Stability of protein formulations: investigation of surfactant effects by a novel EPR spectroscopic technique.
Surfactants are known to stabilize proteins and are often employed as additives in protein formulations. We have developed a method to study the interaction of these formulation additives with proteins by using the partitioning behavior of a spin label. In protein-free formulations, 16-doxyl stearic acid partitions into micelles above the critical micelle concentration (CMC) of the surfactant and gives rise to composite electron paramagnetic resonance (EPR) spectra composed of spectra from "free" label and "rotationally hindered" label. We compute the fraction of micelle-associated label by factor analysis and generate a label partition curve. When protein is added to the formulation, surfactant-protein aggregates form at concentrations below the surfactant's CMC. Partitioning of the label into these aggregates causes the EPR spectrum to reflect hindered rotation of the label at lower surfactant concentrations than in the protein-free solutions. A simple model of label partitioning shows that these partitioning shifts can be correlated to the surfactant:protein binding stoichiometry. We have studied the interactions of various non-ionic surfactants like Brij and Tween with recombinant human growth hormone and recombinant human interferon-gamma and obtained corresponding binding stoichiometries. These binding stoichiometries match those obtained by other techniques. This technique offers a new method for estimating the protein:surfactant binding stoichiometries.
Bam
N B
NB
Department of Chemical Engineering, Yale University, New Haven, Connecticut 06520, USA.
Randolph
T W
TW
Cleland
J L
JL
D002626:Chemistry, Pharmaceutical; D003497:Cyclic N-Oxides; D004355:Drug Stability; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D008823:Micelles; D011136:Polysorbates / Q000737:chemistry; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D013113:Spin Labels; D013501:Surface-Active Agents / Q000737:chemistry* / Q000378:metabolism
D003497:Cyclic N-Oxides; D008823:Micelles; D011136:Polysorbates; D011506:Proteins; D013113:Spin Labels; D013501:Surface-Active Agents; C032363:16-nitroxystearic acid