Skip to main content
2024 PharmSci 360 Main banner
Icon Legend
This session is not in your schedule.
This session is in your schedule. Click again to remove it.
Back

Manufacturing and Analytical Characterization - Biomolecular

(T1330-11-60) Flow Imaging Microscopy for Monitoring Subvisible and Submicron Particles in Cell and Gene Therapies

Tuesday, October 22, 2024
1:30 PM – 2:30 PM MT
Purpose: Many biotherapeutic drug products contain particles that can have important consequences for product quality and patient safety. Flow imaging microscopy (FIM) is commonly used for characterizing subvisible particles (i.e. particles 2-100 µm in diameter) in biotherapeutic samples, providing particle size and concentration information as well as digital images of each particle. Submicron (0.4-2 µm) particles can also be analyzed using a variation of this technique. FIM is a well-established technique for characterizing subvisible particles in therapeutic protein drug products. This approach can also be used to characterize particles in cell & gene therapy drug products, including those using chimeric antigen receptor (CAR) T-cells, viral vectors like adeno-associated viruses (AAVs), and non-viral vectors like lipid nanoparticles (LNPs). This information can help researchers monitor particle formation and control the product stability and quality issues particles can be related to. This study demonstrates how flow imaging microscopy (FIM) can be used to monitor particles present in cell & gene therapies, using a lipid nanoparticle (LNP) formulation as an example. In this study, an LNP formulation was exposed to different accelerated stability stresses to generate particles. Two FIM instruments as well as dynamic light scattering (DLS) were then used to compare the impact of these stress conditions on the particles present in the formulation

Methods: An LNP formulation in phosphate-buffered saline (PBS) was centrifuged to remove any large particles initially present in the sample. Two 5 mL aliquots of the resulting solution were taken. One 5 mL aliquot was exposed to an accelerated freeze-thaw stress in which samples underwent five freeze-thaw cycles. Each freeze-thaw cycle consisted of a -20°C freeze followed by a room-temperature thaw. The other 5 mL aliquot was exposed to an accelerated heating stress in which the sample was heated to 60°C for four hours. The stressed solutions were then analyzed using two FIM instruments: a FlowCam 8100 instrument used to analyze subvisible particles 2-100 µm in size and a FlowCam Nano instrument for analyzing submicron particles 0.4-2 µm in size. DLS measurements of the nanoparticles in these samples were also obtained using a Dynapro NanoStar.

Results: The impact of the different stress conditions on formulation stability varied between the investigated size ranges. In the submicron size range, higher particle concentrations but similar particle sizes were observed in the freeze-thaw stressed sample than in the heating-stressed sample. In the subvisible size range, the heating-stressed sample contained higher particle concentrations as well as larger particles. Likewise, the DLS data suggested that the heating stress also contained larger particles.

Conclusion: The three analytical techniques indicated that heating stress was the more damaging of the two stresses as it caused a larger portion of single LNP particles to aggregate into larger subvisible particles. This demonstrates how FIM in conjunction with other techniques can be used to monitor LNP stability in accelerated stability studies. The particle information it provides can be used to assess the impact not only of stress conditions but also formulation and container-closure components on particle generation in these samples. Similar approaches can also be used for monitoring the stability of viral vector formulations used in gene therapies and some cell therapies. The particle information FIM provides can help researchers monitor and optimize the stability of their cell and gene therapies, leading to safer, more effective treatments for patients.

Acknowledgements: Yokogawa Fluid Imaging Technologies is the manufacturer of the FlowCam instruments used as part of this study.


Particle size distributions obtained by the two FIM instruments on stressed LNP samples. Rows indicate the sample being analyzed, while columns indicate the instrument used in the measurement. Histogram bars indicate the total concentration of particles in each size bin. The curve above the bars indicates the cumulative particle size distribution for that sample. The text displayed on each figure indicates the median particle size in each dataset.


Particle size distributions obtained via DLS for the unstressed and stressed LNP samples.


Collages of particle images captured by FIM as part of the analysis. Rows indicate the sample being analyzed, while columns indicate the instrument used in the measurement.. Boxed images are images of particles that are potentially LNP dimers.