The use of HIC and IEX chromatography in the purification of plasmid DNA

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Technical Note The use of HIC and IEX chromatography in the purification of plasmid DNA Summary Gene therapy, or the introduction of genes into cells to prevent or fight diseases, consists of two components: a vector, for example a viral vector or lipid nanoparticle; and a payload, a custom made nucleic acid sequence. The vector then delivers the payload to patients to achieve a therapeutic effect. The two most common nucleic acid payloads used in gene therapies are messenger RNA (mRNA) and plasmid DNA (pDNA). Plasmid DNA (pDNA) is a fundamental component for the production of the viral vectors themselves, as it is used to code for the production of the particles, and is also linearised to provide a template for the mRNA products. Plasmid DNA (pDNA) are small circular double stranded DNA molecules found in bacteria which aredistinct from genomic DNA and able to replicate separately. Recombinant DNA methods are used to splice genes into plasmids, which subsequently replicate making copies of the spliced gene..Conformations of pDNA include open-circular (oc) pDNA and supercoiled (sc) pDNA. The sc isoform is the choice in DNA vaccination and gene therapy because of stability and antigenicity considerations. It is therefore important to develop purification methods which separate scDNA from ocDNA. Purification of pDNA from bacterial cells is an important step in the process. During the purification of plasmids the bacterial cells are lysed, releasing DNA and cellular components. These cellular components are subsequently removed, and the DNA-containing lysate is further processed to separate the pDNA from the genomic DNA.

Technical note: DEAE and Hexyl PuraBead® HF

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Introduction The use of both hydrophobic interaction chromatography (HIC) and Ion exchange chromatography (IEx) in DNA purification is well documented in the literature. Astrea Bioseparations adsorbent portfolio includes both HIC resins and IEx resins on proprietary PuraBead® HF agarose base matrix. The HIC resins consist of phenyl, butyl, hexy and octyl hydrophobic ligands. IEx resins are both anion exchangers (Q and DEAE) and cation exchangers (SP and CM). This application note details the use of IEx chromatography for purification of plasmid DNA, and the evaluation of HIC PuraBead® HF adsorbents for a pDNA polishing step. In summary, DEAE PuraBead® HF was used for an pDNA primary capture step and Hexyl PuraBead® 6HF used to separate scDNA and ocDNA with a step elution at both 1 mL and 8 mL column scale.

Materials and Methods Optimising pDNA capture using DEAE PuraBead® HF in RoboColumns® A capture step was performed on pDNA feedstock using DEAE PuraBead® HF in RoboColumns®. 15 g of E.Coli expressed pDNA (6.8 Kb sequence coding for an anti-CD19 antibody) was taken through Alkaline Lysis and RNAse treatment to remove RNA. The supernatant was buffer exchanged into four different conditions (EQ buffer with 0, 0.1, 0.2 or 0.3 M NaCl) and loaded in quadruplicate, onto 16 x RoboColumns® packed with 200 µL DEAE PuraBead® HF. • EQ Buffer: 50 mM Tris pH 7.2 10 mM EDTA containing either 0, 0.1, 0.2 or 0.3 M NaCl • Elution 50 mM Tris pH 7.2 10 mM EDTA 600 mM NaCl A BioMek automated handling system was used and the residence time was 1.1 minutes. Elution fractions were desalted and pooled for subsequent pDNA purification. Primary capture of pDNA using DEAE PuraBead HF 5mL pre-packed column 22 mL of E.Coli lysate was buffer exchanged into 50 mM Tris pH 7.2 10 mM EDTA, then loaded onto the 5 mL prepacked PuraBead® DEAE HF column. The column was loaded with 924 μg pDNA/mL of adsorbent, a total load of 4.32 mg pDNA. Buffers Loading: 50 mM Tris pH 7.2 10 mM EDTA Elution: 50 mM Tris pH 7.2 10 mM EDTA 600 mM NaCl Clean in place: 0.5 M NaOH Separation of scDNA and ocDNA using Hexyl PuraBead® HF – 1 mL column Hexyl PuraBead® 6HF was evaluated for the separation of scDNA from ocDNA, with the aim of binding all the pDNA and isolating the scDNA peak during elution.

Technical note: DEAE and Hexyl PuraBead® HF

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The first experiment involved a 0.5 cm diameter 1 mL column of Hexyl PuraBead® HF. Equilibration was performed using 2 M Ammonium Sulphate (Buffer A : 50 mM Tris, 5 mM EDTA, 2 M Ammonium Sulphate pH 8.0) • The total load was 0.23 mg of gWiz pDNA 4.7Kb in Buffer A at a 5-minute residence time. • 100% gradient step to Buffer B (50 mM Tris, 5 mM EDTA pH 8.0) was used to elute the pDNA. • A CIP of 0.5 M NaOH, 30 % Isopropanol was used. Two peaks were collected, desalted and ran on 1 % Agarose gel electrophoresis. Separation of scDNA and ocDNA using Hexyl PuraBead® HF – 8 mL SNAP® column Hexyl PuraBead® HF was packed at 10 cm bed height in 1 cm diameter SNAP® column (8 mL column volume). Buffers used were as follows: • Equilibration Buffer: 50 mM Tris, 5 mM EDTA, pH 8.0, 2 M Ammonium Sulphate • Elution Buffer: 50 mM Tris, 5 mM EDTA pH 8.0 The column was loaded with 0.88 mg of gWiz pDNA 4.7Kb at a 5-minute residence time in equilibration buffer, followed by elution, with fractions collected across the elution peak. The CIP step used 0.5 M NaOH, 30 % Isopropanol. Elution fractions were desalted, pooled and ran on a 1 % agarose electrophoresis gel.

Technical note: DEAE and Hexyl PuraBead® HF

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Results and Discussion Results: Purification of pDNA using DEAE PuraBead® HF – RoboColumns® The table below shows the pDNA loading and recovery at the different conditions tested on the DEAE PuraBead® HF RoboColumns®. Condition EQ buffer + 0M NaCl EQ buffer + 0.1M NaCl EQ buffer + 0.2M NaCl EQ buffer + 0.3M NaCl

Load pDNA (mg/mL resin) 4.77 4.35 4.17 4.33

% Recovery 36 10 13 10

1 % agarose electrophoresis gel of the desalted fractions from the four replicates tested at each condition is given below.

0 M NaCl

0.1 M NaCl

0.2 M NaCl

0.3 M NaCl

In this ion exchange process, the lowest salt condition gives the highest recovery. Where it is possible to buffer exchange the pDNA containing lysate, e.g. by diafiltration, or desalting, using low salt conditions will allow the highest load onto the resin. Using diluted lysate (equivalent to the higer salt conditions up to 30 mS/cm ) it’s still possible to recover purified pDNA and lowering the load will ensure the best recovery.

Technical note: DEAE and Hexyl PuraBead® HF

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Results: Purification of pDNA using DEAE PuraBead® HF – 5 mL Pre-packed column The chromatogram of the 5 mL DEAE PuraBead® HF column run is given below, showing a sharp elution peak for the pDNA fraction.

3.43 mg of pDNA was recovered in the elution fraction, as measured by 260 absorbance on Nanodrop, which equated to a 74 % recovery of loaded pDNA. Results: Separation of scDNA and ocDNA using Hexyl PuraBead® HF – 1 mL column For the 1 mL column experiment, two elution peaks were collected, desalted and run on 1 % agarose gel, shown below.

These conditions demonstrated good purifiation of the scDNA at small scale using the 1 mL Hexyl PuraBead® HF column.

Technical note: DEAE and Hexyl PuraBead® HF

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Results: Separation of scDNA and ocDNA using Hexyl PuraBead® HF – 8 mL column For the 8 mL SNAP® column experiment the chromatogram and 1 % agarose gel are given below.

Results showed a large elution peak. All fractions were desalted and run on 1% agarose gel electrophoresis.

Excellent separation of the different isoforms of pDNA were seen, with ocDNA in the non bound fraction and scDNA in the elution fraction.

Technical note: DEAE and Hexyl PuraBead® HF

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Conclusions DEAE PuraBead® HF and Hexyl PuraBead® HF adsorbents from Astrea Bioseparations were utilized for two separate applications in pDNA purification, namely initial purification of pDNA and separation of scDNA and ocDNA. Both processes are essential to produce functional pDNA. • For initial purification of pDNA, DEAE PuraBead® HF gave good binding and recovery of pDNA in RoboColumns®. A 5 mL prepacked column can process 3.43 mg of purified pDNA in 1 hour. Or 0.68 g/L/hr. A 450 mm diameter, 10 cm bed height Evolve® column packed with DEAE PuraBead® HF will process 1 g batches of plasmid in one hour. • For further polishing of pDNA feedstock containing scDNA and ocDNA, Hexyl PuraBead® HF was shown to give good separation at 1 mL column scale. • In addition, scalability of the polishing step was demonstrated using a SNAP® column packed to 10 cm bed height at 10 mm diameter; this process could also be scaled to Evolve® process columns as required. These applications highlight the effectiveness of the use of both Ion Exchange and Hydrophobic Interaction Chromatography in the purification of plasmid DNA. For more information on IEX and HIC PuraBead® HF adsorbents please visit our website: https://www.astreabioseparations.com/

Technical note: DEAE and Hexyl PuraBead® HF

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