Part 2: Chromatography Purification for pDNA
Plasmid DNA (pDNA) is essential to the production of advanced modalities. Discover how chromatography techniques support efficient pDNA purification strategies.
This article is posted on our Science Snippets Blog
Plasmid DNA, or pDNA, is a large and more robust molecule than mRNA. It can withstand reasonably harsh treatments and still be an intact and functional plasmid. However, small lesions in the pDNA can dramatically affect the molecule’s shape, size, and conformation. Intact pDNA is supercoiled, which is a very condensed and tightly packed state. However, if the pDNA is nicked, it will open up the plasmid, much like unraveling a ball of string. If the circular plasmid is broken completely, then it will linearize. This will also impact the physical properties that can affect purification.
The size of pDNA can also represent a challenge in the purification process, especially at large scales. pDNA is bigger than most biological molecules that are purified using chromatography. The size means that many traditional chromatography matrixes will have a low capacity to bind and purify pDNA, particularly at high flow rates. Furthermore, at very high concentrations of pDNA, the resulting high viscosity means that back pressures are high.
Knowing Which Impurities May be Present Helps Establish Your Process Steps
Since pDNA is produced in bacterial cells, a lot of cellular debris can be released if the lysis is not performed carefully. Along with all the internal cellular components, such as genomic DNA and proteins, components of the E. coli cell membrane are well-known endotoxins. As the name suggests, endotoxins are highly toxic and extremely immunogenic, making them problematic for most biological and clinical applications. Endotoxins must be completely removed during the purification process.
Other contaminants include mRNA and genomic DNA, which have similar properties to pDNA, making them challenging to separate.
The clinical use of pDNA in the production of CRISPR-Cas9 therapies and viral vectors makes removing impurities very important. Safety is a big concern with these types of therapies both in terms of bacterial host impurities and potentially contaminating genetic material. If genomic DNA from bacteria, mRNA, or pDNA with the wrong sequence finds its way into such therapeutic products, they will not be approved for commercial use.
Understanding the Properties that Aid Separation is Critical
The negative charge of pDNA means that ion-exchange chromatography (IEX) can be used as the initial capture step in purification processes. Changing the salt concentration will allow the separation of most cellular debris and even genomic DNA and mRNA.
IEX can be used to remove most of the contaminants in a pDNA preparation, except for the different plasmid forms such as linearized plasmid and open circular variants. Frequently, these are removed by hydrophobic interaction chromatography (HIC), as linear, open circular, and supercoiled pDNA have different hydrophobic properties at different concentrations of ammonium sulfate. At high concentrations of ammonium sulfate, supercoiled pDNA binds more strongly to hydrophobic chromatography matrices than linear and open circular pDNA. Multimers and DNA aggregates are even more hydrophobic, and will elute later than the supercoiled DNA, making HIC an excellent option for the second step of pDNA purification.
Affinity chromatography (AC) can also be useful for purifying pDNA. There are a few different affinity ligands that strongly and reversibly interact with the pDNA, allowing thorough removal of impurities before eluting the target pDNA. Arginine beads, diminazene aceturate, and zinc finger proteins have all been used as affinity partners to purify pDNA.
Matrix of Choice
| Large channels in the chromatography matrix allow pDNA molecules to pass through intact. Discover more about the features of monoliths and membranes Due to the large size of pDNA, monoliths and membranes are the preferred matrices for purification. Suitable ligands exist on these matrices for the chromatography modes that provide efficient isolation from cellular components. They also have large-enough channels sizes to handle high viscosities associated with high concentrations of pDNA. Back pressures are lower with monoliths and membranes compared with beaded resins due to their lower bed heights and wider channels. These two chromatography matrices types also allow faster flow rates, avoiding the diffusion limitations associated with beaded resins. |
Recovering High Yields of pDNA with Chromatography
Effective downstream production processes maximize the recovery of pDNA, which is the starting point for manufacturing many advanced therapies. Learn more about how chromatography methods can be optimally applied to the production of pDNA and other advanced modalities
Comparing Chromatography for Advanced Modalities
pDNA | mRNA | Viral Vectors | |
|---|---|---|---|
| |||
Purpose | Carries genetic material | Codes for protein | Carries genetic material into cells |
Use in advanced therapies | - |
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Vaccines | - |
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Cell and gene therapy |
| - | - |
Raw material | |||
Purification Challenges | |||
Large size |
| ||
Shear sensitive |
|
| - |
Very diverse |
|
|
|
Separation | |||
Separation of empty and full capsids requires high resolution methods | - | - |
|
Typical Impurities | From E coli: | Carry over from IVT reaction: | From cell cultures:
|
Properties that aid separation | Negative charge | Negative charge | Net charge created by surface proteins on the virus
|
Matrix of choice | Monoliths and membranes | Monoliths | Monoliths and membranes |
