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

Importance of the topic

2D-LC-MS techniques improve the separation and identification of protein-based impurities in therapeutic biologics by coupling orthogonal chromatographic methods with high-resolution mass spectrometry. This capability is critical for ensuring product safety, efficacy, and regulatory compliance in biopharmaceutical development.

Objective and study overview

The study presents a flexible multiple loop heart-cut 2D-LC-MS workflow implemented in a biopharmaceutical R&D laboratory. The primary goal was to confirm the identity of impurities in a ~25 kDa therapeutic protein using top-down MS, by combining reversed-phase separation in the first dimension with size-exclusion chromatography in the second to remove TFA and preserve MS sensitivity.

Methodology and Instrumentation

• Chromatographic setup:
  • 1D: RP-LC on a Waters Acquity UPLC Peptide BEH C18 column (2.1×50 mm, 1.7 µm) using 0.05 % TFA in water (A) and 0.04 % TFA in acetonitrile (B), gradient 50–90 % B at 0.6 mL/min.
  • 2D: Isocratic SEC on a Tosoh UP-SW2000 column (4.6×150 mm, 2 µm) with 0.1 % formic acid in water/acetonitrile at 0.35 mL/min to remove TFA prior to MS.
• Instrument platform:
  • Thermo Scientific Vanquish Horizon Simple Switch 2D-LC system with multiple loop heart-cutting and dual split sampler for independent 1D or 2D injections.
  • Orbitrap Fusion Tribrid mass spectrometer for top-down MS/MS using HCD, CID, and ETD fragmentation with a 600 m/z isolation window and 60 000 resolution.
  • Control and data processing via Chromeleon CDS, Xcalibur, and BioPharma Finder software.

Main results and discussion

The 1D RP separation reproducibly resolved the main protein and two impurity peaks across eight injections. Each impurity was heart-cut (48 µL) into MP35N loops, transferred to SEC in the 2D dimension, and analyzed by MS while diverting residual TFA to waste. Top-down fragmentation achieved ~55 % and ~45 % sequence coverage for the two impurities, confirming modification sites previously identified in bottom-up peptide mapping.

Benefits and practical applications

• Single-instrument flexibility enables seamless switching between multi heart-cut 2D-LC-MS, 1D-LC-MS, and dual 1D-LC-UV modes without hardware changes, improving lab efficiency and ROI.
• SEC-based TFA removal enhances MS signal intensity and accuracy while maintaining first-dimension resolution.
• Supports both top-down and bottom-up workflows on the same platform for comprehensive protein characterization.

Future trends and opportunities

Advancements are expected in automated method development, expanded loop capacity for higher throughput, integration of additional orthogonal separation modes (e.g., ion exchange), and intelligent software-driven decision-making. Dual-channel LC platforms with independent control will further streamline complex biologics analysis in quality control and research.

Conclusion

The multiple loop heart-cut 2D-LC-MS approach provides a robust, adaptable solution for in-depth impurity profiling of therapeutic proteins. By leveraging Simple Switch technology and high-resolution MS, laboratories can achieve high-quality data with minimal downtime and hardware reconfiguration.

Reference

1. Pirok BWJ; Stoll DR; Schoenmakers PJ. Recent developments in two-dimensional liquid chromatography: Fundamental improvements for practical applications. Anal Chem 2019, 91(1):240–263.
2. Stoll DR; Maloney TD. Recent Advances in Two-Dimensional Liquid Chromatography for Pharmaceutical and Biopharmaceutical Analysis. LCGC North America 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 a convenient 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.