Targeted nano LC-MS Quantification of Rituximab in Human Bio-Matrix with High-Resolution Accurate Mass MS

Importance of the Topic

Monoclonal antibodies such as rituximab are widely used in oncology and immunology. Accurate quantification in complex human matrices is crucial for pharmacokinetics, quality control and therapeutic monitoring. High-resolution nano LC-MS methods address challenges posed by low drug concentration and matrix interferences.

Study Goals and Overview

This study aimed to develop a sensitive and reproducible targeted nano LC-MS workflow for quantifying rituximab in human cell lysate. The authors combined data-dependent acquisition (DDA), targeted selected ion monitoring (tSIM) and parallel reaction monitoring (PRM) on a quadrupole-Orbitrap platform to cover a dynamic range from amol to fmol.

Methodology and Instrumentation

• Sample preparation: Rituximab was digested with SMART Digest kit and reduced with TCEP. Tryptic peptides were spiked into HeLa cell digest at ratios from 1:100 to 1:100 000.
• Liquid chromatography: UltiMate 3000 RSLCnano with ProFlow flow meter; EASY-Spray 75 µm × 50 cm PepMap C18 column operated at 300 nL/min.
• Mass spectrometry: Thermo Scientific Q Exactive HF with EASY-Spray source. Methods included DDA for peptide discovery, tSIM for high-sensitivity quantification and PRM for selectivity.

Key Results and Discussion

• DDA profiling identified a limited set of rituximab-specific peptides with highest response for FSGSGSGTSYSLTISR and QIVLSQSPAILSASPGEK.
• Retention time repeatability was < 0.2 % RSD, enabling scheduled monitoring.
• tSIM quantification showed linear response over four orders of magnitude (amol to fmol level) with R² > 0.99.
• PRM provided additional selectivity and sensitivity, detecting rituximab at a 1:75 000 ratio to HeLa proteins.

Benefits and Practical Applications

The developed workflow offers:

• High sensitivity and selectivity for therapeutic antibody quantification in complex bio-matrices.
• Robust retention time control for multiplexed assays.
• Applicability in biopharmaceutical QC, pharmacokinetic studies and clinical monitoring.

Future Trends and Possibilities

• Extension to other monoclonal antibodies and biotherapeutics by selecting unique peptide markers.
• Integration with immuno-enrichment to further lower detection limits.
• Automation and scheduling improvements for high-throughput analysis in regulated environments.
• Adoption of next-generation high-resolution instruments for faster acquisition and deeper multiplexing.

Conclusion

Targeted nano LC-MS using tSIM and PRM on an Orbitrap system enabled reproducible, sensitive and selective quantification of rituximab in complex human cell lysate. The approach spans amol to fmol levels and supports routine bioanalysis of therapeutic monoclonal antibodies.

References

• Samonig M., Swart R. LC-UV-MS Peptide Mapping Development for Easy Transfer to LC-UV QA/QC. Application Note 1134. Thermo Fisher Scientific. 2015.
• UniProtKB - P01857 (IGHG1_HUMAN). http://www.uniprot.org/uniprot/P01857