Accelerated development of a SEC-HPLC procedure for purity analysis of monoclonal antibodies using design of experiments.

ABSTRACT

The complex structure of biopharmaceutical products poses an inherent need for their thorough characterization to ensure product quality, safety, and efficacy. Analytical size exclusion chromatography (SEC) is a widely used technique throughout the development and manufacturing of monoclonal antibodies (mAbs) which quantifies product size variants such as aggregates and fragments. Aggregate and fragment content are critical quality attributes (CQAs) in mAb products, as higher contents of such size heterogeneities impact product quality. Historically, SEC methods have achieved sufficient separation between the high molecular weight (HMW) species and the main product. In contrast, some low molecular weight (LMW) species are often not sufficiently different in molecular mass from the main product, making it difficult to achieve appropriate resolutions between the two species. This lack of resolution makes it difficult to consistently quantify the LMW species in mAb-based therapeutics. The following work uses a design of experiments (DoE) approach to establish a robust analytical SEC procedure by evaluating SEC column types and mobile phase compositions using two mAb products with different physiochemical properties. The resulting optimized procedure using a Waters™ BioResolve column exhibits an improved ability to resolve and quantify mAb size variants, highlighting improvement in the resolution of the LMW species. Additionally, the addition of L-arginine as a mobile phase additive showed to reduce secondary interactions and was beneficial in increasing the recoveries of the HMW species.