Stability of Proteins

One of the major problems that a biochemical engineer will encounter is that of the stability of protein materials. The biological function of the molecule is determined by its secondary and tertiary structures and if these are upset irreversibly then the protein becomes denatured. Denaturation can occur under relatively mild conditions as proteins are usually stable over only very narrow ranges of pH (e.g. 5-8) and of temperature (e.g. 10-40°C). The boiling of an egg illustrates this point. In its uncooked form the white is a slimy clear protein solution, but under acidic conditions, or when put in boiling water, the solution gels to white solid. 

The stability of tertiary structure can be appreciated by the relative strengths of these associations as compared with that of a covalent bond, as discussed in Appendix 5.1. 

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Steadman B L, Thompson K C, MIddaugh C R, Matsuno K, Vrona S, Lawson E Q and Lewis R V 1992 The effects of surface adsorption on the thermal stability of proteins Bioteoh. Bioengng. 40 8-15... 

Y-Y Shi, AE Mark, C Wang, F Fluang, FIJC Berendsen, WF Van Gunsteren. Can the stability of protein mutants be predicted by free energy calculations Protein Eng 6 289-295,... 

Protein engineering is now routinely used to modify protein molecules either via site-directed mutagenesis or by combinatorial methods. Factors that are Important for the stability of proteins have been studied, such as stabilization of a helices and reducing the number of conformations in the unfolded state. Combinatorial methods produce a large number of random mutants from which those with the desired properties are selected in vitro using phage display. Specific enzyme inhibitors, increased enzymatic activity and agonists of receptor molecules are examples of successful use of this method. 

The factors accounting for the stabilization of protein conformation are shown in... 

Protein modification by the covalent attachment of ubiquitin chains serves as a signal to mark proteins for the degradation by a multicatalytic proteinase complex called the proteasome. Thus the ubiquitin proteasome system (UPS) controls the stability of proteins in a... 

Recent publications on the thermal stability of proteins organized in dense solid films, deposited by LB (Nicolini et al. 1993, Facci et al. 1994, Erokhin et al. 1995) and by self-assembling (Shen et al. 1993), leave several questions unanswered, hi particular, it is still not completely clear which parameter is responsible for this phenomenon. Two main factors are discussed when speaking about induced thermal stabihty, namely, decreased water content and molecular close packing (Nicolini et al. 1993). It seems that both of them work in parallel, and unfortunately it is difficult to settle directly which one plays the dominant role. 

This comparative study pointed out molecular close packing as a key parameter responsible for the thermal stability of proteins in films. In the case of BR, this close packing is reached due to the nature of the sample, while LB organization seems to be a more general procedure, for the same goal can be reached for practically any type of protein sample. The last statement was even confirmed by the comparison of the thermal behavior of extracted separated BR in self-assembled and LB films. It was found that BR in LB films is more stable for this kind of sample. The results will be reported in detail elsewhere. 

The comparison of the results on enhanced thermal stability of proteins in LB films reported here and those already published again underlines that molecular close packing is a critical parameter responsible for this phenomenon. 

Formulation strategies for stabilization of proteins commonly include additives such as other proteins (e.g., serum albumin), amino acids, and surfactants to minimize adsorption to surfaces. Modification of protein structure to enhance stability by genetic engineering may also be feasible, as well as chemical modification such as formation of a conjugate with polyethylene glycol. 

T. J. Ahern and M. J. Manning, Stability of Protein Pharmaceuticals, Part A, Chemical and Physical Pathways of Protein Degradation, Plenum Press, New York, 1992. 

A variety of approaches exist for stabilizing proteins, for example, chemical modification, immobilization, and site-directed mutagenesis [95,96], but these techniques are not within the scope of this chapter. The focus here will be on stabilization of proteins via formulation development. The principal formulation strategy is to stabilize the protein using clinically acceptable additives (excipients) or through the use of suitable pharmaceutical-processing technologies. 

Globular proteins are known to act as polymeric stabilizers of protein structure in solution. Wang and Hanson [106] review the mechanisms of protein stabilization by serum albumin, and it has been included in... 

Stabilization of proteins against those degradative processes with retention of structure and function through removal of water requires an understanding of the process of lyophilization or freeze-drying. 

M. A. Hanson and S. K. E. Rouan, Introduction to formulation of protein pharmaceuticals, in Stability of Protein Pharmaceuticals, Part B In Vivo Pathways of Degradation and Strategies for Protein Stabilization (T. J. Ahem and M. C. Manning, eds.), Plenum Press, New York, 1992, pp. 209-233. 

The presence of a solvent, especially water, and/or other additives or impurities, often in nonstoichiometric proportions, may modify the physical properties of a solid, often through impurity defects, through changes in crystal habit (shape) or by lowering the glass transition temperature of an amorphous solid. The effects of water on the solid-state stability of proteins and peptides and the removal of water by lyophilization to produce materials of certain crystallinity are of great practical importance although still imperfectly understood. 

Sah H (1999) Stabilization of proteins against methylene chloride/water interface induced denaturation and aggregation. J Control Release 58 143-151... 

The use of discrete PEG spacers in the construction of biotinylation compounds not only increases the water solubility of the modification reagent itself, but significantly increases the hydrophilicity and stability of proteins modified with them. Even when high modification levels... 

Proteins may be stabilized by encapsulation in polyanhydride microspheres. Stability of proteins with respect to water-induced aggregation has been demonstrated to be a function of polymer hydrophobicity for insulin and bovine somatotropin as model proteins (Ron et al., 1993). Encapsulation and enzymatic activity of a variety of other proteins encapsulated in P(SA FAD) was studied by Tabata et al. (1993). 

Chi, E.Y., Krishnan, S., Randolph, T.W., and Carpenter, J.F. 2003. Physical stability of proteins in aqueous solution mechanism and driving forces in nonnative protein aggregation. Pharmaceutical Research 20(9), 1325-1336. 

Lee, B. and Vasmatis, G. 1997. Stabilization of protein structures. Current Opinion in Biotechnology 8, 423-428. 

Arakawa, T., Prestrelski, S.J., Kenney, W.C., and Carpenter, J.F. 2001. Factors effecting short-term and long-term stabilities of proteins. Advanced Drug Delivery Reviews 46, 307-326. 

Interfacial interaction between silicone and protein/starch microparticle, 3 and the use of polysiloxanes having hydrophilic groups for the stabilization of proteins against denaturation, 4 were studied. 

There have been many attempts to improve protein stability and protein properties, utilizing methods such as random mutagenesis, directed evolution, and rational protein design approaches. In general, these methods are far from straightforward and can be time-consuming. In addition, the stabilization of proteins without loss of function is not a trivial problem. 

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