THE EFFECT OF BENZYL ALCOHOL ON RECOMBINANT HUMAN INTERFERON-GAMMA

Purpose. The goal of this study was to investigate the conformational change and aggregation of recombinant human interferon-gamma (rhIFN-ga mma) 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 ef fect of benzyl alcohol on the secondary and tertiary structure of rhIF N-gamma in succinate and acetate buffers was studied using far-UV and near-UV circular dichroism spectrophotometry, respectively. Dynamic li ght scattering was employed to detect aggregate formation due to the i nteraction of benzyl alcohol with rhIFN-gamma. Results. The addition o f benzyl alcohol at 0.9% (w/v) in various liquid rhIFN-gamma formulati ons induced changes in circular dichroism (CD) spectra of the protein in the near-UV region, while the CD spectra in the far-UV region remai ned unaltered. There were gradual decreases in ellipticity with time t hroughout the near-UV CD spectra. The decreases in near-UV ellipticity induced by benzyl alcohol were accompanied by the formation of high m olecular weight aggregates as measured by dynamic light scattering. Lo ss in near-UV ellipticity was accelerated at lower protein concentrati on and by increasing buffer or benzyl alcohol concentration. It was al so faster in succinate than in acetate buffer. Formulation ionic stren gth did not affect the CD spectral changes in both the near- and far-U V regions. Conclusions. Interaction between benzyl alcohol and rhIFN-g amma is formulation dependent. Protein concentration, buffer species, buffer concentration, and preservative concentration play a significan t role in determining the extent of the interaction and consequently t he stability of the product.