Correlation between Dynamic Light Scattering and Size Exclusion High Performance Liquid Chromatography for Monitoring the Effect of pH on Stability of Biopharmaceuticals.

Abstract

Aggregate formation is a major problem affecting both safety and efficacy of biopharmaceuticals and is associated with protein immunogenicity. Size exclusion high performance liquid chromatography (SE-HPLC) has always been the gold standard technique for detection and determination of protein aggregates. However, large protein aggregates may be filtered off and build up on top of the column leading to deterioration in column performance. Moreover, low-affinity protein aggregates may dissociate during analysis and thus not detected. On the other hand, dynamic light scattering (DLS) is a simple and non-destructive technique that can detect high molecular weight physical and chemical aggregates in their native environment. Here, three model biopharmaceutical proteins of different physicochemical properties were selected; quadrivalent human papillomavirus virus like particles vaccine (HPV VLP, physically assembled subunit vaccine, 55kDa), pegylated Interferon (PegIFN, pegylated non-glycosylated protein, 31.3kDa) and Pegylated Erythropoietin (PegEPO, pegylated and glycosylated protein, 60kDa). Samples were subjected to forced degradation conditions previously shown to lead to aggregate formation (pH 4.0, 8.0 and 10.0, at 37°C for 24h) and samples were analyzed using DLS and SE-HPLC. Generally, good agreement between the results of DLS and SE-HPLC was noted, regardless of the differences in physicochemical properties of the studied biopharmaceuticals. Results showed that aggregate formation was not detected in some cases by SE-HPLC and the decrease in the concentration of the monomeric forms indicated that such aggregates might have been filtered off the column. Although no single techniques can reveal all aspects of protein stability, DLS can serve as a screening tool to detect aggregate formation and cross-validate SE-HPLC results during batch release testing. Owing to its simplicity and low-sample volume requirements, DLS can be used even by hospital pharmacists to confirm absence of protein aggregates immediately before drug administration.