Precise Determination of Protein Molecular Weight using the Agilent 1260 Infinity Multi-Detector GPC/SEC System

Significance of the topic

Protein size-exclusion chromatography (SEC/GPC) combined with multi-detection is a robust analytical approach to determine absolute molecular weight, hydrodynamic radius, and relative concentration of proteins. This capability is vital for characterizing biotherapeutics, monitoring aggregation, and ensuring consistency in quality control workflows.

Objectives and Study Overview

This study demonstrates the integration of the Agilent 1260 Infinity Multi-Detector GPC/SEC System with a Bio-inert Quaternary LC platform to achieve precise protein MW and size determinations. The focus is on analyzing proteins from 11 kDa (cytochrome C) to 670 kDa (thyroglobulin), assessing method sensitivity, calibration strategies, and correlation with literature values.

Methodology

• Chromatographic conditions: PBS buffer (pH 7.4), 0.8 mL/min flow, 35 °C column compartment, and 4 °C autosampler.
• Detection scheme: tetra detection using RI, UV (280 nm), dual-angle light scattering (15° and 90°), and viscometer signals.
• Calibration: single-point calibration employing a BSA monomer peak with known concentration and dn/dc, UV extinction coefficient, and intrinsic viscosity settings for detectors.
• Data processing: triple-distribution algorithm combining concentration, LS, and viscometer data to compute MW, hydrodynamic radius (Rh), and relative concentration.

Used Instrumentation

• Agilent 1260 Infinity Bio-inert Quaternary Pump and Autosampler
• Agilent 1290 Infinity Thermostat and Thermostatted Column Compartment
• Agilent 1260 Infinity Diode Array Detector VL (bio-inert flow cell)
• Agilent 1260 Infinity Multi-Detector Suite: RID, Viscometer, Dual-Angle LS Detector
• Columns: Two Agilent Bio SEC-3 (7.8 × 300 mm, 3 µm, 300 Å)
• Software: Agilent GPC/SEC Software v1.2 with multi-detector upgrade

Main Results and Discussion

The system delivered high signal-to-noise separation of BSA monomer and oligomers. Calibration based on a single BSA monomer standard effectively aligned inter-detector delays and response factors. Analysis of six proteins yielded MW values in excellent agreement with literature (R2 = 1.00), and Rh determinations correlated strongly (R2 ≈ 0.99). Dual-angle LS extended sensitivity for high-mass aggregates. Thyroglobulin aggregates were resolved, with MW assignments corresponding to dimer, tetramer, and larger oligomers, and their relative concentrations quantified (monomer ~76 %, aggregates ~24 %).

Benefits and Practical Applications

• Absolute MW and size determination without extensive column calibration
• Sensitive detection across a broad MW range (11–670 kDa)
• Quantification of aggregation critical for biopharmaceutical quality control
• Streamlined workflow leveraging single calibration standard

Future Trends and Potential Applications

Expanding this multi-detection SEC approach may include higher MW species and complex biologics such as antibody–drug conjugates. Automation and advanced data analytics, including machine learning, can further enhance throughput and predictive aggregation profiling. Integration with orthogonal structural techniques (e.g., mass spectrometry, light-based scattering platforms) will advance comprehensive protein characterization.

Conclusion

The Agilent 1260 Infinity Multi-Detector GPC/SEC System coupled with a bio-inert LC platform provides an efficient, accurate method for protein MW and size analysis. The tetra detection configuration affords robust calibration, high sensitivity, and reliable quantification of monomers and aggregates, making it well-suited for research and biopharmaceutical quality assurance.

Reference

• Cleaver G. Precise Determination of Protein Molecular Weight using the Agilent 1260 Infinity Multi-Detector GPC/SEC System. Application Note, Agilent Technologies, Inc., 2015.