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 i...

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Bibliographic Details
Published in:Biotechnology and bioengineering 2009-06, Vol.103 (2), p.329-340
Main Authors: Mannall, Gareth J, Myers, James P, Liddell, John, Titchener-Hooker, Nigel J, Dalby, Paul A
Format: Article
Language:English
Subjects:
Biological and medical sciences
Biotechnology
Egg Proteins
Fundamental and applied biological sciences. Psychology
high-throughput screening
IGF-1
Insulin-Like Growth Factor I - chemistry
Insulin-Like Growth Factor I - metabolism
lysozyme
mixing
Muramidase - chemistry
Muramidase - metabolism
Protein Conformation
Protein Folding
refolding
ultra scale-down
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Summary: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. Biotechnol. Bioeng. 2009;103: 329-340.
ISSN:0006-3592
1097-0290
DOI:10.1002/bit.22245