Daniel E.
Mitchell‡
a,
Alice E. R.
Fayter
a,
Robert C.
Deller
a,
Muhammad
Hasan
a,
Jose
Gutierrez-Marcos
b and
Matthew I.
Gibson
*ac
aDepartment of Chemistry, University of Warwick, Coventry, CV47AL, UK. E-mail: M.i.gibson@warwick.ac.uk
bSchool of Life Sciences, University of Warwick, Coventry, CV47AL, UK
cWarwick Medical School, University of Warwick, CV47AL, UK
First published on 8th November 2018
Proteins are ubiquitous in molecular biotechnology, biotechnology and as therapeutics, but there are significant challenges in their storage and distribution, with freezing often required. This is traditionally achieved by the addition of cryoprotective agents such as glycerol (or trehalose) or covalent modification of mutated proteins with cryoprotectants. Here, ice recrystallization inhibiting polymers, inspired by antifreeze proteins, are used synergistically with poly(ethylene glycol) as an alternative to glycerol. The primary mechanism of action appears to be preventing irreversible aggregation due to ice growth. The polymer formulation is successfully used to cryopreserve a range of important proteins including insulin, Taq DNA polymerase and an IgG antibody. The polymers do not require covalent conjugation, nor modification of the protein and are already used in a wide range of biomedical applications, which will facilitate translation to a range of biologics.
Conceptual insightsThe current method used in biochemical, molecular biology, protein engineering and therapeutic labs for freezing proteins is often based on adding organic solvents to modulate ice formation and stabilise the proteins. Our work takes a disruptive approach, using antifreeze-protein mimetic polymers, which are extremely potent inhibitors of ice growth, to enable protein cryostorage and eliminate the need for organic solvents whilst recovering active protein. In recent years covalent polymer/protein conjugates have been successfully used to freeze proteins, but this can be complex, reduce protein function and crucially generates a new molecular entity which must be tested. In our unique approach, we identified that irreversible protein aggregation due to ice crystal growth is a major cause of cryo-damage and that if we prevent this, the proteins retain activity. We have formulated polymer additives which modulate ice growth and enable quantitative recovery of a range of important proteins without needing conjugation. This work shows that biomimetic materials chemistry can be applied to a real clinical problem to generate unique solutions which will enable new therapies. |
Antifreeze (glyco)proteins (AF(G)P)s from polar fish provide protection against ice formation and growth in hypothermal conditions and have the most potent ice recrystallization inhibition (IRI) activity known.24 Synthetic IRI-active compounds developed by Ben et al.25 have been shown to enhance cellular recovery during cryopreservation.26 Gibson and co-workers have developed synthetic polymers as mimetics of AF(G)Ps,27,28 which have potent IRI activity and can be employed in solvent-free cryopreservation.29–32 The most potent IRI-active polymer to date is PVA (poly(vinyl alcohol)). PVA is a particularly appealing additive as it is widely used in pharmaceutical formulations and is an FDA approved food additive.33 Mitchell et al. found that the reversible aggregation of gold nanoparticles during freezing could be modulated by addition of PVA.34 By slowing the rate of ice growth, the effective surface area of the ice crystals is increased (i.e. more small crystals) and hence the nanoparticles could not approach each other to aggregate. We therefore hypothesized that IRI-active compounds might prevent protein aggregation during ice-growth induced stress, which normally leads to denaturation/deactivation. This effect could in turn be exploited in the solvent-free storage proteins.
β-Gal was frozen at −20 °C for 3 days in the presence of trehalose (as a positive control), PVA or various polymers (HES, PVP, PEG) which are known to have no IRI activity.28,29 Activity was tested after thawing at 20 °C (Fig. 1D).
As expected, trehalose protected β-Gal activity during freezing.9 All other additives failed to protect when used individually, apart from PEG which gave some protection (Fig. 1D). It should be noted that PEG has known cryoprotectant properties at sufficiently high concentrations, making it the ideal choice for a synergistic cryoprotectant.36 When PEG (100 mg mL−1) and PVA (1 mg mL−1) were combined, a synergistic cryoprotective effect was observed, reaching values equivalent to trehalose (Fig. 1D). The combination of PEG/PVA appears to be unique since PVP/PVA mixtures were no different from individual additives. Variable concentration studies (ESI†) show that the PEG concentration could be lowered (at constant [PVA]) to as low as 50 mg mL−1 (∼5 wt%) without affecting recovery, but below 30 mg mL−1 there was no protection. We have previously reported that a secondary water soluble polymer is required when using IRI-active polymers for cellular cryopreservation, agreeing with the observations here.29 PEG has widely been found to have a stabilization effect on various proteins, and is used to improve delivery in pharmaceutical applications.37 Several studies suggest that PEG chains can interact with the protein surface reducing solvent accessible area,38 and prevent unfolding and aggregation through molecular crowding.39 These stabilization effects would also be useful in cryopreservation, preventing aggregation caused by reduced liquid water volume and any denaturing caused by low temperatures.
We hypothesised that the role of PVA in enhancing protein cryostorage may be due to its IRI-activity. To test this hypothesis, we tested another IRI-active polymer (p(ampholyte)) developed in our laboratory. Because p(ampholyte) is less IRI active, we employed a higher concentration to achieve equal IRI activity comparable to that of PVA at 1 mg mL−1. Under identical conditions, we found that p(ampholyte) and PVA displayed identical cryoprotectant properties (Fig. 2A). This data corroborates the hypothesis that ice growth is a cause of protein deactivation and that the rational design of IRI-macromolecules may be an effective strategy for the discovery of new protein stabilizers and be complementary to small molecular cryoprotectants which function by different mechanisms. To investigate if preventing protein aggregation was a critical factor for protein cryoprotection, we employed dynamic light scattering (DLS). When β-Gal was freeze/thawed in phosphate-buffered saline (PBS) alone, large aggregates could be seen (>500 nm in diameter) (Fig. 2B). Similarly, large aggregates were also observed when only PVA polymers were employed. Conversely, PEG/PVA mixtures prevented all freeze-induced aggregation (Fig. 2B).
Fig. 2 (A) Protein freeze/thaw recovery upon addition of p(ampholyte) following β-Gal storage for 3 days at −20 °C, as % of fresh, unfrozen protein. Concentrations as in Fig. 1, but p(ampholyte) used at 30 mg mL−1. (B) DLS analysis of β-Gal aggregation post freezing compared to fresh protein (black line). (C) β-Gal activity after 1 hour incubation in the cryostorage solutions without freeze/thaw; (D) β-Gal recovery after 4 weeks storage at −80 °C. Error bars are S. D. from minimum of 3 repeats. |
Our bio-inspired macromolecules have some advantages over small-molecule cryoprotectants as they have low cytotoxicity, high biocompatibility (FDA approved/GRAS status for several applications),33,40 and at equal mass concentrations have lower molar concentration thus imposing less osmotic stress. To confirm that our polymers are passive additives, we incubated β-Gal with different concentrations of cryoprotectants and tested activity (Fig. 2C). Glycerol solutions significantly impaired protein function or the assay output, but PEG, PVA and trehalose had little impact on protein function, thus indicating that they are biologically inert and may not need removing before use, providing an alternative to solvent based approaches, where dialysis is typically required post-thaw.
Since under standard laboratory conditions proteins are normally stored at −80 °C through long periods, we decided to test the levels of protein activity at this temperature. The PEG/PVA formulation enabled recovery of β-Gal activity comparable to that of trehalose (Fig. 2D).
To ensure these observations were not unique to a single enzyme we set out to study a range of other proteins using this methodology. Glucose oxidase (GOx) is widely used in sensing, food industry and in molecular biology41 whilst hyperthermophylic DNA polymerase from Thermus aquaticus (Taq) is commonly used in diagnostics and research for the amplification of DNA through the polymerase chain reaction (PCR) that underpins modern genomic analyses.42 We found that GOx was relatively stable after freeze/thawing in only PBS, however, addition of IRI-active polymers did increase the recovered enzymatic activity (Fig. 3A). To assess the activity of Taq after freeze/thawing, we used quantitative PCR (qPCR) and determined the number of cycles to reach a threshold level; fewer cycles means more activity. We observed that PEG/PVA improved stability to the extent that it was comparable to fresh recombinantly-expressed enzyme (Fig. 3B).
Encouraged by the above results we proceeded to more closely reproduce laboratory or clinical settings where protein samples are often removed and replaced from/into a freezer, a freeze/thaw cycle assay was developed as a robust test of the technology. To enable continual monitoring of the same sample through many freeze/thaw cycles, recombinant green fluorescent protein (GFP) was used. Upon denaturation, the fluorescence decreases providing a convenient readout. Fig. 3C shows the recovery of fluorescence following 6 freeze (−20 °C) and thaw (27 °C) cycles. For PEG alone, there was a dramatic reduction in fluorescence with only 15% function retained after 6 cycles. Addition of 1 mg mL−1 PVA to PEG was found to be optimal, enabling >75% activity retention after 6 freeze/thaw cycles. Higher concentrations of PVA were found to be detrimental as was the use of higher molecular weight PVA (23 kDa) (Fig. 3D). We hypothesize that this is due to dynamic ice shaping – a common side effect of antifreeze proteins, which is known to compromise cell cryopreservation.32,43 This highlights the delicate balance of IRI-activity with ice shaping and that the actual polymer composition is crucial to success.
To determine if this methodology could be used for therapeutic proteins as well as those described above, an antibody (rabbit IgG) and insulin were both tested. Antibodies are widely used in diagnostics (e.g. ELISA) and molecular biology as well as in emerging therapeutics, but maintaining their function upon storage is challenging. Rabbit IgG purified extract was stored at −20 °C for 3 days and function determined using an ELISA-based assay. As with all the other proteins tested the PEG/PVA mixture greatly enhanced IgG activity (>80%) and the recovery level was superior to that of trehalose (Fig. 4A). Insulin, a biologic therapeutic essential for managing diabetes, is deactivated upon liquid storage by simple agitation or by irreversible aggregation. Dynamic light scattering was therefore employed to probe the prevention of irreversible insulin aggregation upon freeze/thaw using a range of conditions (Fig. 4C). Again the PEG/PVA formulation prevented aggregation more so than PVA alone. Different molecular weight PEGs were considered, 4 kDa and 2 kDa, showing that 4 kDa PEG specifically, in combination with PVA protects insulin from aggregation and thus inactivity. This implies certain molecular weights are important for a solvent-free, polymer only cryopreservation formulation.
Footnotes |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c8mh00727f |
‡ Present address: Department of Chemistry, University of Sheffield, Sheffield, S3 7HF. |
This journal is © The Royal Society of Chemistry 2019 |