E.coli DNA testing to support CGT development

This article is based on a poster originally authored by ACROBiosystems.

Plasmid DNA (pDNA) is widely used in various clinical, commercial, and research and development bioprocessing applications, most notably as a gene delivery vector in gene and cell therapy.

Cell and gene therapy products rely highly on purity testing throughout manufacturing. This is also a key part of quality control measures. For example, pDNA produced in Escherichia coli (E. coli) cell lines must adhere to regulatory guidelines in terms of the levels of E. coli host cell DNA (HCD)/residual DNA (resDNA). These guidelines are provided by organizations such as WHO and FDA.

The limit of quantitation (LOQ) for most commercial E. coli resDNA qPCR kits currently stands at 30 fg/µL.

ACROBiosystems has developed a highly sensitive E. coli resDNA assay kit with an LOQ of just 3 fg/µL. This powerful kit ensures precise E. coli host-cell DNA quantitation in plasmid DNA (pDNA) preparations.

This kit allows researchers to ensure accurate results, even when working with smaller sample quantities for resDNA analysis – a common concern when dealing with precious gene delivery vector products.

Methods and materials

E. coli DNA from an E. coli standard cell line was used for each sample and measured 18 times using the Thermo Fisher Qubit 4.0 fluorometer. This enabled the determination of the mean concentration (M) and standard deviation (S) for this plasmid DNA sample (GMP grade).

Regents used

• resDetect^™ E. coli resDNA Quantitative Kit (qPCR) (Cat. No. OPA-O002)
• resDetect^™ resDNA Sample Preparation Kit (Magnetic Beads) (Cat. No. OPA-R005)

Instruments used

• Applied Biosystems 7500 Real-Time PCR system with Real-Time PCR Software v2.4
• Thermo Fisher’s DynaMag^™-2

Bioanalytical method validation: ICH Q2 (R2) and USP <509>

Validation of the resDetect^™ kits was performed per the criteria set out in USP<509> and ICH Q2 (R2) guidelines. This process requires the assessment of linearity, limit of quantitation, repeatability, specificity, and quantitation of plasmid DNA.

Results

Linearity

In the example presented here, linearity was determined from a minimum range of six different standard curve positive control nucleic acid concentrations. The R² (the square of the correlation coefficient, r) and slope are reported for each generated standard curve. It is important to note that R² should be greater than or equal to 0.98 for standard curves.

Figure 1. Linearity range using the E. coli residual DNA quantitative Kit(qPCR). Image Credit: ACROBiosystems

Figure 1 shows typical analysis results acquired with Standard 1 (300 pg/µL) to 6 (3 fg/µL). This includes the standard curve of the 10-fold dilution series. PCR efficiency was determined to be 99.851%, while the R² associated with these standard solutions was determined to be 0.999.

Limit of quantitation

Quantitation was performed using three replicates of 3 fg/µL E. coli DNA samples on different days, using a standard curve. Each replicate was completed by a different operator.

The mean DNA concentration was measured to determine, calculate, and report the standard deviation and %CV. It was necessary for the standard curve to meet the following specifications:

• R² ≥ 0.98
• Eff%: 90~110%
• Slope: -3.1~-3.8

The %CV of 3 fg/µL replicates should be ＜20% for quantitation.

The assay described here could accurately quantify E. coli DNA while adhering to an assay limit of quantitation of 3 fg/µL (Table 1). The data presented confirms the high sensitivity of the resDetect^™ E. coli kit.

Table 1. The quantify value of 3 fg/μL E. coli DNA. Source: ACROBiosystems

Assay specificity

It was noted that the E. coli qPCR assay was unaffected by the presence of unrelated DNA (CHO DNA and HEK293 DNA) and continued to remain within the specified recovery range.

Table 2. The recovery result of assay specificity. Source: ACROBiosystems

Consistent performance

Consistent performance was observed across the expected range of DNA fragment sizes. Figure 2 confirms that the R² and slope of all standard curves met the specified requirements.

Figure 2. The standard curves of DNA fragment. Image Credit: ACROBiosystems

Quantification of plasmid DNA (pDNA) sample

Three pDNA samples were tested using the E. coli qPCR assay. In these cases, spike recovery was between 99% and 107%.

Summary and discussion

The validation study outlined in this article showcases the resDetect^™ E. coli assay’s compliance with the USP<509> E. coli resDNA validation guidelines.

The results highlight the assay’s sensitivity, specificity, robustness, and precision. The resDetect^™ E. coli assay confidently fulfils all requirements outlined in USP<509> around detecting E. coli resDNA during gene and cell therapy manufacturing processes.

References and further reading

1. Kim, J., Koo, B.-K. and Knoblich, J.A. (2020). Human organoids: model systems for human biology and medicine. Nature Reviews Molecular Cell Biology, (online) 21(10), pp.571–584. https://doi.org/10.1038/s41580-020-0259-3.
2. Zhao, Z. (2022). Organoids. Nature Reviews Methods Primers, (online) 2(1), pp.1–21. https://doi.org/10.1038/s43586-022-00174-y.

Acknowledgments

Produced from materials originally authored by ACROBiosystems.

About ACROBiosystems

ACROBiosystems is a cornerstone enterprise of the pharmaceutical and biotechnology industries. Their mission is to help overcome challenges with innovative tools and solutions from discovery to the clinic. They supply life science tools designed to be used in discovery research and scalable to the clinical phase and beyond. By consistently adapting to new regulatory challenges and guidelines, ACROBiosystems delivers solutions, whether it comes through recombinant proteins, antibodies, assay kits, GMP-grade reagents, or custom services. ACROBiosystems empower scientists and engineers dedicated towards innovation to simplify and accelerate the development of new, better, and more affordable medicine.

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