Identify Optimal Biologic Candidates: Low Volume, Early Stage Developability Assessment with Aura PTx System

Importance of the topic

Early-stage developability screening for biologic candidates requires sensitive, low-volume methods that directly measure subvisible particles, a critical quality attribute linked to immunogenicity, efficacy, and shelf life. Traditional particle techniques demand large sample volumes and are impractical in early discovery. The Aura PTx System provides a high-throughput, low-sample-volume approach to directly quantify subvisible particles and to interrogate single-particle properties with fluorescence membrane microscopy, enabling faster, evidence-based candidate selection and formulation triage.

Objectives and study overview

The study aimed to demonstrate the Aura PTx System’s capability to perform comprehensive developability assessments with minimal sample usage. Three proteins (A, B, C) were evaluated across a platform of 14 industry-relevant buffer/excipient conditions (including acetate, citrate, tris, and glycine systems) to:

• Rank candidates by subvisible particle formation (ECD ≥2 µm).
• Assess measurement robustness at low volumes.
• Use fluorescence membrane microscopy (FMM) and Thioflavin T (ThT) staining to characterize particle composition and morphology at the single-particle level.

Key operational metrics: 40 µL per well (quadruplicate), a 168-well sweep, and total runtime under three hours; the authors also report capability at volumes down to 5 µL per condition in the platform context.

Methodology

Proteins were formulated from lyophilized powder to 0.1 mg/mL in the buffer/excipient matrix designed to cover stresses encountered during viral clearance, elution, storage (air–water interfaces), freeze/thaw, and buffer exchange (UF/DF/TFF). The buffer panel varied buffer type, pH, ionic strength, NaCl content, and stabilizing additives. Each condition was measured in quadruplicate on the Aura PTx System to quantify subvisible particle counts (≥2 µm ECD) and to collect fluorescence-stained images for FMM analysis.

Used instrumentation

• Aura PTx System (particle counting and imaging platform optimized for low-volume, high-throughput subvisible particle analysis).
• Aura+ System referenced as related instrumentation for the platform family.
• Particle Vue Software for image gallery review and single-particle analysis.
• Fluorescent dye Thioflavin T (ThT) for staining proteinaceous aggregates.
• Fluorescence membrane microscopy (FMM) modality integrated into Aura for single-particle fluorescence readout.

Main results and discussion

• Overall ranking: Protein A emerged as the most stable across citrate, tris, and glycine buffers; Protein B was the least stable (particularly in citrate, tris, and glycine); Protein C showed intermediate behavior and comparable stability to A in acetate buffer.
• Buffer dependence: If screening had been limited to acetate buffer, Protein B might have been misidentified as favourable. However, in more aqueous/alkaline buffers (citrate/tris/glycine), Protein B produced dramatically higher subvisible counts (up to ~420-fold increases reported in some comparisons).
• Quantitative performance: The full sweep (168 wells, quadruplicate measurements) used 40 µL per well and took under three hours. Measurement reproducibility was high, with %CVs generally below 10% for most conditions.
• Single-particle insights: FMM with ThT staining produced high-contrast fluorescent images showing round, ThT-positive particles for Protein B in glycine buffer, consistent with hydrophobic, proteinaceous aggregates. Single-particle morphology plus ThT binding helped infer probable aggregation mechanisms and particle composition beyond simple counts.

Benefits and practical applications of the method

• Low sample consumption enables early-stage triage when material is limited.
• Direct measurement of subvisible particles addresses a central stability CQA that many surrogate low-volume techniques (SEC, DLS, DSF) do not predict reliably.
• High throughput allows broad buffer/excipient sweeps to reveal buffer-dependent instability that might be missed in limited-condition screens.
• Single-particle fluorescence imaging adds composition and morphology context, improving mechanistic understanding and informing formulation or engineering decisions.

Limitations and considerations

• ThT fluorescence selectively reports certain protein aggregate types (amyloid-like fibrils or accessible beta-structures); not all particle types will stain equivalently, so orthogonal methods remain recommended for comprehensive characterization.
• Instrument performance and interpretation rely on appropriate staining protocols, imaging thresholds, and image analysis settings; users should validate these parameters for new molecule classes.
• The study used model conditions and three proteins; broader molecule classes and concentrations should be screened to generalize performance across modalities (mAbs, fragments, multispecifics, fusion proteins).

Future trends and potential applications

• Integration of low-volume particle analytics with automated liquid handling and design-of-experiment workflows to accelerate formulation optimization and candidate ranking.
• Combining single-particle fluorescence signatures with machine learning to classify aggregate types, predict degradation pathways, and link particle profiles to immunogenicity risk models.
• Expanded multiplexed staining strategies to discriminate proteinaceous aggregates from silicone oil, polysorbate micelles, and other excipient-derived particles.
• Regulatory alignment: wider adoption of low-volume, direct subvisible particle measurement may influence early-stage acceptability criteria and increase confidence during lead selection.

Conclusion

The Aura PTx System enables robust, high-throughput developability screening with minimal sample volume while directly measuring subvisible particle burdens and providing single-particle compositional insight via FMM. In this study, the platform distinguished clear stability differences among three candidate proteins across a 14-condition buffer/excipient matrix and identified Protein A as the most developable overall. The approach reduces risk and resource expenditure by revealing buffer-dependent instabilities early in development and by supplying mechanistic information to guide formulation or engineering interventions.

References

1. Lobo SA, et al. Stability Liabilities of Biotherapeutic Proteins: Early Assessment As Mitigation Strategy. Journal of Pharmaceutical and Biomedical Analysis. 192:113650. 2021.
2. Garripelli VK, Wu Z, Gupta S. Developability Assessment for Monoclonal Antibody Drug Candidates: A Case Study. Pharmaceutical Development and Technology. 26(1):11–20. 2021.
3. Carpenter JF, et al. Overlooking Subvisible Particles in Therapeutic Protein Products: Gaps That May Compromise Product Quality. Journal of Pharmaceutical Sciences. 98(4):1201–1205.