Validation of a Flow Cytometry Based Binding Assay for Evaluation of Monoclonal Antibody Recognizing EGF Receptor

Cedeño-Arias, Mercedes; Ramírez, Javier Sánchez; Blanco-Santana, Rancés; Rengifo-Calzado, Enrique

doi:10.3797/scipharm.1104-18

Cited by 10 publications

(4 citation statements)

References 12 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…While FC allows detection of specific Ag expression on cells in single-cell suspension through interaction with fluorophore-labeled Ab and has been utilized for many years for diagnosis and management of human disease (21)(22)(23), certainly the methodology could be applied for analysis of biological activity of an Ab labeled for NIR imaging. Recently, validation of an FC-based binding assay was reported for evaluation of FITC-conjugated Nimotuzumab, mAb specific for epithelial growth factor receptor (EGFR), which has been approved for cancer therapy (24). In the present study, an FC assay was developed that utilizes excitation and emission detection wavelengths in the NIR spectrum, and tested alongside the Lindmo cell-binding assay, which conventionally requires the use of radioactive material.…”

Section: Discussionmentioning

confidence: 99%

Quantifying multimodal contrast agent biological activity using near‐infrared flow cytometry

Hall

Aldrich

Azhdarinia

et al. 2012

Contrast Media & Molecular

View full text Add to dashboard Cite

Prior to imaging agent use in preclinical studies and clinical diagnostics, biological activity must be validated. The Lindmo assay has been used conventionally to quantify radiolabeled antibody (Ab) immunoreactivity, although published findings suggest it does not provide consistently accurate results. We developed and tested a near-infrared (NIR) flow cytometry (FC) method for quantifying biological activity of a dual-labeled Ab for use as a multimodal contrast agent in small animal and human positron emission tomography and NIR fluorescence imaging. Antibody specific for epithelial cell adhesion molecule was conjugated to DOTA-NHS-ester, labeled with IRDye 800CW and further labeled with (64)Cu or nonradioactive Cu prior to reacting with human prostate cancer cells for testing by the Lindmo or FC method, respectively. Immunoreactivity of the dual-labeled agent was found to be 76.4 ± 15.7% by the Lindmo assay. When tested with and without Cu labeling using NIR FC, the biological activity was found to be 73.1 ± 7.7 and 79.4 ± 8.1%, respectively. No significant differences were found between these activity levels (p > 0.05), supporting NIR FC as an alternative method for measuring immunoreactivity and demonstrating that Cu labeling does not significantly affect the agent's ability to bind to its target. Biological activity was significantly reduced when the NIR dye-to-protein ratio was increased 3- to 4-fold in agent preparations when tested by FC and the Lindmo assay. In summary, NIR FC is an alternative with similar specificity and sensitivity, and greater reproducibility relative to the Lindmo assay for quantifying biological activity of NIR fluorophore-labeled, multimodal imaging agents.

show abstract

Section: Discussionmentioning

confidence: 99%

Quantifying multimodal contrast agent biological activity using near‐infrared flow cytometry

Hall

Aldrich

Azhdarinia

et al. 2012

Contrast Media & Molecular

View full text Add to dashboard Cite

show abstract

“…Typically, flow cytometry‐based assay validation procedures are “fit‐for‐purpose” in that the goal is to assess assay performance primarily in conditions applicable to the study 50. However, there is also a standard set of parameters characterized for most flow cytometry‐based RO assays including assessments of intra‐ and inter‐assay precision and sample stability 51. Precision measures are designed to assess the relative reproducibility of a given result within and between assays performed on the same specimen(s).…”

Section: Considerations When Developing Flow Cytometry‐based Ro Assaysmentioning

confidence: 99%

Receptor occupancy assessment by flow cytometry as a pharmacodynamic biomarker in biopharmaceutical development

Liang¹,

Schwickart²,

Schneider³

et al. 2015

Cytometry Part B Clinical

View full text Add to dashboard Cite

Receptor occupancy (RO) assays are designed to quantify the binding of therapeutics to their targets on the cell surface and are frequently used to generate pharmacodynamic (PD) biomarker data in nonclinical and clinical studies of biopharmaceuticals. When combined with the pharmacokinetic (PK) profile, RO data can establish PKPD relationships, which are crucial for informing dose decisions. RO is commonly measured by flow cytometry on fresh blood specimens and is subject to numerous technical and logistical challenges. To ensure that reliable and high quality results are generated from RO assays, careful assay design, key reagent characterization, data normalization/reporting, and thorough planning for implementation are of critical importance during development. In this article, the authors share their experiences and perspectives in these areas and discuss challenges and potential solutions when developing and implementing a flow cytometry‐based RO method in support of biopharmaceutical drug development. © 2015 The Authors Cytometry Part B: Clinical Cytometry Published by Wiley Periodicals, Inc.

show abstract

“…The use of accurate and well-characterized assays for testing biological products is vital for their development as therapeutic drugs (ICH, 1999, Cedeńo-Arias et al, 2011. Additionally, the assay which is employed to indicate biological activity of any biotechnological drug "should be based on the intended biological effect which should, ideally, be related to the clinical response" (EMEA, 2008).…”

Section: Introductionmentioning

confidence: 99%

Research paper Validation of a flow cytometry-based assay for monitoring complement-dependent cytotoxicity of an anti-CD20 biosimilar candidate antibody

Blanco¹,

Cedeño-Arias²,

Garcìa-Perez³

et al. 2014

bta

View full text Add to dashboard Cite

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.

show abstract

Validation of a Flow Cytometry Based Binding Assay for Evaluation of Monoclonal Antibody Recognizing EGF Receptor

Cited by 10 publications

References 12 publications

Quantifying multimodal contrast agent biological activity using near‐infrared flow cytometry

Quantifying multimodal contrast agent biological activity using near‐infrared flow cytometry

Receptor occupancy assessment by flow cytometry as a pharmacodynamic biomarker in biopharmaceutical development

Research paper Validation of a flow cytometry-based assay for monitoring complement-dependent cytotoxicity of an anti-CD20 biosimilar candidate antibody

Contact Info

Product

Resources

About