Renewal of the air-water interface as a critical system parameter of protein stability: aggregation of the human growth hormone and its prevention by surface-active compounds.
Soluble proteins are often highly unstable under mixing conditions that involve dynamic contacting between the main liquid phase and a gas phase. The recombinant human growth hormone (rhGH) was recently shown to undergo aggregation into micrometer-sized solid particles composed of non-native (mis- or unfolded) protein, once its solutions were stirred or shaken to generate a continuously renewed air-water interface. To gain deepened understanding and improved quantification of the air-water interface effect on rhGH stability, we analyzed the protein's aggregation rate (r(agg)) at controlled specific air-water surface areas (a(G/L)) established by stirring or bubble aeration. We show that in spite of comparable time-averaged values for a(G/L) (≈ 100 m(2)/m(3)), aeration gave a 40-fold higher r(agg) than stirring. The enhanced r(agg) under aeration was ascribed to faster macroscopic regeneration of free a(G/L) during aeration as compared to stirring. We also show that r(agg) was independent of the rhGH concentration in the range 0.67 - 6.7 mg/mL, and that it increased linearly dependent on the available a(G/L). The nonionic surfactant Pluronic F-68, added in 1.6-fold molar excess over rhGH present, resulted in complete suppression of r(agg). Foam formation was not a factor influencing r(agg). Using analysis by circular dichroism spectroscopy and small-angle X-ray scattering, we show that in the presence of Pluronic F-68 under both stirring and aeration, the soluble protein retained its original fold, featuring native-like relative composition of secondary structural elements. We further provide evidence that the efficacy of Pluronic F-68 resulted from direct, probably hydrophobic protein-surfactant interactions that prevented rhGH from becoming attached to the air-water interface. Surface-induced aggregation of rhGH is suggested to involve desorption of non-native protein from the air-water interface as the key limiting step. Proteins or protein aggregates released back into the bulk liquid appear to be essentially insoluble.
Research Center Pharmaceutical Engineering , Graz, Austria.
Johanna
J
Wiesbauer
Ruth
R
Prassl
Bernd
B
Nidetzky
D000388:Air; D019382:Human Growth Hormone / Q000737:chemistry*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions / Q000187:drug effects; D020442:Poloxamer / Q000494:pharmacology; D055550:Protein Stability / Q000187:drug effects; D013501:Surface-Active Agents / Q000494:pharmacology*; D014867:Water / Q000737:chemistry*
D013501:Surface-Active Agents; D014867:Water; D020442:Poloxamer; D019382:Human Growth Hormone