Characterization of Protein–Excipient Microheterogeneity in Biopharmaceutical Solid-State Formulations by Confocal Fluorescence Microscopy

Abstract

Protein-stabilizer microheterogeneity is believed to influence long-term protein stability in solid-state biopharmaceutical formulations and its characterization is therefore essential for the rational design of stable formulations. However, the spatial distribution of the protein and the stabilizer in a solid-state formulation is, in general, difficult to characterize because of the lack of a functional, simple, and reliable characterization technique. We demonstrate the use of confocal fluorescence microscopy with fluorescently labeled monoclonal antibodies (mAbs) and antibody fragments (Fabs) to directly visualize three-dimensional particle morphologies and protein distributions in dried biopharmaceutical formulations, without restrictions on processing conditions or the need for extensive data analysis. While industrially relevant lyophilization procedures of a model IgG1 mAb generally lead to uniform protein-excipient distribution, the method shows that specific spray-drying conditions lead to distinct protein-excipient segregation. Therefore, this method can enable more definitive optimization of formulation conditions than has previously been possible.

In Situ Characterization of the Microstructural Evolution of Biopharmaceutical Solid-State Formulations with Implications for Protein Stability

“…Consequently, there are no significant microstructural features in the corresponding range of 300 to 1600 Å, such as distinct protein-rich clusters or other microheterogeneities. The longest length scales probed by SANS are comparable to the limit of resolution of the confocal fluorescence microscopy imaging performed previously for similar formulations, which is approximately 1500 Å or 0.15 μm. Hence, we conclude that mAbs in these lyophilized formulations are distributed uniformly on length scales from that of the protein to that of the particle, and that there are no microheterogeneities or evidence of any significant population of aggregated protein.…”

“…Lyophilization and spray-drying are vastly different drying processes, which lead to very different particle morphologies as well as differences in protein–excipient microheterogeneity on the particle scale . For example, certain spray-dried formulations show an increase in protein concentration toward the exterior of the particles, while proteins in lyophilized formulations are generally homogeneously distributed on the particle scale (Figure ).…”

“…An overview of the composition and process conditions of each sample is provided in Table . A heat-stressed formulation (F3) was obtained by maintaining a regular lyophilized mAb1 formulation (F1) at 110 °C for 5 h. As an example of the particle-scale morphologies and protein distributions in typical lyophilized and spray-dried formulations, scanning electron microscopy (SEM) and confocal fluorescence microscopy (CFM) images of the two Fab1 formulations (F4 and F5), obtained as described previously, are included in Figure . The spray-drying, lyophilization, moisture content determination, and size-exclusion chromatography (SEC) also follow the methodologies described previously but are reiterated here for completeness.…”

“…Protein stability can be informed by the multiscale structural characterization of solid-state formulations, which encompasses knowledge of (1) the morphology and size of the solid-state particles, (2) the protein-stabilizer microheterogeneity, and (3) the presence of protein clusters or aggregates. Protein degradation has been shown to be more likely at the particle surface and is determined by the particle morphology, size, and protein distribution within the solid-state particles. ,,− These factors also influence the reconstitution efficiency and the release rate during sustained drug-release applications. , Our previous research probed the three-dimensional morphology and protein-stabilizer microheterogeneity on particle (micrometer) length scales using confocal fluorescence microscopy (CFM) . That work demonstrated that, while particle-scale microheterogeneity is significant in spray-dried formulations, it is generally not observed in lyophilized formulations.…”

Microstructure, Quality, and Release Performance Characterization of Long-Acting Polymer Implant Formulations with X-Ray Microscopy and Quantitative AI Analytics

“…API-NaOH (Fig. 5b) had dominantly spherical particles with internal voids 36 and a lower degree of agglomeration (arrow), while API-NH 4 OH (Fig. 5e) showed dominantly solid particles with no voids visible at the current resolution, and a higher degree of agglomeration.…”