Thermo Scientific Biopharmaceutical Characterisation Compendium

Importance of the Topic

• Comprehensive characterization of biopharmaceuticals is essential to guarantee safety, efficacy, and regulatory compliance of monoclonal antibodies and related protein therapeutics.
• Key quality attributes such as aggregation, charge heterogeneity, primary sequence integrity, glycosylation profiles, and subunit composition directly impact drug performance and patient safety.
• Integration of advanced chromatographic workflows and high-resolution mass spectrometry enables deeper insight into complex biotherapeutics.

Objectives and Overview

• This compendium presents a toolbox of validated analytical techniques, workflows, and technologies for biopharmaceutical characterization delivered through collaboration between Thermo Fisher Scientific and NIBRT.
• Five major areas are addressed: aggregate analysis, charge variant characterization, peptide mapping, glycan profiling, and intact/subunit analysis.
• High-throughput and multi-attribute strategies demonstrate streamlined data acquisition, method transfer, and cloud-based instrument application deployment.

Methodology and Instrumentation

• Size-exclusion chromatography (SEC) on MAbPac SEC-1 columns with inert UHPLC systems to monitor monomer, dimer, and higher aggregates under high-salt mobile phases.
• Cation-exchange chromatography (CEX) using salt and pH gradients with MAbPac SCX-10 stationary phases and CX-1 pH buffers to resolve acidic and basic charge variants.
• Automated peptide mapping using SMART Digest immobilized proteases (trypsin, chymotrypsin), KingFisher Duo robotics, and Vanquish UHPLC-MS to confirm sequence and track post-translational modifications.
• Glycan analysis by enzymatic N-glycosidase release, fluorescent labeling (2-AA, 2-AB, 2-AMAC), and separation on HILIC (Accucore Amide-150), AXH-1, or AXR-1 hybrid columns with FLD and Orbitrap MS confirmation.
• Middle-up and top-down subunit workflows with IdeS and EndoS digestion, followed by HRAM intact mass analysis to quantify Fc core afucosylation and compare originator vs. biosimilar products.
• Dual-channel UHPLC for simultaneous orthogonal analyses, enhancing throughput and method flexibility.

Key Results and Discussion

• SEC methods on narrow-bore UHPLC columns achieved high resolution of mAb aggregates with minimal on-column interaction and excellent column stability over hundreds of injections.
• pH-gradient CEX delivered improved peak capacity and easier method optimization compared to traditional salt gradients, with reproducible batch-to-batch performance.
• Automated SMART Digest protocols reduced manual handling, minimized digestion-induced modifications, and delivered 100% sequence coverage across multiple analysts and days.
• Ultra-fast HILIC glycan methods compressed run times to 2.5 minutes for batch-to-batch glycoprofiling, preserving glycan patterns under different cell culture conditions.
• Hybrid AXH-1 and AXR-1 columns extended separation power for charged and isomeric glycans, with MS-driven identification at <3 ppm mass error.
• IdeS/EndoS subunit analysis precisely quantified Fc afucosylation and detected minor sequence variants, facilitating biosimilar comparability studies.

Benefits and Practical Applications

• Validated, end-to-end workflows reduce development timelines and simplify technology transfer into quality control labs.
• Automation and cloud-deployed eWorkflows ensure reproducibility, minimize human error, and support large-scale screening.
• Multi-attribute platforms enable concurrent analysis of aggregates, variants, peptides, glycans, and intact proteins on the same instrument series.
• High-throughput configurations and dual-channel systems support rapid process development, clone selection, and batch comparability for both innovator and biosimilar molecules.

Future Trends and Applications

• Integration of artificial intelligence and advanced data analytics to automate peak assignment and trend monitoring.
• Miniaturized and microflow LC-MS modalities to further reduce sample and solvent consumption.
• Expansion of multiplexed subunit and glycoform screening with high-speed UHPLC and multi-detector configurations.
• Broader adoption of cloud-based instrument apps to streamline method updates and global standardization.

Conclusion

• The collaborative toolbox from Thermo Fisher Scientific and NIBRT provides a comprehensive suite of robust, high-resolution analytical methods for biopharmaceutical characterization.
• These workflows deliver high data quality, reproducibility, and throughput, supporting critical decision-making from early process development through final product release.

Použitá instrumentace

• Thermo Scientific Vanquish Flex and Vanquish Horizon UHPLC systems
• Thermo Scientific Q Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer
• MAbPac SEC-1, MAbPac SCX-10, GlycanPac AXH-1, AXR-1, Accucore Amide-150 HILIC, and Acclaim VANQUISH C18 columns
• SMART Digest enzyme kits and KingFisher Duo/Prime purification systems
• Thermo Scientific Chromeleon CDS and BioPharma Finder software