Studying the morphology of lyophilized protein solids using X-ray micro-CT: effect of post-freeze annealing and controlled nucleation

Abstract

The objective of this study was to determine how different techniques used during the freezing step of lyophilization affect morphology of the dried protein solids. Aqueous solutions containing recombinant human albumin, trehalose, and sodium phosphate buffer were dried after their freezing by shelf-ramp cooling, immersion in liquid nitrogen, or controlled ice nucleation. Some shelf-frozen solutions were heat treated (annealed) before the vacuum drying. We used three-dimensional (3D) X-ray micro-computed tomography (micro-CT) and scanning electron microscopy (SEM) to study the morphology of solids. The X-ray micro-CT images of the lyophilized microporous solids showed traces of varied size and structure ice crystals that were comparable to corresponding SEM images. A post-freeze heat treatment and a controlled nucleation both induced larger ice crystal ghosts in the solids. The variations in the structure of walls surrounding ice crystals, formed by the different freezing procedures, should affect the water vapor transition during the primary and secondary drying. Some solids also showed higher-density layer in the upper surface. Overall, the simple sample preparation procedures and the ample morphological information make the X-ray micro-CT appropriate for analyzing lyophilized pharmaceuticals.

Fig. 1

The X-ray micro-CT and SEM images of a quarter-sized piece of a lyophilized solid containing rHA, trehalose, and sodium phosphate buffer. An aqueous solution in flat-bottom glass tube was frozen by immersion in liquid nitrogen before drying under vacuum

Fig. 2

Horizontal and vertical section of X-ray 2D micro-CT images of freeze-dried solids containing rHA, trehalose, and sodium phosphate buffer. The solids were dried after shelf-ramp freezing, post-freeze heat treatment, or freezing by controlled nucleation

Fig. 3

An air view of 3D micro-CT image showing the upper surface of a freeze-dried solid containing rHA, trehalose, and sodium phosphate buffer obtained by drying of a post-freeze heat-treated solution

Fig. 4

The small field-of-view X-ray micro-CT 2D images of freeze-dried solids containing rHA, trehalose, and sodium phosphate buffer. The solutions were dried after the original solutions were frozen by shelf-ramp cooling, post-freeze heat treatment, or by controlled nucleation. Number of ice-trace compartments in 15 2D images (circular area, 1-mm diameter) of the particular solids was also included (average ± s.d.)

Fig. 5

The small field-of-view X-ray micro-CT 3D images and SEM images of lyophilized solids containing rHA, trehalose, and sodium phosphate buffer dried after freezing by several methods