Design of Experiments (DOE)
When multiple factors affect the outcome of a process, there are several ways to optimize the process. Traditionally, researchers have varies one parameter, while keeping the others constant and observed the impact on the process. In the next step, the first parameter is held constant (often at the observed optimum condition), while a second parameter is varied. This iterative process is repeated until all parameters have been optimized. The significant drawback of this technique is that the process can be “trapped” in a local optimum condition and the researcher will not observe the global optimum. A better way to optimize parameters is through by testing an organized, statistically-derived matrix of variables that ensures that the full parameter space is sampled. Several software packages are available to assist the researcher in selecting the best matrix of experimental conditions. Click here for an article in Nature Methods "High-throuphput screening: designer screens".
Design of Experiments for Quality by Design Programs for Biologicals
With an overall goal of improving drug safety and efficacy, the FDA is spearheading Quality by Design initiatives to improve drug manufacturing processes. Fully understanding the critical quality attributes (CQAs) of the biological product and the critical process parameters (CPPs) that can be used to control overall product quality requires very large experimental studies that wouldn’t be practical without employing Design of Experiment approaches.
Design of Experiments for Enzymatic Assay Optimization
Optimizing enzymatic assays involves determining the right buffer, detergent, salt, and cation combination. The process of finding the best conditions is very tedious, requiring many individual experiments to test each combination of the reagents. Design of Experiment software can help eliminate some of the experiments by selecting reagent combinations that will result in optimal conditions, while at the same time, check for interferences between reagents.
Caliper Instruments to Accelerate Design of Experiment Studies
The Caliper Sciclone G3 Liquid Handler can be used to prepare the different conditions created in the DesignExpert design of experiment software (See application note). As an example, six different buffer conditions, 5 different detergents, several salt and cation concentrations might be chosen as variables. Creating the buffer mixtures required to assess each condition can be automated on the Sciclone G3, as can the subsequent addition of the substrate and enzyme to the plate, incubation, and termination of the reaction by the addition of EDTA. Some key benefits of using the Sciclone for DOE include:
• The Sciclone G3 prepares 384 unique reactions in about 2.5 hours
• 2-3 reagent plates can be prepared in an 8-hour day
• Reagent plates are stable for 24 hours when sealed and stored at 4°C
• Local and global maxima are easily identified, allowing optimization of reactions while meeting specific criteria
• Unexpected interactions between reagents are revealed
• Use of the optimization platform allows rapid development of mobility-shift kinase assays
• The process is easily adaptable to a plethora of assay formats and technologies
The LabChip GXII, an automated platform for rapid analysis of proteins and nucleic acids, has proven to be an enabling technology for DOE studies, particularly for Quality by Design programs for biological therapeutics, where it can be deployed as a Process Analytical Technology (PAT). As an automated replacement technology for SDS-PAGE gels, the LabChip GXII provides high throughput analysis of titer, sizing, and purity analysis of proteins. The LabChip GXII can also be coupled with the Twister II Plate Handler for longer walk away time and can be integrated into a Staccato Workstation for complete automation of sample preparation and analysis.
In the case of kinase, phosphatase, protease, or other enzymatic classes amenable to mobility shift assay format, Caliper’s LabChip EZ Reader platform can be used to analyze the experimental results. Data from the LabChip platform is easily imported into DOE software for detailed analysis. The LabChip EZ Reader can also be coupled with the Twister II Plate Handler for longer walk away time and can be integrated into a Staccato Workstation for complete automation of sample preparation and analysis.
