Protein-based contaminants detection

A number of different techniques may be used to characterize protein-based biopharmaceutical products, and to detect any protein-based impurities that may be present in that product (Table 7.2). Analysis for non-protein-based contaminant is described in subsequent sections. 

We have developed an analogous, but more robust system which is not necessarily constraint by the aforementioned limitations. The obvious extension has been to couple an affinity-based separation with mass spectrometry. Hutchens et al. have shown that affinity probe surfaces can be ust to capture specific protein ligands allowing detection by laser desorption mass spectrometry (. The limitations to their technique have been that the surface area for ligand capture is quite small and salt (or detergent) contaminants are still problematic. Perfusive affinity resins, on the other hand, provide a tremendous surface area for binding. The nature and composition of the solvents required for affinity chromatography, however, are not directly compatible with mass spectrometric analysis. 

A green fluorescent protein-based Pseudomonas fluorescens strain biosensor was constructed and characterized for its potential to measure benzene, toluene, ethylbenzene, and related compounds in aqueous solutions. The biosensor is based on a plasmid carrying the toluene-benzene transcriptional activator (Stiner and Halverson, 2002). Another microbial whole-cell biosensor, using Escherichia coli with the promoter luciferase luxAB gene, was developed for the determination of water-dissolved linear alkanes by luminescence (Sticher et al., 1997). The biosensor has been used to detect the bioavailable concentration of alkanes in heating oil-contaminated ground-water samples. 

The initial emphasis in analytical biotechnology was on broad safety concerns that translated into detection of host-cell components such as DNA, endotoxins, Escherichia colt proteins, and retroviral contamination.2 The detection of these impurities requires development of high-sensitivity assays that are based primarily on antibodies [e.g., enzyme-linked immunosorbent assay (ELISA) for E. coli proteins) or radioactivity (e.g., dot-blot assays for DNA detection). New developments are focused on low-sensitivity detection, characterization, and removal of undesirable target sequence variants. Bioseparations play an important role even after a product has been isolated and shown to contain a low level of contaminants for initiation of clinical studies. The focus shifts to achievement of a reproducible, large-scale manufacturing process. At this stage, analytical methods provide essential informa-... 

Pathogen identification and characterization systems are dominated by the same two technological approaches as those used for environmental sensors supporting contamination avoidance (1) antibody-based systems in which the identification of a pathogen is made by a characteristic reaction between the pathogen and a protein product of the immune system, or (2) nucleic acid methods in which a pathogen s DNA is amplified and detected. 

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