The functionalisation and application of microporous micro-capillary films for the chromatographic purification of biomolecules
Creators: name: Kouyoumdjian, Arthur Jean Michel
Publisher: name: Apollo - University of Cambridge Repository
Year: 2019
Subjects: Chromatography
Micro-capillary films
ion-exchange

description: Microporous walled micro-capillary films (MMCFs) are porous polymer films with embedded capillaries. MMCFs have been found to be suitable low-cost chromatography substrates capable of tolerating high flowrates, which suggested they might be a solution to the growing bottleneck in the downstream purification of biopharmaceuticals. However, MMCFs have been mainly tested for binary separations of model proteins and broader capabilities of this technology remain largely unknown. The experimental work presented in this thesis focused on developing MMCFs functionalised with different ligands and their subsequent testing as chromatography media for bioseparations. MMCFs were functionalised to form weak anion (MMCF-DEAE) and weak cation-exchangers (MMCF-gCM and MMCF-CA). Emphasis was placed on low-cost functionalisation methods amenable to single-use applications. To confirm the addition of functional groups on the membranes, a comprehensive characterisation routine was implemented. This included the use of spectroscopy, electron microscopy, elemental analysis and pH titration. The binding performance of these weak ion-exchangers was further assessed by static and dynamic adsorption of model proteins. Next, the functionalised MMCFs were tested for bioseparations with binary and complex mixtures, the latter being more relevant for industrial applications. It was found that MMCFs could selectively separate similarly charged biomolecules using optimised elution strategies. Further, it was observed that MMCF-gCM could capture lysozyme from chicken egg white at a near twenty-fold purity increase compared to the feed. Similarly, this weak cation-exchanger could recover antibodies from unfiltered mammalian cell lysate. The recovered biomolecules were then injected onto MMCF-DEAE to remove nucleic acid impurities in subtractive chromatography mode. Finally, MMCFs were explored for the first time as affinity chromatography supports. Bovine serum albumin (BSA) and Protein A were covalently coupled to the substrate and tested for the capture of relevant analytes. Indeed, it was found that MMCF-BSA could bind bilirubin and MMCF-ProtA could be used to recover antibodies. Given the high cost of Protein A, a cheaper synthetic ligand was coupled onto MMCFs and observed to successfully bind antibodies. Overall, this work has furthered the applications of MMCFs for bioseparations, demonstrating their great versatility and robustness. Furthermore, it opened the field for affinity-based MMCFs, which could have numerous applications in both research and industry. Extensive characterisation methods presented here will greatly simplify future studies with these membranes. While the binding capacity of the developed ion-exchangers was typically two orders of magnitude higher than non-porous MCFs (NMCFs), the low yield achieved with MMCFs currently precludes them from commercial applications. However, optimisation of MMCFs, as outlined in the future work, could make this support more commercially viable and leverage the numerous advantages offered by its unique geometry.
description: External Research Studentship from Trinity College, University of Cambridge