Separating and purifying method for new recombinant human interferon alpha2b

Abstract

The present invention relates to the separation and purification method of new recombinant human interferon alpha-2b. The separation and purification method includes the following steps: crushing recombinant human interferon alpha-2b colibacillus as engineering bacillus, dissolving in urea solution, Tris-HCl washing, cracking with guanidine hydrochloride, boric acid renaturing, regulating pH value, dissolving in salt solution, chromatographic separation with hydrophobic column M1, chromatographic separation with CM-Sepharose column, chromatographic separation with hydrophobic column M2, and chromatographic separation with S-100 molecular sieve. The present invention has less purification steps and integrated purification process, and may be used in large scale automatic production.

Description

technical field [0001] The invention relates to a method for splitting and purifying biological proteins, and more specifically, relates to a novel method for separating and purifying recombinant human interferon alpha 2b. Background technique [0002] Recombinant human interferon α2b has the functions of broad-spectrum anti-virus, anti-tumor, inhibiting cell proliferation and improving immune function. The combination of interferon and cell surface receptors induces cells to produce a variety of antiviral proteins, inhibits virus reproduction in cells, and improves immune function, including enhancing the phagocytosis of macrophages, enhancing the cytotoxicity and natural killing of lymphocytes to target cells cell function. [0003] The purification methods of recombinant human interferon α2b reported in the literature mainly fall into two categories, affinity chromatography and non-affinity chromatography. The purity of the recombinant human interferon α2b obtained by a...

AI Technical Summary

Problems solved by technology

[0006] For this purpose, the existing industrialized production techniques of recombinant human interferon α2b all adopt processes such as salting-out and ultrafiltration desalination, salt concentration gradient elution and dialysis, and their common problems are: (1) low renaturation efficiency : The traditional dilution renaturation method dilutes the sample tens of times or even hundreds of times, which will increase the volume of the sample sharply, which brings great difficulties to the subsequent separation and purification, and requires a large amount of renaturation during the renaturation process container; dialysis is time-consuming and requires multiple changes of dialysis solution

The common disadvantage of these two methods is that the protein will aggregate during the renaturation process and produce a large amount of precipitation, the renaturation efficiency is low, the activity recovery rate of the protein is usually only 5-20%, and the protein solution after renaturation contains a large amount of Miscellaneous proteins need to be further separated and purified; (2) The process route is cumbersome and the production cycle is long: In the traditional separation and purification process of recombinant proteins, most of the classic soft gel separation media are used. Due to the large particles of this medium, The separation efficiency is poor, so it is often necessary to use a variety of different modes of chromatographic operations to purify the target protein in order to obtain the target protein with a purity that meets a certain standard

In addition, the pressure resistance of this chromatographic medium is very poor, and it can only be operated at a low flow rate, and the separation and purification time is long; the separation and purification steps and the long separation time make the mass recovery rate and activity recovery rate of the protein very low. low; and in the traditional recombinant protein production process, protein renaturation and purification are two independent unit operations in the production process, which also restricts production efficiency to a large extent; (3) high production costs and large investment in equipment : Because refolding and separation and purification are carried out separately, and there are many separation and purification steps, each step needs to have matching equipment, resulting in large equipment investment and high production cost. With the increase of production scale, this drawback will become more and more serious. more serious