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A washing buffer for ion exchange chromatography for preparing human blood coagulation factor VIII and its application

A technology of ion exchange chromatography and washing buffer, which is applied in the direction of factor VII, blood coagulation/fibrinolysis factor, peptide preparation method, etc., can solve the problem that it is difficult to ensure that the visible foreign matter of the product is qualified, the molecular weight of FⅧ is large, and the preparation process requirements of FⅧ are relatively low. Advanced problems, to achieve good industrial application prospects, high activity yield, good resolubility effect

Active Publication Date: 2020-02-11
CHENGDU RONGSHENG PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] FⅧ has a large molecular weight, complex structure, and is easy to inactivate. Moreover, since it needs to be directly injected into the human body during use, the requirements for visible foreign matter in the product are strict, so the requirements for the preparation process of FⅧ are relatively high.
When using the existing process to prepare FⅧ, the activity loss is relatively large, especially after 72 hours of dry heat treatment at 80°C, the activity loss is serious; at the same time, it is difficult to ensure that the visible foreign matter in the product is qualified
[0006] The current preparation process usually adds protective agents containing albumin or sugar alcohols or complex components in the final preparation of FⅧ to ensure a high activity yield of the product and qualified visible foreign matter, but the effect is not ideal and the cost increases

Method used

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  • A washing buffer for ion exchange chromatography for preparing human blood coagulation factor VIII and its application
  • A washing buffer for ion exchange chromatography for preparing human blood coagulation factor VIII and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1 Preparation of human blood coagulation factor VIII of the present invention

[0047] The preparation process of human coagulation factor VIII of the present invention is shown in figure 1 .

[0048] The preparation method is as follows:

[0049] Ⅰ. Pre-chromatographic treatment

[0050] (1) Using fresh frozen plasma as raw material, melt the plasma, centrifuge to prepare cryoprecipitate, dissolve the cryoprecipitate in 0.02M Tris buffer, precipitate with 30% polyethylene glycol, centrifuge after adsorption by aluminum hydroxide, and obtain the supernatant;

[0051] (2) After the supernatant was clarified and filtered at 0.45 μm, Tween-80 (polysorbate 80) and tributyl phosphate were added to make the final concentrations 1% and 0.3%, respectively, and treated at 25°C±1°C for 6 hours, and the process was completed. For the first virus inactivation (that is, SD virus inactivation), the sample must be loaded on the column.

[0052] Ⅱ. Ion exchange chromatog...

Embodiment 2

[0062] Embodiment 2 Preparation of human blood coagulation factor VIII of the present invention

[0063] The preparation method is as follows:

[0064] Ⅰ. Pre-chromatographic treatment

[0065] With embodiment 1.

[0066] Ⅱ. Ion exchange chromatography

[0067] (1) Prepare buffer

[0068] The balance buffer includes the following components: 0.01M sodium citrate, 0.001M calcium chloride, 0.121M glycine, 0.016M lysine hydrochloride, 0.06M sodium chloride; adjust pH to 6.5;

[0069] The washing buffer includes the following components: 0.01M sodium citrate, 0.001M calcium chloride, 0.121M glycine, 0.016M lysine hydrochloride, 0.121M sodium chloride; adjust pH to 6.5;

[0070] The elution buffer includes the following components: 0.01M sodium citrate, 0.001M calcium chloride, 0.121M glycine, 0.016M lysine hydrochloride, 0.25M sodium chloride; adjust pH to 6.5;

[0071] (2) Use Toyopearl DEAE 650M as the gel for the chromatographic column filler; put the sample to be loaded o...

Embodiment 3

[0075] Embodiment 3 Preparation of human blood coagulation factor VIII of the present invention

[0076] The preparation method is as follows:

[0077] Ⅰ. Pre-chromatographic treatment

[0078] With embodiment 1.

[0079] Ⅱ. Ion exchange chromatography

[0080] (1) Prepare buffer

[0081] Equilibrium buffer includes the following components: 0.01M sodium citrate, 0.001M calcium chloride, 0.121M glycine, 0.016M lysine hydrochloride, 0.16M sodium chloride; adjust pH to 7.5;

[0082] The washing buffer includes the following components: 0.01M sodium citrate, 0.001M calcium chloride, 0.121M glycine, 0.016M lysine hydrochloride, 0.129M sodium chloride; adjust pH to 7.5;

[0083] The elution buffer includes the following components: 0.01M sodium citrate, 0.001M calcium chloride, 0.121M glycine, 0.016M lysine hydrochloride, 0.35M sodium chloride; adjust pH to 7.5;

[0084] (2) Use Toyopearl DEAE 650M as the gel for the chromatographic column filler; put the sample to be loaded o...

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Abstract

The invention discloses a washing buffer solution for ion-exchange chromatography for preparation of an FVIII (human coagulation factor VIII). The washing buffer solution contains sodium chloride with the final concentration being 0.121-0.129 M. The invention further provides an application of the washing buffer solution. The invention further provides a method for preparing the FVIII and a prepared product. The washing buffer solution can better maintain the activity of the FVIII, the activity yield of the FVIII is high, the purity is high, the visible foreign matters are qualified, the stability is good, the formula of the washing buffer solution is simple, the cost is low, and the washing buffer solution has good industrial application prospect.

Description

technical field [0001] The invention belongs to the field of blood products, in particular to a washing buffer for ion exchange chromatography for preparing human blood coagulation factor VIII and its application. Background technique [0002] Human coagulation factor VIII (Human coagulation factor Ⅷ, FⅧ) is an important endogenous coagulation factor, and the deficiency of FⅧ will lead to a severe hereditary bleeding disease - hemophilia A. Hemophilia A is the most common coagulation factor deficiency disease with the highest incidence rate. The most effective treatment method is to infuse the patient with plasma products rich in FⅧ to achieve the purpose of prevention and treatment. Therefore, FⅧ is a life-saving drug for patients with hemophilia A. [0003] FⅧ is made from healthy human plasma, which is separated, purified, virus-removed and inactivated, and freeze-dried. According to the 2015 edition of the Chinese Pharmacopoeia, the specific activity of human blood coa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/755C07K1/18
CPCC07K14/755
Inventor 牟蕾李伟鲁涛王黔川余伟
Owner CHENGDU RONGSHENG PHARMA