Effect of benzyl alcohol on recombinant human interleukin-1 receptor antagonist structure and hydrogen-deuterium exchange

Abstract

Benzyl alcohol, a preservative commonly added to multidose therapeutic protein formulations, can accelerate aggregation of recombinant human interleukin-1 receptor antagonist (rhIL-1ra). To investigate the interactions between benzyl alcohol and rhIL-1ra, we used nuclear magnetic resonance to observe the effect of benzyl alcohol on the chemical shifts of amide resonances of rhIL-1ra and to measure hydrogen-deuterium exchange rates of individual rhIL-1ra residues. Addition of 0.9% benzyl alcohol caused significant chemical shifts of amide resonances for residues 90-97, suggesting that these solvent-exposed residues participate in the binding of benzyl alcohol. In contrast, little perturbation of exchange rates was observed in the presence of either sucrose or benzyl alcohol.

Keywords: Excipients; NMR spectroscopy; Protein aggregation; Protein formulation; Protein structure.

Figure 1.

Recombinant human interleukin-1 receptor antagonist ¹H–¹⁵N-HSQC spectra collected with (red) and without (black) 0.9% benzyl alcohol at 25°C. Buffer in each case contained 10 mM sodium citrate, 140 mM sodium chloride, pH 6.5.

Figure 2.

Recombinant human interleukin-1 receptor antagonist ¹H–¹⁵N-HSQC spectra collected with (red) and without (black) 0.5 M sucrose at 25°C. Buffer in each case contained 10 mM sodium citrate, 140 mM sodium chloride, pH 6.5.

Figure 3.

Minimum rhIL-1ra chemical shifts for the addition of 0.9% benzyl alcohol (open squares) or 0.5 M sucrose (closed diamonds) to buffer at 25°C, 0.9% benzyl alcohol to buffer at 37° C (open circles). 63 Gly and 64 Gly show significant shifts (i.e., >0.08 ppm) upon addition of 0.5 M sucrose to the buffer. The prominent peak caused upon addition of 0.9% benzyl alcohol is caused by residues 90 Ser, 91 Glu, 93 Arg, 96 Asp, and 97 Lys.

Figure 4.

Mapping of chemical shift changes on the structure of rhIL-1ra. Numbers on the crystal structure label residues that shifted significantly (i.e., >0.08 ppm). Residues colored red (90 Ser, 91 Glu, 93 Arg, 96 Asp, and 97 Lys) shifted significantly upon addition of 0.9% benzyl alcohol to buffer at 25°C. Residues colored blue (63 Gly and 64 Gly) shifted significantly upon addition of 0.5 M sucrose to buffer at 25°C. The rhIL-1ra crystal structure was obtained from the protein data bank (keyword: 1ILR) and displayed using Pymol software (Portland, OR).

Figure 5.

Recombinant human interleukin-1 receptor antagonist ¹H–¹⁵N-HSQC spectra collected at 25°C (black) and 37°C (red). Buffer in each case contained 10 mM sodium citrate, 140 mM sodium chloride, pH 6.5.

Figure 6.

Minimum rhIL-1ra chemical shifts caused by increasing temperature from 25°C to 37°C. Closed squares represent rhIL-1ra chemical shifts observed in buffer alone. Open circles represent the effect of the temperature change on chemical shifts for rhIL-1ra residues in buffer plus 0.9% benzyl alcohol. In both cases, 62 His, 63 Gly, and 64 Gly exhibit the greatest temperature-induced shifts.

Figure 7.

Minimum rhIL-1ra chemical shifts at 37°C upon addition of 0.9% benzyl alcohol to buffer, calculated using Eq. 4. Red residues, minimum chemical shifts greater than 0.1 ppm; orange residues, minimum chemical shifts between 0.06 and 0.1 ppm; yellow residues, minimum chemical shifts between 0.03 and 0.06 ppm,; white residues, minimum chemical shifts less than 0.03 ppm; and black residues, unassigned.

Figure 8.

Global HX for rhIL-1ra at 25°C from the sum of all observed ¹H–¹⁵N-HSQC cross-peaks as a function of the remaining peak volume (intensity). The peak intensities were normalized to the first recorded spectra at 10 min after the exchange was initiated. Open circles, rhIL-1ra in buffer only (10 mM sodium citrate, 140 mM sodium chloride, pH 6.5); closed squares, rhIL-1ra in buffer with 0.5 M sucrose; and triangles, rhIL-1ra in buffer with 0.9% benzyl alcohol. Each series displays the HX from one experiment at each solution condition.

Figure 9.

Hydrogen–deuterium exchange rate constants (k_obs) for individual rhIL-1ra residues at 37°C. Residues colored red were fully exchanged before the first data point at 10 min, residues colored orange were exchanged during the 10–120 min observation time, and yellow residues did not appreciably exchange (i.e., <10%) during the 10–120 min observation time. Unassigned residues are colored black.

Figure 10.

A hydrophobicity plot for rhIL-1ra generated on the Kyte–Doolittle scale (http://expasy.org/cgi-bin/protscale.pl) with a window size of seven, using a linear weight variation model. On this scale, values greater than zero represent hydrophobic regions and values less than zero represent hydrophilic regions.