What can MaxPeak Premier Columns do for your protein SEC analysis?

Significance of the Topic

Size exclusion chromatography (SEC) is a cornerstone technique for the characterization of protein therapeutics, including monoclonal antibodies, antibody–drug conjugates and fusion proteins. High resolution, reproducibility, and minimized surface interactions are critical to detect aggregates, fragments and other size variants that affect safety and efficacy in biopharmaceutical development and quality control.

Objectives and Study Overview

This application note evaluates the performance of Waters MaxPeak Premier columns for protein SEC analysis. The goals are:

• To demonstrate universal method applicability across multiple protein classes.
• To compare recovery, sensitivity and reproducibility against traditional high-performance surface columns.
• To illustrate a platform SEC method that reduces development time and overcomes system dispersion constraints.

Methodology

Protein separations were performed using SEC columns with different particle sizes (1.7 µm and 2.5 µm) and pore sizes (250 Å). Mobile phases included 2× phosphate-buffered saline (PBS) or AutoBlend™ Plus mixtures (phosphate/NaCl). Analyses were conducted at moderate flow rates (0.3–0.75 mL/min) and ambient to elevated column temperatures (25–35 °C). Proteins tested comprised rituximab, infliximab, panitumumab, trastuzumab emtansine (ADC), abatacept (Fc-fusion) and certified reference material NISTmAb.

Used Instrumentation

• Columns: XBridge™ Premier SEC 250 Å, 2.5 µm, 7.8×300 mm; ACQUITY™ Premier Protein SEC 250 Å, 1.7 µm, various dimensions (4.6×150, 4.6×300, 7.8×150, 7.8×300 mm).
• LC System: ACQUITY UPLC™ H-Class Bio with dispersion <10 µL.
• Detectors: UV absorbance at 214 nm and 280 nm.
• Mobile Phases: 2×PBS (20 mM phosphate, 276 mM NaCl, 5.4 mM KCl, pH 7.4); AutoBlend™ Plus phosphate/NaCl buffer.

Key Results and Discussion

Performance surfaces mapped UV signal across salt concentrations (50–400 mM NaCl) and pH (5.8–7.6) for rituximab. MaxPeak Premier columns consistently showed higher recovery and signal intensity than standard high-performance surface columns, indicating reduced nonspecific adsorption. Separations of multiple biotherapeutics demonstrated baseline resolution of monomer, aggregates and fragments within 5–11 min. Platform methods on both 1.7 µm and 2.5 µm particles provided robust performance under system dispersion limits, supporting high-throughput and method transfer.

Benefits and Practical Applications

• Enhanced recovery and sensitivity for trace variants through hybrid inorganic/organic surface chemistry.
• Reduced method development time by using a unified SEC platform across protein classes.
• High reproducibility and batch-to-batch consistency qualified with mAb Size Variant standards.
• Scalability for QC and process development with flexible particle sizes and column dimensions.

Future Trends and Opportunities

Advancements in column surface technology will further lower analyte losses and broaden compatibility with mass spectrometry. Integration of online mixed-mode and SEC methods can streamline higher-order structure analyses. Emerging buffer systems and automated blending will optimize workflows, while next-generation fast-LC instrumentation may shorten run times without sacrificing resolution.

Conclusion

Waters MaxPeak Premier SEC columns deliver a universal, high-performance platform for protein size variant analysis. By minimizing surface interactions and maintaining robust separations across diverse biotherapeutic classes, these columns support efficient QC, process monitoring and research applications.

Reference

No external literature cited.