Protein-based contaminants

This family generally needs good surface preparation, except for a few special formulations and the heat-cured, toughened variants, which have proved very tolerant of contamination. Protein-based preserving oils on the surface, however, pose difficulties. 

The clinical significance of protein-based impurities relates to (a) their potential biological activities and (b) their antigenicity. Whereas some contaminants may display no undesirable biological activity, others may exhibit activities deleterious to either the product itself (e.g. proteases that could modify/degrade the product) or the recipient patient (e.g. the presence of contaminating toxins). 

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. 

Because of antibody-based selectivity, ELISAs are capable of handling samples that are impure or only semipurihed. It is possible to perform ELISAs in a variety of matrices. This is in contrast to other methods such as HPLC that require relatively pure material. During the development and validation of the ELISA method, it needs to be demonstrated that the ELISA is not affected by interfering substances that could be in the test sample, such as buffers, salts, contaminating proteins, and excipients. It also needs to be demonstrated that the conjugated antibody does not bind nonspecihcally to the coated solid phase. 

For protein-based drugs, filtration via a 0.2 pm filter is an effective way to achieve sterilization. Factors that determine the filtration efficiency include integrity of the filter, pressure, temperature, how rate, contact time of material with the filter, pH, and viscosity. Validation of filters should include chemical compatibility of the filter with the product and possibility of contaminant from the filters leaching into the product. 

To date, most approved protein-based drugs are for therapeutic or replacement therapies. They are recombinant versions of natural proteins such as insulin and erythropoietin. Their characteristics and functions are relatively well defined and known. The next phase of biopharmaceuticals, such as antibodies and vaccines, is more complex and requires more tests and characterizations. Controls for the reliability, contamination, and fidelity of expression systems will be high on the agenda in the coming decade. 

The CE-SDS method is a size-based separation technique generally applicable to proteins from 10 to -200—300kDa. The specificity is generally tested against the formulation buffer and any other possible contaminant proteins. There is usually no interference from the formulation buffer with the assay. For samples that contain contaminant proteins with a hydrodynamic size of 10—200kDa, the method is not specific. 

Similar to protein-based biopharmaceuticals, the standard battery of genotox-icity studies is not considered to be relevant for cell-based therapies unless there is a specific cause for concern regarding the nature of any expressed products that would indicate a potential interaction directly with DNA or other chromosomal material [50,52], The conduct of genotoxicity studies for assessing the genotoxic potential of process related contaminants is also not considered to be appropriate [50],... 

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. 

Ammonium sulfate precipitation (see Note 2) or fractional precipitation is based on the solubility of the particular immunoglobulin. Solubility is illustrated by salt precipitation. It is a convenient first step that allows the reduction of the large volume of the starting material and the precipitation of the desired protein. Contaminant proteins can be trapped or co-precipitated with the target protein, so other methods must follow the ammonium... 

Although there exists a vast number of protein structures in the databases, there is as yet no rational procedure to crystallize a particular protein. The procedure is still mainly based on a trial and error approach. The crystallization process itself is one of which the protein is slowly and orderly precipitated from a solution. As a general rule, the purity of the protein is the most important factor to be dealt with before attempting to crystallize a protein. If possible, care should be taken not only to remove contaminant proteins, but also to remove any structurally heterologous populations in the purified protein sample. This may be achieved by discarding tail... 

Biochemists extract the contents of cells and separate the constituents based on differences In their chemical or physical properties, a process called fractionation. Of particular interest are proteins, the workhorses of many cellular processes. A typical fractionation scheme Involves use of various separation techniques In a sequential fashion. These separation techniques commonly are based on differences in the size of molecules or the electrical charge on their surface (Chapter 3). To purify a particular protein of interest, a purification scheme Is designed so that each step yields a preparation with fewer and fewer contaminating proteins, until finally only the protein of Interest remains (Figure 1-22). 

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