SIZE-EXCLUSION CHROMATOGRAPHY (SEC) OF PEPTIDES, PROTEINS, AND AAVs

Importance of Topic

Size‐exclusion chromatography (SEC) is the gold‐standard technique for monitoring noncovalent aggregation (HMWS) and degradation fragments (LMWS) in biotherapeutic proteins. As SEC peak volumes shrink with the adoption of smaller‐particle and narrower‐bore columns, dispersion outside the column (extra‐column band broadening) increasingly degrades separation efficiency and quantitative accuracy. Understanding how LC system dispersion interacts with column parameters is essential for designing robust, high‐throughput protein SEC methods.

Goals and Overview

This study systematically evaluates the effects of LC system extra‐column dispersion on SEC separations of monoclonal antibodies (IgG) and related protein standards. It examines how particle size, column internal diameter, and column length influence peak resolution, sensitivity, and quantitative reliability under varying dispersion conditions. The aim is to guide chromatographers in selecting optimal SEC column formats compatible with their LC platforms and analytical requirements.

Methods and Instrumentation

• Systems: Waters ACQUITY UPLC H-Class Bio, ACQUITY Arc Bio, and Alliance HPLC.
• Detectors: UV absorbance at 280 nm (proteins) and 273 nm (caffeine tracer) to measure dispersion.
• Extra‐column dispersion (5σec) measured by injecting a small caffeine peak without a column, sampling at 4.4% of peak height.
• Columns: SEC columns packed with BEH diol‐bonded particles—1.7 µm (2.1 × 150 mm, 4.6 × 150 mm, 4.6 × 300 mm), 2.5 µm and 3.5 µm (7.8 × 150 mm, 7.8 × 300 mm).
• Mobile phase: 20 mM sodium phosphate, 350 mM NaCl, pH 6.8, unless noted.

Main Results and Discussion

■ System dispersion volumes ranged from ~9 µL to >50 µL depending on system configuration and tubing.
■ For large‐bore columns (7.8 mm I.D.), extra‐column dispersion had minimal impact on resolution of HMWS (dimer vs. monomer) even up to ~40 µL dispersion.
■ Narrow‐bore, small‐particle columns (4.6 mm I.D., 1.7 µm) generated small peak volumes; resolution of HMWS and LMWS1 fragments degraded sharply as dispersion exceeded ~15 µL on 150 mm length or ~40 µL on 300 mm length.
■ 300 mm length, 4.6 mm I.D., 1.7 µm columns provided best HMWS and LMWS resolution when 5σec ≤ 25 µL; LMWS quantitation became unreliable above that dispersion.
■ 7.8 mm I.D. columns packed with 2.5 µm particles delivered robust, reproducible separations less sensitive to dispersion (up to ~60 µL), at the cost of moderate run‐time increase.
■ Tandem 7.8 × 300 mm columns improved LMWS1 resolution significantly for systems with higher dispersion.
■ A mid‐range 2.5 µm particle size in 7.8 mm I.D. format strikes a balance: improved efficiency over 3.5 µm, lower backpressure than 1.7 µm, and reduced dispersion sensitivity compared to narrow‐bore UHPLC.

Benefits and Practical Applications

• Provides clear guidelines for matching SEC column format (particle size, I.D., length) with LC system dispersion limits.
• Improves confidence in quantitative aggregate and fragment measurements by controlling dispersion effects.
• Enables high‐throughput HMWS analysis on small‐particle, narrow‐bore columns when used on low‐dispersion UHPLC systems.
• Offers robust alternative SEC formats (wide‐bore, mid‐particle size) for HPLC systems with higher dispersion.

Future Trends and Potential Uses

• Development of low‐dispersion UHPLC and HPLC systems to fully exploit high‐efficiency SEC columns for protein characterization.
• Expanded use of mid‐particle‐size SEC columns (2.5 µm) on a wider range of instruments for both HMWS and LMWS analysis.
• Integration of on‐line multi‐detection (MALS, MS) with optimized SEC separations to provide molecular weight and structural information.
• Further miniaturization and column hardware innovations to reduce extra‐column dispersion and analysis time.

Conclusion

Extra‐column dispersion is a critical, often overlooked, factor in SEC method performance, especially for narrow‐bore, small‐particle columns used for protein aggregate and fragment analysis. By measuring system dispersion and matching column geometry to instrument dispersion capabilities, chromatographers can achieve optimal resolution, sensitivity, and quantitation. Wide‐bore, mid‐particle‐size SEC columns (2.5 µm, 7.8 mm I.D.) provide a practical, robust solution for systems with higher dispersion, while narrow‐bore, sub‐2 µm columns deliver maximum efficiency on low‐dispersion UHPLC platforms.

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