Gram-Scale Synthesis of Site-Specific Antibody-Drug Conjugates Using AJICAP Second-Generation Technology

Chemical site-specific conjugation technology utilizing immunoglobulin-G (IgG) Fc-affinity reagents is a versatile and promising tool for producing next-generation antibody-drug conjugates (ADCs). Our research group recently reported a novel Fc-affinity peptide-mediated conjugation method, termed AJICAP second-generation. This technology, based on thioester chemistry, produces site-specific ADCs without aggregation. Herein, we report further investigations into the AJICAP second-generation technology. By varying the parameters of the peptide conjugation step, it was found that this reaction is feasible under a wide range of reaction conditions. All synthetic intermediates of the AJICAP-ADCs were sufficiently stable, indicating that each synthetic step is a possible holding point in ADC manufacturing. The Lys248- and Lys288-conjugated ADCs were prepared on a gram-scale using two different Fc-affinity peptide reagents, employing a scale-down manufacturing approach involving tangential flow filtration (TFF). The total product yield was > 80%, and ultimately, 13.2 g of trastuzumab-Lys248-MMAE and 1.26 g of trastuzumab-Lys288-MMAE were obtained with high drug to antibody ratios (DARs). The results strongly indicate that the AJICAP second-generation method is a robust and practical approach for the manufacture of ADCs.