SEC Method Transfer from Diol Chemistry to Polymer-Coated Silica

Význam tématu

Size exclusion chromatography is a cornerstone technique for characterizing protein and antibody aggregates in biopharmaceutical development. Reliable assessment of aggregate content ensures drug safety and efficacy, supports regulatory compliance, and helps maintain product quality during manufacture and storage.

Cíle a přehled studie / článku

This application note evaluates the transfer of established SEC methods from diol-bonded columns to modern polymer-coated silica columns. It compares the performance of four column chemistries using protein and peptide standards to identify optimal conditions for aggregate analysis and method robustness.

Použitá metodika a instrumentace

Chromatographic system and components

• Agilent 1260 Infinity bio-inert quaternary pump and autosampler
• Agilent thermostatted fraction collector and column compartment
• Agilent 1260 Infinity diode array detector at 220 nm

Columns and standards

• AdvanceBio SEC 300 Å and 130 Å polymer-coated silica (2.7 µm)
• ZORBAX GF-250 (4 µm) and GF-450 (6 µm) diol-bonded silica
• Protein standards: thyroglobulin, IgG dimer/monomer, ovalbumin, myoglobin, angiotensin II
• Insulin from bovine pancreas (5.7 kDa) for method transfer

Mobile phase optimization

• Phosphate buffer concentrations from 50 to 600 mM at pH 7.0
• Salt concentration varied from 50 to 500 mM NaCl
• Flow rate adjusted for equal linear velocity (1.0 mL/min for AdvanceBio, 1.2 mL/min for ZORBAX)
• Column temperature 25 °C, chiller at 4 °C

Hlavní výsledky a diskuse

AdvanceBio SEC 300 Å delivered baseline separation of large protein standards with highest resolution between IgG monomer and dimer and low tailing for angiotensin II. The 130 Å column excelled in resolving smaller proteins and peptides, achieving baseline separation of all five standard peaks and optimal insulin peak shape. Diol-bonded ZORBAX columns required higher buffer concentrations, produced broader peaks, and showed poorer resolution and tailing, particularly for small peptides. The smaller 2.7 µm particles in AdvanceBio columns provided improved efficiency despite higher backpressure within instrument limits.

Přínosy a praktické využití metody

Using polymer-coated silica SEC columns reduces nonspecific interactions, enhances peak resolution across a broad molecular weight range, and shortens analysis time. Lower mobile phase consumption and earlier elution translate into cost savings, increased throughput, and simplified method transfer for aggregate quantitation in pharmaceutical QC and R&D.

Budoucí trendy a možnosti využití

Future developments may include high-throughput SEC workflows, integration with mass spectrometry for detailed aggregate profiling, and tailored stationary phases for novel biologics. Continued refinement of buffer chemistries and column technologies will support more robust analysis of increasingly complex therapeutic modalities.

Závěr

Polymer-coated silica SEC columns outperform traditional diol-bonded columns for aggregate and peptide analysis, offering superior resolution, faster separations, and reduced nonspecific binding. Method transfer to AdvanceBio SEC 130 Å and 300 Å enhances biopharmaceutical workflow efficiency and data reliability.

Reference

• Hong P, Koza S, Bouvier ESP. Size-Exclusion Chromatography for the Analysis of Protein Biotherapeutics and their Aggregates. J Liq Chrom Rel Technol. 2012;35(20):2923–2950.
• Lagassé HA et al. Recent advances in therapeutic protein drug development. F1000Res. 2017; Feb 7.
• Lau JL, Dunn MK. Therapeutic peptides: Historical perspectives, current development trends, and future directions. Bioorg Med Chem. 2018;26(10):2700–2707.
• Aggregation of Monoclonal Antibody Products: Formation and Removal. BioPharm Int. 2013;26(3).
• ADC Targets Fail Because of Aggregation Problems. PharmTech. 2017; Nov 14.