Meet Big Tuna: automated, versatile buffer exchange

Importance of Buffer Exchange in Biologics Development

Buffer composition plays a pivotal role in determining the conformational, chemical and colloidal stability of therapeutic proteins. Traditional manual buffer exchange methods are laborious, inconsistent and not scalable, creating bottlenecks in formulation screening and stability optimization. Automating this process can accelerate biologics development by improving throughput, reproducibility and process control.

Objectives and Overview of the Study

This application note introduces Big Tuna, an automated platform for high-throughput buffer exchange using pressure-based ultrafiltration/diafiltration (UF/DF). Key goals include:

• Demonstrate buffer exchange for low-volume (100–450 µL) and high-volume (0.45–8 mL) samples
• Compare performance of two filter plate formats (Unfilter 96 and Unfilter 24)
• Evaluate throughput, precision in buffer removal, fill volume consistency and final protein concentration
• Assess advanced workflows: concentrate after exchange, dilute-and-filter and filter-and-refill methods

Methodology

Human IgG samples were prepared at concentrations ranging from 10 to ~100 mg/mL in PBS (pH 6.8). Samples were pipetted into either a 10 kDa Unfilter 96 or Unfilter 24 plate and loaded into Big Tuna. The automated workflow alternates three steps:

1. Pressure-driven UF/DF with gentle orbital mixing
2. Ultrasonic volume measurement in each well
3. Addition of new buffer to achieve the user-defined exchange per cycle

Exchange parameters such as total percent exchange (up to 99 %), percent volume removed per cycle and final concentration targets were set in the software. Protein concentration before and after exchange was measured by A280 on a Lunatic instrument using the hIgG-specific E1% value.

Instrumentation Used

• Big Tuna automated buffer exchange system (Unchained Labs)
• Unfilter 96 and Unfilter 24 ultrafiltration plates (10 kDa MWCO)
• Lunatic spectrophotometer for A280 protein quantification

Main Results and Discussion

1. Unfilter 96 (450 µL wells): Total 96.3 % average exchange in 3 cycles (~20 min each), final concentration 10.4 ± 0.2 mg/mL, fill volume 457 ± 9 µL. 3× concentration to 150 µL achieved in one 1 min cycle (final 31.9 ± 2.5 mg/mL). Total runtime: 3.5 h.
2. Unfilter 24 (8 mL wells): 97.7 % average exchange in 3 cycles (~58 min each), final concentration 10.9 ± 0.1 mg/mL, fill volume 8 001 ± 73 µL. 3× concentration to 2 667 µL in two cycles (final 32.2 ± 0.5 mg/mL). Total runtime: 4.4 h.
3. Filter-and-Refill (4 mL wells, 99.6 mg/mL start): Achieved 96.9 % exchange over 12 cycles (~110 min each) with 25 % removal per cycle. Final concentration maintained at 100.9 ± 1.6 mg/mL, fill volume 3 993 ± 25 µL. Runtime: 24.6 h.
4. Dilute-and-Filter (4 mL wells, 99.7 mg/mL start): Initial dilution to 8 mL enabled higher flow rates, achieving 97.0 % exchange in 5 cycles (~115 min each) with 50 % removal per cycle. Final concentration 98.7 ± 1.3 mg/mL, fill volume 4 009 ± 18 µL. Runtime: 11.4 h.

Benefits and Practical Applications

• High throughput: up to 96 low-volume or 24 high-volume samples per run
• Precise control of buffer removal and refill improves consistency across wells
• Flexible workflows accommodate high-concentration proteins by reducing viscosity (dilute-and-filter) or preserving sample volume (filter-and-refill)
• Integrated volume sensing and software-driven cycle adjustment ensure uniform exchange
• Reduces hands-on time and manual variability in formulation development and stability screening

Future Trends and Possibilities

As biologics pipelines expand, automated platforms like Big Tuna can integrate with liquid handlers and analytical instruments for end-to-end workflows. Further enhancements may include:

• On-line monitoring of protein aggregation during exchange
• Adaptive protocols based on real-time viscosity measurements
• Integration with mass spectrometry desalting
• Extension to other macromolecules (nucleic acids, viruses, nanoparticles)

Conclusion

Big Tuna delivers versatile and automated buffer exchange solutions for a range of sample volumes and protein concentrations. By combining pressure-based UF/DF, ultrasonic volume sensing and customizable workflows, it overcomes the limitations of manual methods, offering improved throughput, precision and reproducibility for biologics formulation and stability studies.

Reference

Unchained Labs. (2019). Application Note: Meet Big Tuna: Automated, Versatile Buffer Exchange.