(W1430-02-09) Exploring the Future Potentials of High-Resolution Mass Spectrometry for Regulated Bioanalysis

Purpose: With increasingly complex drug modalities as well as a need for more sensitive methods, technologies that can complement immunoassays are desired. Multi-dimensional, low-flow chromatography coupled to high-resolution mass spectrometry (HRMS) has been shown to be advantageous for instances where a high degree of sensitivity or greater selectivity is required. Equally, such advanced LCMS techniques can mitigate the need for and/or quality critical reagents typically required for definitive bioanalytical methods used in support of regulated studies. This poster describes the practical application of LC-HRMS to address greater sensitivity or selectivity and in doing so, exemplifies the future impacts of this technology to regulated bioanalysis consistent with the challenges of increasingly complicated drug modalities.

Methods: A hybrid immunoassay – nano-flow liquid chromatography – HRMS method was developed for quantitation of a bispecific antibody as the immunoassay platform could not achieve the sensitivity needed. The hybrid method utilized a bottom-up workflow with bead based immunocapture followed by nanoLC – HRMS. This methodology and workflow has, and can further be expanded, for other drug modalities that utilize a capture antibody and need greater sensitivity than the immunoassay platform alone. Separately, a critical reagent free nanoLC – HRMS method was developed for quantitation of a peptide hormone when no capture antibody was available.  After a solid phase extraction, the instrument method utilized multi-dimensional low flow liquid chromatography including size-exclusion chromatography (SEC) coupled to HRMS.  This workflow has also been further expanded for other peptide hormones that need greater selectivity and sensitivity.

Results: Acceptable method performance was demonstrated with both the hybrid immunoassay – nanoLC – HRMS and critical reagent free nanoLC – HRMS method. The resulting LLOQ was 60X more sensitive with the hybrid immunoassay – nanoLC – HRMS method than the immunoassay alone. The method was fully validated as per the current regulatory guidance and the accuracy and precision statistics are described. With the peptide hormone case study, the critical reagent free method was able to achieve 5 pg/mL sensitivity. The method has demonstrated other peptide hormones can be added with small changes to the method as no capture antibody is used. The method was validated fit for purpose and the accuracy and precision statistics are included.

Conclusion: HRMS coupled to multi-dimensional low flow chromatography has demonstrated successfully achieving the required sensitivity and selectivity needed. This technology could be applied to future challenges when sensitivity or selectivity is required, or when critical reagents are not available. The plug and play capability of the critical reagent free nanoLC – HRMS method for peptide hormones has been shown to be a useful tool for interchangeability of peptide hormones for quantification and can serve as another analytical tool for gaining selectivity and sensitivity.

Bispecific Antibody Accuracy and Precision in Hybrid Immunoassay – nanoLC – HRMS method

Bispecific Antibody Hybrid Immunoassay – nanoLC – HRMS method

Peptide Hormone Accuracy and Precision from Critical Reagent Free Method