Insight into Temperature Dependency and Design of Experiments towards Process Development for Cysteine‐Based Antibody‐Drug Conjugates

Abstract

In the synthetic organic community, antibody‐drug conjugates (ADCs) have become a popular topic of interest. The field is expanding beyond cytotoxic drug synthesis, and into other bioconjugation applications to have a greater impact on drug discovery and development. Bioconjugation chemistry is a complex domain for synthetic chemists, and there are limited published reports that can serve as guidance to overcome technical gaps between small molecule synthesis and large molecule bioconjugation to make ADCs. Here, we report a thorough evaluation of the temperature dependency for the tris‐(2‐carboxyethyl)‐phosphine (TCEP) reduction of antibody interchain disulfide bonds, which is a crucial step for creating cysteine‐conjugated ADCs. This investigation was conducted using three different antibodies with different isoelectric points, and under equivalent reaction conditions. Additionally, an initial stability assessment of the resulting ADCs was performed and revealed appropriate material storage conditions. The experimental data described herein can be a useful guide for synthetic organic chemists designing future ADC preparation processes.

Chemical Site-Specific Conjugation Platform to Improve the Pharmacokinetics and Therapeutic Index of Antibody–Drug Conjugates

“…This stochastic ADC was synthesized from trastuzumab by TCEP-mediated partial reduction of the interchain disulfide bonds followed by conjugation with MC-VC-MMAE to a DAR of 4 in the same manner as commercially approved ADCs Adcetris, Polivy, and Padcev (Figure 4d). 25,26 The stochastic MMAE-ADC 6 was compared against the similar AJICAP-MMAE-ADC 4B in efficacy (Figure 4c), safety (Figure 5), and PK (Figure 6) studies. The optimal DAR for an ADC depends on a large number of complex variables, including the in-part expression level of the target antigen, hydrophobicity of linker-payload, and payload class.…”

“…In addition to the lysine-based stochastic ADC, an AJICAP-ADC was also compared to a cysteine-based stochastic ADC modeled after a well-established established structural format in the ADC field. This stochastic ADC was synthesized from trastuzumab by TCEP-mediated partial reduction of the interchain disulfide bonds followed by conjugation with MC-VC-MMAE to a DAR of 4 in the same manner as commercially approved ADCs Adcetris, Polivy, and Padcev (Figure d). , The stochastic MMAE-ADC 6 was compared against the similar AJICAP-MMAE-ADC 4B in efficacy (Figure c), safety (Figure ), and PK (Figure ) studies. The optimal DAR for an ADC depends on a large number of complex variables, including the in-part expression level of the target antigen, hydrophobicity of linker-payload, and payload class. , Stochastic ADCs produced by cysteine conjugation are a mixture of different compounds with varying DARs (DAR = 0, 2, 4, 6, and 8 are typically the major species, and there are several forms of these ADCs with regard to payload placement on the antibody).…”

Recent Advances in Drug–Antibody Ratio Determination of Antibody–Drug Conjugates

“…A high concentration of salts in the mobile phase typically has negative effects on peak resolution, which may result in low reproducibility. Introduction of isopropyl alcohol (IPA) 20) or acetonitrile (ACN) 21) as an organic modifier sometimes improves peak shape, resolution, and separation.…”

Physical characteristics comparison between maytansinoid-based and auristatin-based antibody-drug conjugates

“…Conjugates derived from antibody heavy chains (HCs) showed the same phenomenon as light chain derivatives. Additionally, we also synthesized trastuzumab-MC-VC-PAB-MMAE with DAR = 1.9 by mild reduction conditions (reaction temperature: 4°C) [13].…”