title: A sensitive multidimensional method for the detection, characterization, and quantification of trace free drug species in antibody-drug conjugate samples using mass spectral detection.
abstract: Conjugation processes and stability studies associated with the production and shelf life of antibody-drug conjugates (ADCs) can result in free (non-conjugated) drug species. These free drug species can increase the risk to patients and reduce the efficacy of the ADC. Despite stringent purification steps, trace levels of free drug species may be present in formulated ADCs, reducing the therapeutic window. The reduction of sample preparation steps through the incorporation of multidimensional techniques has afforded analysts more efficient methods to assess trace drug species. Multidimensional methods coupling size-exclusion and reversed phase liquid chromatography with ultra-violet detection (SEC-RPLC/UV) have been reported, but offer limited sensitivity and can limit method optimization. The current study addresses these challenges with a multidimensional method that is specific, sensitive, and enables method control in both dimensions via coupling of an on-line solid phase extraction column to RPLC with mass spectral detection (SPE-RPLC/MS). The proposed method was evaluated using an antibody-fluorophore conjugate (AFC) as an ADC surrogate to brentuximab vedotin and its associated parent maleimide-val-cit-DSEA payload and the derived N-acetylcysteine adduct formed during the conjugation process. Assay sensitivity was found to be 2 orders more sensitive using MS detection in comparison to UV-based detection with a nominal limit of quantitation of 0.30 ng/mL (1.5 pg on-column). Free-drug species were present in an unadulterated ADC surrogate sample at concentrations below 7 ng/mL, levels not detectable by UV alone. The proposed SPE-RPLC/MS method provides a high degree of specificity and sensitivity in the assessment of trace free drug species and offers improved control over each dimension, enabling straightforward integration into existing or novel workflows.
authors.affiliation: a Waters Corporation, 34 Maple Street , Milford , MA , 01757 , USA.
authors.forename: Robert E
authors.initials: RE
authors.lastname: Birdsall
authors.affiliation: a Waters Corporation, 34 Maple Street , Milford , MA , 01757 , USA.
authors.forename: Sean M
authors.initials: SM
authors.lastname: McCarthy
authors.affiliation: b IRPF, Center d'Immunologie Pierre Fabre , St Julien-en-Genevois , France.
authors.forename: Marie Claire
authors.initials: MC
authors.lastname: Janin-Bussat
authors.affiliation: c IRPF, Center de R&D Pierre Fabre , Toulouse , France.
authors.forename: Michel
authors.initials: M
authors.lastname: Perez
authors.affiliation: b IRPF, Center d'Immunologie Pierre Fabre , St Julien-en-Genevois , France.
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Haeuw
authors.affiliation: a Waters Corporation, 34 Maple Street , Milford , MA , 01757 , USA.
authors.forename: Weibin
authors.initials: W
authors.lastname: Chen
authors.affiliation: b IRPF, Center d'Immunologie Pierre Fabre , St Julien-en-Genevois , France.
authors.forename: Alain
authors.initials: A
authors.lastname: Beck
mesh_terms: D000111:Acetylcysteine / Q000737:chemistry*; D056148:Chromatography, Reverse-Phase; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D013058:Mass Spectrometry; D055550:Protein Stability; D000068878:Trastuzumab / Q000737:chemistry*
chemical_list: D005456:Fluorescent Dyes; D000068878:Trastuzumab; D000111:Acetylcysteine
keywords: 2DLC; ADC; AFC; NAc-linker mimic; SPE/RPLC/MS; antibody-drug conjugate; antibody-fluorophore conjugates; drug mimic; free drug; maleimide-linker-drug mimic; multidimensional chromatography; solid phase extraction