Characterization of Subvisible Particulate Matter in Biopharmaceuticals by Flow Imaging Method

Importance of the Topic

Subvisible particulate matter in biopharmaceuticals poses a significant risk to product efficacy and patient safety. Aggregates and particles can trigger immunogenic responses and affect drug performance. Regulatory guidance from USP and JP highlights the necessity of sensitive detection and detailed characterization of these particles, driving the adoption of advanced analytical techniques.

Objectives and Study Overview

This work demonstrates the use of a dynamic flow imaging method to characterize subvisible particles in a model protein solution. The aim was to compare particle populations generated under heat and mechanical stress, using minimal sample volumes and achieving high imaging efficiency.

Methodology and Instrumentation

A freeze-dried human immunoglobulin was reconstituted in pH 5.0 citrate–phosphate buffer at 1 mg/mL and filtered through a 100 nm syringe filter. Two stress conditions were applied:

• Heat stress: sample heated at 80 °C for three minutes with a heat block
• Stirring stress: sample stirred for ten minutes with a PEEK resin stirring plate

Measurements were carried out with the iSpect DIA-10 Flow Imaging system under the following conditions:

• Frame rate 8 frames per second
• Imaging efficiency 97 percent
• Sample volume 50 microliters, flow rate 0.1 mL per minute
• Image threshold set to 220

The instrumentation included the iSpect DIA-10 dynamic particle image analysis system, a precision heat block, PEEK stirring plate and 100 nm syringe filter.

Main Results and Discussion

Heat-stressed samples produced a higher number of small particles below 2 micrometers, while stirring stress favored particles in the 2 to 10 micrometer range. Total particle concentrations were approximately 6.7×10^5 and 3.9×10^5 particles per milliliter for heat and stirring stress, respectively. Morphological analysis revealed elongated string-like aggregates under stirring and more compact shapes under heat. Threshold adjustments enabled reliable detection of 2 micrometer polystyrene latex beads, confirming method sensitivity.

Benefits and Practical Applications

The flow imaging approach offers high sensitivity to transparent and subvisible particles, image-based classification for shape and size analysis, and minimal sample consumption. This makes it suitable for quality control of biopharmaceutical formulations, formulation development and comparability studies during process stress testing.

Future Trends and Potential Applications

Advancements may include integration with machine learning for automated classification, real-time in-line monitoring of bioprocess streams, expanded regulatory acceptance of imaging methods, and application to a broader range of biologics and complex formulations. Continuous method standardization could further optimize particle characterization across the industry.

Conclusion

The iSpect DIA-10 dynamic particle imaging system effectively characterized subvisible particulate matter in protein solutions under different stress conditions. High imaging efficiency, small sample requirements and robust morphological data underscore its utility in biopharmaceutical analysis and quality assurance.

Reference

• Susumu Uchiyama Toward the Proper Biophysical Characterization of Aggregates in Biopharmaceuticals Yakugaku Zasshi Journal of the Pharmaceutical Society of Japan 138 1503-1507 2018
• Kiyoshi M et al Collaborative Study for Analysis of Subvisible Particles Using Flow Imaging and Light Obscuration Journal of Pharmaceutical Sciences 108 832-841 2019
• United States Pharmacopeia 1788.3 Flow Imaging Method for the Determination of Subvisible Particulate Matter
• Supplement II to the Japanese Pharmacopoeia 18th Edition G3-17-182 Evaluation Method of Insoluble Particulate Matter in Biotechnological Products by Flow Imaging Method