Knock out same-time protein concentration and quality with Stunner

Importance of the Topic

Reliable assessment of protein concentration and aggregation state is essential in biopharmaceutical research and quality control. Early detection of aggregation or degradation prevents costly downstream failures in assay development, structural studies, and formulation screening. Integrating concentration and size measurements into a single workflow preserves limited sample volumes and accelerates project timelines.

Study Objectives and Overview

This application note presents a unified protocol using the Stunner platform to simultaneously quantify protein concentration via UV/Vis spectroscopy and evaluate particle size distribution through dynamic light scattering (DLS). The goal is to demonstrate high-throughput, low-volume analysis of nine representative proteins covering a wide molecular weight range.

Methodology and Instrumentation

The Stunner system combines:

• Micro-volume UV/Vis spectrophotometer with wavelength-specific scattering correction
• Back-scattering dynamic light scattering module
• Two-pathlength microplate (0.1 and 0.7 mm) accommodating 2 µL samples
• Software for automated sample-blank grouping, custom analyte/buffer definitions, and 21 CFR Part 11 compliance (optional)

Sample preparation involved reconstituting lyophilized proteins (RNase A, IgG, ovalbumin, conalbumin, aldolase, thyroglobulin, insulin, lysozyme, BSA) in PBS pH 7.4 to 5 or 10 mg/mL. Each sample was loaded in octuplicate, and DLS settings were configured for four 5-second acquisitions per well.

Main Results and Discussion

The combined workflow measured protein concentrations matching expected values across a 0.02–200 mg/mL dynamic range. Key findings include:

• BSA and insulin samples showed single, narrow DLS peaks (PDI < 0.1), confirming monodispersity.
• Proteins such as RNase A, conalbumin, and aldolase exhibited elevated PDI (> 0.2) and multi-peak intensity distributions, indicating partial aggregation.
• The mass distribution analysis quantified monomer versus aggregate populations, revealing trace aggregates (~0.5 % by mass) even in nominally pure samples.

The UV/Vis module applied a wavelength-specific Rayleigh scattering correction to A280 readings, ensuring accurate concentration determinations without manual dilutions.

Benefits and Practical Applications

By consolidating quantification and sizing into one 60-minute, 96-sample run, Stunner delivers:

• Ultra-low sample consumption (2 µL) with minimal handling
• Elimination of dilution steps and cuvette cleaning
• High sensitivity for low-concentration proteins (down to 0.02 mg/mL)
• Rapid identification of aggregation or formulation instability

This approach is ideal for early-stage biotherapeutic screening, formulation optimization, and quality control laboratories.

Future Trends and Opportunities

Emerging developments are set to enhance integrated protein analytics:

• Automated liquid-handling integration for walk-away workflows
• Expanded multi-wavelength DLS and fluorescence modules for richer characterization
• Advanced data analytics and machine learning to predict aggregation risk
• Miniaturization and multiplexing for simultaneous multi-parameter assays

These advances will further reduce sample requirements and deliver deeper insights into protein behavior.

Conclusion

The Stunner platform streamlines protein quality assessment by uniting UV/Vis concentration measurements with DLS-based sizing in a single, high-throughput protocol. This dual analysis conserves sample material, shortens analysis times, and provides comprehensive data on concentration, hydrodynamic diameter, and polydispersity, enabling more informed decision-making in biopharmaceutical research.

Reference

• Unchained Labs. Knock out same-time protein concentration and quality with Stunner. Application Note, Rev A, 2019.