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 m2/m3), 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.