Thermo Scientific Vanquish Flex UHPLC System

Importance of the Topic

Biotherapeutic proteins such as monoclonal antibodies (mAbs), antibody–drug conjugates (ADCs) and fusion proteins are increasingly complex and demand rigorous analytical characterization to ensure safety, efficacy and regulatory compliance. Ultra-high performance liquid chromatography (UHPLC) platforms need to deliver high sensitivity, reproducibility and flexibility to handle workflows from aggregation screening and glycan profiling to intact mass analysis and peptide mapping.

Objectives and Study Overview

This article presents the design principles, performance evaluation and application workflows of the Thermo Scientific Vanquish Flex UHPLC System tailored for biopharma characterization. It reviews key attributes of the system architecture and column chemistries, and demonstrates its capability for:

• Size-exclusion chromatography (SEC) to quantify protein aggregates.
• Glycan analysis by both fluorescence labeling and charged aerosol detection.
• Ion-exchange chromatography (IEX) for charge variant profiling.
• Peptide mapping with high-resolution separations and MS integration.
• Intact protein and ADC analysis via hydrophobic interaction chromatography (HIC) and MS coupling.

Methodology and Instrumentation

The Vanquish Flex UHPLC features a fully biocompatible fluid path, sample pre-compression, ceramic valves and temperature-controlled column compartment (5–120 °C, ±0.05 °C). Columns include:

• MAbPac SEC-1 for high-resolution aggregate screening under non-denaturing conditions.
• GlycanPac AXR-1 and AXH-1 for mixed-mode glycan separations.
• MAbPac SCX-10 for pH-gradient charge variant analysis.
• Acclaim RSLC C18 and Accucore Vanquish for peptide mapping.
• MAbPac RP and HIC columns for intact protein and ADC Drug-Antibody Ratio (DAR) determination.

Integration with Thermo Scientific Dionex Chromeleon CDS and one-click AppsLab workflows streamlines data acquisition, compliance (21 CFR Part 11/GxP) and reporting.

Main Results and Discussion

Performance benchmarks demonstrated:

• Retention time stability with sample pre-compression yielding RSD < 0.1% in repeated injections.
• Low carryover and high sensitivity across UV, FLD and CAD detectors.
• Efficient baseline separation of trastuzumab monomer and aggregates in < 10 min.
• Fluorescence-labeled N-glycan profiling with precision and CAD-based label-free detection over four orders of magnitude dynamic range.
• Fast pH-gradient IEX separations of mAbs in under 2 min without loss of resolution.
• High-throughput peptide mapping achieving 100% sequence coverage in < 30 min gradients with HRAM MS identification.
• Intact ADC DAR measurement in < 4 min with reproducible peak shapes.

Benefits and Practical Applications

The platform’s modular design and tool-free Viper fittings ensure rapid maintenance, long column life and minimal downtime. Expanded plate capacity and optional robotic Charger module enable high throughput. Seamless coupling to mass spectrometers supports detailed structural elucidation of PTMs and glycoforms, serving R&D, QC and process development laboratories.

Future Trends and Opportunities

As biotherapeutics evolve toward multispecific antibodies and novel conjugates, demands will grow for even greater sensitivity, more complex gradients and advanced data analytics. Emerging applications include micro-flow UHPLC, ion mobility separations and AI-driven method optimization within compliant CDS environments. Increasingly, label-free detection and automated sample prep will accelerate workflows.

Conclusion

The Vanquish Flex UHPLC System delivers robust, high-performance separations with exceptional retention time precision, flexibility across detector modalities and seamless instrument integration. Its comprehensive column portfolio and software ecosystem address the full spectrum of biotherapeutic characterization needs, helping laboratories accelerate method transfer and maintain regulatory compliance.

Used Instrumentation

• Thermo Scientific Vanquish Flex UHPLC System
• Thermo Scientific Dionex Chromeleon Chromatography Data System
• Thermo Scientific Bio LC Columns: MAbPac, GlycanPac, ProPac, Accucore Vanquish
• Thermo Scientific SMART Digest™ sample preparation kits
• Thermo Scientific Orbitrap and Q Exactive mass spectrometers
• Charged Aerosol Detector (CAD), Fluorescence Detector (FLD), Diode Array Detector (DAD)