IMCS Poster Presentation Downloads
(1216-A) An automated plasmid purification protocol – avoiding ancillary equipment and reducing manual intervention
Abstract: Adoption of automated liquid handler workflows has become a primary means to address the ever-increasing demand for the new biotherapeutics. Plasmid purification remains a critical step in the development of novel therapeutics, initial product development, and enzyme engineering. A traditional approach for plasmid purification is to use silica columns in either an individual spin column format or in a 96-well plate. These approaches work well, but require frequent manual intervention, and sufficient centrifuge space. Throughput is limited by time, centrifuge space, and balance requirements. In this work, a pipette-based dispersive solid phase extraction (dSPE) is utilized to purify plasmid DNA. While this functionality exists on automated liquid handlers in the form of packed column beds, magnetic bead purifications, and positive-pressure manifold plates, the workflow presented herein requires no additional off-deck steps, is fully automated, and provides comparable yields and quality. By using a loosely packed resin, we aimed to avoid the pitfalls of other automated methods such as channeling (packed beds and positive pressure manifolds), difficult liquid handling and resultant poor washing (magnetic beads), and on-deck manipulations such as plate movement, centrifugation, and shaking (all methods).
We systematically purified pDNA from bacterial lysates containing plasmids of different sizes (3262 – 8484 bps) using our automated method employing loosely-packed silica tips. The effectiveness of our method was benchmarked against commercially available spin plates and magnetic bead kits. Our method demonstrated higher overall recoveries when compared to magbeads and comparable yields when compared to the manual spin plate, yielding pDNA amounts between 8.92±0.62 µg and 12.28±0.24 µg. Moreover, the resulting purity compared well to alternative methods, with 260/280 and 260/230 ratios of 1.85 and 2.03 across all samples.
Overall, yields and purity were comparable to the spin-plate method. Time from sample isolation to purification was under one hour for up to 96 samples, comparable to the spin-plate method and faster than the magbead workflow (75 minutes). This work represents an adaptation of a traditional plasmid purification workflow for automated liquid handlers. Challenges such as carryover and buffer selection when the purification is automated in pipette tips are identified and how these challenges were overcome are discussed. The workflow can process 96 samples quickly (<60 minutes), while maintaining high yields (>10 µg) and excellent purity.
(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.
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