title: Quantitative Conjugated Payload Measurement Using Enzymatic Release of Antibody-Drug Conjugate with Cleavable Linker.
abstract: As antibody-drug conjugate (ADC) design is evolving with novel payload, linker, and conjugation chemistry, the need for sensitive and precise quantitative measurement of conjugated payload to support pharmacokinetics (PK) is in high demand. Compared to ADCs containing noncleavable linkers, a strategy specific to linkers which are liable to pH, chemical reduction, or enzymatic cleavage has gained popularity in recent years. One bioanalytical approach to take advantage of this type of linker design is the development of a PK assay measuring released conjugated payload. For the ADC utilizing a dipeptide ValCit linker studied in this report, the release of payload PF-06380101 was achieved with high efficiency using a purified cathepsin B enzyme. The subsequent liquid chromatography mass spectrometry (LC/MS) quantitation leads to the PK profile of the conjugated payload. For this particular linker using a maleimide-based conjugation chemistry, one potential route of payload loss would result in an albumin adduct of the linker-payload. While this adduct's formation has been previously reported, here, for the first time, we have shown that payload from a source other than ADC contributes only up to 4% of total conjugated payload while it accounts for approximately 35% of payload lost from the ADC at 48 h after dosing to rats.
authors.affiliation: Medicine Design, ‡Biomedicine Design, and §Pfizer Essential Health, Pfizer Inc. , Eastern Point Road, Groton, Connecticut 06340, United States.
authors.forename: Brian
authors.initials: B
authors.lastname: Rago
authors.affiliation: Medicine Design, ‡Biomedicine Design, and §Pfizer Essential Health, Pfizer Inc. , Eastern Point Road, Groton, Connecticut 06340, United States.
authors.forename: L Nathan
authors.initials: LN
authors.lastname: Tumey
authors.affiliation: Medicine Design, ‡Biomedicine Design, and §Pfizer Essential Health, Pfizer Inc. , Eastern Point Road, Groton, Connecticut 06340, United States.
authors.forename: Cong
authors.initials: C
authors.lastname: Wei
authors.affiliation: Medicine Design, ‡Biomedicine Design, and §Pfizer Essential Health, Pfizer Inc. , Eastern Point Road, Groton, Connecticut 06340, United States.
authors.forename: Frank
authors.identifier: 0000-0001-6018-2131
authors.initials: F
authors.lastname: Barletta
authors.affiliation: Medicine Design, ‡Biomedicine Design, and §Pfizer Essential Health, Pfizer Inc. , Eastern Point Road, Groton, Connecticut 06340, United States.
authors.forename: Tracey
authors.initials: T
authors.lastname: Clark
authors.affiliation: Medicine Design, ‡Biomedicine Design, and §Pfizer Essential Health, Pfizer Inc. , Eastern Point Road, Groton, Connecticut 06340, United States.
authors.forename: Steven
authors.initials: S
authors.lastname: Hansel
authors.affiliation: Medicine Design, ‡Biomedicine Design, and §Pfizer Essential Health, Pfizer Inc. , Eastern Point Road, Groton, Connecticut 06340, United States.
authors.forename: Xiaogang
authors.initials: X
authors.lastname: Han
mesh_terms: D000818:Animals; D001592:Benzyl Alcohols / Q000737:chemistry / Q000378:metabolism; D002401:Cathepsin B / Q000378:metabolism*; D004151:Dipeptides / Q000737:chemistry / Q000378:metabolism; D015195:Drug Design; D018796:Immunoconjugates / Q000737:chemistry / Q000378:metabolism* / Q000493:pharmacokinetics*; D008297:Male; D008301:Maleimides / Q000737:chemistry / Q000378:metabolism; D017207:Rats, Sprague-Dawley
chemical_list: D001592:Benzyl Alcohols; D004151:Dipeptides; D018796:Immunoconjugates; D008301:Maleimides; C456563:Z-valine-citrulline-p-aminobenzylalcohol; D002401:Cathepsin B