Have it all with protein quant & sizing on Stunner

Significance of the Topic

High-throughput, low-volume protein quantification and biophysical sizing are essential for accelerating biologic development and ensuring product quality. Traditional methods often require large sample volumes, extensive dilutions, costly reagents and lengthy protocols, introducing variability and slowing workflows. A streamlined platform combining UV/Vis absorbance with multi-angle light scattering offers rapid, accurate, and multiplexed analysis on just 2 µL of sample, enabling early detection of aggregation, precise concentration determination and reliable molecular weight estimation.

Objectives and Study Overview

This application note demonstrates how the Stunner system integrates protein concentration measurement, rotating angle dynamic light scattering (RADLS) and multi-angle static light scattering (MALS) to:

• Quantify protein concentration without standard curves or dyes
• Determine hydrodynamic size, polydispersity and particle count
• Estimate molecular weight of monodisperse proteins
• Detect and monitor aggregation in monoclonal antibodies under stress

All analyses are performed in under two minutes per 2 µL sample using a 96-well format.

Methodology and Instrumentation

Sample Preparation and Standards:

• Polystyrene size standards (41 nm and 202 nm, NIST traceable) diluted in water and mixed at 20:1 ratio
• Lyophilized proteins (BSA, conalbumin, IgG, ovalbumin, RNase A) reconstituted in PBS at 5 mg/mL, filtered (0.1 or 0.02 µm)
• Monoclonal antibody (mAb1) at 10 mg/mL in sodium succinate/trehalose buffer; aliquots heated to induce aggregation and mixed to prepare 0%, 1% and 10% aggregate content

Instrumentation and Settings:

• Stunner platform integrating UV/Vis, RADLS and MALS on 2 µL samples
• RADLS acquisition: 7 angles, 5 acquisitions, 1 s each, automatic angle selection and outlier exclusion
• Buffers blanked using water, PBS or succinate buffer with specified viscosity and refractive index values

Key Results and Discussion

Size Standards and Polydispersity:

• 41 nm and 202 nm standards measured with Z-average diameters matching nominal values; CVs of 2.9% and 2.1%; PDI <0.1
• 20:1 mix yielded intermediate Z-average diameter and increased PDI >0.2, illustrating heterogeneity
• Regularization analysis produced consistent intensity, mass and number distributions, revealing relative contributions of small and large particles

Protein Concentration and Sizing:

• UV/Vis absorbance at 280 nm quantified five proteins within 2% of target concentration, CV <1%
• Multi-angle intensity distributions distinguished proteins by molecular size and count (e.g., IgG vs ovalbumin at equal mass concentration)

Molecular Weight Determination:

• Rayleigh ratio extrapolated to 0° via Guinier plots for uniform proteins
• Molecular weights of BSA, conalbumin, IgG, ovalbumin and RNase A determined within ±5 kDa of reference values

Aggregation Detection in mAb1:

• UV/Vis concentration remained constant despite aggregation, while RADLS revealed decreasing particle count with increasing aggregate content
• Intensity and mass distributions of 0%, 1% and 10% aggregated samples showed emerging high-size peaks correlating with aggregate fraction
• Time-course heat stress (80 °C) indicated onset of aggregation after 10 min, with complete aggregation by 30 min

Benefits and Practical Applications

The Stunner platform enables:

• Rapid, dilution-free protein concentration using intrinsic absorbance
• Precise, multi-angle sizing and polydispersity assessment in 2 µL samples
• Accurate molecular weight estimation without separation or calibration standards
• Sensitive detection and quantification of protein aggregation for formulation screening and forced-degradation studies

Future Trends and Potential Applications

Integration of high-throughput UV/Vis and multi-angle light scattering paves the way for:

• Automated process analytical technology (PAT) in downstream bioprocessing
• Real-time monitoring of formulation stability and aggregation kinetics
• Expanded analysis of nanoparticles, lipid-based carriers and other macromolecular assemblies
• Regulatory compliance support through standardized, traceable measurements

Conclusion

The Stunner system combines UV/Vis absorbance, RADLS and MALS in a single, plate-based workflow to deliver fast, accurate protein quantification, sizing, molecular weight analysis and aggregation assessment on just 2 µL of sample. This comprehensive, dilution-free approach accelerates biologics development, improves assay reproducibility and enhances decision-making in research, formulation screening and quality control.

Reference

Unchained Labs. Application Note: Protein quant & sizing on Stunner. Rev A. 2024.