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Novel method used for determining biological activity of recombinant human growth hormone fusion protein

A technology of biological activity and fusion protein, which is applied in the field of determination of biological activity of recombinant human growth hormone fusion protein, can solve the problems of non-compliance, large coefficient of variation, complicated operation, etc., and achieve small variation, simple operation, and short experiment time Effect

Pending Publication Date: 2019-08-06
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 GH bioassay methods included in the Chinese Pharmacopoeia are the pituitary rat body weight method and the rat tibia method without questioning. Both methods involve the detection of rat living body, except for the 3R that does not meet animal protection and animal welfare. In principle, the experiment itself is complicated to operate and has a large coefficient of variation, so it is urgent to replace the animal in vivo method with in vitro cytology method.

Method used

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  • Novel method used for determining biological activity of recombinant human growth hormone fusion protein
  • Novel method used for determining biological activity of recombinant human growth hormone fusion protein
  • Novel method used for determining biological activity of recombinant human growth hormone fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Screening of HEK293 cell lines stably expressing SGG1 and GHR receptors

[0035] 1. Materials and methods

[0036] 1.1 cells

[0037] HEK293 cells were derived from ATCC.

[0038] 1.2 Reagents and materials

[0039] Human growth hormone receptor (GHR) plasmid was purchased from Origene; ViaFect TM Transfection reagents were purchased from Promega; DMEM medium, 1640 medium, fetal bovine serum (FBS) and (G-418) was purchased from GIBCO; hygromycin B was purchased from Suleibao Biotechnology Co., Ltd.; Britelite plus luciferase substrate was purchased from PerkinElmer; white bottom transparent 96-well plate was purchased from CORNING; GH-Fc fusion protein and the plasmid containing the SGG1 response element were retained by the Recombinant Drug Department of the Chinese Academy of Food and Drug Control.

[0040] 1.3 Instrument and analysis software

[0041] A microplate reader was purchased from Molecular devices, model SpectraMax M5; SoftMax Pro and Graph...

Embodiment 2

[0053] Example 2 Methodological optimization of GH-Fc fusion protein biological activity assay

[0054] 1. Optimization of the reaction solution Adjust the cell concentration with 1640, 1640 culture medium containing 0.5% FBS and 10% FBS respectively, according to 3×10 4 Add the cells per well into a 96-well plate, culture at 37°C, 5% CO2 for 12-18 hours, add an equal volume of double-diluted GH-Fc fusion protein for stimulation, continue to cultivate for 4-6 hours, discard the reaction solution, and add 60 μL Britelite plus fluorescence Fluorescence value of luciferase was detected after fully shaking for 5 min, and four-parameter curve was fitted ( image 3 ). In view of the principle of minimizing the influence of non-specific substances in the serum on the activity, combined with the SNRs of the three reaction solutions (Table 2), the culture solution containing 1640 was selected as the activity detection reaction solution.

[0055] Table 2 Optimization of reaction solut...

Embodiment 3

[0070] Example 3 Methodological verification of GH-Fc fusion protein biological activity assay

[0071] 1. Exclusiveness

[0072] This method is aimed at the biological activity of the GH-Fc fusion protein. Therefore, different varieties of Fc fusion proteins are used to verify its specificity, including Fc fusion proteins of EPO, VEGFR, IL15 and GLP1. According to the experimental conditions determined in 1-4 in Example 2, the biological activities of the above four Fc fusion proteins were determined.

[0073] see results Figure 7 , this method has no dose-response curves for Fc fusion proteins of EPO, VEGFR, IL15 and GLP1, indicating that this method is not applicable to other drugs except GH-Fc fusion proteins, indicating that the specificity of this method is better.

[0074] 2. Precision

[0075] The precision of the method was evaluated using the experimental conditions determined in Example 2. Four samples of GH-Fc fusion protein were taken for activity determinati...

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Abstract

The invention relates to a method for determining the biological activity of recombinant human growth hormone Fc fusion protein through a luciferase reporter gene method. The method utilize a STAT5 reaction element (SGG1) and GHR plasmid to obtain a SGG1 luciferase reporter gene with stable expression and a cell strain of GHR after the transfection of a HEK293 cell; the expression of the downstream luciferase report gene of the SGG1 reaction element can be activated after growth hormone Fc fusion protein is added; and the biological activity of the growth hormone Fc fusion protein can be determined according to a fluorescence signal value fitting four-parameter curve. The detection method which is easy in operation, high in sensitivity, small in variable coefficient and high in accuracy can be established for the biological activity determination of the growth hormone Fc fusion protein; and the method has important guidance effects on the research and development of the growth hormoneFc fusion protein and the quality control in production.

Description

technical field [0001] The invention relates to the field of biological activity detection of therapeutic recombinant drugs, and establishes a fast, simple and accurate method for measuring the biological activity of recombinant human growth hormone (Growth hormone, GH) fusion protein. Background technique [0002] GH is a pituitary-derived protein that participates in the regulation of metabolism during growth and development. Its deficiency can lead to growth retardation in children and growth hormone deficiency syndrome in adults. Recombinant human GH can correct the above symptoms. Due to the short half-life of GH, daily subcutaneous injections are required, which greatly increases the suffering of the treater. In order to prolong the half-life, long-acting recombinant protein drugs have become the mainstream of the research and development of biotech drugs; technically speaking, long-acting drugs mainly include two strategies: chemical modification (PEGylation) and fusi...

Claims

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Application Information

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IPC IPC(8): C12Q1/6897C12Q1/02C12N15/85
CPCC12Q1/6897G01N33/5008C12N15/85C12N2800/107
Inventor 饶春明王军志姚文荣范文红于雷秦玺史新昌刘兰
Owner NAT INST FOR FOOD & DRUG CONTROL
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