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Biophysical Characterization and Structure of the Fab Fragment from the NIST Reference Antibody RM 8671



Ioannis Karageorgos, Elyssia S. Gallagher, Connor Galvin, David Travis Gallagher, Jeffrey W. Hudgens


Monoclonal antibody (mAb) pharmaceuticals are the fastest-growing class of therapeutics with a wide range of clinical applications. To assure their safety, these protein drugs must demonstrate highly consistent purity and stability. Key to these objectives is a set of higher order structure measurements validated by calibration to reference materials. To this end, we describe the preparation, characterization, and crystal structure of the Fab fragment prepared from the NIST Reference Antibody RM 8671 (NISTmAb). NISTmAb is a humanized IgG1 antibody produced in mammalian cell culture and purified by standard biopharmaceutical production methods, developed at the National Institute of Standards and Technology (NIST) to serve as a standard reference material. The Fab fragment was derived from NISTmAb through papain cleavage followed by protein A based purification. The purified Fab fragment was characterized by size exclusion chromatography (SEC), multi-angle light scattering (MALS), electrophoresis, mass spectrometry, and x-ray crystallography. The crystal structure at 0.2 nm resolution includes four independent Fab molecules with complete light chains and heavy chains thru Cys 223, enabling assessment of conformational variability and providing a well-characterized reference structure for research and engineering applications.


antibody, crystal structure, papain, physical chemical characterization, Fab, IgG1?, NISTmAb, reference protein, reference material.


Karageorgos, I. , Gallagher, E. , Galvin, C. , Gallagher, D. and Hudgens, J. (2017), Biophysical Characterization and Structure of the Fab Fragment from the NIST Reference Antibody RM 8671, Biologicals, [online],, (Accessed April 24, 2024)
Created September 28, 2017, Updated October 12, 2021