A pre-concentration and online solid phase extraction setup for the LC-MS analysis of therapeutic protein mixtures

Importance of the Topic

Solid-phase extraction combined with reversed-phase liquid chromatography and high-resolution mass spectrometry enables precise analysis of therapeutic protein mixtures. Automated online trapping and elution reduce sample handling, improve repeatability, and prevent contamination that often occurs in manual workflows.

Objectives and Study Overview

This work demonstrates a fully automated UHPLC setup for pre-concentration, desalting, and separation of a five-protein mixture, including monoclonal antibodies, using a trap-and-elute approach. Key aims:

• Implement an online SPE protocol with a MSPac DS-10 desalting cartridge and MAbPac RP analytical column
• Optimize temperature, flow rates, and valve switching for robust protein trapping and separation
• Evaluate performance on a Thermo Scientific Vanquish UHPLC system coupled to a Q Exactive HF Orbitrap mass spectrometer

Methodology and Instrumentation

Protein samples (alpha-lactalbumin, cytochrome c, myoglobin, ribonuclease A, rituximab) were prepared in acidic aqueous solution and loaded onto the trap column with 0.1 % TFA at 0.3 mL/min for 2 min. A two-position/6-port valve switched the trap into the analytical flow path for back-flush elution using a 0.1 % FA water/acetonitrile gradient at 0.1 mL/min. High column temperature (70 °C) proved critical for efficient trapping and sharp peak shapes.

Instrumentation Used

• Vanquish Flex UHPLC: quaternary low-pressure loading pump and binary high-pressure analytical pump
• MPac DS-10 desalting trap cartridge and MAbPac RP 1 × 100 mm analytical column
• Biocompatible 2p6p column switching valve with Viper capillaries
• Q Exactive HF Quadrupole-Orbitrap mass spectrometer with HESI-II source
• Chromeleon CDS and BioPharma Finder software for data acquisition and processing

Key Results and Discussion

Elevated trapping and separation temperature (70 °C) yielded baseline-resolved chromatograms for all five proteins. Room-temperature trapping led to loss of the rituximab signal. Representative mass spectra acquired at 120 000 and 15 000 resolution provided isotopic resolution and accurate intact mass measurement. Deconvolution revealed protein masses within 5 ppm of theoretical values, including four major glycoforms of rituximab.

Benefits and Practical Applications

The automated online SPE method offers:

• High throughput with minimal manual intervention
• Improved repeatability and reduced contamination risk
• Efficient enrichment of low-abundance proteins
• Direct injection of complex formulation buffers

It is well suited for biopharmaceutical R&D, quality control, and characterization of therapeutic proteins.

Future Trends and Opportunities

Advances in trap material chemistries and microfluidic valve designs will further reduce sample volumes and cycle times. Integration with multi-attribute methods and data-independent acquisition may extend online SPE applications to peptide mapping and post-translational modification profiling. Coupling with AI-driven method optimization can accelerate method development.

Conclusion

This study validates a robust, fully automated online SPE workflow for intact protein analysis, leveraging Vanquish UHPLC and Q Exactive HF MS. The trap-and-elute approach enhances throughput and reproducibility while delivering high mass accuracy and resolution.

References

1. Dillon TM, Bondarenko PV, Rehder DS, et al. J Chromatogr A. 2006;1120:112–120.
2. Thermo Scientific Technical Note: Influence of Column Temperature on Reversed-Phase Chromatography of an Intact Antibody. 2015.
3. Fekete S, Rudaz S, Veuthey JL, Guillarme D. J Sep Sci. 2012;35:3113–3123.