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Method for improving separation efficiency, purity and biological specific activity of blood coagulation factor VIII and analog thereof by using affine aqueous two-phase system

A technology for coagulation factors and separation efficiency, which is applied in the preparation methods of peptides, chemical instruments and methods, organic chemistry, etc., can solve problems such as failure, and achieve the effect of simple method, universal and flexible system, and improved partition coefficient and separation efficiency.

Inactive Publication Date: 2012-07-18
汪志友
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  • Abstract
  • Description
  • Claims
  • Application Information

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Examples

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Embodiment 1

[0024] This example takes L4-Mal-PEG as an example to demonstrate how to prepare a characteristic hydrophilic polymer compound modified by a sulfhydryl group of a small peptide ligand with specific affinity for blood coagulation factor VIII.

[0025] Take 4.2 mg of polyethylene glycol graft Mal-PEG (MW about 3600 Da), and prepare an appropriate amount of L4 in a buffer solution with pH=4-8, stir at room temperature for 12-48 hours, remove the reaction solution, dilute, and place In the Amicon Ultra-4 ultrafiltration tube (MWCO=1000Da), remove low-molecular impurities, then use water as the eluent, pass through the HiTrap Desalting gel column, and freeze-dry the passed column liquid to obtain the product L4-Mal-PEG. Save for later.

Embodiment 2

[0027] This example shows how to extract and purify gene recombinant coagulation factor VIII from non-secretory and expressing coagulation factor VIII CHO cells by affinity aqueous two phase. A certain amount of CHO cells that have non-secreted and expressed coagulation factor VIII were collected by centrifugation and resuspended in 20 ml of phosphate buffered saline with pH = 8.0 and a concentration of 20 mM. In this case, the L4-Mal-PEG / phosphate system was used for purification ; or resuspended in 20ml of Tris-HCl buffer with pH=8.0 and a concentration of 20mM (containing about 0.14mg of recombinant coagulation factor VIII), in this case the L4-Mal-PEG / dextran system was used for purification. Cells were lysed by sonication and centrifuged to remove cellular debris or residue. Add desired L4-Mal-PEG, potassium phosphate, or / dextran to the sample for reconstitution buffer. Gently stir the solution in a beaker or other container (to reduce the generation of DNA fragments tha...

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Abstract

The invention discloses a simple and quick method for improving separation efficiency, purity and biological specific activity of blood coagulation factor VIII and an analog thereof. The method is based on application of an affine aqueous two-phase technology, i.e., a characteristic hydrophilic macromolecular compound, preferably polyethylene glycol (PEG), forms a stable aqueous two-phase system with phosphate, glucan or other second component in aqueous solution; and one of core technologies is that: covalent linkage of the PEG and the blood coagulation factor VIII specific affine small peptide ligand is realized. The blood coagulation factor VIII is preferably located in a characteristic hydrophilic macromolecular compound phase connected to the affine small peptide ligand; and most of protein (or hybrid protein) from cell extract tends to move to another phase. By changing existence of target protein or polypeptide in one phase or another phase, the protein or polypeptide is efficiently purified with high purity.

Description

field of invention [0001] The invention relates to a method for improving the efficiency and biological activity of the separation and purification of blood coagulation factor VIII and its analogues, which belongs to the field of biopharmaceuticals, and the key is to use the characteristic of covalently linking blood coagulation factor VIII specific affinity small peptide ligands The hydrophilic macromolecular compound, preferably polyethylene glycol (PEG), makes the surface of the characteristic hydrophilic macromolecular compound have a specific affinity binding site for coagulation factor VIII. This specific affinity binding site not only makes the coagulation factor VIII have a strong affinity adsorption interaction with the characteristic hydrophilic polymer compound, but also determines that the coagulation factor VIII can be enriched and dispersed in the affinity two-phase aqueous phase. Water polymer compound phase (upper phase). The method of the present invention ca...

Claims

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Application Information

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IPC IPC(8): C07K1/107
Inventor 汪志友
Owner 汪志友
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