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

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