Scale-Up 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 with low 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. The overall product yield was >80%, and ultimately, 13.2 g of trastuzumab-Lys248-MMAE and 1.26 g of trastuzumab-Lys288-MMAE were obtained with target drug to antibody ratios (DARs). Moreover, ADCs were synthesized at various scales, and it was verified that the DAR and aggregation rates could be replicated consistently across different scales. The results strongly indicate that the AJICAP second-generation process is a robust and practical approach for the manufacture of ADCs.