A Multi-Tiered Analytical Approach For the Analysis and Quantitation of High-Molecular-Weight Aggregates in a Recombinant Therapeutic Glycoprotein

Abstract

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.

A Review Size-Exclusion Chromatography for the Analysis of Protein Biotherapeutics and Their Aggregates

“…[15,16,125,126] During the development and application of an SEC method for the analysis of a biotherapeutic protein orthogonal techniques such as sedimentation velocity analytical ultracentrifugation (SV-AUC), asymmetrical flow field flow fractionation (AF4), and dynamic light scattering (DLS) may be needed to confirm that the SEC method is providing an accurate representation of the forms and level of aggregates in the protein sample.…”

The critical role of mobile phase composition in size exclusion chromatography of protein pharmaceuticals

“…It cannot be overemphasized that except in the rare circumstance that using the formulation buffer as the mobile phase gives 100% sample recovery then SEC alone cannot resolve this dilemma—if recovery is less than 100%, or if the mobile phase differs from the buffer for the samples being injected, then SEC cannot prove that aggregates were not filtered out, or dissociated or induced by the mobile phase. Thus some cross‐validation of SEC methods for aggregate analysis is nearly always needed using orthogonal methods such as analytical ultracentrifugation, batch‐mode static or dynamic light scattering (not SEC‐light scattering), or field‐flow fractionation 6, 14, 27, 100–102…”

Detection of high-molecular-weight amyloid serum protein complexes using biological on-line tracer sedimentation

“…AUC is a solution-based technique that monitors solute redistribution in a centrifugal field and requires no sample preparation or separation matrix. The sedimentation velocity method is routinely used in biotechnology to monitor aggregates in protein therapeutics [20; 21; 22; 23; 24] and offers a valuable alternative to more frequently employed strategies such as size exclusion chromatography [20]. A key advantage of sedimentation velocity is the use of minimally diluted samples (where dilutions are only due to the sector shape of the sample holder) and the elimination of stationary chromatographic phase which may affect data interpretation due to matrix effects.…”