D ESIGN T EAM : Forrest Harrington Alexandre Lessis Ashley Mattison Jason McDermott Michael Zabbo

1. Plasmid DNA PurificationCapstone Design Project Sponsored by: Ed Harlow Harvard Medical School Advised by: Professor Jeff Ruberti Northeastern University DESIGN TEAM: Forrest Harrington Alexandre Lessis Ashley Mattison Jason McDermott Michael Zabbo

2. Problem Statement The Harlow laboratory at Harvard Medical School would like to automate their plasmid DNA purification process to increase throughput, improve purity, and reduce cost. Our focus was to design and demonstrate the feasibility of an improved single unit operation which meets the purity and yield requirements, setting the stage for scale-up to meet the throughput and cost requirements.

3. Harlow Lab and DNA Research • The goal of the Harlow lab is to understand gene function by using shRNA to see the phenotype associated with the loss of gene function. • This is expected to lead to an improved understanding of biochemical processes and ultimately the development of new drugs. • Genes are “transcribed” to create mRNA, which produce proteins. The Harlow lab uses shRNA, a form of RNAi, to prevent the production of the protein (“protein expression”). • Scientists analyze the impact of the loss of the protein. • Up to 100,000 genes are needed to complete one full genome screen • (20,000 genes in human genome) x (5 tests each)

4. Why Plasmid DNA? • In order to have large quantities of each gene to be researched, a gene is inserted into a plasmid DNA, and the plasmid is inserted into an E. coli bacterium, which can be grown easily and quickly • In order to analyze the plasmid, it must be extracted from the bacteria, or “purified” • Meaning 100,000 purifications required per genome screen Gene to be studied Chromosomal DNA Plasmid DNA E. coli bacterium

5. Start with bacteria grown in 96-well plates Centrifuge (First separation step) Add & mix Solution 1 Add & mix Solution 2 Add & mix Solution 3 Centrifuge (Second separation step) Transfer to lysate- clearing plate and centrifuge Capture plasmid DNA Current Purification Process - A “Mini-prep” 16 min • Separate out bacteria in centrifuge • Draw off supernatant (throw away 96 pipette tips) • 3 Step alkaline lysis • Add Solution #1(throw away 96 pipette tips) • Resuspends bacteria • Prevent degradation • Add Solution #2(throw away 96 pipette tips) • Breaks open cell wall to release plasmid • Very alkaline • Add Solution #3(throw away 96 pipette tips) • Neutralizes mixture • Precipitates out everything but plasmid • Very acidic • Separate out plasmid in centrifuge • Draw off plasmid in solution (throw away 96 pipette tips) • Transfer to lysate-clearing plate (throw away first plate) • Centrifuge to clean plasmid • Collect plasmid in standard well plate (throw away lysate-clearing plate) 6 min 6 min 2 min 16 min 18 min TOTAL over 1 hour

6. Current Problems • Time Consuming • 8 hours to do 800 purifications by mini-prep • Cuts into research time • Heavily dependent on human interaction • Constant loading of centrifuges and transferring of liquids • Uses disposable materials • Consumable plastics and chemicals cost about $60,000 per genome screen Disposables used for one run of 96 samples

7. Design Goals • Walk-Away Automation • Streamline the current process to minimize human interaction • Redirects focus from preparation to research • More research can be performed at a lower cost to the lab • Increase throughput to purify 10,000 plasmid DNA samples per week • Lower costs • Reduce disposable materials • Reduce cost per sample by at least 50% • Improve purity of samples • No cell debris in purified sample: only plasmid

8. Constraints • Continue to use 1-2-3 alkaline lysis process • Doesn’t use proprietary methods or expensive chemicals • Use standard dimensions of 96-well plate • Easily integrated with common laboratory robotics • Maintain consistent yield 96-well plate Base is 5” x 3 3/8” Plate picture: www.hamptonresearch.com/products/productdetails.aspx?cid=10&sid=158&pid=453

9. Start with bacteria grown in 96-well plates Centrifuge (First separation step) Filter (First separation step) Add & mix Solution 1 Add & mix Solution 2 Add & mix Solution 3 Centrifuge (Second separation step) Filter (Second separation step) Transfer to lysate- clearing plate and centrifuge Capture plasmid DNA Streamlining the Process • Replace Centrifugation • Centrifuge is difficult to automate • Vacuum filtration • Limited to 14.7 PSIG (atmospheric) • Uses proprietary filter plates (plates with filters built in) • Centrifuge filters • Uses filter plates • Same difficulties as centrifugation • Positive pressure filtration • Attributes of filtration with no pressure limitations • Very fast • Filtration does not need lysate clearing step

10. Filter Requirements • Efficiently remove E. coli bacteria from growth media • Efficiently remove cellular debris allowing passage of plasmid DNA • Low protein binding • Withstand 30 PSIG without damage (assuming proper support) • Chemically compatible with alkaline lysis solutions • Restrict lateral flow through membrane • Low cost • Available in sheet form

11. Filters Tested Polyether Sulfone • Precise fiber pore structure • Low protein binding • High lateral flow rate Track Etched • Vertically etched pores eliminate lateral flow cross-contamination • Extremely accurate pore sizing • Lowest protein binding • Different material options Polyether Sulfone Polycarbonate Track Etched Polyester Track Etched Filter pictures: http://www.sterlitech.com/products.htm

12. Multiple test columns have been manufactured Bacteria is added and pressurized gas is applied Filter Testing Test Column

13. Progression of Filtration Testing • Filtration of bacteria from growth media • Initial testing used PES membrane • Clogging of filter was overcome using Celpure a filtration aid • Testing of PCTE membrane cut time down to 90 sec • Pressure and time trends enabled selection of parameters • Filtration of cellular components from plasmid DNA • Track etched membranes were tested • Time and pressure were varied to select parameters • Analysis of yield and purity proved comparable • Start to finish filtration • The two filtration steps were run successfully in series • The use of one filter to accomplish both filtrations succeeded • Filtration was run against centrifugation and analyzed

14. Celpure® Added to Eliminate Clogging • Filter clogging was prevalent • Hindered data collection • Introduced to Celpure® • Powdered filtration aid (diatomaceous earth) • Acts as a pre-filter • P300 Celpure® • Filtering 0.4 – 0.6 µm particles Celpure® Celpure picture: http://www.advancedminerals.com/celpure.htm

15. Progression of Filtration Testing • Filtration of bacteria from growth media • Initial testing used PES membrane • Clogging of filter was overcome using Celpure a filtration aid • Testing of PCTE membrane cut time down to 90 sec • Pressure and time trends enabled selection of parameters • Filtration of cellular components from plasmid DNA • Track etched membranes were tested • Time and pressure were varied to select parameters • Analysis of yield and purity proved comparable • Start to finish filtration • The two filtration steps were run successfully in series • The use of one filter to accomplish both filtrations succeeded • Filtration was run against centrifugation and analyzed

16. Yield Comparison

17. Purity Filtering Centrifugation Kilobase Pairs 23 9.4 6.6 4.4 2.2 2.0 DNA Classification Genomic nicked linear supercoiled • Supercoiled DNA represents plasmid DNA • Genomic and Chromosomal DNA trace is negligibly small

18. Filtration Parameters • First Filtration: Extract bacteria from growth media • 0.2µm Polycarbonate track etched membrane • 10mg Celpure per well • 30 PSIG • 90 seconds • Second Filtration: Remove cellular debris • 0.2µm Polycarbonate track etched membrane • 10mg Celpure per well • 30 PSIG • 30 seconds • Uses same filter for both steps Track Etched Membrane http://www.2spi.com/catalog/spec_prep/grease-coated-membrane-filters.shtml

19. Filtration Efficiency • Approximately 70% decrease in process time • Down from 64 minutes to 20 minutes • 20 minutes per plate equates to 24 plates per day, or 2,304 samples per 8-hour day • Surpassed our goal of 2,000 Current process Proposed process

20. Breakthroughs in Filtration Testing Testing has proved that : • Two different filtration processes are possible with the use of one filter • Celpure, a cheap and simple to add solution, is capable of preventing clogging at all stages of filtration • Filtration has decreased the purification process time by 70%, or 44 minutes • Filtration is equally comparable to centrifugation in both DNA yield and purity

21. Designing a Reusable Plate • Design a custom reusable 96-well plate to allow for more efficient fluid handling and to reduce the cost of consumables • Similar to a filter plate, but with replaceable filters • Need to seal each well properly to prevent cross-contamination

22. 96 Piston Design

23. Single Piston Design

24. Pressurized Air Supply Pressurized air inlet

25. Component Assembly

26. Component Assembly

27. Material Selection: Gaskets • Five elastomeric materials • Isobutylene-isoprene rubber (IIR) • Chloroprene rubber (CR) • Ethylene-propylene diene monomer rubber (EPDM) • Important material properties • Chemical compatibility • The chemicals used in alkaline lysis can be very harmful to elastomers • Erosion resistance • The repeated usage during automation degrades material properties over time • Compression set • Gas impermeability • -Styrene-butadiene rubber (SBR) • - Nitrile rubber (NR) • EPDM has been chosen as the gasket material

28. Compression Requirement Fb = (π/4)G2P + 2b πGmP Fb ≈ 400 lbs Fb = total load for operating conditions • Using a pressure of 30 PSIG • Takes into account the compression needed to seal the interface as well as containing the hydrostatic end force • Four securing points on assembly • 100 lbs force on each securing point

29. ANSYS Analysis of Clamped Tabs • Integrity of design analyzed with 200 lbs (SF=2) 316L Stainless Steel Max Displacement: 0.159 e-7 in

30. Leak Rate Analysis • Finish of custom well plate would be made with tolerance of +/- 0.001 in • Low amount of leak due to • Open area of filter 99.982% of total flow area • Distance to be filtered • Across filter is = 0.0127mm • Along leak path = 0.991 mm • For the 1.5 ml filtered • Flow across filter = 1.4997 ml • Flow through leak path = 0.263 µl

31. Cost Analysis Cost of consumables for 96-well plate Primary separation only:isolating bacteria, alkaline lysis, and capturing plasmid • Automated process would cut consumables cost of primary separation in half

32. Future Work • Final, toleranced prototypes will be manufactured/machined to specifications • Testing will be performed on the 96 through hole filtration assembly • Consistency among wells • Cross-contamination • Leaks • A thorough integration into an automated sequence

33. Accomplishments • Testing showed that filtration is very plausible for an automated process • The same 0.2 µm filter can be used in both separation steps • Track etched membranes speed up flow and lessen the chance for cross-contamination • Celpure® was found to nearly eliminate clogging • Process time was reduced by 70 %, providing the potential for throughput of at least 2,304 samples per 8-hour day • Goal was 2,000 per day • Consumables costs can be cut in half • Labor dramatically reduced, also • Designed an assembly which is automatable • Calculations showed gaskets will seal properly