Plasmid DNA (pDNA)
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.
Challenges
Recovering high yields of pDNA can be tricky owing to their size, shear sensitivity, and similarity to some contaminants.
- High-density cell cultures are often tricky to monitor, maintain, and harvest.
- Shear forces can damage pDNA and degrade precipitated genomic DNA, contaminating the plasmid extract.
- Large plasmids (>6kb) are challenging to purify using traditional methods such as resins, as they cannot successfully diffuse through the pores.
- Separating pDNA from contaminants (including unwanted plasmid isoforms) to obtain a homogeneous product can be technically demanding, often requiring multiple chromatography steps.
pDNA Production Process
From E. coli to Purified Plasmid
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.
pDNA Process Flow
Host bacteria are expanded to produce millions of copies of the pDNA
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.
Your Need:
A scalable production platform to optimize cell growth and maximize plasmid yield in a simple and reliable manner
- Robust, controllable, and efficient production
- A sterile seed train to avoid cross-contamination
- High-speed feeding pumps
Our Unique Solution:
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).
- A unique and robust film across the process to optimize cell growth conditions
- Integration of a wide range of process analytical technology (PAT), including and
- Flexibility and full scalability from Ambr® to stirred tank reactor (STR) for rapid process development
- Mycap CCX – a cell culture expansion system that allows you to grow and passage cells without ever opening a flask or entering a biosafety cabinet
- Ambr® 250 single-use multi-parallel bioreactors that can be adapted for microbial fermentation to accelerate process development.
- Single-use hollow fiber cartridges within our support the harvest of cells from high-density cultures.
Host cells are lysed to release the pDNA
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.
Initial removal of cell debris from pDNA
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.
Your Challenge
Optimal pDNA clarification solutions that separate impurities from even the highest density cell lysates
- Tools must be able to handle highly viscous solutions
Our Unique Solution:
Sartorius provides a broad range of tangential flow filtration (TFF) consumables, covering applications from process development to large-scale commercial production. Our hollow fiber modules offer a fully scalable platform for the clarification of cell lysates, especially under high cell density environments. are recommended for medium or lower cell concentrations.
- Membrane offering from 3 kD to 750 kD molecular weight cut-off and from 0.1 μm to 0.65 μm pore size
- Gamma-irradiated; Single-use and ready-to-use, saving time and costs associated with equipment set-up
- Particularly effective for viscous lysates from high cell density cultures
- The Sartopore® 2 family provides the broadest range of PES membrane combinations that perfectly adapt to all product types for the lowest filtration costs.
- Sartorius provides a broad range of TFF systems, covering applications from process development to large-scale commercial production. Our solutions have the option to implement functionally closed solutions with single-use and gamma-irradiated flow paths.
Sample volume is reduced and small molecules are removed from the buffer.
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.
Your Challenge:
Tailored concentration solutions for large, sensitive molecules
- Limited shearing to minimize degradation and poor recovery
- Maximum recovery of even the largest constructs
Our Unique Solution:
Sartorius offers a strong portfolio of products suitable for the concentration/diafiltration of biomolecules. Our hollow fiber single-use flow assemblies represent a fully scalable system for concentrating pDNA
- Range of sizes to support the filtration of large plasmids (up to 750 kb). Research recommends between 30 kD and 750 kD MWCO.
- Gamma-irradiated and ready-to-use, reducing process times
- Low shear operations
- The family includes the broadest range of PES membrane combinations that perfectly adapt to all types of products for the lowest filtration costs
Isolation of plasmid DNA
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.
Your Need:
pDNA capture processes that are sensitive, low shear, and suitable for large molecules.
- Efficient and gentle removal of impurities to avoid degradation
- Capture methods without size restrictions
Our Unique Solution:
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
- Concentrates pDNA and removes the majority of host cell proteins and other nucleic acids.
- High dynamic binding capacity for pDNA
- Low shear forces maximize pDNA recovery
- Our detailed, straightforward protocol supports the development of a robust process
- The Allegro single-use chromatography system is designed for pilot, clinical, and commercial production. The flexible modular design enables column and membrane operation with isocratic or gradient set up options
Separation of plasmid isoforms to produce homogeneous 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
Your Need:
Efficient and robust polishing chromatography solutions that produce homogenous pDNA extracts reliably
- Methods must be sensitive enough to separate plasmid isoforms
Our Unique Solution:
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™
- Separates open circular (OC) and linear pDNA isoforms from supercoiled (SC) pDNA
- High dynamic binding capacity and low shear maximizes pDNA recovery
- Offers flexibility and scalability
- The Allegro single-use chromatography system is designed for pilot, clinical, and commercial production. The flexible modular design enables column and membrane operation with isocratic or gradient set up options
Sample volume is reduced and small molecules are removed from the buffer.
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.
Your Challenge:
Tailored concentration solutions for large, sensitive molecules
- Limited shearing to minimize degradation and poor recovery
- Maximum recovery of even the largest constructs
Our Unique Solution:
Sartorius offers a strong portfolio of products suitable for the concentration/diafiltration of biomolecules. Our hollow fiber single-use flow assemblies represent a fully scalable system for concentrating pDNA
- Range of sizes to support the filtration of large plasmids (up to 750 kb). Research recommends between 30 kD and 750 kD MWCO.
- Gamma-irradiated and ready-to-use, reducing process times
- Low shear operations
- The family includes the broadest range of PES membrane combinations that perfectly adapt to all types of products for the lowest filtration costs
Isolated pDNA is subject to sterile filtration to prevent microbial contamination
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.
Your Need:
Sterile filtration solutions applicable to large, shear sensitive pDNA molecules
- Suitable for viscous solutions
Our Unique Solutions:
Sartorius offers the most advanced sterile filters for your application. The family includes the broadest range of PES membrane combinations that perfectly adapt to all types of products for the lowest filtration costs
- Available with 0.2 or 0.45 µm final membrane pore size
- Integrated into filter transfer set for closed single-use applications
- is the ideal choice for final filling applications. The unique surface modification of the PES membrane provides the lowest adsorption, facilitating high product yield and secure wetting for reliable integrity testing.
- Sartocheck 5 is the benchmark for integrity testing of sterilizing grade filters. The unit is designed to provide intuitive, industry-leading data security and integrity with the highest quantitative risk management (QRM) standards for testing
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.
Your Challenge
An analytics solution providing a comprehensive evaluation of the pDNA sample
- Rapidly provide a complete picture of the final product
- Avoid labor-intensive methods
Our Unique Solution:
Sartorius provides complete solutions to monitor and control your pDNA samples.
Our are intended for fast and reproducible HPLC monitoring and quantitation of plasmid DNA, as well as the separation of different plasmid DNA isoforms.
- Primarily designed for the in-process and final control of pDNA samples at various stages of the purification process,
- Can also be used to determine the presence of RNA
- Rapid, high-resolution analysis
- Flow-independent separation
- High sensitivity and reproducibility
- The PATfix™ HPLC system gives you “at-line” analysis for the control of impurities and critical quality components using HPLC fingerprinting
CIMmultus® HiP2 Plasmid Process Pack™
CIMmultus® HiP2 Plasmid Process Pack™
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).
- Efficient - removes the majority of contaminants (RNA, genomic DNA, host cell proteins, and endotoxins) and isolates supercoiled pDNA
- Rapid - processing is completed in just a few hours, increasing productivity and minimizing manufacturing costs. A well-defined protocol helps you develop robust purification procedures quickly.
- Scalable - the unique properties of the monolithic columns facilitate seamless transfer between production levels, from small-scale laboratory to large-scale industrial purification of pDNA.
Increasing Productivity of pDNA Downstream Processing
Learn more about how monoliths perform polishing chromatography during pDNA purification
Purity
Excellent removal of all impurities produces high purity and homogeneous pDNA samples, including isoform separation
Productivity
Perform rapid processing without compromising on recovery and capacity, even for large plasmids
Plug and Play
Toolbox contains two ready-to-use columns and a detailed protocol for quick and easy incorporation into your process
Products in the CIMmultus® HiP2 Plasmid Process Pack™
CIMmultus® DEAE
Anion-Exchange Chromatography
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
How Does it Work?
Chromatogram of pDNA extract applied to CIMmultus® DEAE. pDNA is efficiently isolated from RNA and other contaminants.
CIMmultus® C4 HLD
Hydrophobic Interaction Chromatography
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
How Does it Work?
Chromatogram of captured pDNA applied to CIMmultus® C4 HLD. The remaining contaminants are removed, and SC pDNA is efficiently isolated from OC pDNA.
