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1.  Issues and Challenges of Subvisible and Submicron Particulate Analysis in Protein Solutions 
The AAPS Journal  2012;14(2):236-243.
The analysis of particulates has been a longstanding challenge in biopharmaceutical drug product development and quality control because the active constituents themselves may form particulate matter as a degradation product that may be difficult to quantify. These analytical challenges were met with success as long as the definition of particulate matter remained well within the capabilities of the instruments and methods used to measure it. The current testing as per USP <788> for parenterals at ≤100 mL stipulates that the sample “passes” the test if the average number of particles present does not exceed 6,000 per container at ≥10 μm and does not exceed 600 per container at ≥25 μm. The new challenge, posed by regulatory direction and academic research, is to count and to characterize subvisible particulates that are ≤10 μm with the goal of providing higher resolution information about the particulate levels and potential consequences of this product quality attribute in vivo. The present discussion focuses on two parallel efforts: (a) to develop a model system for protein subvisible particulates in samples with high protein concentrations and (b) to evaluate the capabilities and limitations of different technologies available (at the time these studies were conducted) for subvisible and submicron particle (<1 μm in diameter) sizing and counting. Our findings illustrate the importance of using appropriate instrumentation that is adapted to the characteristics of the samples to be analyzed. Any sample manipulation to meet the capabilities and to accommodate the limitations of the analytical technique should be carefully evaluated.
Electronic supplementary material
The online version of this article (doi:10.1208/s12248-012-9335-8) contains supplementary material, which is available to authorized users.
doi:10.1208/s12248-012-9335-8
PMCID: PMC3326173  PMID: 22391789
light-scattering methods for particle characterization; particle analysis in high-concentration protein solutions; particle formation; particle size and distribution analysis; submicron particle characterization; subvisible particle characterization
2.  Characterization of Particles in Protein Solutions: Reaching the Limits of Current Technologies 
The AAPS Journal  2010;12(4):708-715.
Recent publications have emphasized the lack of characterization methods available for protein particles in a size range comprised between 0.1 and 10 μm and the potential risk of immunogenicity associated with such particles. In the present paper, we have investigated the performance of light obscuration, flow microscopy, and Coulter counter instruments for particle counting and sizing in protein formulations. We focused on particles 2–10 μm in diameter and studied the effect of silicon oil droplets originating from the barrel of pre-filled syringes, as well as the effect of high protein concentrations (up to 150 mg/ml) on the accuracy of particle characterization. Silicon oil was demonstrated to contribute significantly to the particle counts observed in pre-filled syringes. Inconsistent results were observed between different protein concentrations in the range 7.5–150 mg/ml for particles <10 μm studied by optical techniques (light obscuration and flow microscopy). However, the Coulter counter measurements were consistent across the same studied concentration range but required sufficient solution conductivity from the formulation buffer or excipients. Our results show that currently available technologies, while allowing comparisons between samples of a given protein at a fixed concentration, may be unable to measure particle numbers accurately in a variety of protein formulations, e.g., at high concentration in sugar-based formulations.
doi:10.1208/s12248-010-9233-x
PMCID: PMC2977008  PMID: 20953747
biopharmaceuticals; protein; sub-visible particles
3.  Characterization of Particles in Protein Solutions: Reaching the Limits of Current Technologies 
The AAPS Journal  2010;12(4):708-715.
Recent publications have emphasized the lack of characterization methods available for protein particles in a size range comprised between 0.1 and 10 μm and the potential risk of immunogenicity associated with such particles. In the present paper, we have investigated the performance of light obscuration, flow microscopy, and Coulter counter instruments for particle counting and sizing in protein formulations. We focused on particles 2–10 μm in diameter and studied the effect of silicon oil droplets originating from the barrel of pre-filled syringes, as well as the effect of high protein concentrations (up to 150 mg/ml) on the accuracy of particle characterization. Silicon oil was demonstrated to contribute significantly to the particle counts observed in pre-filled syringes. Inconsistent results were observed between different protein concentrations in the range 7.5–150 mg/ml for particles <10 μm studied by optical techniques (light obscuration and flow microscopy). However, the Coulter counter measurements were consistent across the same studied concentration range but required sufficient solution conductivity from the formulation buffer or excipients. Our results show that currently available technologies, while allowing comparisons between samples of a given protein at a fixed concentration, may be unable to measure particle numbers accurately in a variety of protein formulations, e.g., at high concentration in sugar-based formulations.
doi:10.1208/s12248-010-9233-x
PMCID: PMC2977008  PMID: 20953747
biopharmaceuticals; protein; sub-visible particles
6.  A critical review of analytical ultracentrifugation and field flow fractionation methods for measuring protein aggregation 
The AAPS Journal  2006;8(3):E580-E589.
Analytical ultracentrifugation (AUC) and field flow fractionation (FFF) are 2 important biophysical methods for measuring protein aggregates. Both methods can separate protein monomer from its aggregate forms under a broad range of solution conditions. Recent advances in instrumentation and data analysis, particularly in the field of analytical ultracentrifugation technology, have significantly improved the capability and sensitivity of these biophysical methods for detecting protein aggregates. These advances have resulted in an increased use of these methods in the biopharmaceutical industry for characterization of therapeutic proteins. However, despite their many advantages over conventional methods, the difficulty in the use of the instrumentation and the complexity of data analysis process, have often hampered the widespread use and proper interpretation of data. This article reviews the recent progress in both technologies, and a few case studies are also presented to discuss their advantages and limitations.
doi:10.1208/aapsj080367
PMCID: PMC2761065  PMID: 17025276
Analytical ultracentrifuge; sedimentation velocity; field flow fractionation; protein aggregates

Results 1-6 (6)