Fast track formulation pH screening with automated buffer exchange on Big Tuna

Significance of the topic

The stability of biologic molecules is critically dependent on buffer conditions, including pH, ionic strength, and excipients. Traditional methods for pH screening and buffer exchange are time-consuming, labor intensive, and prone to variability. Automating buffer exchange accelerates formulation development, improves reproducibility, and enhances throughput for biologics research and development.

Study objectives and overview

This study demonstrates the use of the Big Tuna automated buffer exchange system to perform a pH screening of a monoclonal antibody (mAb). Twelve histidine buffer formulations spanning pH 6.3 to 7.4 were prepared and the mAb was exchanged into each buffer in duplicate wells of an Unfilter 24 plate. The goal was to evaluate system performance, sample consistency, and throughput for high-throughput pH screening.

Methodology and instrumentation

The mAb stock at ~45 mg/mL was loaded into a 10 kDa Unfilter 24 plate with 4 mL per well. Twelve 20 mM histidine buffers (pH 6.3–7.4 in 0.1 increments) were placed on the Big Tuna deck. The buffer exchange protocol targeted 96% total exchange per pool, with 33% volume removed per cycle. The system alternated pressure-driven ultrafiltration, orbital mixing, volume measurement via ultrasonic sensor, and diafiltration. Final volume was set to 4 mL per well to maintain a 45 mg/mL target concentration. Big Tuna Client software managed protocol execution and data export.

Instrumentation Used

• Big Tuna automated buffer exchange system
• Unfilter 24 plate (10 kDa regenerated cellulose filtration)
• Ultrasonic volume sensor integrated in Big Tuna
• Stunner concentration measurement device
• Big Tuna Client software for experimental control and data export

Key results and discussion

All 24 buffer exchange processes were completed in approximately 7.1 hours, with an average cycle time of 21 minutes. Final well volumes averaged 4,004 ± 15 µL, meeting the 4 mL target. Average percent exchange across wells was 97.9% (range 96.5–99.1%), exceeding the 96% target. Final mAb concentrations averaged 45.3 ± 0.3 mg/mL, indicating high recovery and precise volume control. Big Tuna’s real-time adjustment of pressurization cycle duration compensated for flow rate differences, ensuring consistent exchange across all conditions.

Benefits and practical applications

• Significantly reduced hands-on time compared to manual methods
• High reproducibility and uniform sample handling
• Increased throughput for parallel buffer screening up to 24 samples per run
• Real-time control of exchange parameters for precise volume and concentration maintenance
• Adaptability to different sample volumes and membrane formats

Future trends and potential applications

Advancements may include integration of real-time analytical sensors (e.g., UV, fluorescence), expansion to 96-well formats for larger screens, and coupling with machine learning algorithms to predict optimal buffer conditions. Automation platforms like Big Tuna could be applied to broader biologic modalities, formulation stability testing, and high-throughput process development.

Conclusion

The Big Tuna automated buffer exchange system effectively conducted a high-throughput pH screen for a monoclonal antibody, achieving consistent buffer exchange, precise volume control, and high protein recovery. This automation approach streamlines formulation screening, supports informed decision-making in biologics development, and reduces development timelines.