Stabilization of proteins in solid form

Abstract

Immunogenicity of aggregated or otherwise degraded protein delivered from depots or other biopharmaceutical products is an increasing concern, and the ability to deliver stable, active protein is of central importance. We review characterization approaches for solid protein dosage forms with respect to metrics that are intended to be predictive of protein stability against aggregation and other degradation processes. Each of these approaches is ultimately motivated by hypothetical connections between protein stability and the material property being measured. We critically evaluate correlations between these properties and stability outcomes, and use these evaluations to revise the currently standing hypotheses. Based on this we provide simple physical principles that are necessary (and possibly sufficient) for generating solid delivery vehicles with stable protein loads. Essentially, proteins should be strongly coupled (typically through H-bonds) to the bulk regions of a phase-homogeneous matrix with suppressed β relaxation. We also provide a framework for reliable characterization of solid protein forms with respect to stability.

Keywords: Aggregation; Dynamic stabilization; Lyophilization; Protein stability; Water substitution.

Fig. 1

2^nd Derivative FTIR data in the Amide I region: top) Solution spectra at top, spectra from freeze-dried proteins without stabilizers at bottom. PFK = phosphofructokinase; γ-IFN = gamma interferon; G-CSF = granulocyte-colony-stimulating factor. “r” is a spectral correlation coefficient [42,43]. bottom) Freeze-dried hGH spectra and rate constants for aggregation at 40 °C. The 1658 cm⁻¹ band indicates α helix content. Curve 1: 50% heat-denatured then freeze-dried. Curve 2: no excipient; 3: HES (1:1 protein: excipient); 4: stachyose (1:1); 5: trehalose (1:1); 6: trehalose (1:6) [44]. Reproduced with permission

Fig. 2

Correlation between spectral width and degradation rates: a) Linear ordinate and best linear fits to data. hGH data plotted against Δw_1/2 of 1658 cm⁻¹ α helix marker band [44,46], and IgG data plotted against Δw_1/2 of 1640 cm⁻¹ β sheet marker band [39,53]. b) Same data as in a), but plotted against logarithmic ordinate. Dashed lines are guides to the eye.

Fig. 3

Correlations between T_g, structural relaxation time $({\tau }^{{\beta }_{\mathit{KWW}}})$, and stability: Aggregation in sucrose formulations of an IgG1 antibody. Data from reference [84].

Fig. 4

Correlation between stability and β relaxation processes: a) Enzyme degradation rates at 23 °C in >100 plasticized and antiplasticized sugar-glasses. The solid lines are best fits to the data and yield a slope near 1. Inset: T–T_g for most of the glasses shown in the main figure. b) Aggregation and chemical degradation rates of proteins freeze-dried in sucrose or trehalose-based glasses plotted as a function of <u²>⁻¹. Each symbol represents a distinct temperature – protein – disaccharide combination. See reference [76] for details. Reproduced with permission.

Fig. 5

Myoglobin aggregation after storage for 360 days at 25 °C and 40 °C a) Correlation with fraction of rapidly exchanging protons by HDX. b) Correlation with FTIR spectral signature of α helix content [114]. Reproduced with permission

Fig. 6

Correlation of hGH aggregation after 16 weeks at 50°C with fraction of protein in the interfacial region. Formulations are in stabilizer indicated, at 2% of total solids. Data points represent different freeze drying methods. Data taken from reference [46].