Rapid quantification of maleimide in bioconjuated samples using a novel colorimetric method (TECH2P.761)

A variety of crosslinking reagents with a maleimide group are widely used for crosslinking proteins to proteins, or proteins to other macromolecules. A key challenge in maleimide crosslinking technology is the lack of rapid and sensitive methods for quantifying the amount of maleimide groups in a biopolymer. Maleimides can be directly measured spectrophotometrically at 302 nm. However, the small extinction coefficient of maleimide renders this approach insensitive. In addition, the measurement is non-specific due to the fact that proteins have absorbance at the same wavelength. To address this unmet need, a rapid colorimetric method was developed to quantify maleimide group by using a novel maleimide sensor with the maximum absorbance at ~780 nm. The maleimide sensor can react with maleimide groups with high selectivity and sensitivity. After reaction with the maleimide sensor, the maleimide-linked sample was purified through a single spin column to remove the excess sensor, and the absorption spectrum was further measured. The ratio of maleimide to protein in the maleimide-linked samples was calculated by the ratio of absorbance at 780 nm to the absorbance at 280 nm. Based on this newly developed method, number of maleimide groups can be measured rapidly and precisely for a biopolymer (such as antibodies).