Protein Stabilization at Low Temperatures (Freeze-thawing and Freeze-drying)

Freeze-thawing and freeze-drying are the two methods commonly used for long-term storage of proteins and during transportation of proteins. For storage, some proteins are frozen and then thawed before use. During freeze-thawing and freeze-drying, proteins have to endure stresses such as low temperatures and formation of ice, which destabilize them. It has been found that many proteins are not stable against these stresses (Carpenter et al., 1986; Carpenter et al., 1987; Carpenter and Crowe, 1988a&b). A number of compounds have been examined for their effects on retention of enzyme activity upon

freeze-thawing and freeze-drying (Carpenter and Crowe, 1988b). During freeze-thawing, compounds that stabilize proteins in their solution state, protect proteins against low temperatures, and those compounds that are known to destabilize or denature proteins in solution, enhance damage due to freeze-thawing. On the other hand, it has been found that only sugars protect proteins from damage due to desiccation (Carpenter et al., 1987; Carpenter and Crowe, 1988a; Carpenter and Crowe, 1989). In the sections below, the effect of freeze-thawing and freeze-drying on proteins has been explained further.

2.2.4.1 Freeze-thawing of proteins

For long-term storage, proteins are often frozen and then thawed before use. During this process, proteins are exposed to critical stresses such as low temperature and formation of ice. Cold denaturation has been accounted for the inactivation of many proteins during free-thawing (Becktel and Schellman, 1987; Privalov, 1990). During freezing, protein molecules are excluded from the ice crystals and are subjected to chemical and physical changes that occur in the non-ice phase. With the formation of ice, the concentration of all solutes increases and if the solutes present are destabilizing, then the concentrating effect can lead to protein denaturation (von Hippel et al., 1969). Also, if the protein solution is made up of a buffer such as sodium phosphate, then during freezing the solution can undergo a dramatic decrease in the pH, resulting in destabilization of the protein (Chilson et al., 1965; van den Berg and Rose, 1969). Thus, the combination of factors arising during freeze-thawing, low temperatures and high destabilizing salt concentration, can damage the protein. The duration of exposure of a protein to these conditions can also influence the degree of damage. This has been proved by the finding that the loss of enzyme activity during freezing and thawing is often inversely correlated with the cooling and warming rates (Chilson et al, 1965; Whittam and Rosano, 1973). The perturbations that have been shown to destabilize the protein can be protein-specific. However, it is widely agreed that any factor that alters protein stability in non-frozen aqueous solution will tend to have the same qualitative effect during freeze-thawing (Arakawa et al., 2001). Also, it has been shown that increasing the protein concentration

will increase the stability of the protein during freeze-thawing (Carpenter and Crowe, 1988b). To prevent the damage that occurs to the protein during freeze-thawing, a number of cryoprotectants have been employed. The most common cryoprotectants are sugars, polyols, synthetic polymers and amino acids (Carpenter and Crowe, 1988b). Several mechanisms have been proposed to explain the effect of these cryoprotectants on proteins. The only mechanism that seems to be applicable to most of the known compounds is the preferential exclusion mechanism proposed by Timasheff and co- workers described in Section 2.2.1 of this chapter. Most of these compounds have been shown to be preferentially excluded from the surface of protein, and the compounds like urea and guanidine-HCl, which are known to preferentially bind to the proteins, have been found to cause additional damage during freeze-thawing (Carpenter and Crowe, 1988b; Arakawa et al., 1990b). Arakawa et al. (2001) proposed that the destabilizing conditions that may arise during freeze-thawing, such as concentration of solutes and alterations in solution pH, can be viewed simply as other types of perturbation induced in solution. Thus, the basic thermodynamic principles governing protein stability in the frozen state is not different from those observed in non-frozen aqueous systems (Arakawa et al., 2001).

However, there is a group of compounds, whose effect on proteins in non-frozen aqueous system, do not correlate with their effect during free-thawing (e.g. PEG and MPD), as these compounds destabilize proteins at slightly higher temperatures (Section 2.2.1). In fact, PEG has been found to be a very effective cryoprotectant (Carpenter and Crowe, 1988b). This kind of effect has been attributed to the temperature dependence of hydrophobic interactions between proteins and co-solvents. At low temperatures, these solutes are preferentially excluded and the hydrophobic interactions are weaker, whereas at relatively higher temperatures (>25°C), the hydrophobic interactions become stronger and it leads to preferential binding (Arakawa et al., 1990b).

2.2.4.2 Freeze-drying of proteins

Freeze-drying or lyophilization is a method used in preparation of protein products, when the proteins are not sufficiently stable for long-term storage as aqueous solutions. The process of lyophilization consists of freezing the protein solution followed by drying in vacuum. It is known that the second step of lyophilization, that is drying, takes place in two phases: primary drying, which removes frozen water through sublimation, and secondary drying which removes non-frozen ‘bound’ water (Arakawa et al., 2001). Freeze-drying often destabilizes proteins due to the conformational instability of many proteins when subjected to freezing and subsequent dehydration stresses (Crowe et al., 1990). In contrast to free-thawing, where freezing is the only stress, freeze-drying poses two stresses, freezing and drying. Thus, to protect proteins against freeze-drying, both these fundamentally different stresses have to be overcome.

Several additives have been tried in order to protect proteins against freeze-drying. However, the solutes that provide stabilization are sugars and polyols. The mechanism by which these solutes provide protection is not completely understood. Clearly, co-solute stabilization during freeze-drying is more complex than that of cryoprotection (Crowe et al., 1990). Many effective cryoprotectants fail to stabilize proteins during dehydration. It has been suggested that the mechanism of solute-induced stabilization during dehydration is fundamentally different from that for proteins in aqueous or frozen systems (Carpenter and Crowe, 1989). They have also suggested that the thermodynamic principles that explain protein stabilization by preferential exclusion are not applicable for freeze-drying as the solvent itself is removed from the system.

Carpenter and Crowe (1989), in their study of effect of carbohydrates during freeze- drying of proteins, have suggested that these solutes protect proteins because they hydrogen-bond to the dried protein acting as a water substitute, when the hydration shell of protein is removed. They used Fourier transform infrared spectroscopy (FTIR) to study the interactions between dried proteins and carbohydrates. They proved that hydrogen bonding does occur between proteins and carbohydrates during drying, suggesting that

such bonding may be a requisite for labile proteins to be preserved during drying. Their results are illustrated in Figure 2.6.

Figure 2.6: Infrared spectra. (Spectrum A) Trehalose freeze-dried alone. (Spectrum B) Freeze-dried trehalose + 0.3 g of lysosome/g of trehalose. (Spectrum C) Freeze-dried trehalose + 0.1 g of BSA/g of trehalose. (Spectrum D) Hydrated trehalose. (Arakawa et al., 2001)

Figure 2.6 illustrates the results of trehalose and protein interactions in the dried state. From the figure it is evident that the spectra for trehalose dried in the presence of either lysozyme or bovine serum albumin (BSA) are remarkably similar to that for hydrated trehalose, but very different from the spectrum of crystalline trehalose. Thus, they concluded that proteins form hydrogen bonds with the polar groups in the sugar, serving the same role for dried trehalose as water does for hydrated trehalose.

Based on the results that proteins serve as water substitutes for dried carbohydrates, Carpenter and Crowe (1989) stated that the converse must also be true, which is

carbohydrates serve as water substitutes for dried proteins. To test this hypothesis, they investigated the influence of trehalose on the infrared spectrum of lysozyme. The results are illustrated in Figure 2.7. The effectiveness of trehalose is evident by the similarity of peaks at 1580, 1540 and 1520 cm-1.

Figure 2.7: Amide band region for hydrated lysozyme (dotted line), lysozyme freeze- dried alone (dashed line), and lysozyme freeze-dried in the presence of trehalose (solid line) (Arakawa et al., 2001).

Based on these results, it was proposed that hydrogen-bonding of the sugar to the protein is mandatory for the sugar to preserve dried proteins. This conclusion has been supported by results of Lippert and Galinski (1992), who showed that stabilization of freeze-dried PFK and lactate dehydrogenase (LDH) by compatible solutes, ectoines, depended on the presence of hydroxyl groups on the molecule.

Other investigators have proposed a different mechanism for protection of proteins by carbohydrates during freeze-drying. Franks et al. (1991) proposed that the glass formation in the dried state is responsible for stabilization, which means that the immobilization of the protein and additives in the glassy state results in the protection of the protein from chemical and conformational degradation. Though this is a good hypothesis, there is evidence which argue against it. In a study by Tanaka et al. (1991), they examined the ability of various saccharide monomers and oligomers to protect catalase during freeze-drying. It was observed that the ability of the saccharides to protect the protein decreased with increase in the length of the chain. However, results from Franks et al. (1991) show that with the increase in the length of the chain, saccharides become better glass-formers as they undergo glass transitions at higher temperatures. These results are exactly opposite to the proposed glass formation mechanism.

Of the proposed mechanisms of protection of proteins against freeze-drying, the effect of saccharides, serving as water substitutes for dried proteins, seems to be the most effective explanation. However, the mechanism by which this interaction between protein and the sugar occurs is not completely understood yet.