Subvisible particles are present in every biopharmaceutical drug product and formulation. All formulations will contain some inherent particles or those generated by the drug substance, including protein aggregates and viral vector aggregates. Formulations may also contain inherent particles or those derived from excipients like surfactants and container-closure systems like syringes, vials, and stoppers.
A well-designed formulation can minimize subvisible particles in the final drug product, improving its safety, efficacy, and quality. Flow imaging microscopy (FIM) instruments like FlowCam allow formulation scientists to monitor subvisible particle generation during formulation development, resulting in drug products that produce minimal particles by design.
Subvisible Particles Pose Safety Risks for Patients
Controlling particulate matter is important throughout biotherapeutic development, given their potential to impact efficacy. Subvisible particles have been associated with severe adverse reactions in the clinic, including anti-drug antibody formation, anaphylaxis, and patient fatalities. Inherent particles derived from the drug substance are thought to pose the greatest risk, but all particle types may lead to adverse reactions such as capillary occlusion and inflammation. Formulation design is critical to stabilize the drug substance and minimize particle generation from excipients and container-closure systems. Doing so results in a safer, more efficacious drug product for patients
Formulation Design Helps Ensure Regulatory Compliance
Given their safety risks, the United States, European, and Japanese Pharmacopeias limit the number of subvisible particles in approved biopharmaceuticals. For example, USP <788> limits the number of particles ≥10 and ≥25 μm in biotherapeutics and other parenteral drug products released in the US. Formulations designed with subvisible particle generation in mind will more consistently meet pharmacopeia standards, reducing the number of withheld batches and improving overall product quality.
Traditional techniques provided limited subvisible particle information
To optimize the subvisible particle content in a formulation, researchers often need to measure several attributes of the particles in a sample. Particle concentration and size distribution data allow researchers to compare particle levels quantitatively between formulations. Measurements that provide information on particle type, such as morphology or composition, can then be used to determine the sources of those particles—data that can guide further changes to the formulation to reduce particle concentrations. Historically, researchers have had access to several techniques for measuring subvisible particles in candidate formulations. However, many of these techniques only provide some of the information about particles necessary to inform formulation decisions. Particle counters like light obscuration (LO) provide accurate particle concentration and size data but do not capture information about the types and sources of particles in a sample. Microscopy methods like membrane microscopy capture morphology information but offer less representative particle concentration and size distribution measurements due to their low throughput and often invasive sample preparation.
The Power of Flow Imaging Microscopy
Flow imaging microscopy (FIM) combines the high throughput particle measurements available via LO with the morphology information captured via microscopy methods. These measurements capture brightfield microscopy images of particles in a liquid sample flowing through a microfluidic flow cell.
FIM accurately measures the concentration and size distribution of most particle types in biotherapeutic formulations. This includes translucent particles such as protein aggregates, which are often undercounted and undersized by light obscuration.
Additionally, the high-quality particle images captured by FlowCam contain morphology information, providing valuable insight into particle origin. Using image processing features such as VisualAI, scientists can determine the types of particles present in a formulation and track changes in the concentration of each type as they continue to test and refine their drug product.
USP <1788> recommends FIM as an orthogonal technique to light obscuration and membrane microscopy to characterize subvisible particles—especially during development.
Designing Stable Formulations with Flow Imaging Microscopy
FIM allows researchers to measure the subvisible particle content in candidate formulations following real-time or accelerated stability studies. The particle concentration and size distribution data it measures can be used to quantitatively compare the stability of different formulations to determine which is more stable. Using FlowCam LO, researchers can simultaneously collect light obscuration data with their FIM measurements to determine if the particle content in each formulation meets pharmacopeia guidelines like USP <788>.
The particle morphology data captured during these studies illuminates the sources of particles in different formulations. Analyzing the image data can reveal particle sources such as polysorbate degradation or an incompatible container-closure system. Changes in protein, viral vector, and nonviral vector aggregate morphologies between samples can suggest how the drug substance may be degrading in different formulations or under different conditions. This insight can suggest further changes to the formulation to improve drug substance stability or eliminate a particle source.
Interested in FIM with FlowCam?
Flow imaging microscopy has become a cornerstone of modern quality assurance in the biopharmaceutical industry. FlowCam helps researchers maximize the stability and quality of their biotherapeutic formulations, ensuring compliance with regulatory guidelines and improving the safety and efficacy of each batch of drug product.
