An announcement from Halo Labs (Philadelphia, PA) promoting a poster—“HORIZON® High Throughput Low Volume Subvisible Particle Analysis”—caught my eye. The instrument has a 96-well plate with filter membranes in place of the standard wells. To start, each well of the plate is imaged with a photomicroscope in the Horizon instrument. This is the “B” for “Backgrounded” in BMI. Individual samples are then applied to the wells, which are dried and imaged a second time. The backgrounded image is then subtracted from the second, which provides images of the particles alone. This works surprisingly well, with improved precision compared to flow imaging technology. Percent CVs for BMI are typically tighter than 8% compared to 30% for liquid imaging of a polydisperse sample. Throughput is about 2 hours for a 96-well membrane plate. Sample volume required is less than 25 µL.
The company explains that images obtained from flow imaging often suffer from poor background definition since the refractive index difference Δη between protein samples and the aqueous matrix is small (less than 0.1 RI). In contrast, imaging a dry sample uses air as the matrix, which has a very low RI. Thus, the contrast is large, especially at the periphery of the particle. In short, size estimates obtained from the Horizon are more accurate and reproducible than those achieved using wet techniques, especially for particles in the 2–10 µm range.
Protein aggregation continues to be a concern for regulators. Focusing on protein products, the FDA recommends that “Strategies to minimize aggregate formation should be developed as early as feasible in product development,” with a particular focus on the HORIZON High Throughput Low Volume Subvisible Particle Analysis size range delta = Δη (see FDA Guidance for Industry: Immunogenicity Assessment for Therapeutic Protein Products; https://www.fda.gov/downloads/drugs/guidances/ucm338856.pdf).
Robert L. Stevenson, Ph.D., is Editor Emeritus, American Laboratory/Labcompare; e-mail: [email protected]
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