Biophysical characterization and structure of the Fab fragment from the NIST reference antibody, RM 8671

Abstract

Monoclonal antibody 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 higher order structure measurements validated by calibration to reference materials. We describe preparation, characterization, and crystal structure of the Fab fragment prepared from the NIST Reference Antibody RM 8671 (NISTmAb). NISTmAb is a humanized IgG1κ antibody, produced in murine cell culture and purified by standard biopharmaceutical production methods, developed at the National Institute of Standards and Technology (NIST) to serve as a 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 SDS-PAGE, capillary gel electrophoresis, multi-angle light scattering, size exclusion chromatography, 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 through Cys 223, enabling assessment of conformational variability and providing a well-characterized reference structure for research and engineering applications. This nonproprietary, publically available reference material of known higher-order structure can support metrology in biopharmaceutical applications, and it is a suitable platform for validation of molecular modeling studies.

Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) Centroid Data Measured between 3.6 °C and 25.4 °C for the Fab Fragment of NISTmAb

“…The deuterium content of peptic peptide fragments were measured by mass spectrometry. [1][2][3] These data were reported by fifteen laboratories, which conducted the measurements using orbitrap and Q-TOF mass spectrometers. The cohort reported ≈ 78,900 centroids for 430 proteolytic peptide sequences of the heavy and light chains of NISTmAb, providing nearly 100 % coverage.…”

“…To improve the veracity of peptide identifications, the operator observes MS/MS data for the eluting peptides. With reference to the known sequence of fab fragment of NISTmAb [2] peptide ion identification software analyzes these data and proposes an amino acid sequence, charge state (z), and confidence rank for each peptide ion. The list of retention times and sequences becomes the filter through which peptide ions are selected for HDX-MS analyses.…”

“…Each laboratory conducted proteomics studies on the Fab fragment of NISTmAb (PDB: 5K8A) [1][2][3] and performed three HDX-MS runs. Here, the term "run" is defined by the procedures and capabilities of the laboratory equipment.…”

“…The test protein for the HDX-MS interlaboratory comparison was the Fab fragment that is enzymatically cleaved from Candidate RM 8670 (Lot #5F1b), which has the same structure as the Fab of the NIST IgG1κ reference material, NISTmAb RM8671. [1][2][3] Material used for this study contained a small fraction of free Fc fragment. Triple-angle light scattering data indicated that the Fab fragment of NISTmAb sample was in a monomeric state [2,3].…”

“…[1][2][3] Material used for this study contained a small fraction of free Fc fragment. Triple-angle light scattering data indicated that the Fab fragment of NISTmAb sample was in a monomeric state [2,3]. Testing showed that the protein is unaltered by the mechanical shocks associated with shipping [2].…”