Development of a spectroscopic assay for bifunctional ligand-protein conjugates based on copper.
A simple, non-radioactive method for the determination of ligand-to-protein ratio (L/P) for novel ligand-antibody conjugates has been developed based on an exchange equilibrium with the purple Cu(II) complex of arsenazo III. The method requires a UV/Vis spectrometer and has been verified for monoclonal antibody Herceptin conjugates of a variety of ligand modalities, including common macrocyclic compounds NOTA and TETA, and with a new bifunctional tachpyridine (1H-Pyrrole-1-butanamide,N-[4-[[(1alpha,3alpha,5alpha)-3,5-bis[(2-pyridinylmethyl)amino]cyclohexyl](2-pyridinylmethyl)amino]butyl]-2,5-dihydro-2,5-dioxo-(9CI)). The spectroscopically derived values for L/P were verified by titration of the ligand-antibody conjugate with 64Cu. In each case, the value obtained by UV/Vis spectroscopy matches that found by radiolabeling. The method is rapid, taking less than 30 minutes with each ligand in this study.
Brady
Erik D
ED
Radioimmune and Inorganic Chemistry Section, Radiation Oncology Branch, National Cancer Institute, NIH, Bethesda, MD 20892, USA. bradye@mail.nih.gov
Chong
Hyun-Soon
HS
Milenic
Diane E
DE
Brechbiel
Martin W
MW
D000911:Antibodies, Monoclonal / Q000737:chemistry; D061067:Antibodies, Monoclonal, Humanized; D002855:Chromatography, Thin Layer; D003300:Copper / Q000737:chemistry*; D003301:Copper Radioisotopes; D003514:Cyclohexylamines / Q000737:chemistry*; D007202:Indicators and Reagents; D008024:Ligands; D008301:Maleimides / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D019275:Radiopharmaceuticals / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet; D000068878:Trastuzumab
D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D003301:Copper Radioisotopes; D003514:Cyclohexylamines; D007202:Indicators and Reagents; D008024:Ligands; D008301:Maleimides; C496697:N-(4-((3,5-bis((2-pyridinylmethyl)amino)cyclohexyl)(2-pyridinylmethyl)amino)butyl)-2,5-dihydro-2,5-dioxo-1H-pyrrole-1-butanamide; D011506:Proteins; D019275:Radiopharmaceuticals; D003300:Copper; D000068878:Trastuzumab