Immunoassays impurities detected using

Cell Culture-Derived Media-Derived Protein Impurities. Immunoassays can detect low impurity levels (<1 ppm).4 The ELISA is probably one of the most sensitive analytical methods. If bovine serum is used as a media component, then testing should include ELISAs for bovine serum albumin (BSA), bovine transferrin, bovine fetuin, and bovine IgG. Often hormones and growth factors, such as insulin or insulinlike growth factor, are used as media components. ELISAs should be used to detect and quantitate these residuals in the various production steps as well as in the final product. There are commercially available antibodies to most commonly used media components. If proprietary media components are used, then the same investment in time and effort is required for the production of specific antibodies, as described above for host cell impurities. 

Most recombinant biopharmaceuticals are produced in microbial or mammalian cell lines. Thus, although the product is derived from a human gene, all product-unrelated contaminants will be derived from the producer organism. These non-self-proteins are likely to be highly immunogenic in humans, rendering their removal from the product stream especially important. Immunoassays may be conveniently used to detect and quantify non-product-related impurities... 

The validity of any statement about the purity of a protein is directly linked to the quality of the analytical method used. The validation of immunoassay systems to detect protein impurities in rDNA pharmaceuticals must be achieved by careful production and characterization of the assay reagents. The studies presented here demonstrate that the blank run approach is reasonable for the isolation of reference materials and that high quality broad spectrum antisera can be produced to these mixtures. Significant improvements in assay sensitivity approaching the ppb level are attainable and should provide the methods to further improve product purity. 

This part will mainly focus on host cell proteins (HCPs) quantification. Process-specific HCP assays are in general targeted to be in place prior to the initiation of phase III clinical trials. Immunoassays are the most specific and sensitive techniques available for detecting and quantifying protein impurities. There are two methods commonly employed to quantify protein impurities in biopharmaceuticals enzyme-linked immunosorbent assays (ELISA) and immunoligand assays (ILA). Both methods are able to detect very low ppm level of impurities. ELISA have been developed to measure host protein impurities in a number of recombinant proteins including human growth hormone (Anicetti et ah, 1986), insulin (Baker et al, 1981) and staphylokinase (Wan et ah, 2002). ILA assays have been used to detect protein impurities in recombinant bovine somatotropin (Whitmire and Eaton,... 

For this type of ELISA, the higher the sample antigen concentration, the weaker the analytical signal. Competitive ELISA is highly specific as well as sensitive, since two antibodies are used and the sample antigen is selectively captured and detected. This immunoassay also is suitable for crude or impure sample solutions since purification of the antigen is not required. 

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