Currently the compendial method for quantifying subvisible particles equal to or greater than 10 µm and 25 µm uses light obscuration (LO), which is internationally harmonized in the U.S., European, and Japanese Pharmacopoeia. However, numerous reports have indicated that subvisible particles smaller than 10 µm could elicit immune responses.
The Food and Drug Administration (FDA) has published the guidance on the immunogenicity assessment for therapeutic protein products, wherein they recommend assessment of subvisible particles below 10 µm. The FDA recommends the characterization of subvisible particles during the product life cycle using orthogonal methods, of which flow imaging (FI) is one.
In this study, the size and count of 3 different subvisible particle preparations were analyzed using LO and FI (MFI and FlowCam) in 12 laboratories in an attempt to clarify the consistency of subvisible particles, and the analytical performance of each instrument. Three types of subvisible particles were shared across 12 laboratories and analyzed for their sizes and counts. Results were compared between the methods, FI and LO, inter-laboratories, and inter-instruments (FlowCam and MFI).
It was determined that FI may be a viable alternative to LO, as it yields number of, and detailed morphological properties of subvisible particles. FI has been shown to be more sensitive than LO for highly transparent particles such as protein. Moreover, FlowCam provided a relatively higher number of particles compared with MFI, and consistent results were obtained using the instrument from the same manufacturer in all three samples.
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Pictured above: intravenous immunoglobulin as imaged by the FlowCam 8000 imaging particle analyzer, and the MFI.
