How to optimize the production of Plasmid DNA for cell and gene therapy?

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How to optimize the production of Plasmid DNA for cell and gene therapy?

How to optimize the production of Plasmid DNA for cell and gene therapy? High-quality plasmid DNA is a key component in the production of cell and gene therapy, so it is in great demand.

This leads to the need to optimize production to meet the quantity and quality requirements required for the production of therapeutic drugs. Plasmid DNA (pDNA) faces some challenges due to its large size, shear sensitivity, high viscosity, and the similarity between pDNA and impurities present in the production process. Therefore, understanding all aspects of the process is critical to successful mass production.

With high demand and high quality requirements, it is very important to find domain experts who can clarify key optimization opportunities for pDNA and answer production questions. At this “Ask the Experts” meeting, we convened a panel of experts to answer questions about the production of plasmid DNA in cell and gene therapy applications.

How to deploy Plasmid DNA production for cell and gene therapy?
Dr. Nargisse El-Hajjami, Deputy Director of Development, Cell and Gene Therapy Division of EMEA

Dr. Nargisse El Hajjami is a molecular microbiologist and biotechnology expert with 10 years of experience in scientific research, process development and biological production engineering. Her current responsibilities are to focus on cell and gene therapy business development by leading strategic plans, supporting go-to-market strategies, and supporting customers in establishing, developing and optimizing their knowledge and production processes.

How to deploy Plasmid DNA production for cell and gene therapy?
Laurens Vergauwen, Process Development Scientist

Laurens graduated from Leuven University in Belgium with a master’s degree in industrial chemical engineering. He is an expert in downstream processing and supports customers in the development and optimization of various downstream purification technologies (chromatography, TFF, clarification, aseptic and virus filtration). Being able to work with different types of manufacturers, he has a strong understanding of various biopharmaceutical purification strategies including plasmid DNA.

Question 1 Are there any key media components that can increase productivity?

There are several parameters that can affect the productivity of bacteria, such as the selection of the main cell bank, growth rate, medium, feeding rate, and appropriate growth conditions and parameters, such as pH, osmotic pressure, optical density (DO), and temperature.

General media components include carbon sources, nitrogen sources, magnesium sulfate, dipotassium phosphate and monopotassium phosphate, and minerals. Minimal and semi-defined media can be used. With minimal media, highly reproducible batches can be obtained. On the other hand, since complex ingredients (such as yeast extract) provide growth factors, amino acids, purines and pyrimidines, semi-defined media can support higher cell densities. As a carbon source, glucose is traditionally used because it is highly metabolized and inexpensive. However, glucose may cause overproduction of acetate (Crabbe tree effect). This can be avoided by (partially) replacing glycerol with glucose. Glycerin also helps reduce the maximum specific growth rate.

Nitrogen is usually supplied by adding complex ingredients such as yeast extract, amino acids or peptone. Magnesium sulfate and potassium phosphate are added to provide sources of magnesium, sulfur, potassium, and phosphorus (phosphate also acts as a buffer). Other trace minerals are present in the complex components of the medium, or can be added through specific trace mineral solutions. The C:N ratio has a greater impact on the plasmid yield. It is recommended to test different ratios, for example from 2:1 to 8:1. The optimal ratio is different for each media type.

Question 2 My plasmid DNA is unstable. We want to try to improve the stability. Do you have any suggestions for this?

On the one hand, it is important that the sequence of the gene (insert) and plasmid (vector) you are interested in is well optimized. However, some plasmids are inherently unstable. For example, if the insertion is very large and/or contains reverse tandem repeats. For large genes, a small vector should be chosen. However, if this situation already exists, it is likely that the plasmid will need to be generated with a low copy number (this is also true in the case of inverted tandem repeats).

On the other hand, adjusting growth conditions (such as growing at a lower temperature), selecting alternative hosts, optimizing process conditions, and selecting appropriate buffers to process, prepare, and store pDNA can also help improve its stability. DNase contamination and pH have a great influence on the stability of pDNA. DNA enzymes can degrade and digest double strands of DNA, while extreme pH can destroy, denature or even change the sequence of pDNA. Therefore, choosing the right buffer and solution for your pDNA is the basis for long-term product stability. One of the best choices is Tris buffer and EDTA, because Tris buffer allows pH control to stabilize pDNA, while EDTA chelate inhibits DNAse activity. For storage, pDNA is usually stored at -20°C to -80°. It can be stable for many years, or it can be stable at 4° or at room temperature, but for a short time.

Question 3 Why are GMP plasmids so expensive and in short supply?

GMP production is generally a more costly process. There are many reasons for this. The raw materials used in the GMP production process have higher purity and higher prices. A dedicated production area (such as a clean room) is required. The cleaning method needs to be verified, and the final GMP product has a high purity, which has been confirmed by a series of validated analytical methods.

The reason for the short supply is that in addition to vaccines and cancer treatment applications, the global cell and gene therapy market has grown substantially. According to reports, in the past two years, the number of new drugs tested has grown significantly, and a variety of drugs are obtaining commercial distribution licenses. Because of this growth, contract manufacturers that provide GMP-grade plasmids are struggling to keep up with the increasing demand.

The recent pandemic has exacerbated this problem because many candidate vaccines are under development, including DNA vaccines and mRNA vaccines, where plasmid DNA is the starting material for the in vitro transcription of mRNA.

Question 4 How to choose the best plasmid for my gene?

There are two main types of vectors, cloning vectors and expression vectors. Cloning vectors are ideal for producing many gene replications. If the goal is to express the gene of interest, an expression vector is required. For cloning vectors, the key is copy number (depending on ori), selectable marker and cloning site. Generally, high copy numbers are preferred. Note that when the gene is toxic to the cell, or the plasmid is unstable, a low copy number may be advantageous. The selection marker allows the identification of positive transformants. In most cases, these will be drug resistance markers, but they are also used as malnutrition markers. It is important to check whether the vector contains a suitable cloning site for insertion. To date, most vectors contain multiple cloning sites, which makes the vector likely to be compatible with the selected restriction endonuclease.

The expression vector contains some additional sequences related to expression, such as promoter, ribosome binding site, terminator, tag or fusion protein. Some of these sequences are host-specific. Therefore, the expression vector needs to be compatible with the selected host organism (such as mammals, insects, etc.).

Question 5 We see that the DNA yield is very low, do you have a good plan?

In order to have a good yield of plasmid DNA (pDNA), the best solution is to have a robust overall process in which every step is well optimized. The purification of pDNA is challenging due to its large size, high negative charge, high viscosity, and contaminants similar to pDNA (open-circle pDNA, genomic DNA, high molecular weight RNA). In addition, large plasmids are sensitive to shear stress, further complicating purification. In order to purify supercoiled pDNA (the ideal form of treatment/transfection) with high yields, a well-optimized downstream process is required. Our company provides solutions and capabilities for each step of the downstream process to ensure the best pDNA yield and purity (as shown in Figure 1). The precautions for the operation of each downstream unit are discussed below.

Figure 1: Integrated workflow of pDNA purification from harvest to final filling

Cell harvest

The cell harvest step uses centrifugation or microfiltration TFF. When batch volumes (<10>1000 L) need to be processed, centrifugation is usually more cost-effective. When using centrifugation, special attention should be paid to the high shear forces generated by large-scale centrifugation. Microfiltration can be performed with an open channel, flat-plate TFF device, such as ProstakTM cassette filter with Durapore® 0.1 or 0.2μm microfiltration membrane or Pellicon® stearic pore cassette® V screen or Biomax® 1000 kD V screen ultra Filter membrane.

The open feed channel creates a gentle flow path for cell retention, resulting in low shear and can be used to handle viscous and/or high solids feeds. When using such a membrane shut-off valve, it is important to use a dual pump (permeation control) TFF system. The TFF harvest step usually includes 2-5 times the volume concentration, followed by 3-5 times the volume of refiltration, in order to wash out the waste medium components and extracellular impurities before further downstream purification.

TFF is usually harvested at low transmembrane pressure (TMP; 3–5 psi) and ΔP (<7 psi) to control permeate flux. Hollow fiber modules are also suitable, but due to its non-linear scalability, it may be accompanied by scaling issues.

Typical operating parameters of microfiltration TFF:
How to optimize the production of Plasmid DNA for cell and gene therapy?

Cell lysis

The methods used for cell lysis can be divided into two categories: chemical (alkali, detergent, enzyme, osmotic shock) and physical mechanical (heating, shearing, stirring, ultrasonic and freeze-thaw) lysis. Alkaline lysis (NaOH at pH ~ 12) accompanied by detergents such as sodium dodecyl sulfate (SDS) and Triton® X-100 is the most common method. The optimization of lysis culture time directly affects the quality and quantity of plasmid DNA. Longer culture time can lead to irreversible plasmid DNA and shear degradation of genomic DNA. The key is to use effective but not too aggressive mixing in the alkaline lysis step to ensure that there are no extreme pH values that can cause irreversible plasmids or degrade the plasmids due to excessive shearing. Mobius® disposable mixers are very effective for batch cracking.

In the alkaline lysis method, cells are processed in a specific, narrow pH range (usually around pH 12). At this time, genomic DNA will be irreversibly denatured, while the pDNA double strand remains intact (pH range of 12.0 to 12.5). The optimum pH value varies with the type of plasmid and host bacteria. The deviation of pH value from the optimal value of more than 0.1 may affect the yield, so it is very important to strictly control the pH value range during the alkaline hydrolysis process. When the pH>12.5, pDNA will undergo irreversible denaturation. If the pH is too low, the genomic DNA will not be completely denatured, and the downstream purification process may be more complicated. The incubation time for standard alkaline lysis is quite short, usually within 5 minutes.

After alkaline hydrolysis, the pH value is neutralized. This is usually achieved by adding a high-salt buffer, such as sodium acetate or potassium acetate with a concentration of 0.7M–3.0M and a pH of 5–7.5, in the presence of detergent (0.2-1% sodium lauryl sulfate) In this case, add/do not add 1.0–1.5% calcium chloride (to promote RNA precipitation). Polyethylene glycol (PEG) and polyethyleneimine can also be added to the neutralization buffer to promote the precipitation of genomic DNA. Uniform mixing during neutralization and precipitation is essential to maintain the quality of pDNA.

Purify

The operation of the clarification device of the pDNA process should be able to remove the solid content in the feed stream. It can be clarified by centrifugation and/or normal flow filtration. The traditional clarification method is centrifugation. When using centrifugation, attention should be paid to shear stress, which may damage the structure of the supercoiled plasmid, resulting in a decrease in yield. Centrifugation may also require a second clarification step. Clarification using conventional flow filtration is a modern method. When using this method, untreated, pre-treated or pre-clarified feed streams can be used. Pretreatment has a significant impact on the clarification and filtration capacity, and it must be carefully selected while considering the scalability of the process. The pretreatment program includes the use of gravity sedimentation and separation, Polygard® Chromium 1µm/Polygard® Chromium 50µm, bag filter, stainless steel mesh filter, paper filter, and centrifugal separation. Polygard’s capacity® CR filters are usually in the range of 0.55-8 liters/inch.

The recommended filter settings for the clarification step are as follows:

How to optimize the production of Plasmid DNA for cell and gene therapy?

The expected capacity of the filter depends to a large extent on whether pretreatment is carried out:
How to optimize the production of Plasmid DNA for cell and gene therapy?

The typical recovery when using Clarisolve® is also a Millistac+® filter >90%. The use of salt-containing buffers can increase the recovery rate. After using SHC sterile filter of Millipore Express®, the clarification is generally between 400-650L/m2.

Tangential Flow Filtration (TFF)

After clarification, you can choose the TFF step. This will allow concentration and diafiltration to exchange the medium into a buffer suitable for downstream chromatographic steps. By concentrating before chromatography, the chromatographic loading time can be shortened. In addition, RNA, small molecule genomic DNA and small molecule proteins can be removed during the TFF process, which also helps prevent contamination of the chromatography resin.

Some key considerations when performing TFF include:

The shear sensitivity of plasmids requires careful adjustment of process parameters to prevent plasmid damage.
Membrane fouling, resulting in production loss.
The high-salt buffer promotes plasmid compression, which may allow passage through small pores and result in loss of yield.

These problems can be avoided by carefully selecting recommended open channel membranes, such as Pellicon® 2 Ultracel® or Biomax® 100 or 300 kDa C screens. By using open channels, the shear stress can be reduced. In addition, it is also important to minimize the processing time through the appropriate film size. When using these wider membrane shut-off valves, it is important to control the permeate flux by using a dual pump (permeation control) TFF system. This will help prevent membrane fouling.

The typical TFF process parameters are:

Chromatographic purification

For chromatographic purification, anion exchange chromatography (AEC) and hydrophobic interaction chromatography (HIC) are commonly used. Both of these techniques have been used for capture or intermediate purification/polishing, and are often used in combination. When using AEX as the pDNA capture step, it is beneficial to spike the clarified lysate with NaCl (120-250mm). This will eliminate RNA interference and improve the binding ability of pDNA. The optimal salt concentration of the supplement needs to be determined in advance, for example, by batch analysis in the form of a microtiter plate to measure the plasmid binding capacity when the concentration of sodium chloride is increased. This can be done for different types of resin/membrane adsorbers. The figure below shows this principle of Fractogel® EMD DEAE (M) and Fractogel® EMD DMAE (M):

(Original lysate before replenishment: pH 5.0, 67 mS/cm)

Recommended resins for plasmid purification and their respective properties:

Due to the large size of the plasmid, a typical low binding capacity was observed on commercially available resins. This is because most AEX media were originally designed for protein purification. Since plasmid molecules are much larger than proteins, they cannot enter the small pores, resulting in low binding capacity and slow mass transfer. Because Fractogel® and Eshmuno® resins contain tentacle technology, they generally have a higher binding capacity than other available resins. Natrix® Q is based on membrane technology and contains large convection holes. The latter promotes mass transfer and improves binding capacity (5-10mg/mL). Due to the membrane technology, a very short residence time (<0.2 minutes) is required. Because of these advantages, coupled with its single-use format, it has been widely adopted.

All four resins mentioned in the table above can be used to capture pDNA. However, only Fracture® EMD DEAE (M) and Fracture ® EMD-DMAE (M) resins are very suitable for plasmid DNA due to their moderate binding capacity and fluidity and good resolution of medium particle size (d50:48-60). The intermediate purification or polishing μm) can effectively remove residual impurities such as RNA and endotoxin.

Examples of HIC resins are Capto™ PlasmidSelect and Toyopearl® Butyl. The HIC step can be located before or after the AEX step. When the HIC step is the first chromatographic step, the eluate can be directly processed into Fractogel® resins, because the binding of pDNA to these AEX resins can tolerate the presence of high concentrations of ammonium sulfate (pDNA binding ability is not negatively affected).

An example of an experi

Final concentration and sterilization grade filtration

After chromatography, the sample is concentrated and refiltered in the selected buffer. The conditions are the same as those described in the tangential flow filtration section. Due to the large size and viscosity of the final product, the sterile filtration of pDNA is challenging. In addition, large plasmids can be shear sensitive and may be damaged during manipulation. Some key considerations include:

Salt concentration: When the salt concentration is increased, the plasmid DNA tends to be tighter. The latter can increase yield and filtration capacity.
Membrane type: Generally, when using PES base membrane, higher filtration capacity and flux can be obtained. PES-based membranes are also less damaging to larger plasmids (less shearing force). It is recommended to use Millipore Express® SHC for this operation.
Plasmid purity: Compared with open-circular plasmids, supercoiled plasmids have better filtration performance. Therefore, the yield and filtration capacity will increase with the increase in purity.
Determine the filtration end point: It is found that membrane fouling is related to production loss. Optimizing the filtration end point can increase the yield.
Feed flow or pressure has no significant effect on filtration capacity or output. However, high driving forces should be avoided to reduce potential shear stress.

Question 6 What kind of tests should we do to evaluate the quality of plasmid DNA? How does quality affect carrier production?

The quality and purity of plasmid DNA are critical to successful transfection. The focus is on phenol, endotoxin and sodium chloride. Phenol and endotoxin are harmful to cells, and salt can interfere with lipid complexation, resulting in decreased transfection efficiency.

To determine the quality, you can evaluate (but not limited to):

DNA concentration (OD 260 nm)
DNA purity (OD 260/280 nm or UV scan)
Endotoxin (limulus reagent test)
Osmotic pressure
Residual genomic DNA (agarose gel electrophoresis or qPCR)
Residual RNA (agarose gel electrophoresis)
PH
Ccc monomer content (HPLC or agarose gel electrophoresis)

Through our biocompatibility testing services, we provide a wide range of tests to help you evaluate the quality of plasmid DNA, from biosafety testing requirements (sterility, endotoxin, host cell protein, host cell DNA, sequencing, plasmid conformation) To product characteristics detection (molecular weight (ID) and mass spectrometry (MS), purity – SEC/IEX/RP (depending on the size of pDNA), concentration – A260/280)

Question 7 We are using benzoase treatment to remove the plasmid. Is this your suggestion or is there a better solution?

Removal of DNA is one of the key challenges in viral vector production. In order to meet the regulatory requirements of <10 ng DNA/dose and DNA size <200 bp, a good strategy is needed. A typical strategy consists of at least three different technologies:

DNA digestion using endonuclease
Tangential flow filtration to remove fragmented DNA
Chromatographic purification

Currently, Benzonase® endonuclease is considered the industry standard for DNA digestion in vector processing. It is a highly efficient endonuclease that can degrade all forms of RNA and DNA (single-stranded and double-stranded). One unit of Benzonase® endonuclease can degrade approximately 37µg-DNA to 3-8 base pairs in 30 minutes. Benzonase® endonuclease offers three different quality grades of endonucleases to meet the widest possible processing and cost requirements. For example, the highest purity grade is Benzonase® endonuclease for enhanced safety, which is GMP production and animal source free. Because it is part of Emprove®, it is backed by quality files that can help you quickly respond to regulatory challenges.

In order to optimize Benzonase® endonuclease, a small DoE experiment is recommended. The three parameters that need to be checked are concentration, incubation time and temperature. Examples of DoE settings can be found here. It is also best to check which step in the process is best to use Benzoylase® endonuclease, for example before cell lysis, after cell lysis or after the clarification unit.

There are several benefits of using endonucleases to digest DNA:

Limit viral nucleic acid complexes (due to changes in pI and/or retention time, making purification unpredictable), thereby increasing yield.
Protect downstream equipment from DNA contamination
Reduce viscosity

Question 8 Can you review the purification method of plasmid DNA? Do you have any good suggestions?

Question 5 above discusses and proposes different options and suggestions for purifying plasmid DNA. For more information and data on the optimal parameters of pDNA purification, you can refer to our white paper “Scalable Purification of Plasmid DNA: Strategies and Considerations for Vaccine Production”.

Question 9 How much raw materials are needed for production?

In terms of plasmid production, when using high replication number plasmids, an optimized fermentation process with a cell density in the range of 40-60 g/L can obtain a pDNA yield of about 1-2 g/L. For viral vector production, each 1L bioreactor requires 0.5 mg of pDNA to transiently transfect viral vectors (AAV or lentivirus). The 1L transfected bioreactor usually produces the 1E14 viral genome of AAV and the 3E9 viral genome of lentivirus.

Question 10 When do I need to invest in GMP plasmids? When can the research be conducted?

For the production of viral vectors, it is likely that FDA regulations will soon recommend the use of GMP-compliant plasmids in clinical batches. Plasmid is a key material attribute because it can affect the safety and quality of the final product. Therefore, it is strongly recommended to start using GMP-compliant plasmids when producing clinical batches (starting from the first phase). In addition, once the regulatory agency adjusts supervision, research-grade plasmids can be used for process development and preclinical batches. In nucleic acid vaccines, cancer therapy or gene therapy, pDNA is directly injected into the human body and requires GMP-grade plasmids.

Question 11 How to reduce the production cost of viral vectors, especially the cost of plasmid DNA?

There are several parameters that can affect the cost of viral vector production, such as cell line selection (adhesion and suspension) and process strategies (single use and multi-use solutions). The data shows that when the suspension culture and adherent culture are selected, the upstream cost can be reduced by 57%.

The disposable solution eliminates the need for cleaning and cleaning verification, and allows for greater flexibility and increased production capacity. Specifically, the cost reduction of pDNA production can be achieved through a combination of efficient downstream processing and one-time solutions. Question 5 discusses product recommendations for obtaining high yields in downstream processing.

Question 12 Do you recommend outsourcing or in-house production of plasmid DNA?

Today, if not all, most pDNA production is outsourced to specialized producers and CMOs. As the demand for pDNA in the biopharmaceutical industry continues to grow, the demand for suitable production capacity and expertise is also increasing, which increases the need to establish long-term solutions to meet future market needs and through investment in internal pDNA Production ensures the integrity of supply.

The cost of outsourcing is quite high and requires highly specialized knowledge and production facilities. At the same time, building a pDNA production facility with the required expertise and capabilities is also an expensive investment. Both options have pros and cons. To decide whether to produce plasmid DNA in-house or outsource, a strong cost evaluation study is required for each option, taking into account the required expertise and the availability of facilities to meet the company’s short- and long-term Vision.

For example, compared with companies that use pDNA as the final product of vaccines or direct gene transfer therapy, companies that use pDNA as a raw material for viral vector production or mRNA synthesis will have different visions and needs. Outsourcing can benefit from the required capabilities and quality, efficiency, productivity and faster time to market. However, it is important to carefully select partners based on their on-site experience, quality management system, timetable, and successful supervisory inspection history. Outsourcing work is an extension of your business, so it is very important to find suitable partners with similar values to your company.

(source:internet, reference only)

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How to optimize the production of Plasmid DNA for cell and gene therapy?

How to optimize the production of Plasmid DNA for cell and gene therapy?

Question 1 Are there any key media components that can increase productivity?

Question 2 My plasmid DNA is unstable. We want to try to improve the stability. Do you have any suggestions for this?

Question 3 Why are GMP plasmids so expensive and in short supply?

Question 4 How to choose the best plasmid for my gene?