A Multi-Tiered Analytical Approach For the Analysis and Quantitation of High-Molecular-Weight Aggregates in a Recombinant Therapeutic Glycoprotein
- 9 May 2009
- journal article
- Published by Springer Science and Business Media LLC in The AAPS Journal
- Vol. 11 (2), 335-341
- https://doi.org/10.1208/s12248-009-9108-1
Abstract
In this study, we have investigated sedimentation velocity ultracentrifugation (AUC-SV), size exclusion chromatography (SEC), and circular dichroism (CD) methods for the detection and quantitation of protein aggregates using recombinant acid alpha-glucosidase (rhGAA) as a model. The results of this study showed that the formation and molecular weight distribution of rhGAA aggregated species were dependent upon the formulation conditions as well as the storage or stress conditions used to induce aggregation. The utility of CD as a probe for non-native, aggregated species was affirmed, as this method was sensitive to rhGAA aggregation levels of ≤4%. An extensive evaluation of AUC-SV variability was performed using nine levels of spiked rhGAA aggregate that were analyzed on six occasions. Based on our data, the precision of the AUC-SV results increased with increasing levels of aggregate, with a mean RSD of 37.2%. The limit of quantitation (LOQ) for the AUC-SV method, which was based on a Precision criterion of RSD <20%, was determined to be ≥3% aggregated rhGAA. The Precision and LOQ of the SEC method, determined using the same rhGAA sample set, was found to be 3.8% and ≥0.2%, respectively. In general, there was good agreement between the levels of aggregated rhGAA determined using the AUC-SV and SEC methods, with a slight positive bias noted for the AUC-SV results. These studies emphasize the value of applying multiple, well-characterized analytical tools in the evaluation of therapeutic protein aggregation.Keywords
This publication has 17 references indexed in Scilit:
- A Bayesian Approach for Quantifying Trace Amounts of Antibody Aggregates by Sedimentation Velocity Analytical UltracentrifugationThe AAPS Journal, 2008
- Biochemical and pharmacological characterization of different recombinant acid α-glucosidase preparations evaluated for the treatment of Pompe diseaseMolecular Genetics and Metabolism, 2008
- Effects of Solution Conditions, Processing Parameters, and Container Materials on Aggregation of a Monoclonal Antibody during Freeze-ThawingJournal of Pharmaceutical Sciences, 2008
- Studying the formation of aggregates in recombinant human granulocyte-colony stimulating factor (rHuG-CSF), lenograstim, using size-exclusion chromatography and SDS-PAGEActa Pharmaceutica, 2008
- Quantitation of Aggregate Levels in a Recombinant Humanized Monoclonal Antibody Formulation by Size-Exclusion Chromatography, Asymmetrical Flow Field Flow Fractionation, and Sedimentation VelocityJournal of Pharmaceutical Sciences, 2007
- Role of analytical ultracentrifugation in assessing the aggregation of protein biopharmaceuticalsThe AAPS Journal, 2006
- A critical review of analytical ultracentrifugation and field flow fractionation methods for measuring protein aggregationThe AAPS Journal, 2006
- Effects of protein aggregates: An immunologic perspectiveThe AAPS Journal, 2006
- Antibody Response to Aggregated Human Interferon Alpha2b in Wild-type and Transgenic Immune Tolerant Mice Depends on Type and Level of AggregationJournal of Pharmaceutical Sciences, 2006
- Size-Distribution Analysis of Macromolecules by Sedimentation Velocity Ultracentrifugation and Lamm Equation ModelingBiophysical Journal, 2000