Proteins characterization tests

The final, purified bulk product is thoroughly characterized and compared to reference standards established by the manufacturer for new molecular entities or available from the United States Pharmacopoeia (USP) or the World Health Organization s National Institute of Biological Standards and Control. Characterization tests of proteins may include biologic potency assays, chromatographic assays, gel... 

Very few immunosensors are commercially available. The commercial immunosensors are either the detector or bioanalyzer types. The PZ 106 immunosensor from Universal Sensors Inc. (New Orleans, LA) has been used as a detector to measure antibody-antigen reaction. Ohmicron (Newtown, PA) developed a series of pesticide immuno-bioanalyzers that have been used in field tests. Pharmacia Biosensor USA (Piscataway, NJ) recently introduced BIAcore immunodetection system. A combination of a unique flow injection device and surface plasmon resonance (SPR) detection technique provides a real time analysis. A carboxylmethyldextran layer added to plasmon generating gold film is a hydrophobic, activatable, and flexible polymer that provides high antibody and low non-specific bindings. System demonstration at the Institute of Food Technologists (IFT) 1994 meeting in Atlanta drew attention of food scientists. It should easily be adapted for food protein characterization. 

Cytochrome c (cyt. c) has become a major protein for testing new approaches and techniques in protein science.1 This is partly due to the venerable position that cytochrome c holds in the field of biochemistry since it was isolated and characterized more than 70 years ago. Cyt. c was one of the first proteins to be sequenced,2 and to have its X-ray structure determined in 1967.3 Cyt. c also has the advantage of stability and a spectroscopically distinct heme group. More than 23,000 articles mentioning cyt. c were published between 1945-2002 (ISI Web of Science). Here, we describe an approach to tetraphenylporphyrin-based protein surface receptors and the characterization of their interactions with the principal target cyt. c. 

IEF is used as a purity test and for protein characterization, comparing it with the relative position of an authentic band of the target protein. It can also be used as a method to evaluate the stability of a biological product. Small changes such as the deamination of an amino acid will cause a change in the pi and a modification of the band pattern. 

The norms for medicinal production are particularly stringent. Biological products are composed of complex molecules, produced by cell lines with a relatively recent history, and difficult to characterize. Tests performed only on the final product do not guarantee consistency of production. The purification procedures should be planned and validated for the removal of potential contaminants from diverse sources cells, culture media, equipment, and reagents used in the purification or even degradation products derived from the protein itself. There are examples of products with unexpected risks that have caused serious problems such as blood contamination by HIV-1 virus between 1980 and 1985 (Bloom, 1984) or the presence of residual infectious viruses in the poliomyelitis vaccine due to inefficient inactivation (Lubiniecki et al., 1990). 

Later, Busto et al. used synthesized Fe-labeled transferrin to determine individual transferrin isoforms in human serum samples for the species-specific mode. The stability of the prepared proteins was tested for 1 week and no iron exchange had occurred. They concluded that the results are in good agreement with other calibration methods, e.g. the species-unspecific method however, the species-spedfic mode can offer better precision. Hoppler et al. also synthesized and characterized Fe-labeled Phaseolus vulgaris ferritin for isotope dilution analysis. " ... 

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. 

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