In cell and gene therapy manufacturing, high-quality plasmid DNA is a key component. The demand of high-quality plasmid DNA has increased and this has led to the need to optimize the manufacturing and the quality required for use in the manufacture of therapeutics. However, the plasmid DNA (pDNA) manufacturing faces several challenges like:

• Its large size.
• The shear sensitivity.
• The high viscosity.
• The similarities between the pDNA and the impurities present during manufacturing.

This makes important to understand all areas of the process to successful pDNA manufacturing at a large scale. In this article we are going to answer the questions on pDNA production for cell and gene therapy applications.

Let´s start!

1. How do I select the best plasmid for my gene?

There are two main types of vectors:

• Cloning vectors: They are ideal for the generation of many copies of your gene, that is why, the key aspects are the copy number (depending on the ori), selectable marker, and the cloning sites. Generally, a high copy number is preferred, but when the gene is toxic for the cell, or when the plasmid is unstable, a low copy number might be more favorable. The selectable marker allows for the identification of a positive transformant. Most of the time these will be drug-resistance markers, but also auxotrophic markers are used. It is important to check whether the vector contains a cloning site which is suitable for the insert. Right now, most vectors contain a multiple cloning site, which makes it likely that the vector is compatible with the selected restriction enzyme.
  On the other hand, if the goal is to express the gene of interest, an expression vector is needed.
• Expression vectors: They contain some additional sequences related to the expression, e.g., promotor, ribosome binding site, terminator, tag or fusion protein. Some of these sequences are specific to the host organism. Therefore, the expression vector needs to be compatible with the chosen host organism (e.g. mammalian, insect, E. coli).

53 Biologics is expert on Expression vectors

2. Are there any key media ingredients that improve productivity?

We have to consider several parameters that can affect the bacterial productivity like the master cell bank selection, the growth rate, the culture medium, the feeding rate as well as the appropriate growth conditions and parameters like pH, osmolarity, optical density and temperature.

Generally, the media composition consists of:

• A carbon source, where glucose is conventionally used or glycerol that can also aid in supporting reduced maximum specific growth rates.
• A nitrogen source, that is typically supplied by adding complex components such as yeast extract, casamino acids, or peptones.
• Magnesium sulfate and potassium phosphates, that are added in order to provide a magnesium, sulfur, potassium and a phosphorous source (the phosphates also function as buffering agent).
• Minerals that are present in the complex components of the medium or can be added via specific trace mineral solutions.

You can use minimal or semi-defined media. With minimal media, highly reproducible batches can be obtained, but semi-defined media can support higher cell densities as the complex components (e.g. yeast extract) supply growth factors, amino acids, purines and pyrimidines.

3. How much starting material is required for production?

For plasmid production, an optimized fermentation process with cell densities ranging from 40-60 g/L can achieve pDNA yields around 1-2 g/L when a high-copy number plasmid is used.

For viral vector production, 0.5 mg of pDNA is required for transient transfection of viral vector (AAV or lentivirus) per one 1 L bioreactor. 1 L of transfected bioreactor typically yields 1E14 viral genomes for AAV and 3E9 viral genomes for lentivirus.

4. My pDNA is unstable, what we could try to improve stability?

You have to take into account these aspects:

• On one hand, it is important that the sequence of both your gene of interest (insert) and plasmid (vector) are well optimized. For example, if the insert is very large and/or contains inverted tandem repeats, you have to choose a small vector.
• On the other hand, changing the growth conditions (e.g. growing at lower temperature), selecting an alternative host, optimizing the process conditions as well as choosing the right buffers for processing, formulating and storing your pDNA, can also help improving its stability.
• Another aspect that have great impact on pDNA stability is DNAse contamination and pH . DNAse can degrade and digest the DNA double strands while extreme pH can break, denature and even change the pDNA sequence. That is why, selecting the right buffer and solution for your pDNA is fundamental for preserving the stability of your product for a long time.
• For storage, the pDNA is usually stored at -20°C to – 80°C where it can stay stable for years. For short period, you can storage at 4°C or at room temperature.

5. Why is GMP plasmid so expensive and in short supply?

The reason why GMP pDNA production is more expensive is because:

• The raw products used during GMP manufacturing are of higher purity, which comes at higher price tags.
• Dedicated production areas are required (e.g. cleanrooms).
• Validation is needed for cleaning methods
• Validation analytical methods are needed to confirm that the final GMP product is of high purity.

Another aspect that affects the cost is due to the short supply. In the last two years a big increase has been reported in the number of investigational new drugs and multiple drugs are being licensed for commercial distribution. Due to this increase, contract manufactures which provide GMP-grade plasmids have difficulties to keep up with the increasing demand. The recent pandemic has exacerbated the problem, as a lot of vaccine candidates are being developed, including DNA-based vaccines and mRNA-based vaccines where the plasmid DNA is required as starting material for the in vitro transcription process of mRNA.

6. When do you need to invest in GMP plasmid?

In clinical studies (starting from phase 1), it is highly recommended to start using GMP compliant plasmids. It is increasingly likely that the FDA regulations will soon recommend the use of GMP compliant plasmids for clinical batches, because plasmids are a critical material attribute, as it can impact the safety and quality of the final product.

On the other hand, for process development and pre-clinical batches, research-grade plasmids can be used.

7. What kind of testing should we be doing to evaluate our pDNA quality and in what ways can quality affect vector manufacturing?

The quality and purity of plasmid DNA is of high importance for a successful transfection.

In order to determine the quality, one could assess (but not limited to):

• DNA concentration (OD 260 nm)
• DNA purity (OD 260/280 nm or UV-scan)
• Endotoxin (LAL test)
• Osmolality
• Residual genomic DNA (agarose gel electrophoreses or qPCR)
• Residual RNA (agarose gel electrophoresis)
• pH
• CCC monomer content (HPLC or via agarose gel electrophoresis)

Also, you have to watch outs phenol, endotoxins and sodium chloride. Phenol and endotoxins can be harmful for the cells and salts can interfere with lipid complexing, which results in a decrease in transfection efficiency.

At 53 Biologics, we offer a wide range of testing to help you assess the quality of your plasmid DNA

8. How can we reduce the cost of our viral vector manufacturing?

The costs viral vector manufacturing can be affected by several parameters, such as the choice of cell line (adherent versus suspension) and the process strategy (single-use versus multi-use solutions).

Data suggest that the upstream costs can be lowered by up to 57% when opting for suspension culture versus adherent cultures. Single-use solutions eliminate the need for cleaning and cleaning validation and allow for higher flexibility and increased manufacturing capacity.

9. What is more recommending, outsourcing or manufacturing pDNA internally?

Both options have pros and cons and making decision about manufacturing your pDNA in house or outsourcing it would require a strong cost assessment study for each option taking into consideration the availability of the required expertise and facility in alignment with the company short- and long-term vision.

For example, a company using pDNA as raw material for viral vector production or mRNA synthesis would have a different vision and need compared to a company using pDNA as a final product for vaccine or direct gene transfer therapy. In other words, outsourcing the core activity of a company is different from outsourcing part of the process.

The cost of doing it in house can be high because you will require highly specialized expertise people and manufacturing facility for pDNA production.

Outsourcing enables to benefit from expertize of specialized manufacturers like us.  However, it is important to carefully select the partner based on the experience they have on the field, quality management systems, timelines and successful regulatory inspection history. Outsourced work is an extension of your business, so it is important to find the right partner with similar values as your own company.

With the increasing demand of pDNA within the biopharma industry, demand for appropriate manufacturing capacity and expertise is also increasing. This raises the need to build long-term solutions to be ready to cover future market needs and ensure supply integrity by investing in in house pDNA manufacturing.

53 Biologics is expert on plasmid DNA Production for Cell and Gene Therapy. Would you like to speak with one of our experts? Let´s talk!

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