BioLC columns and accessories

Importance of the topic

Comprehensive profiling of therapeutic proteins is essential for ensuring product quality, safety, and efficacy. Advanced chromatographic techniques allow detailed analysis of intact proteins, subunits, aggregates, charge variants, glycoforms, and peptides.

Objectives and study overview

This application note introduces Thermo Fisher Scientific’s BioLC column portfolio designed to address the full spectrum of biopharmaceutical characterization tasks. The text surveys column chemistries and formats optimized for affinity capture, hydrophobic interaction, size exclusion, reversed-phase, ion-exchange, HILIC, and mixed-mode separations.

Methodology and instrumentation

Innovative stationary phases were evaluated on standard LC-UV and LC-MS platforms. Column families include:

• Affinity: MAbPac Protein A for rapid antibody titer determination.
• HIC: MAbPac HIC-10, HIC-20, and HIC-Butyl for intact proteins, aggregates, fragments, and ADC variants.
• HILIC and mixed-mode: Accucore 150 Amide-HILIC and GlycanPac AXH-1/AXR-1 for released glycan profiling by fluorescence.
• Size-exclusion: SurePac Bio 550 SEC MDi and MAbPac SEC-1 for aggregate and AAV analysis.
• Reversed-phase: MAbPac RP, ProSwift RP, Hypersil GOLD Peptide for intact/subunit and peptide mapping.
• Ion-exchange: ProPac 3R SCX/SAX, MAbPac SCX-10/RS, ProPac WCX/XE, ProPac Elite WCX for charge variant separation.
• Nucleic acids: DNAPac RP, PA200/PA200RS, DNASwift SAX for oligonucleotide purity and length variants.

Instrumentation used

Typical setups employ UHPLC systems coupled to UV detectors (214, 260, 280 nm) and fluorescence or mass spectrometers. Guard cartridges and high-pressure inert hardware ensure minimal sample interaction and robust performance.

Main results and discussion

BioLC columns demonstrated high resolution and reproducibility across multiple workflows:

• Affinity capture yielded sharp elution profiles for mAb titer analysis.
• HIC phases resolved monomer, aggregates, oxidation variants, and DAR species with clear peak separation.
• HILIC and mixed-mode columns resolved over 80 glycan species, differentiating neutral, sialylated, and high-mannose structures.
• Size-exclusion columns separated AAV monomer and aggregates within a 10-minute window and maintained stability over repeated injections.
• Reversed-phase phases enabled intact mass profiling, enzymatic subunit analysis, and reproducible peptide maps suitable for MAM workflows.
• Ion-exchange columns achieved baseline separation of acidic and basic charge variants, including C-terminal lysine truncations and pI shifts, with minimal method development time using pH or salt gradients.
• Oligonucleotide columns provided n-1 resolution and efficient separation of mixed-base sequences and failure products under rapid gradients.

Benefits and practical applications

The BioLC column family streamlines biopharma analytics by offering:

• Robust, scalable methods across QC and R&D.
• High throughput and minimal carryover.
• Broad solvent compatibility for LC-UV and LC-MS.
• Reproducible batch-to-batch performance due to monodisperse and solid-core particles.
• Preformatted pH gradient buffers accelerating charge variant method setup.

Future trends and opportunities

Emerging demands in biotherapeutic design—bispecifics, gene therapies, and ADCs—will drive further refinement of column chemistries for enhanced selectivity. Integration with automated sample prep, multi-attribute detection via tandem MS, and AI-driven method optimization are expected to expand throughput and data depth.

Conclusion

Thermo Fisher Scientific’s BioLC columns provide a unified solution for comprehensive protein and oligonucleotide analysis. Their diverse chemistries and formats deliver high resolution, reproducibility, and ease of use, supporting critical QC workflows and advanced biotherapeutic research.