Lyophilized protein aqueous solution

Solid state forms of macromolecules are a common method for preparing protein pharmaceuticals, due to stability advantages. Lyophilization is often utilized for proteins and peptides because it limits the moisture content and increases protein stability. Proteins are routinely lyophilized from aqueous solutions, but lyophilization from co-solvent conditions have also been documented (Santos et al., 2001). 

The simplest way to prepare a biocatalyst for use in organic solvents and, at the same time, to adjust key parameters, such as pH, is its lyophilization or precipitation from aqueous solutions. These preparations, however, can undergo substrate diffusion limitations or prevent enzyme-substrate interaction because of protein-protein stacking. Enzyme lyophilization in the presence of lyoprotectants (polyethylene glycol, various sugars), ligands, and salts have often yielded preparations that are markedly more active than those obtained in the absence of additives [19]. Besides that, the addition of these ligands can also affect enzyme selectivity as follows. 

There are three commonly used methods [145] to incorporate enzymes in RMs (i) injection of a concentrated aqueous solution, (ii) addition of dry lyophilized protein to a reverse miceUar solution, and (iii) phase transfer between bulk aqueous and surfactant-containing organic phases. Figure 3 shows schematically the three enzyme solubiHzation methods. The injection and dry addition techniques are commonly used in biocatalytic appHcations, the latter being well suited to hydrophobic proteins [146]. The phase transfer technique is the basis for extraction of proteins from aqueous solutions. 

These spectra, taken at variable temperatures and a small polarizing applied magnetic field, show a temperature-dependent transition for spinach ferredoxin. As the temperature is lowered, the effects of an internal magnetic field on the Mossbauer spectra become more distinct until they result at around 30 °K, in a spectrum which is characteristic of the low temperature data of the plant-type ferredoxins (Fig. 11). We attribute this transition in the spectra to spin-lattice relaxation effects. This conclusion is preferred over a spin-spin mechanism as the transition was identical for both the lyophilized and 10 mM aqueous solution samples. Thus, the variable temperature data for reduced spinach ferredoxin indicate that the electron-spin relaxation time is around 10-7 seconds at 50 °K. The temperature at which this transition in the Mossbauer spectra is half-complete is estimated to be the following spinach ferredoxin, 50 K parsley ferredoxin, 60 °K adrenodoxin, putidaredoxin, Clostridium. and Axotobacter iron-sulfur proteins, 100 °K. 

There are no published studies which examine the significance of pH shifts on quality attributes of freeze-dried formulations of small molecules. However, Costantino et al. (14) reported that lyophilized organic compounds containing protein functional groups (amino-, carboxylic-, and phenolic-) exhibit pH memory that is, the ionization state of the solid, as reflected by the FTIR spectrum, is similar to that of the aqueous solution from which the compound was freeze dried. 

Bovine serum albumin (BSA), fraction V, B-lactoglobulin, gum arabic, and alginic acid, low viscosity, were purchased from Sigma Chemical Co. (St. Louis, MO). The proteins were used without further purification. Aqueous solutions of gum arabic and alginic acid were filtered successively through cellulosic membrane filters of 5.0 fan and 0.45 fan pore size to remove insoluble particulate matter. The filtrate was frozen and lyophilized prior to use. 

It is essential that pooled fractions be lyophilized after each RP-HPLC step. This is because the protein is denatured in water-acetonitrile-TFA, so to obtain a clean, unimolecular interaction of the polypeptide with the column there should be no aggregation. The sample is loaded in aqueous solution. 

The purified proteins are dialyzed extensively against two changes of 1% acetic acid and finally against 0.1% trifluoroacetic acid They can be stored as lyophilized powders at -20°C or -80°C and are stable at room temperature for a few days. It is very important to dissolve them in H20 and not buffers or medium, since on lyophilization they form the trifluoroacetate salt, but once in aqueous solution at low pH can be adjusted to the required physiological pH. We routinely dissolve them in H20 at a concentration 10-fold higher than required, and dilute 10-fold into the required buffer or medium. This does not pose a problem even for micromolar concentrations, since all of the chemokines that we have worked with are soluble at 1.25 mM or 10 mg/mL. 

Lyophilization, also called freeze-drying, is the process of subliming a solvent, usually water, with the object of recovering the solid that remains after the solvent is removed. This technique is extensively used to recover heat- and oxygen-sensitive substances of natural origin, such as proteins, vitamins, nucleic acids, and other biochemicals from dilute aqueous solution. The aqueous solution of the substance to be lyophilized is usually... 

An important parameter for maintaining protein activity under non-aqueous conditions is the pH memory of the protein. The half-life of subtilisin in DMF was directly related to the pH of the aqueous solution from which the protein was lyophilized, where increased activity was observed as the pH was increased from 6.0 to 7.9 (Schulze and Klibanov, 1991). The pH memory of the protein from its last aqueous state can be attributed to the ionization state of the functional groups upon lyophilization, precipitation or other isolation methodology (Zaks and Klibanov, 1988a Constantino et al., 1991 Guinn etal., 1991 Klibanov, 1997). Therefore, ionization of the protein, as well as solvent elfects, must be considered. 

Proteins in solid state can be found in different forms, lyophilized or in frozen aqueous solution. Under irradiation, lyophilized proteins mostly aggregate, as was shown for egg-white lysozyme irradiated at room temperature (13, 14, 15, 16). On the contrary, irradiation of frozen protein solutions gives rise to fragmentation (11, 16 and references therein). Rupture of the N-Ca bond of the polypeptide chain seems to be responsible for fragmentation, as it was observed for homopolymers irradiated at 77K (17). 

Physical gels are formed when water is added to a lyophilic polymer but in insufficient amounts to completely dissolve the individual chains. Various polysaccharides such as pectin, carrageenan, and agarose, and proteins such as gelatin form physical gels in aqueous solution. These types of gels are usually reversible, i.e., they can be formed and disrupted by changing the pH, tanperature, and other solvent properties. 

In simple coacervation or gelification (500 pm to 2 mm), an emulsion of oil in an aqueous solution of a polymer/substance able to form a gel, is prepared. By changing pH, tanperature, or adding salts, the substance will precipitate around the drops (alginate/CaClj gelatine hot/cooled oil). Then particles are separated and dried. Essential oils in zein (proteins) nanospherical particles (100 nm) were prepared by phase separation, and then lyophilized (Parris et al., 2005). 

The amount of moisture sorbed by the lyophilized protein sample is directly related to the activity of DjO vapor phase (i.e., RH) to which the protein is exposed. To maintain constant RH throughout the experiment, solid-state amide HX is usually carried out inside a sealed desiccator. The desired RH can be easily maintained by using a suitable saturated salt solution [62]. Some commonly used salts and the resulting %RH are LiCl (11% RH), KCjH30 (23% RH), MgCl (33% RH), I C03 (43% RH), and NaCl (75% RH) [60]. The RH values correspond to the RH over aqueous solutions of these salts at room temperature the humidity over D O is assumed to be identical. To control the rate of moisture sorption and prevent powder collapse in formulations containing hygroscopic excipients... 

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. 

Crude Solid. The simplest way to use enzymes in organic solvents is to suspend a precipitate or a lyophilisate. The enzyme does not need to be of high purity, but some care should be taken during the preparation. In aqueous solution, the enzyme has an optimal pH, dictated by the ionization state under which the amino acids involved in the catalysis must be to allow activity. The solid enzyme must be in the same ionization state when used in organic solvents (15). For this purpose, it is important to precipitate the enzyme or lyophilize it from a solution buffered at this pH. This applies to the other forms of solid enzyme preparations. The other important point is the drying of the preparation. It has been observed that the secondary structure of proteins can be affected by lyophilization (16). This can be avoided by the use of lyoprotectants such as sorbitol (17) or salts such as KCl (18). 

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