Protein/Ice Interaction: High-Resolution Synchrotron X-ray Diffraction Differentiates Pharmaceutical Proteins from Lysozyme

Abstract

Protein/ice interactions are investigated by a novel method based on measuring the characteristic features of X-ray diffraction (XRD) patterns of hexagonal ice (Ih). Aqueous solutions of four proteins and other solutes are studied using high-resolution synchrotron XRD. Two pharmaceutical proteins, recombinant human albumin and monoclonal antibody (both at 100 mg/mL), have a pronounced effect on the properties of ice crystals, reducing the size of the Ih crystalline domains and increasing the microstrain. Lysozyme (100 mg/mL) and an antifreeze protein (1 mg/mL) have much weaker impact on Ih. Neither of the proteins studied exhibit preferred interactions with specific crystalline faces of Ih. It is proposed that the pharmaceutical proteins interact with ice crystals indirectly by accumulating in the quasi-liquid layer next to ice crystallization front, rather than directly, via a sorption on ice crystals. This is the first report, to the best of our knowledge, of major difference in the protein/ice interaction between non-antifreeze proteins. Another important finding is a detection of a second (minor) population of ice crystals, which is tentatively identified as a high-pressure form of ice, possibly IceIII or IceIX. This finding highlights a potential role of mechanical stresses in freeze-induced destabilization of proteins.

Figure 1.

Examples of XRD patterns at 100 K for solutions containing iAFP (b) and other proteins and low molecular weight solutes (a). The intensities are normalized to the most intense peak. See Table 1 for the composition of the samples, and Materials and Methods for the description of 1^st and 2^nd experiments.

Figure 2.

Full width at half maximum (FWHM) of Ih peaks as a function of the diffraction angle. Results from the 1^st and 2^nd experiments are presented in panels (a) and (b), respectively.

Figure 3.

Enlarged portions of representative X-ray diffraction patterns. (a) Top to bottom: 5% sucrose, 10% sucrose, mAb, rHA, lysozyme, mAb+sucrose+ PS80; (b) Top to bottom: iAFP 0.5 mg/ml; iAFP 1 mg/ml in water; iAFP in histidine; ice experimental. The arrows point to broad peaks which are observed in all patterns.

Figure 4.

Schematic presentation of freezing behavior of solutions of proteins. The red circles represent protein molecules. iAFP (a), which can directly interact with ice by sorption on growing ice crystals, is distinguished from non-AFP proteins, which are either partitioning predominantly in the QLL (mAb and rHA, (c)) or in the freeze-concentrated solution, FCS (lysozyme, (b)). The FCS can be either liquid, if the sample temperature is above the corresponding glass transition temperature, Tg, or solid. See Table S1 (Supporting Information) for the Tg values. The QLL has been reported to be present in a wide temperature range from the equilibrium ice melting point down to 200 K.

Figure 5.

An example of experimental X-ray diffraction patterns of a frozen iAFP solution (0.5 mg/ml) overlaid with theoretical patterns of IceIII (a) and IceIX (b). Red arrows show broad peaks without matching Ih peaks.