Webinar: Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA

The life science business of Merck KGaA,
Darmstadt, Germany operates as
MilliporeSigma in the U.S. and Canada.
Effective and Efficient Design
of a Downstream Purification
Process for Plasmid DNA
March 24, 2022
Nargisse El Hajjami, Ph.D. Eng.
Senior Consultant, Novel Modalities Bioprocessing Strategy Operationalization
Global Lead for mRNA Technology
Laurens Vergauwen, Eng.
EMEA Process Development Scientist,
Global Vaccine and Viral Therapies PD Focal Point Lead
Thomas Elich, Eng.
Manager MSAT Americas Purification Process Engineering

MilliporeSigma is the U.S. and
Canada Life Science business
of Merck KGaA, Darmstadt,
Germany.

Agenda
1
2
3
Introduction to plasmid DNA - Nargisse
El Hajjami, Ph.D. Eng.
pDNA purification challenges &
considerations - Laurens Vergauwen
Case study - Thomas Elich
4 Summary

Circular double helix DNA molecules, naturally found in bacteria, intracellular replicated
Introduction
What are Plasmids?
MW Size
Plasmid DNA 4 MD* 200 nm**
mAb 0.2 MD* 10 nm**
* Mega Dalton (6 kb Plasmid)
** Dynamic range
Supercoiled plasmid is recognized by regulatory authorities as the most therapeutically effective
Carsten Voß, 2007
CCC form I: covalently closed circles,
supercoiled, fully intact,
wound around itself
▪ Large size 1.5 – 150 kb
▪ Poly anion, highly negatively charged
▪ Sensitive to mechanical stress
▪ Various topological forms
OC form: one strand nicked/broken,
less compact, totally relaxed
Linear form: both strands broken,
free ends
Characteristics
5 Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA - March 2022

The importance of plasmid DNA
The key-role of pDNA in various applications
Viral Vectors Non Viral Vector
In vitro
Transcription
Linear DNA
Plasmid
DNA
mRNA
plasmid
DNA
MRNA Vx & Tx
mRNA in vitro
transcription
- Delivery:
- - Liposomal
- - Nanoparticles
- - Electroporation
Plasmid DNA
Vx & Tx
The 1st DNA-based Vx
approved for Covid19

What is your application for plasmid DNA? (Select all that apply)
1. Viral vectors production
2. Template for mRNA in vitro transcription
3. Plasmid DNA-based vaccines / therapeutics
4. Raw material for other applications
5. Others
Poll Question #1

✓ Certificate of analysis
✓ Shake-flask/high density
fermenter
✓ Alkaline lysis
✓ Chromatographic purification
✓ Master/working cell bank E. coli
✓ Purity (A260/280 1.8-2.0)
✓ >85% supercoiled (HPLC)
✓ Host protein <1% (ELISA)
✓ Host gDNA <1% (PCR)
✓ Endotoxins <10EU/mg (LAL or rabbit
pyrogenicity test)
✓ Full traceability of materials
✓ Stability studies
✓ ISO 5 validated clean rooms in
GMP suites
✓ Complete QA & QC oversight
✓ >90% supercoiled
✓ Host protein & gDNA <<0.1%
✓ BSE/TSE risk analysis (if animal
products used)
✓ Environmental monitoring
Plasmid DNA manufacturing
GMP-LIKE
RESEARCH USE ONLY
GMP
Highly documented
+ use of clean rooms
+ EM
Least documentation, no
endotoxin measurement
Different grades of plasmid DNA are produced

Advantages
 Simple design
 Easy to manipulate/optimize
 Rapid production & formulation
 Reproducible, large-scale
production
 Requires only BL1 safety level
 Temperature stable, long shelf
life
 Different applications
 Attractive market
General considerations
Plasmid DNA potential & limitations
Disadvantages as Tx or Vx
 High production cost
 Poor gene transfer efficiency
 Estimated that for every 1000
plasmid molecules only one
reaches the cell and is
expressed.
 Low immunogenic potential of
bacterial DNA
 Possible insertional mutagenesis
Challenges
 Lack of adequate infrastructure
 Production scale-up efficiency
 Removal of impurities and non
desired pDNA forms need
careful process design
 High concentration needed (mg
level)
 Delivery and protection of DNA
from degradation
 Complex process
Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA
9

Plasmid DNA Manufacturing
Key Process Considerations
Process
Considerations
• Batch Vs Fed-batch
• Plamsid DNA sequence design
• Final cell density
• Cell harvest- Centrifugation Vs TFF
• Pressure/shear
• Cell lysis – physical Vs chemical
treatment
• Volume
• Surface Area
• Pore Size
• Media
• Flow Rate
• Pressure/shear
• Clarification – Depth filtration Vs
centrifugation
• Size exclusion Vs absorption
mechanisms
• Mixing speed
• Pre-treatment steps (Rnase
treatment
• Volume
• Media
• Purification mode
• Nature of contaminants
• Pressure/Shear
• Viscosity
• Final Filtration
• Mixing speed
• Duration of alkaline lysis

Purification Challenges &
Considerations

Typical pDNA process flow
pDNA purification challenges & considerations
Thaw Cells Fermentation Cell Harvest Cell Lysis
Clarification
Concentration
& Diafiltration
(UF/DF)
Concentration
& Diafiltration
(UF/DF)
Purification with
Chromatography
(1-2 steps)
Storage
Sterile
Filtration

Unique challenges of pDNA purification
 Similarity of product and contaminants (genomic DNA (gDNA), endotoxin, RNA, plasmid isoforms)
leads to low resolution separation.
 Feed often highly viscous, complicating downstream processing.
 Shear sensitivity
 Lack of platform process and integrated solutions

Cell harvest - Centrifugation Vs Filtration
 Cost effective at batch volumes
<5 L and >500 L
 Special attention is needed for high
shear generated in large scale
centrifugation
 More process development required
for scale up
Centrifugation Microfiltration Tangential Flow
Filtration (MF-TFF)
 Open-channel, flat-sheet TFF
devices (1000 kD, 0.1 μm, 0.2 μm)
work well in this application.
 Lower capital cost
 Linear scalability
Pellicon® 2 ProstakTM

Bacterial cells
suspended in
starting buffer
Alkaline solution
with detergent is
added to lyse
bacterial cells
Neutralization with
potassium acetate
Cell Lysis - Alkaline lysis is commonly used
Principle
• Alkaline condition plus detergent solubilizes the cell walls and the
alkaline environment denatures gDNA
• Sodium Dodecyl Sulfate (SDS) commonly used as the detergent
• Reaction neutralized with acid after short incubation
Challenges
• Both alkaline and shear from mixing can damage supercoiled
plasmid DNA
• Sudden increase of viscosity will impact mixing efficiency
Solutions
• Optimize mix speed, incubation time, and chemical
concentrations
• Mobius® single-use mixers

Clarification - The complex nature of lysed cells
 Lysis output can be challenging for clarification
− Complex mixture of cell debris, pDNA, genomic DNA, RNA and HCP
− High solid load
− High viscosity
− Alkaline lysis leads to two-phase separation
− Froth phase (top) – cell debris and genomic DNA
− Lysate phase (bottom) – pDNA, RNA, HCP
 Pretreatment can be performed to enhance further clarification,
options include:
− Gravity separation of top and bottom phase (commonly used)
− Centrifugation
− RNase digestion
− Ca2+ precipitation of RNA
Note: When using gravity separation, yield losses up to 20% were reported
Froth phase
Lysate phase

Clarification - Centrifugation Vs Filtration
 Capable of handling high solid load
 May require secondary clarification
 More process development required for
scale up
 Attention: High shear can damage
supercoiled pDNA
Centrifugation Normal flow filtration
 Depth filtration based on size exclusion
and adsorption
 Robust process
 Linear scalable
 Attention: + charged filtration aid may
interact with pDNA

Clarification - Depth filter selection
 Lysate is highly plugging and viscous in nature due to cell debris
− Depth filters with pore structure >0.5 µm rating work well
− Apply low flow rates 100-150 LMH
• Media with lower adsorptive properties are preferred (dependent on media type and density)
Clarisolve®
Depth Filters

TFF - Tangential Flow Filtration
 First TFF step (optional): After clarification and before chromatography
− Concentrate plasmid to reduce loading time and complete buffer exchange for chromatography steps
− Removal of impurities: RNA, small size gDNA fragments and protein
 Second TFF step: After chromatography and before sterile filtration
− Achieve final concentration and exchange into formulation buffer

TFF - Devices and watch outs
 Typical MWCO include 30, 100, 300 kD
− Pellicon® 2 with Biomax® or Ultracel® C screen
− Pellicon® Capsule available in 30 kD Ultracel® membrane
− Single-use format
− Gamma sterilized
− Eliminated carry-over and cleaning validation
 Watch outs
− High viscosity → D or V screen may be needed to reach high concentration
− Shear stress
− Flat sheet devices can be operated at lower flux compared to hollow fiber
Pellicon® Capsule
Pellicon® 2

Chromatography - Common approaches
 Goal: Separate supercoiled (ccc) plasmid from oc-/linear isoforms and residual impurities (HCP, nucleic
acid, endotoxin) by charge, size or hydrophobicity
 Combination of Anion exchange and Hydrophobic interaction
Anion Exchange Chromatography (AEX)
• Applicable for capture, intermediate and polishing
• Weak AEX resins give highest recovery and selective impurity removal
• Separate plasmid from proteins, RNA and gDNA and removing endotoxin
• Separation of plasmid isoforms difficult
Hydrophobic Interaction Chromatography (HIC)
• Works by salt promoted binding (≈ 2.5 M NH4SO4)
• Separate isoforms: Supercoiled pDNA is less hydrophobic than RNA, oc- and linear- plasmid forms
and denatured gDNA
1
2

Membrane Chromatography
• Improved hydraulic performance due to large
convective pores and low bed height
• Large accessible surface for Plasmids enabling for
improved binding capacity (5 - 10 mg/mL)
at very short residence times < 0.2 min
• High productivity due to short cycling time
• Single use format, principally scalable
Chromatography - Two technologies to consider
Resin Chromatography
• Low binding capacity due to large Plasmid size
limiting diffusion into beads restricting binding to the
resin surface only (< 3 mg/mL)
• High pressure drops due to elevated viscosity feed
stream resulting in flow limitations and long
processing times (2-8 min RT typical)
• Re-use needed for economic feasibility
• Flexible installation – pack as much as needed
• Generally better selectivity than membranes
Natrix® Q membrane
Fractogel®
DEAE/DMAE
(wAEX)

pDNA sterile filtration optimization parameters
 Sterile filtration can be challenging due to the large size of pDNA, shear sensitivity, and viscosity.
 Internal data shows that the following parameters are important:
Optimization Parameter Yield Capacity
Product
integrity
Salt concentration X X
Supercoiled pDNA content (purity) X X
Filtration endpoint X
Membrane type – PVDF or PES X – PES X - PES
pDNA concentration X X
Feed flux or pressure X
Higher concentrations → lower
radius of gyration
Higher purity gives better performance
Membrane fouling leads to lower yield
Can affect Plasmid integrity (shear)
Millipore Express® SHC best performer
Needs further investigation

Integrated solutions for pDNA purification

What is your involvement with plasmid DNA manufacturing?
1. CMO that manufactures & sells pDNA.
2. Company that purchases pDNA from CMO for my process.
3. Company that currently purchases pDNA from CMO, but considering to bring in-house.
4. Company that produces pDNA in-house.
5. Just here to learn!
Poll Question #2

Case study
Downstream process development for high
productivity purification of plasmid DNA
Tom Elich1, Jaime De Souza1,2, Herb Lutz3, John Cyganowski4
1. Technical and Scientific Solutions, MilliporeSigma, Burlington, MA
2. Formerly with MilliporeSigma
3. Ambassador to the Future, Center of Excellence MilliporeSigma, Burlington, MA
4. Downstream Customer Applications, MilliporeSigma, Burlington, MA

TFF cell harvest Lysis
Chromatography
Formulation
Final Filter
Storage
Plasmid DNA Case Study
Process Overview
Fermenter Clarification
Salt Addition
• 20 hr fed-batch E.Coli
• ~100 OD600 endpoint
• Dry cell wt: 20- 40 g/L
• Wet cell wt: 100-120 g/L
• 2x conc. to ~200 g/L wet cell wt.
• 2x DF cell wash
• Freeze
• Release plasmid w/NaOH & SDS
• Quench w/K-Acetate
• Phase separate floating debris
• Depth filter bottom layer
• NaCl
• Bind/elute AEX
• Clear RNA, HCP, Endotoxin
• 5x DF buffer exchange

TFF cell harvest Lysis
Chromatography
Formulation
Final Filter
Storage
Fermenter Clarification
Salt Addition
• 20 hr fed-batch
• ~100 OD600 endpoint
• Dry cell wt: 20- 40 g/L
• Wet cell wt: 100-120 g/L
• 2x conc. to ~200 g/L wet cell wt.
• 2x DF cell wash
• Freeze
• Release plasmid w/NaOH & SDS
• Quench w/K-Acetate
• Phase separate floating debris
• Depth filter bottom layer
• NaCl
• Bind/elute AEX
• Clear RNA, HCP, Endotoxin
• 5x DF buffer exchange
Discussed in this presentation – Feasibility at 3L fermenter scale
Process Overview

Objective:
Retain, concentrate, and wash E. coli cells (2 x 0.5µm); permeate water
& impurities
Materials:
1000KD Biomax® polyethersulfone membrane retains 0.018 mm spheres,
V screen for high viscosity & particles
Schematic of a 2-pump TFF system
Methods:
Membrane preparation:
• Water flush: 20L/m2
• Clean-in-place recirculation: 0.2N NaOH, 20L/m2 single pass
• Buffer flush: 20L/m2 10mM Tris, 1mM EDTA, pH 8 (TE)
Cell concentration:
• Tank volume reduction with permeate to waste; cells are retained.
Cell wash
• Constant volume diafiltration adds TE buffer at same rate as permeate removal
Cell recovery
• Drain tank and flush retentate line and flush tank
Cell Harvest by Tangential Flow Filtration (TFF)
• Tangential flow operation provides high capacity
• Permeate pump has higher capacity than TMP control

0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
18.0
20.0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
1.0 1.4 1.8 2.2
Flux
(LMH)
TMP
(psi)
Concentration Factor
TMP Flux
Critical flux testing1
• 0.1 m2 scale down cassette
• Ramp up pump 2 until TMP is unstable
• Critical flux was identified as 20 LMH
• 80% of the critical flux (~16 LMH) for operation
Cell concentration
• TMP increased from 1.5 psi to 4 psi due to
viscosity; flux was reduced to 11 LMH.
• 2.1x concentration factor (volume
reduction) achieved
0
0.5
1
1.5
2
2.5
3
3.5
0 5 10 15 20 25
TMP
(psi)
Time (min)
10LMH
20LMH
15LMH
0
2
4
6
8
10
12
14
16
18
20
0
5
10
15
20
25
0.0 0.5 1.0 1.5 2.0 2.5
Flux
(LMH)
TMP
(psi)
Diavolume
TMP
Flux
Cell wash
• 2 diavolumes (tank volumes)
exchanged with TE buffer
• Wash out impurities (media
components, HCP, nucleic acids, etc.)
Cell Harvest by Tangential Flow Filtration (TFF)
• 2.1x VCF achieved at 13 LMH average flux
• 2 diavolume wash at average flux of 12 LMH
1 Field 1995

Lysis Buffer Screening
1) Lyse: Add lysis buffer (NaOH/detergent) to
suspension in 1:1 volume ratio and incubate
2) Quench: Add neutralization buffer (3M
potassium acetate, pH 5.5) in 1:1 volume
ratio
3) Recover: Centrifuged at 12,000 x g for 30
minutes and 0.45 mm filtered
4) Assay: PAGE or Quant-iT dsDNA assay
Test Matrix
Parameters: [NaOH] added, [detergent added], lysis time
Meacle 2004
NaOH
Concentration
Detergent
Concentration
Incubation Time
0.05 M
0.075 M
0.1 M
0.125 M
0.15 M
1% SDS 0 (negative control)
1 min
5 min
10 min
60 min
Cell Alkaline Lysis- Test Procedures

Cell Alkaline Lysis - Assay Results
Plasmid DNA Case study
5-minute lysis, 1% SDS, variable NaOH
Plasmid
RNA
Operating setpoints for lysis:
• 5-10 min
• 0.1-0.15 M NaOH
• 1% SDS
1. Resuspension
2. 0.15M NaOH, 1% SDS – 5 min
3. 0.125M NaOH, 1% SDS – 5 min
4. 0.1M NaOH, 1% SDS – 5 min
5. 0.075M NaOH, 1% SDS – 5 min
6. 0.05M NaOH, 1% SDS -5 min
1 2 3 4 5
1. 0.1N NaOH, 1% SDS, 0 min
2. 0.1N NaOH, 1% SDS, 10 min
3. 0.1M NaOH, 1% SDS, 60 min
4. Unfiltered control, 0.15M NaOH,
1% SDS, 5 min
5. Ladder
Plasmid
RNA
0.1M NaOH, 1% SDS, variable time
0
10
20
30
40
50
60
70
80
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
ng/uL
dSDNA
in
solution
Sample #
QuantiT dsDNA [ng/uL] – dsDNA
specific
1. 0.150 M NaOH, 1% SDS – 5 min
2. 0.125 M NaOH, 1% SDS – 5 min
3. 0.100 M NaOH, 1% SDS – 5 min
4. 0.075 M NaOH, 1% SDS – 5 min
5. 0.050 M NaOH, 1% SDS – 5 min
6. 0.150 M NaOH, 1% SDS – 1 min
7. 0.125 M NaOH, 1% SDS – 1 min
8. 0.100 M NaOH, 1% SDS – 1 min
9. 0.075 M NaOH, 1% SDS – 1 min
10. 0.050 M NaOH, 1% SDS – 1 min
5 min 1 min
Confirmation by Quant-iT dsDNA assay

• Multi-layer depth filters
• Clarisolve® 60HX: 60 mm nominal, PP material
• Millistak+® C0HC: 2 mm nominal, DE material
• Scalable, single-use “Pod” format
• Use in series with sterilizing filters
• Batch CSTR reactor
• Single-use
• Neutralize & allow
impurities to
flocculate, ~15 min
after lysis & quench
• Pump out bottom
layer without
disturbing
• Millipore Express® SHC
• Cast PES membrane
• Sterilizing grade
• Single-use
33
1. NaOH induced 2-phase
flocculation
2. Depth filtration of bottom phase 3. Sterile filtration
Lysate Clarification

• E. coli lysate, 1% SDS 0.1N NaOH
• Open Clarisolve® 60HX offers low pressure
drop ; turbidity breakthrough at 300 L/m2
• Tighter Millistak+® C0HC filtrate turbidity
<2NTU protects subsequent sterile filter
Filter Train 1
Clarisolve®
60HX
Millipore
Express® SHC
Filter Train 2
Clarisolve®
60HX
Millistak+®
C0HC
Millipore
Express® SHC
0
500
1000
1500
2000
2500
3000
3500
0 100 200 300 400
J
Throughput (L/m²)
(LMH)
Filter Train 1 Filter Train 2
0
1
2
3
4
5
6
7
8
0.0
0.5
1.0
1.5
2.0
2.5
0 100 200 300 400
Turbidity
(NTU)
Pressure
Throughput (L/m2)
(psig)
Filter Train 2
pressure
turbidity
Filter Train 1
pressure
turbidity
Depth Filter Sizing
Constant flow test (Pmax™, Tmax)
Monitor pressure rise & turbidity
Sterile Filter Sizing
Constant pressure test (Vmax™)
Watch flux decay
Lysate Clarification
Filter Train 1: 98% yield, ~2x more sterile filter area
Filter Train 2: 82% yield due to charged C0HC filter

Natrix® Q Chromatography Membrane
Anion Exchange Capture Chromatography
➢ Porous hydrogel chromatography membrane
• A high surface area containing quaternary amine ligand.
• Convective flow path: high capacity at 6 second residence time.
• Open pore structure suitable for large molecule purification.
➢ Disposable device format
• 0.2 mL, 15 mL, 115 mL, 460 mL devices.

AEX Capture Chromatography - Test Overview and Methods
Test 1
No NaCl
added
Test 2
35mM
NaCl
Addition
Test 3
75mM
NaCl
Addition
Step Mobile Phase
Membrane
Volumes
Flowrate
Equil
1M K-Acetate + 150 mM NaCl, pH 5.0
(75 mS/cm)
50 MV 10 MV/min
Load Clarified, sterile filtered lysate pH 5.2
11 mg pDNA/mL
membrane
10 MV/min
Wash
1M K-Acetate + 150 mM NaCl, pH 5.0
(75 mS/cm)
20 MV 10 MV/min
Elute 100 mM Tris, pH 9 + 1M NaCl 50 MV 5 MV/min
CIP 1M NaOH + 2 M NaCl 20 MV 10 MV/min
Impact of NaCl supplementation
on RNA clearance
Capture pDNA while impurities (RNA) flowthrough
Clarified lysate conditions:
• 6.5 kbp pDNA, 24 µg/mL titer. 1.5M K-acetate buffer, pH 5.3, 86.9 ms/cm
• Nucleic acid content: 3.8% pDNA, 96.2% RNA. Endotoxin content: 380,000 EU/mg pDNA
Analytics:
• DNA and RNA content assessed by HPLC (Tosoh DNA-NPR method)1
• Endotoxin content assessed by Charles River Endosafe assay
1 Urthaler 2005

AEX Capture Chromatography - Results
35 mM NaCl supplementation offers best
balance of capacity, purity, recovery:
• Capacity = 8 mg pDNA/mL membrane
• Nucleic acid purity = 77% pDNA
• pDNA recovery = 88%
• Endotoxin content = 3,100 EU/mg pDNA
Can RNA and endotoxin purity be further
improved with an alternative wash strategy?
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0 35 75
NaCl Supplementation (mM)
pDNA Binding Capacity (mg pDNA/mL membrane)
pDNA purity (% of total nucleic acids)
pDNA Recovery
11
8
2
46%
100%
77%
88%
36%
61%
37
Impact of Salt Supplementation on Capacity, Purity, Recovery

AEX Capture Chromatography- Wash Strategy
Step Mobile Phase
Equilibration 1M K-Acetate + 150 mM NaCl, pH 5.0 (75 mS/cm)
Load Clarified, sterile filtered lysate pH 5.2 + 35mM NaCl
Wash 1M K-Acetate + 150 mM NaCl, pH 5.0 (75 mS/cm)
Elute 100 mM Tris, pH 9 + 1M NaCl
CIP 1M NaOH + 2 M NaCl
Step Mobile Phase
Equilibration 1M K-Acetate + 150 mM NaCl, pH 5.0 (75 mS/cm)
Load Clarified, sterile filtered lysate pH 5.2 + 35mM NaCl
Wash 1M K-Acetate + 150 mM NaCl, pH 5.0 (75 mS/cm)
Detergent
Wash
0.1M Tris, 10mM NaCl, + 0.5% detergent, pH 7.5
EDTA Wash 0.1M Tris, 10mM NaCl, + 2mM EDTA, pH 7.5
Elute w/EDTA 100 mM Tris, 1M NaCl + 2mM EDTA, pH 9
CIP 1M NaOH + 2 M NaCl
Nucleic Acid Content Endotoxin Content Cycle Time
Feed Conditions 4% DNA, 96% RNA 380,000 EU/mg N/A
Elution w/ Control wash
(measured from eluate pool) 77% DNA, 23% RNA 3,100 EU/mg 55 min
Elution w/ Detergent wash
(measured from eluate pool) 95% DNA, 5% RNA 500 EU/mg 65 min
Control Wash Detergent Wash
Results
38

Membrane Chromatography Resin Chromatography
AEX Capture Chromatography- Membrane Vs Resin
100 L batch of clarified lysate, 3.6 g pDNA
Binding Capacity 8 g/L
Membrane Volume 0.46 L
Flow Rate 4.6 LPM
Step Time 1.1 hr
Cycles 1 cycle
Productivity 7.3 g pDNA/L/hr
Binding Capacity 3 g/L
Resin Volume 1.18 L
Flow Rate 0.3 LPM
Step Time 9.9 hr
Cycles 1 cycle
Productivity 0.3 g pDNA/L/hr
Natrix® Q membrane is 24x
more productive than resin

What is your level of interest in membrane chromatography for pDNA?
1. Not interested – resin chromatography works well for me.
2. Interested to evaluate for my process.
3. Currently using in my process.
4. Not enough knowledge to comment.
Poll Question #3

0
20
40
60
80
100
120
140
160
180
5 LMM 6 LMM 7 LMM 8 LMM
Permeate
Flux
(LMH)
Critical Flux
0
5
10
15
20
25
30
35
40
45
50
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0.0 1.0 2.0 3.0 4.0 5.0
Flux
(LMH)
TMP
(psi)
Diavolume (X)
Diafiltration
TMP Flux
• Pellicon® 2 cassette retains plasmids; passes water,
ionic and low MW impurities
• 300KD Ultracel® Membranes (regenerated cellulose membrane)
retains ~10KD DNA, C screen for moderate viscosities
• Tangential flow w/permeate pump for high capacity
• Constant volume DF at 4 LMM feed flow with
buffer (10mM Tris, 1mM EDTA, 10mM NaCl, pH 8.0)
• Declining TMP due to reduced viscosity w/ionic strength over
5DV
• 96% yield
• Critical flux: high 110-170 LMH depending on feed flow
for 0.1 m2 test device, pick 75% (80-125 LMH)
• Dilute formulation: no concentration
Diafiltration Buffer Exchange using TFF

Cell harvest
4.0m2 Pellicon® 2
Biomax® MF-TFF 1000 kDa
V-screen
Lysis
0.15N NaOH, 1% SDS
5 min incubation
200L Mobius® mixer
Chromatography
460mL Natrix® Q membrane
1 cycle
Formulation
3.0m2 Pellicon® 2 cassette
Ultracel® membrane UF-TFF
300kDa C-screen
Final Filter
Storage
Proposed Scaled Up Process Flowsheet
Fermenter
100L
Clarify
1.65m2 Clarisolve® 60HX
2x30 in. Millipore Express® SHC
Capsule
Salt Addition
+35mM NaCl
83% yield
3.2 g plasmid

1
pDNA is a rapidly growing
therapeutic modality for
use with viral vectors,
mRNA, gene therapy.
Summary
2
Feasibility of high productivity
pDNA purification was
demonstrated at lab scale:
MF-TFF harvest, alkaline
lysis, depth filter clarification,
chromatography membrane,
and UF formulation. 3
Implementation at larger
scales requires verification
studies for lysis mixing &
flocculation, membrane
module manifolding, etc.
4
Additional purification
steps may be investigated
to enhance performance,
i.e. CaCl2 precipitation,
isoform separation, final
product concentration,
etc.
Acknowledgements
The authors thank Greenlight BioSciences for their assistance in providing valuable discussions,
feedstocks, and assays.
IP Disclaimer/ At the current time, MilliporeSigma is not aware of any potentially relevant third-party patents, however, a full freedom-to-operate analysis has not
been completed and is the responsibility of the party interested to use pDNA for plasmid purification.

References
1. JD Watson, et. al., Recombinant DNA, Scientific American books, 1992
2. M Gagnon, Purification of nucleic acids, BIA separations, Slovenia, 2020
3. DR Latulippe, Biotechnol. Bioeng. 107(2010), 134
4. S Levy, et. al., “Biochemical engineering approaches to the challenges of producing pure plasmid DNA”, TIBTECH, July 2000, Vol 18
5. RW Field, et. al., “Critical flux concept for microfiltration fouling”, J Member Sci, 100(1995)259-72
6. H Lutz, Ultrafiltration for bioprocessing, Woodhead Publishing, Cambridge, UK 2015
7. FJ Meacle, et. al., “Impact of Engineering Flow Conditions on Plasmid DNA Yield and Purity in Chemical Lysis Conditions”, Biotechnol.
Bioeng., 87(2004)
8. MilliporeSigma, Millistak+® Pod Disposable Depth Filter Performance Guide, Lit No. PF1119EN00 Rev. B 2013
9. MilliporeSigma, Depth Filters at a Glance, Lit. No. PB1900EN00, Ver. 3.0, 2017
10. MilliporeSigma, Filter Sizing Methods, Lit. No. AN1512EN00, Rev 3, 2000
11. MilliporeSigma, Plasmid DNA Downstream Process, Lit. No. MS WP7523EN, Ver 0.1, 2021
12. Urthaler et. al., “Improved downstream process for the production of plasmid DNA for gene therapy”, Acta Biochimica Polonica, 2005.

thomas.elich@milliporesigma.com
Laurens.Vergauwen@merckgroup.com
Nargisse.el-Hajjami@merckgroup.com
Thomas Elich
Laurens Vergauwen
Nargisse El Hajjami
Thank You!
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the vibrant M, Mobius, Pellicon, Natrix, Millistak+, Clarisolve, Millipore Express, Biomax, Ultracel, Prostak, Fractogel,
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