Overview of LC-MS Quantitative Solutions for Biotherapeutic Analysis

Importance of the Topic

Quantitative analysis of biotherapeutic molecules is vital across development stages, ensuring precise dosing, safety and efficacy. Complex biological matrices and low analyte levels necessitate robust workflows that combine sensitivity, selectivity and throughput.

Objectives and Study Overview

This overview presents SCIEX LC-MS solutions for biotherapeutic quantification, aiming to address challenges in sensitivity, specificity, dynamic range, reproducibility and sample throughput. The study surveys various workflows such as immunoaffinity-LC-MRM, microflow LC-MRM, differential mobility separation, MRM3, high resolution accurate mass spectrometry, MRMHR, intact protein analysis and SWATH acquisition.

Methodology

• Immunoaffinity-LC-MRM: Captures target proteins via antibody beads, enabling high sensitivity and minimal matrix interference in a streamlined workflow with QTRAP 6500+ or Triple Quad 6500+ instruments.
• Microflow LC-MRM: Improves sensitivity for limited-volume samples using the M5 MicroLC system and OptiFlow Turbo V source, achieving LLOQs down to 1 ng/mL for trastuzumab emtansine.
• Differential Mobility Separation: Uses SelexION® technology to separate isobaric species in the gas phase, significantly improving signal-to-noise for challenging peptides such as exenatide and PN1944.
• MRM3 Workflow: Provides extra selectivity by monitoring secondary fragment ions on QTRAP systems, reducing interferences in protein isoform quantitation (e.g., CYP450 3A5) and lowering LLOQs to ~1 fmol.
• High Resolution Accurate MS: Employs SCIEX X500B QTOF and TripleTOF® systems for precursor-based quantitation of large peptides (e.g., RTP004), achieving LLOQs of 15.6 ng/mL without MS/MS optimization.
• MRMHR Workflow: Acquires high resolution MS/MS spectra for targeted precursors, enabling post-acquisition extraction of optimal fragment ions and improved limits of detection for oligonucleotides.
• Intact Protein Quantification: Combines immunocapture with high resolution MS for monoclonal antibody analysis, using extracted ion chromatograms, deconvolution or automated data reconstruction to differentiate glycoforms.
• SWATH Acquisition: Applies data independent acquisition for comprehensive host cell protein profiling, with ProteinPilot™ for library generation and PeakView™/MultiQuant™ for targeted quantitation of HCPs.

Instrumentation Used

• SCIEX Triple Quad™ and QTRAP® 6500+ Mass Spectrometers
• OptiFlow™ Turbo V Source and M5 MicroLC System
• SelexION® Differential Mobility Separation Device
• SCIEX X500B QTOF and TripleTOF® Mass Spectrometers
• SWATH® Data Independent Acquisition Technology

Main Results and Discussion

• Immunoaffinity-LC-MRM achieved a linear range over four orders of magnitude for trastuzumab quantification with r² ≥ 0.995 and LLOQ of 5 ng/mL in rat plasma.
• Microflow LC-MRM with trap-elute increased peak area > 5× and S/N > 3×, lowering LLOQ to 1 ng/mL for trastuzumab emtansine with CV < 15% across 4.5 orders of magnitude.
• DMS integration yielded dramatic S/N improvements for low-abundance peptides (5 pg/mL exenatide) and complex peptides like PN1944 in SIM mode.
• MRM3 workflows reduced background interferences in high-homology proteins, enabling CYP450 3A5 peptide quantitation at 1.2 fmol on column with CV < 3%.
• HRAMS precursor quantitation of RTP004 achieved an LLOQ of 15.6 ng/mL and linearity to 8000 ng/mL (r = 0.998) without MS/MS parameter optimization.
• MRMHR post-acquisition fragment selection improved LOD and LOQ for oligonucleotides, overcoming unpredictable matrix interferences and reducing method development time.
• Intact protein workflows detected trastuzumab glycoforms at 50 ng/mL, with quantitation from 100 to 50 000 ng/mL and clear glycoform chromatograms after data reconstruction.
• SWATH-based HCP analysis provided reproducible profiling across purification stages, supporting comprehensive monitoring of host cell protein impurities.

Benefits and Practical Applications

• Enhanced sensitivity and selectivity for diverse biotherapeutic classes
• Streamlined sample preparation and reduced analysis time
• Broad dynamic range and robust quantitation in challenging matrices
• Versatile workflows adaptable to peptides, proteins, ADCs, oligonucleotides and intact biotherapeutics
• Scalable solutions for preclinical, clinical and biomanufacturing QC roles

Future Trends and Applications

Advances in microflow chromatography, ion mobility, high resolution acquisition and data processing automation will continue to push LLOQs lower and expand multiplexing capabilities. Emerging strategies such as SWATH-based quantitation and intact protein profiling will facilitate deeper characterization of biotherapeutics and impurities in process development and clinical monitoring.

Conclusion

SCIEX offers a comprehensive suite of LC-MS workflows and instrumentation to meet the analytical challenges of biotherapeutic quantification. By integrating immunoaffinity capture, microflow LC, ion mobility, high resolution acquisition and advanced data analysis, these solutions deliver high sensitivity, specificity, throughput and robustness across a wide range of molecule classes.

References

• Zhang F et al. Quantification of Trastuzumab in Rat Plasma using an Improved Immunoaffinity-LC-MS/MS Method. SCIEX Technical Note.
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• Xiong L et al. Ultra-Sensitive Quantification of Trastuzumab Emtansine in Mouse Plasma using Trap-Elute MicroLC MS Method. SCIEX Technical Note.
• Baghla R et al. Sub-Picogram Level Quantitation of Desmopressin in Small Volumes of Human Plasma Using a Trap-Elute MicroLC-MS System. SCIEX Technical Note.
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• Knapman T et al. Quantification of Large Oligonucleotides using High Resolution MS/MS on the TripleTOF 5600 System. SCIEX Technical Note.
• Li Y et al. Quantitation of Intact Therapeutic Protein in Plasma Matrix by LC-MS. SCIEX Technical Note.
• Xiong L et al. Universal Data-Independent LC-MS/MS Workflow for Host Cell Protein Characterization and Quantification in Biopharmaceutical Product Purification Process. ASMS 2017 Poster.