Quantitation of reduced disulfide groups in monoclonal antibodies using 5-iodoacetamidofluorescein: A novel size exclusion-HPLC technique

The interference of stannous in the quantitation of sulfhydryl in pretreated MAb using the Ellmanʹs method is described. A HPLC method which effectively overcomes the interference by stannous ion in measuring sulfhydryls by the commonly used Ellmanʹs technique has been developed. The method involves reacting the reduced monoclonal antibody with 0.1 M EDTA followed by the sulfhydryl specific reagent 5-iodoacetamidofluorescein and incubating the mixture at 37°C for 1.0–1.5 h. The 5-IAF labeled protein is separated from the unbound free 5-IAF and its secondary degraded products by size exclusion HPLC using a TSK G3000 SWXL column. The method allows quantitation of MAb sulfhydryl present as monomeric MAb, aggregates and fragments and is a major improvement over the spectroscopic techniques.