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Process for protein isolation

A protein and solid separation technology, applied in the direction of animal/human protein, albumin peptide, serum albumin, etc.

Inactive Publication Date: 2008-01-23
AVT PLASMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In virtually all existing preparations, Factor IX in the supernatant I is usually further purified using an affinity chromatography step using Heparin Sepharose, but since the entire method is based on Cohn fractionation , so there are still significant limitations in the preparation efficiency

Method used

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  • Process for protein isolation
  • Process for protein isolation
  • Process for protein isolation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0150] Example 1 α-1-Protease Inhibitor, Antithrombin III, Fibrinogen, Immunoglobulin Protein and Albumin Separation

[0151] step 1. Solid separation medium: Sepharose-tungsten carbide microspheres reacted with 2-mercaptonicotinic acid. The size distribution of the agarose-tungsten carbide microspheres ranges from 40-120 microns, with an average diameter of 70 microns. The density of the microspheres was 2.9 g / ml (FastLine UFC NNSDW cat. No.: CS48, UpFront Chromatography A / S, Copenhagen, Denmark.). The microspheres were loaded into an EBA column (FastLine 100, UpFront Chromatography A / S, Copenhagen, Denmark) (diameter 10 cm; bed height set at 50 cm; set bed volume = 3.926 L). Linear at 7.5 cm / min with 2.5 column volumes (9.8 L) of 40 mM sodium citrate (pH 4.5) then another 2.5 column volumes (9.8 L) of 40 mM sodium citrate (pH 5.0) at 25 °C The flow rate is balanced.

[0152] Step 2. 2 L of thawed human plasma was diluted 1:2 with 4 L of water, and 6 liters of the d...

Embodiment 2

[0158] Example 2 Alpha-1-Protease Inhibitors, Albumin, Immunoglobulin and Fibrinogen separation, which also includes washing steps

[0159] The solid separation medium used in this example is the same as that used in Example 1.

[0160] step 1. At a temperature of 21°C, an EBA column (FastLine 20, UpFront Chromatography A / S, Copenhagen Denmark) (diameter 2 cm; bed height is 25 cm; set bed volume = 78.5 mL).

[0161] Step 2. 39.3 mL of thawed human plasma was diluted 1:2 with 78.5 mL of water, and 117.8 mL of the diluted plasma solution of thawed human plasma was applied to the column. The pH of the diluted plasma solution was adjusted to pH 5.0 with 1 M HCl before use on the column, and the loading ratio was 1.5 liters of plasma solution per liter of resin.

[0162] Step 3. After loading the diluted plasma solution, the column was eluted with 3.3 column volumes (259.2 mL) of elution buffer 1 containing 10 mM sodium citrate, pH 5.0. Unbound proteins, lipids and oth...

Embodiment 3

[0168] Example 3 α-1-Protease Inhibitors, Albumin, Transferrin, Immunoglobulin and Isolation of Fibrinogen

[0169] The solid separation medium used in this example is the same as that used in Example 1.

[0170] step 1. Equilibrate an EBA column (FastLine 20, UpFront Chromatography A / S, Copenhagen Denmark) (diameter 2 cm; bed height is 25 cm; set bed volume = 78.5 mL).

[0171] Step 2. 39.3 mL of thawed human plasma was diluted 1:2 with 78.5 mL of water, and 117.8 mL of the diluted plasma solution containing thawed human plasma was applied to the column. The pH of the diluted plasma solution was adjusted to pH 5.0 with 1 M HCl before use on the column, and the loading ratio was 1.5 liters of plasma solution per liter of resin.

[0172] Step 3. After loading the diluted plasma solution, the column was eluted with elution buffer 1 containing 9.4 column volumes (738.3 mL) of demineralized water. Unbound proteins, lipids and coagulation factors including 100% alpha-1...

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Abstract

In one aspect, the present invention is directed to a process for isolating proteins from a solution comprising proteins, wherein the solution is selected from the group consisting of: crude bood plasma, blood serum, cryosupernatant derived from plasma, fractionated human plasma, cryoprecipitate derived from plasma and recombinant broths. The process involves providing a solid separation medium having the formula: M-S-L wherein M is a matrix backbone, S is an optional spacer arm, and L is a ligand which is mercaptonicotinic acid, contacting the solid separation medium with the solution comprising the proteins, such that at least one of the proteins becomes reversibly bound bound to said solid separation medium. At least one elution step is then performed to selectively elute at least one protein fraction from the solid separation medium. In another aspect, the present invention is directed to a process for isolating Factor VIII and / or Factor IX.

Description

technical field [0001] The present invention relates to methods for isolating proteins from biological sources. More specifically, the present invention relates to methods for isolating proteins from blood. Background technique [0002] Plasma is one of nature's most valuable raw materials, and proteins purified from plasma are essential to the life and health of many individuals worldwide. Most of these proteins were produced by Cohn cold ethanol precipitation fractionation. The method was developed by Dr. Edwin Cohn of Harvard University in the early 1940s. Cohn found that proteins in plasma could be separated according to their precipitation characteristics under different conditions (pH, ionic strength, protein concentration, temperature and ethanol concentration). By varying these parameters, different proteins were precipitated in a step-wise manner. The Cohn technique has been used in the plasma industry for decades, but the method has some limitations in terms of...

Claims

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Application Information

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IPC IPC(8): C07K1/14C07K1/22C07K14/81C07K14/745C07K14/755C07K14/765C07K16/00B01D15/08
Inventor A·利赫姆
Owner AVT PLASMA
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