Protein purity, assessment

Protein Purity Is Assessed by Polyacrylamide Gel Electrophoresis (PAGE)... 

Because of the speed and high resolution of CZE separations as well as the small sample volumes required to yield information about complex protein samples, CE is increasingly being used to assess protein purity in multistep purification protocols in laboratory, pilot plant, and process scales. Similarly, it is being considered as a candidate for monitoring fermentation. 

Purity and homogeneity of the purified protein is assessed by macromolecular exclusion chromatography, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The later technique, developed by Karas and Hillenkamp, ionizes and separates proteins on the basis of their mass-to-charge ratio (Karas and ffillenkamp, 1988). 

Methods for assessing protein purity must quantitate the amount of target protein relative to the amount of contaminant(s) in the sample. This requires two separate analytical methods one for the taiget protein and one for the total protein including contaminants. For example, with enzymatic proteins, assessment of functional purity typically relies on kinetic assays in which the amount of substrate consumed or product produced per unit time is proportional to the amount of enzyme present. The total activity of the sample is calculated and compared to the total amount of protein to give the specific activity of the sample. Methods for determining total protein are discussed in Chapter Bl. An increase in specific activity at a purification step reflects a loss of contaminant proteins. 

Although electrophoresis is the method of choice for assessing protein purity, it is not frequently used as a purification step, at least not yet. This is because of the small amounts that can usually be analyzed and because of the denaturing conditions that are often used in electrophoretic analysis. 

Galactosidase was purified as described previously (17), except for several slight modifications. An 800 mL gradient from 0.09 M to 0.18 M NaCl was us to elute the protein from the DEAE column. The active fractions from the DEAE elution were pooled, precipitated with ammonium sulfate, and then applied to an FPLC Superose 6 size exclusion column into the appropriate assay buffer. Protein purity was assessed by SDS-PAGE, and the enzyme concentration was determined using an extinction coefficient of 2.09 cm /mg at 280nm(18). 

Capillary electrophoresis (CE) is a modern analytical method that is being extensively applied to the characterization of biotechnology-derived products like peptides and proteins [1], Due to its ease of automation and facilitating the development of reproducible routine analysis, CE seems to be well suited for the quality control of biotechnological products, including process monitoring, purity assessments. 

Intact mitochondria were isolated by the method of Johnson and Lardy with the modifications previously published. Mitochondrial outer membranes were isolated by the method of Parsons et and their purity assessed as described previously. Protease treatment of intact mitochondria and protection experiments were carried out as described previously. " Briefly, this method consisted of incubating the mitochondria (5mg/ml), and outer membranes (1 mg/mL) with Nagarse (5 t.g/mL) at 37°C for lOmin after which the proteolytic activity was stopped by addition of 200 pi of 20% (w/v) BSA/mL of incubation volume plus 40mL of ice-cold isolation medium. After centrifugation (5,600 xg for lOmin), the mitochondria were resuspended (4mg/mL) in isolation medium and used as indicated. Protein determination was by a biuret method. In some experiments intaet mitoehondria or the isolated outer membranes were first ineubated... 

Refractometry is a technique used to detect the concentration of binary mixtures based on differences in their refractive index. Besides concentration, it is also a simple method to quantify purity. Salinity of brine or sucrose concentrations in grapes or syrup are two typical applications. Urine-specific gravity is measured for drug diagnostics and plasma protein is assessed by refractometry in veterinary medicine. 

Purity determination is an essential component of the assessment of the quality of any drug. However, the purity determination of biopharmaceuticals is not as straightforward as that of small-molecule pharmaceuticals, since biopharmaceuticals are structurally complex and have a wide range of potential impurities. Approaches usually involve the judicious choice of a combination of methods that enable the detection and quantitation of impurities from which an overall purity assessment can be derived. As mentioned previously, proteins undergo degradation or modification through a number of pathways. Most of the more common variants encountered in protein preparations... 

Deyl, Z. Miksik, I. Eckhardt, A. Preparative procedures and purity assessment of collagen proteins. J. Chromatogr. B, 2003, 790, 245-275. 

Standardization and Testing". RequHemeats are geaerally specified within Hceases Hi the United States, and include a variety of Hi-process tests to assess purity, safety, and potency of the iadividual components and potency and safety of the final product. Potency is standardized by determining the size of the conjugate and the quantitative amount of saccharide that is bound to the carrier protein. General safety and immunogenicity is assessed Hi animals. 

SDS polyacrylamide gel electrophoresis (SDS-PAGE) represents the most commonly used analytical technique in the assessment of final product purity (Figure 7.1). This technique is well established and easy to perform. It provides high-resolution separation of polypeptides on the basis of their molecular mass. Bands containing as little as 100 ng of protein can be visualized by staining the gel with dyes such as Coomassie blue. Subsequent gel analysis by scanning laser densitometry allows quantitative determination of the protein content of each band (thus allowing quantification of protein impurities in the product). 

Kundu et al.64 used MEKC conditions to assess the purity of two recombinant proteins a cytomegalovirus-CMP-KDO synthetase fusion protein expressed in E. coli and a hepatitis C viral protein expressed in CHO cells. Proteins were prepared in a 10-mM Tris-1% SDS buffer (pH 8.5) and analyzed in a 10-mM borate-100-mM SDS buffer (pH 9.5) in uncoated capillaries. The level of impurities, which varied with the method of protein production, agreed within 5% with results obtained by densitometric scanning of SDS-PAGE gels of the same materials. 

C. A. S. Barnes, D. E. Clemmer Assessment of purity and screening of peptide libraries by nested ion mobdity-TOFMS identification of RNase S-protein binders. Anal. Chem. 2001, 73, 424-433. 

Characterization of Purified Proteins Assessing purity, 182, 555 determining size, molecular weight, and presence of subunits, 182, 566 amino acid analysis, 182, 587 limited N-terminal sequence analysis, 182, 602 peptide mapping, 182, 613 analysis for protein modifications and nonprotein cofactors, 182, 626 protein crystallization, 182, 646. 

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