title: Chemical de-conjugation for investigating the stability of small molecule drugs in antibody-drug conjugates.
abstract: Antibody-drug conjugates (ADCs) offer new therapeutic options for advanced cancer patients through precision killing with fewer side effects. The stability and efficacy of ADCs are closely related, emphasizing the urgency and importance of gaining a comprehensive understanding of ADC stability. In this work, a chemical de-conjugation approach was developed to investigate the in-situ stability of the small molecule drug while it is conjugated to the antibody. This method involves chemical-mediated release of the small molecule drug from the ADC and subsequent characterization of the released small molecule drug by HPLC. The feasibility of this technique was demonstrated utilizing a model ADC containing a disulfide linker that is sensitive to the reducing environment within cancer cells. Five reducing agents were screened for use in de-conjugation; tris(2-carboxyethyl) phosphine (TCEP) was selected for further optimization due to its high efficiency and clean impurity profile. The optimized de-conjugation assay was shown to have excellent specificity and precision. More importantly, it was shown to be stability indicating, enabling the identification and quantification of the small molecule drug and its degradation products under different formulation pHs and storage temperatures. In summary, the chemical de-conjugation strategy demonstrated here offers a powerful tool to assess the in-situ stability of small molecule drugs on ADCs and the resulting information will shed light on ADC formulation/process development and storage condition selection.
authors.affiliation: Small Molecule Analytical Chemistry and Quality Control, Small Molecule Pharmaceutical Sciences, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, United States.
authors.forename: Tao
authors.initials: T
authors.lastname: Chen
authors.affiliation: Assay Development and Technology, Bioanalytical Sciences, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, United States.
authors.forename: Dian
authors.initials: D
authors.lastname: Su
authors.affiliation: Small Molecule Analytical Chemistry and Quality Control, Small Molecule Pharmaceutical Sciences, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, United States.
authors.forename: Jason
authors.initials: J
authors.lastname: Gruenhagen
authors.affiliation: Small Molecule Analytical Chemistry and Quality Control, Small Molecule Pharmaceutical Sciences, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, United States.
authors.forename: Christine
authors.initials: C
authors.lastname: Gu
authors.affiliation: Small Molecule Analytical Chemistry and Quality Control, Small Molecule Pharmaceutical Sciences, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, United States.
authors.forename: Yi
authors.initials: Y
authors.lastname: Li
authors.affiliation: Small Molecule Analytical Chemistry and Quality Control, Small Molecule Pharmaceutical Sciences, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, United States.
authors.forename: Peter
authors.initials: P
authors.lastname: Yehl
authors.affiliation: Small Molecule Analytical Chemistry and Quality Control, Small Molecule Pharmaceutical Sciences, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, United States.
authors.forename: Nik P
authors.initials: NP
authors.lastname: Chetwyn
authors.affiliation: Small Molecule Analytical Chemistry and Quality Control, Small Molecule Pharmaceutical Sciences, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, United States. Electronic address: medley.colin@gene.com.
authors.forename: Colin D
authors.initials: CD
authors.lastname: Medley
mesh_terms: D061067:Antibodies, Monoclonal, Humanized / Q000032:analysis / Q000737:chemistry*; D002626:Chemistry, Pharmaceutical / Q000379:methods*; D004355:Drug Stability; D006801:Humans; D018796:Immunoconjugates / Q000032:analysis / Q000737:chemistry*; D000922:Immunotoxins / Q000032:analysis / Q000737:chemistry
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D018796:Immunoconjugates; D000922:Immunotoxins
keywords: Antibody drug conjugate; Chemical de-conjugation; High performance liquid chromatography; Small molecule stability