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Determination method and application of biological activity of recombinant human epidermal growth factor

A technology for epidermal growth factor and biological activity, applied in the field of biological activity determination of recombinant human epidermal growth factor, can solve the problems of cumbersome biological determination method, long experimental period, poor accuracy, etc., and achieves convenient implementation and high accuracy , Quality controllable effect

Active Publication Date: 2011-11-30
BIOTECH PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In view of the cumbersome bioassay method, long experimental cycle, poor accuracy, and large errors (Liu Lan et al., Chinese Journal of Biological Products, 2002; 15(3): 175-176), and consume a lot of manpower and material resources, the quality of medicines will be greatly reduced in the future. Bioassays in standards will be gradually replaced by advanced physical and chemical methods, or other methods that can ensure the safety and effectiveness of clinical medication, or only exist in the production research stage

Method used

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  • Determination method and application of biological activity of recombinant human epidermal growth factor
  • Determination method and application of biological activity of recombinant human epidermal growth factor
  • Determination method and application of biological activity of recombinant human epidermal growth factor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Recombinant human epidermal growth factor activity determination method

[0036] The method of measuring EGF activity using human lung adenocarcinoma cell line H-125 (from Cuban Molecular Immunology Center) includes the following steps:

[0037] (1) The cell culture laboratory cultures a certain amount of H-125 cells, and the cell viability should generally not be less than 70%. Centrifuge at 4°C and 1100 rpm for 5 minutes. Carefully discard the supernatant, use the remaining small amount of liquid to dissolve the cell pellet, and then slowly add about 10 mL of PBS and mix well. Centrifuge again at 4°C and 1100 rpm for 5 minutes, carefully discard the supernatant, use the remaining small amount of liquid to dissolve the cell pellet, slowly add about 10 mL of PBS, and mix. After centrifugation at 4°C and 1100rpm for 5 minutes, carefully discard the supernatant, use the remaining small amount of liquid to dissolve the cell pellet, adjust the cell suspension of the ...

Embodiment 2

[0049] Example 2 The reproducibility of the biological activity of epidermal growth factor measured by flow cytometry

[0050] Methodological validation of the relative biological binding activity of Nimotuzumab using the same flow cytometry method:

[0051] The method for measuring the relative biological activity of Nimotuzumab using the human lung adenocarcinoma cell line H-125 (from the Cuban Molecular Immunology Center) includes the following steps:

[0052] (1) Prepare 50μg / mL nimotuzumab reference diluent and sample diluent, then dilute 8 dilutions with monoclonal antibody diluent to 0.050μg / mL;

[0053] (2) Add 20 μL of reference diluent and sample diluent of different concentrations to each centrifuge tube, and the subsequent steps are the same as in Example 1;

[0054] (3) Data processing: computer program (four-parameter regression) is used for processing, and then the half effective binding concentration (EC) of the nimotuzumab reference product and sample 50 ) To obtain the...

Embodiment 3

[0086] Example 3 Comparison of biological activities of different recombinant human epidermal growth factors

[0087] EGF component 1 is the C-terminal deletion of arginine and leucine of human EGF (EGF of 51 amino acid residues)

[0088] EGF component 2 is the C-terminal deletion of arginine of human EGF (EGF of 52 amino acid residues)

[0089] The biological activity of EGF was determined according to the steps of Example 1. The test data are shown in Table 6.

[0090] Table 6 Comparison of biological activities of different rEGF

[0091] EGF component 1

EGF component 2

Minimum

10.00

10.00

Max

279.1

279.1

LogED 50

-0.5096

-0.4883

Slope (Hillslope)

-0.7530

-0.7530

ED 50 (ug / mL)

0.3093

0.3249

Curve Fit R 2

0.9987

0.9976

[0092] the result shows:

[0093] ED 50 (EGF component 1)=0.3093ug / mL

[0094] ED 50 (EGF component 2) = 0.3249ug / mL

[0095] The biological activity of EGF component 2 relative to EGF component 1 is 95.2% (EC 50 It is inversely ...

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Abstract

The invention discloses a method for measuring the biological activity of a recombined human epidermal growth factor (rhEGF) and application of the method. The method comprises the following steps of: blocking the combination between the EGF and the EGFR by an antibody to indirectly measure the biological activity of EGF and EGF receptor (EGFR) combination by a competitiveness-restrained flow cytometry; processing experimental data by adopting a computer program (four-parameter regression) to acquire half effective inhibition concentration (EC50) of an EGF standard substance and an EGF samplerespectively and thus obtaining the relative biological activity of the EGF sample; and calculating the biological activity of the EGF sample according to the biological activity (IU / mL) of the EGF standard substance. In the method, the test period is only one day from sample injection except cell pre-culturing; a germfree environment is not needed during test; and compared with the biological activity measurement method for the rhEGF in the Chinese Pharmacopoeia, the method has the advantages that: the method is more convenient to realize and the quality is more controllable.

Description

Technical field [0001] The invention relates to the field of measuring the biological activity of cell growth factor, and more specifically to a method for measuring the biological activity of recombinant human epidermal growth factor (EGF) and its application. Background technique [0002] Human epidermal growth factor (EGF) is a 53-amino acid residue polypeptide with a molecular weight of 6045 Daltons. In in vivo and in vitro experiments, EGF stimulates epithelial and mesenchymal cell proliferation through receptors (epidermal growth factor receptor, EGFR) on the cell membrane. [0003] The current general recombinant human epidermal growth factor biological activity determination method adopts the cell proliferation method / MTT colorimetric method ("Chinese Pharmacopoeia", 2010 edition three, appendix page 74), this method is based on recombinant human epidermal growth factor in mice The growth of embryonic fibroblasts (Balb / c 3T3 cells) has a stimulating effect. The growth stat...

Claims

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Application Information

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IPC IPC(8): G01N33/577
Inventor 何伟李先钟喻志爱林峰白先宏
Owner BIOTECH PHARMA CO LTD
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