Formaldehyde-mediated aggregation of protein antigens: Comparison of untreated and formalinized model antigens

A formaldehyde-mediated aggregation pathway (FMAP) is suggested as being pr imarily 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 ribonucle ase A (RNase) as model antigens; both protein antigens were formalinized ac cording to a method commonly used to detoxify bacterial toxins. To clearly delineate the FMAP from other aggregation mechanisms, the aggregation kinet ics and mechanism of both unmodified antigens (BSA and RNase) and formalini zed antigens (f-BSA and f-RNase) were evaluated. We report that formaldehyd e treatment introduces more rapid and extensive aggregation in antigens und er conditions that favor the FMAP (i.e., 80% relative humidity and 37 degre esC), Consistent with formaldehyde-mediated crosslinking, f-antigen aggrega tes were covalent and non-disulfide-bonded, whereas BSA aggregates were dis ulfide-linked and RNase even did not aggregate under the same conditions. C oincorporation of amino acids (histidine and lysine), which strongly intera ct with formaldehyde, as well as prior antigen reduction with cyanoborohydr ide, significantly inhibited f-BSA aggregation, but showed no selective eff ect on BSA aggregation, Mechanistic analysis of f-BSA aggregates, inhibitio n studies, and similar reactivity of F-BSA with TT all confirmed the existe nce of the FMAP at moisture levels intermediate between the dry and solutio n state. This study demonstrates the potential for covalent reactions betwe en formalinized protein antigens and neighboring chemical or biochemical sp ecies even after formalization, and provides a general approach to inhibit the FMAP, (C) 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 70: 607-517, 2 000.