# Transfer Learning Analysis for Subvisible Particle Flow Imaging of Pharmaceutical Formulations
## 3 Results and Discussion  
### Particle FIM Imaging  
Sample FIM images of seven classes of particles are shown in Figure 2. Glass is one of the most commonly used containers in injectable pharmaceutical processes. Glass particles are mainly formed because of the breakage of glass vials during filling or subsequent storage and transportation. In the glass particle solution prepared from the vial, the most noticeable characteristics of the glass particles (Figure 2a) are their relatively sharp edges and their translucent nature. Cap fibers are derived from bouffant caps, which are typically extrinsic particles that may be introduced into the filled drug product solution through environmental contamination during gowning and filling operations. Particle images (Figure 2b) show that the cap fibers were primarily a flexible combination of long strips which are partly transparent. The rubber closures matched the glass vials used to seal  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Lay** & **Layer Type** & **No. of Features** & **Feature Size** & **Activation** & **Input Shape** & **Output Shape** \\ \hline
1 & Convolutional & 64 & \(3\times 3\) & ReLU & \(150\times 150\times 3\) & \(150\times 150\times 64\) \\
2 & Convolutional & 64 & \(3\times 3\) & ReLU & \(150\times 150\times 64\) & \(150\times 150\times 64\) \\
3 & Max  
them. These particles enter the pharmaceutical formulation primarily during the filling process or during subsequent preservation and transportation. The image of the rubber closure particles prepared by crushing the rubber closures (Figure 2c) shows black, irregularly shaped opaque blocks with flocculent-like edges. Protein aggregation is a major source of particles in pharmaceutical formulations. Protein particles may originate from a variety of processes in the manufacturing of pharmaceutical products, such as unexpected high-temperature exposure during production, shear stress during the freeze-thaw process of drug substances, the mixing of compounded bulk drug products, bulk transfer via a peristaltic pump or gas pressure, filtration, and filling. The protein particles were prepared by simulating shaking stress in a magnetic shaker, and the most notable features of the protein particles (Figure 2d) were their translucency and diverse morphologies. Silicone oil is commonly used as a lubricant in various containers (e.g., vials, pre-filled syringes, cartridges, and plungers). Silicone oil microdroplets may appear in pharmaceutical formulations because of the leachability of these container surfaces. Figure 2e shows distinctly spherical microdroplets with translucent centers. Silicone tubing is commonly used for solution transport and solution filling, and may form silicone tubing granules due to flaking during transport. The silicone tubing particles formed by crushing (Figure 2f) were irregular blocks, either translucent or opaque. As shown in the images, each of the six particles displayed more than two different morphologies. For example, silicone oil had two morphologies: a bright center within a dark circle, and a dark center within a bright white ring. This is due to the depth of field of the 10\(\times\) lens (approximately 10 \(\upmu\)m) being much smaller than the depth of the flow cell (80 \(\upmu\)m). According to the working principle description, based on hydrodynamic action, particles tend to move downwards along two cross-sections within the flow cell; approximately half of the particles are not in the focal plane, which is one of the most important sources of image diversity of the same particles. The FlowCam images of the polystyrene beads (Figure 2g) show black spheres with white centers. In this study, these beads were extracted only in the focal plane because of their limited practical significance, and were mainly used as a standard particle reference to demonstrate the feasibility of the CNN model. Polystyrene beads were used as standard particles for FlowCam to adjust the focus as a reference in the flow cell.