Ligand binding assays (LBAs) used for large molecule drug development can necessitate conjugation of a drug to one or more different tags. To help ensure that LBAs utilizing conjugated drug reagents perform consistently over time and provide high quality data, each lot of conjugated material should be functionally and biophysically characterized prior to being incorporated into the LBA. One potential source of lot-to-lot variability is aggregation, which may be inherent to the drug, induced by the conjugation process and/or handling of the reagents.
SEC-MALS is a superior method for assessing the aggregate profiles of LBA reagents. Sample consumption for other available methods, such as analytical ultracentrifugation (AUC), is higher than SEC-MALS. This can be problematic in cases where very limited amounts of LBA reagent samples are available for testing. SEC-MALS is also higher throughput than AUC. Additionally, AUC sensitivity is confined by the concentration and UV extinction coefficient of the sample. In contrast, very low levels of high molecular weight (HMW) aggregates in LBA reagents can be detected using SEC-MALS because light scattering (LS) signal is proportional to the concentration and molar mass of the sample.
SEC-MALS is also preferable to SEC for this work because the molar masses of aggregate peaks are readily confirmed without additional complexities associated with SEC. Using SEC alone requires column calibration using MW standards (which must be performed in a separate run). Furthermore, SEC-derived molar masses can be inaccurate because variable/unpredictable nonspecific interactions between the sample and the base matrix of the column can occur, resulting in retention time shifts that do not correlate with molar mass.
