Hereceptin Inhibition of Proliferation Bioassay

BioOutsource are industry experts in the development, optimization, and qualification of cell based bioassays, offering cGMP and GLP-compliant testing services. Using this experience, we have created a range of biosimilar off-the-shelf assays to assess the characterization and comparability of Herceptin (trastuzumab) molecules to offer the Herceptin inhibition of proliferation assay.

Herceptin (trastuzumab) is a monoclonal antibody that has been developed to target HER2 in cancer patients. It is a humanized mouse IgG1k monoclonal antibody that specifically binds to the extracellular domain IV of HER2. A combination of trastuzumab with standard chemotherapy has been shown to achieve a better response rate than chemotherapy alone.

The human epidermal growth factor receptor 2 (ERBB2/HER2) is a member of the human epidermal growth factor family and is overexpressed in 20 to 30% of breast cancer cases. HER2 is a ligandless receptor, but is activated by dimerization with itself or other receptors. When functioning normally the HER2 pathway promotes cellular proliferation, survival, migration, invasion, and differentiation. However, when overexpressed cellular proliferation increases due to constant proliferative signals and consequently tumours are formed. In addition the overexpression of HER2 causes deactivation of checkpoints, further increasing cellular proliferation. These negative effects can be counteracted when HER2 is inhibited.

The Herceptin inhibition of proliferation assay measures the ability of the Fab region of Herceptin to bind to HER2 and inhibit the proliferation of cells. The principle of the assay is determining a relevant cell line which will express the HER2 protein, in this case BT474 cells. The cell plates are incubated for a time period optimized for receptor expression, following which a dilution series of Herceptin is prepared and added to the BT474 cells. The binding of Herceptin to the HER2 protein on the surface of the cells results in an arrest of the cell cycle at G1 phase and consequently a reduction in cellular proliferation. After an optimized time period the metabolic activity of the cells is measured using a non-toxic reagent which is proportional to the number of living cells. The assays are performed using the appropriate controls and the samples can be reported as a relative potency measurement against a designated reference standard.

Where required, rapid assay qualification and/or validation can be performed using the customer’s material.

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