Cold denaturation of monoclonal antibodies.
The susceptibility of monoclonal antibodies (mAbs) to undergo cold denaturation remains unexplored. In this study, the phenomenon of cold denaturation was investigated for a mAb, mAb1, through thermodynamic and spectroscopic analyses. Tryptophan fluorescence and circular dichroism (CD) spectra were recorded for the guanidine hydrochloride (GuHCl)-induced unfolding of mAb1 at pH 6.3 at temperatures ranging from -5 to 50 degrees C. A three-state unfolding model incorporating the linear extrapolation method was fit to the fluorescence data to obtain an apparent free energy of unfolding, DeltaG(u), at each temperature. CD studies revealed that mAb1 exhibited polyproline II helical structure at low temperatures and at high GuHCl concentrations. The Gibbs-Helmholtz expression fit to the DeltaG(u) versus temperature data from fluorescence gave a DeltaC(p) of 8.0 kcal mol(-1) K(-1), a maximum apparent stability of 23.7 kcal mol(-1) at 18 degrees C, and an apparent cold denaturation temperature (T(CD)) of -23 degrees C. DeltaG(u) values for another mAb (mAb2) with a similar framework exhibited less stability at low temperatures, suggesting a depressed protein stability curve and a higher relative T(CD). Direct experimental evidence of the susceptibility of mAb1 and mAb2 to undergo cold denaturation in the absence of denaturant was confirmed at pH 2.5. Thus, mAbs have a potential to undergo cold denaturation at storage temperatures near -20 degrees C (pH 6.3), and this potential needs to be evaluated independently for individual mAbs.
Lazar
Kristi L
KL
Early Stage Pharmaceutical Development, Genentech, Inc., San Francisco, CA, USA.
Patapoff
Thomas W
TW
Sharma
Vikas K
VK
D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D003080:Cold Temperature; D003198:Computer Simulation; D004356:Drug Storage; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry* / Q000378:metabolism; D008956:Models, Chemical; D011487:Protein Conformation; D011489:Protein Denaturation; D055550:Protein Stability; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G