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A scaledown system for the rapid development of commercial inclusion body protein refold steps

Myers, James Philip; (2004) A scaledown system for the rapid development of commercial inclusion body protein refold steps. Doctoral thesis (D.Eng), UCL (University College London). Green open access

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Many therapeutic proteins, currently in development or manufacture, are expressed as inclusion bodies. Proteins must be refolded from the inclusion body into its active form. This refolding step can be difficult to develop and can give a low yield. This project was designed, in partnership with Avecia Biologies, with the purpose of improving the way in which commercial protein refold steps are developed in order to reduce development costs, manufacturing costs and time-to-market of new protein pharmaceuticals. Development of protein refold steps for commercial manufacture was investigated. Methods for quantifying refolding yield were evaluated. It was determined that refold development experiments could best be improved by decreasing the amount of material required for experiments, increasing throughput of the experiments and designing the experiment efficiently. Techniques for performing refold reactions in microwells were developed and their usefulness was demonstrated with experiments to improve a refold step for IGF-1. Experiments using only 60mg of protein gave a refold step yield of 50% (up from 26%) and a 4-fold decrease in refold volume, using 20%v/v propylene glycol, 0.25M arginine, 0.25M GdnHCI as refold buffer additives. Techniques were developed for performing rapid refold development experiments using laboratory automation, in particular a pipetting robot. The efficiency of automated microwell refold reactions was demonstrated with refold optimisation experiments, using lysozyme as an example protein. These experiments, which used only 130mg of protein and could be performed by one scientist in just two days, identified refold conditions giving a 58% increase in step yield. Another automated microwell-scale refold experiment using trypsinogen as the example protein established (in 1 day, using 3.1 mg protein) that a 13% increase in yield could be achieved by including 20mM PEG300 and lOmM pentanol in the refold buffer. The application of these techniques in a commercial bioprocess development laboratory was further considered.

Type: Thesis (Doctoral)
Qualification: D.Eng
Title: A scaledown system for the rapid development of commercial inclusion body protein refold steps
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Pure sciences; Applied sciences; Protein refold steps
URI: https://discovery.ucl.ac.uk/id/eprint/10099116
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