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Distribution of Average Aggregate Density from Stir-Stressed NISTmAb Protein
Richard E Cavicchi1, Laura A Philips2, Fook C Cheong2
1Bioprocess Measurements Group, National Institute of Standards and Technology, Gaithersburg, MD 20899, United States.
Journal of Pharmaceutical Sciences
|March 23, 2022
Summary
Protein aggregate density varies with size, impacting antibody therapeutics. This study quantifies this heterogeneity using microscopy and holography, revealing porous structures with decreasing density as aggregates grow.
Area of Science:
- Biophysical characterization of protein aggregates.
- Pharmaceutical analysis of antibody therapeutics.
Background:
- Protein aggregates in antibody therapeutics can affect drug performance.
- Spatially averaged density (ρp) is a key parameter for characterizing protein particles.
- Understanding aggregate heterogeneity is crucial for therapeutic efficacy.
Purpose of the Study:
- To quantify the density distribution of protein aggregates of varying sizes.
- To investigate the relationship between aggregate size and density.
- To compare density measurements obtained by fluorescence microscopy and video holography.
Main Methods:
- Protein aggregates were generated from fluorescently labeled monoclonal antibody (NISTmAb) via stir-stressing.
- Particle density was measured using fluorescence microscopy, analyzing equivalent circular diameter (ECD) and fluorescence intensity.
- Video holography was employed as an orthogonal method for density measurements.
Main Results:
- Protein aggregates exhibited high porosity.
- Median density decreased from 1.07 g/cm³ to 1.02 g/cm³ as aggregate size increased from 0.9 μm to 6 μm.
- Density distributions were broad and asymmetrical, with significant variation within specific size ranges (e.g., 1.005 g/cm³ to 1.1 g/cm³ for 2.5 μm ECD particles).
Conclusions:
- Aggregate density is size-dependent and heterogeneous.
- The porous nature of aggregates influences their measured density.
- These findings aid in characterizing and understanding the impact of protein aggregates in biotherapeutics.
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