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.
The epidermal growth factor receptor (EGF-R) is an important growth regulator of epithelial cancer cells, overexpressed by several human tumors and scantly detectable in most normal tissues. The introduction of monoclonal antibodies (Mabs) and more recently engineered humanized Mabs have greatly expanded the therapeutic potential of this modality of cancer treatment. The present study was designed to compare the specificity of the murine and humanized anti-EGF-R Mabs. Biotinylated Mabs were tested in samples of fetal and adult normal and neoplastic tissues by ABC peroxidase method. All fetal tissues studied were positive for both Mabs, showing 2 different staining patterns, one homogeneous and finely granular in cytoplasm and another grosser with intense labeling in both membrane and cytoplasm. A similar recognition pattern was exhibited in adult normal tissues, where an intense reactivity was also evidenced in skin, tongue, gastrointestinal tract, renal tubules, and breast gland epithelium. In tissues from genitourinary and central nervous system, a faint staining was demonstrate, whereas those from cardiovascular and lymphoid tissues proved to be negative. These Mabs exhibited a heterogeneous and strong membrane and cytoplasm staining in neoplastic cells from lung, breast, and head and neck cancer. On the basis of these results, we conclude that the humanized (h-R3) and murine (egf/r3) anti-EGF-R Mabs show a very similar immunohistochemical pattern of recognition of fetal, adult, and neoplastic tissues. Also h-R3 Mab is a novel candidate for the development of an immunotherapeutic approach suitable for the treatment of tumors with EGF-R overexpression.
Appropriate validation of any bioassay to be used in the characterization of biological products is critical. In this study, we report a validation study of a flow cytometry-based assay to measure the complement-dependent cytotoxicity (CDC) of a biosimilar candidate monoclonal antibody (Mab) directed to CD20 antigen, as indicative of its biological activity. The assay was validated by examining: assay robustness, specificity, repeatability and intermediate precision. It demonstrated to be robust for all factors evaluated. It also showed a high level of specificity and was found to be free of interference through the validation process. The degree of precision (Cvs < 7%) obtained in this study was satisfactory. The presented work demonstrated that a flow cytometry-based cytotoxicity assay is a suitable method in the lot to lot quality control monitoring of the culture supernatant and an active pharmaceutical ingredient of 1B8 Mab.
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