A method for rapid determination of biological activity of IL-2 protein drug and anti-CD25 antibody drug

A biological activity, IL-2 technology, applied in the field of rapid determination of the biological activity of IL-2 protein drugs and anti-CD25 antibody drugs, can solve the problem of increasing economic burden, unable to assess the applicability of stable indicators, and unable to reflect downstream signaling pathways and other problems to achieve stable and reliable test results, avoid the possibility of cell contamination, and shorten the experimental period.

Active Publication Date: 2021-10-01
NAT INST FOR FOOD & DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the methods used to detect the biological activity of basiliximab, daclizumab and related biosimilars include competitive ELISA and flow cytometry. When the biological activity of the anti-CD25 monoclonal antibody is detected by flow cytometry, the downstream signaling pathway cannot be reflected, and thus the applicability of the stable indicator for the release test cannot be evaluated; when the biological activity of the anti-CD25 monoclonal antibody is detected by flow cytometry, relevant equipment and a large amount of consumables are required. Businesses increase the financial burden

Method used

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  • A method for rapid determination of biological activity of IL-2 protein drug and anti-CD25 antibody drug
  • A method for rapid determination of biological activity of IL-2 protein drug and anti-CD25 antibody drug
  • A method for rapid determination of biological activity of IL-2 protein drug and anti-CD25 antibody drug

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1 Construction and screening of monoclonal cell lines expressing STAT5-Luciferase's monoclonal cell line 1, experimental materials

[0073] C8166 cells are derived from ATCC; PLV-STAT5-PGK-ZENCIN is purchased in Ji Man Biotechnology Co., Ltd .; IL-2 is purchased in BiosystemsAcro; Bali antibody (anti-CD25 monoclonal antibody drug) is a monoclonal antibody chamber (Novarta) The Bright-Glo luciferase kit is purchased in Promega.

[0074] 2, slow virus transduction

[0075] C8166 cells were transduced by a slow viral packaging method, and 50 μl of constructed lentiviral plasmid was added to 96-well plates, and 50 μl of density was added to 1 × 10 in the same holes. 6 A / mL C8166 cells, 50 μl of 10% FBS + 1640 medium and 50 μl of density of 10% by 1 × 10 6 The C8166 cells of / ml were used as a blank control, and centrifuged at a rotational speed of 1000 rpm for 30 minutes, and after the incubation was incubated for 30 minutes. 100 μl of the slow viral plasmid was adde...

Embodiment 2

[0082] 2. Comparison of different effects cells

[0083] 1, experimental materials

[0084] C8166 cells are derived from ATCC; PLV-STAT5-PGK-Zencin is built in Ji Man Biological Engineering Co., Ltd .; IL-2 is purchased in BiosystemsAcro; Bali antibody (anti-CD25 monoclonal antibody drug) is a monoclonal antibody chamber storage (Novartis BRIGHT-GLO luciferase kits were purchased in Promega.

[0085] 2, slow virus transduction

[0086] HDLM-2, Mo7e cells were transduced using a slow viral packaging method, and 50 μl of constructed lentiviral plasmid was added to 96-well plates, and 50 μl of the density was 1 × 10 in the same holes. 6 The HDLM-2, Mo7e cells of / ml, adding 50 μl of 10% FBS + 1640 medium and 50 μL of 50 μL of a density of 50 μL of 1 × 10. 6 A / ml of HDLM-2, Mo7e cells were used as a blank control, and centrifuged at a rotational speed of 1000 rpm for 30 minutes, and after the incubation was incubated for 30 minutes. 100 μl of lentiviral plasmid was added to the 24-...

Embodiment 3

[0093] Example 3 Verification of Detection Method

[0094] 1. Methodology compared to traditional IL-2 protein drug test methods

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Abstract

The invention discloses a method for rapidly measuring the biological activity of an IL-2 protein drug and an anti-CD25 antibody drug. The method constructs C8166 effector cells transfected with a lentivirus (pLV-STAT5-Luc-PGK-Zencin), and passes through After screening, a monoclonal cell line stably expressing the STAT5 reporter gene was obtained, the expression of the reporter gene was stimulated and activated with IL-2 protein drug, and the signal pathway stimulated and activated by IL-2 was blocked with anti-CD25 antibody drug, and the signal value was fitted according to the measured signal value The biological activity of IL-2 protein drug and anti-CD25 antibody drug was determined by four-parameter curve. The detection method provided by the invention can evaluate the relevant indications of drugs on the downstream of cell signals, does not require any cells or other components derived from human primary tissues, has stable color development, short experimental period, simple operation, and low cost.

Description

Technical field [0001] The present invention belongs to the field of biological activity detection of biopharmaceutics, and in particular, there is a biological activity method for rapid determination of IL-2 protein drugs and anti-CD25 antibody drugs. Background technique [0002] Treatability monoclonal antibody (simply antibody) as an important biotechnology product, has the advantages of high specificity, strong targeted, efficacy, etc., is the height of single B cell clones, only for a certain specific Antigenic epitope, currently, in the treatment of tumors, autoimmune diseases, infectious diseases, infectious diseases and transplant rejection rejection, significant efficacy. In 2018, the sales of monoblocks accounted for 55.3% of the global biopharmaceutical market. In addition, in the first 20 drugs in 2019, 13 biopharmaceuticals accounted for 9 seats. At present, monoba is one of the most important segments of global pharmaceuticals, and has also become a global medical ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/66C12N5/10C12N15/65C12N15/867G01N21/76
CPCC12N5/0694C12N15/65C12N15/86C12N2510/00C12N2740/15043C12Q1/66G01N21/763
Inventor 王兰段茂芹于传飞张峰刘春雨付志浩王文波郭莎郭潇黄璟徐苗王军志
Owner NAT INST FOR FOOD & DRUG CONTROL
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