Fast and Scalable Separation of Antibody Aggregates Using Cuboid Size Exclusion Chromatography

The presence of aggregates is highly undesirable in monoclonal antibody formulations. Antibody aggregates are typically associated with toxicity and undesirable immune reactions. Size exclusion chromatography (SEC) is widely used to analyze antibody samples to determine their aggregate content. However, SEC is not commonly used for the preparative separation of antibody aggregates. The main reasons for this are low speed and low loading capacity and hence low productivity and poor scalability with currently available equipment. Currently used aggregate removal techniques, such as ion exchange and hydrophobic interaction chromatography, are sensitive to solution conditions, such as buffer type and salt concentration, and this may restrict their usage only to certain stages in the purification process. SEC, which is less sensitive to the buffer type, might have some advantage here. In a recent paper, the use of cuboid SEC devices for increasing speed and scalability in SEC-based separations has been proposed. In this paper, we discuss the use of a similar cuboid SEC device for the preparative separation of antibody aggregates. This device allowed SEC-based separation to be carried out using a significantly lower bed height and greater cross-sectional area than that possible with conventional cylindrical columns. In this study, the cuboid SEC device was challenged with high loading volumes of concentrated antibody samples, and the separations were carried out at high flow rates. The effects of flow rate, sample loading volume, and concentration were systematically studied using a polyclonal human immunoglobulin sample with high aggregate content. The suitability of using this technique for separating monoclonal antibody (mAb) aggregates from monomers was then demonstrated. This cuboid SEC method would make it possible to remove mAb aggregates at stages in the purification process where currently used techniques may not be suitable.