Process Development and Structural Characterization of an Anti-Notch 3 Antibody–Drug Conjugate

The development of a process for the preparation of a conventional anti-Notch 3 antibody–drug conjugate (ADC) is described. The initial reaction conditions used for the conjugation of an auristatin payload to an anti-Notch 3 monoclonal antibody led to the formation of an ADC mixture with a significant level of aggregates. Further process optimization studies resulted in the identification of reaction conditions for formation of the conjugate with a low level of aggregates. The temperature of the antibody reduction step was found to have an impact on the formation of aggregates in the ADC mixture. Differences in the antibody reduction temperatures also caused changes in the distribution of conjugated payload on the ADC species. Stability studies of anti-Notch 3 ADCs prepared by two processes differing in the antibody reduction temperature showed subtle differences in their aggregation propensities. The aggregates produced in the crude ADC reaction mixture could be separated from the desired monomer on the hydroxyapatite column under mild conditions without significantly impacting the average drug loading of the purified ADC.