Conjugations to Endogenous Cysteine Residues

Abstract

Interchain disulfide bonds of antibodies can be reduced by agents such as TCEP or DTT to form reactive cysteine residues. These endogenous cysteines are used for conjugation to biologically active drugs either directly or via linkers to prepare antibody drug conjugates (ADCs). The anti-notch 3 ADC described here is being evaluated in the early clinical development program as a potential treatment for a variety of cancers. The ADC is composed of an IgG1 mAb that is conjugated by endogenous cysteines to a cytotoxic microtubulin inhibitor via a maleimide-containing linker. The endogenous cysteine residues are produced by partial reduction of the mAb with TCEP reducing agent. The conjugation results in the formation of a mixture of 2, 4, 6, and 8 loaded ADC species. In addition to the desired product, several product-related impurities such as aggregates are generated during the conjugation reaction. The product- and process-related impurities are separated from the monomeric ADC by column chromatography and ultrafiltration-diafiltration techniques. The temperature of TCEP reduction step has an impact on the level of aggregates produced in the reaction. The temperature also impacts the isomeric composition of the 4 loaded ADC species.