Complementary analysis in a biopharmaceutical research laboratory with a 2D-LC-MS instrument for flexible operation

Significance of the Topic

Two-dimensional liquid chromatography coupled with high-resolution mass spectrometry has become essential for resolving complex biopharmaceutical samples and accurately characterizing low-level protein impurities. By integrating orthogonal separation mechanisms with precise fraction transfer, 2D-LC-MS enhances analytical resolution and confidence in therapeutic protein quality control.

Goals and Overview of the Study

This application demonstrates a multi heart-cut 2D-LC-MS workflow aimed at confirming the identity of protein-based impurities in a 25 kDa therapeutic protein. The approach combines top-down mass spectrometry for intact protein analysis with comparison to prior bottom-up peptide mapping to validate impurity assignments.

Methodology

The experimental design featured heart-cutting between reversed-phase and size-exclusion chromatography:

• First dimension: RP-LC on a C18 column using trifluoroacetic acid to separate the main protein and two unknown impurities.
• Fraction collection: up to five 48 µL cuts captured in MP35N loops via a 2-position/6-port valve and dual 6-port valves.
• Second dimension: SEC with formic acid modifier to remove TFA and isolate protein peaks.
• Top-down MS: targeted MS/MS using higher-energy collisional dissociation, collision-induced dissociation, and electron transfer dissociation over a 600 m/z window.
• Data control: Chromeleon CDS for LC-UV control, Xcalibur for MS acquisition, and BioPharma Finder for sequence analysis.

Instrumentation

An integrated Thermo Scientific Vanquish platform was employed:

• Vanquish Horizon Simple Switch 2D-LC system with dual split sampler and independent flow paths.
• Vanquish Flex system base with binary pumps and column compartments for both dimensions.
• 2-position/6-port and 6-position/7-port bio-compatible valves equipped with 50 µL and 250 µL loops.
• Diode array detectors for UV monitoring and an Orbitrap Fusion Tribrid mass spectrometer for top-down analysis.

Results and Discussion

The RP–SEC 2D workflow delivered highly reproducible separations across eight consecutive injections, with consistent retention and peak shapes. Heart-cut fractions produced single, well-defined protein peaks in the SEC traces, effectively diverting late-eluting TFA to waste. Top-down fragmentation of two impurity fractions achieved sequence coverages of 55 % and 45 %, confirming modification sites and aligning with bottom-up peptide mapping results.

Benefits and Practical Applications

This flexible system offers clear advantages:

• Seamless switching between multi heart-cut 2D-LC, single-dimension LC-MS, and dual 1D-LC-UV modes without hardware reconfiguration.
• Efficient removal of TFA via SEC to maintain MS sensitivity and reduce signal suppression.
• Optimized laboratory footprint and maximized mass spectrometer utilization.

Future Trends and Applications

Wider adoption of 2D-LC in routine biopharmaceutical workflows is expected as instruments become more accessible. Future developments may include faster fraction interfaces, alternative orthogonal chemistries, increased automation, and integration with advanced data analytics to further improve impurity profiling and regulatory compliance.

Conclusion

The multi heart-cut 2D-LC-MS strategy provides a robust, high-resolution platform for top-down characterization of therapeutic protein impurities. Simple Switch technology enables rapid transitions between operational modes, ensuring reliable MS detection and efficient instrument use without replumbing.

References

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2. Stoll DR, Maloney TD. Recent advances in two-dimensional liquid chromatography for pharmaceutical and biopharmaceutical analysis. LCGC North Am. 2017;35(9):680–687.
3. Grübner M, Greco G. Flexible HPLC instrument setups for double usage as one heart-cut 2D-LC system or two independent 1D-LC systems. Thermo Fisher Scientific Technical Note 73298;2019.
4. Grübner M, Steiner F. Complementary dual LC as an alternative to multiple heart-cut 2D-LC for samples of medium complexity. Thermo Fisher Scientific Technical Note 73184;2019.
5. McGillicuddy N, et al. Simultaneous analysis of monoclonal antibodies using a novel dual-channel UHPLC instrument and orthogonal chromatography. Thermo Fisher Scientific Application Note 72599.