Modulation of antigenicity related to changes in antibody flexibility upon lyophilization.
Lyophilization is frequently used to increase the shelf-life of biopharmaceuticals 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 electron microscope allowed the MMA 383 Fab and Fc moieties to be resolved. By averaging the single antibodies, the angle between the Fab moieties can be calculated. Non-lyophilized antibodies displayed a wider range of shapes than their reconstituted, lyophilized counterparts. Accordingly, the angle between 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 antibodies. These were also more resistant towards thermal denaturation/aggregation. Circular dichroism spectra detected temperature-dependent differences between the two antibody types in the 236 nm region. The subtle but reproducible structural changes induced by lyophilization may be related to the loss of in vivo immunogenic properties.
Taschner
N
N
Maurice. E. Müller Institute for Microscopy, Biozentrum of Basel University, Basel, CH-4056, Switzerland.
Müller
S A
SA
Alumella
V R
VR
Goldie
K N
KN
Drake
A F
AF
Aebi
U
U
Arvinte
T
T
D000888:Antibodies, Anti-Idiotypic / Q000737:chemistry* / Q000276:immunology* / Q000648:ultrastructure; D000918:Antibody Specificity / Q000276:immunology*; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D005454:Fluorescence Polarization; D005612:Freeze Drying; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000276:immunology / Q000648:ultrastructure; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000276:immunology / Q000648:ultrastructure; D007700:Kinetics; D017348:Microscopy, Electron, Scanning Transmission; D008958:Models, Molecular; D018583:Pliability; D011489:Protein Denaturation; D020836:Protein Structure, Quaternary; D013050:Spectrometry, Fluorescence; D013696:Temperature
D000888:Antibodies, Anti-Idiotypic; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments