Plasmid DNA process intensification with an innovative nanofiber adsorbent C. Childers, E. Pawlowska, K. Moulson, B. Galarza, J. Fletcher, C. Daye, M. Hummersone, and I. Scanlon Astrea Bioseparations, Horizon Park, Barton Road, Comberton, Cambridge, CB23 7AJ, UK
4
Abstract
To meet the need for increased manufacturing capacity of quality plasmid DNA (pDNA), Astrea Bioseparations has developed a novel adsorbent, pDNAHERO®, that removes common size and pressure limitations. Unlike conventional chromatography matrices, pDNAHERO® has an electrospun nanofiber structure, consisting of an expansive flowpath with no dead ends to trap product and reduce flow. This not only allows for increased processing speeds but also can also facilitate the processing of larger plasmids (tested with as large as 34 kb).
Purity and recovery of linearized DNA DNA mg/mL*
mL
Total DNA (mg)
Pre-linearization
0.19
21
4.0
HIC-adsorbent elution pool
0.68
5
3.4
Here we demonstrate a scalable pDNA manufacturing process that supports a high quality of impurity clearance in a condensed timeframe. Data from 1 mL development-scale units will be compared to 100 mL process-scale units to demonstrate robust impurity clearance and processing times no matter the scale of the run. The flexibility of this process will also be highlighted through a discussion of linear plasmid DNA processing to meet the demand for mRNA manufacturing. We anticipate this nanofiber adsorbents will revolutionize the speed of pDNA manufacturing to provide CDMOs another well-defined pathway for pDNA production and further support the demand for novel therapies that rely on the supply of pDNA, such as RNA, viral vectors, and other advanced therapy medicinal products.
Process step: Capture
0.025
600
0.02
400
0.015
200
0.01
0
0.005 100
150
200
Elution pool
30
5
0.15
250
0
pDNAHERO®100 polish
2.5 2
200
1.5
150
1
100
0.5
50
0
0 18000
0
2000
4000
6000
8000
10000
12000
14000
16000
Protein measured by Bradford total protein
2000
200
1500
150 1000
100
500
50
0
0 -5
• 336 mg of pDNA from the DEAE elution pool was loaded in one cycle.
250
250
Volume (mL)
Removal of protein: 99%
Result
300
mS/cm
800
50
24
2500
Conductivity (mS/cm)
0.03
Delta column pressure (MPa)
0.035
1000
0
Total protein (mg)
30
Polishing with pDNAHERO®100
AVV helper 11 kb primary capture on pDNAHERO®100 UV (AU)/Delta pressure (bar)
UV (mAU)/Conductivity (mS/cm)
1200
Chrom.1 UV 2_260 mAU
mL
805
The pDNAHERO®100 (100 mL bed volume) on a pilot-scale chromatography system effectively reduced impurities at high flow rates.
pDNAHERO® allows fast processing times with low operating pressure with increasing scale
-200
Protein (µg/mL) Linearization mixture
5
AVV helper 11 kb primary capture on pDNAHERO®1
*Based on densitometry
Process step: Polish
mAU
1
Yield of linearized DNA: 85%
0
5
10
15
20
• pDNAHERO®100 was cleaned efficiently with 1 M NaOH in reverse flow for 1-2 minutes.
25
Minutes UV 260
• Due to limit of the system pump, this example run was carried out at ~18 s residence time. The run time was still less than 35 minutes in total, including cleaning and re-equilibration.
Conductivity
Volume (mL)
Chrom.1 Cond mS/cm
Chrom.1 DeltaC pressure MPa
dP1
UV 260(AU)
UV 280(AU)
C2 (mS)
Adsorbent size
System
Flow rate
Running time
pDNAHERO®1
AKTA Avant 150
5 mL/min
60 minutes
pDNAHERO®100
Watson Marlow peristaltic pump and single use* PendoTECH UV, conductivity, and pressure sensors
500 mL/min
40 minutes
6
High level of process-related impurities are removed Process step
E. coli DNA ug/mg *
Endotoxin Eu/mg pDNA ~ E. coli HCP ug/mg pDNA#
Residual RNA ug/mL+
Lysate
2
(Post-CaCl2 and dilution for load on DEAE)
6.00
17625
11620.68
10.7
Post-DEAE pool
0.53
<25
18.72
28.7
Post-pDNAHERO®
0.17
2
2.25
0
DNA recovery is maintained across scales DNA recovery from pDNAHERO 1
*
®
+
Starting load, nucleic acid
Average yield per cycle
St. dev
Recovery
2.33 mg
1.71 mg
0.06, n=2
73%
Measured by qPCR Measured my NCS endosafe reader LAL kit Measured by RiboGreen® post-DNASE treatment ~
7
DNA recovery from pDNAHERO®100 Starting load, nucleic acid
Average yield per cycle
St. dev
Recovery
166 mg
125 mg
N/A
75%
Final sc pDNA purity 95%
95
12
90
10
8 6
1
2
3
4
80 75
4
70
2
65
0
60 DEAE elution pDNAHERO®100 elution
5
GMP-production scale
Chromatogram oflinear linearDNA DNApurification purification Chromatogram of UV 2_260
0.08
mS/cm
350
0.07
300
0.06
250
0.05
200
0.04
150
0.03
100
0.02
50
0.01
Pressure (MPa)
0.09
400
0
0 50
100
150
200
mL
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Conclusions
• pDNAHERO®1 to produce 1-3 mg per cycle • pDNAHERO®10 to produce 10-30 mg per cycle • pDNAHERO®100 to produce 100-300 mg cycle
• The enzymes and protein used in the linearization are removed in the flow-through.
450
Lysate
Analytical methods: Isoforms measured by CIMAC QA monolith on analytical HPLC
Process-development scale
• Bound DNA was eluted using a step to TE.
UV 1_280
4
0 DEAE elution pDNAHERO®100 elution
2: Purified pDNA (mixture of sc/oc)
5: Elution from HIC adsorbent
0.1
6
DEAE elution
pDNAHERO®100 elution
3x purification runs using pDNAHERO 100 based on 3x 1 kg paste lysis batches from a single 100 L fermenter
4: Flow-through from HIC adsorbent
500
8
2 Lysate
3: DNA post-linearization with HIND III
• The purified plasmid was buffer exchanged into RO water; 4 mg was linearized with HIND III, adjusted to 2.25 M ammonium sulphate, and bound to the HIC adsorbent.
0
85
% isoform
10
1: Ladder
• pDNAHERO®1 (1 mL of adsorbent) produced 6 mg of a 6 kb plasmid sequence from E. coli lysate.
UV (mAU) conductivity (mS/cm)
14
12
®
AGE analysis of linearization reaction
METHOD
Multimer pDNA
100
% isoform
% isoform
Purifying linearized plasmid on pDNAHERO
®
sc pDNA
14
Lysate
3
Measured by ELISA
Product-related impurities removed from 8 kb pDNA process oc pDNA
Process: Preparation of linearized plasmid for mRNA production
#
MPa
• All steps run at 5 mL/min, including 0.5 M NaOH CIP • Total process took 45 minutes • Delta column pressure remains below 0.2 bar through the process
• Produce 1 g of purified plasmid, with multiple (6-8) cycles (pDNAHERO®100) in a single shift due to low process time