FlowCam for Protein Aggregation Analysis in Therapeutics Development 

Protein aggregate analysis involves detecting, sizing, and characterizing protein particles that form when therapeutic proteins degrade. This analysis is critical because protein aggregates pose significant risks for patient safety, potentially leading to treatment failure and other dangerous adverse reactions. 

Several analytical techniques are available for characterizing protein aggregates, each offering different types of data and suited to various stages of development:

• Light Obscuration: A common quality control method that counts and sizes particles typically above 1 µm based on light blockage. While fast and automated, it provides no visual information to distinguish particle types, potentially leading to inaccurate classification of contaminants.

• Membrane Microscopy: Involves visual inspection of particles using a standard brightfield microscope after using membrane filtration to remove the formulation buffer. It provides qualitative morphological information but is labor-intensive and can bias the number, sizes, and types of particles detected.

• Dynamic Light Scattering (DLS): Estimates nanoparticle size distribution in solution by measuring fluctuations in scattered light intensity. DLS is rapid and sensitive, but it struggles with samples containing a polydisperse particle population, especially those with a high concentration of subvisible particles. It also does not distinguish between different particle types in a sample.

• Multi-Angle Light Scattering (MALS): Often used as a detector for methods like size-exclusion chromatography and field-flow fractionation. MALS provides detailed molecular weight and size information for monomeric proteins and small, "soluble" protein aggregates. However, protein formulations can exhibit significant differences in subvisible and visible protein aggregate content even with very little changes in this size range.

• Flow Imaging Microscopy: Integrates microscopy with continuous flow analysis, capturing high-resolution images of thousands of particles per minute as they flow through the instrument. This approach balances the speed of light obscuration with the detailed imaging of manual microscopy for effective, accurate analysis.

FlowCam flow imaging microscopes can detect and image particles from 300 nm to 1 mm, depending on the selected instrument model. This broad detection capability makes FlowCam ideal for visualizing and quantifying particles across both subvisible and visible size ranges, as well as submicron sizes.

FlowCam instruments support USP <788>, <787>, <789>, and even <790> compliance by offering more sensitive detection of protein aggregates and other sources of particulate matter than the compendial methods. Minimizing the particle content of a protein drug product measured by FlowCam improves the likelihood of that therapy meeting USP requirements when measured via the compendial methods. Additionally, when a drug product fails to meet USP requirements, FlowCam provides particle morphology information that can determine and resolve the source of any unexpected particles.

Flow imaging microscopy is a recommended orthogonal technique for subvisible particle monitoring via USP <1788>. With FlowCam LO, it is even possible to capture FIM data alongside compendial light obscuration measurements, streamlining subvisible particle monitoring to USP <1788> recommendations.

FlowCam is particularly helpful in the later stages of formulation design, when comprehensive, quantitative particle characterization becomes critical. Its measurements are used to quantify the stability of protein drug products during accelerated, real-time, and in-use stability studies. It is also often used as a secondary analytical tool in quality control testing, complementing primary lot release tests to diagnose unexpected changes in particle content and ensure that each batch contains a consistent, safe amount of particles.

Key features of protein aggregate particles include their shape and coloration. Protein aggregates typically appear as irregular, somewhat transparent particles. In contrast, silicone oil droplets appear as perfectly spherical droplets with more pronounced coloration.

FlowCam's VisualAI™ software can automatically classify these particle types with over 90% accuracy, reducing subjectivity in particle identification.

FlowCam can be used independently or as an orthogonal or complementary analytical method to provide more complete characterization of the particles in a sample. It provides labs with important information about the size, shape, and type of protein aggregates and contaminants, complementing techniques that measure other aspects, such as molecular weight or concentration. Together, these tools give a fuller picture of a formulation’s stability and quality.