Co-lyophilization

Basically, there are three ways to tune enzyme enantioselectivity by means of additives (i) the additives are placed in the reaction medium together with the organic solvent, the enzyme, and the reagents (ii) the additives are co-lyophilized with the biocatalyst before use in the organic solvent (iii) the additives are complexed with the substrates before their transformation in the organic medium. 

Crown ethers are heterocyclic chemical compounds that, in their simplest form, are cyclic oligomers of ethylene oxide. The essential repeating unit of any simple crown ether is ethyleneoxy, i.e., —CH2CH20—, which repeats twice in dioxane and six times in 18-crown-6. Crown ethers can activate enzymes for use in organic solvents through two methods (a) direct addition of 18-crown-6 to the reaction solvent [93], or (b) co-lyophilization of the enzyme with 18-crown-6, the latter being the most effective [94, 95]. 

When 18-crown-6 was co-lyophilized with a-chymotrypsin, a 470-fold activation was seen over the free enzyme in the transesterification of APEE with 1-propanol in cyclohexane (Scheme 3.2) [96]. There was a low apparent specificity for the size and macrocyclic nature of the crown ether additives, suggesting that, during lyophilization, 18-crown-6 protects the overall native conformation and acts as a lyoprotectant. To examine this global effect, FTIR was used to examine the effect of crown ethers on the secondary structure of enzymes. In one study [98], subtilisin Carlsberg was shown to retain its secondary structure in 1,4-dioxane when lyophi-lized in a 1 1 ratio with 18-crown-6. In addition, examination of FTIR spectra from varying incubation temperatures indicated that an increase in crown ether content in the final enzyme preparation resulted in a decreased denaturation temperature in the solvent, indicating a more flexible protein structure. 

The lyophilization of enzymes from solutions containing salts or amphiphilic compounds is known to increase the activity in organic media by up to several orders of magnitude. Thus, the transesterification activity of a-chymotrypsin was increased 82-fold by co-lyophilization with pentaglyme [75]. The colyophilization of lipases and (poly ethylene)glycol (PEG) led to an enhanced transesterification activity in various ionic liquids [76, 77]. 

Zeng, X.M., Martin, G.P., Marriott, C. Effects of molecular weight of polyvinylpyrrolidone on the glass transition and crystallization of co-lyophilized sucrose. Int. J. Pharm. 218, 63-73, 2001... 

Davidson, P., Sun, W.Q. Effects of su-crose/raffinose mass ratios on the stability of co-lyophilized protein during storage above Tg. Pharm. Res. 18, 474-479, 2001... 

Figure 5.1 Transesterification activity of (a) 0.025 or (b) 0.3 mg of lipase BC co-lyophilized with different amounts of sucrose ( ), trehalose (x) and mannitol ( ). Activity was tested in toluene by measuring the rate of 1-...

In the case of sugars, which are largely employed in the pharmaceutical field as lyoprotectants for the formulation of proteins used as drugs, it was found that FT-IR spectra of lipase BC co-lyophilized with these additives are more similar to that of lipase BC lyophilized without sugars than to that of the enzyme in water (the comparison of spectra was done on the basis of correlation coefficients between the infrared amide I band) [14]. An analysis of the relative area of the... 

Skujins and McLaren (1967) co-lyophilized urease and [ CJurea. The rate of reaction, determined by the level of C02, was measured as a function of water content. Onset of enzyme reaction occurred at 0.6 relative humidity. The samples contained a 25 1 weight ratio of urea to urease. Sorption isotherms measured separately for enzyme and urea showed that below 0.75 relative humidity the urea adsorbed no water, and thus that the enzyme changes reflected adsorption of water by the urease. From the sorption isotherm for urease, 0.6 relative humidity corresponds to 0.15 h. 

Raffinose has been shown to accumulate in organisms that can survive extreme desiccation, and has therefore been examined as an excipient in stabilizing co-lyophilized protein and labile preparations during storage at elevated temperatures. 

Biliaderis, C.G., Lazaridou, A., Mavropoulos, A., and Barbayiannis, N. Water plasticization effects on crystallization behavior of lactose in a co-lyophilized amorphous polysaccharide matrix and its relevance to the glass transition, Int. 

Al-Azzam W, Pastrana EA, Griehenow K. Co-lyophilization of bovine serum albumin (BSA) with poly(ethylene glycol) improves efficiency of BSA encapsulation and stability in polyester microspheres by a solid-in-oil-in-oil technique. Biotechnology Letters. 2002 24 1367-1374. 

Morita T, Horikiri Y, Suzuki T, Yoshino H. Preparation of gelatin microparticles by co-lyophilization with poly(ethylene glycol) characterization and application to entrapment into biodegradable microspheres. Int J Pharm. 219 (1-2) 127-137,2001. 

Rate effects may not be chemical kinetic ones. Benson and co-worker [84], in a study of the rate of adsorption of water on lyophilized proteins, comment that the empirical rates of adsorption were very markedly complicated by the fact that the samples were appreciably heated by the heat evolved on adsorption. In fact, it appeared that the actual adsorption rates were very fast and that the time dependence of the adsorbate pressure above the adsorbent was simply due to the time variation of the temperature of the sample as it cooled after the initial heating when adsorbate was first introduced. 

A second motivation to include CE methods is the excellent performance of chiral CE, which is often the first choice technique to separate stereoisomers. Such method can be used complementarily to avoid potential co-elution of isomers or related products, e.g., degradation products, with similar chromatographic properties. Practically, one can fractionally collect the peak volume, lyophilize it, and dissolve the resulting mass in an appropriate solvent. The pre-concentrated sample can then easily be analyzed with a selective and efficient CE method. Another option is to develop an on-line coupling between HPEC and CE to facilitate the analysis. " ... 

Figure 4 SEM micrograph (15,000X) of a lyophilized particle dispersion prepared by co- precipitation.

The amorphous state of the lyophilized solids was verified by powder X-ray diffractometry (Rigaku Denki Co., Ltd., Danvers, MA), and their Ca/P04 ratios after dissolution in HCl were determined by atomic absorption (AAS, Perkin Elmer, Norwalk, CT) and UV spectrophotometric [6] (Varian Analytical Instruments, Palo Alto, CA) measurements of Ca + and PO4, respectively. Dissolution of the ACP fillers was studied by kinetically following the changes in Ca + and PO4 concentrations in continuously stirred HEPES-buffered (pH = 7.4) solutions adjusted to 240 mOsm/kg with NaCl at 37°C. All solutions initially contained 0.8 mg/mL of the ACP filler. 

Furthermore, the type of enzyme formulation (free enzyme, immobilized enzyme, or whole cells) plays a key role in determining the progress of the overall reaction. For most applications, lyophilized enzyme powders have been used with good results presumably they dissolve into the liquid phase. When poorly soluble products are formed, the enzyme can be recovered by washing with water [52]. For co-factor-dependent reactions permeabilized cells may be used [44]. When using immobilized enzymes, it has been demonstrated that the chemical nature and the pore size of the support are very important parameters to consider [8, 41]. 

---