Imagine seeing bits of glass, rubber, or metal floating in a vial of medicine that is about to be injected into your arm. Small shards and scraps from the manufacturing process or storage containers sometimes creep into liquid-based therapeutics, posing significant health risks.1 The problem is that these particles are too tiny to be visible to the naked eye.

Protein therapeutics add another layer of complexity to the problem. Components in protein therapeutics may form aggregates in response to pharmaceutically-relevant time scales or under stress conditions. Prolonged storage, container choice, shipping and handling, changes in air, light, temperature, or changes in state from solid to liquid, which occurs during thawing, may induce the formation of subvisible protein aggregates.2 With so many possibilities for aggregation and particle contamination, it is no surprise that subvisible particle size ranges by over one million-fold!3

Scientists use manual microscopy and membrane microscopy to detect and characterize subvisible particle number and shape in protein therapeutics, but such technologies are time consuming. More frequently, scientists use light obscuration technology to quickly assess subvisible particles, while creating a digital record of particle assessment. However, light obscuration only detects particle number and size. Flow imaging microscopy allows scientists to detect subvisible particles and visually characterize particles so that they can differentiate protein aggregates from manufacturing debris.

Early generations of microscopy and volumetric technologies detect large particles ranging from 10 – 25 µm in size. However, even a submicron-sized aggregate can trigger an immune reaction that renders the protein activity of a drug inactive. In some cases, an immune reaction to a therapeutic version of a protein may even cross-react and neutralize the patient’s endogenous protein.2 Indeed, current research suggests that submicron-sized aggregates pose a greater threat for inducing unwanted immunological responses than larger aggregates.4

In addition to detecting particles in the 10 – 25 µm range, current regulatory guidelines encourage researchers to look for particles in the 2 – 10 µm range,5 which can be readily accomplished by many flow imaging microscopy platforms on the market. While optimal for detecting smaller subvisible particles in the 10 µm – 600 µm range, few flow imaging particle analyzers detect submicron particles.

In a recent study, scientists observed a decrease in the total number of subvisible particles after isothermal incubation. By observing the dynamics of both small and large subvisible particles, they determined that the decrease in particle number was due to a redistribution of abundant smaller particles into larger aggregates following incubation. This highlights the importance of simultaneously observing both submicron and micron-sized particles with a single instrument.1

The FlowCam® Nano (Nano-Flow Imaging®) from Yokogawa Fluid Imaging is the first instrument of its kind to capture highly resolved images of both nano and micro particulates in drug suspensions, allowing researchers to simultaneously assess particle morphology and dynamics. Using FlowCam Nano, scientists can detect and visually characterize a broad range of subvisible particles from as large as 10 µm to as small as 300 nm. It is also sensitive enough to assess differences in colloidal stability of protein therapeutics in different formulations and conditions. The FlowCam® Nano provides a convenient and robust platform to visualize and evaluate a broad range of subvisible particles, enabling scientists to more accurately assess drug formulations and enhance therapeutic safety. 

References

  1. D. Chou, “The next frontier in subvisible particle analysis: new tools and opportunities,” BioPharma Asia: Manufacturing, 2020.
  2. J. F. Carpenter, “ Overlooking subvisible particles in therapeutic protein products: gaps that may compromise product quality,” Journal of Pharmaceutical Science, 98(4):1201-1205, 2009.
  3. S. K. Singh, et. al., “An industry perspective on the monitoring of subvisible particles as a quality attribute for protein therapeutics,” Journal of Pharmaceutical Science, 99:3302-3321, 2010.
  4. G. Kijanka, et. al., “Submicron size particles of a murine monoclonal antibody are more immunogenic than soluble oligomers or micron size particles upon subcutaneous administration in mice,” Journal of Pharmaceutical Sciences, 107(11):2847 – 2859, 2018. 
  5. S.K. Singh, et. al., “Particulate Matter in Sterile Parenteral Products,” in Sterile Product Development, eds. P. Kolhe, M. Shah, and N. Rathore, Vol. 6 of AAPS Advances in the Pharmaceutical Sciences Series. New York, NY: Springer, 2013.