To Hofmeister and beyond: an improved understanding of protein solubility and stability

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Proteins are used increasingly widely, in pharmaceutical and diagnostic formulations, cosmetics, industrial processes, and detergents. A major problem is that proteins have limited solubility and/or stability in liquid formulations, leading over time to loss of quality, reduced benefit, wasted cost (especially as the protein is usually the most expensive part), and a lack of reproducibility and predictability. Similar problems abound in other spheres: for example many proteins give poor analytical spectra, cloudiness or haze in beverages, and cannot be crystallised, because of difficulties in keeping them in solution. There is thus a major need to improve stability without losing solubility or vice versa. The main approach used is to add high concentrations of ions and other molecules to the protein solution (known generally as excipients). Currently the selection of excipients is usually tackled on an ad hoc basis, using high-throughput testing of different solution conditions, and there is very little theoretical rationale that could be used as a basis for a more rational, cheaper and quicker route to improved performance. This proposal aims to provide a more rational basis for understanding and improving stability and solubility.We recently proposed a model that explains how salts added to a protein solution affect its solubility and stability, by competing with the protein for water molecules. Different salts compete better or worse than the protein, and therefore have different effects. The effects have been known for over 100 years and are generally called the Hofmeister effect, but there remains considerable disagreement over the explanation. This matters, because once we understand the physical basis for the Hofmeister effect, we can exploit it to develop better solution conditions. Our model was based largely on measurements using a spectroscopic technique known as NMR.The model differs in important ways from the current standard models. We will therefore start by using NMR to compare our model to the most popular of these, to eliminate the most significant likely objection to our model and clear the ground. Our model emphasises the role of water molecules in mediating changes in solubility and stability: in particular, it predicts that ions that stabilise proteins necessarily make them less soluble, and vice versa. If this is strictly true, then it places severe limits on what we can do to (for example) improve stability without compromising solubility. We shall therefore explore ways of getting round the problem. First, we shall investigate whether mixtures of ions behave simply in an additive way. To the extent that they do not, we might be able to exploit a window of opportunity. A particularly interesting pair of ions is a mixture of the two amino acids arginine and glutamate, which seem to work in a different way and are apparently 'special'. We shall investigate how they work, whether they are in fact different, and whether combining them with more typical Hofmeister ions helps. We will also investigate our suspicion that increasing the size of a protein makes the effect of added ions weaker: if true, this is useful information, since it guides the way we would go about stabilising different proteins.There are many organisms that can grow in high salt concentrations. Normally, high salt reduces either solubility or stability or both: so how do these organisms manage to grow successfully? The answer is that they produce high concentrations of specific small molecules to balance the high external ionic strength. We shall investigate how these molecules work, and whether they behave in the same way as 'normal' ions. If they work in different ways, then these can be exploited.Finally we will study whether external pressure can be used as an alternative way to stabilise proteins, and thus whether pressure would be a useful variable. The outcome will be a rational toolbox to guide solution conditions.