SIMI: A Different Approach to Revealing Dangerous Subvisible Impurities

Significance of the Topic

Biopharmaceutical products are highly vulnerable to contamination by subvisible extrinsic particles, which pose serious risks to patient safety and can lead to severe immunogenic responses, tissue damage, and costly product recalls. Recent incidents, including mRNA vaccine recalls due to steel particulates and numerous recalls from glass delamination and residual crystals, highlight the urgent need for rapid and reliable detection methods during drug manufacturing and quality control.

Objectives and Study Overview

This application note introduces Side Illumination Membrane Imaging (SIMI) as an innovative technique integrated into the Aura membrane imaging platform. The study aims to demonstrate how SIMI complements established methods—backgrounded membrane imaging (BMI) and fluorescence membrane microscopy (FMM)—to accurately differentiate rigid extrinsic particles from compliant biological aggregates and inherent drug-related crystals.

Methodology and Instrumentation

SIMI employs oblique darkfield illumination on a membrane plate to enhance side scattering from high refractive index, mechanically rigid particles. Key elements include:

• Oblique illumination source parallel to membrane surface to generate side-scattered light.
• Relative SIMI intensity scale with membrane baseline set to zero; positive values indicate protruding rigid particles, negative values for absorbing or low-index materials.
• Combination of three imaging modes on Aura instruments: BMI for isotropic scattering contrast, SIMI for enhanced side scatter detection, and FMM for fluorescence labeling of biological aggregates.

Key Results and Discussion

SIMI was applied to distinguish:

• Protein aggregates vs. ETFE mimetic particles of similar size and morphology. SIMI yielded strong positive signals from rigid ETFE, while protein aggregates remained nearly invisible.
• Glass delamination particles, which are transparent under brightfield but exhibit high side scatter in SIMI due to rigidity.
• Polypropylene particulates shed from vials and tubing, readily detected by SIMI for particles >5 μm.
• Extraneous fibers with rod-like morphology and strong SIMI signal, in contrast to biological particles.
• Residual magnetic Dynabeads in CAR-T cell products. Negative SIMI signal enabled clear separation from cells and polystyrene beads.
• Intrinsic drug crystals (e.g., dipyridamole), which display characteristic angular shapes and robust SIMI contrast compared to amorphous particles.
• Composite particles combining extrinsic and intrinsic materials, where SIMI delineated rigid components within mixed aggregates.

Benefits and Practical Applications

Integrating SIMI with BMI and FMM on a single platform offers:

• High specificity for identifying dangerous extrinsic contaminants without relying on complex machine learning.
• Rapid, real-time particle characterization during bioprocess development and release testing.
• Enhanced detection of low-contrast materials such as glass and plastics across biologic and small molecule formulations.

Future Trends and Opportunities

Advancements may include:

• Automated classification algorithms leveraging multimodal imaging data.
• Real-time inline monitoring of manufacturing streams for particle surveillance.
• Expansion of SIMI principles to other scattering-based modalities, improving detection in complex media.

Conclusion

SIMI is a valuable tertiary imaging mode that complements brightfield and fluorescence microscopy to deliver orthogonal information for robust identification of subvisible extrinsic and intrinsic particles. Its implementation in Aura systems streamlines high-throughput particle analysis, enhancing safety and quality control in pharmaceutical development.

References

1. Jiao N., et al. Characterization of Subvisible Particles in Biotherapeutic Prefilled Syringes. Journal of Pharmaceutical Sciences. 109(1):640–645 (2021).
2. Pharmaceutical Technology. Japan: 1.6 Million Moderna Vaccine Doses Recalled; Novavax Replacement Deal Signed (2021).
3. SiO2 Glass-Related Recalls in Pharma. Scribd (2025).
4. Fierce Pharma. Mylan and Hospira Both Recall Injected Drugs After Particulate Discovered in Vials (2020).
5. FDA News. Hospira Recalls Cancer Drug After Finding Carboplatin Crystals (2020).
6. Evident. What Is Darkfield Microscopy? (2025).
7. National Institute of Standards and Technology. Development and Interlaboratory Evaluation of a NIST Reference Material RM 8634 for Subvisible Particle Standards (2024).
8. Kiyoshi M., et al. Collaborative Study for Analysis of Subvisible Particles Using Flow Imaging and Light Obscuration. Journal of Pharmaceutical Sciences. 108(2):832–841 (2019).