title: Validation of a rat pheochromocytoma (PC12)-based cell survival assay for determining biological potency of recombinant human nerve growth factor.
abstract: A method has been validated, according to the Guidelines of the International Conference on Harmonization (ICH), for precise quantitation of the biological activity of recombinant human nerve growth factor (rhNGF) for lot release testing. The assay is based on the survival of a subclone of rat pheochromocytoma PC12 cells (PC12-CF) in response to rhNGF. Cell survival is measured by monitoring the reduction, by living cells, of the alamarBlue dye into a red form which is highly fluorescent. The assay is simple, has high throughput (performed in 96-well microtiter plates) and shows reproducible dose-response curves in the concentration range of 0.2-50 ng/ml. The method was validated for its linearity, accuracy, precision, robustness, and to meet current regulatory requirements. The assay demonstrated good linearity, yielding a coefficient of determination of 0.9902. Sample recovery studies demonstrated an accuracy ranging from 96 to 98%. The repeatability of the assay and intermediate precision had coefficients of variation (CV) of <9%. The assay was stability-indicating since it was able to detect changes in rhNGF samples degraded by protease treatment and in a number of isolated rhNGF variants. Robustness was demonstrated by the relative insensitivity of the assay to small deliberate changes in key method parameters. The validation data, provided in this manuscript, indicate that the newly described bioassay for rhNGF is robust, accurate, precise, and suitable for lot release potency testing of rhNGF.
authors.affiliation: Quality Control Biochemistry, Genentech, South San Francisco, CA 94080, USA. helene@gene.com
authors.forename: H
authors.initials: H
authors.lastname: Gazzano-Santoro
authors.forename: A
authors.initials: A
authors.lastname: Chen
authors.forename: B
authors.initials: B
authors.lastname: Casto
authors.forename: H
authors.initials: H
authors.lastname: Chu
authors.forename: E
authors.initials: E
authors.lastname: Gilkerson
authors.forename: V
authors.initials: V
authors.lastname: Mukku
authors.forename: E
authors.initials: E
authors.lastname: Canova-Davis
authors.forename: C
authors.initials: C
authors.lastname: Kotts
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002470:Cell Survival / Q000187:drug effects*; D004396:Coloring Agents; D004305:Dose-Response Relationship, Drug; D005453:Fluorescence; D005470:Fluorometry; D006801:Humans; D020932:Nerve Growth Factor / Q000494:pharmacology*; D010078:Oxazines; D016716:PC12 Cells; D010673:Pheochromocytoma / Q000473:pathology; D051381:Rats; D011994:Recombinant Proteins / Q000494:pharmacology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014966:Xanthenes
chemical_list: D004396:Coloring Agents; D010078:Oxazines; D011994:Recombinant Proteins; D014966:Xanthenes; C005843:resazurin; D020932:Nerve Growth Factor