title: Development of a sensitive potency assay to measure the anti-proliferation effect of an anti-HER2 antibody.
abstract: For therapeutic antibodies that inhibit the growth of cancer cells, proliferation assays that measure cell number changes after the antibody treatment are often used to determine the potency of the antibody. Two of the most commonly used non-radioactive readout systems for proliferation assays, the ATP bioluminescence assay and the fluorescent dye Alamar Blue assay, were initially tested as potency assays an anti-HER2 antibody. Due to the slow growth of the target cells, these assays only produced less than 3-fold difference after 5 days of antibody treatment. BrdU incorporation-based proliferation assay, which differentiates proliferating cells from arrested cells, was developed, and showed superior sign-to-background ratio. Colorimetric, chemiluminescent, and DELFIA readouts were compared for BrdU incorporation assays, and DELFIA-based assay was further optimized using a Design of Experiment (DoE) approach. The final DELFIA-based BrdU incorporation assay demonstrated superior signal-to-background ratio, robustness, accuracy, and precision, and represented significant improvement over traditional proliferation assays.
authors.affiliation: Analytical Development, Biogen Idec, Inc., Cambridge, MA 02142, USA. Electronic address: Xiaohui.Lu@biogenidec.com.
authors.forename: Xiaohui
authors.initials: X
authors.lastname: Lu
authors.affiliation: Analytical Development, Biogen Idec, Inc., Cambridge, MA 02142, USA.
authors.forename: Svetlana
authors.initials: S
authors.lastname: Bergelson
mesh_terms: D000255:Adenosine Triphosphate / Q000032:analysis / Q000096:biosynthesis; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D001973:Bromodeoxyuridine / Q000276:immunology / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D005163:Factor Analysis, Statistical; D005456:Fluorescent Dyes; D015870:Gene Expression; D006801:Humans; D007118:Immunoassay / Q000379:methods* / Q000592:standards; D008163:Luminescent Measurements; D010078:Oxazines; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000276:immunology; D015203:Reproducibility of Results; D059629:Signal-To-Noise Ratio; D014966:Xanthenes
chemical_list: D000255:Adenosine Triphosphate; D000911:Antibodies, Monoclonal; D001973:Bromodeoxyuridine; D005456:Fluorescent Dyes; D010078:Oxazines; D018719:Erb-b2 Receptor Tyrosine Kinases; D014966:Xanthenes; C005843:resazurin; C508053:ERBB2 protein, human
keywords: BrdU incorporation assay; DELFIA; Potency assay