| Autor: |
Yang J; Department of Chemical and Biomolecular Engineering, University of Notre Dame, Notre Dame, Indiana 46556, United States., Ostafe R; Molecular Evolution, Protein Engineering and Production Facility, Purdue Institute for Inflammation, Immunology and Infection Diseases, Purdue University, West Lafayette, Indiana 47907, United States., Welch CJ; Indiana Consortium for Analytical Science & Engineering (ICASE), 410 W. 10th St., # 1020H, Indianapolis, Indiana 46202, United States., Verhalen B; Corteva Agriscience, 8325 NW 62nd Ave, Johnston, Iowa 50131, United States., Budyak IL; Biopharmaceutical Research and Development, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285, United States., Bruening ML; Department of Chemical and Biomolecular Engineering, University of Notre Dame, Notre Dame, Indiana 46556, United States.; Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States. |
| Abstrakt: |
Therapeutic monoclonal antibodies (mAbs) provide effective treatments for many diseases, including cancer, autoimmune disorders, and, lately, COVID-19. Monitoring the concentrations of mAbs is important during their production and subsequent processing. This work demonstrates a 5 min quantitation of most human immunoglobulin G (IgG) antibodies through capture of mAbs in membranes modified with ligands that bind to the fragment crystallizable (Fc) region. This enables binding and quantitation of most IgG mAbs. Layer-by-layer (LBL) adsorption of carboxylic acid-rich polyelectrolytes in glass-fiber membranes in 96-well plates allows functionalization of the membranes with Protein A or a peptide, oxidized Fc20 (oFc20), with high affinity for the Fc region of human IgG. mAb capture occurs in <1 min during the flow of solutions through modified membranes, and subsequent binding of a fluorophore-labeled secondary antibody enables quantitation of the captured mAbs using fluorescence. The intra- and inter-plate coefficients of variations (CV) are <10 and 15%, respectively, satisfying the acceptance criteria for many assays. The limit of detection (LOD) of 15 ng/mL is on the high end of commercial enzyme-linked immunosorbent assays (ELISAs) but certainly low enough for monitoring of manufacturing solutions. Importantly, the membrane-based method requires <5 minutes, whereas ELISAs typically take at least 90 min. Membranes functionalized with oFc20 show greater mAb binding and lower LODs than membranes with Protein A. Thus, the membrane-based 96-well-plate assay, which is effective in diluted fermentation broths and in mixtures with cell lysates, is suitable for near-real-time monitoring of the general class of human IgG mAbs during their production. |
