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A kind of preparation method of anti-human thymus stromal lymphopoietin (tslp) monoclonal antibody concentrated solution

A monoclonal antibody and lymphocyte technology, applied in the biological field, can solve problems such as process failure, concentration polarization out of control, and reduced sliding performance of the drug-filled syringe, and achieve good clinical results

Active Publication Date: 2022-05-20
QYUNS THERAPEUTICS CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In some extreme cases, a gel-like substance may even form, which brings great challenges to the ultrafiltration membrane and the ultrafiltration equipment itself, such as the decrease in the tangential flow rate caused by the rapid increase in the pressure difference during the final concentration, and the concentration difference Polarization gets out of control until protein precipitation clogs the membrane, which inevitably leads to reduced recovery or process failure
On the other hand, even if the final high-concentration protein solution is obtained by improving the equipment or membrane type, it is difficult to put it into actual clinical application, because it needs to be sucked by disposable sterile syringe or prefilled needle for subcutaneous administration The final packaging form, and too high viscosity will reduce the sliding performance of the filled syringe, so that it cannot be manually pushed into the subcutaneous
Another difficulty in concentrating high-concentration monoclonal antibody solutions by ultrafiltration is that protein samples tend to aggregate to form soluble aggregates when highly concentrated, and further aggregate to form protein precipitates

Method used

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  • A kind of preparation method of anti-human thymus stromal lymphopoietin (tslp) monoclonal antibody concentrated solution
  • A kind of preparation method of anti-human thymus stromal lymphopoietin (tslp) monoclonal antibody concentrated solution
  • A kind of preparation method of anti-human thymus stromal lymphopoietin (tslp) monoclonal antibody concentrated solution

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Experimental program
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preparation example Construction

[0058] The application provides a method for preparing an anti-human thymus stromal lymphopoietin (TSLP) monoclonal antibody concentrated solution, comprising the following steps:

[0059] Step 1: Concentrating the solution containing anti-human thymus stromal lymphopoietin (TSLP) monoclonal antibody to obtain the first concentrated sample;

[0060] Step 2: replacing the first concentrated sample with a buffer solution to obtain a replacement concentrated solution;

[0061] Step 3: mixing the concentrated replacement solution and the mother liquor of the amino acid protecting agent to obtain a mixed solution, and performing ultrafiltration and concentration on the mixed solution to obtain a second concentrated sample;

[0062] The anti-human thymus stromal lymphopoietin (TSLP) monoclonal antibody comprises three heavy chain complementarity determining regions (CDR-H1, CDR-H2 and CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2 and CDR-L3), whe...

Embodiment 1

[0151] Embodiment 1 Preparation of anti-human TSLP monoclonal antibody QX008N

[0152] Purchasing human thymic stromal lymphopoietin (hTSLP) 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 TSLP and having human TSLP inhibitory activity of monoclonal antibodies. The cell supernatant was detected by Binding ELISA and Blocking ELISA, and the target clone was selected. The above immunization and screening processes are entrusted to commercial companies.

[0153]Seven clones were selected for recombinant expression and sequenced. It has been determined that 8G2 has the best cell neutralizing activity. Therefore, the 8G2 clone was humanized. Use NCBI IgBlast to carry out homology comparison of human IgG germline sequence (Germline), select IGHV3-66*01 as the heavy chain CDR transplantation template, and clone the CDR re...

Embodiment 2

[0158] The mensuration of embodiment 2 equilibrium dissociation constant (KD)

[0159] The affinity between QX008N(HZD8G2-57) and human TSLP was detected by Biacore T200, 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 instrument's own analysis software to select the 1:1 binding model in the Kinetics option for fitting, and calculate the antibody's association rate constant ka, dissociation rate constant kd, and dissociation ...

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Abstract

The application discloses a method for preparing a concentrated solution of anti-human thymus stromal lymphopoietin (TSLP) monoclonal antibody, comprising the following steps: Concentrate the solution to obtain the first concentrated sample; Step 2: Replace the first concentrated sample with a buffer solution to obtain a replacement concentrated solution; Step 3: Mix the replaced concentrated solution and the mother liquor of the amino acid protective agent to obtain a mixed solution, and the The above mixed solution was concentrated by ultrafiltration to obtain a second concentrated sample. The preparation method described in this application can reduce the viscosity of high-concentration antibody drug solution and improve the stability. The method is simple and easy, and can be scaled up for production, which can ensure the high purity of the sample and obtain a high recovery rate.

Description

technical field [0001] The present application relates to the field of biotechnology, in particular to a method for preparing a concentrated solution of anti-human thymus stromal lymphopoietin (TSLP) monoclonal antibody. Background technique [0002] Therapeutic biological products are a fast-growing market with numerous R&D pipelines, and at the same time bring more innovative therapies to patients. For some autoimmune-related diseases that are difficult to treat with traditional small molecule drugs, biological drug targeted drugs provide more choices. On the other hand, in order to reduce the cost of clinical use of biological agents and improve patient compliance, the dosage form of biological agents has gradually changed from freeze-dried dosage form to aqueous injection dosage form, and the route of administration has also changed from intravenous administration to subcutaneous injection dosage form. Since the dosage of monoclonal antibody injection is usually in the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/395A61K9/08A61K47/18A61K47/22A61P37/02A61P11/06C07K16/24C07K1/36C07K1/34C07K1/22C07K1/18
CPCC07K16/244A61K9/08A61K9/0019A61K47/183A61K47/22A61P37/02A61P11/06C07K2317/56C07K2317/565A61K2039/505Y02P20/55
Inventor 薛刚朱华杰戴长松李帅许芹郭彩明陈卫吴亦亮
Owner QYUNS THERAPEUTICS CO LTD
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