Optimization of Flow Imaging Microscopy Setting Using Spherical Beads with Optical Properties Similar to Those of Biopharmaceuticals

• DOI: 10.1016/j.xphs.2023.10.007
• Publication Date: 2023-10-07T15:07:30Z
• Authors: Takaaki Kurinomaru; Kimitoshi Takeda; Megumi Onaka; Yuki Kuruma; Keiji Takahata; Kayori Takahashi; Hiromu Sakurai; Akira Sasaki; Naohiro Noda; Shinya Honda; Risa Shibuya; Tomohiko Ikeda; Rio Okada; Tetsuo Torisu; Susumu Uchiyama
• Keywords: Bead; Particle (ecology)

Flow imaging microscopy (FIM) is widely used to characterize biopharmaceutical subvisible particles (SVPs). The segmentation threshold, which defines the boundary between particle and background based on pixel intensity, should be properly set for accurate SVP quantification. However, thresholds are often subjectively empirically set, potentially leading variations in measurements across instruments operators. In present study, we developed an objective method optimize FIM threshold using poly(methyl methacrylate) (PMMA) beads with a refractive index similar that of biomolecules. Among several candidate were evaluated, 2.5-µm PMMA most reliable size number, suggesting bead analyzed by could objectively determine measurements. concentrations measured highly consistent indicative concentrations, whereas decreased increasing threshold. optimal where was closest differed instrument black-and-white camera color camera. Inter-instrument differences acid-stressed recombinant adeno-associated virus (AAV) protein aggregates successfully minimized setting optimized specific instrument. These results reveal can aid determining more appropriate evaluate SVPs FIM.