Subvisible Particles: Why Don’t I Just Filter My Biologic Samples?

Importance of the Topic

Biologic drug products often contain subvisible particles that can impact safety and efficacy. Filtration is routinely used to remove aggregates, but inherent instability of proteins and lipid nanoparticle formulations can lead to new particle formation over time. Assessing particle generation after sterile filtration is critical for quality control and long-term stability of therapeutics.

Goals and Overview of the Study

This study evaluated the effect of a single 0.2 µm filtration step on subvisible particle counts in three biologic formulations: two therapeutic proteins and a lipid nanoparticle (LNP) model. Particle levels were measured immediately before and after filtration, then monitored at day 1, week 1 and week 2 to quantify regeneration of subvisible aggregates.

Methodology and Instrumentation

Sample Composition and Preparation

• Protein 1 and Protein 2: two commercially available therapeutic proteins.
• LNP formulation: mimics an mRNA vaccine carrier with PEG-lipid, cholesterol, DSPC and RNA at final lipid concentration of 0.9 mg/mL and RNA at 45 µg/mL; average diameter 110 nm by dynamic light scattering.

Filtration and Storage

• Single pass through a 0.2 µm PVDF syringe filter.
• Filtrate stored at 4 °C and sampled at specified time points.

Particle Analysis

• Instrument: Aura System using backgrounded membrane imaging.
• Software: Particle Vue version 4.2.
• Measurement: four replicates per condition, 40 µL per well; counting particles >2 µm.

Main Results and Discussion

Initial Filtration Efficiency

• Over 90 percent of subvisible particles (>0.2 µm) removed by a single filtration step.
• Residual particles above filter threshold indicate imperfect clearance and need for multiple filtration passes.

Particle Regeneration Over Time

• Protein 1 showed the fastest increase: from 1 500 to >10 000 particles/mL over two weeks (>400 percent rise).
• Protein 2 doubled its particle count in two weeks, suggesting sensitivity to filtration-induced stress.
• LNP sample increased from 750 to 4 000 particles/mL over two weeks, indicating slower but significant aggregation.

These findings underscore that filtration does not guarantee long-term particle stability and that formulation properties govern aggregation kinetics.

Benefits and Practical Applications

Implementing rapid imaging-based particle analysis after sterile filtration enables:

• Routine monitoring of subvisible particle regeneration in biologic formulations.
• Optimization of filtration protocols to minimize aggregate carryover.
• Improved formulation development by identifying stability liabilities early.
• Quality assurance in bioprocess workflows for proteins and lipid nanoparticles.

Future Trends and Applications

Advances in the field are likely to include:

• Integration of high-throughput imaging platforms with machine learning for automated particle classification.
• Real-time monitoring of particle formation during bioprocessing.
• Development of novel membrane materials and multi-stage filtration strategies for complex biologics including cell therapies.
• Standardization of SVP stability assays for regulatory compliance.

Conclusion

A single sterile filtration step effectively reduces subvisible particles but does not prevent new aggregate formation over time. Continuous particle monitoring using low-volume, high-throughput imaging is essential to ensure the long-term stability and safety of biologic drug products.

Reference

• Zydney AL New developments in membranes for bioprocessing a review Journal of Membrane Science 2021 620 118804
• Saggu M et al Extended characterization and impact of visible fatty acid particles a case study with a mAb product Journal of Pharmaceutical Sciences 2021 110 1093–1102