Preparation Of Antibody-drug Conjugates

In recent years, the antibody-drug conjugates (ADC) has been a new hot spot inanti-cancer antibody drug research and received extensive attentions. ADC usuallyconsists of antibody, joint (linker) and drug. Many methods have been described forthe conjugation of drugs to monoclonal antibodies. The presence of a discrete numberof readily reducible disulfides in the common IgG subtypes presents a convenientopportunity for conjugation to cysteine residues with thiol-reactive drug-linkers.Conjugates prepared by a straightforward two-step reaction scheme involving thereduction of the antibody disulfides to the desired number of average thiols perantibody, followed by addition of the drug-linker, ideally with a maleimidofunctionality for rapid and selective reaction.Previous investigations of mAb disulfide biochemistry did not demonstratewhether there was selective reduction of particular mAb interchain disulfides, in partbecause of the inability to fractionate the reduction reaction into subpopulationstogether with the low resolution of the analytical techniques employed. By comparingthe size exclusion chromatography and polyacrylamide gel electrophoresis(SDS-PAGE) through experiments, the non-reducing SDS-PAGE method can get abetter separation of a thioether linked light chain the free light chain and the freeheavy after disulfide reduction. In addition, this method may help to identify hecontent of each fragment. Time-dependent reduction of antibody disulfide bondsusing dithiothreitol (DTT) and TCEP was used to characterize the structure of IgG.Using this approach, the disulfide bond between a light chain and a heavy chain wasdetermined to be cleaved faster than the disulfide bonds between two heavy chains.Reaction temperature, pH, and the ratio of the reduction of the reducing agent arethree factors to influence the antibody reduction. Though response surfaceexperimental design, the molar ratio of the reducing agent has significant effect onyield of HC-LC, secondly the reaction temperature, finally the reaction pH.Ultimately we determined the optimal temperature is37°C, reaction pH is between7-8. And subsequently by changing the ratio of antibody with a reducing agent, fromnine kinds of different reaction parameters reducing agent, the experimental evidencesuggests that DTT, Glu mercapto ethylamine, Ac-Cys and TCEP have some selcctive reduction of particular mAb interchain disulfides. When DTT/IgG=2, cysteamine/IgG=50, the TCEP/IgG=0.5and Ac-Cys/IgG for=3, the HC-LC proportion is30.4%,22.0%,26.5%,25.8%and29.5%.The reduction products were conjugated with a maleimido drug. The resultingconjugates are readily characterized using hydrophobic interaction chromatogram andR—HPLC methods.The optimal reation conditions of ADC prepared by partialreduction with average drug loading of4was obtained.