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

Abstract

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.

Bispecific Antibodies Produced via Chemical Site-Specific Conjugation Technology: AJICAP Second-Generation

“…In contrast, the AJICAP method establishes a stable covalent bond with the Fc peptide reagent, ensuring that antibody modifications. In particular, the achieved scale-up in ADC synthesis reinforces the efficacy of this conjugation technique for the production of site-specific antibody conjugates …”

“…In particular, the achieved scaleup in ADC synthesis reinforces the efficacy of this conjugation technique for the production of site-specific antibody conjugates. 17 In previous studies, our research group successfully enhanced the therapeutic index of site-specific ADCs. 11 The stability of these ADCs was confirmed by rat PK studies, and the newly formed thiol-maleimide bond, specifically linked to the Lys248 position of the antibody Fc region, was stable.…”

The Evolving Landscape of Antibody–Drug Conjugates: In Depth Analysis of Recent Research Progress

“…Affinity peptides have been used to control the location of conjugation with antibodies; 184 for example, binding of a peptide-containing acylating agent to the Fc domain of an antibody directs the acylation reaction to nearby residues 185 and the conjugation method has been performed on gram scale using a peptide-substituted thioester. 186 Cleavage of the peptide yielded a thiol available for further reaction.…”

“…Acylating agents can form esters with tyrosine, threonine, or serine residues, but the esters are not stable; thus, if not prevented, some of the acylating agents will be consumed, decreasing the amount of drug attached to the antibody. Affinity peptides have been used to control the location of conjugation with antibodies; for example, binding of a peptide-containing acylating agent to the Fc domain of an antibody directs the acylation reaction to nearby residues and the conjugation method has been performed on gram scale using a peptide-substituted thioester . Cleavage of the peptide yielded a thiol available for further reaction.…”