In-line circular dichroism and fluorescence for real-time monitoring of monoclonal antibody structure during affinity...

Future implementation of continuous processing within the biopharma industry is contingent on having real-time analytics and associated process control systems in situ in accordance with Quality by Design (QbD) manufacturing principles. In this work, circular dichroism (CD) and fluorescence (F) are applied as in-line sensors to measure structural changes of a monoclonal antibody (mAb) during acid-induced and thermally mediated elution from affinity chromatography columns. In three case studies in-line CD/F revealed: (i) the extent of unfolding of late eluting protein can be greater than that induced by exposure to acidic buffer alone; (ii) conformational changes can be modulated by supplementing elution buffers with additives; and (iii) thermal elution under isocratic neutral pH binding conditions preserves native-state mAb protein structure. The structural dimension that in-line CD/F measurements provide for chromatographic analysis has the potential to improve our understanding of detrimental unfolding events induced by processing operations. In addition, the speed with which data can be collected and interpreted makes these tools promising candidates for real-time release testing (RTRT) within continuous manufacturing.