Development of an equilibrium immunoassay using electrochemiluminescent detection for a novel recombinant protein product and its application to pre-clinical product development

In order for a biotechnology derived protein product to be considered 'well characterized', a thorough understanding of the pharmacokinetic and metabolic fate of the product is essential. Specifications for such products need to be based on historical data obtained in the pre-clinical, clinical and manufacturing experience through the use of specific, accurate, precise and validated assays. Typically, assays such as ELISA or RIA that have the disadvantages of limited dynamic range, matrix interactions and hazardous waste generation are used to gather this data. We present data in this abstract that demonstrates the utility of an equilibrium immunoassay that uses electrochemiluminescent detection in the assessment of the pharmacokinetic and metabolic fate of a biotechnology derived product based on either the human (AMG1h) or murine (AMG1m) protein sequence. The assay uses biotinylated antibody in a sandwich format with antibody labeled with the N-hydroxysuccinimide ester of a ruthenium (II) tris-bipyridine chelate for detection in the Origen(TM) System. Streptavidin-coated paramagnetic beads are used to capture the antibody-antigen-antibody sandwich complex and facilitate electrochemiluminescent detection. The assay standard curve for AMG1h ranges from a lower limit of quantitation (LOQ) of 2.5 ng ml(-1) to an upper limit of quantitation (ULQ) of 2000 ng ml(-1) with accuracy and precision of not greater then 15% CV and deviation from nominal over the range. The corresponding LOQ (0.5 ng ml(-1)) and ULQ (200 ng ml(-1)) values determined for AMG1m displayed similar accuracy and precision. In addition, we demonstrate that the assay as performed is insensitive to matrix effect up to addition of 7% of the to tal reaction volume. General guidelines for developing similar electrochemiluminescent based assays and their applications, advantages and limitations will be discussed. (C) 1997 Elsevier Science B.V.