Scalable purification workflow of plasmid DNA using a novel 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
3
Abstract
Plasmid DNA (pDNA) is a critical raw material for many gene therapy treatments, whether as an intermediate to produce viral vectors, as a template for the IVT manufacture of mRNA, or as the payload of the therapeutic itself. The growth of gene therapy development has resulted in increasing demand for pDNA. Process intensification is critical to serve this need. The challenges of large-scale manufacturing at speed, such as increasing pressure across the chromatography column, can inhibit productivity. To mitigate these problems, we developed processing workflow consisting of a high flow resin with a next generation hydrophobic adsorbent, comprised of a composite electrospun nanofiber with a wide flowpath with no dead ends. Commercial processes generally employ two-step chromatography using ion exchange chromatography (IEX) and hydrophobic interaction chromatography (HIC). Here we demonstrate a two-stage process using pre-packed IEX resin (DEAE PuraBead® HF) as a capture step and a pre-packed hydrophobic nanofiber adsorbent for polish (pDNAHERO®), both at process development and pilot scale.
Polishing with pDNAHERO® 1
Method • Loading conditions for the elution pool from the DEAE capture step: dilution in 4 M ammonium sulphate in TE to give 2.85 - 3 M ammonium sulphate. • Column equilibration: 3 M ammonium sulphate in TE. • Loading: residence time of 12 s (5 mL/min on pDNAHERO® 1). • Elution: target pDNA was eluted by lowering the concentration of ammonium sulphate to 0.6 M (or lower) with TE. This could be done with a step elution to 100% TE to reduce salt concentrations. This polish step removes process related and product related impurities.
Result The pDNAHERO® 1 polishing step completely removed the measurable amounts of RNA remaining in the postDEAE elution pool (the broad peak at 6 minutes). The Capillary electrophoresis analytics from each process step of an 8 kb pDNA sequence, purified by DEAE PuraBead® HF in a 50 mL pre-packed Evolve®R column and pDNAHERO® 1 is shown below.
1
Capture of pDNA on DEAE PuraBead® HF 1 mL column
800 700
500 400 300 200
• This clarified lysate was loaded onto DEAE PuraBead® HF pre-packed columns 0.7 cm ID x 2.5 cm bed height (1 mL column volume) equilibrated with 50 mM Tris pH 8 with 10 mM EDTA (TE), and eluted with 1 M NaCl in TE.
100 0
1
Residence time, minutes 35 mS/cm
2
10 mS/cm
1 mL DEAE binding pDNA at 35 mS/cm and 2 min res’ time 800 180 160 Load
600
Elution from 3 repeats run 1% agarose gel
Flow-through fractions 1% agarose gel
140
500
120
400
100
300
80
200
60
100
40
0 -3
20 17
37
-100
57
77
97 0
mL UV 260
Conductivity
mS/cm
700
mAU
Clarified lysate
• The clarified lysate was treated with CaCl2 to remove RNA and was divided into two samples diluted to either 10 mS/cm or 35 mS/cm using RO water.
600
• The two feedstocks were run in triplicate at 1- and 2-minutes residence time. Loading and recovery were measured by UV 260 spectrophotometry.
Average yield per cycle
St. dev
Recovery
2.33 mg
1.71 mg
0.06, n=2
73%
4
Scaling up: Polishing with pDNAHERO® 100
The above process was scaled up with the pDNAHERO® 100 (100 mL bed volume) on a pilot scale chromatography system capable of running at high flowrates. pDNAHERO® 100 polish
2500
Result
300
• 336 mg of pDNA from the DEAE elution pool was loaded in one cycle.
250
2000
200
1500
150 1000
100
500
50
0
0 -5
0
5
10
15
20
Scaling up: pDNA capture on DEAE PuraBead® HF 15 mL column
Column size (mL)
Residence time (minutes)
pDNA recovered µg/mL
St. dev (µg)
1
1
147.0
2.7
1
2
185.0
5.3
15
3
2920.0
34.6
Conductivity
DNA recovery from pDNAHERO® 100
Method The pDNAHERO® 1 (1 mL bed volume) has a capacity of 3-4 mg of pDNA, so to create a process that size, and scale the resin column appropriately, we combined three 5 mL DEAE PuraBead® HF pre-packed columns and purified the same 4.7 kb sequence, from clarified lysate conditioned to 35 mS/cm post-CaCl2 precipitation in triplicate at 3 minutes residence time.
• 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. • pDNAHERO® 100 was cleaned efficiently with 1 M NaOH in reverse flow for 1 - 2 minutes.
25
Minutes UV 260
2
#
Starting load, nucleic acid
Average yield per cycle
St. dev
Recovery
336 mg
187.5 mg
0.7 n=2
56%
166 mg
125 mg
N/A
75%
#
Optimized load to improve yield
5
Result
Impurity clearance: Post-polish pDNA passes necessary regulatory requirements* Capture
Polish
AEX: DEAE PuraBead®
HIC: pDNAHERO®
Removal of endotoxin, RNA and HCP
This confirms that the 15 mL column capture step is appropriately sized for the subsequent 1 mL polish step.
Process step
Summary
E. coli DNA ug/mg
*
Removal of residual impurities
Endotoxin Eu/mg pDNA
• At process development scale to produce ~1 - 3 mg of purified plasmid (15 mL DEAE PuraBead®, and pDNAHERO® 1).
6.00
17625
11620.68
10.7
0.53
<25
18.72
28.7
Post-pDNAHERO®
0.17
2
2.25
0
Measured by qPCR ~ Measured my NCS endosafe reader LAL kit # Measured by ELISA + Measured by RiboGreen® post-DNASE treatment *
References:
• www.fda.gov Guidance for Industry, Considerations for Plasmid DNA • Vaccines for Infectious Disease Indications • www.ema.europa.eu EMA Guidelines relevant for advanced therapy medicinal products
Any data or results provided are only examples and do not provide any guarantee of similar results in future. The products of Astrea Bioseparations may be covered by or for use under one or more patents: astreabioseparations.com/patents All trademarks, trade names, trade dress, product names and logos are the property of Astrea UK Services Ltd or their respective owners. © 2024 Astrea Bioseparations Ltd. All rights reserved
Residual RNA ug/mL+
Post-DEAE pool
• To process 1 g in a single step, a 500 mL pDNAHERO® could be run at flow rates of 150 L/hr to which would take less than 1 hour of machine time.
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E. coli HCP ug/mg pDNA#
(Post-CaCl2 and dilution for load on DEAE)
• This process can be scaled to produce 100 – 300 mg in one cycle
Next steps
~
Lysate
• We have developed an effective scalable process for pDNA.
• With multiple cycles, 1 g of purified plasmid could be processed on pDNAHERO® 100 in a single shift. Including set up and clean down of the skid.
Post-pDNAHERO® 1 elution
Starting load, nucleic acid
Result Modifying the loading conductivity successfully blocked RNA binding to the DEAE column. 0.9 mg/mL of nucleic acid bound to the DEAE PuraBead® HF at 10 mS/cm but AGE analysis showed that over 50% of the recovered nucleic acid was RNA. At 35 mS/cm however, the RNA did not bind and was present in the flowthrough fraction as shown to the left.
Post-DEAE PuraBead® HF elution DNA recovery from pDNAHERO® 1
mAU
Binding capacity µg/mL
900
-23
Method • A 4.7 kb pDNA sequence was expressed in E. coli and the cells lysed with a typical alkaline method.
1000
mS/cm
Binding capacity for nucleic acids at different residence times