title: Physicochemical stability of the antibody-drug conjugate Trastuzumab-DM1: changes due to modification and conjugation processes.
abstract: In the manufacture of the antibody-drug conjugate Trastuzumab-DM1 (T-DM1), the lysine residues on the antibody trastuzumab (Tmab) are modified to form the intermediate Tmab-MCC (T-MCC) and then conjugated with the drug DM1. Our goal is to understand the effects of modification and conjugation steps on the physicochemical stability of the antibody. The structural stability of Tmab relative to its modified and conjugated forms was assessed, employing thermally induced stress conditions to formulations containing Tmab, T-MCC, and T-DM1. DSC, SEC, CE-SDS, and LC-MS were used to study the stability of Tmab, T-MCC, and T-DM1 to thermal stress. The DSC thermograms show a decrease in melting temperature for the CH2 transition, in the order Tmab > T-MCC > T-DM1. As per SEC analysis, a significant increase in level of aggregation was detected in T-MCC (∼32%) and T-DM1 (∼5%) after 14 days at 40 °C. Tmab did not show significant aggregate formation. CE-SDS and LC-MS data demonstrate that the aggregation in the case of T-MCC is largely covalent and involves mechanisms other than formation of intermolecular disulfide cross-links. The aggregation observed for T-MCC was significantly inhibited upon addition of amino acids with nucleophilic side chains containing thiol (Cys) and hydroxyl moieties (Ser, Tyr). The covalent aggregation observed for T-MCC and the ability of nucleophilic amino acids, particularly Cys, to inhibit it indicate that the maleimide moiety in the MCC linker may react to form intermolecular covalent cross-links between T-MCC molecules, possibly through a Michael addition mechanism. In addition, DSC results demonstrate that the conjugation of the drug moiety DM1 to Tmab results in destabilization of the CH2 domain of the antibody.
authors.affiliation: Late Stage Pharmaceutical and Processing Development, Early Stage Pharmaceutical Development, and Protein and Analytical Chemistry, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA. wakankar.aditya@gene.com
authors.forename: Aditya A
authors.initials: AA
authors.lastname: Wakankar
authors.forename: Maria B
authors.initials: MB
authors.lastname: Feeney
authors.forename: Javier
authors.initials: J
authors.lastname: Rivera
authors.forename: Yan
authors.initials: Y
authors.lastname: Chen
authors.forename: Michael
authors.initials: M
authors.lastname: Kim
authors.forename: Vikas K
authors.initials: VK
authors.lastname: Sharma
authors.forename: Y John
authors.initials: YJ
authors.lastname: Wang
mesh_terms: D000080044:Ado-Trastuzumab Emtansine; D000596:Amino Acids / Q000737:chemistry; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D061067:Antibodies, Monoclonal, Humanized; D000972:Antineoplastic Agents, Phytogenic / Q000737:chemistry* / Q000378:metabolism; D002626:Chemistry, Pharmaceutical; D002853:Chromatography, Liquid; D003432:Cross-Linking Reagents / Q000737:chemistry; D004355:Drug Stability; D019075:Electrophoresis, Capillary; D018796:Immunoconjugates / Q000737:chemistry / Q000378:metabolism; D008301:Maleimides / Q000737:chemistry; D008453:Maytansine / Q000031:analogs & derivatives / Q000737:chemistry* / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry; D053719:Tandem Mass Spectrometry; D013696:Temperature; D013997:Time Factors; D000068878:Trastuzumab
chemical_list: D000596:Amino Acids; D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D000972:Antineoplastic Agents, Phytogenic; D003432:Cross-Linking Reagents; D018796:Immunoconjugates; D008301:Maleimides; D013438:Sulfhydryl Compounds; D008453:Maytansine; C043592:maleimide; D000068878:Trastuzumab; D000080044:Ado-Trastuzumab Emtansine