Towards on-column monitoring of protein purification by ATR-FTIR and Raman Spectroscopy

Lead Research Organisation: Imperial College London
Department Name: Life Sciences


Biopharmaceuticals, proteins used as drugs, are an emerging area in the treatment of a range of diseases including rheumatoid arthritis and cancer. While these therapeutics have been shown to be very effective, they are also extremely expensive (~£10,000s/treatment), due to the complex, large scale production processes required. The most expensive step is Protein A affinity capture. Previous research by our group has utilised a specialized technique called Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy to explore the causes of Protein A resin decay. The research outlined in this proposal aims to further characterise binding capacity decay using this approach and also develop the approach to make this a tool that will be useful to scientists in an industrial bioprocessing environment. Our direct monitoring approach will provide much greater control into Protein A purification and has the potential to extend resin life time, saving production costs.
Description We have developed a system which allows us to spectrophotometrically assess changes on resin beads used for antibody purification in real time. Additional analysis of the system is currently underway. We have used this to analyse resin obtained from different parts of a used pilot scale Protein A column used to isolated therapeutic antibodies. The findings show that there is a difference in teh binding capacity loss of the resin as a result of use depending on the position within teh column. Resin at the inlet exhibits a lower binding capacity relative to resin at the outlet. Importantly ATR-FTIR analysis showed that this loss of binding capacity was not due to a loss in Protien A ligand and is likely due to the irreversible binding of contaminants to the resin. Further work using MS analysis has subsequently confirmed that mAB binds irreversibly to the column. A paper describing this analysis has been published (Beattie et al, 2021).
We have completed Raman spectroscopic analysis on the same resin samples. This has revealed differences in the way in which mAb binds to different depths of unused and used resin samples. This analysis confirmed our earlier ATR-FTIR findings regarding reduced binding capacities of used resin and additionally has allowed insight into the precise nature of the irreversibly bound contaminants that result in loss of binding capacity over time. A manuscript describing these results is just in teh final stages of preparation.
Subsequent analysis is applying the Raman approaches developed to the real-world issues of efficient purification of therapeutic mAbs in collaboration with our industrial partner, GSK. We anticipate a further manuscript will result from this study.
Exploitation Route Detection of contaminant binding and leaching from Protein A columns significantly reduce the efficacy of this step in the isolation of theraprutic antibodies. Thus this approach has the potential to be usefully applied in industry settings. We are currently trialling the value of our approaches to industrial purification protocols in collaboration with GSK.
Sectors Education,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

Title Prediction of protein A dynamic binding capacity by confocal Raman spectroscopy 
Description Protein A affinity chromatography is a key step in isolation of biotherapeutics (BT), including monoclonal and bispecific antibodies. Dynamic binding capacity (DBC) analysis assesses how much BT will bind to a protein A column. Importantly the DBC reduces with column usage, effectively reducing the amount of recovered product overtime. Drug regulatory bodies mandate specific DBCs for BT isolation and so this feature is carefully monitored in industrial purification pipelines. HPLC, that reports on the concentration of BT loaded at which there is significant breakthrough of BT in the flowthrough from the column is normally used. HPLC gives accurate assessment of DBC and how this changes over time but only reports on protein concentration, requires calibration for each new BT analysed and is an off-line technique. Our study utilised Raman spectroscopy, and revealed that this approach is at least as effective as both HPLC and UV chromatogram methods at monitoring DBC of Protein A resins. In addition to reporting on protein concentration, the chemical information in the Raman spectra provides information on the aggregation status and protein structure, providing an extra quality control to any industrial bioprocessing pipeline. Importantly in combination with PLS analysis, Raman spectroscopy can be used to determine DBC of a BT without any prior calibration. Here we performed the Raman analysis off-line in a 96-well plate format, however it is eminently feasible to include this in-line removing the need for sampling. We have clearly demonstrated the power of Raman spectroscopy as a significantly improved approach to DBC monitoring in industrial pipelines. 
Type Of Material Technology assay or reagent 
Year Produced 2023 
Provided To Others? No  
Impact The manuscript describing this approach is currently being drafted and will be submitted soon. No impact as yet but we anticipate that this approach will streamline monitoring of dynamic binding capacities in industrial biotherapeutic pipelines and make monitoring the quality of the product more effective. 
Description Collaboration with GSK 
Organisation GlaxoSmithKline (GSK)
Country Global 
Sector Private 
PI Contribution We will supervise a BBSRC/GSK funded PhD student to continue aspects of the work started during the BRIC project
Collaborator Contribution They have provided the studentship funding and will also host the student for periods of time during the PhD.
Impact None as yet, the student only starts in Oct 2018.
Start Year 2018