Jackson, N.B.; (2011) Microscale approaches to the rapid evaluation and specification of microfiltration processes. Doctoral thesis , UCL (University College London).

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Abstract

A high throughput method for the study of normal flow microfiltration operations has been established using a custom designed 8-24 well filter plate (0.8 cm2) and a commercial 96-well Multiscreen filter plate (0.3 cm2). Integration of this new approach with a typical robotic platform has enabled automation of the experimental procedure. Membrane resistance data can be quantified using either filter plate. The accuracy of these measurements has helped to determine that plate position does not affect experimental results and applied pressure difference does not vary across either plate. Each of the two filter plate designs has been used to demonstrate that cell condition following fermentation, buffer type and media composition are all important factors influencing the specific cake resistance of E.coli TOP10 cells. The microscale method therefore allows parallel quantification of the impact of upstream process conditions on microfiltration performance. The custom filter plate, optimised for bioprocess studies, allows multiple membrane types to be evaluated on a single plate and the measurement of both permeate and retentate masses to ensure against cross-contamination or loss. Lower variation in specific cake resistance values is seen in the custom filter plate compared with the commercial filter plate. These automated microscale normal flow microfiltration techniques have also been combined with factorial experimentation to identify the key factors and interactions which influence the protein transmission and specific cake resistance during filtration of an E.coli and protein mixture. Results indicated pH and ionic strength were important factors. The pH and ionic strength interaction was further investigated using response surface methodology and a window of operation was generated showing the pH (5.5 ± 0.1) and ionic strength (153 ± 8 mM) values necessary to achieve a protein transmission above 95% and a specific cake resistance below 80 × 1012 m.kg-1. The custom microwell filter plate cake resistance and transmission data from the response surface models scaled up by a factor of 17 to conventional laboratory scale equipment, showing that the optimum conditions achieved in the microwell could be replicated at a larger scale. In addition to this, experiments at the laboratory scale confirmed the optimum indentified by the custom microwell filter plate. This demonstrated that the combination of experimental design and the custom microwell filter plate is capable of investigation, optimisation and scale-up of a complex separation process. Finally, the approaches established here have been expanded to a whole process sequence for the purification of plasmid DNA. A non-chromatographic process sequence, which might be used in industrial practice, involving 7 consecutive processes (4 filtration steps) has been run with 72 combinations of 8 different factors in parallel, collecting hundreds of scaleable data points. The key filtration challenges were identified as the lysis clarification and the removal of lipid removal agent (LRA). Once again the important interactions and trends were identified using microscale experimentation. A major discovery was the filter aid effect of the adsorbent; increasing LRA concentration showed a dramatic reduction in specific cake resistance. This trend was repeated in larger scale devices with different filter formats at high area (150X) and volumetric (2000X) scale-up factors. This shows that the microscale techniques developed in this thesis are capable of determining quantitative, scaleable data for early stage evaluation of whole microwell process sequences.

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