Completely Automated Plasmid Prep & Opmized Protein Separaon Using PhyTip Columns Shadie Nimri, Carrie Huynh, Lee Hoang, Chris Suh, Doug Gjerde, PhyNexus Inc., San Jose CA Introducon Characterizaon of therapeuc candidates requires that proteins are well purified and enriched post expression. The process requires scale up to sufficient quanes of material and processing in a me consuming manner using expensive chromatography equipment. As developments in funconal and analycal assays increase throughput and reduce the amount of protein required for analysis, efficient small-volume purificaon would provide high-value informaon to researchers in earlier stages of drug discovery and development. Recent advances in the area of miniaturized high-throughput tools for protein enrichment and desalng eliminate bolenecks associated with tradional techniques. With high-performance, parallel small scale separaons, it is now possible to obtain more data, with less sample and in a completely automated format. Demonstrang the versality of the technology, its use is also compable with plasmid purificaon. While many of the concepts used to automate protein purificaon are relevant for plasmid DNA purificaon from bacteria cell pellets (miniprep), new challenges arise. Complete, walk-away automaon requires capturing plasmid DNA from a complex cell lysate composed of cell debris, precipitated proteins and genomic DNA. While the major strategy for low-throughput, manual miniprep has been to clear the sample by centrifugaon, this strategy is not feasible for automaon. Processing the samples in the presence of these parculates remains the major challenge to any automaon strategy. The technology presented here show a new strategy for automang mini, midi, maxi, mega and giga preps that are robust and reproducible, yielding low endotoxin DNA suitable for transfecons and sequencing. PhyTip ® Columns: Design and Operaon The PhyTip ® column has been designed to provide high-performance protein plasmid purificaon in a format that allows for complete automaon while maintaining a high level of control over the separaon process. The high capacity disposable micro-columns are confined within the body of plasc pipee ps by encasing the resin between two inert screens situated at the end of the ps. The unique design contributes virtually no dead volume to the column, resulng in extremely efficient processing of small sample volumes. In addion, the low backpressure of the columns facilitates processing using a low-cost automated pipeng system. Applicaons Sample is introduced to the column at the end of the p. The sample is cycled back and forth mulple mes unl equilibrium is reached. We call this Dual-Flow Chromatography. Flow-through, wash and eluon fracons are retained in the original discrete wells throughout the process allowing for sample tracking and process monitoring. PhyTip columns are compable with liquid handling robots from PhyNexus, Hamilton, Tecan, Beckman, Dynamic Devices, Agilent, Perkin Elmer and Rainin PureSpeed. The chromato- graphic media is contained between two inert hydrophilic frit screens Transient transfecon and DNA sequencing Plasmid DNA encoding GFP was purified using Lysate Direct PhyTip columns. Samples were tested for transfecon efficiency as well as sequencing quality. 50 ng of plasmid DNA were transfected into COS7 cells using three different transfecon reagents (Fugene 6, Fugene HD, and TransIT LT1). Transfecons were carried out as per manufacturers’ suggested protocol. Ninety six hours aſter transfecon, GFP posive cells were counted and mean fluorescence calculated for each method using an IncuCyte instrument. Sequencing was carried out on an ABI 3730xl. Sequence was analyzed using Sequence Scanner version 1.0 soſtware resulted in a 784 connuous read length, of which 754 base pairs had a QV ≥ 20. (Top) Sequence peaks from 275 to 336 bases are shown. (Boom) Sequence peaks from 544 to 606 bases. **Data courtesy of K. Billeci and T. Di Ioia Salvador , Genentech. PhyTip columns shown in 200 µL & 1000 µL p size: 5, 20, 10, 20, 40, 80, 160 and 320 µL resin beds Dual flow chromatography can also be applied to the process of plasmid purificaon and allow for the automaon of mini preps on various liquid handling instruments; midi on the PhyNexus MEA; maxi, mega, and giga preps on the Autoplasmid MMG. Process Development & Opmizaon Small scale columns using less sample to test up to 96 condions in parallel for process development. 24 condions (capture, wash, eluon) tested for each of 4 clones. Yield measured by ELISA; acvity measured by SPR. Sample Prep for Protein Analycs Reproducible results for CE-SDS-LIF of rMabs that are comparable to the manual NAP-5 with 10 mes less sample. **Data courtesy of Will McElroy, Genentech, “Automaon of Sample Preparaon for CE-LIF of rMAbs with a Roboc Purificaon System,” CASSS, 2006 Micro-scale purificaon columns provide high-performance separaon from small sample volumes eliminang the requirement to scale-up sample preparaon procedures, resulng in reduced sample consumpon and me associated with previous purificaon methods. The availability of mulple column bed sizes provides a range of capacies suitable for delivering purified protein samples suitable for a wide range of downstream applicaons including analycal, funconal, and structural studies. The automated process can be set up to purify proteins from sample sizes ranging from 100 µL to 50 mL. The versality of the PhyTip columns allows automaon of plasmid mini, midi, maxi, mega, and giga preps. Generang sequencing and transfecon quality DNA. Sample Prep for Glycan Profiling HPLC data showing reproducibility of PhyTip columns and comparison with nylon filter using porcine thyroglobulin, LU, luminescent units **Prater et al., “Automated sample preparaon facilitated by the PhyNexus MEA purificaon system for oligosaccharide mapping of glycoproteins,” Analycal Biochemistry, (2007) 369 (2), 202-9 Summary