Drying protein solutions

Films or membranes of silkworm silk have been produced by air-drying aqueous solutions prepared from the concentrated salts, followed by dialysis (11,28). The films, which are water soluble, generally contain silk in the silk I conformation with a significant content of random coil. Many different treatments have been used to modify these films to decrease their water solubiUty by converting silk I to silk II in a process found usehil for enzyme entrapment (28). Silk membranes have also been cast from fibroin solutions and characterized for permeation properties. Oxygen and water vapor transmission rates were dependent on the exposure conditions to methanol to faciUtate the conversion to silk II (29). Thin monolayer films have been formed from solubilized silkworm silk using Langmuir techniques to faciUtate stmctural characterization of the protein (30). ResolubiLized silkworm cocoon silk has been spun into fibers (31), as have recombinant silkworm silks (32). 

Like the carbodiimide method, the mixed anhydride method results in an amide complex (Table 5, Figure 17). The acid-containing hapten is dissolved in a dry, inert, dipolar, aprotic solvent such as p-dioxane, and isobutyl chloroformate is added with an amine catalyst. The activated mixed anhydride is chemically stable and can be isolated and characterized. The aqueous protein solution is added to the activated acid and the pH is maintained at around 8.5. A low temperature (around 10 °C) is necessary during the reaction to minimize side reactions. 

Most proteins are not sufficiently stable in aqueous solution to allow formulation as a sterile solution. Instead, the protein is freeze-dried and reconstituted before use. Development of a freeze-dried protein formulation often requires special attention to the details of the freezing process (potential pH shifts and ionic strength increase with freezing) as well as to potential loss of activity with drying. Formulation additives, such as sugars and polyhydroxy compounds, are often useful as cryoprotectants and lyoprotectants. Residual moisture may also be critical to the stability of the dried preparation [33],... 

With mixing, add a quantity of the mono(lactosylamido) mono(succinimidyl)suberate in dry DMF to the protein solution to result in a 10-20 fold molar excess of reagent over the amount of protein present. Depending on the desired application for the lactosyl-modified protein, several different molar ratios of reactant-to-protein may have to be tried to optimize the resulting modification level. 

Add the protein solution to the dried, iodinated Bolton-Hunter reagent. 

As the temperature is lowered further, the viscosity of the unfrozen solution increases dramatically until molecular mobility effectively ceases. This unfrozen solution will contain the protein, as well as some excipients, and (at most) 50 per cent water. As molecular mobility has effectively stopped, chemical reactivity also all but ceases. The consistency of this solution is that of glass, and the temperature at which this is attained is called the glass transition temperature Tg-. For most protein solutions, Tg- values reside between -40 °C and -60 °C. The primary aim of the initial stages of the freeze-drying process is to decrease the product temperature below that of its Tg- value and as quickly as possible in order to minimize the potential negative effects described above. 

Fig. 1.52. Schematic model of CPA action in protein solutions during freezing and freeze drying. (Fig. 10 from [1.36]). Top row Without CPA the hydrate water of the ovalbumin has migrated into the ice and the freed valences are exposed to the influence of the environment. Second row With CPA a part of the hydrate water of the proteins becomes replaced by CPA molecules. These, together with the remaining water molecules and the protein molecule, form a quasi (replacement) hydrate layer.

The starch fraction was washed initially with 0.02% NaOH, the extract being added to the protein solution (Figure 2). The starch was then slurried in distilled water, separated by sedimentation, and dried at 30°C. The combined protein extracts were adjusted to pH 4.5 with IN HC1 and the whey separated from the curd by centrifugation in the basket centrifuge (1100 x g). The protein curd was washed twice with water adjusted to pH 4.5, then resuspended at pH 7.0 using IN NaOH. The proteinate was freeze-dried. 

The reactions were carried out in each case with a 0-1 per cent protein solution in phosphate buffer (pH 6 8), to which the radioactive phosphorofluoridate was added as a concentrated solution in dry ethanol. At the end of the reaction time, the product was dialysed for 20 hr. against running water, and precipitated at 0° by addition of two volumes of acetone. The precipitate was spun off and washed at —5° with ethanol and ether, and dried in air or over sulphuric acid. Samples of 25-50 mg. of dry powder were used for radioactivity determinations, and compared with a standard prepared by hydrolysing a weighed amount (ca. 1 mg.) of the phosphorofluoridate in n sodium hydroxide, neutralizing and drying. 

As the first step, a Ni(II) metal complex of HPMA copolymer was formed and purified [35]. It was then mixed with protein solutions to form dried films, which were rehydrated to investigate the swelling profile and stimuli sensitivity. 

Pipet about 5-10 MBq (about 250 000-500 000 dpm) ofSoln. Ainto a 4-ml test tube. Carefully vaporize the solvent by a gentle stream of dry nitrogen. Add 10 pi of protein solution, containing 2-5 pg of protein in Soln. B, and agitate the tube in an ice bath for 30 min. Add 0.5 ml Soln. C and continue shaking at 0 °C. Fill up to 1 ml with Soln. D after an additional 5 min. Separate the labeled protein from the unreacted reagent as described in Protocol 6.4.1. 

EXAMPLE 4.4 Extent of Hydration of a Protein Molecule from Intrinsic Viscosity Measurements. Suppose an aqueous solution of a spherical protein molecule shows an intrinsic viscosity of 3.36 cm3 g 1. Taking p2 = 1.34 g cm 3 for the dry protein, estimate the extent of hydration of the protein. 

Weigh out dry protein and prepare a stock solution at a concentration of 3 mg/ml in the same solvent as used for the sample protein. Store up to 3 months at -20°C. 

After chromatography, a protein solution can be prepared for storage either by drying or by stabilization in solution with an excipient. We will now discuss both options. 

Spray drying, employed preferentially for inexpensive enzymes on a mass scale, evaporates water by spraying the protein solution through a nozzle at high temperature, utilizing the Bernoulli effect. As contact times are short (on the order of less than 1 s), the enzyme is not deactivated. Not much modeling has been performed on this operation. 

Lyophilization employs sub-zero temperatures in combination with very low pressure to withdraw water from the protein solution typical values would be -80 °C and 1-3 mbar. In these conditions, as the water phase diagram reveals, water sublimes, leaving a fluffy porous enzyme powder. As typical run times are on the order of a day, lyophilization is not the method of choice for large-scale enzyme drying operations. On a laboratory scale, however, lyophilization is a very effective method. 

Table 3.3 Comparison of T and Tc forthree protein solutions (Table 1 from [3.70]) and recovery of protein activity after freeze-drying (%) (Table 2 from [3.70])...

The sample of 0.01-0.02 ml is applied as a spot either by means of a micropipette or, more easily, by means of a small thin paper disk impregnated with the protein solution. The application zone is slightly dried beforehand to avoid diffusion and to favor rapid uptake of the sample. To obtain each fraction as an isolated spot and with little trailing along the migration path, the immediate uptake of the entire microspot by the application zone of the substrate is important. If the application... 

Figure 14.9 Microcontact printing of proteins.93 A protein solution is incubated on the top of an elastomeric stamp. After drying, the stamp is brought into conformal contact with the glass substrate and transfer of proteins occurs only in the places of contact between the stamp and the substrate.

Rabouille, Cortassa, and Aon[81 dried protein, glycoprotein, or polysaccharide containing brine solutions that resulted in dendritic-like fractal patterns. The fractal dimension, D = 1.79, was determined for the pattern afforded by an ovomucin-ovalbumin mixture (0.1 M NaCl). Similar D values were obtained for dried solutions of fetuin, ovalbumin, albumin, and starch the authors subsequently suggest that fractal patterning is characteristic of biological polymers. 

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