An ideal approach involves using commercially available payload-likers (Figure in supporting information, SI). ready for conjugation at a later stage, although this requires a two-step conversion process from native antibodies. This can streamline production while maintaining adherence to GMP standards and ensuring safety. The "AJICAP-M" method, described herein, utilizes an optimized affinity reagent with an internal N-hydroxysuccinimide ester (Figure ). This method efficiently produces site-specific ADCs using readily available payload linkers in two steps (antibody modification followed by payload-linker conjugation by click chemistry). AJICAP-M can modify two distinct sites (Lys248 and Lys288), and the resulting site-specific ADC was subjected to in vivo efficacy and pharmacokinetic evaluations. Furthermore, this technology has been applied to continuous-mode ADC production, achieving rapid site-specific conjugation within 5 min, thus addressing the limitations of the existing traceless conjugation techniques and marking significant advancements in ADC development. Traceless affinity conjugation targets lysine residues in native antibodies using acyl transfer reagents linked to self-releasing peptides. The primary challenge is to maintain stable reactivity at room temperature while avoiding heating, particularly with commonly used functional groups such as thioesters and carbonates. To address the reactivity and stability issues in antibody modification, we developed a highly reactive reagent using N-hydroxysuccinimide (NHS) ester, capable of facilitating modification under mild conditions. We incorporated an NHS moiety internally and introduced an azidophenyl group . This modification was designed to increase steric hindrance around the carbonyl group of NHS. Consequently, the modified NHS moiety exhibits increased resistance to hydrolysis . This balance between enhanced stability against hydrolysis and preserved reactive capability is significant, as it ensures the effectiveness of the NHS moiety in forming stable amide bonds with lysine residues. The reagent, synthesized for Lys248 modification, exhibited exceptional stability through multiple freeze-thaw cycles in DMF solution and showed efficient reactivity with native antibodies, completing the modification process within one hour at ambient temperature. This is detailed for the optimized AJICAP-M reagent (1) (Figure ). Furthermore, we developed an alternative AJICAP-M peptide reagent (2) that targets Lys288 on antibodies using a different affinity peptide sequence (Figure ). In our recent study, we streamlined the synthesis of peptide reagents, which traditionally involved a multi-step procedure . Using Traut's reagent, we developed an efficient one-pot method that encompasses three distinct reactions (Figure ). This approach employs a versatile base peptide compatible with various conjugation methodologies, including AJICAP and AJICAP-M. The adaptability of this base peptide is pivotal because it enables the synthesis to be finely tuned to meet the specific requirements of each antibody and the corresponding mechanism of action of the ADC. AJICAP-M conjugation compatibility was evaluated through using two different antibodies.