(W1030-06-36) Novel Technical Breakthrough: qPCR with Reported LLOQ of 8 Copies / mg DNA in a 20 µL Reaction

Purpose: The current FDA guidance on Quantitative Polymerase Chain Reaction (qPCR) method requires the reported lower limit of quantitation (LLOQ) to be less than 50 copies of the target of interest per microgram of total DNA input. As BMV guidelines state that the LLOQ samples must exhibit a %RE within 20% of the nominal value and a CV < 20%, each qPCR assay must be highly optimized to meet regulatory requirements. In a qPCR run, a standard curve with 6-8 calibrators is used to generate a semi-log plot of CT value vs. expected copy number, which is then used to calculate the copy number of the target gene). These calibrators are usually made by preparing a stock vector DNA solution with known mass concentration and converted copy number concentration and then serially diluting it to produce standards with the desired copy numbers. Which diluent is used when creating these calibrators varies considerably, and includes such solutions as nuclease water, TE buffer, animal/human genomic DNA solution, vendor-manufactured PCR reaction buffer, salmon sperm DNA buffer, yeast tRNA solution, etc. Usually, this diluent is fit for purpose. For example, gene or cell therapy studies often use human blood aliquots as clinical samples, and therefore background human genomic DNA would be present after DNA extraction. Consequently, diluting/spiking a target gene plasmid into human genomic DNA when making standards and QCs would properly best mimic the chemical environment of clinical samples.

Methods: Here, we report a novel protocol of qPCR that achieves exceptionally high sensitivity, precision, and accuracy, by using human genomic DNA digested with restriction enzymes as the diluent to make standards and QCs, at the concentration of 125 ng/µL. 8 mL of each standard/QC was used as template in multiplex qPCR reactions with a total volume of 20 µL; in total, 1 microgram of human genomic DNA was used as the background of the reaction. Moreover, we included the amplification of a second control gene present in the human genome, and whose reported CT value is both proportional to the concentration of human gDNA and to target gene concentration present in each standard and QC.

Results: Surprisingly, we found that such high levels of human genomic DNA background in a qPCR reaction stabilized data reproducibility, maintaining accuracy and precision well within BMV guidelines even at extremely low concentrations of the target vector [Figure 1]. We have internally validated the method and achieved a sensitivity of 8 copies of the target of interest per microgram of total DNA input, far better than the current industry standard, with amplification efficiencies well within a commonly acceptable range (90-110%) [Table 1] and with a reported %CV and %RE of less than 5% [Table 2].

Conclusion: The establishment of this method represents a significant technical breakthrough in qPCR method development, promising significantly higher accuracy, precision, and sensitivity than generally available and potentially offering a reliable and cost-effective option to meet FDA guidelines.

References: 1. US Department of Health and Human Services (FDA). Guidance for industry: bioanalytical method validation. (2018).
www.fda.gov/regulatory-information/search-fda-guidance-documents/bioanalytical-method-validation-guidance-industry.
2. EMA. Guideline on bioanalytical method validation. (2011).
www.ema.europa.eu/en/documents/scientific-guideline/guideline-bioanalytical-method-validation en.pdf
3. Hays A, Islam R, Matys K, Williams D. Best practices in qPCR and dPCR validation in regulated bioanalytical laboratories. AAPS J.
24(2), 36 (2022).
4. Ma H, Bell KN, Loker RN. qPCR and qRT-PCR analysis: regulatory points to consider when conducting biodistribution and vector
shedding studies. Mol. Ther. Methods Clin. Dev. 20, 152–168 (2020).

Acknowledgements:The authors declare all rights reserved.

Figure 1. Amplification Curve of 9 Calibrators of Duplex qPCR Assay

Table 1. Linear Regression of 9 Calibrators of the Duplex qPCR Assay

Table 2. CT (Cycle Threshold) Values of Transgene and Control Gene of 9 Calibrators of the Duplex qPCR Assay.