Use this URL to cite or link to this record in EThOS:
Title: Chromatography resin characterisation to analyse lifetime and performance during biopharmaceutical manufacture
Author: Nweke, M. C.
ISNI:       0000 0004 7229 2452
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2018
Availability of Full Text:
Access from EThOS:
Full text unavailable from EThOS. Please try the link below.
Access from Institution:
This thesis, completed in collaboration with Eli Lilly & Co., aims to understand and assess the structural and mechanical changes that occur as agarose-based chromatography resins are exposed to different bioprocessing conditions in an attempt to explore the mechanisms by which different resins age. By understanding this, there is significant potential for facilitating timely and improved decisions in large-scale chromatographic operations, maximising resin lifetime whist maintaining acceptable column performance. Scanning electron microscopy (SEM) was used to image the structural properties of nine widely used agarose-based chromatography resins before use while pressure-flow analysis was used to characterise the mechanical properties of the same fresh resins. The results showed that the Capto family had the highest critical velocities (Capto Adhere- 492, Capto Q- 477 cm/hr), whilst Sepharose CL-6B, Sepharose 4 Fast Flow and Sepharose CL-4B had the lowest critical velocity values (283, 204, 149 cm/hr respectively). There were practical limitations in using the pressure-flow technique alone to for mechanical characterisation, including the large quantity of chromatography resin and buffers and the stringent criteria required to pack a column. Dynamic mechanical analysis (DMA) was therefore developed as a novel technique in this field to address these limitations and allowed for further mechanical characterisation based on the viscoelastic properties using 1ml of resin. The technique was applied to the nine studied resins and correlated with the results obtained using the pressure-flow technique. The same trends were observed – The Capto family showed the highest resistance to deformation (Capto Adhere- 2.7, Capto Q- 1.92 1/%min-1) through to Sepharose CL-6B, Sepharose 4 Fast Flow and Sepharose CL-4B which exhibited the lowest slurry resistances (0.59, 0.4, 0.3 1/%min-1 respectively). These results indicate that DMA can be used as a small volume, high-throughput technique, relative to pressure-flow analysis, for the mechanical characterisation of chromatography media. The structural imaging and mechanical testing tools developed in this study were then applied to measure changes in resins that had undergone lifetime studies. These studies expose the resins to repeated cycles of use to understand how they age in a particular bioprocess, enabling decision making about their use. The first set of experiments exposed the resins to the cleaning cycle only, whilst in the second set of experiments, the resins had been used for lifetime studies in the production of monoclonal antibodies (termed ‘aged’ resins). The results indicated that MabSelect (highly cross-linked protein A resin) and Q-Sepharose High Performance (anion exchange cross-linked resin) appeared to show similar mechanisms of aging. Their matrices showed agarose fibre breakage with increased exposure to process conditions. In the case of Capto Adhere (highly cross-linked multimodal anion exchange resin) and MabSelect Xtra (highly cross-linked protein A resin), the mechanism of aging appeared to be associated foulants coating the surface fibres. The results indicate that the interaction of CIP reagents and foulants (as opposed to CIP reagents alone) cause the greatest impact on the structural integrity of the resins. Pressure-flow and DMA characterisation were used to examine the mechanical properties of the cycled resins to provide the first systematic study of these issues. The results showed that fresh resins were consistently more robust than either of the cycled resins but the greatest mechanical differences were observed between fresh resins and aged resins, which corroborated the structural analysis data. Statistical analysis was performed with one way ANOVA to determine whether DMA could be independently used to assess the impact of process conditions on the mechanical properties of chromatography media and the results show a >80% certainty that DMA can be employed for this purpose.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available