Purification of linearized DNA for use as an in vitro transcription temple
mRNA therapeutics, such as those created for the COVID-19 pandemic, are manufactured using an enzymatic reaction referred to as an in vitro transcription (IVT) reaction. The desired sequence is expressed as a plasmid from E. coli. Super coiled and open circular DNA are digested enzymatically to give a linear template. This IVT reaction uses the linearized plasmid to create multiple copies of the mRNA sequence required.
Purity of the linearized template is critical, since any remaining host cell DNA impurities or other nucleic acid contaminants will give rise to off-target mRNA products, which consume costly reagents and are difficult to remove.
We propose a process whereby the primary capture of pDNA is carried out on an ion exchange resin. In this case, DEAE PuraBead® HF is loaded with the clarified lysate conditioned to a high enough conductivity so that only the DNA species is bound, and the RNA is removed in the flow-through. The elution pool from the AEX step, can then be desalted, linearized enzymatically, and loaded onto an orthogonal purification step using a hydrophobic adsorbent, the pDNAHERO® device. The linear DNA is eluted from the column using a TRIS EDTA buffer.
AEX capture
1. Alkaline lysis process
2. Calcium chloride precipitation
3. Diafiltration into 50 mM TRIS, 10 mM EDTA, pH 7.5, 300 mM NaCl
4. Adjust to 35–37 mS/cm
5. Load onto DEAE PuraBead® HF
Conditions for AEX capture
Equilibration buffer A1: 50 mM TRIS, 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. Either select a pre-packed column or pack an appropriately sized column with DEAE PuraBead® HF, according to the technical user guide. Connect to the MPLC system.
2. Equilibrate the column in equilibration buffer A1.
3. Charge column with 5 CVs of buffer B1.
4. Equilibrate 10 CVs of buffer A1.
5. Dilute or buffer-exchange your clarified lysate with equilibration buffer to a conductivity of 35–37 mS/cm. This ensures the RNA is in the flow-through.
6. Load the sample to the column with a two-minute residence time. If using multiple 5 mL pre-packed columns joined together, a lower flow rate (three-minute residence time) may be needed to control the system pressure. We recommend determining the ideal load by loading until pDNA breakthrough is observed in the fractions of flow-through on agarose gel, approximately 1 mg/mL of total pDNA.
7. Wash the column with 20 CV of buffer A1.
8. Perform a two-step elution using 10% and then 30% B1 to wash away residual RNA and SDS contaminants.
9. Elute and collect pDNA with 10 CV of 100% buffer B1.
10. Strip the column with 10 CV of A2 in reverse flow.
11. Column can be cleaned and stored as per the technical user guide.
Purifying linear DNA on the pDNAHERO®
Desalt the elution pool, linearize, and then purify on the pDNA HERO® .
Conditions for HIC bind-and-elute purification of linear DNA
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. Condition the linearized material to 2.25 ammonium sulphate by mixing with 3 M ammonium sulphate in TE.
4. 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, which should be maintained below 0.6 MPa; residence time does not affect binding.
5. Continue loading until DNA breakthrough is observed to establish binding capacity, estimated ~4–5 mg/mL linear DNA.
6. We recommend running a breakthrough study as described above, collecting flow-through fractions, and running AGE gels on those to observe the volume in which the target starts to be observed in the flow-through. For process loading, apply approximately 70% of that volume.
7. Wash the column with 10 CV of buffer A1.
8. Elute and collect using 10 CV steps at 10%, 80%, and 100% buffer B.
9. Strip the column with RO water and 1 M NaOH, 10 CV each.
10. Column can be cleaned and stored as per the technical user guide.
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
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)
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 cGMP grade devices.