterized in that, In step S3, the concentration of hydrochloric acid is 0.05-0.15 mol / L.
7. The method for preparing human coagulation factor VIII according to claim 1, characterized in that, In step S7, the clarification filtration involves first using a 2μm filter membrane for initial filtration, followed by filtration using a 0.45-0.55μm filter membrane.
8. The method for preparing human coagulation factor VIII according to claim 1, characterized in that, In step S8, the stabilizer is a mixture of sucrose and trisodium citrate, and the inactivator is a mixture of tributyl phosphate and polysorbate-80.
9. The method for preparing human coagulation factor VIII according to claim 1, characterized in that, In step S8, the water bath temperature is 24-26℃.
10. A human coagulation factor VIII obtained by the preparation method according to any one of claims 1-9.