title: Measurement of in vivo drug load distribution of cysteine-linked antibody-drug conjugates using microscale liquid chromatography mass spectrometry.
abstract: Analysis of samples containing intact antibody-drug conjugates (ADC) using mass spectrometry provides a direct measurement of the drug-load distribution. Once dosed, the drug load distribution changes due to a combination of biological and chemical factors. Liquid chromatography-mass spectrometry (LC-MS) methods to measure the in vivo drug load distribution have been established for ADCs containing native disulfide bonds (lysine-linked or cysteine-linked). However, because of an IgG reduction step in conjugation processes, using LC-MS to analyze intact cysteine-linked ADCs requires native conditions, thus limiting sensitivity. While this limitation has been overcome at the analytical scale, to date, these methods have not been translated to a smaller scale that is required for animal or clinical doses/sampling. In this manuscript, we describe the development of ADC specific affinity capture reagents for processing in vivo samples and optimization of native LC-MS methods at a microscale. These methods are then used to detect the changing drug load distribution over time from a set of in vivo samples, representing to our knowledge the first native mass spectra of cysteine-linked ADCs from an in vivo source.
authors.affiliation: Seattle Genetics Inc., 21823 30th Drive Southeast, Bothell, Washington 98021, United States.
authors.forename: Shawna Mae
authors.initials: SM
authors.lastname: Hengel
authors.forename: Russell
authors.initials: R
authors.lastname: Sanderson
authors.forename: John
authors.initials: J
authors.lastname: Valliere-Douglass
authors.forename: Nicole
authors.initials: N
authors.lastname: Nicholas
authors.forename: Chris
authors.initials: C
authors.lastname: Leiske
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Alley
mesh_terms: D000906:Antibodies / Q000737:chemistry*; D002850:Chromatography, Gel / Q000379:methods*; D003545:Cysteine / Q000737:chemistry*; D018796:Immunoconjugates / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*
chemical_list: D000906:Antibodies; D018796:Immunoconjugates; D003545:Cysteine