(1216-A) An automated plasmid purification protocol – avoiding ancillary equipment and reducing manual intervention.
Monday, February 5, 2024
12:00 PM – 1:00 PM EST
Location: Exhibit Halls AB
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.