Determination of free sulfhydryl contents for proteins including monoclonal antibodies by use of SoloVPE

Free sulfhydryls are important properties of protein products including monoclonal antibodies (mAbs). Here, a new technology, variable pathlength extension (SoloVPE), is employed to quantify the amount of free sulfhydryl in monoclonal antibodies (mAbs) using the well-known Ellman reagent. Briefly, the unbound thiols (free sulfhydryls) of proteins including mAbs react with Ellman reagent to produce a 2-nitro-5-thiobenzoate (TNB2−) which is detected at visible wavelength of 412 nm and quantified. The method does not require dilution of antibody samples, is simple, reproducible and takes less than one hour to complete. Values obtained by the new method are compared to literature values from traditional UV or fluorescence methods with agreements. Qualification and trending data over two years of method utilization in our labs support that assay variability is minimal with an intermediate precision of relative standard deviation (RSD) ≤ 10 % and a limit of quantification (LOQ) of 0.1 mol/mol, which is sufficient to measure free sulfhydryl content in proteins including mAbs.