Protein-based treatments are rapidly becoming a crucial component of modern medicine. The presence of subvisible particles (SbVPs) in these medications is a critical quality attribute (CQA) that is closely monitored because of potential risks to product stability, quality, bioavailability, and patient safety. However, conventional optical methods make characterizing SbVPs difficult.
Proteinaceous SbVPs have a low refractive index, making detection challenging and limiting the capacity to assure compliance with CQA requirements outlined in compendial procedures and published in recognized pharmacopeias.
A recent US Pharmacopeia (USP) "Stimuli to the Revision Process" article emphasizes the importance of orthogonal approaches such as Flow Imaging Microscopy (FIM) to distinguish silicone oil droplets (SiOPs) from protein aggregates.1
Although light obscuration (LO) is the most widely used method for measuring subvisible particulate matter in parenteral medicinal formulations, it often fails to detect low-contrast particles.
In contrast, FIM collects high-resolution images for direct measurement, enabling thorough morphological characterization and precise particle-type distinction.
Image Credit: Yokogawa Fluid Imaging Technologies
Addressing the regulatory and technical gaps in compendial methods
The USP Stimuli article discusses the challenges in characterizing SbVPs in protein therapies (USP <787>), parenteral medication products (USP <788>), and therapeutic injections/ophthalmic solutions (USP <1788>).
LO measures particles by detecting how much light they block as they pass through a laser, then sizes each particle based on the shadow it casts. However, it is ineffective at detecting low-contrast particles such as protein aggregates and SiOPs because of their low refractive index.
As prefilled syringes (PFS) lubricated with silicone oil have emerged as the preferred primary container for biologics, it is becoming increasingly important to determine whether and how SiOPs interact with therapeutic proteins, potentially influencing stability, aggregation behavior, and overall product quality, a challenge that cannot be met solely by LO.
This study shows how FIM enhances traditional LO workflows by providing the visual and quantitative evidence required to bridge these gaps.
The findings are provided in two separate study arms. In the first arm, FlowCam, a FIM instrument, demonstrates how morphological data can be used to count, measure, and categorize flick-generated SiOPs separately from freeze-thaw (FT)- stressed human IgG (hIgG) aggregates, which are studied individually and in defined ratio metric blends.
Silicone-oil-lubricated PFS present a unique challenge: FT stress can cause both protein aggregation and SiOP shedding, resulting in mixed low-refractive-index particle populations that compendial LO consistently underestimates.
The second arm of this investigation addresses this challenge by assessing the FT stability of hIgG in silicone-oil-lubricated PFS using FlowCam LO, which allows for simultaneous FIM and LO measurements.
Because PFS act as both storage containers and delivery devices, International Council for Harmonization (ICH) guideline Q5C2 and combination product stability expectations require FT stress testing in the anticipated final container-closure system, not just the bulk solution.
By incorporating FlowCam into the analytical workflow, manufacturers receive the comprehensive insights needed to identify the core causes of particle generation and develop effective mitigation methods when drug products exceed compendial limitations.
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References
- USP Stimuli Article: Addressing Subvisible Silicone Oil Droplets – Industry Challenges, Analytical Strategies, and USP’s Rationale for a New General Informational Chapter
- International Council for Harmonization. Quality of Biotechnological Products: Stability Testing of Biotechnological/Biological Products, Q5C. ICH Harmonized Tripartite Guideline, Step 4 version, 30 November 1995.
- Cohen, J. (1988). Statistical power analysis for the behavioral sciences. Journal of the American Statistical Association, 73(363), p.680. DOI:10.2307/2286629. https://utstat.toronto.edu/~brunner/oldclass/378f16/readings/CohenPower.pdf.
About Yokogawa Fluid Imaging Technologies, Inc.
At Yokogawa Fluid Imaging Technologies our mission is to bring clarity and focus to the study of subvisible particles in the life sciences. With our FlowCam technology, we are committed to developing innovative hardware and software to monitor Earth’s water bodies and drinking water supply for hazardous algae, to help make vaccines and injectable medicines safer, and to improve product quality in a variety of applications.
FlowCam instruments combine the benefits of digital imaging, flow cytometry, and microscopy into a single solution - Flow Imaging Microscopy (FIM). Beyond traditional particle sizing and counting, FIM's image-based analysis allows for comprehensive characterization of subvisible API aggregates and contaminants in biopharmaceuticals, mammalian cells, microplankton, emulsions, and advanced materials. FlowCam instruments have been deployed in over 50 countries, supporting research, development, and environmental monitoring in the life sciences, materials research, and industrial applications.
In the video below you will learn about flow imaging microscopy and how FlowCam works to provide count, size, characterization, and digital images of subvisible particles.
FlowCam Explainer Video - What is Flow Imaging Microscopy?
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