Virtually all biotherapeutics contain subvisible particles, objects that are roughly 2-100 μm in diameter. Effective subvisible particle monitoring strategies are critical in ensuring the safety and quality of these drug products. This article focuses on subvisible particles in protein therapies, their impact on product quality and safety, and effective strategies to monitor them. Similar considerations also apply to particles in other types of biotherapeutics such as cell and gene therapies.
Particles in Protein Therapeutics
A core challenge in developing protein drug products is the limited stability of the active ingredient in solution. Stresses that samples are exposed to during manufacturing, storage, and use can trigger protein aggregation—especially when samples are mishandled. Other components of the drug product can also degrade to generate particles, including excipients like polysorbate as well as container-closure components such as vials and syringes. Identifying the types and sources of particles in a sample is useful as it allows researchers to make targeted changes to the formulation and manufacturing process to control the particle concentration in the final drug product.
Controlling protein aggregation and other subvisible particle sources is a critical part of biotherapeutic development to ensure product safety. While the features of protein therapies that promote adverse reactions are unknown, particles in protein therapies have been associated with adverse drug reactions in the clinic. This includes the formation of anti-drug antibodies and, in extreme cases, anaphylaxis and patient fatalities. As a result, particles are often treated as a risk factor for adverse reactions in the clinic even if their role in these reactions is unknown.
Particles also pose a product quality risk. Like other parenteral drug products, protein therapies and other biopharmaceuticals are subject to pharmacopeia limits on subvisible particle content such as USP <788> and EP2.9.19. Careful subvisible particle measurements during formulation design and as part of process monitoring can help researchers minimize their concentration in the final drug product to ensure that every batch of drug product meets pharmacopeia requirements.
Light Obscuration (LO)
Historically, light obscuration (LO) has been used to monitor protein therapies for subvisible particle content. LO measurements involve flowing a sample past a light source and using the shadows cast on a detector to count and size subvisible particles. These measurements are highly established in pharmaceutical development; researchers can compare LO data from their samples against decades of historical LO data. It is also the preferred compendial technique to meet the subvisible particle monitoring requirements in USP <788> and its equivalents in other pharmacopeias.
LO has several limitations as a technique for monitoring particles in protein formulations. Many common particles such as protein aggregates are highly transparent, blocking less light than opaque particles of the same size and shape. The measurement principle of LO often results in particles in biotherapeutics being undercounted and undersized. LO also does not provide any information about particle morphology that could help researchers identify particle types and control particle sources.
Flow Imaging Microscopy (FIM)
Flow imaging microscopy (FIM) has become a well-established technique for monitoring subvisible particles in protein formulations. In FIM, a liquid sample passes through a microfluidics channel and the particles present are digitally imaged using brightfield microscopy. The resulting images can be processed to determine the particle count, concentration, and size distribution in a sample. Since FIM uses images to count and size particles rather than light blockage, its measurements are less strongly impacted by particle transparency than LO. This results in more accurate particle measurements on most biopharmaceuticals.
Also, FIM provides an image of each particle and various measurements of particle morphology. Researchers can use the data to identify and quantify important particle types in their samples such as protein aggregates and silicone oil droplets. It can also be used to detect and identify anomalous particles in a sample such as metal flakes from degrading process equipment. This information can help researchers design stable protein formulations and monitor manufacturing processes to ensure samples contain a consistently low number of subvisible particles, helping minimize the safety and quality risks of the therapy.
Pairing FIM with Orthogonal and Complementary Measurements
The subvisible particle measurements captured by FIM complement those provided by orthogonal techniques for subvisible particle characterization. For instance, FIM and LO are often used as a pair of orthogonal techniques as recommended by USP <1788>. Combining these methods allows researchers to obtain accurate particle measurements and morphology information from FIM while still being able to compare samples against historical data and the compendial limits via LO.
FlowCam LO is a useful instrument for researchers interested in performing both orthogonal measurements. This instrument captures FIM and LO measurements in series using only a single sample aliquot. In addition to streamlining the analysis, FlowCam LO also allows for direct comparisons between FIM and LO measurements. The instrument is ideal for researchers wishing to bridge historical LO data with modern FIM particle measurements.
FIM also pairs well with complementary techniques for particle characterization. Complementary techniques are often critical in characterizing protein aggregation given the wide size range of aggregates that are typically present in samples. One common approach is to pair FIM with dynamic light scattering (DLS). DLS provides size information from nanoparticles too small to be analyzed with FIM. As nanoparticle-sized protein aggregates can agglomerate to form larger subvisible aggregates, monitoring smaller particles can help detect and mitigate aggregation preemptively. In return, FIM can be useful in monitoring larger subvisible particles outside of the size range for DLS and determining their source. Monitoring larger particles can also be useful for analyzing highly aggregated samples that can pose challenges for DLS measurements.
Flow imaging microscopy is an effective technique for monitoring subvisible particles in protein drug products. The sensitive particle detection and morphology information it provides allows researchers to accurately analyze protein aggregates and other relevant particle types in their samples. The information pairs effectively with that available from complementary particle characterization techniques like LO and DLS. FIM measurements allow researchers to better understand, monitor, and control the particles in their protein drug products as well as other biotherapeutics, ensuring the safety and efficacy of these life-saving treatments for patients.