Purpose: The pharmaceutical industry has recently been focusing on factors affecting immunogenicity of biotherapeutics. Particles represent an immunogenic risk to patients; however, the risk will vary depending on particle type and size. Silicone oil particles are commonly found in biotherapeutics as it is used a primary lubricant for syringe barrels. While the immunogenicity of silicone oil has been previously studied, the field is still investigating the patient risk of si oil/protein agglomerates. A challenge in investigating this topic is identifying instances of silicone oil/protein interactions to further study immunogenicity of these populations. The work presented here establishes a qualitative method for assessing conditions and materials that can be used to further study the immunogenicity of protein adsorbed to silicone oil.
Methods: A qualitative method for determining conditions that lead to silicone oil and protein interactions was established. This method involves fluorescently labeling protein and silicone oil with proteostat and BODIPY, respectively. Samples were visualized under a fluorescent microscope using a 50x objective. Samples were also run under microflow imaging (MFI) as a direct comparison to current particle characterization techniques. MFI results were additionally analyzed in LINK software using filters capable of separating populations of silicone oil and non-silicone oil particles.
Results: Using the method developed in this work, multiple forms of protein/silicone oil interactions were able to be detected. Figure 1 shows 3 varying cases. In the top there is a large protein aggregate with silicone oil droplets embedded in the aggregate. The middle example shows a small aggregate adsorbed to the surface of the protein. The bottom example, which would be undetectable by MFI, shows protein adsorbed to the outer surface of a si oil droplet. Representative images from MFI are shown in Figure 2. They further confirm that large protein aggregates adsorbed to silicone oil are visible by MFI, however, smaller aggregates and protein coated silicone oil are not detectable with the current technology. Therefore, fluorescent microscopy provides additional information to MFI analysis.
Conclusion: This work provided a qualitative method for assessing protein-silicone oil interactions. Fluorescent labeling of protein and silicone oil allows for a microscopic method to differentiate conditions leading to silicone oil alone, silicone oil adsorbed to large protein aggregates, and silicone oil with a small layer of protein adsorbed to it. Future studies regarding immunogenicity of protein adsorbed to silicone oil can utilize this method to determine conditions with varying levels of protein adsorption to silicone oil. This will allow for a relationship between protein adsorption on silicone oil and immunogenicity.