Keeping pace with the increasing demand for high quality drug candidates in pharmaceutical research: Development of a new two-step preparative tandem high performance chromatographic system for the purification of antibodies

Abstract

In the area of biological drug development, high throughput (HT) technologies are key to identifying the most promising therapeutic candidate in a time-efficient and market-competitive manner. While efficient cloning and expression methods exist, HT downstream processing mainly relies on liquid handling workstations applying miniaturized chromatography columns or resin-based 96-well plates to shorten process development time. However, there is still a lack of generic, preparative chromatographic methods devoid of aggregates and endotoxins with sufficient throughput. The only truly generic antibody purification strategy including an efficient dimer removal consists of Protein A capture followed by size exclusion chromatography (SEC) as a polishing step. Other polishing methods, including IEX, HIC, and CHT, require an antibody-specific fine tuning. However, standard preparative SEC setups tend to be rather time-consuming, and so limit throughput.In this work, we devised a unique chromatography setup enabling an unattended two-step purification of IgGs on the milligram scale directly from 35 mL clarified cell supernatants, processing up to 48 samples in 44.0 h. By introducing a silica-based SEC column, preparative SEC could be accelerated. By further developing an HT two-step preparative Protein A/alternating column regeneration SEC system using Agilent 1260 Infinity LC components, mAbs can be purified generically by two chromatographic steps in 55 min. In this way, by using a 2-position/10-port valve and two quaternary pumps, two SEC columns can be run in parallel, excluding the cleaning and equilibrating phase from the actual cycle time. By further applying a third pump, the Protein A step can be run independently, resulting in a time-optimized process nesting. By introducing a CETAC ASX-520 autosampler, 48 samples can be run automatically without any user intervention over two working days. The developed system is highly reproducible for all tested human IgG1 antibodies, easily generating milligram scale material sufficient for full characterization of the antibodies and for their use in in vitro and in vivo activity assessments.