pDNA Production
Plasmid DNA (pDNA) represents a critical starting point for many genetic engineering pursuits, including the development of recombinant proteins, viral vectors, and advanced biotherapeutics. Continued progress in gene therapy and DNA/RNA-based therapeutics has led to a growing demand for reliable pDNA production processes suitable for clinical applications.
Plasmids are circular molecules of DNA found naturally occurring in bacteria. They replicate independently (episomal) from the bacterial genomic DNA, a property that makes them ideally suited as a vector for genetic engineering. Purified pDNA can be used as a raw material in processes such as the production of adeno-associated (AAV) and lentiviral vectors, in vitro transcription (IVT) reactions during mRNA production, and direct gene transfer.
Sartorius provides various tools for efficient manufacturing throughout the production of pDNA. Explore the process of plasmid DNA manufacturing from bacteria to final filtration below, and discover the solutions that steer you through every step.
Recovering high yields of pDNA can be tricky owing to their size, shear sensitivity, and similarity to some contaminants.
The typical steps required to isolate plasmid DNA
Our two-step platform for pDNA purification
The gene of interest is inserted into the pDNA, which is introduced into host bacteria (typically E. coli) in a process called transformation, usually by electroporation. The host cells replicate during fermentation, making copies of the plasmid and producing high yields of the desired sequence. Once the cells have reached the desired density, they are harvested, and the pDNA is extracted, typically by alkaline lysis. The lysate is then clarified, and the pDNA is purified using chromatography. Finally, the plasmid is then concentrated and filtered before being subject to quality control measures to ensure the product is of sufficient purity.
The aim of upstream processing in plasmid production is to generate as much pDNA as possible using a population of host cells. Typically, E. coli are used due to their ability to proliferate and produce high yields of the pDNA rapidly. A high-producing clone is selected and used to form the seed train, which provides adequate cell numbers to inoculate the production bioreactor. Following cultivation, cells are harvested, usually by centrifugation.
A scalable production platform to optimize cell growth and maximize plasmid yield in a simple and reliable manner
Sartorius delivers a range of solutions for microbial fermentation at all production scales.
Biostat® D-DCU is a compact bioprocess system with vessel sizes from 10 to 200 L, meeting the requirements for high-density fermentation of E. coli (>50 OD).
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The objective of cell lysis is to release pDNA and remove cell debris and genomic DNA. Once the host cells are harvested, the cell pellet is lysed with NaOH solution and subsequently neutralized with potassium acetate. The resulting precipitate contains impurities, including host cell proteins and DNA.
The material collected from alkaline lysis contains pDNA as well as contaminants from the host cells. Clarification purifies pDNA from high-density cell cultures, removing cell debris and impurities.
Optimal pDNA clarification solutions that separate impurities from even the highest density cell lysates
Sartorius provides a broad range of tangential flow filtration (TFF) consumables, covering applications from process development to large-scale commercial production. Our Green Line hollow fiber modules offer a fully scalable platform for the clarification of cell lysates, especially under high cell density environments. Hydrosart® 0.2µm Microfilter membranes are recommended for medium or lower cell concentrations.
The concentration of pDNA, contaminant removal, and buffer exchange steps are essential in pDNA production but can be challenging due to the large size and shear sensitivity of pDNA. The latest innovations combine low-shear operations and fully closed processing to allow the highest recovery in a contained system, creating an ideal environment for pDNA manufacturing.
Tailored concentration solutions for large, sensitive molecules
Sartorius offers a strong portfolio of products suitable for the concentration/diafiltration of biomolecules. Our Green Line hollow fiber single-use flow assemblies represent a fully scalable system for concentrating pDNA
As a biopharmaceutical product, pDNA is subject to stringent requirements for purity, efficacy, and yields. pDNA capture is a critical step in the downstream process, removing most contaminants while concentrating pDNA.
pDNA capture processes that are sensitive, low shear, and suitable for large molecules.
The HIP² Plasmid Process Pack™ is an end-to-end solution for pDNA purification developed by expert scientists at Sartorius.
CIMmultus® DEAE columns represent the first step of our plug and play platform for effective purification of pDNA
An additional polishing chromatography step is required to separate pDNA isoforms, usually to isolate supercoiled (SC) pDNA from open circular (OC) and linear DNA. Pharmaceutical pDNA has stringent requirements of purity, homogeneity, and efficacy, and SC pDNA is the most stable, efficient, and biologically active form
Efficient and robust polishing chromatography solutions that produce homogenous pDNA extracts reliably
The HIP² Plasmid Process Pack™ from Sartorius is a comprehensive pDNA purification solution. CIMmultus® C4 HLD columns form the second step of the HIP² Plasmid Process Pack™
The final sterile filtration step is critical to prevent microbial contamination and ensure patient safety. Innovative risk mitigation strategies will protect the integrity of the entire operation from the last filtration step to storage in single-use bags.
Sterile filtration solutions applicable to large, shear sensitive pDNA molecules
Sartorius offers the most advanced sterile filters for your application. The Sartopore® 2 family includes the broadest range of PES membrane combinations that perfectly adapt to all types of products for the lowest filtration costs
Analytics are necessary to determine the presence of impurities and quantify the product throughout the pDNA purification process. The information provided supports process control, robustness, and high product quality.
An analytics solution providing a comprehensive evaluation of the pDNA sample
Sartorius provides complete solutions to monitor and control your pDNA samples.
Our CIMac™ pDNA 0.3 mL Analytical Columns are intended for fast and reproducible HPLC monitoring and quantitation of plasmid DNA, as well as the separation of different plasmid DNA isoforms.
We created a ready-to-use pDNA purification pack to help scientists develop their own pDNA purification process and transfer it to production. The HiP2 platform facilitates the efficient recovery of pDNA in just two chromatography steps (anion exchange chromatography followed by hydrophobic interaction chromatography).
Learn more about how monoliths perform polishing chromatography during pDNA purification
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Excellent removal of all impurities produces high purity and homogeneous pDNA samples, including isoform separation
Perform rapid processing without compromising on recovery and capacity, even for large plasmids
Toolbox contains two ready-to-use columns and a detailed protocol for quick and easy incorporation into your process
Using weak anion-exchange chromatography, CIMmultus® diethylamine (DEAE) concentrates pDNA and removes the majority of contaminating host cell proteins, mRNA, and genomic DNA.
Performs the initial capture in the two-step plasmid process pack
Can be used to process clarified lysates or partially purified plasmids from other purification methods
High capacity for pDNA molecules, which are concentrated in a single peak
Most impurities do not bind (with the exception of RNA, which elutes after the first increase of NaCl)
pDNA size is not a limiting factor
For pDNA larger than 11 kb, we recommend 6 μm channels
Chromatogram of pDNA extract applied to CIMmultus® DEAE. pDNA is efficiently isolated from RNA and other contaminants.
The CIMmultus® C4 high ligand density (HLD) butyl-modified monolithic column eliminates remaining genomic DNA, endotoxins and open circular (OC) and linear pDNA isoforms, isolating supercoiled (SC) pDNA.
Performs the polishing step in the two-step plasmid process pack
pDNA binding promoted under high salt concentrations
The remainder of non-DNA contaminants are removed
Facilitates the separation of supercoiled pDNA from other isoforms
pDNA purification can be performed in both bind-elute and sample displacement modes
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Chromatogram of captured pDNA applied to CIMmultus® C4 HLD. The remaining contaminants are removed, and SC pDNA is efficiently isolated from OC pDNA.
Learn how mRNA molecules are manufactured and how our tools can help optimize your process at every step.
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