Tue H. Nguyen

The Effect of Benzyl Alcohol on Recombinant Human Interferon-γ

AbstractPurpose. The goal of this study was to investigate the conformational change and aggregation of recombinant human interferon-gamma (rhIFN-γ) as a result of interaction between benzyl alcohol and the protein. The effects of buffer concentration, buffer species, ionic strength, rhIFN-γ 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-γ 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-γ. Results. The addition of benzyl alcohol at 0.9% (w/v) in various liquid rhIFN-γ 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-γ 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.

Replacing succinate with glycolate buffer improves the stability of lyophilized interferon-γ

Abstract Lyophilization is commonly used to dry protein pharmaceuticals to enhance their shelf life. During the freezing step of this process, significant events (e.g. pH shifting) can occur in the uncrystallized, liquid portion which influence the stability of the product. Herein, we present evidence of such an effect and the impact on the quality of recombinant human interferon-γ (IFN-γ) lyophilized from mannitol-containing succinate buffer at pH 5. In the frozen matrix, we hypothesize that the monosodium form of succinic acid crystallized, as evidenced by electrical resistance data, affecting the buffer systems ability to maintain pH, as probed by Fourier-transform infrared (FT-IR) spectroscopy. The latter indicated that the succinate buffer lyophilized from aqueous solution at pH 5 exhibited an ionization state corresponding to that of some 1–2 pH units lower. In exploring the implications for stability, we found that IFN-γ exhibited a marked bioactivity loss during aqueous incubation at pH 3 compared with pH 5. This loss correlated with (reversible) unfolding of the IFN-γ molecule at low pH, as determined by both FT-IR spectroscopy and circular dichroism. We also examined the stability of IFN-γ following lyophilization from pH 5 in two different buffer systems, succinate and glycolate. The latter, which appeared to minimize the freeze-induced pH shifting, exhibited superior solid-state stability upon 4-week incubation at 25°C. Both samples had a similar cake structure (based on X-ray diffraction and differential scanning calorimetry) and had the same residual moisture content. The data suggest that the difference in stability was a consequence of the freeze-induced pH shifting in the succinate buffer system, resulting in a more perturbed (solid-state) structure for IFN-γ. This is consistent with our FT-IR spectroscopic analysis of the lyophilized protein.