Validation of a flow cytometry based binding assay for evaluation of monoclonal antibody recognizing EGF receptor

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.

Keywords: Binding Assays; Method validation; Monoclonal antibody; Nimotuzumab.

Fig. 1.

A typical dose-response curve for a FACS-binding assay data set is shown. Graph in (A) shows % of binding and in (B) mean of fluorescence intensity of nimotuzumab on cell surface EGFR in two different tumor cell lines.

Fig. 2.

Half-normal probability plot for the effects estimated in Plackett-Burman design (from Table 2) with the identification of margin of error (ME). Variable: % of binding of nimotuzumab evaluated by flow cytometry on A 431 (A) and H125 (B) cell lines respectively.

Fig. 3.

Binding of nimotuzumab to EGFR expressed on A431 cell line. The graphs represent a comparison between native and degraded-mAbs. [A], % of binding and [B], mean intensity of fluoresce of degraded isotype control ( ), degraded nimotuzumab ( ), native nimotuzumab ( ), or native FITC-conjugated nimotuzumab ( ), antihuman/FITC was used as secondary Ab when non-conjugated mAb was evaluated. [C], Results of competition binding assay, concentrations from 0.01 to 50 μg/mL of native and degraded mAbs were incubated for 30 min at 4°C in the presence of FITC-Conjugate nimotuzumab (2.5 μg/mL).