title: The impact of trisulfide modification of antibodies on the properties of antibody-drug conjugates manufactured using thiol chemistry.
abstract: Antibody-drug conjugates (ADCs) are promising biotherapeutic agents for the treatment of cancer. The careful monitoring of critical quality attributes is important for ADCs' development, manufacturing and production. In this work, the effect of the presence of a trisulfide bond in the monoclonal antibody (mAb) conjugated to DM4 cytotoxic payload through a disulfide-bond linker sulfo-SPDB (sSPDB) was investigated. Three lots of antibody containing variable levels of trisulfide bonds were used. The identity and levels of trisulfide bonds were determined by liquid chromatography/ mass spectrometry (MS)/MS analysis. The antibodies were conjugated to sSPDB-DM4 to generate ADCs. Further analysis indicated that the drug-to-antibody ratio (DAR) value, a critical quality attribute, slightly increased for the conjugates made from antibody containing higher levels of trisulfide bond. Also, higher fragmentation levels were observed in the conjugates with more trisulfide bond. Detailed characterization by MS revealed that a small amount of DM4 payload was directly attached to inter-chain cysteine residues by disulfide or trisulfide bonds. Overall, our investigation indicated that the trisulfide bond present in the mAb could react with DM4 during the conjugation process. Therefore, the presence of trisulfide bonds in the antibody moiety should be carefully monitored and well controlled during the development of a maytansinoid ADC.
authors.affiliation: a Analytical and Pharmaceutical Science Department , ImmunoGen Inc. Waltham , MA , USA.
authors.forename: Renpeng
authors.initials: R
authors.lastname: Liu
authors.affiliation: a Analytical and Pharmaceutical Science Department , ImmunoGen Inc. Waltham , MA , USA.
authors.forename: Xuan
authors.initials: X
authors.lastname: Chen
authors.affiliation: a Analytical and Pharmaceutical Science Department , ImmunoGen Inc. Waltham , MA , USA.
authors.forename: Junia
authors.initials: J
authors.lastname: Dushime
authors.affiliation: a Analytical and Pharmaceutical Science Department , ImmunoGen Inc. Waltham , MA , USA.
authors.forename: Megan
authors.initials: M
authors.lastname: Bogalhas
authors.affiliation: a Analytical and Pharmaceutical Science Department , ImmunoGen Inc. Waltham , MA , USA.
authors.forename: Alexandru C
authors.initials: AC
authors.lastname: Lazar
authors.affiliation: a Analytical and Pharmaceutical Science Department , ImmunoGen Inc. Waltham , MA , USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Ryll
authors.affiliation: a Analytical and Pharmaceutical Science Department , ImmunoGen Inc. Waltham , MA , USA.
authors.forename: Lintao
authors.initials: L
authors.lastname: Wang
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000737:chemistry; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry*; D015202:Protein Engineering / Q000379:methods*
chemical_list: D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D018796:Immunoconjugates
keywords: Antibody-drug conjugates (ADC); LC-MS; drug-to-antibody ratio (DAR); sSPDB-DM4; trisulfide bond