Structural analysis of a therapeutic monoclonal antibody dimer by hydroxyl radical footprinting.

Hydroxyl radical footprinting is a covalent labeling strategy used to probe the conformational properties of proteins in solution. We describe the first application of this high resolution technique for characterizing the structure of a therapeutic monoclonal antibody (mAb) dimer. As monitored by size-exclusion chromatography (SEC), therapeutic mAbs typically contain small amounts of a dimer species relative to the primary monomeric form in its drug substance or drug product. To determine its structural orientation, a sample enriched in an IgG1 mAb dimer was oxidized by hydroxyl radicals generated by exposure of the aqueous solution to synchrotron X-rays in millisecond timescales. The antibody monomer that served as a control was oxidized in a similar fashion. The oxidized samples were digested with trypsin and analyzed by RP-UHPLC-MS. The footprinting data show that peptides displaying decreased rates of oxidation (i.e., regions of increased protection) in the dimer are localized in the light and heavy chains of the Fab domain. The interface region for the monomers comprising the dimer was thus inferred to be between their Fab arms, allowing us to model two possible theoretical dimer orientations: a head-to-head, single arm-bound Fab-to-Fab dimer, and a head-to-head, double arm-bound Fab (') 2-to-Fab (') 2 dimer. Lower resolution fragment-SEC analysis of the dimer and monomer samples treated with papain or FabRICATOR® enzyme provided complimentary evidence to support the Fab/Fab orientation of the IgG1 dimer.

Figure 1: IgG1 homology structure with mapped protection levels. (A) Model of IgG1 constructed using 1BJ1 and 1IGY crystal structures. Colored according to the relative protection levels derived from oxidative labeling. Yellow indicates regions with decreased protection in the dimer. Green indicates regions with increased protection in the dimer. Uncolored areas indicate regions which show no relative difference in protection levels between the dimer and monomer. The blue region represents the Asn-linked Fc glycans typically found in IgG1 therapeutic mAbs. (B) Space-filled cartoon model of the IgG1 with the heavy chains colored purple and light chains colored red. The light chains of an IgG1 are on opposite planes with respect with each other. The heavy chains in the Fab region are also on opposite planes.

Figure 2: Proposed possible orientations of the IgG1 dimer. Cartoon representations of proposed possible dimer orientations based on the hydroxyl radical footprinting data. The bottom mAb has heavy chains in gray and light chains in black. The top mAb has heavy chains in light blue and light chains in dark blue. Head-to-head orientation allows for the light and heavy chains of the dimer to associate with each other in the interface region. (A) Cartoon depiction of a head-to head, a single arm bound Fab-to-Fab dimer. (B) Cartoon depiction of a head-to head, double arm-bound Fab'2-to-Fab'2 dimer.

Results from:
Deperalta G, Alvarez M, Bechtel C, Dong K, McDonald R, Ling V.
MAbs. 2013 Jan 1;5(1):86-101. doi: 10.4161/mabs.22964. Epub 2012 Dec 17.
PMID: 23247543 [PubMed - in process]
PMCID: PMC3564890 [Available on 2014/1/1]