The potential immunogenicity of particles ranging from nanometer to micron sizes in biotherapeutics has been a growing area of discussion between regulators and industry. Both the USP and Ph.Eur. currently require biopharmaceutical products to quantify subvisible particles larger than 10 µm and 25 µm, respectively. However, clinical and laboratory data have yet to conclusively define thresholds for immunogenicity.
Due to the complex milieu of subvisible particles, sample preparation and the method of analysis can affect the accuracy of:
- Particle Sizing
- Particle Counting
- Particle Classification
Consequently, multiple orthogonal techniques are strongly recommended for subvisible particle analysis. Integrity Bio, Inc. offers multiple orthogonal techniques for subvisible particle characterization:
- Light Obscuration
- Flow Imaging Microscopy
- Dynamic Light Scattering
USP <788> and USP <789> particle testing via light obscuration uses a HIAC liquid particle counter for quantitation of particles greater than or equal to 10 µm and 25 µm. However, there are limitations with this technique:
- Cannot distinguish between proteinaceous and non-proteinaceous particles (air bubbles, silicone oil, etc.)
- Difficulty in detecting translucent particles
Flow Imaging Microscopy
Flow imaging microscopy can be performed using either Micro-Flow Imaging™ or FlowCam™.
- Captures digital images of particles
- Can acquire particle counts and morphological data
- Highly sensitive technique able to detect translucent particles
- Can screen for particles of specific morphologies or screen against particles that are not of interest, such as air bubbles or silicone oil droplets
Dynamic Light Scattering
Dynamic light scattering is another orthogonal technique for subvisible particle analysis.
- Measures particle mass, size, and size distribution of molecules and particles