Isoelectric impurities

Purification. Enzyme purity, expressed in terms of the percent active enzyme protein of total protein, is primarily achieved by the strain selection and fermentation method. In some cases, however, removal of nonactive protein by purification is necessary. The key purification method is selective precipitation of the product or impurities by addition of salt, eg, sodium sulfate, or solvent, eg, ethanol or acetone by heat denaturation or by isoelectric precipitation, ie, pH adjustments. Methods have been introduced to produce crystalline enzyme preparations (24). 

Capillary zone electrophoresis (CZE) is the most common electrophoretic separation technique due to its simplicity of operation and its flexibility. It is the standard mode for drug analysis, identification of impurities, and pharmacokinetic studies. Other separation modes, such as capillary isotachopho-resis (CITP), micellar electrokinetc chromatography (MEKC), capillary electrochromatography (CEC), capillary gel electrophoresis (CGE), capillary isoelectric focusing, and affinity capillary electrophoresis (ACE), have then-advantages in solving specific separation problems, since the separation mechanism of each mode is different. 

An example of isoelectric focusing is shown at the left below. A mixture of seven proteins (and some impurities) was applied to a polyacrylamide gel containing a mixture of polyprotic compounds called ampholytes. Each end of the gel was in contact with a conducting solution and several hundred volts were applied across the length of the gel. The ampholytes migrated until they... 

The purification of glutamine cyclotransferase from papaya latex has been carried out by Messer and Ottesen (116). A batch procedure was used for the removal of impurities by passage of a papaya latex extract through a thin layer of carboxymethyl-Sephadex the active protein was separated by selective elution. Additional purification was achieved by chromatography on a column of carboxymethyl-Sephadex and by gel filtration on Sephadex G-100. The purified enzyme was homogeneous by the criteria of paper electrophoresis, ultracentrifugation, and gel filtration on Sephadex G-100 columns and chromatography on carboxy-methyl- or DEAE-Sephadex. The electrophoretic behavior of the enzyme indicates that it is a basic protein with an isoelectric point near... 

The surface of uncalcined aluminas consists of amphoteric hydroxyl groups that are even less acidic than those in silica gel. Electrophoretic measurements by Stigter et al. (90) show that the isoelectric point for alumina is attained at a pH of 9, much higher-than the value of 2 obtained in the case of silica gel. Thus, hydroxylated aluminas are relatively non-acidic, unless they are promoted with acid-producing impurities. 

CE has many separation modes that are beneficial to protein impurity analysis. Within the many thousands of potential protein impurities in a recombinant product there will be several that have only minor physicochemical differences from the drug product. The application of different CE modes can potentially resolve these impurities. CE methods can be divided into four principle modes that are applicable to recombinant protein impurity analysis capillary zone electrophoresis, capillary isoelectric focusing, capillary gel electrophoresis, and micellar electrokinetic capillary chromatography. Each mode will be discussed briefly. Since the technology is so young and still very exploratory, CE methods are developed empirically for specific separations. It is difficult to provide standard protocols for CE impurity analysis. Instead, protocols that can be used as a starting point for impurity analysis will be provided as well as the citation of examples of impurity analyses from the literature to provide additional sources of protocols for interested readers. 

Capillary isoelectric focusing (CIEF) separates proteins based on differences in their isoelectric points. CIEF has been used to separate product-related impurities of recombinant proteins, mainly deamidated species, such as those of human growth hormone.71... 

Abrasive particles are a key component in CMP slurry. The most commonly used abrasive particles include silica, alumina, ceria, zirconia, titania, and diamond. Table 21.1 listed a set of information on each type of abrasive particles such as density, microhardness, and isoelectric points (lEP). It is important to point out that the specific values for these properties depend highly on the preparation techniques and the specific states of the samples. The values listed in the table represent an average of the most commonly reported data. For example, the isoelectric point for silica is a function of the number of hydroxyl groups, type and level of adsorbed species, metal impurity in the solid matrix, and the treatment history of the materials [1]. There are three major types of silica according to their preparation methods fumed, colloidal, and precipitated. The common sources for obtaining these abrasive particles are listed in Table 21.2. As examples, some of the more specific information on... 

With its determination carried out at constant ionic strength the pH, value, so obtained, provides an assessment of the acidity of the amphoteric ion exchanger. This pH, relatable to the average value of pKj and pK2 with Eq. (16), is very close to the value of the isoelectric point 0 )- Paries [105] has pointed out that the relationship between the lEP of a solid surface and the valency-effective ionic radii, when corrected for crystal field effects, coordination, hydration, and other factors, is quite good. He also has indicated that the broad probable lEP range characteristic of a cation oxidation state may be selected from the data in Table 2 as shown below. It is known that the lEP for amphoteric oxides is affected by the presence of impurities, crystallinity and the chemical species under investigation. 

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