(T1330-06-36) A GxP-Qualified dPCR-Based RCL Assay to Quantify Replication Competent Lentivirus in Bioanalytical Samples

Purpose: Lentiviral vectors are increasingly being used in the development of gene/cell therapy products for various diseases (HIV, cancer, Parkinson’s etc). These viral vectors, although inherently replication incompetent and cannot generate additional viruses and therefore, cannot cause unwanted transductions. However, in rare instances, the lentiviruses regain their replication competency through recombination events and can infect non-cancerous cells, thereby producing untoward effects. Contamination of replication incompetent pools with replication competent viruses is also a possibility during the manufacturing process. Therefore, FDA had mandated the testing of replication competent lentivirus (RCL) in vector-based gene therapy products during the manufacture as well as after the patients have received the therapy (FDA, 2020). To ensure a highly sensitive and accurate assay that is compliant with regulatory agency’s guidance, our company has developed and qualified a dPCR-based RCL assay under GMP and GLP regulations.

Methods: We have developed and qualified a duplex dPCR-based RCL assay, including DNA extraction from human whole blood, to detect the presence of any RCL in bioanalytical samples. There are numerous advantages to dPCR assay which can be leveraged in RCL detection, such as: (i) absolute quantification of target gene without the need for a calibration curve or amplification efficiency, (ii) improved reproducibility (precision) and detection of samples with low target concentrations, (iii) higher tolerance to PCR inhibitors. DNA extraction: Human genomic DNA (hgDNA) was extracted from human whole blood using KingFisher Flex automated DNA extraction system. Blood input volume ranges from 0.2 – 2 ml per well in a 24-well deep-well plate, with a final elution using 200µL of AE buffer. DNA extraction recovery rate was estimated by spiking blood samples with VSV-G gBlock (our positive control). Extracted hgDNA yield and quality was measured by Qubit and Nanodrop. dPCR: Given the importance of the envelope gene (VSV-G) in the generation of lentivirus, and its ease of detection, VSV-G is our preferred target for detecting RCL, and RPP30 gene works as the reference gene. In a duplex dPCR assay, probe for VSV-G will be FAM-labeled and for RPP30 will be HEX-labeled. Firstly, we developed and qualified the duplex dPCR assay on Bio-Rad’s QX200 ddPCR™ platform under GMP and GLP regulations. The qualification process was carried out by 2 analysts, using 2 instruments and performed on different days. All ddPCR™ reactions were run as triplicates in the final ddPCR™ wells and, only reactions with >10,000 droplets/well were considered for analysis. When the droplet count is less than 10,000 in a well, the technical repeat must be omitted. No more than 1 technical repeat is omitted from each dilution point. Negative control in these experiments was AE buffer only (with neither hgDNA nor gBlock). Secondly, our RCL assay with a range of target concentrations was also run on another digital PCR platform, the QIAcuity Four (Qiagen), using a 24-well 26k plate and a 96-well 8.5k plate. A dedicated Qiagen dPCR master mix was used, but the primers/probes and cycling conditions were the same as that for the Bio-Rad ddPCR™ system.

Results: The yield from automated DNA extraction using KingFisher Flex from human whole blood was linear from 0.2 – 2ml/well sample input volume and had a gBlock recovery rate of >70%. Our RCL assay (qualified using Bio-Rad QX200 system) was linear in a dynamic range of 0.25-5000 copies/µL with a slope = 1 and R2 = 0.99, in the presence of 200ng of hgDNA as the background, with the accuracy and precision meeting or surpassing the current industry standards for RCL assay (Wissel, Poirier, Satterwhite et al, 2022; Hays, A., et al, 2024). We have determined our LoB as 0 copies/µL for NTC reactions using AE Buffer. Our RCL assay has a LLoQ of 0.5 copies/µL and LoD of 0.25 copies/µL, both of which are more sensitive than that offered in leading commercial kits and by other competitors in the market. Our RCL assay performed equally well on QIAcuity Four dPCR platform. The results showed that the assay was linear at a broad range of 0.25-2500 copies/µL in 26k and 8.5k plates with a slope = 1 and R2 = 0.999. With the 26k plate on QIAcuity, we achieved valid partition number of >25,000 per well. This high partition number helped further improve the sensitivity of the assay, achieving LoD as low as 0.05 copies/µL.

Conclusion: The dPCR-based RCL (VSV-G) assay developed and qualified by our company exemplifies exceptional robustness, sensitivity, and competitiveness. This state-of-the-art assay is compatible with multiple dPCR platforms and has been meticulously qualified under stringent GMP/GLP regulations. It meets and exceeds the recommended guidelines for detecting and reporting replication-competent lentiviruses in bioanalytical samples, ensuring unparalleled accuracy and reliability.

References: 1. Wissel, M., Poirier, M., Satterwhite, C. et al (2022). Recommendations on qPCR/ddPCR Assay Validation by GCC, Bioanalysis,14:12, 853-863, DOI: 10.4155/bio-2022-0109.
2. FDA CBER. (2020). Testing of Retroviral Vector-Based Human Gene Therapy Products for Replication Competent Retrovirus During Product Manufacture and Patient Follow-Up - Draft Guidance for Industry.
3. Verichek ddPCR™ Replication Competent Lentivirus Kit User Guide, Bio-Rad.
4. Hays, A., et al (2024). Recommendations for Method Development and Validation of qPCR and dPCR Assays in Support of Cell and Gene Therapy Drug Development. AAPS Journal, 26(1). https://doi.org/10.1208/s12248-023-00880-9
5. Tusneem, N. et al (2021). Quantitation of Replication Competent Viruses using ddPCR™. Association for Molecular Pathology 2022 Annual meeting & Expo, Phoenix, AZ. November 3-5, 2022.
6. ICH (2005). Validation of Analytical Procedures: Text and Methodology Q2(R1) .
7. FDA CBER, CDER, ICH (2016). Q7 Good Manufacturing Practice Guidance for Active Pharmaceutical Ingredients – Guidance for Industry
8. FDA HHS (2014). Title 21 of the U.S. Code of Federal Regulations Part 58 —Good Laboratory Practice for Nonclinical Laboratory Studies.