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

Experiment description

Materials. Monoclonal IgG1 antibody (>95% purity, Cat # I5154) and silicone oil (Dow Corning Corporation "200," 1000 cSt) was obtained from Sigma-Aldrich (Oakville, ON, Canada). Dulbecco's phosphate buffered saline (without calcium chloride and magnesium chloride, pH 7.2) was obtained from Gibco (Carlsbad, CA). Filtered (0.22 μm Durapore membrane filters, Millipore, MA) de-ionized water was used to make all solutions.

Methods. Silicone oil emulsion. A silicone oil suspension of 1.5% (w/w) in phosphate buffered saline (PBS) buffer was prepared by gravimetric dilution in a 50 mL polypropylene centrifuge tube. The solution was sonicated for 30 minutes in a Bransonic 1200 (Branson, CT, USA) ultrasonicator (50/60 Hz) ice-bath to create an emulsion. The silicone oil emulsion was diluted into a buffer solution within 10–15 min to avoid agglomeration and subsequent phase separation after completion of sonication. Over the duration period of the experiments, the resulting emulsion was verified to be stable in concentration and size distribution over time and with dilution. The silicone oil emulsion was diluted with PBS buffer at a concentration of 0.20% (w/v) and analyzed using MFI for particle size, concentration, and various morphological parameters. The sample was pretreated to remove the micro-air bubbles by letting the sample rest for approximately 30 min at 4 C before analysis. Positive control samples containing only filtered PBS (without any silicone oil) were treated in similar manner and found to contain no air bubbles.

Protein particles. Monoclonal IgG1 antibody (1 mg/mL) was selected as the model protein for this study. Subvisible (and visible) protein aggregates were created with the antibody solution using a freeze-thaw method. The freeze-thaw method consisted of repeatedly freezing the protein sample at -80 C for 5 min and immediately thawing it in a warm water bath at 37 C for 5 min. The freeze–thaw cycle was performed five times after the original stock sample thaw. The count, size, and morphological characteristics of the protein particulates in the freeze–thaw treated antibody solution were analyzed using MFI. Positive control samples containing only filtered PBS (without protein), which were treated in a similar manner, were again verified to contain no air bubbles. The resulting concentration of protein particulates/aggregates was verified to be stable for the period of the experiments, as verified by time and dilution stability studies (data not included).

Combining silicone oil and protein particle solutions. The silicone oil emulsion and aggregated protein suspension were generated as outlined above. Protein and silicone oil stock solutions were combined in a 10-mL sample syringe to create concentrations of protein and silicone oil of 0.1 mg/mL and 0.20% (w/v) respectively. The samples were mixed by repeated, gentle pipetting and inspected visually to ensure a homogeneous appearance. Control samples of protein and silicone oil were prepared individually in PBS buffer by substituting the appropriate buffer for the protein and silicone-oil suspension. The test and control samples and their corresponding buffer blanks were prepared in quadruplet.

MFI measurements. Sample fluids were drawn, using a peristaltic pump, from a stirred (600 rpm) 10-mL sample syringe through a 400 μm flow-cell mounted in the HEV setup of an MFI instrument. Low shear forces on the fragile aggregates are ensured through low flow rates, gravity-assisted flow, uniform sample linear velocity, and minimized dead volumes. Sampling efficiency is maximized through flow cell design, material selection, and the use of hydrophobic coatings. Before each sample run, particle-free fluid was flushed through the system to achieve a clean baseline (0 particle counts per mL) and to optimize the illumination at the selected magnification. During the sample run, successive frames were displayed in grayscale or binary mode. These provide immediate visual feedback on the nature of the particle population as well as visual confirmation of the data obtained.

The measurements of particle size or morphology made by MFI are independent of the particle's material type. However, if all or part of a particle lacks sufficient contrast due to sufficiently high transparency, the corresponding particle may be undersized or missed entirely. To measure highly transparent particles such as silicone-oil droplets and highly transparent protein aggregates, the sensitivity of the instrument is designed to use very high threshold values, low noise electronics, and intelligent software algorithms.


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