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CN-122011157-A - Human coagulation factor VIII and preparation method thereof

CN122011157ACN 122011157 ACN122011157 ACN 122011157ACN-122011157-A

Abstract

The application discloses a human coagulation factor VIII and a preparation method thereof, wherein the preparation method comprises the following steps of S1, freezing and centrifuging cold precipitation to obtain an extracting solution, S2, mixing the extracting solution with heparin sodium solution to obtain a dissolving solution, S3, adjusting the pH value of the dissolving solution to be 6.2-6.4 by using hydrochloric acid, controlling the temperature to be 20-30 ℃ to obtain acid precipitation, S4, mixing the acid precipitation with polyethylene glycol to obtain polyethylene glycol precipitation, S5, adding carboxylated ferroferric oxide nano particles into the polyethylene glycol precipitation, incubating, magnetically separating to obtain a first separating solution, S6, centrifuging the first separating solution to obtain a second separating solution, S7, clarifying and filtering the second separating solution, adjusting the pH value to be 6.4-7.4 to obtain a clarified solution, S8, adding a stabilizer and an inactivating agent into the clarified solution, and carrying out water bath heat preservation to obtain a human coagulation factor VIII stock solution. The application has the effect of improving the recovery activity and purity of human coagulation factor VIII.

Inventors

  • YANG FAN
  • CAO LIWEI
  • CAO JING
  • YANG DONGWEI
  • GUO RUI
  • CHEN JIE
  • ZHANG LIANG
  • XIONG XIAOXIA
  • ZHANG WEI
  • WANG BIN

Assignees

  • 国药集团上海血液制品有限公司

Dates

Publication Date
20260512
Application Date
20260227

Claims (10)

  1. 1. A method for preparing human factor VIII, comprising the steps of: s1, performing freeze centrifugation on the cryoprecipitate, and removing supernatant to obtain an extracting solution; s2, mixing the extracting solution with heparin sodium solution, and stirring until the extracting solution and the heparin sodium solution are completely dissolved to obtain a dissolving solution; s3, adjusting the pH value of the solution to be 6.2-6.4 by using hydrochloric acid, uniformly stirring, and controlling the temperature to be 20-30 ℃ to obtain acid precipitation; S4, mixing the acid precipitate with polyethylene glycol, and uniformly stirring to obtain polyethylene glycol precipitate; S5, adding carboxylated ferroferric oxide nano particles into the polyethylene glycol precipitate, incubating, and magnetically separating to obtain a first separation liquid; S6, carrying out centrifugal separation on the first separation liquid to obtain a second separation liquid; s7, clarifying and filtering the second separation liquid, and adjusting the pH value to be 6.4-7.4 to obtain clarified liquid; And S8, adding a stabilizer and an inactivating agent into the clarified liquid, uniformly stirring, and carrying out water bath heat preservation to obtain the human coagulation factor VIII stock solution.
  2. 2. The method for preparing human factor VIII according to claim 1, wherein the shaking speed of incubation in step S5 is 1000-2000rpm.
  3. 3. The method for preparing human coagulation factor VIII according to claim 1, wherein the centrifugal force of centrifugation in step S6 is 10000-20000g and the centrifugal temperature is 15-18 ℃.
  4. 4. The method for preparing human coagulation factor VIII according to claim 1, wherein in the step S1, the temperature of refrigerated centrifugation is 0-4 ℃ and the centrifugation speed is 4500-6500r/min.
  5. 5. The method according to claim 1, wherein in step S2, the concentration of heparin sodium in the heparin sodium solution is 70-90IU/ml, and the volume ratio of the heparin sodium solution to the cryoprecipitate is 1 (1.5-2.5).
  6. 6. The method for producing human factor VIII according to claim 1, wherein the concentration of hydrochloric acid in step S3 is 0.05 to 0.15mol/L.
  7. 7. The method according to claim 1, wherein in the step S7, the clarification filtration is performed by first performing the primary filtration using a2 μm filter membrane and then performing the filtration using a 0.45-0.55 μm filter membrane.
  8. 8. The method according to claim 1, wherein in the step S8, the stabilizer is a mixture of sucrose and trisodium citrate, and the inactivating agent is a mixture of tributyl phosphate and polysorbate-80.
  9. 9. The method for preparing human coagulation factor VIII according to claim 1, wherein the temperature of the water bath is 24-26 ℃ in the step S8.
  10. 10. Human factor VIII obtained by the method of any one of claims 1-9.

Description

Human coagulation factor VIII and preparation method thereof Technical Field The application relates to the technical field of biological products, in particular to human coagulation factor VIII and a preparation method thereof. Background The plasma source human coagulation factor VIII is a key blood product prepared by separating and purifying healthy human plasma, is mainly used for preventing and treating bleeding events of patients with hemophilia A, and is one of the most main alternative therapies of the disease. The product is incorporated into the medical insurance catalogue of China, and patients usually need to take the medicine for life, so that the safety, the effectiveness and the accessibility of the product are of great significance. It is counted that about 5-10 ten thousand people of patients with hemophilia A in China need about 80 ten thousand bottles (counted by 200 IU/bottle) of plasma-derived human coagulation factor VIII every year, but the market supply still has a gap of about 40 ten thousand bottles. Although recombinant FVIII preparations reduce the risk of viral transmission to some extent, several clinical studies have shown that the risk of eliciting inhibitor (antibody) formation is higher than in plasma-derived products, and thus the latter is still clinically irreplaceable. At present, the industrialized preparation process of the plasma-derived human coagulation factor VIII mainly comprises two steps of pre-purification and post-purification. The pre-purification adopts precipitation method such as polyethylene glycol precipitation, glycine precipitation, acid precipitation or aluminum hydroxide gel adsorption, etc., to remove fibrinogen, fibronectin, etc., and the post-purification usually further improves purity by ion exchange chromatography, affinity chromatography or immunoaffinity chromatography, etc. Among these methods, aluminum hydroxide gel adsorption is widely used because it can effectively remove impurities such as prothrombin complex, but this method is liable to cause aluminum ion residue in the final product. The existing technology still faces challenges in balancing the aspects of high recovery rate and high purity, such as high recovery rate but insufficient purity of the acid precipitation method, high purity but low recovery rate of the polyethylene glycol precipitation method, and relatively deficient optimization research of technological parameters (such as centrifugal force and temperature) by taking the centrifugation as a key pretreatment link, and the high-speed centrifugation can cause acid precipitation and polyethylene glycol precipitation to be sheared and dispersed, thereby reducing the purity. Therefore, a process method capable of realizing high activity recovery rate and high product purity is needed in the current preparation technology of plasma-derived human coagulation factor VIII. Disclosure of Invention In order to improve the recovery activity and purity of human blood coagulation factor VIII, the application provides human blood coagulation factor VIII and a preparation method thereof. The application provides a preparation method of human coagulation factor VIII, which adopts the following technical scheme: A method for preparing human coagulation factor VIII, comprising the steps of: s1, performing freeze centrifugation on the cryoprecipitate, and removing supernatant to obtain an extracting solution; s2, mixing the extracting solution with heparin sodium solution, and stirring until the extracting solution and the heparin sodium solution are completely dissolved to obtain a dissolving solution; s3, adjusting the pH value of the solution to be 6.2-6.4 by using hydrochloric acid, uniformly stirring, and controlling the temperature to be 20-30 ℃ to obtain acid precipitation; S4, mixing the acid precipitate with polyethylene glycol, and uniformly stirring to obtain polyethylene glycol precipitate; S5, adding carboxylated ferroferric oxide nano particles into the polyethylene glycol precipitate, incubating, and magnetically separating to obtain a first separation liquid; S6, carrying out centrifugal separation on the first separation liquid to obtain a second separation liquid; s7, clarifying and filtering the second separation liquid, and adjusting the pH value to be 6.4-7.4 to obtain clarified liquid; And S8, adding a stabilizer and an inactivating agent into the clarified liquid, uniformly stirring, and carrying out water bath heat preservation to obtain the human coagulation factor VIII stock solution. By adopting the technical scheme, the application firstly forms a high-efficiency primary purification step by a double precipitation method combining acid precipitation and polyethylene glycol precipitation, and can effectively remove main impurities on the premise of avoiding the adsorption of alumina gel. Further, carboxylated ferroferric oxide nano particles are introduced, protein aggregates can be adsorbed by utilizing the