Method for preparing human prothrombin complex from Cohn blood plasma component III

A technology of human prothrombin and complexes, which is applied in the field of preparation of human prothrombin complexes, can solve the problems of unpredictable effects of protein separation and residual gelatin, reduce the probability of virus contamination, and prevent the activation of thrombin , no cross-contamination effect

Inactive Publication Date: 2016-02-17
上海洲跃生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Existing PCC preparation method is divided into two kinds according to source of raw material, and a kind of is directly from gel adsorption from the plasma of cold gum, and a kind of is from the gel adsorption in the component III after pretreatment; Wherein, the former mostly The classic batch adsorption method is adopted, that is, the swollen gel is added to the cold gelatinized plasma in a certain proportion, and it is adsorbed while stirring. After the adsorption is completed, the plasma is filtered, and the gel adsorbed with PCC is collected for subsequent processing. ;This method will inevitably have part of the colloid remaining in the plasma, which will have an unpredictable impact on the subsequent protein separation. In addition, the PCC produced by this process contains significantly less coagulation factor VII, generally only coagulation factor VII One quarter to one third of IX

Method used

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  • Method for preparing human prothrombin complex from Cohn blood plasma component III
  • Method for preparing human prothrombin complex from Cohn blood plasma component III

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] 1， Component III dissolution: suspend 2kg of component III precipitate in 10kg dissolution buffer, the dissolution buffer formula is 0.02M sodium citrate, 0.15M sodium chloride, pH6.50-6.60; stir for 4 hours to fully dissolve the precipitate ;

[0043] 2， PEG precipitation to remove impurities: add 50% PEG solution to the above suspension until the PEG content in the suspension is 3%, press filter after stirring for 1.5 hours, the filter plate is Supradur50P+1.0μm filter element of PALL company, and the filter plate is pre-used in the steps ( 1) Pre-washing with the dissolution buffer, and collecting a clear filtrate;

[0044] 3. S / D virus inactivation: Add Tween80 to 1.0% (wt%) and TNBP (tributyl phosphate) to 0.3% (wt%) to the above filtrate, stir well, heat up to 24-26°C, and keep warm for 6 Hour;

[0045] 4. Strong anion exchange column chromatography: Cool the above S / D solution to 8°C, and then put it on the Capto-Q column. The column is pre-filled with equilibr...

Embodiment 2

[0053] 1， Dissolution of Component III: Suspend 2kg of Component III precipitate in 20kg of dissolution buffer, the formulation of the dissolution buffer is 0.02M sodium citrate, 0.15M sodium chloride, pH7.40-7.50; stir for 2 hours to fully dissolve the precipitate ;

[0054] 2. PEG precipitation to remove impurities: add 50% PEG solution to the above suspension until the PEG content in the suspension is 7%, press filter after stirring for 0.5 hours, the filter plate is Supradur50P+1.0μm filter element of PALL company, and the filter plate is used in advance 1, the dissolution buffer pre-washed, collected to obtain clarified filtrate;

[0055] 3, S / D virus inactivation: with embodiment one;

[0056] 4. Strong anion exchange column chromatography: cool down the above S / D solution to 20°C, and then put it on the QSepharoseFF column. Sodium,) balance; after loading the column, wash the column with the above equilibration buffer, and then wash the column with the elution buffer...

Embodiment 3

[0063] 1， Dissolution of component III: Suspend 2kg of component III precipitate in 20kg of dissolution buffer, the composition of the dissolution buffer is 0.02M sodium citrate, 0.15M sodium chloride, pH6.90-7.10; stir for 3 hours to fully dissolve the precipitate ;

[0064] 2. PEG precipitation to remove impurities: Add 50% PEG solution to the above suspension until the PEG content in the suspension is 5%, press filter after stirring for 1 hour, the filter plate is Supradur50P+1.0μm filter element of PALL company, the filter plate is pre-used The dissolving buffer described in step 1 is pre-washed, and the clarified filtrate is collected;

[0065] 3, S / D virus inactivation: with embodiment one;

[0066] 4. Strong anion exchange column chromatography: cool the above S / D solution to 15°C, and then put it on the Capto-Q column, which is pre-filled with equilibrium buffer (PH6. Sodium chloride,) balance; after loading the column, wash the column with the above equilibration b...

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Abstract

The invention discloses a method for preparing a human prothrombin complex from a Cohn blood plasma component III. The method comprises the following steps: 1, dissolving the Cohn blood plasma component III; 2, sedimenting polyethylene glycol (PEG), and removing impure protein; 3, conducting S / D viral inactivation; 4, conducting strong anion-exchange column chromatography; 5, conducting ultrafiltration dialysis and concentration; 6, adding a stabilizing agent, and conducting adjustment; 7, conducting virus-removal filtration through nanofilms; 8, conducting sterilization, filtration and subpackage; 9, conducting freeze-drying; 10, conducting dry-heat viral inactivation. According to the method, efficient strong anion-exchange column chromatography is adopted, traditional batch processing gel adsorption is replaced, labor intensity of operation is greatly lowered, cross contamination problems caused by a batch processing method are avoided, meanwhile, in the whole production process, three steps of viral inactivation and removal measures are adopted for products, and the method has the advantages that the flow is brief and simple, the production cycle is short, operation is easy to conduct, and the products are safe and reliable to use.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals and relates to the preparation of blood products, in particular to a method for preparing human prothrombin complex (PCC) from Cohn plasma fraction III. Background technique [0002] Human prothrombin complex (PCC) is a hemostatic preparation for intravenous infusion that promotes blood clotting. PCC products include four coagulation factors II, VII, IX, and X, which are a mixture of four coagulation factors. The four factors of PCC are all glycoproteins, and the molecules all contain special amino acid residues - γ-carboxyglutamic acid (Gla). Gla is an amino acid that can bind calcium ions. Its existence makes PCC have the ability to bind metal ions. nature. After PCC binds to calcium ions, its conformation changes, revealing the characteristics of binding to phospholipid membranes, and then participating in the blood coagulation process. [0003] The four factors included in PCC are all seri...

Claims

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Application Information

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IPC IPC(8): A61K35/16A61K38/36A61P7/04A61P1/16
Inventor 李春洲
Owner 上海洲跃生物科技有限公司
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