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Method for absorbing and separating human prothrombin complex by utilizing expansion bed

A human prothrombin, adsorption and separation technology, applied in the field of biopharmaceuticals, can solve the problems of large gel loss, troublesome operation, high production cost, etc., achieve good adsorption and separation effects, reduce resin loss, and reduce production costs.

Inactive Publication Date: 2010-09-22
成都英德生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The preparation process has the disadvantages of troublesome operation, many procedures, large gel loss, high production cost, open operation, and the risk of cross-contamination affecting product quality.
[0004] The application of expanding bed adsorption to separate target proteins has achieved initial success, and the separation and extraction of target proteins in genetic engineering fermentation products has reached a production scale, but there have been no research reports on the separation and extraction of plasma proteins.

Method used

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  • Method for absorbing and separating human prothrombin complex by utilizing expansion bed
  • Method for absorbing and separating human prothrombin complex by utilizing expansion bed
  • Method for absorbing and separating human prothrombin complex by utilizing expansion bed

Examples

Experimental program
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Effect test

Embodiment 1

[0026] Preparation of plasma raw material:

[0027] Take two bags of plasma (600mL each), put the frozen bagged plasma in a water bath at 23°C, and thaw the plasma after 20 minutes; divide the thawed plasma into four centrifuge cups, put them into BECKMAN J2-HS high-speed In a refrigerated centrifuge, centrifuge at 9000rpm at 2°C for 20min; pour out the supernatant plasma in the centrifuge cup and put it in a plastic bottle.

Embodiment 2

[0029] Such as figure 1 As shown, the process flow of the method for the expanded bed adsorption and separation of human prothrombin complex provided by the present invention is as follows:

[0030] (1) Take out the frozen plasma, clean the outer surface of the plasma bag with water for injection at normal temperature, disinfect with 70% ethanol solution, and rinse with water for injection at normal temperature after disinfection. The bag was broken manually or mechanically, and the plasma was poured into the slurry tank, and the jacket was filled with hot water at 37°C. After the plasma was completely melted, the temperature of the plasma was controlled at 2°C±0.5°C. Use a rotor pump or a diaphragm pump to input the plasma into a continuous flow centrifuge to separate the cryoprecipitate, and collect the supernatant plasma. Frozen plasma was collected from the bodies of qualified plasma donors using a single plasma extractor, and immediately placed in a quick-freezing refri...

Embodiment 3

[0045] Such as Figure 4 , Figure 5 As shown, the optimized elution conditions in the adsorption and step elution purification of expanded bed adsorption separation PCC:

[0046] Put the weighed EBA-CG6 adsorption resin 96g into the expanded bed column (100cm×25mmI.D.), balance it with buffer A first, and control the expansion rate to 1.5. After the expanded bed is stable for 20min, quickly switch the feed solution to Implementation of the prepared plasma in Case 1; first detect the absorption value of the plasma at A280, and continuously monitor the plasma flowing out of the column with an ultraviolet spectrophotometer. Wash in this mode until the UV signal returns to the baseline. The volume of the washing solution is 6 times the column volume; after the resin has fully settled to the lower part of the column, lower the upper distribution head to the top surface of the resin, and first use buffer B1 to elute. The volume is 2 times the column volume; after the eluent peak ...

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Abstract

The invention relates to a method for absorbing and separating a human prothrombin complex by utilizing an expansion bed, comprising the following steps: (a) separating cryoprecitation from melted refrigerated plasma by using a refrigeration type continuous flow centrifuge, and collecting supernate as a raw material; (b) carrying out stable expansion on an expansion bed by using a buffer solution A, wherein the buffer solution A is 0.05mol / L1tris-citric acid with the PH value of 7.0; (c) rapidly switching into a feed solution after the expansion bed is in stable balance, stopping feeding and rapidly switching into the buffer solution A when breakthrough points reaches 5-10%, and washing in an expanded mode; and (d) switching into buffer solutions of 0.4mol / L NaCl and 1.6mol / L NaCl after washing, carrying out gradient elution in a stationary bed mode, and collecting the protein elution peak of the buffer solution of 1.6mol / L NaCl to obtain a prothrombin complex crude product.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, in particular to a method for separating and preparing human prothrombin complexes using a novel expanded bed adsorption separation technology. Background technique [0002] Expanded bed adsorption separation technology was first proposed by Professor Chase of the University of Cambridge in 1992. It uses the physical properties of the adsorbent itself and carefully designs the fluidized bed device to obtain a stable fluidized bed with little back mixing, that is, expanded bed. bed (expanded bed). Subsequent studies have shown that expanded bed adsorption chromatography can directly adsorb and preliminarily purify the target protein from the raw material liquid. It integrates several steps of clarification, concentration and preliminary purification into one unit operation, which reduces operating steps and improves product yield. Efficiency, reducing purification costs and capital investment, s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/22
Inventor 邱家山赵红卫何琦
Owner 成都英德生物工程有限公司
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