Plasmids—Part 2: Perfecting Plasmid Production for IVT mRNA Manufacturing

Plasmids—Part 2: Perfecting Plasmid Production for IVT mRNA Manufacturing
Posted in: Therapeutics

  • Plasmid DNA-Encoding mRNA Is a Key Starting Material for IVT mRNA  
  • cGMP-Quality Plasmid DNA Ensures IVT mRNA Quality 
  • The Large Quantities of Plasmid DNA Needed May Surprise You 

This post is Part 2 of a series of Zone blogs featuring DNA plasmids used to produce in vitro transcribed (IVT) mRNA. Part 1, posted on June 8, 2021, provided historical perspectives on the discovery of plasmids, as well as descriptions of how these circular double-stranded DNAs enabled recombinant DNA technology and now IVT mRNA production. The two growth phases of plasmid applications are reflected in this chart of publications in PubMed indexed to gene therapy and genetic vaccination for two 10-year periods: 1991–2000 and 2012–2021, respectively. 

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PubMed search query ("gene therapy") OR ("genetic vaccination"), 2021 estimate, and chart by Jerry Zon.

This Part 2 follow-up discusses how key steps in the manufacturing of plasmid DNA for IVT mRNA have been perfected in the context of current Good Manufacturing Practice (cGMP) guidelines acceptable to the Food and Drug Administration (FDA) or other national regulatory bodies. The cGMP environment should be implemented independently of the intended use of the DNA product. In all cases, the ability to use plasmid DNA free of any other materials is essential. Contaminating materials include components used in the DNA isolation process and material originating from the host organism used in plasmid propagation and isolation (i.e., residual proteins, RNA, and genomic DNA).

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In Part 1, the general aspects of plasmid DNA manufacturing were briefly summarized in this flow chart of a generic, high-level industrial production process. Now, in Part 2, the various steps in this process will be discussed in more detail, including Quality Control (QC) and examples from published sources. In general, all cGMP plasmid DNA production steps are performed under well-documented conditions and, when appropriate, under controlled environmental conditions. 

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Flow chart of a generic, high-level industrial production process for manufacturing plasmid DNA. Adapted from Urthaler et al. and drawn by Jerry Zon.

As a final introductory comment, this blog is not intended to be comprehensive in scope or depth. Instead, relevant reviews will be cited as supplemental information. Readers interested in regulatory guidelines for cGMP of plasmids for IVT mRNA should consult applicable authoritative sources, such as the FDA Vaccine and Related Biological Product Guidances available at this link and other related documents not discussed herein.

Plasmid Construction and QC

Although they differ in detail, plasmids for IVT mRNA are comprised of certain common functional features (e.g., selection marker, promoter, inserted gene, etc.), and they are generally constructed using well-known methodologies. Nonetheless, plasmid customization based on final application is especially important for the design and sequence optimization of the cDNA that encodes the mRNA of interest. Such optimization has been the subject of recent Zone blogs that can be accessed here. Among the numerous available resources for consultation, the Zone found two exemplary items that provide helpful introductions: 1) a short YouTube video titled Plasmid Vectors, presented by Dr. David Smith from Sheffield Hallam University, and 2) a lengthy PDF titled Plasmids 101: A Desktop Resource, posted by SanDiego.edu.

Because of the need to start with a perfect plasmid, i.e., the plasmid of interest comprised of the exact sequence of DNA bases, the identity must be verified by appropriate QC data (e.g., size, restriction pattern, sequence, etc.) before releasing the plasmid for further processing.

Host Cell Selection

A single appropriate bacterial host strain for all research work or industrial scale pharmaceutical manufacturing does not exist, according to an authoritative review by Schorr et al. titled Production of Plasmid DNA in Industrial Quantities According to cGMP Guidelines. The researchers add that considerable experience in molecular cloning and DNA techniques has been obtained using Escherichia coli (E. coli), as different strains have demonstrated significant qualitative and quantitative differences among all sub-strains tested. Readers interested in such details can consult a review by Gonçalves et al. titled Rational engineering of Escherichia coli strains for plasmid biopharmaceutical manufacturing.

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Using E. coli strain K12, which fulfills most requirements for a safe, well-characterized host strain for DNA production, Schorr et al. outline the typical methods used to transform E. coli K12 host cells with a QC-verified plasmid of interest. This process is then followed by selecting individual colonies for small-scale cell culturing and characterization to choose those with high-plasmid yield and correct plasmid isoform distribution, i.e., supercoiled and not open circle or linear.

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Three forms of plasmid DNA. Taken from commons.wikimedia.org and free to use.

Typically, the selected clone is subjected to two single-colony passages and then checked for identity and absence of microbiological contaminants. Then, the verified clone is used to inoculate a culture and prepare a glycerol stock of the desired number of vials (e.g., 100–500). This stock is called the Master Cell Bank (MCB) and is required for the reproducible inoculation of culture media from the MCB in the subsequent process step and any future manufacturing run. In addition, as with most biological macromolecules, glycerol allows for ultra-low temperature (-80 °C) long-term storage.

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Generally, the MCB is subject to an extensive quality assurance (QA) program that checks for identity, quality, plasmid content, and absence of microbiological contamination. At this stage, an important additional requirement is the complete sequencing of the DNA construct, which enables the exclusion of any difference to the original plasmid and as well as a data backup for post-production sequencing.

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Vials of the MCB are then used to inoculate a fresh culture to typically produce an equally large set of stocks (e.g., 100–500 vials) required for the reproducible inoculation of the fermentation precultures. This second glycerol stock is called the Manufacturing Working Cell Bank (MWCB), and it is subject to the same tests for QA/QC as the MCB.

Growth Conditions 

Bacterial cultures for plasmid production are generally conducted under rigorously controlled conditions, such as the exact composition of culturing medium, a specific culturing vessel and mixing technique, and a controlled atmosphere, temperature, and time. Fermentation processes require different growth media than batch cultures. The ability to monitor growth conditions enables the introduction of essential media components before they are exhausted (feeding) and allows for the maintenance of a constant pH and oxygen supply.

Plasmid production for exploratory investigations or early-stage R&D requires relatively small-to-medium-scale plasmid production. It is typically undertaken using bacteria host cell growth in classic shake flasks (shown here; see also). These flasks may be used to produce plasmids from bacteria like E. coli, and they utilize volumes ranging from 250 mL to 10 L. Typical shake flasks may be made of glass or plastic, with glass flasks being multi-use and plastic flasks intended for single-use applications. Thus, thorough cleaning of glass flasks is required to ensure sterility, and single-use plastic flasks are often not cost-effective options. In addition, the large-bottomed shape of shake flasks requires expansive space in laboratory incubation equipment, which may reduce the efficiency of the plasmid production process. Furthermore, openings at the top of these flasks used for respiration can result in unintended cross-contamination from other flasks or the external environment.

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"Cell culture bags" are commercially available, and they represent an alternative to shake flasks. However, these are intended for mammalian cell culture, and they can be relatively costly for the practical production of plasmids by bacterial cell culture. In addition, the materials that culture bags are manufactured from are so thick that they fail to promote respiration of the cultured cells. Consequently, inexpensive gas-permeable low-density polyethylene (LDPE) bags have recently been reported for plasmid production by bacterial cells. They are sealable to the external environment while permitting respiration of the cultured cells. 

Unsurprisingly, the scale of bacterial cell culturing is much greater for cGMP manufacturing of plasmids used for IVT mRNA production for large clinical trials and approved vaccines or therapeutics. This is now widely recognized in the context of the Pfizer-BioNTech and Moderna IVT mRNA vaccines against COVID-19. In these cases, the hundreds of millions of doses required unprecedented quantities of plasmid DNA. For example, a recent editorial states that "delivering one billion doses of an mRNA vaccine may require the production of in excess of 1 kg of DNA." While this estimate may be somewhat high or low relative to what is actually required for the current mRNA vaccines against COVID-19, the quantity of plasmid DNA is substantial, as it impacts operating scales in all processing steps from plasmid manufacturing to vialing of the vaccine. 

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As a final comment on scale, an expert review states that "[i>

f a fermentor [aka bioreactor>

is available (12–20 L working volume), then it is relatively easy to generate between 400 and 600 g of biomass, which for an average plasmid should equate to between 200 and 300 mg of purified DNA." If this is accurate, then 12,000–20,000 L working volume leads to between 200 and 300 g of purified plasmid DNA. 

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Industrial bioreactor mechanical for aeration and agitation, 3D rendering.

Downstream Processing

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Lipid A bacterial endotoxin, molecular model. Atoms are represented as spheres with conventional color coding: hydrogen (white), carbon (grey), oxygen (red), phosphorus (orange).

The isolation of a biomolecule from the bacterial culture, usually referred to as downstream processing (DSP), requires the separation of plasmid DNA from other undesired components present within the source of the material. These undesirable components include bacterial genomic DNA, RNA, proteins, lipids, lipopolysaccharides (LPS, aka endotoxins), components of the cell wall, and intact bacteria. According to Schorr et al., endotoxins are major contaminants of nucleic acids, especially plasmid DNA preparations. Due to their negatively charged phosphate groups, depicted here, endotoxins tend to co-purify with nucleic acids. Furthermore, LPS contamination of DNA has been shown to have a direct influence on transfection efficiency into many types of cultured cells, and different cells show variable sensitivity to this contamination.

Based on the original rapid, efficient, and scalable plasmid "miniprep" method Ish-Horowicz and Burke published in 1981 (cited more than 2,000 times according to Google Scholar, see Footnote at the end of this blog), a modified alkaline lysis procedure is typically used to isolate plasmid DNA from E. coli cells. By way of an example for isolating 100 mg of plasmid DNA, Schorr et al. start with 60 g of wet weight biomass suspended in 100 mL of Buffer 1 (50 mMTris-HCl, pH 8.0/10 mMEDTA) (TE) in a 5-L glass bottle, followed by addition of 100 mL of Buffer 2 [200 mMNaOH, 1% sodium dodecyl sulfate (SDS)>

, mixing, and incubation at room temperature for 5 min. Then, 100 mL of Buffer 3 (3.0 Mpotassium acetate, pH 5.5) is added, mixed, and incubated for 30 min at room temperature "to allow the flaky white precipitate of sodium dodecyl sulfate (SDS), protein, genomic DNA, and cell residue to rise to the surface," from which the lysate is removed and filtered for subsequent chromatography.

According to Schorr et al., the most important feature of the technique is the aggregation of most of the undesired components mentioned above, which can then be easily removed by centrifugation (research-scale) or floating and filtration (research and industrial-scale). 

Chromatography

There are many different kinds of chromatographic materials, columns, detectors, and pumps that can be used depending on impurities of concern, resolution, scale, and more. According to Ballantyne, however, the four most applicable modes of chromatography for plasmid purification are anion exchange, reverse phase, hydrophobic interaction, and size exclusion, as will be discussed below. The last three are primarily used as a second or polishing step, whereas anion exchange is best suited for the initial capture mode. However, if enough work is put upstream (i.e., selective precipitation and diafiltration), the plasmid generated with anion exchange purification may be of high enough quality for many uses.

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Anion Exchange Chromatography: This mode of separation, usually based on a tertiary or quaternaryamine functional group, is the most widely practiced method of plasmidcapture. The functionalities are usually bound to the support through a predominantlyaliphatic linkage, and they are typically described as weak (e.g., dimethyland diethyl amino) or strong (e.g., trimethyl amino, exchangers). ThepKa of the functional group increases as the degree of substitution and substituentsize increase, and it generally ranges from 9 to 13. Although using the strongest anion-exchange functions is appealing due to increased bindingcapacity, efficient elution can be challenging to achieve because of the greaterstrength of the plasmid-amine interaction. For further details, see the review by Ballantyne.

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Ion-exchange column. Taken from commons.wikimedia.org and free to use.

All species carrying a negative charge at the load pH will interact with the cationic resin to some degree. Of these, the most problematic (depending on the method used to generate the lysate) are large RNAs. Because of the numbers of these molecules that will still be present in most schemes not utilizing RNase A or selective precipitation, problems arise with binding site competition and co-elution. 

According to Ballantyne, considerable amounts of LPS can be removed at the anion exchange step by adding Triton X-114 (1%–2%) nonionic detergent to the clarified feedstock. By mixing, chilling, and adding up to 10% 2-propanol, it is possible to solubilize the detergent completely. The Triton interacts with the hydrophobic portions of the LPS, forming micelle-like structures that prevent the negatively charged regions of the endotoxin from interacting with the cationic substituent of the resin. If this method is going to be employed, resins with hydrophobic backbones need regular low-pH cycles as part of the clean-in-process protocol.

Size-Exclusion Chromatography: This method is cumbersome and slow for purifying plasmid DNA, but it can still produce a final product of exceptional quality, according to Ballantyne. Resins with very high exclusion limits are required, and the plasmid DNA must be applied in concentrated "packets," typically at 2–5% of the void to achieve good resolution. Care needs to be taken that the viscosity of the load is not too high or resolution will suffer. 

Hydrophobic Interaction Chromatography: According to Ballantyne, hydrophobic interaction chromatography (HIC) resins are generally sparsely substituted with hydrophobic pendants such as butyl, octyl, and phenyl groups. In the presence of kosmotropic (order-making) agents such as ammonium sulfate, the altered hydration sphere around the macromolecular species magnifies the hydrophobicity. As such, weak interactions between the solute and the hydrophobic side chains occur. 

Among the species of interest present, the supercoiled (SC) plasmid form is the most electronically dense, and it is, therefore, most resistant to the effect. In terms of plasmid purification, HIC is a frontal mode of chromatography. Combined with low-binding capacity, a significant amount of time should be spent in development to determine the quantities and types of impurities present in the feedstock. There is always a danger that the resin bed will be overwhelmed, and impurities will leach into the purified fraction. However, with prudent adjustment, the appropriate conditions can enable plasmid DNA to flow through unimpeded, while impurities like genomic DNA, LPS, residual proteins, and RNA stay bound. In general, a series of buffers descending in kosmotrope concentration from 0.1 M to 0.2 Mcan be used to fractionate the feedstock effectively. 

Reverse-Phase Chromatography: This mode of purification, abbreviated RPC, relies on the hydrophobicity, or more correctly,the induced hydrophobicity of the macro-molecules present in the mobilephase. The addition of cationic ion-pairing agents such as triethylammoniumacetate (TEAA), tetrabutylammonium phosphate (TBAP), and tetrabutylammoniumchloride (TBAC) causes charge neutralization that leads to interactionbetween the solute molecules and the densely functionalized hydrophobicgroups on the resin surface. The interaction is broken through a combination ofmodified surface tension and polarity brought about by increased levels oforganic solvent (generally ethanol) and subtle changes in other aspects ofmobile phase composition, such as pH and buffer strength. For additional details on factors influencing binding and elution, including the shape and porosity of the resin, see Ballantyne. 

The major roles of RPC in a purification scheme are to further lower endotoxin and enrich the SC plasmid content. Importantly, because it is a retention mode of chromatography, large volumes of feedstock containing highly dilute plasmid can be applied with little influence on the peak resolution, as long as the binding capacity is not exceeded. 

The plasmid DNA-rich fraction can be diafiltered or, with the appropriate changes to elution buffer, the plasmid can be precipitated with 2-propanol. The major drawback to using RPC is the required use of volatile solvents like ethanol at concentrations of up to 25%. 

In concluding this section and by way of a practical example, Silva-Santos et al. reported a 2017 study titled A process for SC plasmid DNA purification based on multimodal chromatography. Briefly, SC plasmid DNA was isolated by multimodal chromatography, using a stepwise NaCl elution method that includes washing of unbound open circle plasmid DNA at 830 mM NaCl, SC plasmid DNA elution at 920 mM, and removal of RNA at 2 M. The method provided baseline separation of isoforms and yielded SC-rich fractions (>90%) virtually free from RNA with levels of genomic DNA (1.3 ± 0.3% µg gDNA/µg SC plasmid DNA) and protein (4.97 ± 1.34 µg/mL) impurities within target specifications. The process was reproducible and performed similarly with differently sized plasmids (2686 bp, 3696 bp, and 10,410 bp). 

Concentration and Purity Analysis

Briefly, the eluted fraction of plasmid DNA is typically either diafiltered and concentrated or precipitated directly and then dissolved in the desired final buffer at the desired concentration using conventional UV spectroscopic optical density (OD) measurements. Purity analysis typically includes a total protein assay by either a colorimetric or mass spectrometric method, standard endotoxin testing, gel electrophoresis, restriction analysis, spectrophotometric quantitation, ratio calculation, bioburden, and clarity inspection. 

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PicoGreen. Taken from commons.wikimedia.org and free to use.

Regarding quantitation of the supercoiled circular (SC) form of plasmid DNA, Levy et al. published a novel fluorescence-based method, termed SCFluo, which takes advantage of the reversible denaturation property of SC forms and the high specificity of the PicoGreen fluorochrome (shown here) for double-stranded DNA. Fluorescence values of those forms capable of reversible denaturation after a 5 min heating and 2 min cooling step are normalized to fluorescence values of total dsDNA present in the preparation. 

For samples with a SC content >20%–30%, good regression fits were obtained when values derived from densitometric scanning of an agarose gel and those derived from the SCFluo method were compared. Levy et al. state that "[t>

he method represents an attractive alternative to currently established methods because it is simple, rapid, and quantitative." During large-scale processing and long-term storage, enzymatic, chemical, and shear degradation may substantially decrease the SC content of plasmid DNA preparations. The SC content of 6.9 kb, 13 kb, and 20 kb plasmid preparations subjected to chemical and shear degradation was successfully quantified using the new method.

Concluding Comments

Because of extensive media coverage of COVID-19 vaccine development, even non-scientists are acutely aware of the importance of manufacturing "mRNA vaccines." Although this awareness does not include technical details, dealing with the pandemic by mRNA vaccine development has led virtually all scientists to gain some knowledge of immunology and IVT mRNA production. The principal focus on the importance of synthetic mRNA has overshadowed if you will, the critical role of plasmid DNA. 

This Part 2 blog, and its Part 1 forerunner, have provided perspectives culminating in timely and reliable production of cGMP-quality plasmid DNA in scalable quantities that span exploratory research through commercialization of IVT mRNA. While it is possible to proceed from "do-it-yourself to hand-off" to a service provider or to use several service providers, the ideal circumstance is to plan for success at the outset and work with one provider. TriLink's tag lines for such service say it all:   

Getting to the clinic is hard enough.

So we've made mRNA manufacturing much easier.

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Your comments are welcomed, as usual.

Please feel free to share this blog with your colleagues or on social media.

Footnote

For historical purposes, the classic plasmid "miniprep" method published by Ish-Horowicz and Burke in 1981 read as follows: 1 mL of a saturated culture is harvested in a 1.5 mL microcentrifuge tube (20 sec in a Beckman Microfuge), resuspended in 100 µL 50mM glucose, 25 mM Tris-HCl pH 8.0, 10 mM EDTA (TE) (Solution I), and incubated for 5 min at 22 °C. 200 µL 0.2 N NaOH, 1% SDS (Solution II; made weekly from a 10 N NaOH stock) is added, mixed gently, and put on ice for 5 min. 150 µL precooled 5 M KOAc pH 4.8 (Solution III; final concentration = 3 M KOAc + 2 M HOAc) is added, mixed gently, and after 5 min on ice, the precipitated protein, dodecyl sulfate, and chromosomal DNA are removed by centrifugation for 1 min. 2 vol. EtOH is added to the supernatant, incubated for 2 min at room temperature, and the nucleic acid is precipitated with a 1 min centrifugation. The pellet is washed with 70% EtOH, dried, and taken up in 50 µL TE. 

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