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Cytotoxicity-based Activity Test Method for Antibody Biotherapeutics

2021-09-17
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Antibody drugs refer to protein drugs containing antibody gene fragments. The specificity, safety and effectiveness of binding to target antigens have enabled them to achieve rapid development in some major clinical diseases. Before studying how antibody drugs affect the human body, it is necessary to investigate the effects of drugs on the interior of cells. Cytotoxicity-based antibody biotherapeutic drug activity testing methods provide directions for the development and quality control of antibody drugs.

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Biotechnology drug activity testing is very important in quality control. The current biological activity analysis methods mainly include in vivo (animal) and in vitro (cell) experiments. The Biology Department of Shanghai Medicilon has extensive experience in the field of in vitro biology, through enzyme level determination, cell level determination, cell biology, biochemistry, in vitro isotope determination, stable cell line establishment, gene knockout, RNAi and MicroRNA Technology, etc., to provide a complete set of biological services.

The quality of drugs should be based on a comprehensive understanding of the biological properties and mechanism of drug molecules, and the corresponding production process should be designed and developed to achieve the expected quality attributes of the drug molecules. Although product quality analysis after the end of the production process is an important part of drug quality control, these tests should not simply reveal the results of the production process, but confirm the expected attributes of the drug. The drug molecule enters the cell and binds to the target in the cell to produce pharmacological effects. In drug research, insufficient target exposure is the main cause of high drug consumption. It is assumed that insufficient exposure of cellular targets will result in lower “drug efficiency”. Insufficient target exposure leads to a high consumption rate in drug experiments, which is an important factor in the failure of clinical drug development.

Cytotoxicity is a simple cell killing event caused by cells or chemical substances, and does not depend on the cell death mechanism of apoptosis or necrosis. Sometimes it is necessary to test the cytotoxicity of specific substances, such as screening drugs through drug activity tests. Cytotoxicity testing is mainly based on changes in cell membrane permeability. MTT and XTT methods are commonly used, using the activity of mitochondrial enzymes to transform specific tetrazolium salts, and then testing with a microplate reader.

There are two pathways for apoptosis, one is the mitochondrial-dependent pathway, and the other is the death receptor-mediated pathway. After tumor necrosis factor-α (tumornecrosisfactor-alpha, TNF-α) binds to the receptor, it can initiate the death receptor-mediated pathway, which makes procaspe-8 self-hydrolyze and activate to form active caspase-8, which then activates caspase3, 6, 7, etc., cause the following cascade reaction, leading to cell apoptosis. The activity determination of recombinant human tumor necrosis factor receptor-antibody fusion protein can be used to inhibit the activation of caspase3/7 in the sensitive cell line U-937 caused by TNF-α.

Test the Activity of  Antibody Biotherapeutics by Detecting the Enzyme Activity of Ldh in the Cell Culture Supernatant

LDH (lactate dehydrogenase) is a stable protein that exists in the cytoplasm of normal cells. Once the cell membrane is damaged, LDH is released outside the cell; LDH catalyzes the formation of lactic acid into pyruvate, and INT (tetrazolium salt) Class) The reaction forms a purple crystalline substance, which can be detected by a 500nm microplate reader. By detecting the activity of LDH in the cell culture supernatant, the degree of cell damage can be judged.

Test Drug Activity by Changing the Luminescence Signal of Luciferase

The activity of the product is reflected by the change of luciferase luminescence signal in the Caspase-Glo3/7 detection kit. In addition, monoclonal antibodies against TNF-α can also use cell lines that are sensitive to TNF-α killing, such as mouse fibroblast L929, mouse fibrosarcoma cell WEHI164, etc. Antibodies can inhibit cell apoptosis induced by TNF-α Function, by detecting the staining of cell survival to evaluate the biological activity of the antibody. In the selection of dyes related to cell survival, dyes including crystal violet, MTT, MTS, CCK-8, etc. have their own advantages and disadvantages.

Other Enzyme Methods

(1) Crystal Violet

Crystal violet is a triphenylmethane dye, often used as a biological dye and a developer of inorganic ions. Although the staining process is relatively simple, it cannot distinguish between dead and living cells and can only reflect the number of cells.

(2) MTT

As the substrate of mitochondrial dehydrogenase, MTT is reduced by living cells into water-insoluble orange-yellow formazan products and deposited in the cells. Dead cells have no such function, so it can indirectly reflect the number of living cells, but MTT reduction products It is insoluble in water and needs to be dissolved before testing.

(3) MTS

MTS reduction product is water-soluble formazan, without washing and dissolving steps, is more convenient to use, and has better repeatability than MTT. The CCK-8 solution can be directly added to the cell sample. The solution is quite stable, has no toxicity to the cells, and can be incubated for a long time. It is a simple, rapid, highly sensitive method for determining the number of living cells in the cytotoxicity or cell proliferation test. Dyes with good repeatability.

With the continuous advancement of biotechnology and the advent of human postgenomics and metabolomics, more and more new targets have been discovered and studied, which expands the range of antigens targeted by antibodies and will surely expand the market for antibody drugs. There will also be more and more new technologies used in monoclonal antibody drug activity testing to achieve accurate, multi-directional, and dynamic analysis of antibody drugs, which provides a new guarantee for the quality of antibody drugs.

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