Water Proton Flow-NMR—A Novel Tool for Real-Time In-Line Process Monitoring in Biomanufacturing

Abstract

Due to serious limitations, such as experiment duration, relaxation parameters, flow-NMR has limited applications to study structure and properties of a solute, especially, in the case of biomacromolecules, or in the manufacturing of biologics. Unlike conventional structural applications of NMR spectroscopy targeted towards complex solute biomacromolecules, its approaches directed to study the interactions between solute and solvent are less explored. However, in aqueous solutions, water molecules bear useful information about the state of solute molecule, in particular, biomacromolecules, e.g., proteins, actively interacting with water molecules. We have already demonstrated that water proton transverse relaxation rate, R2(1H2O), is a powerful indicator of protein aggregation sensitive to a wide range of aggregate sizes. Less is known about flow-NMR potential to explore water-proton interactions. Meanwhile, due to generally high water concentration in aqueous solutions of biomacromolecules and long relaxation times of water, many existing limitations of flow-NMR are lifted when it comes to study water-protein interactions mediated by relaxation. We report here the results obtained by a newly designed low-field benchtop flow-NMR instrument, which can monitor R2(1H2O) within the flow rates ranging from 0 to 50 mL/min. In our experiments, we demonstrated that R2(1H2O) in a flow depends on a flow rate in accordance with theoretically predicted residence time component. We also showed the sensitivity of water flow-NMR to the pH and ionic strength/conductivity of buffered aqueous solutions. R2(1H2O) was also found sensitive to the concentration of proteins in solution in the range from 0 to 50 mg/mL and flow rates from 0 to 50 mL/min. Our results pave the way for the application of water-proton flow-NMR as a noninvasive process analytical technology in continuous biomanufacturing. In a product flow, this unique technique could detect in-line concentration changes and protein aggregates.