QUANTITATION OF MONOCLONAL-ANTIBODIES BY PERFUSION CHROMATOGRAPHY IMMUNODETECTION

The ImmunoDetection PG (protein G) cartridge which is based on perfusion chromatography has been evaluated for the quantitation of murine monoclonal antibody (IgG1) in biological fluids. The results indicate a low intra-day coefficient of variation (C.V.) less than or equal to 5% and a sample analysis time of 5.5 min. A standard curve generated using injection of known amounts of monomeric antibody against the eluted peak area shows linearity (r(2) = 0.99) over the 0.1-100 mu g range. The minimum detectable concentration (MDC) at 280 nm is about 2 mu g/ml which can be decreased to 200 ng/ml if the antibody elution is monitored at 214 nm. Under these conditions the C.V. is less than or equal to 5%. Accuracy of the quantitation is independent of operator, solution composition, sample pH and the ionic strength, all of which are factors to consider in performing immunoassays. Antibody samples containing aggregated and fragmented F-c regions of the antibody will interfere with this method of quantitation. An optimized validated method for the quantitation of antibody is described. This mode of perfusion chromatography could be applied in the biopharmaceutical field due to its speed, efficiency and sensitivity.