Purpose: Focused Ion Beam-Scanning Electron Microscopy (FIB-SEM) is a powerful imaging advancement that enables the characterization of sub-micron features and subsequent construction of three-dimensional models for further quantification. FIB-SEM was utilized in this research to gain an expanded understanding of material and pore distribution within lipid multiparticulates (LMPs), a commercially-precedented intermediate and final dosage form with tunable release rates and excellent patient-centric properties such as good mouth feel. Unlike traditional cross-sectioning and lyophilization techniques for exposing particle interiors, nanometer-scale layers of material are removed with FIB milling, resulting in truly flat surfaces without artifacts such as smearing or breaking. This improvement in sample preparation enables observation of sub-micron structural features otherwise destroyed or unresolved in orthogonal techniques. Repetitive FIB milling followed by SEM imaging produces a stack of images that can be reconstructed into a 3D volume for advanced modeling and quantification of phases.
LMPs typically consist of a crystalline drug dispersed in a hydrophobic lipid matrix with hydrophilic pore former (Figure 1). Compositional and process parameters such as amount of pore former or LMP particle size are selected to tune the drug release rate of LMPs. An annealing step is sometimes required to stabilize release performance; the hypothesis for this change is related to redistribution and form changes of the pore former and/or lipid matrix. In this research, the ability of FIB-SEM to resolve different phases within compositionally-different LMPs enabled an assessment of material and pore distribution before and after annealing.
Methods: A melt spray congeal (MSC) process was used to make a variety of LMPs exhibiting immediate release and/or controlled release of the model compound acetazolamide. The MSC manufacturing included atomization of the formulation melt and collection of the congealed product.
FIB-SEM technology was used to mill portions of whole LMPs, exposing the interior surfaces for analyses by both SE (secondary electron) and BSE (back-scattered electron) SEM. Differences in material density and conductivity resulted in contrast differences, enabling the visual discrimination of phases such as drug, matrix, and voids. A workflow for image analysis based on machine learning was then applied to segment and quantify these various phases for characteristics such as area fraction, domain size, and domain distribution.
Results: FIB-SEM successfully resolved drug domains within LMPs, enabling assessment of drug dispersion through the exposed portion of the particle interior. Characteristics such as drug volume fraction and particle size distribution were approximated by image analysis and compared for different compositional choices (Figure 2). Other domains were attributed as lipid matrix and pores. A reduction of porosity due to annealing was qualitatively imaged and quantitatively characterized (Figure 3). Surprisingly, another type of matrix-related domain was detected at drug particle boundaries. These submicron- to micrometer-scale domains at drug particle boundaries may indicate localized sequestering of pore former and/or presence of an amorphous drug phase.
Conclusion: FIB-SEM is a viable technique for resolving submicron drug particles and porosity within lipid multiparticulates. In addition to the visualization of drug dispersion throughout the LMP, accurate cross sectional imaging enabled quantification of characteristics such as drug volume fraction, porosity, and domain size distribution as a function of compositional and process selections. Three-dimensional FIB-SEM tomography will enable the reconstruction of an interconnected drug domain model including porosity that can be informative in explaining drug release behavior as a function of process and product history.
Edward LaChapelle– Sr. Scientist Product Development, Lonza Pharma and Biotech, Bend, Oregon
Nathaniel Klaus– Lonza Pharma and Biotech, Oregon
Amanda Pluntze– Lonza Pharma and Biotech, Oregon
Kathryn Pugh– Lonza Pharma and Biotech, Bend, Oregon
Matthew Shaffer– Manager Multiparticulate Product Development, Lonza Pharma and Biotech, Bend
Shawn Zhang– Managing Partner, DigiM Solution LLC