Phosphate buffer solutions, freezing

Mann and Mitchell [58] described a simple colorimetric method for estimation of (-D)-penicillamine in plasma. Blood containing 2-50 pg of penicillamine was mixed with 0.1 M EDTA solution in tromethamine buffer solution. 0.1 mL of this solution was adjusted to pH 7.4 and centrifuged. To a portion of the plasma was added 3 M HCL, the mixture was freeze-dried, and a suspension of the residue in ethanol was centrifuged. The supernatant liquid was mixed with tromethamine buffer solution (pH 8.2) and 10 mM 5,5 -dithiobis-(2-nitrobenzoic acid) in phosphate buffer solution (pH 7.0), the mixture was shaken with ethyl ether, and the absorbance of the separated aqueous layer was measured at 412 nm. The mean recovery was 60% (four determinations), and the calibration graph was linear for the cited range. 

Gomez G, Pikal MJ, Rodriguez-Homedo N. Effect of initial buffer composition on pH changes during far-from-equilibrium freezing of sodium phosphate buffer solutions. Pharm Res 2001 18 90-97. 

Standard Preparation Use a commercial reference standard lysozyme of a specified strength from an animal or microbial source in accordance with the origin of the preparation being measured. Measure 50 mg of the reference standard lysozyme into a 50-mL volumetric flask, and dissolve, with stirring, in approximately 25-mL of Sodium Phosphate Buffer Solution. Dilute to volume with Sodium Phosphate Buffer Solution, and mix thoroughly. If desired, freeze aliquots of this Standard Preparation for subsequent assays. Quantitatively transfer 3 mL of the Standard Preparation to a 100-mL volumetric flask, and dilute to volume with Sodium Phosphate Buffer Solution. 

While buffer salt concentrations are not directly measured during freezing, they can be calculated from in situ pH measurements. Temperature-pH-time profiles during freezing reveal the onset of ice crystallization (increase in temperature) followed by Na2HP04 I2H2O crystallization (decrease in pH) in sodium phosphate buffer solutions of initial pH 7.4 and initial concentration in the range of 8-100 mM (Fig. 

Nucleation outcomes from solutions with initially the same composition may vary as a consequence of impurities, rates at which supersaturation was created, thermal histories, experimental techniques employed to detect precipitation, and solution volumes in which nucleation occurred. This is illustrated by comparing results of the selective crystallization of buffer components during freezing from various labora-tories.f The initial salt concentrations and the crystallization behavior of disodium phosphate during the freezing of sodium phosphate buffer solutions are shown in Table 1. Murase et report... 

Murase, N. Franks, F. Salt precipitation during the freeze-concentration of phosphate buffer solutions. Biophys. Chem. 1989, 34 (3), 293-300. 

To prepare I-DOTATATE, 1 mg of DOTATATE was dissolved in 100 mL of 0.02M acetic acid in redistilled water and dispensed into 1 mL fractions in plastic coated vials. These vials were then freeze dried for 24 h and refrigerated for use in further experiments. A vial containing 10 gg of DOTATATE was added to 40 gL of O.IM phosphate buffer solution at pH7.5. A Na l solution at pH7.5 with high specific activity (>2 x 10" Bq/mg) produced in our laboratory using dry distillation of irradiated natural TeO2 powder was used for the labelling studies. 

The freeze-dried products were added 100 /a1 of deionized distilled water, then diluted with 200 fil of 50 mmol dm phosphate buffer solution to determine the residual activity of LDH (Lin and Thomahow, 1992). Residual activities of LDH were assayed at 35° using NADH as a substrate. An enzyme solution (50 /rl) was added to 2 ml of the substrate solution (0.18 mmol dm NADH in a mixture solution of phosphate buffer (50 mmol dm, pH 7.5) and 0.62 mmol dm lithium pyruvate). The absorbance at 340 nm was observed by a Beckman DU-65 spectrometer. 

Freezing of phosphate buffer solutions may affect drug stability by lowering the micro-pH of the condensed aqueous phase. The degradation rate of mitomycin C in frozen phosphate buffer solutions was faster than in solutions.353... 

In aqueous environments, enzymatic activity is sensitive to the pH of the bulk solution. One may therefore suspect that the SCCO2, which is dissolved in the microwater layer of an enzyme in an essentially nonaqueous system, would change the pH of that layer and affect enzyme activity. Kamat et al. clearly showed that this effect is negligible [5]. Increasing the CO2 pressure by a factor of 100 decreases the pH of bulk water by only one unit in an unbuffered system. Enzymes are normally lyophilized from a buffered solution prior to their use as catalysts in SCFs. In lyophilization, the enzyme is first dissolved in water where the pH is adjusted for maximum enzymatic activity. The enzyme/buffer salt solution is then freeze-dried under vacuum to remove almost all of the water. In a typical phosphate buffer solution, the pH may be 7.8. Kamat et al. calculated that at 100 bar CO2 pressure the new pH of the buffer solution would be 7.75, and at 1010 bar the pH would be 7.66. Lyophilization increases the buffer salt concentration in the residual water in the enzyme considerably. Thus, the effect of CO2 on the pH of the remaining microaqueous layer in enzymes becomes even smaller. 

The experimental aqueous matrices included deionized-distilled water, laboratory-prepared phosphate buffer solutions, U.S. mean water (1), and tap waters. U.S. mean water is a solution of specified mineral content which may be prepared and used as a reference standard. These aqueous systems were supplemented with known quantities of Fe, Cu, Zn, Mn, Pd, Ni, Ca, Mg, and K singly and in mixtures. Cations with the exception oiF Fe and K were added as nitrates. Fe and K were added as chlorides. The freezing rate is controlled by the vessel rotation rate and bath temperature. Specific conductance is used as an indicator of the dissolved sohds concentration, although it is recognized that this property is influenced by the hydrogen ion concentration as well as the nature of the dissolved constituents. 

Bj Pivaloyloxymethyl D(—)-Ot-aminobenzylpenicillinate. hydrochloride To a solution of pivaloyloxymethyl D(—)-a-azidobenzylpenicillinate (prepared as described above) in ethyl acetate (75 ml) a 0.2 M phosphate buffer (pH 2.2) (75 ml) and 10% palladium on carbon catalyst (4 g) were added, and the mixture was shaken in a hydrogen atmosphere for 2 hours at room temperature. The catalyst was filtered off, washed with ethyl acetate (25 ml) and phosphate buffer (25 ml), and the phases of the filtrate were separated. The aqueous phase was washed with ether, neutralized (pH 6.5 to 7.0) with aqueoussodium bicarbonate, and extracted with ethyl acetate (2 X 75 ml). To the combined extracts, water (75 ml) was added, and the pH adjusted to 25 with 1 N hydrochloric acid. The aqueous layer was separated, the organic phase extracted with water (25 ml), and the combined extracts were washed with ether, and freeze-dried. The desired compound was obtained as a colorless, amorphous powder. 

For activity assays, proteinase solutions were made fresh daily (10 mg freeze-dried solids in 1 ml pH 10.0 phosphate buffer, 0.1 M). Two ml of the proteinase, 0.5 ml of substrate (azocasein or other proteins in pH 10 buffer), 0.3 ml of 0.1% EDTA, and deionized water were made up to a volume of 3.5 ml. Reaction tubes were incubated in a 40°C water bath for 1 hr, then the reaction was stopped by addition of 1 ml 5% TCA. After removal of the precipitated proteins by centrifugation, absorbance was read at 366 nm (for azocasein) or by the Lowry method (15) for other... 

Neorecormon is one such product. Produced in an engineered CHO cell line constitutively expressing the EPO gene, the product displays an amino acid sequence identical to the native human molecule. An overview of its manufacturing process is presented in Figure 6.7. The final freeze-dried product contains urea, sodium chloride, polysorbate, phosphate buffer and several amino acids as excipients. It displays a shelf-life of 3 years when stored at 2-8 °C. A pre-filled syrine form of the product (in solution) is also available, which is assigned a 2 year shelf-life at 2-8 °C. 

Fig. 3.2.1. Activity recovery vs concentra tion of LDH after freezing in a freezer at -40 °C for 20 h and thawing at room tern perature in different buffer solutions (1) 0.05 M sodium phosphate, pH 7.4 (2) 0.050 M citrate, pH 7.4 (3) 0.05 M Tris, pH 7.4 (part of Figure 2 from [3.70])...

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