title: A size exclusion-reversed phase two dimensional-liquid chromatography methodology for stability and small molecule related species in antibody drug conjugates.
abstract: Antibody drug conjugates (ADCs) are complex therapeutic agents combining the specific targeting properties of antibodies and highly potent cytotoxic small molecule drugs to selectively eliminate tumor cells while limiting the toxicity to normal healthy tissues. One unique critical quality attribute of ADCs is the content of unconjugated small molecule drug present from either incomplete conjugation or degradation of the ADC. In this work, size exclusion chromatography (SEC) was coupled with reversed-phase (RP) HPLC in an online 2-dimensional chromatography format for identification and quantitation of unconjugated small molecule drugs and related small molecule impurities in ADC samples directly without sample preparation. The SEC method in the 1st dimension not only separated the small molecule impurities from the intact ADC, but also provided information about the size variants (monomer, dimer, aggregates, etc.) of the ADC. The small molecule peak from the SEC was trapped and sent to a RP-HPLC in the 2nd dimension to further separate and quantify the different small molecule impurities present in the ADC sample. This SEC-RP 2D-LC method demonstrated excellent precision (%RSD<2.0), linearity (r(2)=0.9999), sensitivity (LOQ of 0.05μg/mL of free drug in ADC sample) and accuracy (95-105% recovery of spiked samples). The 2D-LC method was further utilized to study the stability of an ADC drug product at different temperatures and pHs. Both small molecule degradation products and aggregation of the conjugate were observed in the stability samples and the degradation pathways of the ADC were investigated. This 2D-LC method offers a powerful tool for ADC characterization and provides valuable information for conjugation and formulation development.
authors.affiliation: Small Molecule Pharmaceutical Sciences, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Li
authors.affiliation: Small Molecule Pharmaceutical Sciences, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA.
authors.forename: Christine
authors.initials: C
authors.lastname: Gu
authors.affiliation: Small Molecule Pharmaceutical Sciences, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA.
authors.forename: Jason
authors.initials: J
authors.lastname: Gruenhagen
authors.affiliation: Small Molecule Pharmaceutical Sciences, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA.
authors.forename: Kelly
authors.initials: K
authors.lastname: Zhang
authors.affiliation: Small Molecule Pharmaceutical Sciences, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA.
authors.forename: Peter
authors.initials: P
authors.lastname: Yehl
authors.affiliation: Small Molecule Pharmaceutical Sciences, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA.
authors.forename: Nik P
authors.initials: NP
authors.lastname: Chetwyn
authors.affiliation: Small Molecule Pharmaceutical Sciences, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA. Electronic address: medley.colin@gene.com.
authors.forename: Colin D
authors.initials: CD
authors.lastname: Medley
mesh_terms: D000906:Antibodies / Q000737:chemistry*; D000970:Antineoplastic Agents / Q000737:chemistry*; D002850:Chromatography, Gel / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D056148:Chromatography, Reverse-Phase / Q000379:methods; D018796:Immunoconjugates / Q000737:chemistry*; D013058:Mass Spectrometry
chemical_list: D000906:Antibodies; D000970:Antineoplastic Agents; D018796:Immunoconjugates
keywords: 2D-LC; Antibody drug conjugates