Active dimer of Epratuzumab provides insight into the complex nature of an antibody aggregate

Understanding the intermolecular products of antibodies as a consequence of host-cell expression, aging, and heat-stress can be insightful especially when it involves the development of a stable biopharmaceutical product. The dimerized form of Epratuzumab (an IgG1 antibody) with a molecular mass of ∼300 kDa (twice the monomer antibody molecular weight of ∼150 kDa) was examined to gain a better perspective of its properties pertaining to structure and activity. The nascent dimer was shown to partially dissociate upon incubation at 30°C and 37°C, exhibit no discernable alteration of structure (i.e., secondary or tertiary structure based on CD and 2nd derivative UV spectroscopy), have ∼70% covalent forms (based upon CE-SDS results) and manifest twofold higher activity relative to the active monomer form (on a weight basis the dimer and monomer have equal activity). Interestingly, these properties were not attributed to a single dimer species, but rather to a more complex dimer assembly. The Epratuzumab dimer was digested with papain to reveal three uniquely dimerized aggregates. The relative molar distribution of Fab:Fab, Fc:Fc, and Fab:Fc was found to be 4:3:8, respectively. The data suggest that all three predominantly covalent dimer adducts are capable of full activity, shedding light on their complex nature and showing that their target specificity was unaltered. ESI-MS data indicated the presence of remnant levels of noncovalent dimers for all three dimerized forms. Material aged at 37°C exhibited a similar papain digest molar distribution of the three dimerized forms, except with enhanced chemical heterogeneity and an increase in covalent forms to ∼84%. © 2005 Wiley-Liss, Inc. and the American Pharmacists Association