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Affinity purification method for reducing content of host cell protein in monoclonal antibody production

A host cell protein and monoclonal antibody technology, applied in the biological field, can solve the problems of host cell protein residue and amplification, and achieve the effect of simple and easy affinity process and good stability.

Active Publication Date: 2022-02-18
QYUNS THERAPEUTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In order to solve the problem of host cell protein (HCP) residues in antibody production, the present invention provides a new method for effectively reducing CHO host cell protein (HCP) in antibody purification and production, which can be widely used in antibody affinity purification processes, and the The cost of materials used is low, and the process is easy to scale up

Method used

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  • Affinity purification method for reducing content of host cell protein in monoclonal antibody production
  • Affinity purification method for reducing content of host cell protein in monoclonal antibody production
  • Affinity purification method for reducing content of host cell protein in monoclonal antibody production

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0121] Example 1 Preparation of anti-human interferon alpha receptor 1 monoclonal antibody QX006N

[0122] Purchasing human interferon alpha receptor 1 (IFNAR1) from Shanghai Nearshore Technology Co., Ltd. for immunization of New Zealand rabbits, using B cell cloning technology to obtain antigen-binding specific antibody clones, and then screening for binding to human IFNAR1 and having human IFNAR1 inhibitory activity of monoclonal antibodies. First, the cell supernatant was detected by Binding ELISA, and the clones binding to human IFNAR1 were selected; then, the clones with human IFNAR1 inhibitory activity were selected for detection by HEK Blue IFNα / β reporter gene cell method. The above immunization and screening processes are entrusted to commercial companies.

[0123] 37 clones were selected for recombinant expression and sequenced. It was determined that 362# and 1203# had the best cell neutralizing activity, and the sequences of the two clones were very similar. The...

Embodiment 2

[0129] Embodiment 2 Equilibrium dissociation constant (K D ) determination

[0130] The affinity between QX006N (HZD1203-45-IgG4.1) and human IFNAR1 was detected by BiacoreT200, and all processes were carried out at 25°C. Using a commercial Protein A chip, an appropriate amount of antibody was immobilized by the capture method, so that the Rmax was around 50RU, and the capture flow rate was 10 μl / min. The antigen was serially diluted, the flow rate of the instrument was switched to 30 μl / min, and the concentration flowed through the reference channel and the channel of the immobilized antibody in order of concentration from low to high, and the buffer was used as a negative control. After each association and dissociation, the chip was regenerated with pH 1.5 glycine. Use the analysis software that comes with the instrument to select the 1:1 binding model in the Kinetics option for fitting, and calculate the binding rate constant k of the antibody a , the dissociation rate ...

Embodiment 3

[0135] Example 3QX006N and Anifrolumab neutralize human interferon-induced HEK Blue IFNα / β cell STAT1 / 2 phosphorylation activity

[0136] Use the HEK Blue IFNα / β reporter gene cell line to measure the phosphorylation activity of the intracellular signal molecule STAT1 / 2 mediated by QX006N antagonizing interferon through IFNAR1: the cells in the culture medium were divided into 4×10 per well 4 Cells were added to 96 wells and then incubated at 37°C and 5% CO 2 conditions overnight. Serial dilutions of antibody concentrations ranging from 0 to 5 μg / ml were added to the cells along with 0.2 ng / ml of IFNα.2b. Then at 37 °C and 5% CO 2 Cultivate under conditions for 24 hours, collect the cell culture supernatant and add 10% QUANTI-Blue TM Detection reagents at 37°C and 5% CO 2 React under the conditions for 1 hour, then detect the OD630nm value and draw the dose-effect curve, and then analyze the antagonistic activity of the antibody. The experimental results show that QX006N c...

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Abstract

The invention discloses an affinity purification method for reducing the content of host cell protein in monoclonal antibody production, which comprises the following steps: balancing an affinity chromatography medium by using a first equilibrium buffer solution to obtain a balanced affinity chromatography medium, and combining monoclonal antibody fermentation liquor with the balanced affinity chromatography medium; and then carrying out intermediate pre-elution and elution, and then carrying out final elution to remove host cell protein so as to obtain a monoclonal antibody, wherein the monoclonal antibody is a separated monoclonal antibody against human interferon alpha receptor 1 (IFNAR1), and comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2 and CDR-H3) and three light chain complementary determining regions (CDR-L1, CDR-L2 and CDR-L3). The method is simple and easy to implement, amplification purification production can be carried out, the cell fermentation supernate does not need to be subjected to pretreatment, the elution sample yield is high, meanwhile, the HCP residual quantity is kept at a low level (the residual control quantity is not higher than 0.1%), and therefore the pressure for removing HCP in the subsequent purification step is relieved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an affinity purification method for reducing host cell protein content in the production of anti-human interferon alpha receptor 1 monoclonal antibody. Background technique [0002] Affinity purification is a very critical process step in the production process of antibody drugs. This process captures and concentrates the antibodies in the fermentation broth to realize the first step of rough purification of antibodies. During mass fermentation of genetically engineered cell lines such as Chinese hamster ovary cells (CHO), the cells undergo apoptosis and lysis during different physiological cycles, releasing host cell protein (HCP). HCP refers to protein components derived from host cells, including host cell structural proteins and transforming proteins (growth-promoting proteins secreted by cells). HCP may not only induce the body to produce anti-HCP antibodies and cause allergic ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C07K1/22
CPCC07K16/2866C07K2317/565C07K2317/56C07K2317/51C07K2317/515C07K2317/92C07K2317/76Y02A50/30
Inventor 戴长松李帅郭斌黄文俊戴璐裘浙伟王逸尘何勇梅吴亦亮
Owner QYUNS THERAPEUTICS CO LTD
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