Impact of lyoprotectors on protein-protein separation in the solid state: Neutron- and X-ray-scattering investigation

Polyhydroxycompounds (PHC) are used as lyoprotectors to minimize aggregation of pharmaceutical proteins during freeze-drying and storage.

Lysozyme/PHC mixtures with 1:1 and 1:3 (w/w) ratios are freeze-dried from either H₂O or D₂O solutions. Disaccharides (sucrose and trehalose), monosaccharide (glucose), and sugar alcohol (sorbitol) are used in the study. Small-angle neutron and X-ray scattering (SANS and SAXS) are applied to study protein-protein interaction in the freeze-dried samples.

Protein interaction peak in the freeze-dried mixtures has been detected by both SANS (D₂O-based samples only) and SAXS (both D₂O- and H₂O-based). In the 1:1 mixtures, protein separation distances are similar (center-of-mass distance of approx. 31 Å) between all lyoprotectors studied. Mixtures with a higher content of the disaccharides (1:3 ratio) have a higher separation distance of approx 40 Å. The higher separation could reduce protein-protein contacts and therefore be associated with less favourable aggregation conditions. In the 1:3 mixtures with glucose and sorbitol, complex SANS and SAXS/WAXS patterns are observed. The pattern for the glucose sample indicate two populations of lysozyme molecules, while the origin of multiple SAXS peaks in the lysozyme/sorbitol 1:3 mixture is uncertain.

Protein-protein separation distance is determined predominantly by the lyoprotector/protein weight ratio.

Use of SANS and SAXS improves understanding of mechanisms of protein stabilization by sugars in freeze-dried formulations, and provide a tool to verify hypothesis on relationship between protein/protein separation and aggregation propensity in the dried state.