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Plasmid DNA Purification
Capstone 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
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.
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)
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
Plasmid DNA
Chromosomal DNA
E. coli bacterium
Current Purification Process A “Mini-prep”
Start with bacteria grown
in 96-well plates
Centrifuge
(First separation step)
16
min
Add & mix
Solution 1
6
min
Add & mix
Solution 2
6
min
Add & mix
Solution 3
2
min
Centrifuge
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)
(Second separation step)
Transfer to lysateclearing plate and
centrifuge
18
min
Capture plasmid
DNA
TOTAL
over
1 hour
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
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
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
Streamlining the Process
Start with bacteria grown
in 96-well plates
Centrifuge
(First separation step)
 Replace Centrifugation
Filter
(First separation step)
Add & mix
Solution 1
 Centrifuge is difficult to automate
- Vacuum filtration
Add & mix
Solution 2
 Limited to 14.7 PSIG (atmospheric)
 Uses proprietary filter plates
(plates with filters built in)
Add & mix
Solution 3
- Centrifuge filters
 Uses filter plates
 Same difficulties as centrifugation
Centrifuge
(Second separation step)
Filter
(Second separation step)
Positive pressure filtration
 Attributes of filtration with
no pressure limitations
 Very fast
 Filtration does not need lysate clearing step
Transfer to lysateclearing plate and
centrifuge
Capture plasmid
DNA
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
Filters Tested
Polyether Sulfone
Track Etched
 Precise fiber pore
 Vertically etched pores eliminate
structure
 Low protein binding
 High lateral flow rate
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
Filter Testing
Test Column
 Multiple test columns have been manufactured
 Bacteria is added and pressurized gas is applied
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
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
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
Yield Comparison
DNA Yield Analysis
12000
DNA Yield (ng)
10000
8000
6000
4000
Centrifugation (Control)
Filtration
2000
0
0
5
10
15
20
25
Test Number
DNA Yield (ng)
5961 8895
6543
9700
10921
8068
7798
8540
8443
7869
6867
6891
8384 6894
8517
8648
7903
9011
8690
7905
7471
6285
7441
Filtration 5867 6466
6540
5648
5781
4666
4959
3643
5496
5418
4859
7764
-
Centrifugation
Mean
Standard deviation
Coefficient of variation
Filtration (ng)
5394.82
835.64
6.46
Centrifugation (ng)
7975.38
1117.39
7.14
Purity
Filtering
Centrifugation
DNA
Classification
Kilobase
Pairs
23
9.4
6.6
4.4
2.2
2.0
Genomic
nicked
linear
supercoiled
 Supercoiled DNA represents plasmid DNA
 Genomic and Chromosomal DNA trace is negligibly small
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
Filtration Efficiency
 Approximately 70% decrease in process time
0
- Down from 64 minutes to 20 minutes
10
 20 minutes per plate equates to 24 plates per day,
20
- Surpassed our goal of 2,000
30
Current process
Centrifuge
Proposed process
16
Transfer to custom plate
40
4
Add & mix Solution 1
6
Filter
Add & mix Solution 2
6
Add & mix Solution 1
4
Add & mix Solution 3
2
Add & mix Solution 2
6
16
Add & mix Solution 3
2
Centrifuge
Transfer to lysate plate
2
Centrifuge
16
TOTAL
64
Filter
TOTAL
1.5
50
60
2.5
20
Old
New
70
Process Time (minutes)
or 2,304 samples per 8-hour day
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
Component Assembly
Material Selection: Gaskets
 Five elastomeric materials
- Isobutylene-isoprene rubber (IIR)
- Chloroprene rubber (CR)
- Styrene-butadiene rubber (SBR)
- Nitrile rubber (NR)
- 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
 EPDM has been chosen as the gasket material
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
ANSYS Analysis of Clamped Tabs
316L Stainless Steel
Max Displacement: 0.159 e-7 in
 Integrity of design analyzed with 200 lbs (SF=2)
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
Cost Analysis
Cost of consumables for 96-well plate
Primary separation only: isolating bacteria, alkaline lysis, and capturing plasmid
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)
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
Box LTS 1000 pipet tips
Celpure
(none)
20 mg
$0.22
0.2 µm filter
(none)
1
$4.77
Total (approximate, before volume discounts, w/tips)
$18.45
 Automated process would cut consumables cost of
primary separation in half
$9.25
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
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