HORIZON - High Throughput Low Volume Subvisible Particle AnalysisColby Ashcroft1; Brian DiPaolo1; Gjergji Konica1; Xiangning Li1; Caitlin Wood2; Jane Gosling1; Stephen Voellinger1;

Rick Gordon1; Bernardo Cordovez1; Robert Hart11Halo Labs, 3675 Market Street, Suite 200, Philadelphia, PA 19104

2University of Delaware Department of Chemical and Biomolecular Engineering, Newark, DE 19716

The HORIZON, powered by its novel Backgrounded Membrane Imaging (BMI) technology enables:• Low volume: Requires only 25 μL of sample• High throughput: Completes a full 96 well particle screening assay within 2 hours or 1 min per sample• High dynamic range: Measures particles from 2 µm to 4 mm• Highly reproducible: CVs <10% for polydisperse samples• High contrast for samples with low Refractive Index contrast particles, like protein aggregates • Walk-away automation; Zero carry-over; No air bubbles

Subvisible Particle AnalysisThe HORIZON produces data comparable to other subvisible systems including histograms and images. A comparison of Flow Imaging Microscopy with the HORIZON system is shown here. While trends are similar, the HORIZON counts more particles in each size bin (especially for translucent particles) due to the refractive index advantages of the Back-grounded Membrane Imaging.

System RobustnessRepeatability measurements were carried out using 25 µL samples of polydisperse mock protein particles (ETFE) provided by NIST (n=20), filtered water (n=4), and IgG particles (n=20) purchased from Sigma-Aldrich. ETFE samples were also tested with 6 replicates at 3 different sample volumes. Standard deviations between 5% and 8% were obtained, showing ro-bust, volume invariant behavior for polydisperse samples.

6% CVOn ETFE

High Throughput Formulation ScreeningHuman IgG was diluted into filtered PBS to a concentration of 0.1 mg/mL. This sample was split into 4 tubes, each with a different concentration of polysorbate 80 (0%, 0.06%, 0.12% and 0.18%). Each tube was then placed into a tube-rotator at 15 rpm with aliquots taken out at 6 time points for measurement. 4 different solutions and 6 time points yields 24 condi-tions with each measured with 4 replicates for a total of 96 measurements.

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Introduction

Subvisible particle analysis is a key predictor of protein drug stability and an essential drug product quality metric. However, cur-rent subvisible methods require large volumes of precious protein sample and are labor- and time-intensive, making it almost im-possible to obtain this vital info during late stage candidate screening or early in formulation.

In this poster, Halo Labs presents the HORIZON, a new instrument that uses Backgrounded Membrane Imaging (BMI) to mea-sure subvisible particles. The technique is fully automated, high throughput and uses 1/10th the volume of other techniques. Furthermore, the HORIZON uses a filter plate which eliminates the washing, cleaning and clogging that commonly afflict flow-cell-based systems. BMI is also insensitive to solution refractive index, allowing it to measure translucent protein aggregates easi-ly, has zero cross contamination (carry over) and does not require purge (priming) volumes.

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Backgrounded Membrane Imaging (BMI) has its roots in USP <788>. In the USP method, samples are vacuumed through a filter plate and particles are cap-tured on a flat membrane where they are manually counted. In BMI, this technique has been modern-ized using robotics, image processing and innovative optics. First a background image of the membrane is taken then samples are vacuumed through the fil-ter and reimaged. The background and measure raw images are processed together in order to remove the background texture and only identify the particles present in the sample.

BMI OverviewNot your grandma’s membrane microscopy

Step 4 Data Visualization

Step 3 Measurement and Analysis

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