Lyophilized solids

Fig. 8. N Is XANES spectra of (a) fulvic acid isolated from a glucose-glycine-S-Mn02 system and (b) the lyophilized solid phase (Jokic et al. 2004b). The peaks are assigned to pyridinic (398.6 eV), pyridone (400.7 eV), amide (401.3 eV) and pyrrolic (402.0 eV) moieties.

Mounting mount sections in aqueous medium or balsam for brightfield microscopy or in anti-fade medium for fluorescence microscopy (see Sect. 3.2.2). Notes. A11 incubations are at room temperature unless otherwise noted. Nuclear dyes (DAPI, Hoechst 33342 and Propidium Iodide) supplied as lyophilized solids are usually reconstituted in methanol. The stock solutions (5 mg/ml) are stable for many years when stored frozen at <—20°C and... 

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. 

Aqueous PolyNIPAAM Homopolymer (PolyNIPAAM). To 20 mg NIPAAM dissolved in phosphate buffered saline, 2.3 mg of ammonium persulfate and 9.3 mg of N,N,N, N -tetramethylethylenediamine (TEMED) was added to initiate the free radical polymerization. The mixture was then incubated for 3 hours at room temperature. The polyNIPAAM was isolated by precipitation in 14.3%, by volume, saturated (NH4)2S04 After removal of residual (NH4)zS04 by ion exchange chromatography (Bio-Rad AG501-X8D), polyNIPAAM was stored as the lyophilized solid. 

Of course, it is common (and often desireable) to have both amorphous and crystalline phases present in a freeze-dried formulation. This is particularly relevant to freeze-dried proteins, where the lyoprotectant is present in the amorphous state, and another component, such as glycine or mannitol, is present as a crystalline solid in order to impart mechanical integrity and pharmaceutical elegance to the lyophilized solid. 

Fig. 2. The dependence of the negative enthalpy of solution (soln) of lyophilized solid ribonuclease A on the rado of the number of moles of water contained in the lyophilized solid to the number of moles of ribonuclease A. From Almog and Schrier (1978).

After expression and purification, protein samples are often initially available as lyophilized solids, in which the proteins are correctly folded but in an amorphous arrangement with an isotropic distribution of orientations. This form of the protein may be used for NMR studies but the lack of water can significantly affect the strucmres of the individual protein molecules. One simple way to overcome this is to rehydrate the sample with small amounts of water." The effect can be quite significant, as illustrated in Fig. 2 for a sample of the 76-residue protein ubiquitin. Flash freezing offers the opportunity of preparing solid samples of proteins in which the solution-state structure is largely preserved." " ... 

The effect of storage conditions on the crystalline nature of lyophilized ethacrynate sodium has been reported [37]. Samples of the drug substance were dried to various moisture levels and then stored at either 60°C or 30°C/75% RH. It was found that crystalline drug substance was obtained after short periods of time as long as sufficient levels of water were either present in the initial lyophilized solid or introduced by adsorption of humidity. The crystalline form of the drug was identified as a monohydrate phase. 

Figure 15.2 Typical HX-MS experimental workflow for (a) protein adsorbed on solid surfaces, (b) protein In frozen solution, and (c) protein in lyophilized solid powders. The experimental conditions are shown in the figure...

HX-MS for proteins in lyophilized powders has developed over the past 5 years. Recent studies suggest that the method can provide detailed information on protein conformation, dynamics, and interactions with excipients in lyophilized solids and that HX with mass spectral peak width analysis can be used to screen protein formulations for the presence of nonnative subpopulations. Though the utility of the method for developing lyophilized protein formulations has not been fully tested, early results promote the wider development and application of the method. 

Over the past 25 years, there has been increasing interest in expanding the use of HX-MS. In this chapter, we have reviewed its development and application for proteins in three different environments proteins adsorbed onto solid surfaces, in frozen solutions, and in lyophilized solids. The results have demonstrated the capability of HX-MS to detect and monitor protein conformation and dynamics with high resolution in these environments that differ from bulk aqueous solution. In addition, HX-MS has provided quantitative and site-specific information, addressing many of the limitations of more established techniques such as FTIR and CD spectroscopy. 

Diflerential Scanning Calorimetry (DSC). Calorimetric measurements were carried out by means of a Perkin-Elmer DSC8500 instrument equipped with an Intracooler 2 sub-ambient device and calibrated with purity indium standards. In order to measure the transitions of the nanoparticles, the external block temperature was set at —100 °C. A lyophilized solid powder of nanoparticles was used as sample with weight c.a. 2 mg. The powder was enclosed in aluminum pans and heated from -20 to 200 °C at a rate of 20 °C/min. Bulk sample was measured as received, enclosed in aluminum pans. 

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