Flow Imaging Microscopy for Particle Quantification and Characterization
By: Austin Daniels, PhD, Application Scientist, Yokogawa Fluid Imaging Technologies
Subvisible Particles in Biotherapeutics Impact Product Quality and Safety
Biotherapeutics continue to revolutionize how clinicians treat many human diseases. Protein therapies using molecules like peptides and monoclonal antibodies have become well established in the clinic. Gene therapies using vectors like adeno-associated viruses (AAVs), lentiviruses, and lipid nanoparticles (LNPs) have shown promise for treating genetic disorders and other hard-to-treat conditions. Biologics already account for over 50% of new drug approvals—a number likely to increase with each passing year.
Product safety and efficacy are critical to biotherapeutic development regardless of the active pharmaceutical ingredient (API) used. Therefore, it is essential to monitor attributes of biotherapeutics that can influence their performance in the clinic.
One of these attributes is a therapy’s subvisible particle content, which refers to particles between 2-100 μm in diameter in a sample. Virtually all biotherapeutics contain particles in this size range— regardless of the API size. Many of these particles are inherent or derived from the API, their aggregates, and other degradation products. Protein aggregates and other API aggregates fall in this category. Biotherapeutics can contain intrinsic particles or those from sources within the regular drug product and manufacturing process. Intrinsic particles include those from excipients, such as fatty acid particles from polysorbate, as well as particles from container-closure systems like glass lamellae and silicone oil droplets. Extrinsic particles or those from contaminants such as fibers and skin cells from operators may also be present.
Subvisible particles in biotherapeutics are critical to monitor as they can adversely impact product safety. Inherent particles, in particular, are often of the highest concern as they can affect the biodistribution and overall efficacy of the API1. They have also been associated with immune reactions, antidrug antibody formation, and even patient fatalities2,3. Other intrinsic and extrinsic particle types can cause capillary occlusion or inflammation at the injection site, among other adverse reactions.
Particles can also pose product quality risks. All pharmacopeias limit the amount of subvisible particle content present in approved biotherapeutics. For example, USP <788> sets limits on the number of subvisible particles larger than 10 and 25 μm that can be present in parenteral biologics.
Strategies to analyze and control subvisible particles in biotherapeutics are crucial to ensure that every sample meets pharmacopeia requirements and is safe and efficacious for patients. These strategies involve using appropriate analytical techniques to characterize the particle content of a sample.
Traditional Subvisible Particle Analysis Techniques Offer Limited Particle Data
Given the importance of particles in the clinic, several methods have been developed and used to analyze subvisible particle populations in a sample. However, many of the established methods for analyzing particles only capture information on a few facets of a particle population, such as concentration, size, or structure. The limited information these techniques provide often restricts how useful these methods are for monitoring and controlling subvisible particle populations.
Some particle analysis methods primarily measure particle concentration and size distribution. Light obscuration (LO) is the most commonly used method of this type, but other techniques like laser diffraction, flow cytometry, and electrical sensing zone provide similar data. Each of these methods is useful for quantifying the amount of particle content in a sample which is necessary data to meet pharmacopeia requirements. However, these methods provide limited information about the types of particles present in a sample—information that can indicate how these particles were generated in or introduced to the drug product. As a result, it can be challenging to use these methods to reduce or change the particle content of a sample.
Other methods capture information about the shape and composition of particles in a sample. This category includes many microscopy-based methods, including membrane microscopy and electron microscopy. The information these measurements provide is often necessary to determine the types and sources of particles in a sample which is necessary to control a sample’s particle content. However, most microscopy based methods offer lower throughput than methods like LO, limiting the precision of the particle concentration and size distribution measurements these techniques can offer. It also makes it challenging to quantify the amount of different particle types in a sample, especially if they are present at low concentrations. Additionally, some methods such as membrane microscopy and electron microscopy require significant sample preparation such as dilution, solvent removal, and staining. This preparation can bias the number, types, and sizes of particles detected during the measurements and further reduces the throughput of these methods.
In many research applications, information about the concentration, size, and types of particles in a biopharmaceutical sample is often necessary to accurately monitor and control the particle content in a drug product. Historically, researchers often needed to use multiple orthogonal particle analysis techniques to capture this information. With flow imaging microscopy, researchers can now capture much of the relevant information about the subvisible particle content in a sample with a single technique.
Flow Imaging Microscopy Provides Thorough Subvisible Particle Characterization
Flow imaging microscopy (FIM) instruments like FlowCam measure the subvisible particle content of liquid samples. During FIM measurements, a fluid sample flows through a microfluidic flow cell between a light source and a digital camera. The camera captures images of the sample as it passes through the flow cell— including any particles it contains. The resulting digital images are then processed to isolate images of each particle. Particle images are then further processed to measure particle properties, quantifying each particle’s size, shape, and color.
Like other flow-through particle analysis methods, such as light obscuration (LO) and flow cytometry, FIM accurately measures the concentration and size distribution of subvisible particles in samples. As an imaging-based platform, FIM offers higher sensitivity to important transparent particle types like protein aggregates, which can be challenging to analyze via LO4. FIM simultaneously captures information about the particle morphologies in a sample akin to other microscopic techniques. This information is available qualitatively via the high-quality images of each particle FIM reveals and quantitatively via particle property measurements. Particle morphology data can be used to identify the types of particles in a sample, as different particle types will often appear distinct when imaged5. This information is captured in an automated fashion and at a higher throughput than other microscopy-based approaches, allowing researchers to process the morphology of thousands of particles in samples within a few minutes.
Watch this video to see how flow imaging microscopy works:
CONCLUSION
The combination of count, size, and particle morphology data provided by FlowCam and other flow imaging microscopes is beneficial as it can often indicate the sources of particles in a sample—whether that source is inherent, intrinsic, or extrinsic. Knowing the relevant particle sources allows scientists to make targeted changes to a drug product and its manufacturing process to minimize or otherwise control the particle content of a sample. This optimization can help ensure that each batch of a biotherapeutic contains a low, consistent particle population, helping ensure the quality, safety, and efficacy of the therapy for patients.
Download this article with additional FlowCam images and data here.
REFERENCES
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- Rosenberg AS. Effects of protein aggregates: An immunologic perspective. AAPS J. 2006;8(3):E501-E507. doi:10.1208/aapsj080359
- Kotarek J, Stuart C, De Paoli SH, et al. Subvisible Particle Content, Formulation, and Dose of an Erythropoietin Peptide Mimetic Product Are Associated with Severe Adverse Postmarketing Events. J Pharm Sci. 2016;105(3):1023-1027. doi:10.1016/S0022-3549(15)00180-X
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