Author/Authors :
abolhassani , m Hybridoma Lab. - Department OF Immunology - Pasteur Institute of Iran - Tehran, Iran , abdollahpour-alitappeh, m. Hybridoma Lab. - Department OF Immunology - Pasteur Institute of Iran - Tehran, Iran , razavi-vakhshourpour, s. Hybridoma Lab. - Department OF Immunology - Pasteur Institute of Iran - Tehran, Iran , lotfinia, m. Department of Biochemistry - Pasteur Institute of Iran - Tehran, Iran , jahandideh, s. Department of Biochemistry - Pasteur Institute of Iran - Tehran, Iran , najminejad, h. Hybridoma Lab. - Department OF Immunology - Pasteur Institute of Iran - Tehran, Iran , sepehr, k.s. Department of Immunology - School of Public Health - Tehran University of Medical Sciences - Tehran, Iran , moazami, r. Biotechnology Research Center - Pasteur Institute of Iran - Tehran, Iran , shams, e. Biotechnology Research Center - Pasteur Institute of Iran - Tehran, Iran , habibi-anbouhi, m. National Cell Bank of Iran - Pasteur Institute of Iran - Tehran, Iran
Abstract :
Reduction/alkylation is one of the leading strategies for the development of antibody drug conjugates
(ADCs). Precise control of the reduction process would not only yield a defined number of free thiols per antibody
but also result in development of more homogenous conjugates. Methods: In the present study, we investigated the
effect of various dithiothreitol (DTT) concentrations, temperature conditions, and DTT exposure times on antibody
reduction. After antibody reduction, the Ellman's test and SDS-PAGE analysis were used to evaluate free thiols
produced and confirm the reduction process, respectively. Results: DTT concentration seems to be a potential
factor in the reduction process. Concentrations of 0.1, 1, 5, 10, 20, 50, and 100 mM DTT at 37°C for 30 minutes
resulted in approximately 0.4, 1.2, 5.4, 7, 8, 8, and 8 thiols per antibody, respectively. Conclusion: Optimized sitespecific
conjugation can provide better process control and reproducibility for the development of disulfide-based
ADCs.
