Meet Stunner: The one-shot protein concentration and sizing combo

Importance of the Topic

Characterizing protein therapeutics requires accurate measurement of both concentration and particle size to assess purity, stability, and aggregation propensity. Combining UV/Vis spectroscopy with dynamic light scattering (DLS) in a single platform streamlines workflows, conserves precious samples, and enhances data reliability for research and quality control in biopharmaceutical development.

Study Objectives and Overview

This technical note introduces Stunner, a bench-top instrument that integrates high-speed UV/Vis spectral analysis with DLS. The goal is to demonstrate how Stunner measures protein concentration and hydrodynamic size distribution simultaneously in micro-volumes (2 µL) across a wide dynamic range (0.03–275 OD or 0.02–200 mg/mL). Applications include monomer detection, aggregation screening, kD and B22 determination, high-concentration quantification, and stress-induced aggregation studies.

Used Instrumentation

• Stunner system combining micro-volume UV/Vis spectrophotometer and DLS module
• Microfluidic Stunner Plates with dual path lengths (0.1 and 0.7 mm)
• Integrated software for UV/Vis spectrum acquisition (230–750 nm) and DLS data collection (4 acquisitions × 5 s)
• Homebrew application for custom workflows and mathematical processing

Methodology

Multiple monoclonal antibodies (mAb 1, mAb 2, Adalimumab) were prepared in various buffer formulations and diluted volumetrically. Two microliters of each sample were loaded in triplicate into Stunner Plates. UV/Vis spectra and DLS intensity distributions were recorded simultaneously. Background correction for scattering used wavelength-specific algorithms. kD values were calculated from diffusion coefficients versus concentration. B22 values were determined using PEG40 as a reference standard. Aggregation index was computed from OD350 changes and validated against DLS size and polydispersity index (PDI).

Main Results and Discussion

• Monodispersity: mAb 1 exhibited a monomeric size (~10 nm) with PDI < 0.1, while mAb 2 showed a broad distribution (0.1–0.4 PDI) indicating aggregates.
• High-concentration quantification: Linear response observed from 0.3 to 180 mg/mL with low %RSD, demonstrating reproducibility even at 275 OD.
• kD analysis: Positive kD for formulations without excipients and with sucrose indicated net repulsive interactions; negative kD in arginine and NaCl formulations revealed attractive self-association.
• B22 measurement: Trends matched kD results, with repulsive behavior in control and sucrose, neutral interactions in arginine/NaCl solutions.
• Aggregation stress test: Vortex mixing induced large aggregates in surfactant-free samples (aggregation index 3.76; Z-avg ~355 nm), whereas 0.1% PS 80 suppressed aggregation (index < 0.4; PDI < 0.09).

Practical Benefits and Applications

• Simultaneous concentration and size measurements reduce assay time and sample consumption.
• No manual dilutions or risk of evaporation in micro-volume workflows.
• High throughput: 96 samples measured in under one hour.
• Customizable software (Homebrew) enables bespoke assays and data analysis.
• 21 CFR Part 11 compliance for regulated environments.

Future Trends and Opportunities

The integration of label-free multi-parameter analysis will expand into formulation screening, stability studies, and process development. Automated workflows linked to laboratory information management systems (LIMS) and AI-driven data interpretation promise to accelerate biotherapeutic development. Emerging microfluidic advances and novel scattering methods may further enhance sensitivity and throughput.

Conclusion

Stunner provides a unique one-shot solution for protein concentration and sizing. By merging UV/Vis and DLS in a micro-volume, high-throughput format, it delivers robust data on monomer content, aggregation behavior, and colloidal stability while conserving valuable sample.

References

• Volkin DB, Middaugh CR, Joshi SB, Esfandiary R, Kamerzell TJ. Protein-excipient interactions: mechanisms and biophysical characterization applied to protein formulation development. Adv Drug Deliv Rev. 2011;63:1118-1159.
• Hawe A, Kasper JC, Friess W, Jiskoot W. Structural properties of monoclonal antibody aggregates induced by freeze-thawing and thermal stress. Eur J Pharm Sci. 2009;38(2):79-87.