Plasmids are small double-stranded circular DNA molecules containing extrachromosomal genetic information that exist in archaea, bacteria, and some yeasts. They contain at least one replication origin and can replicate independently. A cell can contain multiple copies of a single plasmid. The genetic information of the plasmid backbone sequence may not be necessary, but at least helps the cell survive in its given environment.

Over the past few decades, plasmid DNA (pDNA) has become one of the most important tools in genetics and molecular biology, and more recently gene therapy and nucleic acid vaccines. In pharmaceutical applications, the use of plasmids includes both direct and indirect ways, such as direct administration of plasmids to patients, that is, the patient will receive the DNA itself, for such applications, including DNA vaccines, complete Good Manufacturing Practice (GMP) is essential requirement because DNA itself is the active pharmaceutical ingredient (API). Another way is the indirect way, that is, the pDNA is not used directly for patient administration, but is used to produce viral vectors or therapeutic proteins for transfected cells, or used as a template to produce mRNA, that is, it is not an API, but produced as raw materials or starting materials, even in this case the plasmid product is subject to specific regulatory requirements (in terms of quality control and process documentation) and compliance with applicable standards.

Plasmid preparation in scientific research laboratories often uses commercially available extraction kits, but this method is not suitable for industrial production that requires several milligrams or several grams, and more importantly, the plasmids prepared by laboratory methods have low reproducibility in terms of purity and safety, which is a prerequisite for pharmaceutical applications. Given the widespread use of pDNA in the development of current advanced therapies, it has become a key material in next-generation biopharmaceutical applications, and the industry's demand for high-quality pDNA is increasing. Therefore, its production must achieve higher efficiency and productivity.

Host cell selection

The selection of host cells is one of the decisive factors in biotechnology, and the production of plasmids is strain/plasmid dependent, so it is very important to select a suitable strain for a fit-for-purpose plasmid. The characteristics of a suitable host strain include the ability of being transformed by plasmid and its feasibility to grow to high biomass. Due to the large amount of available biological information of Escherichia coli (E.Coli) and its use in pDNA production, a variety of strains have been selected for pDNA production based on laboratory use and commercial availability.

Furthermore, since the introduction of pDNA into E. coli results in a metabolic burden, usually reflected in a decrease in growth rate and biomass, and an increase in acetate production. Understanding the mechanism by which plasmid production is affected at the overall physiological level is not a small challenge, but this knowledge can provide useful information for further cell engineering, such as gene knockout or gene overexpression, to alleviate metabolic burden of host organisms. or better understand the host mechanism for pDNA replication, thereby increasing pDNA yield. However, engineering of host cells can also lead to excessive modifications, making it difficult to control and predict the physiological activities occurring in the host. The ability to control host cells is very important in large-scale operations.

For GMP production, master cell banks (MCB) and working cell banks (WCB) need to be established to ensure reproducible large-scale cultivation of bacterial organisms, cell banks need to be fully characterized and meet applicable quality standards, including host identity , quality and yield consistency, purity of cell bank and inspection of containers, and properly stored for subsequent production.

Fermentation strategy

Well-established fermentation techniques for recombinant protein production are equally applicable to the production of pDNA. However, since various therapeutic uses require large quantities of plasmids, there is a need to increase the production efficiency of pDNA. Various approaches have been proposed to improve pDNA productivity, including pDNA volumetric yields and bacterial cell-specific productivity.

Multiple parameters affect bacterial productivity such as master cell bank selection, growth rate, media, feed rate, and appropriate growth conditions and operation parameters such as pH, osmolarity, and temperature.

The currently optimized E.coli fermentation can usually reach 40 - 60g/L biomass, and the pDNA yield can reach 0.5 - 1 g/L. Combining host cell and vector engineering, and further optimizing the fermentation strategy, it may reach > 2 g/L .

Fermentation scale-up requires maintaining optimal and homogeneous reaction conditions to minimize E. coli exposure to stress conditions and improve metabolic accuracy, thereby increasing yield and ensuring consistent product quality. A comprehensive and detailed process characterization is required to identify the most relevant process parameters affecting product yield and quality and keep them as constant as possible during scale-up. Process characterization can be performed with real-time or near-real-time data collected from in-line or at-line monitoring, combined with supporting methods and tools, such as chemometric data analysis and modeling, and elucidation of mixing and flow conditions. To ensure straightforward and successful scale-up, fermentation facilities need to be designed strictly according to scale-up standards to reduce the variability of process conditions in different tanks, while fully considering the highly complex interdependence and interaction between fermentation parameters.

Cell harvest and lysis

Centrifugation is the most common way to harvest E.Coli broth. Under laboratory-scale conditions, it can realize simple and fast process. For production scale, large-scale disc centrifuges or continuous flow centrifuges can be used. The challenge is high capital cost investment, high energy consumption, and manual steps that can be cumbersome and need open operation. Tangential flow filtration is another strategy that is easily scalable and enables continuous closed operation, during which the media is exchanged to the buffer system required for subsequent lysis step.

Mechanical lysis methods used in the production of recombinant proteins can be used for the extraction of nucleic acids. However, the high shear conditions used in protein processing can lead to nucleic acid degradation. So, the most commonly used method of cell lysis here is alkaline lysis, usually performed with ~pH 12, 0.1 - 0.5 N NaOH solution, combined with 0.1-0.2% SDS or Triton X 1-00, to disrupt cells by leveraging the fragility of the cell membrane , and maintain intact pDNA. Scale-up of this method can be challenging, as increased viscosity can lead to shearing of pDNA during mixing. In addition, pH gradients may exist under large-scale conditions, ensuring adequate but not too vigorous mixing. The time of the lysis process can also directly affect the quality and quantity of pDNA. If too long, it may cause irreversible denaturation of pDNA. Therefore, well-designed mixing equipment and carefully controlled lysis parameters are the keys to success.

Clarification

After cell lysis, solids will need to be removed. Due to the viscosity of the lysate, this will be a very critical step. Impurities can be removed using centrifugal or depth filters of specific pore size. More recently, flocculation has been reported as a pretreatment step in filtration operations. There are also strategies to remove contaminants by precipitation using high-salt buffers and chaotropic agents. Usually, in the lysate of bacterial cells after clarification, only about 3% of the content is pDNA, while the remaining 97% is other impurities, including 65% protein, 25% RNA, 4% endotoxin and 3% genomic DNA, this complexity is also the challenge of pDNA downstream process, other challenges include its own large particle size, high viscosity, shear sensitivity and similarity with impurities.

Tangential flow filtration

Tangential flow filtration is a simple and cost-effective pDNA concentration and buffer exchange technology that can be easily scaled up. It can partially remove impurities such as linear DNA, RNA, and endotoxin through the selection of a suitable MWCO, and retain pDNA. One challenge of tangential flow filtration operations is the increasing viscosity of the feed solution during this step and the shear sensitivity of pDNA. And in some cases, due to the structural properties of pDNA or the lower effective particle size due to higher ionic strength, pDNA may pass through membrane pores smaller than its particle size and cause loss. Therefore, given the 3-5X rule of thumb for membrane pore selection, the lower end of this range can be chosen to ensure yield.

Chromatography

The final pDNA must meet regulatory quality requirements, such as being free from host cell proteins, genomic DNA, RNA, and endotoxins, and may require >90% supercoiled pDNA for specific applications, so chromatography steps are included in the pDNA process flow as basic requirement to produce efficient and safe pDNA. Given the complexity of the impurities and their similarity to the target pDNA, a combination of different modes of chromatography strategies can be used.

Anion-exchange chromatography (AEX) utilizes electrostatic interactions between negatively charged plasmids and positively charged stationary phases to selectively separate supercoiled pDNA from open-circular pDNA and other components of the lysate, but biomolecules with similar charge and structure, such as gDNA, endotoxin, and some RNAs will co-purify. A strategy often used in combination with AEX is hydrophobic interaction chromatography (HIC), which leverages the hydrophobicity of single-stranded nucleic acid impurities (RNA, denatured gDNA, and pDNA) and endotoxins to facilitate binding and hinder these impurities through hydrophobic supports , which separates pDNA from endotoxins and single-stranded nucleic acids, but requires high salt concentrations for elution. Affinity chromatography (AC) can obtain high-purity supercoiled pDNA in a single purification step and can ensure that the product is within the standards of regulatory requirements, which utilizes natural biological processes based on molecular recognition, selectively purify supercoiled pDNA, However, the biological ligands they use can be unstable and usually have low binding capacity. Another method that does a good job of removing RNA and protein is size exclusion chromatography (SEC), which takes advantage of the difference in hydrodynamic particle size between pDNA and other impurities, but is challenging to remove gDNA.

Sterile filtration

Sterile filtration is designed to retain bacteria and ensure sterility. The larger particle size, purity after purification, and buffer composition of pDNA can affect filter throughput, resulting in lower flow rates and step yields. Before selecting a filter with a suitable specification, these factors need to be considered comprehensively to maximize the filtration performance of the sterilizing grade.

Storage

pDNA products intended for direct or indirect use in pharmaceutical applications require appropriate storage systems that can monitor and report storage conditions to comply with full GMP requirements. Specific evaluation studies are required to determine optimal storage conditions. For pDNA stored under controlled conditions at -20°C, analysis shows that the ratio of open and supercoiled or covalently closed circular forms remains unchanged, there is no linearization or degradation of pDNA, and transfection of DNA shows expression unchanged, that is, the storage conditions effectively preserve the integrity of the DNA.

General quality requirements

Regulatory agencies and/or purchasers of pDNA will have different quality standards and expectations given the different uses of pDNA, such as raw materials/key starting materials, intermediates, drug substrates, or drug products in drug development, but include DNA concentration, bioburden/sterility, endotoxin (eg <0.1 EU/ug plasmid or <5 EU/kg bw), purity - residual host cell protein (eg undetectable or <0.01 μg/dose), genomic DNA ( Release testing such as <0.05 μg/ug plasmid or <0.01 μg/dose), RNA (if undetectable) – and consistency are common requirements.