{"id":12603,"date":"2023-11-29T08:53:02","date_gmt":"2023-11-29T08:53:02","guid":{"rendered":"https:\/\/www.richter-helm.eu\/?p=12603"},"modified":"2025-11-18T08:46:06","modified_gmt":"2025-11-18T08:46:06","slug":"a-guide-to-the-analysis-of-plasmid-dna","status":"publish","type":"post","link":"https:\/\/www.richterbiologics.eu\/de\/a-guide-to-the-analysis-of-plasmid-dna\/","title":{"rendered":"A guide to the analysis of plasmid DNA"},"content":{"rendered":"\t\t
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Plasmids hold potential across the drug development landscape, most notably as an API \u2013 but how best can they be identified and analysed?<\/h2>\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t
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By Sandra Wenzel at Richter-Helm Biologics<\/em><\/p>\n\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t

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\n\t\t\t\t\t\t\t\t\tPlasmids are circular, double-stranded DNA molecules of various sizes originally found in bacteria, archaea and eukaryotic yeast. They replicate independently of chromosomal DNA and are easily genetically modified. Plasmids can be used for a variety of pharmaceutical purposes: as the active pharmaceutical ingredient (API) in DNA vaccines to prevent viral infections or cancer development; as critical raw material, starting material or ancillary material for viral vector\nproduction; or as template for mRNA-based products. In the latter cases, the plasmid is necessary for the manufacturing of the active drug substance and the quality can strongly affect the purity and potency of the final product. The plasmid itself, however, is not present in the final therapeutic \u2013 it is either transcribed into RNA or translated into protein (eg, virus particles).\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t
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While there are established guidelines regarding quality requirements for plasmid DNA vaccines and viral-\/non-viral gene therapy products, the requirements for release of plasmids used as starting material are not clearly defined.1,2,3 The BioPhorum expert group on Cell and Gene Therapy (CGT) Products has published two papers that provide an excellent foundation for methods and acceptance criteria for the release of plasmids used in CGT production.4,5 A statement published by the EMA also clarified that GMP principles should be applied to the manufacturing steps of plasmids intended as starting material, thus answering the previously open question.6 This article summarises the consensus between the different guidelines and provides an overview of the analytical methods used for the release of plasmid starting material, DNA vaccines and non-viral gene therapy products (see Table 1).<\/p>\n\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/section>\n\t\t\t\t

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Physicochemical properties<\/h2>\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t
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To cover the physicochemical properties of the plasmid material, several compendial methods are usually required for release testing, regardless of the intended use. Visual inspection by qualified operators is used to verify the general appearance of the material. Plasmid solutions are usually clear to slightly opalescent, especially at higher concentrations, and colourless. For drug product material, the absence of visible and sub-visible particles should be demonstrated. The pH is usually dependent on the formulation, which may be adjusted to a physiological range or to meet the requirements of subsequent processes. An optimal pH buffer capacity of the formulation is advantageous
for plasmid stability.<\/p>\n\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/section>\n\t\t\t\t

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Inhalt<\/h2>\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t
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The plasmid DNA content is determined by UV spectroscopy based on the absorption of nucleotides at a wavelength of 260 nm. This method cannot differentiate between plasmid or genomic DNA or residual RNA, and therefore measurements can only be performed on highly pure samples \u2013 otherwise the content will be greatly overestimated. The determination of the A260\/A280 ratio gives a good estimate of the purity with respect to protein or RNA contamination but is highly sensitive to changes in the pH of the solution.<\/p>\n

Measurements should not be performed in unbuffered water but rather in low-salt buffer solutions with a neutral pH. Dissolution of CO2 from the air likely causes differences in the pH of unbuffered solutions, affecting the absorption maximum of DNA. Content determination by anion-exchange chromatography is also widely used and is advantageous for early in-process samples with high levels of contamination by host cell impurities. It should be noted, however, that these measurements are more time-consuming, and the precision of the method may be lower than for UV spectroscopy. <\/p>\n\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/section>\n\t\t\t\t

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Identity testing and cross-contamination <\/h2>\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t
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Identity testing is among the most essential requirements for any starting material, excipient, drug substance or drug product. It should be performed during release testing by the supplier, but also prior to the use in further downstream applications to avoid misuse or interchange of material. The standard procedure is to sequence the entire length of the plasmid with sufficient coverage, followed by comparison to a reference sequence. While the most common approach would be Sanger sequencing, it is not sensitive enough for potential cross-contamination with other plasmids and faces limitations with difficult sequences such as inverted terminal repeats (ITRs) and long terminal repeats (LTRs). For these cases, next-generation sequencing (NGS) is better suited to detect low percentage variants and to sequence past long repetitive regions. Restriction digest is often used as an additional orthogonal method but is not sufficient for the release of the material.<\/p>\n\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/section>\n\t\t\t\t

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Tests and current specifications for plasmids used as starting material, DNA vaccine or gene therapy product<\/p>\n\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/section>\n\t\t\t\t

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Plasmid homogeneity<\/h2>\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t
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Plasmids occur in several conformations, depending on the integrity of the circular, double-stranded DNA. Each conformation can exist in monomeric or multimeric forms. Different electrophoretic mobilities of these forms allow good separation and quantification with different analytical methods.<\/p>\n

A supercoiled, covalently closed circular (CCC) form is the target for all types of plasmid products. The double-strands are fully intact, and the plasmid has a twisted, compact shape. The supercoiled form is expected to have the highest potency and transfection efficiency and is best suited for all further downstream applications (eg, virus or mRNA production). The monomeric and dimeric CCC forms are usually summed to percentage supercoiled.<\/p>\n

DNA strand breaks can occur at any point of the production process, storage, and transport of the final bulk due to shear forces, high temperatures or extreme pH conditions. When single-strand breaks occur in the plasmid, the circular form remains intact, but the twist of
the plasmid is unwound and adopts a relaxed open circular shape. If further breaks occur, the double-strand can break, and the plasmid will linearise. The linearised form increases the risk of integration into the recipient’s chromosomal DNA, leading to a risk of insertional mutagenesis or the spread of antibiotic resistance genes.<\/p>\n

Therefore, a chromatographic polishing step should be introduced to the production process to reduce the linearised form. Careful handling of the bulk and analytical samples is crucial to prevent linearisation.<\/p>\n

Typical analytical methods to determine plasmid homogeneity are capillary gel electrophoresis, HPLC and agarose gel electrophoresis. The use of orthogonal methods is preferred for initial characterisation of the product. It is advised to use reference material for comparison and even to generate open circular and linearised standard material to be able to identify their respective chromatographic peaks.<\/p>\n\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/section>\n\t\t\t\t

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Residual host protein<\/h2>\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t
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Host cell proteins (HCP) summarise a diverse mixture of proteins produced by the host cell, the profile of which can show batch-to-batch variation induced by stress or cell lysis during manufacturing. They can negatively affect product quality, safety and efficacy, posing a risk for immunogenicity, toxicity, biological activity and product stability, and they can interfere with further steps in gene therapy production. Final levels should be as low as possible and are usually determined via a total-protein BCA assay or specific HCP ELISAs.<\/p>\n\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t

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There are three most commonly employed principles in the development of HCP ELISAS:<\/p>\n\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t

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  1. Process-specific assay, where a mock production process with a null cell line is used to generate specific antibodies and HCP standard

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  2. Platform assay, set up in the same way as the process-specific ELISA but used for several products from a given manufacturer derived from the same host

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  3. A commercially available kit, which is not recommended for later clinical stages as the antisera usually derive from a combination of strains and do not reflect the individual manufacturing process and host strain.<\/li>\n<\/ol>\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/section>\n\t\t\t\t
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    It is advised to determine the coverage of the used antibodies with 2D SDS-PAGE and Western Blot.<\/p>\n\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/section>\n\t\t\t\t

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    Residual RNA and genomic DNA<\/h2>\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t
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    Residual RNA and genomic DNA deriving from the host cell consist of fragments of different length and physical forms. They can co-purify with the vector under specific circumstances and sufficient clearance throughout the manufacturing process should be demonstrated.<\/p>\n

    Quantitative PCR (qPCR) is the method of choice for genomic DNA quantification. DNA is typically isolated from the test samples; potentially inhibiting proteins are digested, and a highly conserved genomic sequence is amplified. Primer and probe site homologies between the host genome and the product sequence should be avoided. Thoroughly characterised USP reference standards are available for genomic DNA of Escherichia coli and Chinese hamster ovary (CHO) cell lines for absolute sample quantification.<\/p>\n

    An emerging alternative is digital PCR (dPCR), where the reactions are divided via capillaries, microarrays or oil droplets into tens of thousands of partitions, resulting in partitions with a single or no target molecule at all. PCR amplification occurs up to the endpoint and the number of positive amplifications is counted. Statistical analysis is then performed to calculate the absolute concentration of the target molecule that was present in the original sample. This approach offers several advantages, but it has not yet been established as a standard tool and no agreed-upon methodologies are described in the guidelines. Residual RNA is usually determined via HPLC or SYBR gold staining, but the latter stains RNA, doubleand single-stranded DNA indiscriminately which should be considered for sample preparation. Alternatively, qRT PCR assays targeting a highly conserved host RNA can be used.<\/p>\n\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/section>\n\t\t\t\t

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    \u201cWhile there are established guidelines regarding quality requirements for plasmid DNA vaccines and viral-\/non-viral gene therapy products, the requirements for release of plasmids used as starting material are not clearly defined.\u201d<\/p>\n\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t

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    Parameters regarding the microbiological safety of the product, like bioburden, sterility or endotoxin, are usually measured with widely used compendial methods and are not explained in detail here. Process-related impurities (eg, residual antibiotics or antifoam) may pose a risk to the patient and should be reduced to the lowest possible level. Depending on the application of the plasmid, it might be necessary to establish assays to demonstrate the potency or transfection efficiency of the plasmid. This is highly transgene-dependent and may require the development of different methods, like cell-based bioassays, ELISAs or RT-PCRs. Ideally, a highly sensitive analytical platform is established including a generically validated testing approach covering the above-mentioned methods, being offered by CDMOs across the pharma landscape.<\/p>\n\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/section>\n\t\t\t\t

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    References<\/em><\/p>\n

      \n
    1. WHO Expert Committee on Biological Standardisation (2021) Annex 2: Guidelines on the quality, safety and efficacy of plasmid DNA vaccines. WHO Technical Report Series No 1028, 2021<\/em><\/li>\n
    2. United States Pharmacopeia, General Chapter, <1047> Gene Therapy Products<\/em><\/li>\n
    3. European Pharmacopeia (2010) Monograph 5.14, Gene transfer medicinal products for human use<\/em><\/li>\n
    4. BioPhorum (2020) Raw materials: Cell and gene therapy critical starting material: a discussion to help establish release specifications for plasmids and the bacterial master cell banks used to produce them<\/em><\/li>\n
    5. BioPhorum (2022) Raw materials: Cell and gene therapy critical starting material: Further discussion on plasmids to establish release specifications using a risk-based approach to manage supply<\/em><\/li>\n
    6. European Medicines Agency (2021) Questions and answers on the principles of GMP for the manufacturing of starting materials of biological origin used to transfer genetic material for the manufacturing of ATMPs, EMA\/246400\/2021<\/em><\/li>\n<\/ol>\n\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/section>\n\t\t\t\t
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      Dr Sandra Wenze<\/strong>l is responsible for CDMO projects as Quality Control scientist at Richter-Helm Biologics<\/strong>, where she is in contact with various biopharmaceutical companies and their project-specific analytical requirements. She holds a PhD in Molecular Cell Biology from the Friedrich Schiller University of Jena, Germany, and has profound first-hand experience in the development and validation of analytical methods.<\/p>\n\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/section>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/section>\n\t\t\t\t<\/div>\n\t\t","protected":false},"excerpt":{"rendered":"

      Plasmids are circular, double-stranded DNA molecules of various sizes originally found in bacteria, archaea and eukaryotic yeast. They replicate independently of chromosomal DNA and are easily genetically modified. Plasmids can be used for a variety of pharmaceutical purposes: as the active pharmaceutical ingredient (API) in DNA vaccines to prevent viral infections or cancer development; as critical raw material, starting material or ancillary material for viral vector production; or as template for mRNA-based products.<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"footnotes":""},"categories":[96],"tags":[],"class_list":["post-12603","post","type-post","status-publish","format-standard","hentry","category-produkte-und-technologien"],"acf":[],"yoast_head":"\nA Guide to Plasmid DNA Analysis | Richter BioLogics<\/title>\n<meta name=\"description\" content=\"Plasmid DNA quality control made clear: identity, purity, potency, endotoxin, and sequence integrity to de-risk development and accelerate release.\" \/>\n<meta name=\"robots\" content=\"index, follow, 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