April 10, 2024
Sponsored by Thermo Fisher Scientific
Digital polymerase chain reaction (dPCR) technology is used increasingly for absolute quantification of nucleic acids in research, diagnostics, bioproduction, and environmental testing. In a September 2023 Ask the Expert webinar, Samyuktha Shankar (field application specialist for the Pharma Analytics group with Thermo Fisher Scientific) discussed leveraging dPCR for absolute quantification of lentiviral vector (LVV) samples. Shankar explained how the technique reduces variability and improves both accuracy and analytical sensitivity even when high-background conditions are present.
Shankar’s Presentation
Vector copy number (VCN) is a critical quality attribute (CQA) for assessing integration and safety of transduced cell-therapy products. Because LVVs are used to transduce cells, they are considered to be an active drug-substance ingredient. Regulatory guidelines require drug substances to be tested for identity, purity, potency, safety, and stability.
Once cells have been transduced, they must be tested to ensure safety, quality, and potency of a gene-modified cell therapy product. Among the CQAs for LVVs is vector concentration (titer), an important attribute for strength, quality, and safety testing. Total and infectious particles can be measured. The former informs on vector yields (for process development and optimization); the latter relates to dose. Vector copy numbers must be measured for final products because too-high integration frequencies can pose a safety risk.
A typical LVV manufacturing process includes a series of purification and downstream processing steps. The Applied Biosystems ViralSEQ Lentivirus kit can be used to measure and compare yields across those different phases, and a ViralSEQ Proviral DNA titer kit can be used to measure infectious titers in parallel. Data from both dPCR assays can be correlated seamlessly. The latter can measure infectious titers and vector copy numbers for transduced cells.
Digital PCR quantifies nucleic-acid targets without standard curves by dividing a bulk reaction into thousands of smaller independent reactions. “Bulk PCR” measurements leverage endpoint data to indicate the presence or absence of a target of interest; quantitative PCR (qPCR) uses a standard curve to provide relative quantitative data. Digital PCR requires no standard curve and can provide absolute quantitation of known genetic targets, improving precision and reproducibility over qPCR.
The key is robust, consistent compartmentalization of the bulk PCR reaction. How samples are divided into reaction volumes affects the number, size, and consistency of those individual microreactions, all of which are key elements in data quality. Counting positive and negative microreactions after amplification yields an absolute quantitation of known targets.
How the bulk PCR reaction is subdivided into independent microreactions matters. Most dPCR platforms suffer from significant sample waste (“dead volume”), limited and/or inconsistent compartmentalization, tedious workflow requiring multiple instruments and human intervention, and long turnaround times (over six hours to generate a single data point). Also, most methods provide only endpoint analysis, which increases the potential for false positives.
The Applied Biosystems QuantStudio Absolute Q system is a reagent-efficient instrument with proven consistency of 20,000 microreactions per array. A simplified workflow shortens turnaround times and improves confidence in generated data (Figure 1). Users can analyze 4–16 samples per run, with multiplex capability.
Figure 1: Streamlined Absolute Q workflow.
Both ViralSEQ LVV titer kits leverage Absolute Q technology. The lentivirus physical titer kit provides a one-step real-time dPCR assay for quantitation of genome-containing LVVs. The assay has a broad dynamic range and provides high target specificity based on Applied Biosystems TaqMan detection. Infectious viral titer can be calculated from the number of proviruses measured by genomic assays. The proviral DNA titer kit quantitates provirus in transduced cells. It provides high sensitivity and specificity for proviral DNA using the same detection method. Combining these assays provides a convenient way to compare data for total and infectious titers and measure vector copy number. Both kits are designed for robust performance that facilitates LV analytics in process development and quality control.
The total assay runtime for both assays (including manual or automated sample preparation with an Applied Biosystems PrepSEQ universal nucleic acid extraction kit) is about four hours. Individual steps include reagent preparation, nucleic acid extraction, dPCR reactions, preparation, assay run, and data review. Elution from the sample preparation kit containing extracted nucleic acid runs directly in a dPCR kit on a platform such as the QuantStudio Absolute Q Digital PCR system, with its dedicated software that includes security e-signature and audit-trail features for 21 CFR Part 11 compliance. For enhanced accuracy, the software applies advanced criteria to qualify individual microchambers. Analysis obtained for physical titer shows dye-labeled fluorescence intensity on a y-axis for all microchambers on the x-axis from a given sample. Another plot illustrates the linearity of sample dilution.
Comparing actual concentration results determined by a QuantStudio Absolute Q system, after accounting for serial dilution factors, shows consistency reflecting the method’s high precision and accuracy. Total dilution factor is calculated and then leveraged to determine the lentiviral stock concentration and physical titer. For determination of proviral copy numbers, the positive and negative controls for a dye-labeled proviral target show signal and lack of signal, respectively. The total dilution factor again is used to determine the concentration of proviral copies. Proviral concentration is derived based from dPCR quantitation; proviral copies per cell, multiplicity of infection, and infectious viral titer can be calculated accordingly. Physical and infectious viral titers provide insight into the efficiency of viral packaging and infection.
Questions and Answers
How do I know this assay will work for a specific recombinant LVV platform? The assay targets a conserved region of the long terminal repeat (LTR) sequence and has been tested in silico with LTR sequences on different transfer plasmids, including self-inactivating (SIN) modifications. It should be compatible with most transfer plasmid systems as long as the LTR is not modified specifically.
Can the physical titer assay distinguish between plasmid and viral genomes? The assay isn’t designed to select against plasmid DNA directly, but the protocol includes a DNA step to degrade and remove residual plasmid and host cell DNA.
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The Applied Biosystems ViralSEQ and QuantStudio Absolute Q systems are intended for research use only.
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