Matrix-assisted UV-laser desorption/ionization mass spectrometric analysis of monoclonal antibodies for the determination of carbohydrate, conjugated chelator, and conjugated drug content.

The chemically averaged molecular weights of a variety of native and conjugated monoclonal antibodies, approximately 150,000, were measured by matrix-assisted UV-laser desorption/ionization mass spectrometry. The average mass of the carbohydrate present in a monoclonal antibody was estimated from the difference between the measured mass of the monoclonal antibody and the mass of the protein present in the monoclonal antibody computed from the amino acid translation of the DNA sequence. The loading of chelators and anticancer drugs conjugated to a monoclonal antibody was quantitated from the difference in the measured masses for the conjugated and untreated monoclonal antibody relative to the expected mass change upon conjugation of 1 mol of chelator or drug. The loading results obtained by mass spectrometry were consistent in most cases with measurements obtained by radioactivity trace assay or UV spectrometry. Similar matrix-assisted UV-laser desorption/ionization mass spectrometric studies were also made after reducing untreated and conjugated monoclonal antibodies with dithiothreitol to determine the distribution of carbohydrate and chelator between the light and heavy chains of the molecules. Matrix-assisted UV-laser desorption/ionization mass spectra were used to compute loading values for covalently bound drugs and proteins, while the loading values obtained by use of gel-filtration HPLC and UV spectrometry cannot distinguish between covalently and noncovalently bound drugs and proteins.