Development and Use of Enzyme-Linked Immunosorbent Assays (ELISA) for the Detection of Protein Aggregates in Interferon-Alpha (IFN-α) Formulations

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Abstract
Purpose. Protein aggregates are thought to be involved in the immunogenicity of recombinant proteins in humans. To probe human IFN-α formulations for the presence of soluble protein aggregates, enzyme-linked immunosorbent assays (ELISA) were developed. Methods. For the detection of IFN-α-IFN-α and HSA-IFN-α aggregates, sandwich ELISAs were developed using a monoclonal anti-IFN-α antibody as a capture antibody and the same anti-IFN-α antibody and an anti-human serum albumin (HSA) antibody (HRP-labeled), respectively. Results. Marketed freeze-dried, HSA-containing IFN-α formulations tested in the ELISAs all contained IFN-α-IFN-α and/or HSA-IFN-α protein aggregates, although in varying amounts. These aggregates were predominantly IFN-α dimers and 1:1 conjugates of HSA with IFN-α. Test formulations revealed that aggregation of IFN-α was strongly affected by the presence of pharmaceutical excipients, pH of the formulation, lyophilisation procedure, and storage temperature and time. Conclusions. The ELISAs are rapid, highly specific for aggregates in the presence of both IFN-α and HSA monomers and allow the direct detection of both types of aggregates in formulations in the nanogram range. The new assays will assist the monitoring of the aggregate-inducing processes during IFN-α formulation and storage in an early phase and the development of aggregate-free IFN-α formulations.