Plasmid DNA (pDNA) Manufacturing Process: Downstream Purification

Plasmid DNA (pDNA) is an essential component of, and key technology for production of viral vector, mRNA, and vaccine therapies. The far-reaching potential of novel therapeutic modalities and vaccines that rely on pDNA is driving increased demand and the need for improved production strategies.

• Overview of the Plasmid DNA Manufacturing Process
• General Process for pDNA Manufacturing
• Challenges in pDNA Production
• Designing a Plasmid DNA Downstream Purification Process
• Related Products

Overview of the Plasmid DNA Manufacturing Process

The starting material for pDNA purification after lysis has a complex composition with no more than 3% of the content being pDNA while the remaining 97% represent impurities. Thus, pDNA production presents several challenges. Our plasmid DNA downstream purification website provides you with guidance to optimize and streamline your plasmid DNA downstream process development, in addition to representative data. 

General Process for pDNA Manufacturing

The manufacturing schemes for pDNA were first developed in the mid-1980s and have since relied on well-established traditional production processes, typically fermentation using a microbial source, usually E. coli.

The general downstream process development for pDNA manufacturing includes the following steps:

Cell Harvest, Cell Lysis, Clarification

Cell harvest: After fermentation, the cells are harvested by centrifugation or microfiltration tangential flow filtration (MF-TFF).

Cell lysis: The cell lysis step breaks apart the cell membranes. What remains is a mixture of cellular contents including plasmid DNA, genomic DNA, proteins, RNA, and cell debris. The first step to removing these contaminants includes precipitation and flocculation.

Clarification: Clarification removes solid content that arose during chemical lysis and neutralization from the feed stream.

• Technical Article: Cell Harvest, Lysis, Neutralization and Clarification of Plasmid DNA (pDNA) for mRNA, plasmid-based DNA Vaccines, and Viral Vector applications

Chromatography (1-2 steps)

Chromatography: Anion exchange chromatography and hydrophobic interaction chromatography are the most used chromatography methods for plasmid purification. Size exclusion chromatography can be used as the last chromatography step.

• Technical Article: Chromatographic Purification of Plasmid DNA (pDNA) for mRNA, plasmid-based DNA

Ultrafiltration/Diafiltration (UF/DF)

Ultrafiltration/diafiltration: Tangential flow filtration separates, concentrates, washes, and resuspends the plasmid DNA in an appropriate buffer.

• Technical Article: Ultrafiltration/Diafiltration (UF/DF) in Plasmid DNA Manufacturing

Final Sterile Filtration

Final sterile filtration: Final sterile filtration removes any microbial contaminants that may have been introduced during processing. 

• Technical Article: Sterilizing Grade Filtration Unit Operations for Plasmid DNA Processes

The final bulk pDNA must meet quality specifications set by regulatory agencies and should be free from impurities.

Challenges in pDNA Production

With the growing demand for pDNA in the production of viral vectors, mRNA, and vaccine therapies, upstream productivity must be increased to achieve yield and efficiency goals, ensure robust impurity removal and maximize downstream recovery, all while ensuring patient safety. The starting material for pDNA purification after lysis has a complex composition with no more than 3% of the content being pDNA while the remaining 97% represent impurities. Thus, pDNA production presents several challenges.

Plasmids are Large and Negatively Charged

Production suffers from low productivity of microbial fermentation and the purification process is complicated by the fact that plasmids are quite large and possess a highly negative charge. The bacterial lysate contains contaminants with properties similar to pDNA. Most of the critical impurities are negatively charged (RNA, genomic DNA, endotoxins), similar in size (open circular pDNA, genomic DNA, high molecular weight RNA), and hydrophobic (endotoxins). These similarities can lead to low resolution separation.

Bacterial Lysates are Highly Viscous

Bacterial lysate can be highly viscous. A low flow rate is needed for chromatography, and it can be difficult to achieve the desired concentrations at the final tangential flow filtration (TFF) step.

Plasmids are Sensitive to Mechanical Damage

pDNA is sensitive to mechanical damage, which can lead to changes in its topological form. Plasmid isoforms include supercoiled (fully intact and wound around itself), open circular (one strand is broken and the molecule relaxes) and linear (both strands are broken with free ends). Supercoiled plasmids are recognized as the most therapeutically relevant, and regulatory agencies set expectations for the supercoiled percentage in final drug substance for DNA vaccines.

Designing a Plasmid DNA Downstream Purification Process

Our plasmid DNA downstream purification eBook provides you with guidance to optimize and streamline your plasmid DNA downstream process development, in addition to representative data. We explore Cell Harvest, Lysis, Neutralization and Clarification; Chromatographic Purification; Tangential Flow Filtration (TFF); and Sterile Filtration unit operations.

This guide also includes data from our collaboration with a biotechnology company focused on the design and development of RNA-based therapeutics and products.

Learn more about the Emprove^® Program, helping maintain compliance with current Good Manufacturing Practices.