Agilent AdvanceBio SEC for Peptides, Proteins & ADC Analysis

Význam tématu

Size exclusion chromatography (SEC) is a cornerstone technique for characterizing the size distribution of peptides, proteins and antibody–drug conjugates (ADCs). Minimizing nonspecific secondary interactions between analytes and the stationary phase is essential to preserve sample integrity, improve resolution and ensure accurate quantitation in biopharmaceutical quality control.

Cíle a přehled studie

The primary objective was to evaluate Agilent AdvanceBio SEC columns—with a novel hydrophilic coating—in comparison to a competitive BEH-based SEC column. Two model systems were tested: human growth hormone (hGH) for aggregate and fragment profiling, and a monoclonal antibody ADC for monomer/aggregate quantitation.

Použitá metodika a instrumentace

• Instrument: Agilent 1260 Infinity II Bio-inert LC system
• Software: Agilent OpenLab CDS
• Columns: AdvanceBio SEC (120 Å and 200 Å pore sizes, 1.9 µm particle size)
• Mobile phase for hGH: 150 mM phosphate buffer, pH 7.0; for ADC: 50 mM sodium phosphate with 200 mM NaCl, pH 7.0
• Flow rate: 0.35 mL/min; column temperature: 25 °C; injection volume: 2 µL
• Detection: UV at 220 nm

Hlavní výsledky a diskuse

• Human growth hormone analysis yielded superior monomer recovery and baseline separation of low- and high-molecular-weight species on AdvanceBio SEC. The competitive column showed reduced recovery and merged peaks due to secondary binding.
• Aggregate analysis of a Sigma MAb ADC demonstrated a single, sharp monomer peak on AdvanceBio SEC. The BEH column produced split monomer peaks, complicating accurate quantitation of monomer and dimer fractions.
• The hydrophilic coating on AdvanceBio SEC effectively suppresses nonspecific interactions, delivering higher peak symmetry, lower tailing factors and consistent retention times.

Přínosy a praktické využití

• Reliable aggregate and fragment quantitation supports critical quality attribute (CQA) monitoring of biotherapeutics.
• No additional method development is required for “sticky” samples such as ADCs, saving time and resources.
• Wide applicability across peptides, proteins, ADCs and other complex biologics with minimal compromise to sample integrity.

Budoucí trendy a možnosti využití

• Coupling hydrophilically coated SEC with mass spectrometry for intact protein mass analysis.
• Design of next-generation stationary phases with tailored surface chemistries to further reduce secondary binding.
• Development of microflow and high-throughput SEC formats for limited sample volumes and rapid screening.
• Application to emerging biotherapeutics—fusion proteins, bispecifics and novel conjugates—requiring precise size distribution profiling.

Závěr

Agilent AdvanceBio SEC columns offer a robust solution for size exclusion characterization of proteins and ADCs by effectively eliminating nonspecific secondary interactions. The result is improved recovery, resolution and reproducibility, making these columns a valuable tool for routine biopharmaceutical analysis.

Reference

• No external literature citations were provided in the original document.