vWF (von willebrand factor) activity protection fluid

Abstract

The invention discloses a vWF (von Willebrand factor) activity protection fluid. The vWF activity protection fluid is used in preparation for extracting von Willebrand factors from cryoprecipitated blood coagulation factor VIII waste. In a preparation technology of the von Willebrand factors, glycine is added into a chromatographic buffer solution, and lysine, glycine and albumin are added into protein fluid before freeze drying after chromatography to protect vWF activity. The vWF activity protection fluid has the advantages that by means of adding the glycine into the chromatographic buffer solution, vWF activity loss can be reduced in a chromatographic separation and purification process; the albumin, the lysine and the glycine are added into protein dialysate to serve as freeze-drying protectants, so that von Willebrand factor molecules can be stabilized; the albumin serving as an excellent protein stabilizer is capable of effectively adsorbing protein surfaces; the lysine and the glycine, serving as micromolecular amino acids, are capable of protecting a protein structure, increasing collapse temperature of a finished product and stopping protein damage caused by collapse during freeze-drying, so that biological activity is kept.

Description

technical field [0001] The invention relates to a preparation process for extracting human von Willebrand factor from the waste of blood coagulation factor VIII extracted by cryoprecipitation of human plasma, in particular to a vWF active protection solution, which belongs to the field of biopharmaceuticals. Background technique [0002] vWF is a plasma glycoprotein with multimers ranging in molecular weight from 250 kDa to 20 million kDa, forming the largest known molecular weight soluble protein in plasma. The small molecular weight vWF in plasma is mainly in the form of dimers with a molecular weight of about 500kDa. The multimers of different sizes are of great significance to maintain the normal biological activity of vWF. [0003] Plasma vWF plays an important role in primary hemostasis as it is responsible for the adhesion of platelets to the surface of injured vessels and thus the formation of platelet plugs that contribute to the formation of fibrin crosslinks. In...

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Problems solved by technology

[0008] There is a patent to purify vWF factor components with DEAEFractogel TSK650M (Merck), which has high purity, but the process is relatively complicated, requiring two steps of ion exchange and one step of affinity chromatography, such as "Process for anindustrial-scale preparation of standardized human von Willebrand factor concentrate of very high purity and suitable for therapeutic use" (US5408039)

It has also been reported that CHT chromatography combined with pH 5.4 acid precipitation to separate vWF components has a low specific activity, and while acid precipitation removes fibronectin, it may affect the biological activity of vWF, such as "Production of avonwille brand factor prepartion having great specific activity" (US20070135619A1)

[0009] In addition, there are some processes that use EMDFractogelTMAE (Merck) to purify the VIII / vWF factor complex, but none of them can obtain high-purity vWF alone, and the treatment is not targeted, such as "Preparation containing vWF with hemostatic activity and its preparation method" (CN1425024A), "A Process for recovering high-purity virus-inactivated factor VIII by anion exchange changer chromatography" (US5714590)

[0010] There are also some companies that use genetic recombination methods (monkey kidney cells or CHO cells) to purify vWF. They purify from animal cell culture fluid by anion exchange chromatography, but due to the existence of animal origin antigens, the human body may have Repulsion, such as "Method for isolation of highly pure von Willebrand Factor" (US5854403)

[0011] There is another report of vWF purified by two-step chromatography, but its purity is not high, and its specific activity is relatively low, only 50IU / mg, such as "Processformanufacturing vonWillebrandfactor" (US5252710)

[0012] It can be seen that there are still some problems in the existing vWF purification process: (1) The preparation cycle is long, which is not suitable for large-scale preparation, especially when immunoaffinity chromatography is used for purification, the antibody preparation cycle is long and the gel loading capacity is too long. Low

(2) It is difficult to control the process stability of the VIII / vWF complex product; (3) The vWF product obtained by the recombinant method may have heterogeneous antigens; (4) The separation and purification process is cumbersome

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