Analytical solutions for biopharmaceutical characterization and control

Importance of the topic

Biotherapeutic drugs—such as monoclonal antibodies, antibody–drug conjugates, biosimilars and nucleotide-based therapies—present a level of structural complexity and heterogeneity that far exceeds traditional small-molecule pharmaceuticals. Subtle changes in glycosylation, charge variants, aggregation state or post-translational modifications (PTMs) can profoundly affect safety, efficacy and manufacturability. Advanced, stage-appropriate analytical solutions are essential to fully characterize these molecules, monitor critical quality attributes (CQAs) and accelerate development timelines while ensuring regulatory compliance.

Objectives and overview of the study/article

This whitepaper surveys a comprehensive portfolio of analytical technologies and workflows designed to support every stage of biopharmaceutical development. From high-throughput discovery screening through detailed characterization, process optimization and final quality control, the objective is to demonstrate how fit-for-purpose tools can deliver deeper structural insight, robust monitoring of microheterogeneity and faster product release decisions.

Methodology and instrumentation

A diverse set of orthogonal technologies underpins the presented workflows:

• Mass spectrometry platforms: Orbitrap Eclipse Tribrid (with HMRn and PTCR), Exploris 480/240 with BioPharma options, Q Exactive Plus and UHMR for high-mass native analysis.
• Chromatography systems: Vanquish Horizon, Flex and Duo UHPLC systems; capillary electrophoresis–MS with the ZipChip interface.
• Separation chemistries: MAbPac SEC-1 (aggregate analysis), HIC, RP and SCX columns, DNAPac and DNASwift for oligonucleotides, Accucore amide-HILIC for glycan profiling.
• Ion mobility: FAIMS Pro for enhanced gas-phase selectivity in top-down workflows.
• Software: BioPharma Finder for intact mass, peptide mapping and multi-attribute workflows; Chromeleon CDS for compliance, MAM and New Peak Detection; Proteome Discoverer and HDExaminer for HDX analysis.
• Sample preparation: SMART Digest kits (peptide and oligonucleotide digestion), KingFisher Duo Prime for automated proteolysis, HDX automation with H/D-X PAL autosampler.
• Specialized detection: charged aerosol and pulsed amperometric detection (HPAE-PAD) for sugars and excipients; CAD for polysorbates and non-chromophoric compounds.

Main results and discussion

• Discovery & early screening: Tribrid MS with high-resolution Orbitrap and FAIMS enabled ultra-low-level PTM detection, site-specific CQA mapping and rapid intact mass confirmation.
• Peptide mapping: Vanquish Duo tandem UHPLC doubled throughput, achieving ≤3% RSD across digests with SMART Digest workflows; BioPharma Finder’s deconvolution and annotation ensured correct mass assignments and sequence coverage.
• Intact mass & native MS: Exploris and Q Exactive UHMR systems extended m/z up to 80,000, resolving antibody aggregates (~600 kDa) and ligand-bound complexes, while PTCR simplified spectra for accurate weight determination.
• Charge variant analysis: pH-gradient SCX (CX-1 buffers) provided universal method transfer for mAb isoform separation; coupling to native MS (CVA-MS) delivered simultaneous mass and charge profiling of deamidation and glycoforms.
• Aggregate monitoring: SEC-MS workflows on MAbPac SEC-1 and HIC columns separated monomers, fragments and high-molecular-weight species with <5 min per sample and consistent glycoform distributions at sub-µg levels.
• Glycan profiling: Rapid HILIC-FLD on Accucore 150-amide columns resolved isomeric N-glycans in <2 min; HPAE-PAD on CarboPac columns enabled label-free monosaccharide and sialic acid quantitation.
• Oligonucleotide mapping: DNAPac RP with Vanquish UHPLC and Exploris MS delivered confident sequence confirmation and impurity quantitation at <0.5 ppm levels.
• ADC characterization: Native MS DAR determination and HIC/RP-LC separations resolved drug-load distributions with high mass accuracy (<5 ppm) directly on intact molecules.
• HDX-MS: Integrated platform provided automated labeling, quench, digestion and MS analysis for higher-order structure comparison (originator vs. biosimilar), revealing regional stability differences in minutes.
• High-Resolution Multi-Attribute Method (HR MAM): A standardized UHPLC-HRAM-MS workflow with system suitability test enabled simultaneous monitoring of multiple CQAs, impurity detection and New Peak Detection in a compliance-ready Chromeleon environment.

Benefits and practical applications of the method

• Accelerated decision-making in candidate selection, process optimization and batch release.
• Reduced method development time through universal pH- and ion-exchange gradients, one-click workflows and automation.
• Deeper product understanding by combining orthogonal separation, native MS and top-down fragmentation for intact protein analysis.
• Robust QC compliance via integrated MS and chromatography data systems, audit-ready reports and validated New Peak Detection.
• Scalable throughput from discovery to manufacturing control, maximizing utilization of high-value HRAM MS instrumentation.

Future trends and potential applications

Emerging directions include real-time in-process monitoring with on-line MS, AI-driven data analysis for accelerated CQA prediction, expansion of single-cell and single-virus native MS, and integration of orthogonal multi-omics (proteomics, glycomics, lipidomics) for holistic biotherapeutic characterization and personalized medicine applications.

Conclusion

A coordinated suite of advanced chromatography, mass spectrometry, sample preparation and data management technologies is essential to meet the evolving challenges of biopharmaceutical development. By deploying fit-for-purpose tools at each stage—discovery, characterization, process development and QC—scientists can achieve comprehensive product insights, ensure regulatory compliance and bring life-changing therapies to patients faster and more cost-effectively.

Used instrumentation

• Thermo Scientific Orbitrap Eclipse Tribrid with HMRn and PTCR
• Orbitrap Exploris 480 & 240 MS with BioPharma options
• Thermo Scientific Q Exactive Plus & UHMR Hybrid-Quadrupole-Orbitrap MS
• Vanquish Horizon, Flex and Duo UHPLC systems
• Columns: MAbPac SEC-1, HIC-10, RP-C18, SCX-10, ProPac Elite WCX, Accucore 150-Amide-HILIC, DNAPac RP/PA200, GlycanAssure APTS
• FAIMS Pro high-field ion mobility interface
• 908 Devices ZipChip capillary electrophoresis interface
• KingFisher Duo Prime automation
• SMART™ Digest and PepMap™ RSLC sample preparation kits
• Thermo Scientific BioPharma Finder, Chromeleon CDS and Proteome Discoverer software
• H/D-X PAL autosampler and HDExaminer for HDX workflows
• CarboPac PA20/PA200 Ion Chromatography and Charged Aerosol Detector