Kinetics and thermodynamics of dimer formation and dissociation for a recombinant humanized monoclonal antibody to vascular endothelial growth factor.
The recombinant humanized antibody (rhuMAb) VEGF has a high affinity for vascular endothelial growth factor and is currently being evaluated in clinical trials as a cancer therapeutic. Under acidic pH and low ionic strength conditions, the antibody was predominantly present as monomer. Under physiological conditions, the appearance of significant amounts of a noncovalent, reversible dimer were observed by size-exclusion chromatography. The kinetics and thermodynamics of the reversible self-association for rhuMAb VEGF monomer were investigated as a function of pH, temperature, and ionic strength by size-exclusion chromatography using the concentration jump method. The rate constant for dimer formation ranged 23-112 M(-)(1) min(-)(1) under the conditions studied, values that are significantly lower than those reported in the literature for other proteins that self-associate. The rate constant for dissociation ranged 0.0039-0.021 min(-)(1). Gibbs' free energies, enthalpies, entropies, and activation energies were determined and revealed that dimer formation is optimal at pH 7.5-8.0, which may be reflective of charge shielding occurring near the pI of the protein. There was a negative change in entropy for dissociation (values from -18.1 to -12.8 cal/mol K). In the presence of D(2)O or 1 M NaCl, dimerization was enhanced. The results of the kinetic and thermodynamic analysis of this study indicate that rhuMAb VEGF dimerization occurs primarily through hydrophobic interactions.
Moore
J M
JM
Department of Pharmaceutical R&D, Genentech, Inc., South San Francisco, California 94080, USA.
Patapoff
T W
TW
Cromwell
M E
ME
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016466:CHO Cells; D002122:Calcium Chloride / Q000737:chemistry; D002850:Chromatography, Gel; D006224:Cricetinae; D019281:Dimerization; D016228:Endothelial Growth Factors / Q000276:immunology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D008222:Lymphokines / Q000276:immunology*; D008956:Models, Chemical; D009994:Osmolar Concentration; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism*; D012542:Scattering, Radiation; D012965:Sodium Chloride / Q000737:chemistry; D013696:Temperature; D013816:Thermodynamics; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D000911:Antibodies, Monoclonal; D016228:Endothelial Growth Factors; D008222:Lymphokines; D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D012965:Sodium Chloride; D002122:Calcium Chloride