Evaluation of SEC-MALS Data Quality with Premier Protein SEC Columns

Significance of the Topic

Size exclusion chromatography coupled with multiangle light scattering (SEC-MALS) is a fundamental analytical technique for evaluating the biophysical properties of large biomolecules, including molecular weight distribution, aggregation state, hydrodynamic size, and conformational features. Reliable baseline stability and low noise are critical to ensure accurate characterization of therapeutic proteins and their variants.

Objectives and Study Overview

This study aimed to assess the baseline noise performance of two MaxPeak Premier Protein SEC columns (XBridge Premier Protein SEC 250 Å, 2.5 µm and ACQUITY Premier Protein SEC 250 Å, 1.7 µm) in comparison to established Waters SEC columns. Additionally, the work evaluated the precision of molecular weight determination for the monoclonal antibody trastuzumab using SEC coupled with ultraviolet (UV) detection and MALS.

Methodology

Blank injections were performed after column equilibration with 2× phosphate-buffered saline to measure MALS baseline noise. Trastuzumab (21 mg/mL) was injected neat to determine molecular weight and size variants. Chromatographic parameters included ambient column temperature, 10 °C sample temperature, flow rates of 0.75 mL/min (7.8 mm i.d.) and 0.38 mL/min (4.6 mm i.d.), and injection volumes of 10 µL and 3.5 µL, respectively.

Used Instrumentation

• ACQUITY UPLC H-Class PLUS Bio System
• ACQUITY UPLC Tunable UV (TUV) Detector with 5 mm titanium flow cell (280 nm)
• Wyatt µDAWN MALS detector
• Empower 3 chromatography data system
• XBridge Premier Protein SEC 250 Å, 2.5 µm, 7.8×300 mm
• ACQUITY Premier Protein SEC 250 Å, 1.7 µm, 4.6×300 mm
• ACQUITY UPLC Protein BEH SEC 200 Å, 1.7 µm, 4.6×300 mm
• BioResolve SEC mAb 200 Å, 2.5 µm, 7.8×300 mm
• Diol-bonded silica SEC 250 Å, 5 µm, 7.8×300 mm

Main Results and Discussion

Baseline noise on the Premier Protein SEC columns was 1.5 to 2 times higher than on the BEH 200 Å, 1.7 µm and BioResolve SEC columns, yet remained substantially lower (2.5–4×) than that of the diol-bonded silica SEC column. No significant spikes indicated minimal particle shedding. Signal-to-noise ratios for trastuzumab monomer were consistent with noise trends. All columns effectively separated monomer, high molecular weight species (HMWS), and low molecular weight species (LMWS), though partial coelution of LMWS1 was noted under the monomer peak, and diol-bonded silica showed additional artifacts likely due to pressure pulses.

Molecular weight calculations using Astra software yielded consistent monomer and dimer (HMWS1) values across all columns. Greater variability was observed for low-abundance LMWS1 and LMWS2 peaks, particularly on the diol-bonded silica column, reflecting combined effects of low signal intensity and higher noise.

Benefits and Practical Applications

• Enhanced baseline stability and reduced noise for accurate MALS measurements
• Reliable molecular weight determination of therapeutic antibodies
• Minimal particle shedding under standard operating conditions
• Compatibility with high-throughput UPLC workflows

Future Trends and Potential Applications

Continued innovations in column packing materials and surface treatments are expected to further lower baseline noise and extend column lifetimes at physiological pH. Integration of advanced detection modalities and automated data analysis will streamline SEC-MALS workflows. These developments will support expanded applications in biosimilar characterization, formulation screening, and real-time monitoring of protein aggregation.

Conclusion

MaxPeak Premier Protein SEC columns deliver robust SEC-MALS performance, combining modestly elevated baseline noise relative to BEH-based columns with marked improvements over diol-bonded silica alternatives. They provide consistent molecular weight determinations for monoclonal antibodies and their size variants, making them suitable for routine biophysical characterization in research and quality control environments.

References

1. Some D, Amartely H, Tsadok A, Lebendiker M. Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering (SEC-MALS). J Vis Exp. 2019;(148):e59615.
2. Huang RYC, Wang F, Wheeler M, et al. Integrated Approach for Characterizing Bispecific Antibody/Antigens Complexes and Mapping Binding Epitopes with SEC/MALS, Native Mass Spectrometry, and Protein Footprinting. Anal Chem. 2020;92(16):10709–10716.
3. Koza SM, Chen W. Improving SEC-MALS Data Quality with Ethylene Bridged Hybrid HPLC Size-Exclusion Columns. Waters Application Note. 2018;720006289.
4. Koza SM, Yang H, Yu YQ. Expanding Size-Exclusion Chromatography Platform Method Versatility for Monoclonal Antibody Analysis Using Waters XBridge Premier Protein SEC Columns. Waters Application Note. 2022;720007500.
5. Koza SM, Yang H, Yu YQ. Modern Size-Exclusion Chromatography Separations of Biosimilar Antibodies at Physiological pH and Ionic Strength. Waters Application Note. 2022;720007484.
6. Koza SM, Yang H, Yu YQ. MaxPeak Premier Protein SEC 250 Å Column Lifetimes at Physiological pH for Polysorbate (Tween) Formulated Biosimilar Monoclonal Antibodies. Waters Application Note. 2022;720007523.
7. Yang H, Koza SM, Yu YQ. USP Monograph mAb SEC Method Robustness on the XBridge Premier Protein SEC Column. Waters Application Note. 2021;720007481.