Evaluation of Factors Influencing Antibody Reduction for Development of Antibody Drug Conjugates

Authors
Meghdad Abdollahpour-Alitappeh 1
Majid Lotfinia 2
Sepand Razavi-Vakhshourpour 1
Saeed Jahandideh 2
Hamid Najminejad 1
Koushan Sineh Sepehr 3
Reza Moazami 4
Elnaz Shams 4
Mahdi Habibi-Anbouhi 5
Mohsen Abolhassani 1
1 Hybridoma Lab., Department OF Immunology, Pasteur Institute of Iran, Tehran, Iran
2 Department of Biochemistry, Pasteur Institute of Iran, Tehran, Iran
3 Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
4 Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran;
5 National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran
10.18869/acadpub.ibj.21.4.270
Abstract
Background: 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 site-specific conjugation can provide better process control and reproducibility for the development of disulfide-based ADCs.
Keywords
conjugation
Dithiothreitol
Antibody drug conjugate