(1038-C) Fully automated size exclusion chromatography workflows for biomolecules with Dynamic Devices Lynx, for sample volumes of 100 µL up to 1 mL

Abstract: Size exclusion chromatography (SEC) is vital in biotechnology for separating molecules by size, aiding sample prep through buffer exchange for downstream compatibility, and removing interfering contaminants like salts for subsequent analyses. SEC methods can be manual, using resin-containing spin columns or cartridges, or automated with pre-packed filter plates (96-well plates with resin) loaded by a liquid handler. While both approaches are effective, they have limitations. Automated systems offer a rapid and less labor-intensive option; however, they are more restrictive in terms of sample size and require an off-deck centrifugation step that prevents further integration with other workflows. To address these limitations, IMCS has developed SizeX—a miniaturized SEC column incorporated into a larger pipette tip to accommodate larger sample volumes of up to 1 mL. This new design allows users to load samples via the top of the tips onto a packed resin bed and subsequently push the solution through the resin bed using positive pressure generated by the liquid handler's plunger. By utilizing the pipette tip as a chromatography column, SizeX eliminates the need for centrifuges associated with traditional SEC methods. The objective was to develop an automated, high-throughput SEC workflow with the Dynamic Devices Lynx liquid handling system. This method accommodates sample volumes ranging from 100 µL to 1 mL, making it suitable for a wide range of applications. This workflow consists of a few key steps, including a blowout step to remove the storage buffer, an equilibration step to prepare the resin, sample loading with a dispense step to settle the sample into the resin bed, and a chaser step involving elution buffer loading and a dispense step that yields the final product. Method testing focused on recovering green fluorescent protein (GFP) and IgG at varying concentrations, ranging from 0.1 mg/mL to 1.0 mg/mL, and measuring desalting efficiency using bromophenol blue (BPB) and tartrazine solutions. The average GFP recovery for SizeX100 was 84% ± 3%, and for SizeX1000, 85% ± 4%; for sample sizes of 100 µL and 1 mL, respectively; IgG recoveries were comparable to GFP. Moreover, our desalting experiments showcased salt removal of 98% ± 0.5% and ≥99% for 100 µL and 1 mL samples respectively. Results demonstrated that SizeX enables fully automated, rapid buffer exchange and desalting of purified proteins without using centrifuges or other equipment beyond the liquid handler.