Silicone Microdroplets in Protein Formulations—Detection and Enumeration - Pharmaceutical Technology

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Silicone Microdroplets in Protein Formulations—Detection and Enumeration
The authors describe a novel analytical approach that uses the shape-analysis capabilities of MFI to detect and enumerate silicone oil microdroplets in protein formulations that also contain aggregates of similar size and in a similar concentration.

Pharmaceutical Technology
Volume 33, Issue 1


Figure 1: Silicone oil droplets in IgG1 antibody solution. (ALL FIGURES ARE COURTESY OF THE AUTHORS.)
The experimental protocol was successful in creating repeatable and stable populations of subvisible and visible particles under controlled conditions. All samples were verified for time and dilution stability. The selected monoclonal IgG1 antibody readily forms aggregates and particulates under freeze-thaw conditions.

Figure 2: Generation of protein aggregates in IgG1 antibody solution. (ALL FIGURES ARE COURTESY OF THE AUTHORS.)
Silicone microdroplet characterization. Sonication of the silicone oil sample resulted in the formation of an oil emulsion consisting of microdroplets of varying sizes ranging from 2 to 60 μm with the majority of these particles at approximately 5 μm. Positive control samples containing only filtered PBS buffer were analyzed for the presence of air bubbles and other contaminants. The PBS buffer sample was observed to be particle-free during the analysis, indicating that any air bubbles were removed during sample pretreatment. The homogenous population of siliconeoil droplets was observed to have a well defined circular shape with an aspect ratio >0.85 (see Figure 1). Experiments showed that the deviation from the ideal aspect ratio of 1.0, for circular objects, was the result of noise and pixilation effects at the high instrument sensitivity required to detect and measure the protein aggregates.

Figure 3: Images of typical protein particles. (ALL FIGURES ARE COURTESY OF THE AUTHORS.)
Protein particle generation and characterization. The untreated IgG1 formulation contained a small number of protein particles, believed to have formed during the first sample thawing. As shown in Figure 2, an eight-fold increase in the number of particle counts was observed by MFI in the antibody solution following five freeze–thaw cycles. The protein particles or aggregates were observed to be highly heterogeneous in shape, ranging from small dense fibers to large ribbon-like aggregates. Figure 3 contains representative images of protein particles with a wide range of values for the morphological parameters such as circularity, aspect ratio, and intensity. Air bubbles were not observed in the positive control samples. The results indicate that the freeze–thaw method is suitable for generating protein aggregates in this particular IgG1 formulation.

Figure 4: Protein aggregates in IgG1 antibody solution. (ALL FIGURES ARE COURTESY OF THE AUTHORS.)
It was also observed that, with the increase in size of protein particles, both aspect ratio (see Figure 4) and mean intensity decreased (data not included).


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