Besenyei, G. C.; Tang, A.; Aylott, J. W.; Chauhan, V. M.

Biopharmaceutical manufacturing requires precise pH control during production to ensure product quality and process performance. However, achieving this precision in low-volume, high-throughput automated settings present significant challenges with existing technology, often leading to inefficiencies and inaccuracies. This study introduces fluorescent nanosensors as a novel solution for accurate pH monitoring in micro-scale environments. Employing ratiometric fluorescence measurement, these nanosensors use analyte responsive and reference fluorophores in inert polyacrylamide matrices to perform dynamic measurements over pH 3.5 - 7.5. Nanosensors (35.26 {+/-} 3.66 nm diameter) were synthesised with a neutral or positive surface charge (-5.08 {+/-} 4.05 mV and +12.87 {+/-} 1.25 mV, respectively). An automated workflow, using sacrificial and at line sample analysis, was developed by integrating the nanosensors with a TECANTM automated liquid handling platform and a fluorescence spectrophotometer. The method was verified using in-process samples obtained from a monoclonal antibody purification, to highlight the compatibility of the nanosensors to different buffer systems in a typical biopharmaceutical manufacturing process. We show that pH-sensitive nanosensors can effectively monitor pH through the various stages of purification, demonstrating high accuracy (pH {+/-} 0.23-0.40) even in sample volumes as low as 12.5 L. The application of nanosensors represents a significant advancement in high-throughput scale-down bioprocess development by enabling precise, automated pH adjustment. This study improves the understanding of biopharmaceutical manufacturing, through the application of fluorescent nanosensors, paving-the-way for optimisations in low-volume and high-throughput product production.