A comparative study of the developability of full-length antibodies, fragments, and bispecific formats reveals higher stability risks for engineered constructs

• Published in: mAbs Vol. 16; no. 1; p. 2403156
• Main Authors: Condado-Morales, Itzel, Dingfelder, Fabian, Waibel, Isabel, Turnbull, Oliver M, Patel, Bhargav, Cao, Zheng, Rose Bjelke, Jais, Nedergaard Grell, Susanne, Bennet, Anja, Hummer, Alissa M, Raybould, Matthew I J, Deane, Charlotte M, Egebjerg, Thomas, Lorenzen, Nikolai, Arosio, Paolo
• Format: Journal Article
• Language: English
• Published: United States Taylor & Francis 31.12.2024; Taylor & Francis Group
• Subjects: aggregation; Antibodies, Bispecific - chemistry; Antibodies, Bispecific - genetics; Antibodies, Bispecific - immunology; Antibodies, Monoclonal - chemistry; Antibodies, Monoclonal - genetics; Antibodies, Monoclonal - immunology; Antibody; bispecifics; developability; Fragmentation; fragments; Humans; Protein Engineering - methods; Protein Stability; Single-Chain Antibodies - chemistry; Single-Chain Antibodies - genetics; Single-Chain Antibodies - immunology; Tumor Necrosis Factor-alpha - immunology; antibody formats; developability; Self-association; Antibody; Shelf-life stability; aggregation; Biophysics; Fragmentation; bispecifics; Colloidal stability; scFv; Formulation; fragments
• ISSN: 1942-0862; 1942-0870; 1942-0870
• DOI: 10.1080/19420862.2024.2403156

Engineered antibody formats, such as antibody fragments and bispecifics, have the potential to offer improved therapeutic efficacy compared to traditional full-length monoclonal antibodies (mAbs). However, the translation of these non-natural molecules into successful therapeutics can be hampered by developability challenges. Here, we systematically analyzed 64 different antibody constructs targeting Tumor Necrosis Factor (TNF) which cover 8 distinct molecular format families, encompassing full-length antibodies, various types of single chain variable fragments, and bispecifics. We measured 15 biophysical properties related to activity, manufacturing, and stability, scoring variants with a flag-based risk approach and a recent developability profiler. Our comparative assessment revealed that overall developability is higher for the natural full-length antibody format. Bispecific antibodies, antibodies with scFv fragments at the C-terminus of the light chain, and single-chain Fv antibody fragments (scFvs) have intermediate developability properties, while more complicated formats, such as scFv- scFv, bispecific mAbs with one Fab exchanged with a scFv, and diabody formats are collectively more challenging. In particular, our study highlights the propensity for fragmentation and aggregation, both in bulk and at interfaces, for many current engineered formats.