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Biology activity determination method of monoclonal antibody

A monoclonal antibody and biological activity technology, which is applied in the field of antibody biological activity determination, can solve the problems of poor applicability of anti-EGFR antibodies, increased variation detection results, and low sensitivity, so as to achieve more controllable quality, Wide applicability and high accuracy

Active Publication Date: 2014-05-14
BIOTECH PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the applicability of these two types of cells to anti-EGFR antibodies with different affinities is not strong, and they are only suitable for high-affinity anti-EGFR antibodies, resulting in low sensitivity. Due to some problems such as protein precipitation after staining and low solubility of the product, it is necessary to 10% SDS or 10% DMSO dissolution step increases the chances of variable detection results

Method used

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  • Biology activity determination method of monoclonal antibody
  • Biology activity determination method of monoclonal antibody
  • Biology activity determination method of monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Determination of biological activity of recombinant anti-human epidermal growth factor receptor monoclonal antibody by in vitro proliferation inhibition method

[0036] The method for measuring the activity of anti-EGFR antibody using human lung cancer lymph node metastasis cell line H292 (purchased from Cuba Molecular Immunology Center) includes the following steps:

[0037] (1) Preparation of H292 cell suspension: The cell viability of the H292 cell suspension should not be lower than 85%, and the cells should be counted. In a clean bench, use complete culture medium to make a density of (6-10)×10 4 cells / mL of cell suspension.

[0038] (2) The cell suspension was inoculated in a 96-well plate, 100 μL / well (blank control well plus 100 μL complete culture solution) was placed at 37°C CO 2 cultured in an incubator.

[0039] (3) Prepare Nimotuzumab solutions of different concentrations, dilute the Nimotuzumab solution samples or standards to (300-1000) μg / m...

Embodiment 2

[0051] Example 2: Method verification for measuring the biological activity of anti-EGFR monoclonal antibody by in vitro cell proliferation inhibition method

[0052] The verification of the method for determining the biological activity of nimotuzumab using the H292 cell line of lung cancer lymph node metastasis (purchased from the Cuban Molecular Immunology Center) includes the following steps:

[0053] (1) Specificity: Nimotuzumab is a human IgG1 subclass kappa immunoglobulin, and the same human IgG1 subclass kappa immunoglobulin was used as the isotype control, and the experiment was repeated at least twice. Acceptable standard: the isotype non-EGFR monoclonal antibody should have almost no cross-reaction with H292 cells, and should not show a similar response curve.

[0054] The result is as figure 2 As shown, the isotype control antibody T1h monoclonal antibody has almost no cross-reaction with H292 cells, and does not show a similar reaction curve, indicating that T1h...

Embodiment 3

[0068] Example 3: Biological Activity Comparison of Different Recombinant Anti-EGFR Monoclonal Antibodies

[0069] High affinity antibody: 10 affinity to EGFR -10 ~10 -11

[0070] Medium affinity antibody: 10 affinity to EGFR -8 ~10 -9

[0071] The high-affinity antibody used in this example is cetuximab, and the medium-affinity antibody is nimotuzumab. Carry out anti-EGFR monoclonal antibody biological activity assay according to the step of embodiment 1, test data is shown in the following table:

[0072]

[0073] From the data in the table above, the IC of the high-affinity antibody 50 The value is about 0.0002611, the IC of a medium affinity antibody 50The value is 1.634, indicating that the H292 cell line is sensitive to antibodies with different affinities, and is especially suitable for the determination of the biological activity of antibodies with low to medium affinity. Commonly used cell lines such as MB-468 cell line are not sensitive. IC of Hi...

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Abstract

The invention discloses a biology activity determination method of a monoclonal antibody of a recombined anti-human epidermal growth factor receptor. The method comprises the following steps: incubating human lung cancer-lymph node metastasis cells H292 and anti-EGFR monoclonal antibody with different concentrations; adding a cell counting dye reagent CCK-8; determining the light absorption value of stained vital cells through a microplate reader; processing experiment data through a computer program; calculating the biology activity of an anti-EGFR sample relative to an anti-EGFR standard. According to the invention, a cell strain with the detected cells H292 is applicable to antibodies with different affinities, the cell culture time is short, and the needed cell quantity is less; staining fluid of an MTT colorimetric method is improved, CCK-8 staining fluid is used, the product solubility is good, further solution treatment is not needed, the detection result can be detected at different time after the staining fluid is added, the developing result is stable, and the quality is controllable. The biology activity determined by the method is related to clinical effects, thus meeting the relevant technical requirement of FDA.

Description

technical field [0001] The present invention relates to the technical field of measuring the biological activity of antibodies, and more specifically relates to a method for measuring the biological activity of a recombinant anti-human epidermal growth factor receptor monoclonal antibody. Background technique [0002] Epidermal growth factor receptor (EGFR) is a glycoprotein, which belongs to tyrosine kinase receptor, and is widely distributed on the surface of mammalian epithelial cells, fibroblasts, glial cells, keratinocytes and other cells. Its signaling pathway plays an important role in physiological processes such as cell growth, proliferation and differentiation (Hanley SC, Assouline-Thomas B, Makhlin J, et al. J Endocrinol, 2011, 211(3): 231-239). Studies have shown that EGFR is related to tumor cell proliferation, angiogenesis, tumor invasion, metastasis and inhibition of apoptosis. There are high or abnormal expressions of EGFR in many solid tumors, so EGFR can be...

Claims

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Application Information

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IPC IPC(8): G01N21/31G01N33/577
Inventor 何伟刘永清喻志爱白先宏
Owner BIOTECH PHARMA CO LTD
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