Circular dichroism and fluorescence were used to create a platform for monitoring changes in monoclonal antibodies.
University of Birmingham (England) researchers led efforts to create a continuous platform for measuring structural changes in monoclonal antibodies and similar laboratory-made proteins. Their procedures and findings were published in the Journal of Chromatography A (1).
Monoclonal antibodies (mAbs) are laboratory-made proteins created to stimulate someone’s immune system (2). A key part of mAbs’ success has been the near-uniform approach of their manufacture, thus allowing biopharmaceutical companies to adopt robust platform technologies for industrial scale production and purification. Economic pressures from biosimilars and emerging economies, alongside regulatory guidance to modernize manufacturing methods, have led industry personnel to developed more streamlined and efficient processing operations.
Continuous platforms, which are built off a software development strategy where code changes to an application are released automatically into a production environment, have become a popular approach for processing systems (3). These types of platforms are believed to offer new opportunities for increasing process productivity, significantly reducing facility footprints, and supplying smaller patient populations. However, industry has been hesitant to use these processing methods, mainly due to challenges that come with enacting suitable process monitoring and control. One key test involves managing product critical quality attributes (CQAs), of which there can be up to 30, over a prolonged period within a highly automated and inter-connected operation. With continuous flow operations, there is an inherent risk that disturbances, if not detected in time, might propagate from one unit operation to the next. This new process control burden shows an existing technology gap for process analytical technologies (PAT) capable of directly and autonomously measuring product CQAs from process streams in “real-time.” As such, there is a need for new PATs with the potential to feed into overarching control strategies.
In this study, circular dichroism (CD) and fluorescence (F) were applied as in-line sensors to measure structural changes of a mAb during acid-induced and thermally mediated elution from affinity chromatography columns. CD is the difference in absorption of left and right circularly polarized light, typically with a solution containing the molecules of interest (4). In-line CD detection during high-performance liquid chromatography (HPLC) found its main application in distinguishing enantiomeric peaks and determining purity during the synthesis of optically active chemicals. Alongside F, in-line CD can offer an unexplored platform for observing unfolding events that can occur during elution from Protein A capture chromatography columns.
The researchers converted a high-throughput capillary-based CD/F system designed for bioprocess development tasks into an in-line CD/F detector for monitoring conformational changes of an IgG1 mAb as it elutes from chromatography columns. This exploits the system’s exploiting near-ultraviolet (UV) CD negative exciton couplet feature around 230 – 240 nm8 and fluorescence wavelength maxima as reporters of mAb structural integrity. Control experiments under non-binding conditions were used to validate in-line CD/F data acquisition and show unequivocally that CD/F measurements distinguish different folding states of eluted mAb. The utility of in-line CD/F for reporting transient changes in mAb structure during chromatographic elution from different affinity chromatography media was subsequently demonstrated in three case studies examining the: influence of buffer pH; use of additives; and benefits of eluting mAb by means of a change in temperature under isocratic neutral pH binding conditions.
The method of applying in-line CD and F was deemed to successfully applied to monitor mAbs during affinity purification. The techniques’ abilities to distinguish folding states across an elution profile show that, far from being homogenous, column eluates may contain an array of non-native species that have undergone variable degrees of unfolding. Further, in cases where harsh elution conditions are used, the degree of structural deformation presented by late eluted mAbs can exceed that caused by the acidic buffer alone. There is great potential for applying in-line structural analysis in assessing the processing implications of various elution buffers, resins and mAb species during development stages. These tools can further existing trends and create new ones, while also being extendable to applications for investigating other continuous processing formats.
(1) Moore-Kelly, C.; Rodger, A.; Dafforn, T. R.; Thomas, O. R. T. In-line Circular Dichroism and Fluorescence for Real-Time Monitoring of Monoclonal Antibody Structure During Affinity Chromatography. J. Chromatogr. A 2025, 1757, 466153. DOI:
(2) Monoclonal Antibodies. Cleveland Clinic 2025.
(3) Susnjara, S.; Smalley. I. What is Continuous Deployment? IBM 2024.
(4) Rodger, A.; Marrington, R.; Roper, D.; Windsor, S. Circular Dichroism Spectroscopy for the Study of Protein-Ligand Interactions. Methods Mol. Biol. 2005, 305, 343–364. DOI:
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