Stabilization of a model formalinized protein antigen encapsulated in poly(lactide-co-glycolide)-based microspheres

A formaldehyde-mediated aggregation pathway (FMAP) has been shown to be pri marily responsible for the solid-state aggregation of lyophilized formalini zed protein antigens [e.g., tetanus toxoid (IT) and formalinized bovine ser um albumin (f-BSA)] in the presence of moisture and physiological temperatu re. Coincorporation of the formaldehyde-interacting amino acid, histidine, strongly inhibits the FMAP. The purpose of this study was to test whether p revious solid-state data are applicable toward the stabilization of formali nized antigens encapsulated in poly(lactide-co-glycolide) (PLGA)-based micr ospheres. Formaldehyde-treated bovine serum albumin (f-BSA) and BSA were se lected as a model formalinized protein antigen and a nonformalinized contro l, respectively. As in the solid state, we found that the FMAP was dominant in the aggregation of f-BSA encapsulated in PLGA 50/50 microspheres, where as the aggregation mechanism of encapsulated BSA was mostly converted from thiol-disulfide interchange to an acid-catalyzed noncovalent pathway. The l ack of noncovalent aggregation in encapsulated f-BSA could be explained by its higher thermodynamic stability after formalinization, which inhibits pr otein unfolding. Targeting the FMAP, coencapsulation of histidine and treha lose successfully inhibited the aggregation of f-BSA in microspheres. By co mbining the use of an optimized oil-in-oil (o/o) encapsulation method, coen capsulation of histidine and trehalose, and use of low-acid-content poly(D, L-lactide) (PLA) and poly(ethylene glycol) (PEG) blends, a 2-month continuo us release of f-BSA was achieved with the absence of aggregation. (C) 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association.