Purpose: The goal of this study was to investigate the conformational change and aggregation of recombinant human interferon-gamma (rhIFN-gamma) as a result of interaction between benzyl alcohol and the protein. The effects of buffer concentration, buffer species, ionic strength, rhIFN-gamma and benzyl alcohol concentrations on the dynamics of the interaction in liquid formulations were also examined.
Methods: The effect of benzyl alcohol on the secondary and tertiary structure of rhIFN-gamma in succinate and acetate buffers was studied using far-UV and near-UV circular dichroism spectrophotometry, respectively. Dynamic light scattering was employed to detect aggregate formation due to the interaction of benzyl alcohol with rhIFN-gamma.
Results: The addition of benzyl alcohol at 0.9% (w/v) in various liquid rhIFN-gamma formulations induced changes in circular dichroism (CD) spectra of the protein in the near-UV region, while the CD spectra in the far-UV region remained unaltered. There were gradual decreases in ellipticity with time throughout the near-UV CD spectra. The decreases in near-UV ellipticity induced by benzyl alcohol were accompanied by the formation of high molecular weight aggregates as measured by dynamic light scattering. Loss in near-UV ellipticity was accelerated at lower protein concentration and by increasing buffer or benzyl alcohol concentration. It was also faster in succinate than in acetate buffer. Formulation ionic strength did not affect the CD spectral changes in both the near- and far-UV regions.
Conclusions: Interaction between benzyl alcohol and rhIFN-gamma is formulation dependent. Protein concentration, buffer species, buffer concentration, and preservative concentration play a significant role in determining the extent of the interaction and consequently the stability of the product.