Have it all with protein quant & sizing on Stunner

Importance of the Topic

Reliable and fast measurement of protein concentration, size, molecular weight and aggregation state is essential throughout biologic drug discovery, development and manufacturing. Traditional assays often demand large sample volumes, extensive dilutions, standard curves or dyes, which introduce variability and slow throughput. A platform that consolidates multiple characterization modalities into a single, low-volume assay accelerates decision making and conserves precious samples.

Objectives and Study Overview

This application note evaluates the performance of the Stunner platform in delivering high-throughput quantification and sizing of proteins and nanoparticles from just 2 µL of sample. Key aims include:

• Validating hydrodynamic size and polydispersity against NIST-traceable polystyrene standards
• Measuring protein concentration accurately by UV/Vis absorbance without standard curves or dyes
• Determining molecular weight via multi-angle static light scattering (MALS)
• Assessing aggregation levels in monoclonal antibody samples under thermal stress

Methodology and Experimental Design

Polystyrene sphere standards of 41 nm and 202 nm were diluted and measured in triplicate or higher replicates. Five model proteins (BSA, conalbumin, IgG, ovalbumin, RNaseA) were reconstituted in PBS at 5 mg/mL, filtered, and analyzed in quadruplicate. A monoclonal antibody (mAb1) was prepared at 10 mg/mL, thermally stressed at 80 °C for varying times, and mixed with unstressed material to generate defined aggregation levels (0%, 1%, 10%). Measurements were performed in a 96-well plate format, using 2 µL per well. RADLS acquisition employed automatic angle selection (7 angles, 5 acquisitions of 1 s each), and UV/Vis absorbance at 280 nm provided concentration values without external standards. Buffer viscosity and refractive index corrections were applied.

Used Instrumentation

• Stunner platform integrating UV/Vis spectroscopy, rotating-angle dynamic light scattering (RADLS) and multi-angle static light scattering (MALS)
• 96-well plate sample handling with 2 µL volume per measurement
• 0.1 µm and 0.02 µm syringe filters for sample clarification
• NIST™-traceable polystyrene size standards

Main Results and Discussion

• Size Standards: The 41 nm and 202 nm spheres yielded Z-average diameters within 2% of nominal values and polydispersity indices (PDIs) <0.1. A 20:1 mixture produced a broadened PDI (>0.2) and clear bimodal distributions via regularization analysis.

• Protein Quantification: UV/Vis absorbance at 280 nm delivered concentrations within 2% of the target across five proteins, with <1% coefficient of variation, demonstrating high precision without dye-based assays.

• Molecular Weight Determination: Guinier extrapolation of MALS data provided molecular weights within 5% of reference values (e.g., IgG measured at 150 kDa vs. 150 kDa reference).

• Aggregation Assessment: Monoclonal antibody samples with 0%, 1% and 10% aggregates maintained identical masses by UV/Vis but exhibited decreasing particle counts by RADLS. Intensity and mass distributions clearly resolved aggregated versus monomeric peaks. Thermal stress time courses showed a rapid increase in hydrodynamic size after 10 minutes at 80 °C, with complete aggregation by 30 minutes.

Benefits and Practical Applications

• High­-throughput, low­-volume characterization conserves valuable biologic samples
• Dilution­-free, standard­-free protein quantification reduces assay bias
• Combined RADLS and MALS delivers real intensity, mass and number distributions for head-to-head sample comparisons
• Rapid aggregation profiling supports forced degradation studies and quality control during process development

Future Trends and Possibilities

Integration of machine learning for automated data interpretation, expansion to new light scattering modalities (e.g., advanced angular resolution), and incorporation of microfluidic sample handling may further enhance throughput and sensitivity. Combining this platform with orthogonal techniques such as mass spectrometry or chromatography could yield comprehensive multi­parameter biologic profiles.

Conclusion

The Stunner platform streamlines biologic characterization by delivering accurate protein concentration, size, molecular weight and aggregation information from just 2 µL in under 2 minutes. Its integrated UV/Vis, RADLS and MALS capabilities offer a dilution- and standard-free workflow that accelerates method validation and improves data consistency for researchers and quality control scientists alike.