Modulation of antigenicity related to changes in antibody flexibility uponlyophilization

Lyophilization is frequently used to increase the shelf-life of biopharmace uticals containing antibodies. A case in which an anti-idiotypic antibody, MMA 383, substantially lost its in vivo immunogenic properties although the protein was not degraded, is investigated. The scanning transmission elect ron microscope allowed the MMA 383 Fab and Pc moieties to be resolved. By a veraging the single antibodies, the angle between the Fab moieties can be c alculated. Non-lyophilized antibodies displayed a wider range of shapes tha n their reconstituted, lyophilized counterparts. Accordingly, the angle bet ween the two Fab fragments varied more, indicating greater flexibility. The tryptophan steady-state fluorescence intensity, steady-state fluorescence anisotropy and fluorescence Lifetime, were smaller for the lyophilized anti bodies. These were also more resistant towards thermal denaturation/aggrega tion. Circular dichroism spectra detected temperpture-dependent differences between the two antibody types in the 236 nm region. The subtle but reprod ucible structural changes induced by lyophilization may be related to the l oss of in vivo immunogenic properties. (C) 2001 Academic Press.