Nano and Microscale Peptide Mapping by LC-UV-Orbitrap-Astral Detection of Monoclonal Antibodies with Fraction Collection for Detailed Analysis

Significance of the Topic

The detailed mapping of peptides derived from monoclonal antibodies at nano- and microscale levels is critical for ensuring product quality, verifying primary sequence integrity, and monitoring post-translational modifications in biopharmaceutical development and QA/QC laboratories.

Objectives and Study Overview

This study evaluates a combined LC-UV-Orbitrap-Astral workflow with integrated fraction collection to perform high-sensitivity peptide mapping of tryptic digests from trastuzumab.
Key goals include comparing nanoflow and microflow chromatographic formats, establishing detection limits by UV and MS, assessing data-dependent (DDA) versus data-independent acquisition (DIA) on the Astral detector, and demonstrating fraction collection for offline confirmatory analysis.

Methodology

• Sample Preparation: Trastuzumab was digested using a two-step SMART Digest Trypsin protocol with heat-stable beads and TCEP reduction.
• Chromatographic Conditions:
– Nanoflow: Direct injection onto a PepMap Neo column (75 µm × 15 cm) at 350 nL/min.
– Microflow: μPAC Neo column (5.5 cm, 1.25 µL/min) with variable wavelength UV detection.
• Data Acquisition: DDA and DIA methods were implemented on an Orbitrap Astral mass spectrometer, collecting full-scan MS and MS/MS spectra.
• Fraction Collection: UV-triggered peak collection into microvials (15–22 µL per fraction), followed by weight-based accuracy assessment (RSD 5.9%).

Instrumentation

• Thermo Scientific Vanquish Neo UHPLC system with integrated variable-wavelength UV detector and fraction collector
• Thermo Scientific Orbitrap Astral mass spectrometer with nano-ESI source and EASY-Spray emitter
• Columns: Thermo Scientific PepMap C18 (1 mm × 15 cm), Acclaim PepMap 100 (75 µm × 15 cm), μPAC Neo HPLC (5.5 cm)
• Thermo Scientific Biopharma Finder 5.2, Chromeleon 7.3.1, and TraceFinder 5.1 software for data processing and quantification

Main Results and Discussion

• Detection Limits: Microflow required ~70 pmol injection for full sequence coverage by UV, whereas nanoscale chromatography achieved mapping at 80 fmol (12 ng).
• Separation Performance: Radially elongated pillar architecture enhanced peak capacity within a compact column footprint.
• Acquisition Strategies: Both DDA and DIA on the Astral provided comprehensive peptide identification; DIA exhibited consistent fragment ion ratios across fractions.
• Fraction Collection: Recovered fractions yielded reproducible weights (RSD 5.9%) and enabled high-confidence offline MS/MS analysis.

Benefits and Practical Applications

• High Sensitivity: Enables detailed mapping of antibody digests using low-nanogram sample amounts, ideal for early-stage or orphan drug candidates.
• Workflow Flexibility: Seamless switching between UV detection, fraction collection, and high-resolution MS enhances QA/QC capabilities.
• Data Quality: Coupling UV peak detection with Astral MS ensures accurate monitoring of critical quality attributes and PTMs.

Future Trends and Potential Applications

• Further miniaturization and integration of LC-MS platforms for single-cell proteomics and ultra-low-volume biotherapeutic analysis.
• Advanced data-acquisition algorithms and AI-driven peak picking to improve throughput and reproducibility.
• Emerging column technologies (e.g., open-tubular or 3D-printed microcolumns) to boost separation efficiency in constrained formats.
• Real-time monitoring and feedback control of fraction collection for adaptive workflows in process analytics.

Conclusion

The integrated nano- and microflow LC-UV-Orbitrap-Astral platform with fraction collection offers a robust, sensitive, and versatile approach for monoclonal antibody peptide mapping in QA/QC environments. Both DDA and DIA methods on the Astral detector reliably deliver comprehensive sequence coverage from low-ng sample loads, supporting critical quality assessment and method development needs.

References

1. Analytical Chemistry 2022, 94, 17195–17204.
2. Analytical Chemistry 2015, 87, 7382–7388.