Stabilization and controlled release of formaldehyde-treated protein antigens encapsulated in poly(lactide-co-glycolide) microspheres

A major hurdle in developing a single-dose tetanus vaccine based on poly(lactide-co-glycolide)(PLGA) microspheres is the poor stability of encapsulated tetanus toxoid (TT) in this delivery system. We hypothesized that formaldehyde treatment of the antigen and an acidic microclimate within microspheres are primarily responsible for the instability of TT. The purpose of this thesis was to develop a mechanistic approach for stabilization and controlled release of formaldehyde-treated protein antigens, particularly TT, from PLGA microspheres.; The dissertation consists of two parts. In Part I (Chapters 2–4), the development of a rational approach to stabilize a model formaldehyde-treated protein antigen, formaldehyde-treated bovine serum albumin (f-BSA), in PLGA systems is described. In Chapter 2, by comparing the aggregation kinetics and mechanism of f-BSA and non-formalinized control (BSA), we demonstrated that formaldehyde treatment initiated formaldehyde-mediated aggregation (FMA). FMA can be significantly inhibited by co-incorporation of amino acids and sugars, as well as prior antigen reduction with cyanoborohydride. In Chapter 3, a polymer blend of slow degrading PLA and water-soluble PEG was devised for delivery of acid-labile BSA. One-month continuous release of stable BSA from PLA/PEG blend was demonstrated without aggregation, indicating the absence of a highly acidic microclimate during release. In Chapter 4, FMA of f-BSA was demonstrated to be the dominant instability pathway in PLGA microspheres. By combining the use of PLA/PEG blend and co-encapsulation of histidine and trehalose, continuous release of f-BSA over two months was observed with the absence of FMA.; In Part II (Chapters 5, 6, 7), the applicability of the stabilization approach for delivery of the vaccine antigen, TT, from PLGA microspheres is described. In Chapter 5, amino acids and sugars were found to inhibit FMA of lyophilized TT efficiently. Mechanisms of amino acid inhibition of TT aggregation involved: (i) outcompeting for reactive electrophiles in TT, and (ii) sequestering slowly released formaldehyde from TT, consistent with previous FMA mechanism. In Chapter 6, lysine and sorbitol were identified as stabilizers that not only inhibit the FMA but also help retain TT antigenicity at elevated temperature and moisture. In Chapter 7, by co-encapsulating MgCO₃ (neutralizing acidic microclimate), lysine and sorbitol/trehalose (targeting FMA), TT microspheres were prepared that delivered antigenic TT for 4 weeks with almost complete inhibition of FMA.; Hence, we conclude that (1) formaldehyde-mediated aggregation and acid-induced antigenicity losses are two principal instability pathways of TT in PLGA microspheres; (2) the stabilization approaches developed herein are generally applicable for delivery of formaldehyde-treated antigens from PLGA systems.