Validation of a quantitative flow cytometer assay for monitoring HER-2/neu expression level in cell-based cancer immunotherapy products.
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.
Randlev
Britta
B
Assay Development, Cell Genesys, Inc., 500 Forbes Boulevard, South San Francisco, CA 94404, USA.
Huang
Li-chun
LC
Watatsu
Mitsuko
M
Marcus
Matthew
M
Lin
Andy
A
Shih
Shian-Jiun
SJ
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
D000906:Antibodies; D018719:Erb-b2 Receptor Tyrosine Kinases