Table II.
Overview of Challenges and Solutions: Multiplex Validation, Sample Analysis, and Assay Maintenance
| Method stage | Challenge | Solution |
|---|---|---|
| Method validation | Data handling |
• Use built-in templates • Contract IT resources • Work with instrument/software vendors to assist with solutions |
| All pass/procedure if an analyte system fails? |
• Report data for analytes that pass, repeat run for analytes that do not pass (second run should mask results for passing analytes) • Analyte that does not meet validation acceptance criteria (if repeat analysis fails) should not be included in multiplex sample analysis. • Demonstrate that removal of capture/detect/analyte for failed assay does not change the multiplex assay performance |
|
| Lack of regulatory performance recommendations | Utilize approach based on bioanalytcial methods for biotherapeutics, implementing acceptance criteria prior to in-study analysis, and using fit-for-purpose approach based on intended use of the assay | |
| Sample analysis | Data handling (work list preparation, data management, assay approval, data reporting) |
• Possible use of macros, built-in templates • Contract IT resources • Work with instrument/software vendors to assist with solutions |
| All pass—if you must rerun one analyte, what do you do with data for other passing analytes from first run? | Report data for analytes that pass, repeat run for analytes that do not pass (second run should mask results for passing analytes). Analyte that does not meet sample analysis acceptance criteria (if repeat analysis fails) should not be included in final analysis. | |
| Identical curve fitting |
• Use best fit approach • It is acceptable to use different curve fits/weighting for different analytes; however, for a single analyte continue using same curve fitting after validation |
|
| Lack of regulatory performance recommendations |
• Utilize regulatory requirements that most closely meet the study requirements • FDA Draft Guidance on Bioanalytical Method Validation includes a discussion on biomarkers |
|
| Assay maintenance | Variability in reagent lots |
• Implement a defined process to investigate lot-to-lot variability • Apply a correction factor • Screen multiple lots • Acquire sufficient volumes of an original lot with expiration dating that allows completion of the program |
| Availability of critical reagents |
• Use surrogate molecules if needed • Initiate an analyte/antibody production program in anticipation starting the project • Contact vendors to assist with sourcing • Review research literature for possible academic sources |
Italicized points are unique to multiplexing