Nealon, A.J.; (2006) Microwell methods for fast process development. Doctoral thesis , University of London.

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Abstract

The combination of automated liquid handling robots (LHR) and microwell plates present a possible methodology for processing the large number of potential drug targets produced by drug discovery and combinatorial chemistry programs. Many biological processing operations do not require harsh physical environments and can therefore easily be completed in standard, off-the-shelf microwell supports. The key questions for automated microscale bioprocessing relate to the reproducibility and robustness of each unit operation carried out in a standard microwell support. The utility of an automated liquid handling robot integrated with a microwell plate reader to enable the rapid acquisition of bioprocess kinetic data has been investigated. The relationship between the key parameters for liquid handling accuracy and precision and the sample detection period has been characterised for typical low viscosity (<2.0mPas) aqueous and organic phases and for a high viscosity aqueous phase (60mPas), all exhibiting Newtonian rheology. The use of a simple graphical method enables the suitability of a given automation platform to be assessed once these key parameters have been determined. Leading to a priori prediction of well usage within each microwell plate to maximise productivity. Macro-mixing is commonly ignored by many laboratories when using microwell supports for analytical assays and bioprocess development. A microwell combined with a LHR is modelled using standard jet mixing theory. An inert food dye was used in conjunction with high-speed video technology to capture the stages of macro-mixing. Correlations have been produced for both 96-standard and 96-deep microwells. Thus allowing the prediction of the minimum addition volume needed to complete macro-mixing in a static micro well. Nficro-mixing conditions with a reaction vessel have been shown to effect the product distribution in situations where multiple reaction pathways are available. An assessment of micro-mixing conditions has been made using a competitive chemical reaction system and response surface methodology techniques. The key parameters influencing the micromixing efficiency have been identified for each reactor configuration. Engineering design equations have also been produced allowing the prediction of the micromixing environment from initial operating conditions. Common micromixing efficiencies have been highlighted as a possible scale-up methodology. A number of case studies demonstrating the applicability and implications of each of the three systems on the generation of bioprocess development data have been completed. These have highlighted the effects of reaction rates on the quality of data generated while measuring reaction kinetics, the need to complete macro-mixing within the jet lifespan to ensure quantitative data is produced and strategies for counteracting the effects of poor macro-mixing when high reagent costs prevent the completion of macro-mixing within the jet lifespan. A study of the effects of micromixing efficiency on plasmid DNA yield and quality during the chemical lysis of bacterial cells has also been detailed. This has demonstrated that chemical denaturation of plasmid DNA has an influence on the yield and quality of the plasmid and the micromixing efficiency plays a role in the determination of the denaturation rate. Caveats have also been discussed to suitability of using micromixing efficiency as a scale-up tool from the microscale to bench and pilot scales.

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