The Application of Nuclear Magnetic Resonance Towards Understanding Chromatographic Separations of Proteins
Protein chromatography remains indispensable for biomanufacturing, with multiple, orthogonal chromatographic techniques typically employed for the downstream processing of a single protein of interest. However, the mechanisms underpinning chromatographic separations and, consequently, the causes of atypical protein behaviour are not always fully understood. Nuclear magnetic resonance (NMR) can facilitate the direct study of opaque systems, such as intact chromatography columns, in a non-invasive manner under normal operating conditions, with techniques capable of examining intermolecular interactions and mass transfer phenomena, both of which contribute to the overall efficacy of a chromatographic separation. The separation of the monoclonal antibody NIP228 in a 4 mL Poros 50 HS column was used as the model system. Poros 50 HS is a strong cation exchange resin frequently used in industry, with an average particle size of 50 μm and bi-disperse pore distribution of so-called micro- and macro-pores. NMR behaviours of the buffer mobile phase within the chromatography column were first extensively characterised, including the impact of the structure of the packed porous bed, the surface chemistry of the resin, and the flow rate of the mobile phase on NMR relaxation, as well as diffusive transport of the buffer in the absence of flow within the chromatography system. The NIP228 protein sample was then added to the chromatography column, which was observed to generally enhance relaxation of the water molecules of the buffer mobile phase. The WATERGATE pulse sequence was utilised for the suppression of water signal, enabling the NMR behaviours of other protein sample constituents such as sucrose and NIP228 to be studied. It was then demonstrated how the 1H signal intensity of the resin sulfopropyl ligand measured using diffusion-weighting to gate out water signal and the T2 constant of the in-column water molecules could be used to accurately track the bind-and-elute chromatographic separation of NIP228. When studying mass transfer within the system, T2-T2 measurements proved ineffective for characterising the fast exchange of mobile phase water molecules between the inter- and intra-particle regions of the column; however, this behaviour could be studied using NMR propagator measurements, which also facilitated quantification of other transport parameters such as apparent axial and transverse dispersion coefficients. In addition, the impact of protein adsorption to the stationary phase on in-column transport of the mobile phase was studied, as was the effect of subsequent column sanitisation.
Protein chromatography, NMR, In-column interactions, Mass transfer
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