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

Significance of the Topic

Protein biologics demand accurate concentration and stability profiling to ensure efficacy, safety and manufacturability. By integrating UV/Vis spectroscopy with dynamic light scattering (DLS), researchers can obtain comprehensive data on sample purity, size distribution and interaction behavior using minimal volumes, accelerating development and quality control workflows.

Objectives of the Study

This technical note showcases the Stunner platform, designed to deliver simultaneous protein quantification and sizing in a single micro-volume assay. Key goals include demonstrating:

• High-throughput capability for 96 samples in one hour
• Quantification accuracy across a wide concentration range (0.03–275 OD; 0.3–180 mg/mL)
• Concurrent measurement of hydrodynamic diameter, polydispersity, kD and B22
• Minimal sample consumption (2 µL) and contamination risk

Methodology

Protein samples (monoclonal antibodies, therapeutic mAbs) were prepared in various buffer formulations, including citrate, histidine and excipient-supplemented solutions. Two microliters of each sample were loaded in triplicate onto microfluidic Stunner Plates featuring fixed pathlengths (0.1 and 0.7 mm). Measurements comprised:

• UV/Vis full-spectrum absorbance (230–750 nm) with wavelength-specific background correction
• DLS acquisitions (4 × 5 s) to determine hydrodynamic diameter (Z-Ave) and polydispersity index (PDI)
• Calculation of diffusion interaction parameter kD from diffusion coefficient versus concentration plots
• Determination of second virial coefficient B22 using PEG40 reference scattering intensities
• Aggregation index evaluation under vortex stress via OD350 and DLS particle sizing

Instrumentation

• Stunner platform with integrated UV/Vis spectrometer and DLS detector
• Micro-volume Stunner Plates enabling 2 µL sample analysis and dynamic range up to 275 OD
• Optional 21 CFR Part 11–compliant software and automated plate handling compatibility

Main Results and Discussion

Concentration and Size Distribution:

• Linear quantification from 0.3 to 180 mg/mL with R² > 0.99 and low replicate error
• Monomeric mAb exhibited Z-Ave ≈ 10–12 nm and PDI < 0.1; polydisperse samples showed PDI up to 0.4

Interaction Parameters:

• Positive kD in formulations without excipient or with sucrose indicated net repulsive protein interactions
• Negative kD in arginine and NaCl formulations reflected attractive interactions and higher aggregation risk
• B22 values mirrored kD trends, validating stability ranking: no excipient ≈ sucrose > arginine > NaCl

Aggregation Studies:

• Vortex stress induced rapid aggregation in formulations without polysorbate 80; aggregation index rose from 0.36 to 3.76 and DLS revealed particles >350 nm
• Addition of 0.1% polysorbate 80 prevented aggregation, maintaining low aggregation index and monomeric size profile

Practical Benefits

• Single-shot measurement of concentration, size, and interaction parameters conserves precious sample and reduces hands-on time
• High throughput and micro-volume format support formulation screening, lead selection and QC assays
• No manual dilutions for high-concentration samples minimizes error and variability
• Customizable Homebrew application enables user-defined data processing and background corrections

Future Trends and Opportunities

• Integration with automated liquid handling for full 96-well assays in biologics discovery
• Real-time monitoring of aggregation and stability during upstream and downstream bioprocessing
• Cloud-based data analysis and remote collaboration to support regulatory filings and multi-site research
• Expansion of biophysical modules (e.g., fluorescence detection, temperature ramping) for deeper characterization

Conclusion

The Stunner platform uniquely combines UV/Vis spectroscopy and DLS in a micro-volume, high-throughput format to deliver comprehensive protein characterization. It enables precise concentration measurements, size distribution analysis, polydispersity and interaction parameter determination from low to high concentrations, streamlining workflows in biopharmaceutical research, formulation development and quality control.

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.