Upstream Optimization for Plasmid DNA Manufacturing

Plasmid DNA manufacturing is increasingly needed. The pDNA-based cell and gene therapies treatments emerging in both industry and academia are growing. The need for novel treatment options to combat cancer, metabolic disorders and other infectious diseases is increasing on a global scale. 

 In this article, we will analyze the scalable process workflow solutions available for plasmid DNA manufacturing, from initial R&D investment to clinical testing, and commercialization. We will focus on culture medium and fermentation protocols. 


Culture medium is a critical step in pDNA Manufacturing

Culture medium selection during upstream optimization directly contributes to the quantity and quality of pDNA produced in E. coli. 

 The implementation of various media can result in differences in the specific growth rate, plasmid copy number, stability of the pDNA product and the amount of biomass produced. Therefore, optimizing parameters that contribute to high quantity and quality of pDNA is pivotal to ensuring financial and operational efficiency.  

We have experience generating protocols based on growth parameters focusing on maintaining consistency between batches. Using a blend of consistent nutritional supplements during the early research stages can help to mitigate variability risks during pDNA production. 

In the early stages of research, inoculum development, media development and growth kinetic parameter monitoring allow us to fine-tune their protocols with the end goal of efficiently increasing yields while maintaining target quality parameters. In this phase, safety is a key pillar of regulatory focus. 

The industry trend is to move toward animal origin–free components to avoid safety risks, creating a need for decreased components or even completely chemically defined medium. This allows researchers to eliminate risks that may arise due to unknown components from the originator species. 


Fermentation for pDNA production

When microbial culture processing increases volumetrically, a more complex and controlled environment is needed. Here amplification will move from flask containment to larger scale fermenters in our facilities.

This fermentation equipment, in addition to protocols and monitoring factors, will directly contribute to the amount of pDNA recovered from wet biomass. Not only that, because the success of microbial fermentation processes is also dependent on the growth environment including temperature, pH, dissolved oxygen, agitation, and mixing speed. These process-monitoring components impact the quality and percent yield of the final pDNA product, therefore a shift toward predictive reproducibility between batches can offer time and cost savings. 


At 53 Biologics, we have a GMP manufacturing line of pDNA up to 200 L


An added resource for pDNA production success is using single-use tanks. They help reduce batch turnover activities, eliminating the time-consuming clean-in-place (CIP) and sterilization-in-place (SIP) cycles, helping to decrease costs and increase productivity.  Also, the automation can be used as a process control feature eliminating the need for taking repetitive samples and therefore limiting the potential risk to the bulk mix. 


You might be also interested in reading: Plasmid DNA Production for Cell and Gene Therapy – Q&A



As we mentioned at the beginning of this article, the global demand for novel treatments in the therapeutic areas of cancer, metabolic disorders and other infectious diseases is rising.  

This increase of the demand for reproducible, high-quality, and customizable bioprocessing material has caused a bottleneck in pDNA manufacturing.  

The bulk production of pDNA requires the continuous monitoring of important production parameters. On the other hand, culture medium and fermentation protocols are pivotal in maintaining a high-quality optimal percent yield while minimizing contaminants that will need to be removed during downstream process steps.  

Finding a way to increase manufacturing capacity while maintaining high-quality standards poses a unique challenge. 53Biologics excels in the ability to maintain supply chain integrity while sourcing consistently high-quality raw materials and providing customers with cGMP scale-up support. 


Would you like to contact our team to analyze your plasmid DNA manufacturing? Contact us!


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