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Ice recrystallisation inhibiting polymers prevent irreversible protein aggregation during solvent-free cryopreservation as additives and as covalent polymer-protein conjugates
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Fayter, Alice E. R., Hasan, Muhammad, Congdon, Thomas R., Kontopoulou, Ioanna and Gibson, Matthew I. (2020) Ice recrystallisation inhibiting polymers prevent irreversible protein aggregation during solvent-free cryopreservation as additives and as covalent polymer-protein conjugates. European Polymer Journal, 140 . 110036. doi:10.1016/j.eurpolymj.2020.110036 ISSN 0014-3057.
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Official URL: https://doi.org/10.1016/j.eurpolymj.2020.110036
Abstract
Protein storage and transport is essential to deliver therapies (biologics), enzymes for biotechnological applications, and underpins fundamental structural and molecular biology. To enable proteins to be stored and transported it is often essential to freeze them, requiring cryoprotectants such as glycerol or trehalose. Here we explore the mechanisms by which poly(vinyl alcohol), PVA, a potent ice recrystallisation inhibitor protects proteins during freeze/thaw to enable solvent-free cryopreservation with a focus on comparing mixing, verses polymer-protein conjugation. A panel of poly(vinyl alcohol)s are investigated including commercial, well-defined (from RAFT), and PVA-protein conjugates, to map out PVA’s efficacy. Enzymatic activity recovery of lactate dehydrogenase was found to correlate with post-thaw aggregation state (less aggregated protein had greater activity), which was modulated by PVA’s ice recrystallisation inhibition activity. This macromolecular cryoprotectant matched the performance of glycerol, but at lower additive concentrations (as low as 1 mg.mL−1). It was also demonstrated that storage at −20 °C, rather than −80 °C was possible using PVA as a cryoprotectant, which is not possible with glycerol storage. A second protein, green-fluorescent protein (GFP), was used to enable screening of molecular weight effects and to obtain PVA-GFP bioconjugates. It was observed that covalent attachment of RAFT-derived PVA showed superior cryoprotectant activity compared to simple mixing of the polymer and protein. These results show that PVA is a real alternative to solvent-based protein storage with potential in biotechnology, food and therapeutics. PVA is already approved for many biomedical applications, is low cost and available on a large scale, making it an ideal cryoprotectant formulation enhancer.
Item Type: | Journal Article | |||||||||||||||||||||
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Subjects: | Q Science > QD Chemistry Q Science > QR Microbiology R Medicine > RS Pharmacy and materia medica |
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Divisions: | Faculty of Science, Engineering and Medicine > Science > Chemistry Faculty of Science, Engineering and Medicine > Medicine > Warwick Medical School |
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Library of Congress Subject Headings (LCSH): | Biologicals, Cryobiology, Polyvinyl alcohol | |||||||||||||||||||||
Journal or Publication Title: | European Polymer Journal | |||||||||||||||||||||
Publisher: | Pergamon | |||||||||||||||||||||
ISSN: | 0014-3057 | |||||||||||||||||||||
Official Date: | 5 November 2020 | |||||||||||||||||||||
Dates: |
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Volume: | 140 | |||||||||||||||||||||
Article Number: | 110036 | |||||||||||||||||||||
DOI: | 10.1016/j.eurpolymj.2020.110036 | |||||||||||||||||||||
Status: | Peer Reviewed | |||||||||||||||||||||
Publication Status: | Published | |||||||||||||||||||||
Access rights to Published version: | Open Access (Creative Commons) | |||||||||||||||||||||
Date of first compliant deposit: | 28 October 2020 | |||||||||||||||||||||
Date of first compliant Open Access: | 16 November 2020 | |||||||||||||||||||||
RIOXX Funder/Project Grant: |
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