title: Validation of a quantitative flow cytometer assay for monitoring HER-2/neu expression level in cell-based cancer immunotherapy products.
abstract: GVAX immunotherapy for prostate cancer is comprised of two genetically modified prostate cancer cell lines, CG1940 and CG8711, engineered to secrete granulocyte macrophage-colony-stimulating factor. As part of the matrix of potency assays, CG1940 and CG8711 are tested for the expression level of cell surface HER-2/neu using a quantitative flow cytometer assay. This assay reports the antibody binding capacity value of the cells as a measure of HER-2/neu expression using cells immediately after thawing from cryogenic storage. With optimized cell handling and staining procedure and appropriate system suitability controls, the assay was validated as a quantitative assay. The validation results showed that assay accuracy, specificity, precision, linearity, and range were suitable for the intended use of ensuring lot-to-lot consistency of HER-2/neu expression. Assay robustness was demonstrated using design of experiments that evaluated critical assay parameters. Finally, the assay was successfully transferred to a current good manufacturing practice Quality Control laboratory in a separate facility. Since the overall precision of this assay is better than that of ELISA methods and it can be performed with ease and high throughput, quantitative flow cytometer-based assays may be an appropriate immunological assay platform for Quality Control laboratories for characterization and release of cell-based therapies.
authors.affiliation: Assay Development, Cell Genesys, Inc., 500 Forbes Boulevard, South San Francisco, CA 94404, USA.
authors.forename: Britta
authors.initials: B
authors.lastname: Randlev
authors.forename: Li-chun
authors.initials: LC
authors.lastname: Huang
authors.forename: Mitsuko
authors.initials: M
authors.lastname: Watatsu
authors.forename: Matthew
authors.initials: M
authors.lastname: Marcus
authors.forename: Andy
authors.initials: A
authors.lastname: Lin
authors.forename: Shian-Jiun
authors.initials: SJ
authors.lastname: Shih
mesh_terms: D000906:Antibodies / Q000032:analysis* / Q000627:therapeutic use; D002138:Calibration; D002277:Carcinoma / Q000378:metabolism / Q000628:therapy*; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000592:standards; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D020869:Gene Expression Profiling; D006801:Humans; D007118:Immunoassay / Q000295:instrumentation / Q000379:methods / Q000592:standards; D007167:Immunotherapy / Q000592:standards; D019169:Jurkat Cells; D008297:Male; D011471:Prostatic Neoplasms / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology / Q000378:metabolism; D012680:Sensitivity and Specificity
chemical_list: D000906:Antibodies; D018719:Erb-b2 Receptor Tyrosine Kinases