Non-random conformation of a mouse IgG2a monoclonal antibody at low pH.
The pH dependence of the conformation of a mouse IgG2a, kappa monoclonal antibody (MN12) was investigated by several physical techniques, including fluorescence spectroscopy, near-ultraviolet and far-ultraviolet CD, and electric-field-induced transient birefringence measurements. The intensity of the intrinsic tryptophan fluorescence remained constant in the pH range from 3.5 to 10.0. A conformational alteration in the MN12 molecule was observed in the pH region between pH 3.5 and 2.5, as reflected by a substantial enhancement of the fluorescence quantum yield. This effect was more pronounced at high ionic strengths. The fluorescence emission was unaltered, indicating that the acid-induced conformational state is different from a completely unfolded state. This was confirmed by CD and fluorescence polarisation measurements. Iodide and acrylamide fluorescence quenching studies indicated a gradually increasing accessibility of MN12 tryptophan residues with decreasing pH. At low pH precipitation was observed in the presence of iodide. One rotational relaxation time (0.16-0.18 microseconds) was observed for MN12 by electric-field-induced transient birefringence measurements at low ionic strength. After exposure of MN12 to low pH for 1 h, the relaxation time was increased to 0.23 microseconds; a further increase to 0.30 microseconds was observed after 24 h. The combined results suggest an acid-induced expansion and enhanced flexibility of MN12, which eventually leads to irreversible aggregation.
Jiskoot
W
W
Department of Pharmaceutics, University of Utrecht, The Netherlands.
Bloemendal
M
M
van Haeringen
B
B
van Grondelle
R
R
Beuvery
E C
EC
Herron
J N
JN
Crommelin
D J
DJ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D002942:Circular Dichroism; D005454:Fluorescence Polarization; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000737:chemistry*; D051379:Mice; D009994:Osmolar Concentration; D011487:Protein Conformation; D012995:Solubility; D013050:Spectrometry, Fluorescence
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G