Characterizing Chromatography Media : NMR-based Approaches

Abstract: Liquid chromatography is an essential technique in manufacturing biopharmaceuticals where it is used on all scales from analytical applications in R&D to full-scale production. In chromatography the target molecule, typically a protein, is separated and purified from other components and contaminants. Separation is based on different affinities of different molecules for the chromatographic medium and the physical and chemical properties of the latter determine the outcome. Controlling and designing those properties demand efficient analytical techniques.In this thesis the approach was to develop characterization methods based on nuclear magnetic resonance (NMR) spectroscopy for the assessment of various important physico-chemical properties. The rationale behind this strategy was that the versatility of NMR – with its chemical and isotopic specificity, high dynamic range, and direct proportionality between the integral intensity of the NMR signal and the concentration of spin-bearing atomic nuclei (e.g., 1H, 13C, 31P and 15N) – often renders it a very good choice for both qualitative and quantitative evaluations.These characteristics of NMR enabled us to develop two quantification methods for chromatography-media ligands, the functional groups that provide the specific interactions for the molecules being separated. Furthermore, a new method for measuring the distribution of macromolecules between the porous chromatographic beads and the surrounding liquid was established. The method, which we have named size-exclusion quantification (SEQ) NMR, utilizes the fact that it is possible to assess molecular size distribution from corresponding distribution of the molecular self-diffusion coefficient where the latter is accessible by NMR. SEQ-NMR results can also be interpreted in terms of pore-size distribution within suitable models. Finally, we studied self-diffusion of small molecules inside the pores of chromatographic beads. The results provided new insights into what affects the mass transport in such systems.The methods presented in this thesis are accurate, precise, and in many aspects better than conventional ones in terms of speed, sample consumption, and potential for automation. They are thus important tools that can assist a better understanding of the structure and function of chromatography media. In the long run, the results in this project may lead, via better chromatographic products, to better drugs and improved health.