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 1
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.
Method
A 4.7 kb pDNA sequence was expressed in E. coli and the cells lysed with a typical alkaline method.
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.
• 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.
The two feedstocks were run in triplicate at 1- and 2-minutes residence time. Loading and recovery were measured by UV 260 spectrophotometry.
Scaling up: pDNA capture
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.
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).
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can be scaled to produce 100 – 300 mg in one cycle
• 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. Next steps
• 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.
Result
• 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.
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
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.