Abstract
An ideal test used to characterize a product must be appropriate for the measurement of product quality, manufacturing consistency, product stability, and comparability studies. Flow cytometry has been successfully applied to the examination of antibodies and receptors on membrane surfaces; however, to date, the analytical validation of cytometry based assays is limited. Here we report on the validation of a flow cytometry-based assay used in the evaluation of nimotuzumab binding to cells over-expressing EGFR on cell surface. The assay was validated by examining, assay robustness, specificity, repeatability and intermediate precision. The assay was highly specific, robust for all studied factors except for cell fixation with 1% paraformaldehyde and met criteria for precision with RSD < 2%. In addition the assay has stability-indicating properties evidenced by the ability to detect changes in mAb degraded samples. Most importantly, the assay demonstrated to be useful for its intended use.
Highlights
Most biological products act through some form of binding to another moiety
Based on the method characteristics and requirements of the International Conference on Harmonization (ICH) guidelines, each analytical procedure must be validated with respect to parameters which are relevant to its performance [8, 10]
While mean of fluorescence intensity (MFI) was examined (Figure 1b), the saturating monoclonal antibody (mAb) concentration was of 10–20 μg/mL in both cell lines
Summary
Most biological products act through some form of binding to another moiety. Fluorescence flow cytometry is used in the observations and analysis of the interaction of fluorescently labeled ligands and their cellular receptors. M. Cedeño-Arias et al.: is commonly used to characterize the activity of the product through binding to its specific receptor. When the mechanism of action of a monoclonal antibody (mAb) is to block the binding of ligand to cell surface receptor, in vitro binding assay can be used as surrogate potency test using the therapeutic mAb [1]. The development of accurate and well characterized assays for biological products is vital for their development as therapeutic drug [2]. The appropriate validation of any bioassay used in the characterization of biological products is critical. We report on the validation study (assay robustness, specificity and precision) of the nimotuzumab binding assay by Flow Cytometry
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