Ultra scale-down of protein refold screening in microwells: challenges, solutions and application.
Steps for the refolding of proteins from solubilized inclusion bodies or misfolded product often represent bottlenecks in process development, where optimal conditions are typically derived empirically. To expedite refolding optimization, microwell screening may be used to test multiple conditions in parallel. Fast, accurate, and reproducible assays are required for such screening processes, and the results derived must be representative of the process at full scale. This article demonstrates the use of these microscale techniques to evaluate the effects of a number of additives on the refolding of IGF-1 from denatured inclusion bodies, using an established HPLC assay for this protein. Prior to this, microwell refolding was calibrated for scale-up using hen egg-white lysozyme (HEWL) as an initial model protein, allowing us to implement and compare several assays for protein refolding, including turbidity, enzyme activity, and chromatographic methods, and assess their use for microwell-based experimentation. The impact of various microplate types upon protein binding and loss is also assessed. Solution mixing is a key factor in protein refolding, therefore we have characterized the effects of different methods of mixing in microwells in terms of their impact on protein refolding. Our results confirm the applicability and scalability of microwell screening for the development of protein refolding processes, and its potential for application to new inclusion body-derived protein products.
Mannall
Gareth J
GJ
Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, United Kingdom.
Myers
James P
JP
Liddell
John
J
Titchener-Hooker
Nigel J
NJ
Dalby
Paul A
PA
D004527:Egg Proteins; D007334:Insulin-Like Growth Factor I / Q000737:chemistry* / Q000378:metabolism*; D009113:Muramidase / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding
D004527:Egg Proteins; D007334:Insulin-Like Growth Factor I; D009113:Muramidase