The characterization of particulates in biologics is a relatively new concern that presents unique challenges, compared with the characterization of particulates in classical (i.e., nonbiologic) injectable drug formulations. Dynamic imaging particle analysis represents an exciting new and increasingly popular method for the characterization of particulates in biologics. However, as with any other type of instrumentation, it is important to understand how dynamic-imaging instruments measure things and what factors affect the measurements to properly interpret the results. This article will discuss three of the primary factors to understand when using dynamic imaging particle analysis as a particle-characterization technique for biologics: resolution, thresholding, and image quality.
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Characterization of subvisible particulates in parenterals has been a concern since 1936 and was formally addressed by US Pharmacopeia <788> in 1975 (1). At the time of its implementation, <788> was primarily concerned with foreign matter, such as rubber stopper pieces. The concern was largely mechanical in nature, because these were hard particles that might not be distributed through the blood system easily (2). Although biologics raise the same concerns, they also are subject to protein aggregation, whereby small particles combine to create larger ones. Aggregated proteins are soft particles, so they may be able to pass through restrictions that block hard particles. Aggregated proteins may be more difficult to detect than opaque particles because they are transparent. Although light-obscuration devices specified by <788> detect hard, opaque particles well, they do not always detect or properly characterize soft, transparent particles. This deficiency is well documented (2).
Further complicating the characterization of biologics is the fact that the aggregates can be amorphous and range from strandlike to circular shapes. Because light-obscuration devices calculate size based on an assumption of spherical particles, the size measurements can be highly inaccurate. Finally, because these biologics frequently will be delivered through prefilled syringes, the presence of silicone droplets also can cause inflated particle counts and an overall mischaracterization of the biologic.
Recognizing the above cited limitations of light-obscuration techniques, researchers have begun to look for alternative methods for characterizing subvisible particulates in biologics. One area that has shown great promise is in dynamic imaging particle analysis (2). Dynamic imaging particle analysis systems capture digital microscope images of particles in the biologic as they pass through a flow cell. Each particle captured can be measured using standard image-analysis algorithms. Unlike light-obscuration systems, imaging systems can make various measurements, both morphological and spectral, on each particle, even when the particle is transparent. With all these different measurements available for each particle, one can achieve a detailed description that includes particle shape. This description enables automatic differentiation between particle types, such as protein aggregates and silicone droplets. A block diagram of a typical system is shown in Figure 1.
Figure 1: Block diagram of FlowCAM (Fluid Imaging Technologies) dynamic imaging particle-analysis system. LED is light-emitting diode.