Using continuous chromatography methodology to achieve high-productivity and high-purity enrichment of charge variants for analytical characterization.

Charge variants of biological products, such as monoclonal antibodies (mAbs), often play an important role in stability and biological activity. Characterization of these charge variants is challenging, however, primarily due to the lack of both efficient and effective isolation methods. In this work, we present a novel use of an established, high productivity continuous chromatography method, known as multi-column counter-current solvent gradient purification (MCSGP), to create an enriched product that can be better utilized for analytical characterization. We demonstrate the principle of this separation method and compare it to traditional batch HPLC (high performance liquid chromatography) or FPLC (fast protein liquid chromatography) methods, using the isolation of charge variants of different mAbs as a case study. In a majority of cases, we are able to show that the MCSGP method is able to provide enhanced purity and quantity of samples when compared to traditional fractionation methods, using the same separation conditions. In one such case, a sample prepared by MCSGP methodology achieved 95% purity in 10 hours of processing time, while those prepared by FPLC and HPLC achieved purities of 78% and 87% in 48 and 300 hours of processing time, respectively. We further evaluate charge variant enrichment strategies using both salt and pH gradients on cation exchange chromatography (CEX) and anion exchange chromatography (AEX) resins, to provide more effective separation and less sample processing following enrichment. As a result, we find that we are able to utilize different gradients to change the enrichment capabilities of certain charged species. Lastly, we summarize the identified mAb charge variants used in this work, and highlight benefits to analytical characterization of charge variants enriched with the continuous chromatography method. The method adds a new option for charge variant enrichment and facilitates analytical characterization of charge variants.

Mechanistic insights into viral clearance during the chromatography steps in antibody processes by using virus surrogates.

Viral safety is required for biological products to treat human diseases, and the burden of inactivation and or virus removal lies on the downstream purification process. Minute virus of mice (MVM) is a nonenveloped parvovirus commonly used as the worst-case model virus in validation studies because of its small size and high chemical stability. In this study, we investigated the use of MVM-mock virus particle (MVP) and bacteriophage ΦX174 as surrogates for MVM to mimic viral clearance studies, with a focus on chromatography operations. Based on structural models and comparison of log reduction value among MVM, MVP, and ΦX174, it was demonstrated that MVP can be used as a noninfectious surrogate to assess viral clearance during process development in multiple chromatography systems in a biosafety level one (BSL-1) laboratory. Protein A (ProA) chromatography was investigated to strategically assess the impact of the resin, impurities, and the monoclonal antibody product on virus removal.