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Particle analysis of therapeutic protein formulations with ImageStreamX® Imaging Flow Cytometry and the PROTEOSTAT® Protein Aggregation Assay

Christine Probst, Brian Hall, and David Basiji.
Amnis part of EMD Millipore, 645 Elliott Ave W, Suite 100, Seattle, WA 98119, USA


Featured Product: PROTEOSTAT® Protein Aggregation Assay

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Abstract

The formation of protein aggregates (PA) and other subvisible particles within protein-based drugs is a major concern for the pharmaceutical industry because they can impact drug activity or cause unwanted immunogenicity in patients (1). It is critical to monitor the presence of these particulates to ensure the safety and efficacy of these drugs, especially since their formation can be highly sensitive to production and storage conditions. Currently US and European pharmaceutical regulations require testing of subvisible (>10 um) and visible (>25 um) particles of therapeutics, and recent literature has highlighted the importance of also measuring particles in the 1-10 um size range (2). Another emerging requirement is the need to classify particle type, as multiple contaminants including silicone oil droplets and bacteria may be present in this size range (3). No currently accepted industry method meets the need to simultaneously size, enumerate, and classify particles within this critical 1-100 um size range, thus illustrating the need to develop new approaches for this application (3). In this study we evaluate the potential for the Amnis ImageStreamX MKII® (ISX) imaging flow cytometer to address these criteria and enable comprehensive particulate characterization for therapeutic protein formulations. The major advantage of the ISX is the coupling of flow cytometry with microscopy, which allows particle type to be classified using specific fluorescence stains and quantitative measurement of morphological properties such as size from high resolution imagery. Though traditional flow cytometry has been previously applied for this application (4), it is limited compared to imaging flow cytometry because it cannot directly size particles, is less sensitive for detection of small and translucent particles, and does not measure other potentially important morphological parameters such as shape and contrast.

In this study we used Enzo Life Sciences PROTEOSTAT® Protein Aggregation Standards as a model PA system. PROTEOSTAT® Protein aggregation Standards are the only commercially available PA standards, and provide a useful tool for measuring the linearity and sensitivity of the ISX for PA analysis. Additionally, the PROTEOSTAT® Protein Aggregation assay was used to fluorescently label PAs with high specificity. This simple mix-and-read assay offers high selectivity for PAs using a proprietary fluorescent (FL) probe that displays 20-90 fold brightness enhancement upon binding to the cross-beta spine quaternary structure of aggregated proteins. As silicone oil microdroplets are a common contaminant for PA applications, we further evaluated the ability of ISX to differentiate this contaminant using BODIPY® fluorescent dye. Through this study we determined that the ISX offers multiple advantages for PA analysis compared to currently accepted platforms, including (i.) direct measurement of particle size and other morphological characteristics through quantitative image analysis (ii.) classification of multiple particle types by incorporating additional fluorescent stains, (iii.) collection of statistically robust data sets via rapid image acquisition, (iv.) measurement of absolute particle concentrations using a volumetric syringe pump, and (v.) small minimum sample volume requirement of 20 µL.

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