Investigation of protein/carbohydrate interactions in the dried state. 2. Diffuse reflectance FTIR studies
Introduction
The need to study protein/excipient interactions in the solid state has become increasingly apparent, as freeze-drying has become the preferred process to improve the long-term stability of pharmaceutical recombinant proteins. Upon freeze-drying in the absence of lyoprotectants, changes in the secondary structures of most proteins have been observed. The addition of excipients has been found to both improve the stability of proteins upon freeze-drying and subsequent storage. It has been hypothesized that a direct interaction between the additives and the surface of proteins occur. During the secondary drying phase, the removal of water molecules is believed to be at least partially responsible for protein aggregation. By replacing water molecules with other hydrogen bond forming compounds such as carbohydrates, proteins can be stabilized during the secondary drying phase. This is usually referred to as the water-replacement mechanism. The presence of such protein/carbohydrate interactions has been inferred by infrared spectroscopy (Carpenter and Crowe, 1989, Prestrelski et al., 1993, Kreilgaard et al., 1998, Allison et al., 1999). Changes in the IR spectra of dried proteins compared with those of proteins in solution have been interpreted as either due to significant changes in the secondary structure (Lamba et al., 1983, Poole and Finney, 1983, Poole and Finney, 1984, Prestrelski et al., 1993, Costantino et al., 1995, Allison et al., 1998, Costantino et al., 1998) or simply due to changes in the hydration state (Careri et al., 1980, Rupley and Careri, 1991). Proteins composed of α-helices and mixtures of α-helices and β-sheets have generally shown more dramatic changes upon freeze-drying than those consisting primarily of β-structure (Prestrelski et al., 1993, Costantino et al., 1995, Costantino et al., 1998). Most commonly, a significant decrease in α-helix content is detected, with a simultaneous increase in β-sheet formation (possibly intramolecular in nature). Most FTIR studies (Yang et al., 1987, Costantino et al., 1996, Dong et al., 1996, Carpenter et al., 1998), however, have been conducted on samples in which the protein was pressed at high pressure into a KBr pellet. Dispersing dry protein samples in material such as KBr after fine grinding and compressing this mixture to obtain a transparent disk could result in additional protein structural alterations. Chan et al. (Chan et al., 1996) demonstrated a correlation between the loss of activity (due to the formation of irreversible aggregates) of rh-DNase and the pressure used for the compression of KBr pellets. Additional protein deterioration could occur from partial dissolution of KBr in the presence of residual moisture contained in the freeze-dried samples. Bromide anions are highly chaotropic and their dissolution in residual water hydrating the protein could induce further structural alterations. Diffuse reflectance Fourier transform infrared spectroscopy (DRIFTS) eliminates sample preparation and permits direct determinations of FTIR spectra of solid samples. Spectra obtained from the absorbed fraction of the radiation are, however, characterized by different band intensities compared with those obtained by classical transmission geometry. A correction factor can be applied to DRIFTS spectra which compensates for the artificial enhancement of weaker intensity peaks (Culler, 1993):where R is the absolute reflectance of the absorbing layer relative to that of a non-absorbing material (KBr or KCl), s is the scattering coefficient, and K is the molar absorption coefficient. The reflectance spectrum then is defined as the ratio of the sample concentration to the scattering characteristic of the sample.
Section snippets
Materials
Recombinant human deoxyribonuclease I protein (rh-DNase) and recombinant human insulin-like growth factor I (rh-IGF-I) were produced at Genentech Inc., (South San Francisco, CA). All proteins were provided as excipient free powder after dialysis against pure water or a 10 mM ammonium bicarbonate buffer followed by lyophilization (Costantino et al., 1997, Overcashier et al., 1997). Sucrose, and dextrans 69 and 503 kDa were purchased from Sigma Chemicals, St. Louis, USA, dihydrate trehalose from
rh-DNase-containing mixtures
The FTIR spectrum of rh-DNase in solution was obtained using ATR geometry. Curve fitting of the amide I region (Fig. 1a) revealed the presence of peaks at 1688, 1679, 1666, 1654, 1644, 1630, and 1619 cm−1. The position of these peaks is consistent with previous measurements (Chan et al., 1996, Costantino et al., 1998). The peaks at 1666 and 1654 cm−1 were assigned to short and long α-helices, respectively (Costantino et al., 1998), while signals at 1679 and 1630 cm−1 arose from β-sheets.
Conclusions
Diffuse reflectance infrared spectroscopy provides an attractive alternative method for examination of solid proteins compared with the more commonly used KBr pellet method. The therapeutic proteins rh-DNase and rh-IGF-I undergo significant reversible changes in their secondary structure upon freeze-drying. For both proteins, the presence of intermolecular β-sheets was detected and α-helix content decreased significantly. The addition of carbohydrates inhibited protein secondary structure
Acknowledgements
The proteins used in this study and partial financial support were provided by Genentech, Inc. The authors would like to thank Dr H.R. Costantino for his careful reading of the manuscript.
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