(M1030-02-06) The Importance of Pairing a Matrix with a Nucleic Acid Extraction Approach: Compilation of 66 Bioanalytical PCR (qPCR and dPCR) Validations

Purpose: PCR is a mainstay in the assessment of safety and efficacy of a large and growing number of helpful and widely used drugs – drugs with modalities dependent upon unique nucleic acid signatures inherent to the drug. This is because PCR can sensitively and specifically detect genetic elements (RNA/DNA). Successful nucleic acid extraction of the gene target is foundational to successful PCR’s. Yet to our knowledge, analyses of nucleic acid extraction from >50 validations using a limited number of extraction approaches and matrices from a single lab have not been published.

Methods: This presentation unveils a laboratory’s long history with validating PCRs for various contexts of use (e.g., vector shedding, replication competence & biomarker detection).  And done so in a manner consistent with a recent publication on best practices for validating qPCR and dPCR assays for cell and gene therapy (Hays et al, 2024). More than 80 PCR validations or qualifications performed by our laboratory were examined in this study.  Sixty-six involved 3 nucleic acid extraction platforms which had available and appropriate data for analysis of Precision (all 66) and Limit of Detection (65). The three platforms (easyMAG NucliSENS (eM), KingFisher Flex MVP1 (KF) and QIAgen (QIA)) (Table 1) were tested on 11 Cellular and Acellular Matrices. The Cellular Matrices were BAL, Bone Marrow, Nasal Wash, PBMCs, Saliva, Semen and Whole Blood. The Acellular Matrices were Plasma, Serum, Urine and Swab. Precision (Figure 1) and Limit of Detection (LoD)(Figure 2) were assessed. Depending on context of use, we performed intra- and inter-assay precision from 3 to 5 runs with 3-5 positive levels and 3-5 replicates each. To assess LoD, we typically tested seven levels that were 2-fold separated, 10-20 replicates each. The samples were tested by the full process (extraction and PCR) and the LoD determined by Probit analysis. Key variables examined were Cellular & Acellular Matrices, DNA & RNA targets, qPCR, RT-qPCR & ddPCR and coated magnetic beads & silica columns.

Results: Generally Acellular matrix testing yielded better precision and Limits of Detection than Cellular Matrices (Figures 1 and 2). Acellular Matrices performed best with the eM, a platform for which we had 6 and 8 validations for Swab and Urine, respectively in the analysis. Percent coefficient of variations (%CV’s) ranged from 9 to 24% for swab and urine. While the LoD was very good (88 copies/mL for swab), it was poorer (259 copies/mL) for urine. Using the KF and QIA, LoDs were below 50 copies/mL for serum and plasma (albeit with only one validation performed for each). For Cellular Matrices, QIA had good results. Consistent with the literature (Hays et al, 2024), %CVs and LoD’s were higher than for Acellular Matrices. While  %CV’s ranged from 11% to 34% and the LoDs from 65 to 288 for PMBCs (N=15 Validations) and Whole Blood (N=9).  These values were higher in other cases.  For example, 45% CV (Bone Marrow N=3) and 288 copies/mL (Semen (N=1)) LoD. With regards to comparative performance of the 3 platforms across all matrices, the KF on average had slightly lower LoDs (102 copies/mL) compared to 143 and 131 for eM and QIA, respectively. The amount of concentrating that occurs during extraction and the amount of eluate added to the PCR both impact the amount of nucleic acid added to the PCR which decreases the LoD. KF theoretically adds the most nucleic acids to the PCR. For droplet digital PCR, 3 validations (all in Peripheral Blood Mononuclear Cells (PBMCs)) could be assessed for LoD and Precision. Whereas LoD was highly variable, Precision was generally better than the same matrix for qPCR.

Conclusion: eM performs well with acellular matrices and QIA performs well with cellular matrices. Concerns of other unique matrices, available instrumentation and needs for higher throughput extraction inform laboratorian's decisions as well. Further platform comparisons are needed as there are several other low, medium and high throughput platforms known to perform well in multiple contexts (e.g., MagNA Pure, VERSA and Maxwell).  The poor LoD observed lead us to recommend what others have in the literature.  That is, to avoid using neat urine, but rather a collection kit with neutralization buffer.

References: Hays et al., 2024, Recommendations for Method Development and Validation of qPCR and dPCR Assays in Support of Cell and Gene Therapy Drug Development, AAPS J. Feb 5;26(1):24.

Table 1. Overview of Extraction Platforms. MagNA Pure is not included in this study but included for comparison.

Figure 1. Intra- and Inter-Assay Precision Summary of the Minimum and Maximum %CV values encountered per Extraction Approach per Matrix in 66 Validations that could be assessed for Precision.

Figure 2. Limit of Detection (LoD) by per Extraction Approach per Matrix in 65 Validations that could be assessed for LoD