Purification of super coiled pDNA using orthogonal HIC and AEX bind-and-elute steps
Purifying plasmids for cell and gene therapy vectors requires chromatography systems where sequence size is not a limitation. This method describes a two-step orthogonal purification process that enables efficient pDNA capture from large volumes of lysate and high resolution of supercoiled plasmid DNA (sc pDNA), with consistent performance for both short and long sequences (for example, 34 kb). The capture step is performed with a hydrophobic interaction chromatography (HIC) adsorbent, pDNAHERO®, followed by polish with Q PuraBead® Edge anion exchange chromatography resin.
Methods
pDNA capture
Details of the capture were as follows:
HIC capture
1. Alkaline lysis process
2. Calcium chloride precipitation
3. Diafiltration into 50 mM TRIS, 10 mM EDTA, pH 7.5 (TE)
4. Adjust clarified lysate to 2.85 M ammonium sulphate in TE by dilution with 3 M ammonium sulphate in TE
5. Load onto pDNAHERO®
Conditions for HIC bind-and-elute
Equilibration buffer A1: 3 M ammonium sulphate, 50 mM TRIS, 10 mM EDTA, pH 7.5
Elution buffer B1: 50 mM TRIS, 10 mM EDTA, pH 7.5
CIP A2: RO water A3, 1 M NaOH
Procedure
1. Connect the pDNAHERO® unit, as per the technical user guide.
2. Equilibrate the pDNAHERO® unit in equilibration buffer A1.
3. Load the sample to the column with a twelve-second residence time. It may be possible to run at six-second residence time depending on the pre-column pressure from the system; residence time does not affect binding.
4. Continue loading until DNA breakthrough is observed to establish binding capacity, estimated ~3–4 mg/mL pDNA.
5. We recommend running a breakthrough study as described above, collecting flow-through fractions, and running AGE gels on those to observe the volume at which the target starts to be observed in the flow-through. For process loading, apply approximately 70% of that volume.
6. Wash the column with 10 CV of buffer A1.
7. Elute and collect using 10 CV steps at 10%, 80%, and 100% buffer B. Note that optimization may be required for specific sequence composition or length.
8. Strip the column with RO water and 1 M NaOH 10 CV each.
9. Column can be cleaned and stored as per the technical user guide.
pDNA polish
Conditions for AEX bind-and-elute
Equilibration buffer A1: 50 mM TRIS, 0.6 M NaCl, 10 mM EDTA, pH 7.5
Elution buffer B1: 50 mM TRIS, 10 mM EDTA, 1 M NaCl, pH 7.5
CIP A2: 50 mM TRIS, 10 mM EDTA, 0.5 M NaOH
Procedure
1. Select a pre-packed column or pack an appropriately sized column with Q PuraBead ® Edge according to the technical user guide, and attach to an MPLC system.
2. Equilibrate the Q PuraBead® Edge column in equilibration buffer A1.
3. Charge column with 5 CVs of buffer B1.
4. Equilibrate 10 CVs of buffer A1.
5. Condition the elution from the pDNAHERO® to 55–57 mS/cm.
6. Load the sample to the column with a two-minute residence time. Continue loading until pDNA breakthrough is observed at approximately 500 µg/mL.
7. We recommend running a breakthrough study as described above, collecting flow-through fractions, and running AGE gels on those to observe the volume at which the target starts to be observed in the flow-through. For process loading, apply approximately 70% of that volume.
8. Wash the column with 10 CV of buffer A1 to remove any weakly-bound RNA contamination.
9. Perform a gradient elution from 0–45% B. following with a step to 100% B to establish the elution conditions to separate the isoforms. This can be converted to a step gradient to reduce buffer usage.
10. Strip the column with 10 CV of A2.
11. Column can be cleaned and stored as per the technical user guide.
Materials
pDNAHERO® products
Research use only
pDNAHERO®1 (5–10 mL/min)
pDNAHERO®10 (50–100 mL/min)
Process-ready, suitable for use in GMP manufacturing
pDNAHERO®100 (500 mL/min–1 L/min)
Note: All materials used in the construction of the pDNAHERO®10 are identical to the materials used for the pDNAHERO®100. The assembly, cleaning, and testing processes are all the same. The only additional tests are for the certification and release for the process-ready cGMP grade devices.
Pre-packed columns for all PuraBead® products
Research use only
• 1 mL and 5 mL columns, 2.5 cm bed height
• Up to 3 of these pre-packed columns can be combined to give a larger volume of media, up to 15 mL
Process-ready, suitable for use in GMP manufacturing
Evolve®R columns
• 5 mL and 50 mL columns, 10 cm bed height
Evolve®D columns
• Multiple sizes available: Choose from diameters of 7 cm, 10 cm, 14 cm, and 20 cm to suit various application scales
• Flexible bed heights: Options of 5 cm, 10 cm, 15 cm, or 20 cm for tailored chromatography solutions