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Affinity purification method for reducing content of host cell protein in production of anti-human interleukin-33 monoclonal antibody

A host cell protein, monoclonal antibody technology, applied in the biological field, can solve the problems of low cost of materials, process amplification, host cell protein residue, etc., and achieve good clinical effect, good stability, and the affinity purification process is simple and easy to implement. Effect

Pending Publication Date: 2022-01-11
QYUNS THERAPEUTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the problem of host cell protein (HCP) residues in antibody production, this application discloses a new method for effectively reducing CHO host cell protein in antibody purification and production, which can be widely used in antibody affinity purification processes, and the used The cost of materials is low, and it is easy to scale up the process

Method used

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  • Affinity purification method for reducing content of host cell protein in production of anti-human interleukin-33 monoclonal antibody
  • Affinity purification method for reducing content of host cell protein in production of anti-human interleukin-33 monoclonal antibody
  • Affinity purification method for reducing content of host cell protein in production of anti-human interleukin-33 monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0131] Example 1 Preparation of anti-human interleukin-33 monoclonal antibody QX007N

[0132] Purchasing human interleukin-33 (hIL-33) from Shanghai Nearshore Technology Co., Ltd. was used to immunize New Zealand rabbits. Using B cell cloning technology to obtain antigen-binding specific antibody clones, and then screened out human interleukin-33 binding and human interleukin-33 33 monoclonal antibodies that inhibit activity. First, use Binding ELISA to detect cell supernatants, and select clones that bind to human interleukin-33; then use HEK Blue TM The IL-33 reporter gene cell method was used for detection, and clones with human interleukin-33 inhibitory activity were selected. The above immunization and screening processes are entrusted to commercial companies.

[0133] Twelve clones were successively selected for recombinant expression and sequenced. It was determined that 78# had the best cell neutralizing activity, and 78# was humanized. Use NCBI IgBlast to carry o...

Embodiment 2

[0138] Embodiment 2 Equilibrium dissociation constant (K D ) determination

[0139] Biacore T200 was used to detect the affinity of QX007N(HZD78-70) to human interleukin-33, 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 cons...

Embodiment 3

[0144] Example 3 neutralizes HEK Blue induced by human interleukin-33 TM Activity Detection of NF-κB / AP-1 Signal Transduction in IL-33 Cells

[0145] HEK Blue TM IL-33 cells were generated by stably transfecting human embryonic kidney cells HEK 293 with human IL1RL1 gene, and the responses of TNF-α and IL-1β were blocked, so HEK-Blue TM IL-33 cells specifically respond to IL-33. The binding of interleukin-33 to IL-1RL1 / IL-1RAcP on the cell surface triggers a signal cascade reaction, leading to NF-κB / AP-1 signal transduction and production of secreted alkaline phosphatase (secreted alkaline phosphatase, SEAP), which can be detected Interleukin-33 biological activity or antibody screening.

[0146] Utilize HEK Blue TM The neutralizing activity of QX007N(HZD78-70) to human interleukin-33 was determined in IL-33 cells. HEK Blue TM IL-33 cells at 4×10 per well 4 Cells were plated into 96 wells at 37°C and 5% CO 2 conditions overnight. Dilute the antibody to a concentr...

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Abstract

The invention discloses an affinity purification method for reducing the content of host cell protein in production of an anti-human interleukin-33 monoclonal antibody, which comprises the following steps: balancing before loading: balancing an affinity chromatography medium by using a buffer solution I to obtain the balanced affinity chromatography medium; loading a sample: combining the anti-human interleukin-33 monoclonal antibody fermentation liquor with the balanced affinity chromatography medium; and elution: carrying out pre-elution and final elution on the mixed solution so as to remove host cell protein and obtain the purified anti-human interleukin-33 monoclonal antibody, wherein the anti-human interleukin-33 monoclonal antibody comprises three heavy chain complementarity determining regions and three light chain complementarity determining regions, the three heavy chain complementarity determining regions are CDR-H1, CDR-H2 and CDR-H3, and the three light chain complementarity determining regions are 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 pretreated, the elution sample yield is high, the HCP residual quantity is kept at a low level, and therefore the pressure for removing HCP in the subsequent purification step is relieved.

Description

technical field [0001] This application relates to the field of biotechnology. Specifically, the present application relates to an affinity purification method for reducing host cell protein content in the production of anti-human interleukin-33 (IL-33) 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...

Claims

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Application Information

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IPC IPC(8): C07K16/28C07K1/22
CPCC07K16/2866C07K2317/565C07K2317/56C07K2317/51C07K2317/515C07K2317/76C07K2317/92Y02A50/30
Inventor 戴长松朱华杰李帅徐义超唐宇杰戴璐李梦茹李梦杰何勇梅吴亦亮
Owner QYUNS THERAPEUTICS CO LTD
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