Accelerating monoclonal antibody peptide mapping with high acquisition speed Orbitrap-based MS

Importance of the Topic

Monoclonal antibodies represent a dominant class of biotherapeutics with significant therapeutic impact and commercial importance. Fast and reliable peptide mapping is critical for confirming amino acid sequence, identifying post-translational modifications (PTMs) and ensuring consistent product quality throughout development and manufacturing. High-throughput methods that maintain comprehensive sequence coverage and PTM characterization enable rapid process optimization and support quality control.

Objectives and Overview

This study demonstrates the capabilities of the Thermo Scientific™ Orbitrap Exploris™ 480 mass spectrometer coupled with a Vanquish™ UHPLC system for rapid, high-resolution peptide mapping of the therapeutic antibody golimumab. By employing shortened chromatographic gradients (5–40 minutes) and automated sample preparation, the work aims to maintain 100% sequence coverage, detect and quantify key PTMs, and illustrate the method’s applicability to process development and high-throughput screening.

Methodology

Golimumab samples were subjected to forced oxidation (500 ppm H2O2, 24 h) or analyzed in their native state. Automated digestion was performed using SMART Digest™ magnetic resin and a Thermo Scientific™ KingFisher™ Duo Prime system controlled by BindIt™ software. Peptides were separated on an Acclaim™ VANQUISH™ C18 column (2.1 × 250 mm) using the Vanquish Horizon UHPLC platform under gradients from 5 to 40 min (2–40% acetonitrile in 0.1% formic acid). Data were acquired on the Orbitrap Exploris 480 (MS1 resolution 60 000, data-dependent MS2 Top15 at 15 000) and processed using Chromeleon™ 7.2 CDS and BioPharma Finder™ 3.2 software.

Instrumentation Used

• Vanquish Horizon UHPLC system with Binary Pump H, Sampler HT and Column Compartment H
• Acclaim VANQUISH C18, 2.2 µm, 2.1 × 250 mm column
• Orbitrap Exploris 480 mass spectrometer with BioPharma option
• SMART Digest magnetic trypsin kit and KingFisher™ 96 Deepwell plate
• Chromeleon™ 7.2.10 CDS and BioPharma Finder™ 3.2 software

Main Results and Discussion

Peptide mapping across all gradients achieved 100% sequence coverage for both heavy and light chains, even with a 5-minute separation. Key PTMs were consistently identified and quantified:

• Oxidation at methionine sites (M261 ~100%, M437 ~50%) and lower-abundance sites (M51, M113)
• Deamidation and succinimide formation at N43 (~60% Asn→Asp, ~10% Asu) and low-abundance deamidation at N370
• N-terminal pyroglutamate conversion of HC Q1 (~100%) and absent at LC E1
• C-terminal lysine clipping (~15–20%) evidenced by distinct charge states and retention times
• N-linked glycosylation profile at N306 featuring G0F (~38–43%), G1F (~34–39%), G2F (~9–11%) and minor high-mannose species

High-accuracy mass measurements (<1.5 ppm) and high-quality MS2 spectra enabled unambiguous localization of modifications, even for small mass shifts (e.g., +0.984 Da for deamidation).

Benefits and Practical Applications of the Method

• Shortened analysis times increase throughput for process support and clone or media screening
• Automated digestion and data acquisition ensure reproducibility and reduce hands-on time
• High-resolution, fast scan performance preserves data quality in rapid UHPLC separations
• Comprehensive PTM profiling supports critical quality attribute monitoring during development

Future Trends and Potential Applications

Further enhancements may include:

• Integration of multiplexed or multiplex-augmentation strategies to analyze several samples simultaneously
• Advanced AI-driven data analysis for accelerated PTM discovery and trend prediction
• Expanded workflows for deeper glycan characterization using complementary hydrophilic separations
• Real-time process analytical technology (PAT) applications for in-line monitoring during bioreactor runs

Conclusion

The combination of the Orbitrap Exploris 480 mass spectrometer and Vanquish UHPLC system delivers rapid, high-throughput peptide mapping without compromising sequence coverage or PTM characterization. This robust workflow, supported by automated digestion and compliance-ready software, is well suited for biopharmaceutical process development, quality control, and biosimilar comparability studies.

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