Flow Imaging Microscopy: A New Analytical Capability

As the pharmaceutical industry shifts from chemical compound therapies to engineered biological therapies there is an increasing need to visually confirm subvisible components or unexpected objects (aggregates of liposomes, cellular matrix aggregates, contaminant particles) at the microscopic scale. Flow imaging microscopy captures images of subvisible particles suspended in fluids, and can also provide particle counts, sizes and other metrological parameters.

McCrone Associates has acquired a Yokogawa Fluid Imaging FlowCam 8000 to assist our clients with troubleshooting their subvisible particle concerns associated with new biotherapy solutions. We have added this new capability to our analytical toolbox to evolve our collaborative relationship with our client base and continue providing premier particle identification services.

The results of a particle size distribution analysis conducted with a light obscuration technique can be frustrating when objects larger than the expected size of product components are detected, as in this application: FDA regulations require some manufacturers to screen products for visible and subvisible particulate and identify those unknown or unexpected. Light obscuration methods only provide raw size distribution data, and without any morphological information or visualization of the particles analyzed, the results often lead to more questions about the sample that must be answered, for example: are the measured particles aggregates of formulation components? Are they artifacts such as air bubbles, oil droplets, or foreign contaminant particles?

The FlowCam 8000 utilizes light microscopy and a high-speed digital camera to capture individual images of the particles passing through a flow cell. The flow cell is a quartz chamber that is approximately 1500 µm wide and 300 µm deep (largest flow cell), or smaller. The images captured of each particle are stored in a SQL database along with a variety of length, width, and shape measurements. The immense power of flow imaging microscopy is derived from the ability to combine these images with the size distributions. As the saying goes; a picture is worth a thousand words, but in flow imaging microscopy a size distribution is worth a thousand pictures. The ability to capture particle images also allows a single sample to be reprocessed under a variety of conditions, without expending additional sample material.

This means that the anomalies observed in a light obscuration analysis can be more thoroughly investigated by flow imaging microscopy. Particles can be viewed, selected, and sorted based on their size characteristics including morphological features. This is very useful when investigating potential anomalies because a size range can be selected from the size distribution and the particle images for all the particles within that size range can be viewed. The ability to visualize specific particles throughout the distribution makes it possible to answer questions regarding anomalies, aggregation, and foreign particle contamination. Characteristic images of particle types of interest can also be archived, and compared to past or future samples, as a way of tracking the recurrence of specific contaminants.

Lastly, we can combine flow imaging microscopy with particle identification. As our traditional starting point for particle identification is optical microscopy of the particles of interest, we can search for particles of interest identified by flow imaging microscopy using a light microscope, and prepare them for identification by other methods such as FTIR spectroscopy. By leveraging both flow imaging microscopy and our expertise in particle identification, we improve our ability to answer the challenging questions that often arise in particle investigations.

Contact us to learn more about how we can collaboratively assist with your subvisible particle identification needs using flow imaging microscopy, a new McCrone Associates analytical service capability.