Developing Improved Protein-Like Particle Standards for Subvisible and Visible Particle Analysis

Protein aggregates in protein therapies can be difficult to monitor and measure in protein formulations due to their high transparency and irregular morphology. Many common techniques for monitoring particles in pharmaceuticals can struggle to measure protein aggregates accurately. This includes techniques like light obscuration for subvisible particles (2-100 μm) and visual inspection for visible (>150 μm) particles. Since protein aggregates can pose risks to product quality and safety, researchers must validate how accurately their analytical techniques can monitor these important particles.

To assist with validation, researchers are interested in developing protein-like particle standards, samples containing a known concentration and size distribution of particles with similar physical and optical properties as protein aggregates. Several particle standards have been previously developed for this purpose, including abraded ethylene tetrafluoroethylene (ETFE)¹ and photolithographic SU-8 particles². However, these particles still exhibit some dissimilarities with protein aggregates, such as their sedimentation velocity for ETFE or morphology for SU-8. There is still an opportunity for a more realistic particle standard to be developed.

Amara et al.³ have recently developed a new protein-like particle standard that could be used for validating common subvisible and visible particle monitoring techniques. In “Comparison of Protein-like Model Particles Fabricated by Micro 3D Printing to Established Standard Particles”, the researchers describe a method for 3D printing protein-like particles based on 3D models of protein aggregates. This manufacturing method can generate particles of a precise, user-defined size, including sizes in the subvisible, visible, and “gray zone” (100-150 μm) ranges. It also allows users to easily control the morphology of these particles by adjusting the structure of the 3D model. This results in a monodisperse but representative particle population ideal for method validation.

FlowCam, a flow imaging microscopy (FIM) instrument, was extensively used to validate the morphology and optical properties of the 3D printed particles. FlowCam offers a wide size range (2 to 1000 μm), allowing both subvisible and visible particle standards to be analyzed with a single instrument. Brightfield images are acquired directly in a high-throughput fashion for morphology characterization.

The team used FlowCam to compare the appearance of the 3D printed particle standards to other common particle standards, including polystyrene beads, ETFE, and SU-8 particles, as well as actual protein aggregates. These comparisons were performed using several morphology measurements FlowCam provides in conjunction with a novel Siamese neural network approach. The results indicated that the particles exhibited a similar morphology to protein aggregates but with significantly less variability.

Above: Amara et al. Figure 2³: "Representative images obtained by flow imaging microscopy of protein particles, PS spheres, NIST standard particles and PLMP. All particles were measured using the same flow imaging microscopy settings and suspended in protein matrix formulation." 

Additionally, to assess the transparency of the particles and associated challenges for accurate particle concentration and size, many of these measurements were also performed after suspending the particles in media of different refractive indices, approaching the refractive index of the measured particles. Changing the refractive index matrix the 3D printed particles were suspended in resulted in significant changes in particle intensity as well as a small change in measured particle size. This behavior matches that exhibited by protein aggregates when resuspended in media of different refractive indices.

This article demonstrates that the 3D-printed model particles developed in these studies may be a more viable protein-like particle standard than existing ETFE and SU-8 particles. These improved standards, both for subvisible and visible particulates, will allow researchers to effectively assess and improve the accuracy of their protein aggregate measurements and validate the analytical methods used.

References

1. Ripple D, Telikepalli S, Steffens K, et al. Reference Material 8634 Ethylene Tetrafluoroethylene for Particle Size Distribution and Morphology. NIST Spec Publ 260-193. Published online 2019. https://doi.org/10.6028/NIST.SP.260-193
2. Telikepalli SN, Carrier MJ, Ripple DC, et al. An Interlaboratory Study to Identify Potential Visible Protein-Like Particle Standards. AAPS PharmSciTech. 2023;24(1). doi: 10.1208/s12249-022-02457-9
3. Amara I, Germershaus O, Lentes C, et al. Comparison of Protein-like Model Particles Fabricated by Micro 3D Printing to Established Standard Particles. J Pharm Sci. 2024;Article in Press. doi: 10.1016/j.xphs.2024.04.011