Utilizing design of experiments to characterize assay robustness.

BACKGROUND Design of experiments (DOE) is a systematic approach to assess the effects of many factors on a response of an assay. This paper provides a case study whereby DOE was successfully utilized to evaluate robustness parameters for a ligand-binding assay (LBA). METHODOLOGY A 24-run Plackett-Burman design was developed to investigate factors that may have caused a lack of robustness in this particular LBA. We modeled five main effects and their ten two-way interactions, using the standard curve signal as the response. RESULTS By utilizing DOE, we were able to quickly identify the factors that affected our assay's performance. The lack of robustness was attributed to the handling of the coat reagent. Factors that had an adverse effect on the coat material were vortexing and freeze-thaw cycles. CONCLUSION We recommend that a robustness DOE be conducted prior to the validation of an assay for early identification of critical factors that may impact assay performance.

[1]  Francesca Civoli,et al.  Implementation of design of experiments (DOE) in the development and validation of a cell-based bioassay for the detection of anti-drug neutralizing antibodies in human serum. , 2012, Journal of immunological methods.

[2]  Maneesha Altekar,et al.  Assay optimization: a statistical design of experiments approach. , 2007, Clinics in laboratory medicine.

[3]  Binodh DeSilva,et al.  Key elements of bioanalytical method validation for macromolecules , 2007, The AAPS Journal.

[4]  Binodh DeSilva,et al.  Recommendations for the Bioanalytical Method Validation of Ligand-Binding Assays to Support Pharmacokinetic Assessments of Macromolecules , 2003, Pharmaceutical Research.

[5]  Matthew Troutman,et al.  Optimizing ELISAs for precision and robustness using laboratory automation and statistical design of experiments. , 2008, Journal of immunological methods.

[6]  J. W. Findlay,et al.  Some important considerations for validation of ligand-binding assays. , 2009, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[7]  D Brynn Hibbert,et al.  Experimental design in chromatography: a tutorial review. , 2012, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[8]  Y. Vander Heyden,et al.  Comparison of Plackett-Burman and supersaturated designs in robustness testing. , 2007, Analytica chimica acta.

[9]  Judy Y Shih,et al.  Method transfer for ligand-binding assays: recommendations for best practice. , 2011, Bioanalysis.

[10]  Mark Ma,et al.  Application of multi-factorial design of experiments to successfully optimize immunoassays for robust measurements of therapeutic proteins. , 2009, Journal of pharmaceutical and biomedical analysis.

[11]  Afshin Safavi,et al.  Ligand Binding Assays in the 21st Century Laboratory: Recommendations for Characterization and Supply of Critical Reagents , 2012, The AAPS Journal.

[12]  Wendell C. Smith,et al.  Application of experimental design techniques to optimize a competitive ELISA. , 1996, Journal of immunological methods.

[13]  R. Plackett,et al.  THE DESIGN OF OPTIMUM MULTIFACTORIAL EXPERIMENTS , 1946 .

[14]  Daniel Sikkema,et al.  Complexities of Clinical Assay Development and Optimization Prior to First-in-Man Immunization Trials – A Description of Immunogenicity Assay Development for the Testing of Samples from a Phase 1 Alzheimer's Vaccine Trial , 2008, Journal of immunoassay & immunochemistry.

[15]  S Furlanetto,et al.  How experimental design can improve the validation process. Studies in pharmaceutical analysis , 2003, Analytical and bioanalytical chemistry.

[16]  Lakshmi Amaravadi,et al.  Solution ELISA as a platform of choice for development of robust, drug tolerant immunogenicity assays in support of drug development. , 2011, Journal of immunological methods.

[17]  R. W. Hansen,et al.  The price of innovation: new estimates of drug development costs. , 2003, Journal of health economics.

[18]  Bieke Dejaegher,et al.  Ruggedness and robustness testing. , 2007, Journal of chromatography. A.