B-297 Effortless and Streamlined Workflow for Absolute Quantitation of Therapeutic Monoclonal Antibodies Using Promise Proteomics mAbXmise Kits and TSQ Altis Plus Mass Spectrometer

Abstract

Abstract Background Laboratories continuously seek improved productivity and efficacy for clinical testing, ultimately impacting the turn-around time and sample throughput. Various efforts have been made including rapid and high-throughput testing, automation, and high-end instrumentation. This also applies to therapeutic drug monitoring (TDM) of therapeutic monoclonal antibodies (mAbs). In particular, mass spectrometry (MS) has gained significant popularity in clinical laboratories for TDM of mAbs due to its great versatility to measure such complex biological proteins qualitatively and quantitatively. Here we present the streamlined workflow for the absolute quantitation of therapeutic mAbs using the Promise Proteomics mAbXmise kits and the Thermo Scientific™ TSQ Altis™ Plus mass spectrometer. The mAbXmise kit is the first commercial ready-to-use kit that offers all the necessary reagents for the LC-MS/MS assay including the stable-isotope labeled (SIL) proteins of the target mAbs (SIL-mAbs). The TSQ Altis™ Plus mass spectrometer is the latest generation of the TSQ MS series, offering superior acquisition speeds, sensitivity, selectivity, and robustness for routine and high-volume targeted protein quantitation. Methods The experimental workflow followed the instructions provided by Promise Proteomics with minor modifications (refer to instructions manual—https://customer.mabxmise.com). The kits used in this study include pre-coated plates with a full-length SIL-mAb corresponding to each target mAb sequence as internal standards with purity >95% and isotopic corporation >98%. Thus, the ITDM1 kit contains 2 SIL-mAbs and the OTDM1 kit contains 7 SIL-mAbs pre-coated in each plate. The kits also provide buffer stocks, reagents, 6 calibration standards including zero, and 2 QC controls. Each kit provides 12 strips capable of 96 sample preparation. The calibrators and QC samples were added to the mAbXmise plate and subjected to immunocapture followed by trypsin digestion. LC-MS/MS was performed using a Thermo Scientific™ Vanquish™ Flex UHPLC system hyphenated to TSQ Altis™ Plus mass spectrometer. Results Highly comparable results were observed across six replicates for both OTDM1 and ITDM1 kits with R2 values >0.99 and % RSD of peak areas of peptides <15. After confirming the complete sample preparation of 96 samples, an evaluation of LC-MS analytical performance was performed. The results show great linearity with R2 > 0.998 over measured calibration concentration points for all the OTDM1 and ITDM1 target peptides, respectively. Also, the data support the reproducible LC-MS measurement of the entire plate providing % RSD <10. The accuracy of QC samples was determined to be between 94 and 103% for OTDM1 peptides with a precision of <7% CV and between 95 and 107 % for ITDM peptides with a precision of <9% CV. The response of CAL1 (LLOQ) was 5 times higher than CAL0 for all the target peptides with accuracy between 96 and 112% and precision of <11% CV. Additionally, extremely reproducible retention times were observed from all SIL-mAb peptides with RT difference ±0.03 min during the evaluation. Conclusion The workflow tested in this report generated sensitive and robust data with high confidence, emphasizing the capability of MS-based TDM of mAbs as routine testing.