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Jennifer Markarian is manufacturing editor of Pharmaceutical Technology.
Flow imaging microscopy can be used to identify particulates and their sources.
Subvisible particles (SVP) are a concern for biologic-based drugs. Some technologies can detect but not identify particles. “Knowing that subvisible particles are present-without seeing what they are-can only take you so far. Without an image of the impurity, it may be difficult to figure out where the problem originates,” notes Kent Peterson, CEO of Fluid Imaging Technologies. Pharmaceutical Technology spoke with Peterson about using imaging to identify subvisible particles in parenterals.
PharmTech: In what situations in bio/pharma manufacturing would a manufacturer need to perform imaging for SVP?
Peterson (Fluid Imaging Technologies): SVP are either foreign contaminants or undesirable byproducts of the therapeutic molecules, and they may cause adverse effects and compromise patient safety. SVP can cause immunogenicity, and a SVP count that exceeds the parameters set by the United States Pharmacopeia (USP) can cause capillary occlusion.
Biopharma companies analyze their product from formulation development all the way to patient administration. Formulation scientists can use the imaging capabilities of flow imaging microscopy to study the behavior, including stability and compatibility, of the therapeutic formulations.
SVP may form or be introduced into the formulation at any point during manufacturing or post-manufacturing operations. Research has shown that equipment used during manufacturing, including filters, filling pumps, and tubing, can shed foreign particles into the product. Additionally, particles have been found to form resulting from physical stresses during post-filling operations. Storage containers, such as vials and syringe components, have been found to shed during shipping and storage (1,2). Additionally, agitation during shipping, dropping the syringe or vial, or sending the intravenous (IV) bladder through a pneumatic tube (common in many hospitals) can result in fluid flows and cavitation, which can initiate particle formation/protein aggregation. Exposure to light can cause protein degradation. Extreme temperature changes and accidental freeze/thaw cycles can induce particle formation and/or protein aggregation, and improper IV dilution can cause particle formation. Research has shown that nanoparticles can serve as nucleation points for protein agglomeration when exposed to mechanical stresses, such as agitation during filling pump operation, and can result in a therapeutic unsafe for patient use (3,4).
Regular analysis throughout the manufacturing process can determine the point at which SVP form or are introduced, and analysis following post-fill operations can verify the quality of the product administered to the patient. Flow imaging microscopy enables the user to analyze the morphology of the particulate load, identify the SVP and their likely source, and remediate the issue.
For example, a monoclonal antibody from a well-known drug manufacturer was forming visible particles during the final filling operation. Using flow imaging microscopy, the manufacturer identified that metal shards were being introduced to the product during filling operations. They linked the steel shard shedding to one pump in particular. The ability to see morphological characteristics enabled the manufacturer to identify the particle, target its source, and resolve the problem.
In addition to analyzing SVP during manufacturing and post-manufacturing operations, the manufacturer would also need to perform particle analysis following a recall.
Currently, USP requires that the product meet the particle size and count requirements using light obscuration (LO) or membrane microscopy as means of particle analysis. While both methods are acceptable, LO is the most commonly used method for its rapid results and ease of use, compared to standard membrane microscopy. However, LO does not capture, detect, or report on particle morphology other than particle size. It also often fails to detect small particles. Although particle size and count, as provided by LO, are important parameters to measure, subtle morphological cues can be used to identify the source of the SVP and determine whether it is intrinsic, extrinsic, or inherent based on its identity, and from there determine the point in the process it was introduced.
PharmTech: What are the current industry regulations and how can this technique be used to comply with them?
Peterson (Fluid Imaging Technologies): Industry regulations for protein therapeutics continue to be modified and adapted as our understanding of proteins and our detection technology advances. When USP <788> was published, it required only the enumeration of particles greater than or equal to 10 µm and greater than or equal to 25 µm using LO or standard membrane microscopy (5). Since the publication of USP<788>, however, technology has advanced, and our understanding of proteinaceous particles has deepened. USP <787> was later published to serve as a complementary regulation to USP <788>, allowing smaller sample volumes to be used for analysis (6).
USP <1787> considers our current understanding of particles in protein therapeutics, the dangers of foreign particles and poor characterization, and the inadequacies of using LO as the only characterization method (7). LO only provides particle count and size distribution. It is unable to identify the particle and determine whether it is extrinsic, intrinsic, or inherent. Therefore, USP <1787> recommends alternative methods, including flow imaging microscopy, for improved particle characterization and identification of foreign contaminants in effort to improve product safety.
While LO or standard membrane microscopy analyses are required per USP regulations, flow imaging microscopy is recommended as an additional, orthogonal analysis for method verification and enhanced particle characterization. Particle morphology, captured by images, can be used to determine whether the SVP is inherent, intrinsic, or extrinsic; identify its source; and remediate and refine manufacturing and post-manufacturing operations.
PharmTech: How does dynamic imaging particle analysis allow identification of subvisible particles?
Peterson (Fluid Imaging Technologies): Flow imaging microscopy images and analyzes particles ranging in size from 300 nm to 100+ µm suspended in a fluid in real-time. A liquid sample is introduced into the instrument and pulled through the flow cell using a micro-syringe pump. Objectives of varying magnifications can be installed to optimize imaging particles of various sizes. The flow cell is positioned between the camera and the illumination source. A strobe illumination is used to ‘freeze’ the moving particles in space and capture a clear image of each particle as the sample travels through the flow cell. As the images are captured, intelligent analysis software enumerates particles, analyzes the particulate populations, and measures over 40 different physical parameters from each particle image. Such parameters include length, width, area, diameter, circularity (Hu), elongation, convexity, and many more.
Software filters based on morphological measurements can be built and used to distinguish between particle types (such as silicone oil droplets, bubbles, protein aggregates, glass shards) and enable analysis of each subpopulation. Size bins can be created to analyze particles in specified size ranges, such as the recommended 2–10 µm, and the required greater than or equal to 10 µm and greater than or equal to 25 µm. These images are then visually inspected by the user to identify the likely origin of the particle. Each particle image is saved to a user-built reference library and used to improve the software’s pattern recognition algorithms and improve particle identification.
All particle types within the size range of 300 nm to 100+ µm can be analyzed using flow imaging microscopy. Unlike LO, flow imaging microscopy’s unique light and dark thresholding technology ensures that particles are detected, imaged, and analyzed, regardless of their opacity. Light obscuration instruments rely on the opacity of the particle to generate a shadow. The shadow produces a signal, correlated to the size of the shadow, from which particle size is inferred. Therefore, if the particle and its fluid medium have low contrast, the particles will not produce a strong enough shadow to be detected. This is particularly common with protein aggregates, which are transparent to semi-transparent in nature. Therefore, protein aggregates, due to their transparent nature, often fail to be accurately analyzed and even go undetected when analyzed using LO instruments.
Until recently, flow imaging microscopy could only analyze particles as small as 2 µm. The combination of a blue LED light and patented oil immersion technology of Fluid Imaging Technologies’ FlowCam Nano, released in August 2017, has improved the resolving capabilities of the technology, and now particles as small as 300 nm can be analyzed using flow imaging microscopy.
1. Saller et al., J. Pharm. Sci. 104 (4) 1440-1450 (2015).
2. Gerhardt et al., J. Pharm. Sci. 103(6) 1601-1612 (2014).
3. Tyagi et al., J. Pharm. Sci. 98 (1) 94-104 (2009).
4. Maddux et al., J. Pharm. Sci.106 (5) 1239-1248 (2017).
5. USP, USP General Chapter <788>, “Particulate Matter in Injections,” USP29-NF 24, p. 2722 (US Pharmacopeial Convention, Rockville, MD, 2006).
6. USP, USP General Chapter <787>, “Subvisible Particulate Matter in Therapeutic Protein Injections,” USP 38-NF 33, p. 547 (US Pharmacopeial Convention, Rockville, MD, 2014).
7. USP, USP Informational Chapter <1787>, “Measurement of Subvisible Particulate Matter in Therapeutic Protein Injections,” USP 38-NF 33, pp. 1680-1693 (US Pharmacopeial Convention, Rockville, MD, 2015).
Vol. 42, No. 6
When referring to this article, please cite it as J. Markarian, "Identifying Contamination: Subvisible Particle Imaging," Pharmaceutical Technology 42 (6) 2018.