Advanced SEC-MALS Analysis of Monoclonal Antibodies with the Agilent 1260 Infinity II Multi-Angle Light Scattering Detector

Significance of the Topic

Size exclusion chromatography (SEC) coupled with multi-angle light scattering (MALS) is a cornerstone technique in biopharmaceutical development and quality control. It delivers absolute molecular weight determination and aggregation profiling of therapeutic proteins such as monoclonal antibodies. Accurate aggregate quantification is critical for product safety and regulatory compliance.

Objectives and Study Overview

This application note demonstrates the use of the Agilent 1260 Infinity II MALS detector paired with the Agilent 1290 Infinity II Bio LC System to achieve precise molecular weight measurements of monoclonal antibodies and protein aggregates. Key goals include evaluating chromatographic resolution, comparing UV and MALS sensitivity, and assessing reproducibility.

Methodology

Samples of bovine serum albumin (BSA) and rituximab (MabThera) were analyzed using an AdvanceBio SEC 300 Å, 7.8 × 300 mm column with 150 mM phosphate buffer at pH 7. Prior to injection, buffers and samples were triple-filtered to minimize contamination. Separation was carried out at 0.6 mL/min and 30 °C with UV detection at 280 nm and a 20-angle MALS detector.

Used Instrumentation

• Agilent 1290 Infinity II Bio LC System: Flexible Pump, Multisampler with Sample Thermostat, Multicolumn Thermostat, Variable Wavelength Detector
• Agilent 1260 Infinity II Multi-Angle Light Scattering Detector (20 angles)
• Agilent AdvanceBio SEC 300 Å column (7.8 × 300 mm, 2.7 µm)
• Agilent WinGPC software, version 1.0

Main Results and Discussion

• BSA separation yielded distinct peaks for monomer, dimer, trimer, tetramer, and higher aggregates. MALS-determined masses matched expected multiples of the 66.9 kDa monomer.
• Small-angle MALS signals showed enhanced sensitivity for detecting large aggregates compared to UV absorbance.
• Rituximab analysis produced a monomer mass of 144 kDa with 0.14% RSD over ten injections. Additional peaks revealed a heavy chain fragment (103.4 kDa) and aggregates up to 3.39 MDa.
• An iron-free, biocompatible flow path and high buffer tolerance prevented nonspecific interactions and microbial contamination, ensuring data integrity.

Benefits and Practical Applications

SEC-MALS provides absolute molecular weight and aggregation profiles without reliance on column calibration standards. This enhances characterization workflows, supports regulatory filings, and strengthens quality control of biotherapeutics.

Future Trends and Opportunities

Emerging developments include integration with automated online data analysis, coupling with orthogonal detectors such as viscometers or mass spectrometers, and application to novel biotherapeutic formats like nanobodies and viral vectors. Increased throughput and broader regulatory acceptance are expected.

Conclusion

The combination of an advanced biocompatible LC system with multi-angle light scattering detection delivers highly precise absolute molecular weight measurements and sensitive aggregation detection, critically supporting biopharmaceutical research and quality assurance.

References

• Narhi L. O. et al. Classification of Protein Aggregates. J. Pharm. Sci. 2012, 101(2), 493–498.
• Agilent Technologies. Light Scattering and Size Exclusion Chromatography (SEC) in Biopharma. White paper 5994-6110EN, 2023.
• Wagner C. et al. Biophysical Characterization of Adeno-Associated Virus Vectors Using Ion-Exchange Chromatography Coupled to Light Scattering Detectors. Int. J. Mol. Sci. 2022, 23(21), 12715.
• Van Bruijnsvoort M. et al. Retention Behaviour of Amylopectins in Asymmetrical Flow Field-Flow Fractionation Studied by Multi-Angle Light Scattering Detection. J. Chromatogr. A 2001, 925, 171–182.
• Flores-Ortiz L. F. et al. Physicochemical Properties of Rituximab. J. Liq. Chromatogr. R. T. 2014, 37(10), 1438–1452.