Abstract
Publisher Summary This chapter discusses the analysis of different forms of recombinant human interferons by high-performance liquid chromatography (HPLC). The cloning and expression of the recombinant human leukocyte interferon A (IFN-αA) gene in E. coli and subsequent purification by the use of immobilized monoclonal antibodies has led to the scaled-up production of IFN-αA for human clinical trials. Analysis of the resultant IFN-αA by nonreducing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE) revealed the presence of higher oligomers (dimers, trimers, and tetramers) and at least two monomeric forms. The two major monomeric forms have been termed “slow” and “fast” migrating monomers (SMM and FMM, respectively) to designate their relative mobilities on SDS-PAGE. Although the components of IFN-αA can be separated by SDS-PAGE, HPLC is an alternative method, which may be advantageous for the routine analysis of multiple samples. The HPLC system comprises of two constametric pumps (Models I and IIG), Analytical HPLC is a powerful tool for the evaluation of human leukocyte interferons (IFN-αA, IFN-αD), hybrids (IFN-αA/D), immune interferon (IFN-γ) as well as related synthetic peptide fragments. The three forms of human leukocyte interferon can be resolved, and chromatograms of admixtures can be readily carried out to make positive identification of known impurities.
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