Freeze-drying of proteins with glass-forming oligosaccharide-derived sugar alcohols
Faculty of Pharmaceutical Sciences, Toho University, Miyama 2-2-1, Funabashi, Chiba, 274-8510, Japan.International Journal of Pharmaceutics (Impact Factor: 3.65). 04/2010; 389(1-2):107-13. DOI: 10.1016/j.ijpharm.2010.01.027
Physical properties and protein-stabilizing effects of sugar alcohols in frozen aqueous solutions and freeze-dried solids were studied. Various frozen sugar alcohol solutions showed a glass transition of the maximally freeze-concentrated phase at temperatures (T(g)'s) that depended largely on the solute molecular weights. Some oligosaccharide-derived sugar alcohols (e.g., maltitol, lactitol, maltotriitol) formed glass-state amorphous cake-structure freeze-dried solids. Microscopic observation of frozen maltitol and lactitol solutions under vacuum (FDM) indicated onset of physical collapse at temperatures (T(c)) several degrees higher than their T(g)'s. Freeze-drying of pentitols (e.g., xylitol) and hexitols (e.g., sorbitol, mannitol) resulted in collapsed or crystallized solids. The glass-forming sugar alcohols prevented activity loss of a model protein (LDH: lactate dehydrogenase) during freeze-drying and subsequent storage at 50 degrees C. They also protected bovine serum albumin (BSA) from lyophilization-induced secondary structure perturbation. The glass-forming sugar alcohols showed lower susceptibility to Maillard reaction with co-lyophilized L-lysine compared to reducing and non-reducing disaccharides during storage at elevated temperature. Application of the oligosaccharide-derived sugar alcohols as alternative stabilizers in lyophilized protein formulations was discussed.
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ABSTRACT: The purpose of this review is to demonstrate the critical importance of understanding protein-excipient interactions as a key step in the rational design of formulations to stabilize and deliver protein-based therapeutic drugs and vaccines. Biophysical methods used to examine various molecular interactions between solutes and protein molecules are discussed with an emphasis on applications to pharmaceutical excipients in terms of their effects on protein stability. Key mechanisms of protein-excipient interactions such as electrostatic and cation-pi interactions, preferential hydration, dispersive forces, and hydrogen bonding are presented in the context of different physical states of the formulation such as frozen liquids, solutions, gels, freeze-dried solids and interfacial phenomenon. An overview of the different classes of pharmaceutical excipients used to formulate and stabilize protein therapeutic drugs is also presented along with the rationale for use in different dosage forms including practical pharmaceutical considerations. The utility of high throughput analytical methodologies to examine protein-excipient interactions is presented in terms of expanding formulation design space and accelerating experimental timelines.Advanced drug delivery reviews 07/2011; 63(13):1118-59. DOI:10.1016/j.addr.2011.07.006 · 15.04 Impact Factor
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ABSTRACT: Chloroperoxidase (CPO) is thought to be the most versatile heme-containing enzyme with enormous applications in organic synthesis, biotransformation, pharmaceutical production, and detoxification of environmental pollutants. Any improvement in the stability of this enzyme will greatly enhance its application in the mentioned areas. In the present study, the effects of three polysaccharides (soluble starch, β-cyclodextrin, and dextrin) on the stability of CPO at elevated temperatures (20, 30, 35, 40, and 50 °C) or in aqueous-organic solvents media (methanol, dioxane, DMSO, and DMF) were investigated. An improved catalytic performance of CPO was observed in the presence of a small amount of the three polysaccharides, where dextrin provided the most effective promotion. The changes of enzyme structure and microenvironment around heme in the presence of additives were studied by fluorescence, circular dichroism, and UV-vis spectra analyses, as well as kinetic parameters measurement. A catalytically favorable structure of CPO was induced, including the strengthening of the α-helix structure and more exposure of heme for easy access of the substrate, resulting in an increase of catalytic turnover frequency (k (cat)) and the improvement of affinity and selectivity of CPO to substrate. The results revealed that the introduction of trace soluble starch, β-cyclodextrin, and dextrin (<10 μmol/L) in reaction media was an effective strategy for the enhancement of the thermodynamic and the operational stability of the enzyme, which are promising in view of the industrial applications of this versatile biological catalyst.Applied biochemistry and biotechnology 09/2011; 165(7-8):1691-707. DOI:10.1007/s12010-011-9388-7 · 1.74 Impact Factor
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ABSTRACT: Hepatitis C disease burden is substantially increasing in Egyptian community, it is estimated that prevalence of Hepatitis C virus (HCV) in Egyptian community reach 22% of total population. Recently there is a global alert of HCV cardiovascular complications. To evaluate LV diastolic functions of HCV patients using tissue Doppler Imaging and NTPBNP. 30 HCV patients of 30 years, sex & BMI matched controls were evaluated by PCR, ECG, Echocardiography "conventional Doppler, pulsed wave tissue Doppler (PW-TD), strain rate imaging" & NTPBNP to assess LV diastolic functions. Mean age was 32.8 years ± 5.1 in HCV group, 29.8 years ± 6.6 in control group. Cardiovascular anomalies and predisposing factors were excluded. HCV group has shown significant increase in QTc interval, significant statistical increase in A wave, deceleration time; (p < 0.05), highly significant decrease in tissue Doppler E a (p < 0.001), highly significant decrease in A a (p < 0.001), highly significant increased E/E a ratio (p value < 0.001), significant decrease in E a/A a ratio and significant increase in SRa (p < 0.05). NTPBNP levels showed highly significant increase with mean value 222 pg/ml ± 283 in HCV group and 32.7 pg/ml ± 21.2 in control group (p value < 0.001). The best cut-off value of NTPBNP to detect diastolic dysfunction in HCV group was 213 pg/ml. No statistical differences in SRe/SRa and E/SRe ratios were observed, however they had significant correlation with NTPBNP level and tissue Doppler parameters. The best cut-off value of E/SRe ratio to detect diastolic dysfunction in HCV group was 0.91, with 75% sensitivity and 100% specificity. This data show the first direct evidence that HCV infection causes diastolic dysfunction without any other predisposing factors, probably due to chronic inflammatory reaction with mild fibrosis in the heart. Previous studies did not follow strict inclusion and exclusion criteria that confirm the independent role of HCV to cause diastolic dysfunction. Tissue Doppler was more sensitive to diagnose diastolic dysfunction than conventional Doppler.10/2011; 23(4):217-23. DOI:10.1016/j.jsha.2011.04.005
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