Thermo Bio LC Columns - See your protein therapeutics in high resolution

Significance of the Topic

The complexity of monoclonal antibodies and their conjugates lies in site-specific modifications such as glycosylation, oxidation, deamidation and charge variants. Robust analytical methods are essential to ensure safety, efficacy and lot-to-lot consistency for biotherapeutics during development and production.

Objectives and Study Overview

This whitepaper presents a comprehensive, single-vendor workflow for high-resolution characterization of protein therapeutics. Key goals include reliable titer determination, aggregate quantitation, charge variant profiling, oxidation monitoring, antibody–drug conjugate (ADC) drug-to-antibody ratio analysis, intact/subunit mass confirmation, peptide mapping and glycan profiling.

Methodology and Instrumentation

Multiple chromatographic modes and mass spectrometry tools are integrated into streamlined assays:

• Affinity titer via protein A chromatography for rapid IgG quantification.
• Size-exclusion chromatography (SEC) with hydrophilic-coated media to separate monomer, dimer and higher aggregates.
• Cation and anion exchange chromatography using salt and pH gradients to resolve charge variants with high resolution.
• Hydrophobic interaction chromatography (HIC) to monitor oxidative modifications and ADC drug loading.
• Reversed-phase liquid chromatography (RPC) for intact mass and subunit (LC, HC, Fab, Fc) separations coupled to high-resolution Orbitrap MS.
• Enzymatic peptide mapping with immobilized trypsin for rapid, reproducible sequence coverage.
• HILIC and mixed-mode WAX/RP columns for detailed N-glycan profiling of neutral and charged species.

Key instrumentation includes the Thermo Scientific Vanquish UHPLC systems, UltiMate 3000 inline pH/conductivity monitor, Q Exactive Plus hybrid quadrupole-Orbitrap mass spectrometer and dedicated MAbPac, ProPac and GlycanPac column families.

Main Results and Discussion

Affinity chromatography displayed linear response (R2 > 0.999) for IgG titer across culture extracts. SEC columns achieved baseline separation of monomer and aggregate species in under 20 minutes, with formats ranging from 7.8 to 2.1 mm ID to suit sample throughput or MS compatibility. Charge variant assays on SCX columns demonstrated clear resolution of acidic, main, and basic C-terminal lysine variants using both salt and pH gradients, including ultra-fast (sub-5 min) methods on RS columns. HIC methods resolved oxidized mAb variants and ADC species by drug-to-antibody ratio (DAR 0–8). RPC-MS confirmed intact and subunit masses, identified glycoforms and localized modifications. SMART Digest workflows provided complete peptide maps in less than one hour with excellent retention time precision (RT RSD < 0.01%). GlycanPac and Accucore HILIC columns separated 35–59 2AB-labeled N-glycans with high peak capacity.

Benefits and Practical Applications

These workflows deliver:

• Single-vendor consistency and support.
• High throughput with low backpressure columns and fast gradients.
• Platform methods reducing development time across multiple mAbs.
• Seamless LC-UV and LC-MS integration for orthogonal confirmation.
• Robust, reproducible assays suitable for QC, stability, comparability and process monitoring.

Future Trends and Potential Applications

Emerging directions include real-time buffer pH/conductivity monitoring to predict elution, automated multi-attribute methods combining charge, glycan and peptide analysis in single runs, expanded ADC analytics for site-specific conjugates, and miniaturized UHPLC-MS integration for bioprocess PAT (process analytical technology).

Conclusion

The unified Thermo Scientific biopharma workflow leverages advanced column chemistries and high-resolution instrumentation to comprehensively characterize protein therapeutics. By combining affinity, SEC, IEX, HIC, RPC, peptide mapping and glycan analysis into standardized methods, laboratories can achieve rapid, high-fidelity results that support accelerated development and reliable quality control.

Reference

• Thermo Scientific application notes on mAb titer, aggregation, charge variant, oxidation and ADC analysis.
• Column and instrumentation technical datasheets from Thermo Fisher Scientific.
• Published workflows for immobilized digestion and multi-attribute MS assays.