Purification crystallization

S. Ravaud, H. Watzlawick, R. Haser, R. Mattes, and N. Aghajari, Overexpression, purification, crystallization and preliminary diffraction studies of the Protaminobacter rubrum sucrose isomerase SmuA, Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun., 62 (2006) 74-76. 

Let us consider the crystal growth of the three previous compounds which have retained our attention by their high nonlinear optical susceptibilities (POM, MAP, NPP) plus an additional one R(+)N-methylidene (gem-carbethoxiphenyl) amide of 4-N-dime-thylamino- benzylidene cyanacetlc acid -AMA, of potential interest but not yet investigated from the optical point of view. We look successively into their synthesis, purification, crystal growth and characterization. 

H. Kuhl, J. Kruip, A. Seidler, A. Krieger-Liszkay, M. Bunker, D. Bald, J.A. Scheidig, M. Rogner (2000) Towards structural determination of the water-splitting enzyme. Purification, crystallization, and preliminary crystallographic studies of photosystem II from a thermophilic cyanobacterium. J. Biol. Chem., 275 20652-20659... 

This product can be used without further purification. Crystallization from mixtures of acetonitrile and ether gives colorless, felted needles, m.p. 118-120° (dec.) (Note 5). 

FEson H, Fox A, KeUeher D, Windle HJ, Sanders DA. 2003. Purification, crystallization and preliminary X-ray analysis... 

Habel, J. E., Ohren, J. R and Borgstahl, G. E. (2001). Dynamic light-scattering analysis of full-length human RPA14/32 dimer purification, crystallization and selfassociation. Acta Crystallogr. D 57, 254-259. 

Hirsch, J. A. and Aggarwal, A. K. (1995). Purification, crystallization, and preliminary X-ray diffraction analysis of even-skipped homeodomain complexed to DNA. Proteins 21,268-271. 

Ruppert, M., Panjikar, S., Barleben, L. and Stockigt, J. 2006. Heterologous expression, purification, crystallization and primary X-ray analysis of raucaffricine glu-cosidase, a plant enzyme specifically involved in Rauvolfia alkaloid biosynthesis. Acta Crystallographica Section F-Structural Biology and Crystallization Communications, 62 257-260. 

Turnbull, J.J. et al.. Purification, crystallization and preliminary x-ray diffraction of anthocyanidin synthase from Arabidopsis thaliana. Acta Crystallogr. D, 57, 425, 2001. 

Crystals and X-ray Data Collection. Detailed information concerning protein purification, crystallization, and X-ray data collection can be found in a previous report (Xu et al., 1991) and will be mentioned here in summary form. Recombinant murine apo-ALBP crystallizes in the orthorhombic space group P2j2i2i with the following unit cell dimensions a = 34.4 A, b = 54.8 A, and c = 76.3 A. The asymmetric unit contains one molecule with a molecular weight of 14,500. The entire diffraction data set was collected on one crystal. In the resolution range t -2.5 A, 5115 of the 5227 theoretically possible reflections were measured. Unless otherwise noted the diffraction data with intensities greater than 2a were used for structure determination and refinement. As can be seen in Table 8.2, this included about 96% of the measured data. 

Boeshans KM, Wolf R, Voscopoulos C, Gillette W, Esposito D, Mueser TC, Yuspa SH, Ahvazi B. 2006. Purification, crystallization and preliminary X-ray diffraction of human S100A15. Acta Crystallograph Sect F Struct Biol Cryst Commun 62(Pt 5) 467-470. 

In this dilemma of contradicting interests, COSMO-RS can be a valuable tool for the computational characterization of the solubility behavior of the drug candidate as well as of its dissociation constants. Both are of crucial interest since the small and very expensive amount of compound has to be dissolved and embedded in different solvents and environments for the various steps of purification, crystallization, analysis, and formulation. At present, empirical solubility parameter approaches are often used in order to classify and predict the solubility behavior of the new drug, but despite their poor physical foundation, they have the additional disadvantage that the experimental measurement of the solubility parameters of the new drug consumes time and compound. In contrast, the required DFT/COSMO calculations can be started before the compounds come to the development laboratory, and a COSMO-RS solubility and dissociation screening can be completed—even at optimal computational level— when the work in the development department starts. Furthermore, none of the valuable substance is wasted in this step. 

Over the past fifteen years there have been numerous attempts at reviewing this field (Garrett, 1959 Inokuchi and Akamatu, 1961 Lyons, 1963 Kearns, 1964 Okamoto and Brenner, 1964 Kommandeur, 1965 Gutmann and Lyons, 1967 Le Blanc, 1967 Sharp and Smith, 1970 D. M. Hanson, 1973 Karl, 1974 Goodings, 1976 Inokuchi and Maruyama, 1976) with wide variability in the degree of success achieved. These serve as useful sources of references and also adquately describe most of the methods of measurement, techniques of material purification, crystal growth and electrode fabrication but invariably do not attempt the interpretation of the phenomena in molecular terms nor indicate possible applications. 

Adding up the times of all steps, an industrial scale production takes roughly 3 weeks, of which 2 weeks are devoted to the fermentation and about 1 week is required for the downstream processing. Derivatization at positions 3 and 7 to yield an API is not included. Starting from 7-ACA, these processes may take 1 day each for the derivatization plus the time for purification, crystallization, and drying. The resulting bulk active cephalosporin can then be sterilized and formulated for marketing. 

Sugiura, M., Oikawa, T., Hirano, K., Inukai, T. 1977. Purification, crystallization and properties of triacylglycerol lipase from Pseudomonas fluorescens. Biochim. Biophys. Acta 488, 353-358. 

Lee, K. F., Parales, R., Parales, J. V., Gibson, D. T., and Ramaswamy, S. 2005. Purification, crystallization and preliminary x-ray diffraction studies of the three components of the toluene 2,3-dioxygenase enzyme system. Acta Crystallographica (F Structural Biology and Crystallization Communications), F61 (7), 669-672. 

Himanen, J.-P., and Nikolov, D. B. (2002). Purification, crystallization and preliminary characterization of an Eph-B2/ephrin-B2 complex. Acta Crystallogr. D 58, 533-535. 

Purification crystallized from hexane or from MeOH. 

The first, and often most difficult, step in XRC is the production of 3-D crystals of purified protein. It is important to emphasize the importance of protein purity for successful crystallization because, although it is true that a nonhomogenous mixture may crystallize, it has been noted by some researchers that difficulties in crystallizing a protein may be negated by further purification. Crystallization of membrane proteins is a complicated process, and various approaches have been used, including vapor diffusion, microdialysis, batch crystallization... 

Crystallization is used to separate saturated fats and oleic acid from linoleic acid. If a highly concentrated product is required, the linoleic acid may be crystallized once or repeatedly as the last step in purification. Crystallization is a mild procedure but usually requires the use of a solvent (108) such as acetone or methanol. The use of low boihng point and flammable solvents raises concerns over plant safety, government regulations on manufacturing, and market acceptance of the product. Furthermore, the removal of oleic acid by crystallization in solvent is only possible by lowering the temperature of the liquor to below —40°C (108). To crystallize linoleic acid, the temperature must be reduced to —75 °C. 

Igarashi, M., and Hollander, V. P., Acid phosphatase from rat liver. Purification, crystallization, and properties. J. Biol. Chem. 243, 6084-6089 (1968). 

H. Kawasaki, K. Miyoshi, and K. Tonomura, Purification, crystallization and properties of haloacetate halohydrolase from Pseudomonas sp., Agric. Biol. Chem., 45 543 (1981). 

F. A. Kroger, The Chemistry of Imperfect Crystals, Vol. 1 Preparation, Purification, Crystal Growth, and Phase Theory, 2nd ed., North Holland, Amsterdam, 1973. 

Cordeiro AT, Godoi PH, Delboni LE, Oliva G, Thiemann OH. Human phosphoglucose isomerase expression, purification, crystallization and preliminary crystallographic analysis. Acta Crystaliogr D Biol Crystaliogr 2001 57 592-5. 

---