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 pret reated MAb using the Ellman's method is described. A HPLC method which effectively overcomes the interference by stannous ion in measuring s ulfhydryls by the commonly used Ellman's technique has been developed. The method involves reacting the reduced monoclonal antibody with 0.1 M EDTA followed by the sulfhydryl specific reagent 5-iodoacetamidoflu orescein and incubating the mixture at 37 degrees C for 1.0-1.5 h. The 5-IAF labeled protein is separated from the unbound free 5-IAF and it s 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.