Method for purifying and preparing anti-VEGF antibody fragment

Abstract

The invention relates to a method for purifying and preparing a recombinant anti-VEGF antibody fragment which is periplasmically-expressed in escherichia coli. The key technique is the purification and preparation process and method of an anti-VEGF antibody fragment which is periplasmically-expressed in escherichia coli. When the recombinant antibody is purified, a soluble reinforcing reagent is added in the periplasm extraction process to facilitate the correct folding of the heavy-chain and light-chain of the antibody. Before the antibody fragment is purified, heat treatment is performed to inactivate and deposit a part of host mycoprotein and thus to make the antibody molecule more stable. During the purification process, the two-step purification of cation exchange-hydrophobic chromatography is carried out to produce the antibody protein with purity of more than 95%. The method is easy and convenient to operate, is low in cost and has a high yield, and is suitable for industrialization.

Description

technical field [0001] The invention relates to the field of biotechnology and provides a method for purifying and preparing anti-VEGF antibody fragments. Background technique [0002] With its high specificity, effectiveness and safety, monoclonal antibody drugs have become the pillar varieties of international biotech drugs, accounting for 36% of the total amount of biotech drugs, ranking among the forefront of medical biotechnology products. In 2010, monoclonal antibody drugs continued to lead the global pharmaceutical market with sales of US$48 billion, a year-on-year increase of 20%. From 2000 to 2010, the compound growth rate of the global monoclonal antibody drug market was as high as 32%. The entire antibody product market is showing explosive growth, and it is expected that monoclonal antibody drugs will become the leading product in the development of the global biomedical field in the next ten years. Monoclonal antibody drugs have made outstanding progress in th...

AI Technical Summary

Problems solved by technology

However, in the process of large-scale production, there are some defects in affinity chromatography: 1. Low loading 2. Ligands are easy to fall off 3. Expensive 4. Most affinity chromatography media need to be eluted under acidic conditions Antibody proteins, leading to aggregation, precipitation, and loss of activity of some acid-resistant antibody proteins

The affinity ligand is coupled to the matrix skeleton through a chemical bond, and there is a possibility that the ligand will fall off during each batch of purification, so the product needs to detect the residual amount of the ligand; The amount is further reduced. In order to ensure the processing amount of the sample, it is necessary to replenish and replace the gel regularly, and the cost is increasing; the price of the affinity medium is expensive, which increases the production cost of the product, so in the process of mass production of antibodies and antibody fragments, do not use Affinity chromatography can quickly obtain the recombinant protein required by the Pharmacopoeia, and is a low-cost and efficient purification method

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