Protein phase diagram

Once a range of suitable pH and ionic strength are selected, the effect on protein stability is evaluated for the final selection of an optimal formulation pH. Only when the optimal pH and addition of common salts do not render the desired solubility are other additives considered. This adds to the complexity of the formulation and the challenge of maintaining stability. Recently, an empirical approach to determine protein phase diagrams using various biophysical techniques has been used to facilitate identification of optimal formulation conditions (Fan etal., 1995). 

Smeller, L. (2002) Pressure-temperature phase diagram of hiomo QCVi Q ,Biochimica et Biophysica Acta 1595, 11-29 Lesch, H., Hecht, C., and Friedrich, J. (2004) Protein phase diagrams the physics behind their elliptic shape, Journal of Chemical Physics 121, 12671-12675... 

CASE STUDIES IN LYSOZYME AND THE GENERIC PROTEIN PHASE DIAGRAM... 

Figure 12.16 The generic protein phase diagram as presented by Muschol and Rosenberger (1997). Zone I depicts the region of supersaturation where well formed lysozyme crystals form. Zone II is a region where lysozyme undergoes a rapid liquid-liquid phase separation, with the resulting concentrated lysozyme phase quickly tranforming to the more stable crystalline form. Crystals formed in Zone II are of poor quality. Zone III depicts a region characterized by gel formation, unsuitable for crystal growth.

The basis for the separation is that when two polymers, or a polymer and certain salts, are mixed together in water, they are incompatible, leading to the formation of two immiscible but predominantly aqueous phases, each rich in only one of the two components [Albertsson, op. cit. Kula, in Cooney and Humphrey (eds.), op. cit., pp. 451 71]. A phase diagram for a polyethylene glycol (PEG)-Dextran, two-phase system is shown in Fig. 22-85. Proteins are known to distribute unevenly between these phases. This uneven distribution can be used for the selective concentration and partial purification of the products. Partitioning between the two phases is controlled by the polymer molecular weight and concentration, protein net charge and... 

FIG. 22-85 Phase diagram for a PEG/Dextran, hiphasic, aqueous-polymer system used in liquid-liquid extraction operations for protein separations. Alheiisson, Partition of Cell Particles and Macromolecules, 3d ed., Copyright 1986. Reprintedhy petTTUssion of John Wiley Sons, Inc.)... 

Curtis, R. A. Blanch, H. W. Prausnitz, J. M. Calculation of Phase Diagrams for Aqueous Protein Solutions. ]. Phys. Chem. B 2001, 105, 2445-2452. 

The crystallization process can be illustrated by a phase diagram that shows which state (liquid, crystalline, or amorphous solid [precipitate]) is stable under a variety of crystallization parameters. It provides a means of quantifying the influence of the parameters such as the concentrations of protein, precipitant(s), additive(s), pH, and temperature on the production of crystals. Hence phase diagrams form the basis for the design of crystal growth conditions (McPherson, 1999 Ducruix and Giege, 1992 Ducruix and Giege, 1999 Chayen et ah, 1996 and references therein). 

Ataka, M. (1993). Protein crystal growth An approach based on phase diagram determination. Phase Transitions 45, 205-219. 

Saridakis, E. and Chayen, N. E. (2003). Systematic improvement of protein crystals by determining the supersolubility curves of phase diagrams. Biophys. 84, 1218-1222. 

Elysee-Collen, B. and Lencki, R. (1996). Protein ternary phase diagrams. I. Effect of ethanol, ammonium sulfate and temperature on the phase behaviour of type gelatin. J. Agric. Food Chem., 44, 1651-1657. 

Tolstoguzov, V.B. (2000) Compositions and phase diagrams for aqueous systems based on proteins and polysaccharides. International Review of Cytology, 192, 3-31. 

Figure 7.10 Effect of the thermodynamic incompatibility of otsi/p-casein + high-methoxy pectin (pH = 7.0, / = 0.01 M) on phase diagram of the mixed solutions and elastic modulus of corresponding casein-stabilized emulsions (40 vol% oil, 2 wt% protein), (a) (O) Binodal line for p-casein + pectin solution with critical point ( ) ( ) binodal line for asi-casein + pectin solution with critical point ( ). (b) Complex shear modulus G (1 Hz) is plotted against the pectin concentration (O) p-casein ( ) o i -casein. Dotted lines indicate the range of pectin concentration for phase separation in the mixed solutions. The pectin was added to the protein solution before emulsion preparation. Data are taken front Semenova et al. (1999a).

Lyophilization employs sub-zero temperatures in combination with very low pressure to withdraw water from the protein solution typical values would be -80 °C and 1-3 mbar. In these conditions, as the water phase diagram reveals, water sublimes, leaving a fluffy porous enzyme powder. As typical run times are on the order of a day, lyophilization is not the method of choice for large-scale enzyme drying operations. On a laboratory scale, however, lyophilization is a very effective method. 

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