Quantitation of therapeutic proteins following direct trypsin digestion of dried blood spot samples and detection by LC-MS-based bioanalytical methods in drug discovery

Background: There is considerable interest in the pharmaceutical industry today in both development of therapeutic proteins as viable biopharmaceutical agents as well as the implementation of microsampling techniques, such as dried blood spots (DBS), as an alternative to current sample collection and handling procedures for biological samples generated in drug discovery and development studies. We have demonstrated that these two techniques can be integrated by developing bioanalytical methods that simultaneously determine the concentrations of unique therapeutic protein constructs, using LC-MS-based detection of multiple surrogate peptides following direct trypsin digestion of DBS. Results: Bioanalytical methods were developed for the simultaneous determination of two structurally different therapeutic proteins (PEGylated-Adnectin (TM)-1 MW 11,144 amu and an Fc-fusion protein, MW 67,082 amu) in a single DBS sample using LC-MS-based detection of multiple peptides generated from different regions of the proteins following trypsin digestion. The same methodology was applied to the analysis of DBS samples collected following dosing of a third unique protein (PEGylated-Adnectin-2) to mice. Although these initial DBS methods were slightly less sensitive than those developed specifically for each individual protein in plasma or serum, the generic digestion procedure yielded sufficient accuracy, precision and an extended linear dynamic range to justify their further evaluation in pharmacokinetic, pharmacodynamic and toxicological studies of selected therapeutic proteins following dosing in preclinical discovery studies. Additionally, DBS samples may offer a convenient, generic platform approach for direct enzymatic digestion and sample preparation for LC-MS-based quantitation of proteins. DBS samples prepared for two of the therapeutic proteins were also stable for at least 2 weeks when stored at room temperature. Conclusion: Although the same clarification and interpretation of DBS results will be required (e.g., blood vs plasma levels, hematocrit effects on DBS determinations and red blood cell partitioning) as for small-molecules, there still remains the potential to further develop and expand this strategy with appropriate proteins of interest. While additional studies will be required to validate this approach in specific applications, we have demonstrated the feasibility of using DBS sampling to directly quantify structurally different types of therapeutic proteins in blood in discovery studies and present the potential to simultaneously measure other proteins, such as biomarkers, to augment and integrate data generated from in vivo studies.