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

DESIGN TEAM:Forrest HarringtonAlexandre LessisAshley MattisonJason McDermottMichael Zabbo

Sponsored by:Ed Harlow

Harvard Medical School

Advised by:Professor Jeff Ruberti

Northeastern University

Plasmid DNA PurificationCapstone Design Project

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.

Problem Statement

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)

Harlow Lab and DNA Research

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

Why Plasmid DNA?

Plasmid DNA

Gene to be studied

E. coli bacterium

Chromosomal DNA

Current Purification Process -

A “Mini-prep”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)

16 min

6 min

6 min

2 min

16 min

TOTAL over

1 hour

Centrifuge(Second

separation step)

Centrifuge(Second

separation step)

Capture plasmid DNA

Capture plasmid DNA

Start with bacteria grown in 96-well

plates


plates

Centrifuge(First separation

step)


step)

Add & mix

Solution 1

Add & mix

Solution 1

Add & mix

Solution 2

Add & mix

Solution 2

Add & mix

Solution 3

Add & mix

Solution 3

Transfer to lysate-

clearing plate and centrifuge

Transfer to lysate-


18 min

Current ProblemsTime Consuming

- 8 hours to do 800 purifications by mini-prep

- Cuts into research timeHeavily 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

Design GoalsWalk-Away Automation

- Streamline the current process to minimize human interactionRedirects 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

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 yield96-well plateBase is 5” x 3

3/8”

Plate picture: www.hamptonresearch.com/products/productdetails.aspx?cid=10&sid=158&pid=453

Streamlining the Process

Replace CentrifugationCentrifuge 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 filtrationAttributes of filtration with no

pressure limitationsVery fastFiltration does not need lysate

clearing step

Centrifuge(Second

separation step)

Centrifuge(Second

separation step)

Capture plasmid DNA

Capture plasmid DNA


plates


plates


step)


step)

Add & mix

Solution 1

Add & mix

Solution 1

Add & mix

Solution 2

Add & mix

Solution 2

Add & mix

Solution 3

Add & mix

Solution 3

Transfer to lysate-


Transfer to lysate-


Filter(First separation

step)

Filter(First separation

step)

Filter(Second

separation step)

Filter(Second

separation step)

Filter RequirementsEfficiently remove E. coli bacteria from growth

mediaEfficiently remove cellular debris allowing

passage of plasmid DNALow protein bindingWithstand 30 PSIG without damage (assuming

proper support)Chemically compatible with alkaline lysis

solutionsRestrict lateral flow through membraneLow costAvailable in sheet form

Polyether Sulfone Polycarbonate Track Etched Polyester Track Etched

Filter pictures: http://www.sterlitech.com/products.htm

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

Filter Testing

Multiple test columns have been manufacturedBacteria is added and pressurized gas is applied

Test Column

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

parametersFiltration 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

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 picture: http://www.advancedminerals.com/celpure.htm

Celpure®

Celpure® Added to Eliminate Clogging

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

parametersFiltration 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

Yield Comparison

Filtration (ng) Centrifugation (ng) Mean 5394.82 7975.38

Standard deviation 835.64 1117.39Coefficient of variation 6.46 7.14

DNA Yield Analysis

0

2000

4000

6000

8000

10000

12000

0 5 10 15 20 25

Test Number

DN

A Y

ield

(ng)

Centrifugation (Control)Filtration

DNA Yield (ng)Centrifu-

gation

5961 8895 6543 9700 10921 8068 7798 8540 8443 7869 6867 6891

8384 6894 8517 8648 7903 9011 8690 7905 7471 6285 7441 7764

Filtration 5867 6466 6540 5648 5781 4666 4959 3643 5496 5418 4859 -

Kilobase Pairs

239.46.64.4

2.22.0

Filtering Centrifugation

Purity

Genomic

nicked

linear

supercoiled

DNA Classification

Supercoiled DNA represents plasmid DNAGenomic and Chromosomal DNA trace is

negligibly small

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 stepsTrack Etched Membrane

http://www.2spi.com/catalog/spec_prep/grease-coated-membrane-filters.shtml

Filtration Parameters

Filtration EfficiencyApproximately 70% decrease in

process time- Down from 64 minutes to 20

minutes20 minutes per plate equates to 24

plates per day, or 2,304 samples per 8-hour day

- Surpassed our goal of 2,000

0

10

20

30

40

50

60

70

Process Time (m

inutes)

OldNew

Centrifuge 16

Add & mix Solution 1 6



Centrifuge 16

Transfer to lysate plate 2

Centrifuge 16

TOTAL 64

Transfer to custom plate 4

Filter 1.5




Filter 2.5

TOTAL 20

Current process Proposed process

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

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

96 Piston Design

Single Piston Design

Pressurized Air Supply

Pressurized air inlet

Component Assembly

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 resistanceThe 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

Material Selection: Gaskets

Compression Requirement

Fb = (π/4)G2P + 2b πGmP

Fb ≈ 400 lbs

Fb = total load for operating conditions

Using a pressure of 30 PSIGTakes 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

Integrity of design analyzed with 200 lbs (SF=2)

Max Displacement: 0.159 e-7 in316L Stainless Steel

ANSYS Analysis of Clamped Tabs

Leak Rate AnalysisFinish 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 filteredAcross 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

Cost Analysis

Consumable Current way Proposed process

Qty. Price Qty. Price

Deep-well block for cell growth 1 $2.00 1 $2.00

Lysate clearing microplate (800 ml) 1 $12.40 (none)

Box LTS 1000 pipet tips 0.5 $2.25 0.1 $0.45

Solution I (RNAse) 28.8mL $1.50 28.8mL $1.50

Solution II 28.8mL (negligible) 28.8mL (negligible)

Solution III 28.8mL $0.30 28.8mL $0.30

Celpure (none) 20 mg $0.22

0.2 µm filter (none) 1 $4.77

Total (approximate, before volume discounts, w/tips) $18.45 $9.25

Automated process would cut consumables cost of primary separation in half

Cost of consumables for 96-well plate Primary separation only: isolating bacteria, alkaline lysis, and

capturing plasmid

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

AccomplishmentsTesting 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

cloggingProcess 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 dayConsumables costs can be cut in half

- Labor dramatically reduced, alsoDesigned an assembly which is

automatable- Calculations showed gaskets will seal

properly

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

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