Detection and Characterization of In Vitro Payload-Containing Catabolites of Noncleavable Antibody-Drug Conjugates by High-Resolution Mass Spectrometry and Multiple Data Mining Tools - WuXi AppTec DMPK

This website stores cookies on your computer. These cookies are used to collect information about how you interact with our website and allow us to remember you. We use this information to improve and customize your browsing experience and for analytics and metrics about our visitors both on this website and other media. To find out more about the cookies we use, see our Privacy Policy.

Contact Us

What can we help you find?

Top Searches

PROTAC

ADC

RNA

ADME

OLIGO

Detection and Characterization of In Vitro Payload-Containing Catabolites of Noncleavable Antibody-Drug Conjugates by High-Resolution Mass Spectrometry and Multiple Data Mining Tools

  • Publication

  • Apr 28, 2025

Tingting Cai 1, Liqi Shi 1, Huihui Guo 1, Ruixing Li 1, Weiqun Cao 1, Liang Shen 1, Mingshe Zhu 2, Yi Tao 2


Drug Metabolism and Pharmacokinetic Services, WuXi AppTec, Nanjing, Jiangsu, China (T.C.); Drug Metabolism and Pharmacokinetic Services, WuXi AppTec, Shanghai, China (L.S., R.L., W.C., L.S., Y.T.); Hangzhou DAC Biotechnology Co., Ltd., Hangzhou, China (H.G.); and MassDefect Technologies, Princeton, New Jersey (M.Z.)




Abstract


The formation and accumulation of payload-containing catabolites (PCCs) from a noncleavable antibody-drug conjugate (ADC) in targeted and normal tissues are directly associated with the therapeutic effect and toxicity of the ADC, respectively. Understanding the PCC formation is important for supporting the payload design and facilitating preclinical evaluation of ADCs. However, detection and identification of PCCs of a noncleavable ADC are challenging due to their low concentrations and unknown structures. The main objective of this study was to develop and apply a generic liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method for profiling PCCs in vitro. Noncleavable ADCs, ado-trastuzumab emtansine (T-DM1) and ADC-1, were incubated in liver lysosomes, liver S9, and/or cancer cells followed by data acquisition using LC-HRMS. Profiling PCCs mainly relied on processing LC-HRMS datasets using untargeted precise and thorough background subtraction (PATBS) processing and targeted product ion filtering (PIF). As a result, 12 PCCs of T-DM1 were detected and structurally characterized in human liver lysosomal incubation, a majority of which consisted of 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (MCC)-DM1 and a few amino acids. Additionally, the incubation of ADC-1 in human, rat, and monkey liver S9 and cancer cells generated one major and three very minor PCCs, verifying the payload design. The results demonstrate that PATBS enabled the comprehensive profiling of PCCs regardless of their molecular weights, charge states, and fragmentations. As a complementary tool, PIF detected specific PCCs with superior sensitivity. The combination of the in vitro metabolism systems and the LC-HRMS method is a useful approach to profiling in vitro PCCs of noncleavable ADCs in support of drug discovery programs. SIGNIFICANCE STATEMENT: Profiling in vitro payload-containing catabolites (PCCs) of a noncleavable antibody-drug conjugate (ADC) is important for optimization of the payload design and preclinical evaluation of ADC. However, currently used analytical approaches often fail to quickly provide reliable PCC profiling results. The work introduces a new liquid chromatography high resolution mass spectrometry method for comprehensive and rapid detection and characterization of PCCs released from a noncleavable ADC in liver lysosomes and S9 incubations.

Stay Connected

Keep up with the latest news and insights.

  • Email address*

    * Please check the filled content
  • First name*

    * Please check the filled content
  • Last name*

    * Please check the filled content
  • Company*

    * Please check the filled content

By clicking submit, you consent to WuXi AppTec DMPK collecting and processing the information you provide for our internal purposes, in accordance with our privacy policy.

* Please agree to the Privacy Policy

Thanks for signing up

Help us get to know you better! By customizing your email preferences, we can deliver curated content relevant to you.