Effect of Tween 20 on freeze-thawing- and agitation-induced aggregation of recombinant human factor XIII.
Agitation- and freeze-thawing-induced aggregation of recombinant human factor XIII (rFXIII) is due to interfacial adsorption and denaturation at the air-liquid and ice-liquid interfaces. The aggregation pathway proceeds through soluble aggregates to formation of insoluble aggregates regardless of the denaturing stimuli. A nonionic surfactant, polyoxyethylene sorbitan monolaurate (Tween 20), greatly reduces the rate of formation of insoluble aggregates as a function of surfactant concentration, thereby stabilizing native rFXIII. Maximum protection occurs at concentrations close to the critical micelle concentration (cmc), independent of initial protein concentration. To study the mechanistic aspects of the surfactant-induced stabilization, a series of spectroscopic studies were conducted. Electron paramagnetic resonance spectroscopy indicates that binding is not occurring between Tween 20 and either the native state or a folding intermediate state of rFXIII. Further, circular dichroism spectroscopy suggests that Tween 20 does not prevent the secondary structural changes induced upon guanidinium hydrochloride-induced unfolding. Taken together, these results imply that Tween 20 protects rFXIII against freeze-thawing- and agitation-induced aggregation primarily by competing with stress-induced soluble aggregates for interfaces, inhibiting subsequent transition to insoluble aggregates.
Kreilgaard
L
L
The Department of Pharmaceutics, Royal Danish School of Pharmacy, Copenhagen.
Jones
L S
LS
Randolph
T W
TW
Frokjaer
S
S
Flink
J M
JM
Manning
M C
MC
Carpenter
J F
JF
D002942:Circular Dichroism; D004355:Drug Stability; D005176:Factor XIII / Q000187:drug effects*; D005615:Freezing; D006801:Humans; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy; D011136:Polysorbates / Q000494:pharmacology*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000187:drug effects; D013501:Surface-Active Agents / Q000494:pharmacology*; D013696:Temperature
D011136:Polysorbates; D011994:Recombinant Proteins; D013501:Surface-Active Agents; D005176:Factor XIII