Application of DBS for the quantitative assessment of a protein biologic using on-card digestion LC-MS/MS or immunoassay.

Background: Dried blood spot (DBS) sampling has recently gained popularity in the bioanalysis community for quantitation of small molecules. Since the pharmaceutical industry continues to increase investment in biopharmaceuticals, DBS technologies were investigated to determine if immunoassay and/or LC-MS/MS techniques would be amenable for quantitation of a large protein therapeutic (>70 kDa). Results: Methods were successfully qualified for the protein therapeutic utilizing DBS technology. DBS methods in rat blood were qualified for this therapeutic protein using either immunoassay or enzymatic digestion directly off the DBS card followed by UHPLC-MS/MS separation and detection. Both qualifications were carried out in accordance with current acceptable practices defined by international acceptance criteria. Card selection was critical to both DBS methods. Conclusion: The advantages gained by DBS technology can successfully be applied to the quantitative assessment of biologics. This UHPLC-MS/MS method illustrates that digestion of large molecules directly off blood spot cards allows quantitation of these molecules. In addition, DBS technologies are amenable to immunoassay ana-lysis. The immunoassay was 20-fold more sensitive than the UHPLC-MS/MS method, however the UHPLC-MS/MS assay had a much broader dynamic range.