title: Determination of Antibody-Drug Conjugate Released Payload Species Using Directed in Vitro Assays and Mass Spectrometric Interrogation.
abstract: Antibody-drug conjugates (ADC) are currently an active area of research, focused primarily on oncology therapeutics, but also to a limited extent on other areas such as infectious disease. The success of this type of targeted drug delivery is dependent upon many factors, one of which is the performance of the linker in releasing an active drug moiety under the appropriate conditions. As a tool in the development of linker/payload chemistry, we have developed an in vitro method for the identification of payload species released from ADCs in the presence of lysosomal enzymes. This method utilizes commercially available human liver S9 fraction as the source of these enzymes, and this has certain advantages over lysosomal fractions or purified enzymes. This article describes the characterization and performance of this assay with multiple ADCs composed of known and novel linkers and payloads. Additionally, we report the observation of incomplete degradation of mAb protein chains by lysosomal enzymes in vitro, believed to be the first report of this phenomenon involving an ADC therapeutic.
authors.affiliation: Pharmacokinetics, Dynamics and Metabolism, ∥Worldwide Medicinal Chemistry, and ¶Pharmaceutical Sciences Chemical R&D, Pfizer Worldwide R&D , Groton, Connecticut 06340, United States.|Oncology Research Unit, and ⊥Bioprocess R&D, Pfizer Worldwide R&D , Pearl River, New York 10965, United States.|Worldwide Medicinal Chemistry, and §Global Biotherapeutics Technologies, Pfizer Worldwide R&D , Cambridge, Massachusetts 02139, United States.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Bessire
authors.affiliation: Pharmacokinetics, Dynamics and Metabolism, ∥Worldwide Medicinal Chemistry, and ¶Pharmaceutical Sciences Chemical R&D, Pfizer Worldwide R&D , Groton, Connecticut 06340, United States.|Oncology Research Unit, and ⊥Bioprocess R&D, Pfizer Worldwide R&D , Pearl River, New York 10965, United States.|Worldwide Medicinal Chemistry, and §Global Biotherapeutics Technologies, Pfizer Worldwide R&D , Cambridge, Massachusetts 02139, United States.
authors.forename: T Eric
authors.initials: TE
authors.lastname: Ballard
authors.affiliation: Pharmacokinetics, Dynamics and Metabolism, ∥Worldwide Medicinal Chemistry, and ¶Pharmaceutical Sciences Chemical R&D, Pfizer Worldwide R&D , Groton, Connecticut 06340, United States.|Oncology Research Unit, and ⊥Bioprocess R&D, Pfizer Worldwide R&D , Pearl River, New York 10965, United States.|Worldwide Medicinal Chemistry, and §Global Biotherapeutics Technologies, Pfizer Worldwide R&D , Cambridge, Massachusetts 02139, United States.
authors.forename: Manoj
authors.initials: M
authors.lastname: Charati
authors.affiliation: Pharmacokinetics, Dynamics and Metabolism, ∥Worldwide Medicinal Chemistry, and ¶Pharmaceutical Sciences Chemical R&D, Pfizer Worldwide R&D , Groton, Connecticut 06340, United States.|Oncology Research Unit, and ⊥Bioprocess R&D, Pfizer Worldwide R&D , Pearl River, New York 10965, United States.|Worldwide Medicinal Chemistry, and §Global Biotherapeutics Technologies, Pfizer Worldwide R&D , Cambridge, Massachusetts 02139, United States.
authors.forename: Justin
authors.initials: J
authors.lastname: Cohen
authors.affiliation: Pharmacokinetics, Dynamics and Metabolism, ∥Worldwide Medicinal Chemistry, and ¶Pharmaceutical Sciences Chemical R&D, Pfizer Worldwide R&D , Groton, Connecticut 06340, United States.|Oncology Research Unit, and ⊥Bioprocess R&D, Pfizer Worldwide R&D , Pearl River, New York 10965, United States.|Worldwide Medicinal Chemistry, and §Global Biotherapeutics Technologies, Pfizer Worldwide R&D , Cambridge, Massachusetts 02139, United States.
authors.forename: Michael
authors.initials: M
authors.lastname: Green
authors.affiliation: Pharmacokinetics, Dynamics and Metabolism, ∥Worldwide Medicinal Chemistry, and ¶Pharmaceutical Sciences Chemical R&D, Pfizer Worldwide R&D , Groton, Connecticut 06340, United States.|Oncology Research Unit, and ⊥Bioprocess R&D, Pfizer Worldwide R&D , Pearl River, New York 10965, United States.|Worldwide Medicinal Chemistry, and §Global Biotherapeutics Technologies, Pfizer Worldwide R&D , Cambridge, Massachusetts 02139, United States.
authors.forename: My-Hanh
authors.initials: MH
authors.lastname: Lam
authors.affiliation: Pharmacokinetics, Dynamics and Metabolism, ∥Worldwide Medicinal Chemistry, and ¶Pharmaceutical Sciences Chemical R&D, Pfizer Worldwide R&D , Groton, Connecticut 06340, United States.|Oncology Research Unit, and ⊥Bioprocess R&D, Pfizer Worldwide R&D , Pearl River, New York 10965, United States.|Worldwide Medicinal Chemistry, and §Global Biotherapeutics Technologies, Pfizer Worldwide R&D , Cambridge, Massachusetts 02139, United States.
authors.forename: Frank
authors.initials: F
authors.lastname: Loganzo
authors.affiliation: Pharmacokinetics, Dynamics and Metabolism, ∥Worldwide Medicinal Chemistry, and ¶Pharmaceutical Sciences Chemical R&D, Pfizer Worldwide R&D , Groton, Connecticut 06340, United States.|Oncology Research Unit, and ⊥Bioprocess R&D, Pfizer Worldwide R&D , Pearl River, New York 10965, United States.|Worldwide Medicinal Chemistry, and §Global Biotherapeutics Technologies, Pfizer Worldwide R&D , Cambridge, Massachusetts 02139, United States.
authors.forename: Birte
authors.initials: B
authors.lastname: Nolting
authors.affiliation: Pharmacokinetics, Dynamics and Metabolism, ∥Worldwide Medicinal Chemistry, and ¶Pharmaceutical Sciences Chemical R&D, Pfizer Worldwide R&D , Groton, Connecticut 06340, United States.|Oncology Research Unit, and ⊥Bioprocess R&D, Pfizer Worldwide R&D , Pearl River, New York 10965, United States.|Worldwide Medicinal Chemistry, and §Global Biotherapeutics Technologies, Pfizer Worldwide R&D , Cambridge, Massachusetts 02139, United States.
authors.forename: Betsy
authors.initials: B
authors.lastname: Pierce
authors.affiliation: Pharmacokinetics, Dynamics and Metabolism, ∥Worldwide Medicinal Chemistry, and ¶Pharmaceutical Sciences Chemical R&D, Pfizer Worldwide R&D , Groton, Connecticut 06340, United States.|Oncology Research Unit, and ⊥Bioprocess R&D, Pfizer Worldwide R&D , Pearl River, New York 10965, United States.|Worldwide Medicinal Chemistry, and §Global Biotherapeutics Technologies, Pfizer Worldwide R&D , Cambridge, Massachusetts 02139, United States.
authors.forename: Sujiet
authors.initials: S
authors.lastname: Puthenveetil
authors.affiliation: Pharmacokinetics, Dynamics and Metabolism, ∥Worldwide Medicinal Chemistry, and ¶Pharmaceutical Sciences Chemical R&D, Pfizer Worldwide R&D , Groton, Connecticut 06340, United States.|Oncology Research Unit, and ⊥Bioprocess R&D, Pfizer Worldwide R&D , Pearl River, New York 10965, United States.|Worldwide Medicinal Chemistry, and §Global Biotherapeutics Technologies, Pfizer Worldwide R&D , Cambridge, Massachusetts 02139, United States.
authors.forename: Lee
authors.initials: L
authors.lastname: Roberts
authors.affiliation: Pharmacokinetics, Dynamics and Metabolism, ∥Worldwide Medicinal Chemistry, and ¶Pharmaceutical Sciences Chemical R&D, Pfizer Worldwide R&D , Groton, Connecticut 06340, United States.|Oncology Research Unit, and ⊥Bioprocess R&D, Pfizer Worldwide R&D , Pearl River, New York 10965, United States.|Worldwide Medicinal Chemistry, and §Global Biotherapeutics Technologies, Pfizer Worldwide R&D , Cambridge, Massachusetts 02139, United States.
authors.forename: Klaas
authors.initials: K
authors.lastname: Schildknegt
authors.affiliation: Pharmacokinetics, Dynamics and Metabolism, ∥Worldwide Medicinal Chemistry, and ¶Pharmaceutical Sciences Chemical R&D, Pfizer Worldwide R&D , Groton, Connecticut 06340, United States.|Oncology Research Unit, and ⊥Bioprocess R&D, Pfizer Worldwide R&D , Pearl River, New York 10965, United States.|Worldwide Medicinal Chemistry, and §Global Biotherapeutics Technologies, Pfizer Worldwide R&D , Cambridge, Massachusetts 02139, United States.
authors.forename: Chakrapani
authors.initials: C
authors.lastname: Subramanyam
mesh_terms: D000818:Animals; D002401:Cathepsin B / Q000378:metabolism; D045744:Cell Line, Tumor; D004337:Drug Carriers / Q000737:chemistry* / Q000378:metabolism; D065546:Drug Liberation; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry*; D008099:Liver / Q000166:cytology; D008247:Lysosomes / Q000201:enzymology; D013058:Mass Spectrometry; D051379:Mice; D051381:Rats
chemical_list: D004337:Drug Carriers; D018796:Immunoconjugates; D002401:Cathepsin B