A formaldehyde-mediated aggregation pathway (FMAP) is suggested as being primarily responsible for the aggregation of lyophilized tetanus toroid (TT; a formalinized antigen) in the presence of moisture, The general occurrence of the FMAP was examined by using bovine serum albumin (BSA) and ribonuclease A (RNase) as model antigens; both protein antigens were formalinized according to a method commonly used to detoxify bacterial toxins. To clearly delineate the FMAP from other aggregation mechanisms, the aggregation kinetics and mechanism of both unmodified antigens (BSA and RNase) and formalinized antigens (f-BSA and f-RNase) were evaluated. We report that formaldehyde treatment introduces more rapid and extensive aggregation in antigens under conditions that favor the FMAP (i.e., 80% relative humidity and 37 degreesC), Consistent with formaldehyde-mediated crosslinking, f-antigen aggregates were covalent and non-disulfide-bonded, whereas BSA aggregates were disulfide-linked and RNase even did not aggregate under the same conditions. Coincorporation of amino acids (histidine and lysine), which strongly interact with formaldehyde, as well as prior antigen reduction with cyanoborohydride, significantly inhibited f-BSA aggregation, but showed no selective effect on BSA aggregation, Mechanistic analysis of f-BSA aggregates, inhibition studies, and similar reactivity of F-BSA with TT all confirmed the existence of the FMAP at moisture levels intermediate between the dry and solution state. This study demonstrates the potential for covalent reactions between formalinized protein antigens and neighboring chemical or biochemical species even after formalization, and provides a general approach to inhibit the FMAP,