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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.

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Antibody−drug conjugates (ADCs) consist of a target-specific antibody that is covalently conjugated to a drug via a linker. ADCs are designed to deliver cytotoxic drugs (payloads), specifically to cancer cells, while minimizing systemic toxicity. Conventional cysteine conjugation typically results in the formation of ADC molecules containing a heterogeneous mixture of 2, 4, 6, and 8 drug-loaded species. The drug-to-antibody ratio (DAR) of the mixture represents the weighted average of these species. In this report, we have investigated the impact of the hydrophobicity of payloads and the overall drug loading on the in vitro binding and cytotoxicity of ADC species. Several ADCs were prepared by conventional cysteine conjugation using different payloads. ADC species with different DAR values were purified from the ADC mixture and characterized by standard analytical techniques. These ADC species were evaluated for target antigen binding using an immunoassay, enzyme-linked immunosorbent assay (ELISA). The potency was assessed using a cell-based cytotoxicity assay. These structure−function studies lead to a better understanding of factors that impact the in vitro target binding and cytotoxicity of ADC species. ADC species containing hydrophobic payloads with high DAR were found to have lower target binding by ELISA compared to that of the unconjugated antibody or the heterogeneous reference ADC with DAR ∼4. Under similar assay conditions, the ADCs conjugated to hydrophilic payloads did not show a significant impact on the target binding. The cytotoxic potency of ADC species increased with increasing level of drug loading in the cell-based cytotoxicity assay.