Completely Automated Plasmid Prep & Optimized Protein Separation Using

PhyTip Columns Shadie Nimri, Carrie Huynh, Lee Hoang, Chris Suh, Doug Gjerde, PhyNexus Inc., San Jose CA

Introduction Characterization of therapeutic candidates requires that proteins are well purified and enriched post expression. The process requires scale up to sufficient quantities of material and processing in a time consuming manner using expensive chromatography equipment. As developments in functional and analytical assays increase throughput and reduce the amount of protein required for analysis, efficient small-volume purification would provide high-value information to researchers in earlier stages of drug discovery and development. Recent advances in the area of miniaturized high-throughput tools for protein enrichment and desalting eliminate bottlenecks associated with traditional techniques. With high-performance, parallel small scale separations, it is now possible to obtain more data, with less sample and in a completely automated format. Demonstrating the versatility of the technology, its use is also compatible with plasmid purification. While many of

the concepts used to automate protein purification are relevant for plasmid DNA purification from bacteria cell

pellets (miniprep), new challenges arise. Complete, walk-away automation 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 centrifugation, this strategy is not feasible for

automation. Processing the samples in the presence of these particulates remains the major challenge to any

automation strategy. The technology presented here show a new strategy for automating mini, midi, maxi, mega and

giga preps that are robust and reproducible, yielding low endotoxin DNA suitable for transfections and sequencing.

PhyTip® Columns: Design and Operation The PhyTip® column has been designed to provide high-performance protein plasmid purification in a format that

allows for complete automation while maintaining a high level of control over the separation process. The high

capacity disposable micro-columns are confined within the body of plastic pipette tips by encasing the resin between

two inert screens situated at the end of the tips. The unique design contributes virtually no dead volume to the

column, resulting in extremely efficient processing of small sample volumes. In addition, the low backpressure of

the columns facilitates processing using a low-cost automated pipetting system.

Applications

Sample is introduced to the column at the end of the

tip. The sample is cycled back and forth multiple times

until equilibrium is reached. We call this Dual-Flow

Chromatography. Flow-through, wash and elution

fractions are retained in the original discrete wells

throughout the process allowing for sample tracking

and process monitoring. PhyTip columns are

compatible 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 transfection and DNA sequencing Plasmid DNA encoding GFP was purified using Lysate Direct PhyTip columns. Samples were tested for transfection efficiency as well as sequencing quality. 50 ng of plasmid DNA were transfected into COS7 cells using three different transfection reagents (Fugene 6, Fugene HD, and TransIT LT1). Transfections were carried out as per manufacturers’ suggested protocol. Ninety six hours after transfection, GFP positive 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 software resulted in a 784 continuous read length, of which 754 base pairs had a QV ≥ 20. (Top) Sequence peaks from 275 to 336 bases are shown. (Bottom) 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 tip 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 purification and

allow for the automation of mini preps on

various liquid handling instruments; midi on

the PhyNexus MEA; maxi, mega, and giga

preps on the Autoplasmid MMG.

Process Development & Optimization Small scale columns using less sample to test

up to 96 conditions in parallel for process

development.

24 conditions (capture, wash, elution) tested for each of 4

clones. Yield measured by ELISA; activity measured by SPR.

Sample Prep for Protein Analytics Reproducible results for CE-SDS-LIF

of rMabs that are comparable to

the manual NAP-5 with 10 times

less sample.

**Data courtesy of Will McElroy, Genentech,

“Automation of Sample Preparation for CE-LIF of

rMAbs with a Robotic Purification System,”

CASSS, 2006

Micro-scale purification columns provide high-performance separation from small sample volumes eliminating the requirement to scale-up sample preparation procedures, resulting in reduced sample consumption and time associated with previous purification methods.

The availability of multiple column bed sizes provides a range of capacities suitable for delivering purified protein

samples suitable for a wide range of downstream applications including analytical, functional, and structural studies. The automated process can be set up to purify proteins from sample sizes ranging from 100 µL to 50 mL.

The versatility of the PhyTip columns allows automation of plasmid mini, midi, maxi, mega, and giga preps.

Generating sequencing and transfection 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 preparation facilitated by the PhyNexus MEA purification system for oligosaccharide mapping of glycoproteins,” Analytical Biochemistry, (2007) 369(2), 202-9

Summary