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Published on: December 13, 2019
Quantification and characterization of subvisible proteinaceous particles in opalescent mAb formulations using
Deepak K Sharma1, Peter Oma, Mark J Pollo
1Brightwell Technologies Inc., 115 Terence Matthews Crescent, Ottawa, Ontario, Canada.
Abstract:
Micro-flow imaging (MFI) has been shown to be more sensitive than light obscuration (LO) methods for measuring subvisible proteinaceous particles in protein formulations. Given the potential challenges in detecting particulates in opalescent mAb formulations, the accuracy of MFI to size and count particles in opalescent solutions was investigated and compared to LO and membrane microscopy methods. Proteinaceous monoclonal antibody (mAb) particles, generated either by chemical denaturation or agitation stress, polystyrene and glass particles were used as model systems for measurements in opalescent mAb solutions. The sizing and counting accuracies of MFI were unaffected by the opalescence of the medium. Using glass particles as a model system for proteinaceous particles, MFI was able to detect relatively low particle concentrations (approximately 10/mL) in opalescent solutions. MFI showed excellent linearity (R(2) = 0.9969) for quantifying proteinaceous particles in opalescent solutions over a wide range of particle concentrations (approximately 20-160,000/mL). Analyses of MFI particle image intensities revealed significant differences in the transparency of proteinaceous particles as a function of their size and mode of generation. LO method significantly underestimated proteinaceous particles, particularly those in the 2-10 microm size range. The less opaque proteinaceous particles were relatively more underestimated by the LO method in opalescent solutions.
Insights
Micro-flow imaging (MFI) accurately sizes and counts subvisible protein particles in opalescent monoclonal antibody (mAb) solutions, outperforming light obscuration (LO) methods. MFI is unaffected by solution turbidity, providing reliable particle quantification.
Area of Science:
- Biopharmaceutical analysis
- Particle characterization
- Protein aggregation
Background:
- Subvisible particles in protein therapeutics, particularly monoclonal antibodies (mAbs), are critical quality attributes.
- Opalescent formulations pose challenges for traditional particle sizing methods like light obscuration (LO).
- Micro-flow imaging (MFI) offers potential for improved sensitivity in particle detection.
Purpose of the Study:
- To evaluate the accuracy of MFI for sizing and counting proteinaceous particles in opalescent mAb solutions.
- To compare MFI performance against LO and membrane microscopy in challenging formulations.
- To assess the impact of opalescence on MFI's particle measurement capabilities.
Main Methods:
- Utilized proteinaceous mAb particles (chemically denatured or agitation-stressed), polystyrene, and glass particles as model systems.
- Performed particle analysis in opalescent mAb solutions using MFI, LO, and membrane microscopy.
- Investigated MFI linearity, sensitivity, and accuracy across a wide particle concentration range.
- Analyzed MFI particle image intensities to understand particle transparency variations.
Main Results:
- MFI's sizing and counting accuracy remained unaffected by solution opalescence.
- MFI detected low particle concentrations (approx. 10/mL) in opalescent solutions.
- MFI demonstrated excellent linearity (R² = 0.9969) for quantifying particles from approx. 20 to 160,000/mL.
- LO significantly underestimated proteinaceous particles, especially in the 2-10 µm range, with greater underestimation for less opaque particles.
Conclusions:
- MFI is a robust and accurate method for quantifying subvisible protein particles in opalescent mAb formulations.
- MFI overcomes limitations of LO in turbid solutions, providing more reliable particle characterization.
- Understanding particle transparency via MFI image analysis can further refine biopharmaceutical quality control.
