Purpose: The purpose of this work is to provide an assessment of alternative and fit-for-purpose strategies for LC-MS based quantitation of target proteins in either the toxicokinetic or the efficacy application space. LC-MS/MS technology is commonly used to quantify proteins in biological samples, especially in cases when affinity-based ligand binding assay cannot be used, for example when suitable highly specific antibodies are not available for capture and detection of analyte (Jenkins, R. 2015). The immunoprecipitation with LC-MS/MS approach is not in-scope for this work. The high throughput, though less sensitive method described here applicable for toxicokinetics (TK), is based on enzymatic digestion of a protein pellet, followed by Solid Phase Extraction (SPE). These processed samples are then analyzed by liquid chromatography (LC) coupled to a triple quadrupole mass spectrometry instrument with a rapid binary gradient, to selectively quantify the target protein’s signature peptides. The lower-throughput, and more selective and sensitive method herein, for efficacy marker quantitation, used an alternative bioanalytical strategy. To further enhance sensitivity and selectivity, the sample preparation was performed using a carefully designed fractionation approach, for the digested samples. For the analytical method, we demonstrated the great value for using nanoflow LC coupled to a high-end orbitrap instrument with a high-field asymmetric waveform ion mobility spectrometry (FAIMS) interface operating in parallel reaction monitoring mode (Bourmaud, A. 2016). In this poster we will present an assessment of these two LC-MS/MS approaches under non-GLP conditions for preclinical programs, demonstrating for which applications they are fit-for-purpose. Methods: TK application: Multiple Reaction Monitoring (MRM) method used a triple quadrupole instrument from Sciex. Samples were prepared via protein precipitation prior to enzymatic digestion, followed by SPE for additional clean-up of the plasma/serum samples. The MS method used classical MRM, and the LC method was short to enable fast cycle times. This approach resulted in robust quantitation and high-throughput bioanalysis. Efficacy application: Parallel Reaction Monitoring (PRM) method used a high mass accuracy and resolution Orbitrap Eclipse instrument from Thermo Fisher Scientific. The samples were prepared with fractionation of digested peptides. The MS method implemented FAIMS to reduce the complexity of biological samples, resulting in improved selectivity and sensitivity. The LC method had long, optimized gradients to further improvements in selectivity. These methods were used in a fit-for-purpose manner to meet different needs for preclinical programs. Results: Developed LC-MS/MS MRM and PRM methods were qualified following non-GLP study requirements. For toxicokinetic preclinical programs where sensitivity is not a major concern, but fast cycle times are critical, the MRM based method is typically used, resulting in analysis of up to 200 samples per day. The lower limit of quantitation (LLOQ) of the MRM method achieved 0.25 micro gram per milliliter (ug/mL of plasma) in Non-Human Primate (NHP) plasma. The linearity of standard curves was from 0.25 ug/mL to 1000 ug/mL target protein in plasma using the high-throughput MRM method. For the efficacy preclinical program where sample volume is limited and the target protein could be highly homologous with the endogenous protein, a more selective and sensitive method is needed. The PRM based method was adopted as fit-for-purpose, which led to significant improvements in selectivity and sensitivity. For example, the FAIMS interface alone improved analyte signal to noise about 3-fold compared to the standard interface. Combined sensitivity enhancement of the PRM based method resulted in an LLOQ of 7.13 nanogram per gram (ng/g of total protein) in extract protein of NHP tissues. The linearity of the standard curves was from 7.13 ng/g to 5200 ng/g protein in multiple tissues. About 25 samples per day were analyzed using the PRM approach. Using these methods, absolute levels of human and therapeutic proteins were measured in NHP tissues and plasma. Conclusion: We successfully developed and qualified high-throughput LC-MS/MS MRM and highly sensitive nanoflow LC-MS/MS PRM methods for targeted protein bioanalysis under non-GLP conditions. We established an innovative liquid phase and gas phase fractionation workflow for measuring human protein expression levels in NHP tissues, providing impactful insights for the project team. Both PRM and MRM methods were used for preclinical efficacy and toxicokinetic studies in multiple therapeutic areas. References: Jenkins, R. 2015, Recommendations for Validation of LC-MS/MS Bioanalytical Methods for Protein Biotherapeutics. AAPS Journal, 17(1). https://doi.org/10.1208/s12248-014-9685-5 Bourmaud, A. 2016, Parallel reaction monitoring using quadrupole-Orbitrap mass spectrometer: Principle and applications. Proteomics, 16(15–16), 2146–2159. https://doi.org/10.1002/pmic.201500543
