Meet Big Tuna: automated, versatile buffer exchange

Significance of the Topic

The selection and optimization of formulation buffers are essential for maintaining the conformational, chemical, and colloidal stability of therapeutic proteins. Conventional manual ultrafiltration and diafiltration approaches are time-consuming, inconsistent and scale poorly when high throughput is required. Automating buffer exchange can accelerate biologics development, improve reproducibility and reduce hands-on time.

Objectives and Study Overview

This application note evaluates the performance of the Big Tuna automated buffer exchange platform across multiple volumes and modes. Key goals include:

• Demonstrating high-throughput buffer exchange in 96-well (100–450 µL) and 24-well (0.45–8 mL) formats
• Assessing accuracy of buffer removal and refill percentages
• Comparing standard Filter & Refill to Dilute & Filter workflows for high-concentration, viscous samples
• Validating post-exchange concentration control

Methodology and Instrumentation

Big Tuna employs pressure-based ultrafiltration/diafiltration with gentle orbital mixing to minimize membrane fouling and accelerate flow. An integrated ultrasonic sensor measures well volume in real time to adjust each cycle’s duration, ensuring user-defined percent removal per cycle and total exchange targets are met. Two plate formats were used:

• Unfilter 96: 10 kDa membrane, 96 wells at 100–450 µL each
• Unfilter 24: 10 kDa membrane, 24 wells at 0.45–8 mL each

Used Instrumentation

• Big Tuna automated buffer exchange system
• Unfilter 96 and Unfilter 24 ultrafiltration plates (10 kDa cutoff)
• Lunatic spectrophotometer with A280 application for protein concentration measurement

Main Results and Discussion

Buffer exchange into PBS, pH 6.8, was tested for human IgG at various starting concentrations and volumes:

• 96-well (450 µL): 96% exchange in 3 cycles (~60 min each), average final concentration 10.4±0.2 mg/mL, volume accuracy 450→457±9 µL, total run time 2.6 h
• 96-well + 3× concentration: achieved 29.1 mg/mL target in one concentration cycle, total run time 3.5 h
• 24-well (8 mL): 96% exchange in 3 cycles (~60 min each), final concentration 10.9±0.1 mg/mL, volume 8,000→8,001±73 µL, total run time 4.3 h; post-exchange 3× concentration to 2,680±15 µL
• Filter & Refill (24-well, 4 mL, 99.6 mg/mL): 12 cycles (~110 min each), 97.0% exchange, final concentration 100.9±1.6 mg/mL, total run time 24.6 h
• Dilute & Filter (24-well, 4 mL + 4 mL buffer): 5 cycles (~115 min each), 97.0% exchange, final concentration 98.7±1.3 mg/mL, total run time 11.4 h

Results highlight precise control over removal percentages, consistent volume retention, uniform exchange across wells and significant time savings with Dilute & Filter for viscous samples.

Benefits and Practical Applications

Big Tuna offers:

• Low- and high-volume formats in a single platform
• Customizable exchange parameters: percent removal per cycle, total exchange, final concentration
• Real-time volume monitoring for uniformity and prevention of over-concentration
• Minimal hands-on operation and software-driven experiment design

This flexibility supports formulation screening, stability testing, high-throughput QC and process development in biopharmaceutical research.

Future Trends and Potential Applications

Emerging opportunities include:

• Integration with automated formulation screening and high-content analytics
• AI-driven optimization of exchange protocols tailored to specific proteins
• Expansion to broader membrane chemistries and cutoff ranges
• Inline coupling to downstream analytics (e.g., SEC, MS) for real-time monitoring
• Scalable adaptations for pilot- and manufacturing-scale ultrafiltration

Conclusion

The Big Tuna platform delivers versatile, high-throughput buffer exchange with exceptional control over exchange and concentration parameters. Its automated workflows reduce manual labor, improve reproducibility and accommodate a wide range of sample volumes and concentrations, making it an ideal solution for biopharmaceutical formulation and stability studies.