High-Throughput Fluorescence-Based mAbs Aggregation Analysis Workflow

Significance of the topic

Rapid and reliable assessment of monoclonal antibody aggregation is essential in modern biologics development. Aggregation is a critical quality attribute influencing efficacy and safety. Conventional size exclusion chromatography delivers high resolution but limits throughput at around five minutes per sample. A fluorescence based workflow that reduces analysis time to seconds per sample can accelerate clone screening formulation development and quality control.

Objectives and study overview

This study evaluates a high-throughput fluorescence assay using the Agilent Cary Eclipse Microplate Reader and PEPBOPS dye to estimate high-molecular-weight aggregates in mAb samples. Three mAbs (Rituximab Trastuzumab and NISTmAb RM 8671) were subjected to forced aggregation via pH stress and mechanical shake stress to benchmark fluorescence measurements against size exclusion chromatography.

Methodology and instrumentation used

• Sample preparation: buffer exchange into 50 mM Tris-HCl pH 7.5 transient acidification to pH 2.4–2.6 incubation at 37 °C and vortex induced shake stress followed by centrifugation for insoluble aggregates
• Size exclusion chromatography: Agilent 1260 Infinity II Bio-Inert LC with AdvanceBio SEC column 4.6×300 mm 300 Å 2.7 μm detection at 220 nm and 280 nm
• Fluorescence assay: Agilent Cary Eclipse Microplate Reader white 96-well format PEPBOPS dye compared to Bis-ANS and SYPRO Orange with excitation and emission settings optimized per dye
• Data analysis: blank subtraction normalization to a human serum albumin positive control and regression against SEC determined high-molecular-weight percentages

Main results and discussion

• Acid stress induced increasing HMW aggregates in all three mAbs dependent on pH and incubation time with up to 6% HMW in Rituximab after 20 minutes at pH 2.6
• PEPBOPS exhibited substantially lower background fluorescence with unstressed mAb compared to Bis-ANS and SYPRO Orange enabling higher sensitivity to early aggregation events
• Strong linear correlations (R² ≥ 0.95) were observed between PEPBOPS fluorescence intensity and SEC determined HMW fractions over a range of ~1% to >16%
• Standard error of fluorescence-based estimates remained below ±0.85% demonstrating semi-quantitative accuracy
• Shake stress samples confirmed detection of both soluble and insoluble aggregates
• PEPBOPS addition had minimal impact on aggregation profiles confirming assay non interference
• Calibration derived from innovator Rituximab applied accurately to a biosimilar counterpart with slopes near unity and intercepts near zero

Benefits and practical applications of the method

• Analysis time reduced from minutes to ~5 seconds per sample
• High throughput pre screening tool to prioritize samples for detailed SEC analysis
• Semi quantitative estimation of mAb aggregation suitable for clonal selection formulation screening and comparability studies
• Applicable to biosimilar development supporting regulatory comparability assessments
• Flexible platform enabling rapid method development and transfer between fluorimeters with microplate reader accessory

Instrumentation used

• Agilent Cary Eclipse Microplate Reader G9801AA
• PEPBOPS fluorescent dye Agilent part number 5799-0025
• Agilent 1260 Infinity II Bio-Inert LC system with diode array detector and AdvanceBio SEC column (PL1180-5301)
• OpenLab CDS software version 2.4
• Vivaspin 500 centrifugal concentrators 10 kDa MWCO
• White 96-well microplates Tris HCl buffer HPLC grade salts

Future trends and potential applications

Integration of high-throughput fluorescence aggregation assays into quality by design frameworks and automated screening pipelines can further accelerate biologics development. Extensions to other protein modalities exploration of novel aggregation sensitive probes and coupling with machine learning for predictive analytics represent promising directions. Miniaturization multiplexing and inline process monitoring are potential areas for future innovation.

Conclusion

The combination of the Agilent Cary Eclipse Microplate Reader and PEPBOPS dye provides a rapid sensitive and semi quantitative workflow for mAb aggregation analysis. The method delivers strong correlation with SEC results minimal dye interference and applicability across different mAb formats including biosimilars. Adoption of this high throughput fluorescence approach can enhance productivity in clone selection formulation development and comparability assessments.

References

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