Purification method of anti-VEGF (Vascular Endothelial Growth Facto) type monoclonal antibody

Abstract

The invention relates to the field of protein purification and in particular relates to a purification method of an anti-VEGF (Vascular Endothelial Growth Facto) type monoclonal antibody. The purification method comprises: firstly, capturing by utilizing a composite material chromatography to separate the antibody and almost components in a harvesting solution; furthermore, carrying out fine purification by utilizing hydroxyapatite chromatography, so as to further remove host cell pollutants, aggregates and the like, wherein a composite chromatography material is a composite medium with ion exchange effect and hydrophobic effect. According to the purification method of the anti-VEGF type monoclonal antibody, the content of host cell protein (HCP), DNA (Deoxyribonucleic Acid), polymers and acidic peaks is reduced, so that the aim of remarkably improving the purity of the antibody is realized; the purification method is simple in structure and relatively low in cost.

Description

technical field [0001] The invention relates to protein purification, in particular to a method for purifying anti-VEGF monoclonal antibodies. Background technique [0002] Bevacizumab (bevacizumab, trade name Avastin) is a recombinant humanized monoclonal antibody. Approved by the FDA on February 26, 2004, it is the first drug to inhibit tumor angiogenesis approved for marketing in the United States. The IgG1 antibody can be combined with human vascular endothelial growth factor (VEGF) and block its biological activity through in vivo and in vitro detection systems. Bevacizumab is produced by fermentation and culture of Chinese Hamster Ovary (CHO) expression system. Once the monoclonal antibody of interest is obtained to clarify the fermentation broth, a combination of different chromatographic techniques is usually used to try to combine the target protein with Other proteins produced by the cell are separated. [0003] The traditional purification process obtains monoc...

AI Technical Summary

Problems solved by technology

Protein A column is the first choice for affinity chromatography due to its high selectivity and strong ability to remove impurities. More and more research has begun to focus on solving existing problems with non-Protein A processes

In addition, the three-step process has a low recovery rate, high economic costs, and the high salt used in hydrophobic chromatography has the risk of inactivating the protein. People have begun to pay attention to the use of more efficient two-step chromatography to purify monoclonal antibodies.

[0004] The patent CN200880119331.X applied by Genentech, a bevacizumab monoantigen research unit, uses cation exchange chromatography for antibody purification. As the first step of chromatography, cation chromatography is the biggest bottleneck in the adjustment of the sample, which needs to adjust the pH of the sample On the one hand, the adjustment of the sample increases the volume of the sample, which leads to the increase of the liquid storage tank and the sample loading time. On the other hand, precipitation often occurs during the adjustment process, resulting in the loss of the sample, because the sample contains a large amount of Host cell protein (HCP), which passes through the isoelectric point of HCP during pH adjustment, resulting in precipitation

At the same time, the loading capacity of cations is often lower than that of affinity chromatography, resulting in an increase in cost

[0005] European patent EP1651665 uses MEP to capture and purify non-antibody proteins and fragments similar to antibodies. It uses two-step elution. 35% propylene glycol is added to the first elution, and 50% propylene glycol is added to the second elution. The recovery rate is 85%, but it is not optimized for monoclonal antibodies, and the purity and yield are low after being applied to monoclonal antibodies

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