Method for improving purity of bispecific antibody by purification with magnetic beads

A bispecific antibody and magnetic bead technology, applied in the biological field, can solve problems such as the increase of intermolecular impurities, achieve complete sample elution, save time, and reduce equipment requirements

Inactive Publication Date: 2019-09-24
SHANGHAI WUXI BIOLOGIC TECH CO LTD +2
View PDF3 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although bispecific antibodies have incomparable advantages over monoclonal antibodies, due to the co-expression of up to four specific polypeptide chains or the assembly of extended length polypeptide chains, the recombinant production of IgG bispecific antibodies is usually accompanied by Homodimerization of heavy chains, mispairing of heavy and light chains, unbalanced expression of different chains, and mispartitioning between molecules lead to increased impurities (by-products or aggregates) of the target species
Since some by-products show high similarity to the target bsAb, their removal poses a high challenge to the production process

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for improving purity of bispecific antibody by purification with magnetic beads
  • Method for improving purity of bispecific antibody by purification with magnetic beads
  • Method for improving purity of bispecific antibody by purification with magnetic beads

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1. Application of AmMag Protein A magnetic beads in monoclonal antibody purification and control of endotoxin

[0033] First prepare the AmMag Protein A magnetic beads: (1) Remove 20% ethanol from the storage magnetic beads by magnetic separation; (2) Add 0.1M NaOH to wash the magnetic beads, and remove NaOH by magnetic separation after incubation for 0.5h; (3) Add 0.1 M Tris, pH 7.0 Equilibrate the magnetic beads, repeat washing 3 times; (4) Add three times the volume of magnetic beads 0.1M Tris, pH 7.0 and mix well to obtain an activated magnetic bead suspension.

[0034] Add an appropriate amount of activated magnetic bead suspension to 20ml monoclonal antibody expression supernatant, and incubate on a shaker for 2 hours to fully combine the magnetic beads with the antibody, and then remove the supernatant by magnetic separation. Wash the magnetic beads with 0.1M Tris, pH7.0, repeat 3 times, and then wash with H 2 O wash the beads. Add 1.5ml 0.1M Glycine, pH ...

Embodiment 2

[0040] Example 2. Comparison of AmMag Protein A magnetic beads and traditional media used in the purification of WuXiBody bispecific antibodies

[0041] Firstly, the magnetic beads were washed and activated as in Example 1. At the same time, prepare the traditional GE MabSelect SuRe chromatography column, first wash with 0.5M NaOH for 0.5h, then equilibrate with 0.1M Tris, pH7.0, and then load the sample. The bispecific antibody expression supernatant samples were equally divided into two and purified simultaneously. AmMag Protein A magnetic beads were eluted with 0.1M Glycine pH3.0, and GE MabSelect SuRe chromatography columns were eluted with 0.1M Glycine pH3.5.

[0042] Table 2 compares the purification results of the four tests: the purity of bispecific antibodies purified from AmMag magnetic beads is generally higher than that of samples purified from MabSelect SuRe chromatography columns.

[0043] Table 2: Statistical data of double antibody sample purification

[004...

Embodiment 3

[0045] Example 3. AmMag Protein A is applied to the purification of large-volume bispecific antibody samples

[0046] The structure of the bispecific antibody is relatively complex, especially when the sample is prone to polymerization, it may be difficult to elute when using GEMabSelect SuRe chromatography column for purification. When the BsAb 5 sample was purified using GE MabSelectSuRe chromatography column, almost no product was obtained when eluted with conventional 0.1M Glycine pH3.5, and the eluted product was obtained by changing the elution conditions to use 0.1M Glycine, 0.2M Arginine, pH3.0 Antibody purity is low. After trying to purify with AmMag magnetic beads, although the yield is still low, the purity of the product is high, so the volume is enlarged for production. Firstly, the magnetic beads were washed and activated as in Example 1. In order to make the sample more fully combined with the magnetic beads, the 2L antibody expression supernatant was evenly d...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses a method for improving purity of a bispecific antibody by purification with magnetic beads. The method comprises the steps of mixing magnetic beads coated with activated Protein A with a bispecific antibody containing supernatant to be purified, performing incubation, and after the magnetic beads are sufficiently mixed with the bispecific antibody, removing the supernatant through magnetic separation; firstly, cleaning the magnetic beads with a balanced buffered solution for 2-5 times; cleaning the magnetic beads with water; eluting the cleaned magnetic beads with an eluent, performing magnetic separation, and collecting the eluent; and eluting the separated magnetic beads with the eluent for 1-3 times, and merging the eluent so as to obtain the purified bispecific antibody. The method does not need the clarification operation, so that the time saved, the demand for equipment is reduced, and the bispecific antibody of samples being higher in flux and larger in volume can be purified within a short time.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for improving the purity of bispecific antibodies by using magnetic beads to purify. Background technique [0002] Monoclonal antibodies are produced by hybridomas formed by the hybridization of B lymphocytes and myeloma cells, and the domains formed by their light and heavy chains can recognize and bind specific antigens. Since the FDA approved the first chimeric monoclonal antibody for the treatment of lymphoma in 1997, many antibody drugs have been approved, and many of them have become "bomb drugs" with annual sales exceeding one billion US dollars. Bispecific antibodies are artificially engineered on the basis of monoclonal antibodies, which can recognize two identical or different immunoglobulins on antigenic epitopes at the same time. This feature has significant advantages over traditional monoclonal antibodies . For example, bispecific antibodies that bind two epi...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/46C07K1/14
CPCC07K16/468
Inventor 郁博文刘科梅徐珊珊王芳陈俏梅王卓智李竞顾继杰陈智胜
Owner SHANGHAI WUXI BIOLOGIC TECH CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap