Multi-functional chromatography materials: new designs & applications

Joseph, Stephan (2019). Multi-functional chromatography materials: new designs & applications. University of Birmingham. Ph.D.

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Abstract

Process chromatography proves to be a continual bottleneck in bioprocessing, especially when considered against constraints such as rocketing product titres, complexity and size of emerging bio-products and a drive to reduce cost of goods and waste generation. Incremental changes in mono-functional chromatography media do not appear to be sufficient in coping with these increased demands and opens the door for novel ‘multifunctional’ beaded media, designed from the ground up, to posit a solution.
This work will, in part, demonstrate the creation of bi-layered chromatography resins with size-exclusion and anion exchange functionality and subsequently assess their performance against benchmarked commercial beads of similar structure. Three size exclusion chromatography matrices, differing in agarose content, cross-linker chemistry, particle and pore size distributions, were transformed into bi-layered supports featuring anion exchange functionalised cores and exterior size excluding shells.
The media were evaluated with respect to loss of surface and core binding, utilising finite bath studies allied with confocal scanning laser microscopic imaging. Plasmid DNA, bovine serum albumin and bovine serum albumin nanoparticles were utilised as surface and core binding probes, and the binding selectivities of different bi-layered support materials were compared by means of a simple selectivity ratio (μg of pDNA (or NP) bound per mg of BSA bound); the lower the number the more selective the support. The best performing bi-layered matrix was Superose 6 Prep Grade, when challenged with both plasmid DNA and BSA nanoparticles. Results for other base matrices was dependent on the nanoplex challenge as well as the bi-layering technique employed.
A second challenge addressed in this thesis was the acquisition of representative virus feedstock to road-test chromatography media, which can pose several practical issues with respect to requirements of particle concentration, volume and methods for tracking the species through chromatography media. A major focus has been developing a reliable platform in characterising commercial and in-house manufactured mono-functional and multi-functional chromatography resins, especially in the context of purifying large nanoplexes.
Against this backdrop, this work details a method for the reproducible manufacture of protein nanoparticles using a modified desolvation method; the nanoparticles function as surrogate mimics of virus products with a view to characterising viral chromatographic media.
Initially, BSA nanoparticles were explored and their manufacture optimised to develop a robust and reproducible system for creating defined nanoparticles within a defined size range. The nanoparticles were subsequently characterised for efficiency of manufacture, their morphological appearance, and secondary protein structure composition as well as how well they represented a bona fide viral species, namely adenovirus type V. The approach was then translated to other readily available proteins (e.g. lysozyme, bovine haemoglobin and ovalbumin) to determine the flexibility of the desolvation/coacervation approach and to target a wider range of viral species in terms of their physiochemical properties. The concept here was to develop a “toolbox approach” to develop custom-made nanoparticles to target a variety of viral species. Similar characterisation techniques were applied to the newly formed nanoparticles, as well as novel strategies applied to develop “second-generation nanoparticles” using alternative cross-linking mechanisms to produce multi-component nanoparticles.
The final chapter brings the work full-circle and looks to road-test the chromatography materials manufactured in Chapter II, with a range of sizes of BSA nanoparticles described in Chapter III. The nanoparticles are qualitatively assessed using confocal microscopy alongside quantitative assessment to determine bead performance. As in Chapter II, BSA protein is utilised as a core-binding probe. The nanoparticles are also applied to commercial resins, Q-Sepharose Fast Flow and Capto™ Core 700, both in batch and column scenarios. Of the bi-layered materials, Superose 6 Prep Grade generally outperformed both Sepharose 6 Fast Flow and Superose 12 Prep Grade, revealing differences between the media not shown by the plasmid DNA binding studies. This was generally supported by the complimentary confocal data, which provided an insight into the spatial location and binding kinetics when challenged onto the beads.
It is envisioned that the chromatographic supports manufactured here may be utilised in large scale nanoplex purification, exploiting their utility derived from their bi-functional modality. Furthermore, it is hoped that the protein nanoparticle-viral mimic methodology may be employed in the characterisation of a wider range of purification materials, taking advantage of their low cost and rapid manufacture, as well as their ability to provide valuable purification data which may inform important bioprocessing decisions.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):