Stability testing proteins

Fig. 3.2.7 Left panel Effects of temperature on the luminescence intensity and stability of the protein P from Meganyctiphanes. The initial light intensity was measured with F plus P in 5 ml of 20 mM Tris-HCl/0.15 M NaCl, pH 7.5, at various temperatures. In the stability test, P was kept at the indicated temperature for 10 min, then mixed with 5 ml of 25 mM Tris-HCl/1 M NaCl, pH 7.59, containing F, to measure initial light intensity. Right panel Effect of the concentration of salts on the light intensity of the luminescence of F plus P, in 25 mM Tris-HCl, pH 7.6, at near 0°C. In the case of NaCl, the light intensity decreased to about a half after 10 min. From Shi-momura and Johnson, 1967, with permission from the American Chemical Society.

During the characterization process, hits are typically tested for kinetic solubility and permeability in a model of passive diffusion such as PAMPA [22]. As new compounds are synthesized, additional parameters also need to be considered, such as pZa, chemical and plasma stability, and protein binding. Calculated properties such as MW, clogP, and PSA should also be tracked. 

Quality of biotechnology products, analysis of the expression construct in cells used for production of r-DNA derived protein product Quality of biotechnological products stability testing of biotechnological/Biology products Availability of Draft Guideline on Quality of... 

In addition to the direct absorbance methods, colorimetric methods are suited for relatively pure proteins as purification progresses. They are accurate if calibrated from a standard curve of the test protein reference sample and fast if automated. However, they are not as simple to perform as direct absorbance methods. Hence they are not as suitable for production as direct absorbance methods. The relative simplicity of colorimetric methods makes them more suited to automated formulation and stability studies and total-protein assays of complex mixtures. Microtiter plate versions of colorimetric assays allow for automation and consumption of relatively small sample sizes while requiring little specialized equipment or training. 

Finally, the protein assay for the drug product will also be used for realtime and accelerated stability testing if it has been validated to be stability indicating. A stability-indicating protein concentration method usually translates to a method that can reveal how much protein can be recovered from the dosage form. Many protein instabilities result in precipitation of the protein and adsorption to the container. An instability that results in only a modification of the protein structure but not in loss of protein from solution will not be detected by a sequence-independent protein assay such as a colorimetric assay. 

Physical properties of the protein structure should be considered in designing strategies to achieve stable formulations because they can often yield clues about which solution environment would be appropriate for stabilization. For example, the insulin molecule is known to self-associate via a nonspecific hydrophobic mechanism66 Stabilizers tested include phenol derivatives, nonionic and ionic surfactants, polypropylene glycol, glycerol, and carbohydrates. The choice of using stabilizers that are amphiphilic in nature to minimize interactions where protein hydrophobic surfaces instigate the instability is founded upon the hydro-phobic effect.19 It has already been mentioned that hydrophobic surfaces prefer... 

Commercial wines are commonly tested for protein stability. Wine proteins, upon denaturation by heat or cold, may cause cloudiness and unsightly deposits after bottling. In addition, proteins may combine with iron and copper salts to form flocculate material in bottled wines. The reaction and absorption of proteins on bentonite is an effective means of removing protein from wines (109, 110, 111). Therefore, fining wines... 

Wilde, P.J. and Clark, D.C. 1996. Foam formation and stability. In Methods of Testing Protein Functionality (G.M. Flail, ed.) pp. 110-152. Blackie Academic and Professional, New York. 

Stamper GF, Lambert WJ. Accelerated stability testing of proteins and peptides pH-stability profile of insulinoptropin using traditional Arrhenius and non-linear fitting analysis. Drug Dev Ind Pharm 1995 21 1503-1511. 

First, laboratory testing is conducted to ascertain the stability of the wine. Like tests for protein stability, tests for determining stability and method for correcting instability vary from winery to winery. Berg (34) suggested that a wine stored at — 4° C for four days, without a bitartrate crystalline deposit, may be considered stable. The wines usually are allowed to warm to room temperature before test results are read. Absence of crystals indicates stability. A quantitative method, the concentration product (36), also can be used to evaluate tartrate stability. 

Complementary to the simple adsorption test, the mass balance of the separation could be investigated. This method is more accurate to determine the loss of free drug, which may occur due to variations in sample preparations methods, non-specific protein binding and to metabolism. The latter may also be tested in separate stability tests in plasma or applied matrix. 

An example for an automated stability test in plasma is described by Linget and du Vignaud (1999). Incubations are performed on a 215 Gilson liquid handler. Incubation was done at substrate concentrations of 50 pM on 96 deep well plates. Each incubation tube contained 375 pL of a 200 pM test compound solution (in 0.1 M Tris buffer with 3% BSA, added to assist dissolution of compounds with poor solubility) and 1125 pL of plasma. Samples are taken after incubation times of 0, 1, 2, 3, 4 and 5 min. At each of these time points an aliquot of the incubation mixture was transferred from the incubation tube into a well in a 96 deep well plate containing an equal volume of acetonitrile for quenching by protein precipitation followed by centrifugation of the plates. Supernatants were analyzed by HPLC for metabolic screening. 

Q5C in Cell Lines used for Production of r-DNA derived Protein Products Quality of Biotechnological Products Stability Testing of Biotechnological/ CPMP/ICH/138/95 Step 5... 

Pocock, K.F., Waters, EJ. (2006). Protein haze in bottled white wines how well do stability tests and bentonite fining trials predict haze formation during storage and transport . Aust. J. Grape Wine Res., 12, 212-220... 

A fundamental premise of the entatic state concept is that the metal simply fits into a site that is preformed by the protein and determined by the many interactions which stabilize the protein structure. This idea has been tested by crystallographic studies of apo-forms of azurin, plastocyanin, pseudoazurin and amicyanin. 

Although copper binds tighter than zinc to aU forms of the enzyme tested, zinc stabilizes the protein fold better as judged by solvent-induced denaturation experiments. In addition the dissociation rate constant for zinc is about 100 times slower than copper suggesting the zinc is kinetically trapped once folding has occurred. This may thus be a physiological means by which metal ion specificity is achieved. ... 

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