Tandem UHPLC operation for high-throughput LC-MS peptide mapping analyses

Significance of the topic

High-throughput peptide mapping by LC-MS is critical for biopharmaceutical development and quality control, enabling detailed characterization of protein therapeutics with improved productivity and reproducibility.

Objectives and study overview

• Assess the feasibility of a tandem UHPLC configuration on a Vanquish system to eliminate idle reconditioning time.
• Compare chromatographic performance and throughput between single-column and tandem LC setups using peptide digests.
• Demonstrate application on an Infliximab SMART digest as a model biotherapeutic peptide mapping analysis.

Methodology

• The tandem LC configuration employs two parallel flow paths: one for analytical separation and one for off-line column wash and equilibration.
• Columns are switched via active 2-position 6-port valves, allowing continuous cycling without modifying the analytical gradient.
• Alternating runs on columns A and B reduce total cycle time by up to 60 % compared to sequential single-column operation.
• SMART Digest of Infliximab: 1 µg digest injected on two Acclaim Vanquish C18 columns (2.1 × 250 mm, 2.2 µm) with a 1–45 % acetonitrile gradient over 40 min at 60 °C, 400 µL/min.

Used instrumentation

• Vanquish Tandem UHPLC system with Binary H (or Binary F) analytical pump and Quaternary F reconditioning pump.
• Acclaim Vanquish C18 columns (2.1 × 250 mm, 2.2 µm).
• Q Exactive HF mass spectrometer (resolution 15 000, m/z 140–2000).
• Optional UV detector for monitoring wash and equilibration phases.

Main results and discussion

• Tandem LC setup delivered retention time RSDs below 0.11 % and area RSDs around 2.5 %, comparable to single-column performance.
• Overlay of total ion chromatograms showed consistent peak alignment between columns A and B, with negligible retention shifts.
• The alternating column strategy reduced non-productive reconditioning time, increasing sample throughput by up to 60 % without altering established methods.

Practical applications and benefits

• Enhanced laboratory efficiency for peptide mapping and multiple attribute monitoring in biopharma QC.
• Compatibility with existing LC-MS methods and software (Chromeleon 7.2, SII 1.3) facilitates adoption.
• Maintains chromatographic robustness and data quality while accelerating analytical cycles.

Future trends and applications

• Integration of multi-column parallelization for further throughput gains.
• Expansion to continuous manufacturing and real-time process monitoring.
• Coupling with high-resolution mass spectrometry and advanced data analytics for automated QC.
• Potential for broader adoption in pharmaceutical, clinical, and academic research laboratories.

Conclusion

The tandem UHPLC approach on the Vanquish platform provides a practical solution to improve throughput in LC-MS peptide mapping workflows without compromising chromatographic performance, offering significant advantages for biopharmaceutical analysis.

Reference

• Samonig M, Patzelt S, Paul C, Rühl M, Swart R. Tandem UHPLC operation for high-throughput LC-MS peptide mapping analyses. Thermo Fisher Scientific.