Developing and Validating a Potency Assay for Oncolytic Viruses

 

In the rapidly evolving field of oncolytic virotherapy, the accurate measurement of viral potency is essential for product development, clinical evaluation, and regulatory approval. Potency assays are essential for transitioning oncolytic virus candidates from early discovery through clinical development and commercialization. They must quantify infectivity while aligning with the virus’s mechanism of action, such as selective replication and tumor lysis. Recognizing the limitations of conventional assays such as the tissue culture infectious dose (TCID₅₀) method, scientists at Merck & Co., Inc. have developed and validated a robust, cell-based plaque assay for Coxsackievirus A21 (CVA21), a promising OV currently under clinical investigation (Chamcha et al (2024)).

What are Oncolytic Viruses?

Oncolytic viruses (OVs) are viruses, natural or engineered, that selectively infect and kill cancer cells while sparing healthy tissue (Lin et al (2023)). They take advantage of the weakened antiviral defenses and surface markers of tumor cells to enter, replicate, and trigger cell lysis. This process also releases tumor antigens, helping to stimulate an anti-cancer immune response.

Oncolytic virotherapy uses these viruses as cancer treatments, combining direct tumor cell killing with immune activation (Lin et al (2023)). Some OVs are engineered to express immune-stimulating genes like GM-CSF to boost their effects. This dual action makes them a promising approach in cancer immunotherapy (Malhotra et al (2008)). Several OVs are now in clinical trials for various cancers, and a few—such as Talimogene laherparepvec (T-VEC) for melanoma—have gained regulatory approval.

Oncolytic Cancer Therapy Using Coxsackievirus A21

Coxsackievirus A21 (CVA21), a non-enveloped picornavirus, has emerged as a promising clinical candidate due to its natural tropism for tumor cells expressing ICAM-1 and DAF receptors, mild pathogenicity in humans, and demonstrated efficacy in clinical trials against malignancies like melanoma. As highlighted in the CALM trial, CVA21 (marketed as Cavatak™) achieved durable responses in 21% of late-stage melanoma patients (Andtbacka et al (2015)).

The Critical Need for Potency Assays in Oncolytic Virotherapy

The transition of such therapies from early to late-stage clinical development and eventual commercialization necessitates robust analytical methods to ensure product quality, consistency, and safety. Central to this is the potency assay, critical quality attributes (CQAs) required by regulatory agencies (FDA, EMA, ICH) for lot release, stability testing, and dose determination.

Broader Applicability and Strategic Significance of This Study

Although this study centered on CVA21, its significance extends to the broader domains of oncolytic virotherapy and live viral vaccines. The methodological approach, encompassing parameter optimization, assay qualification, statistical analysis, and robustness testing—offers a versatile framework applicable to other viral platforms. Additionally, the assay’s streamlined workflow, which supports efficient training, assay transfer, and minimal equipment requirements, enhances its suitability for both centralized and decentralized testing environments, thereby supporting global clinical trial execution and regulatory compliance.

This work presents a comprehensive, practical strategy for developing a regulatory-compliant, high-performance cell-based potency assay. It not only advances the clinical development of CVA21 but also sets a benchmark for potency assay development across the oncolytic virus and live viral vector fields.

Methodical Development: Optimization, Qualification, and Validation

The rigor of assay development is exemplified in the phased approach employed by the authors:

  1. Optimization Phase: An I-optimal response surface DOE was implemented to explore how four critical parameters, cell seeding density, virus adsorption time, overlay volume, and cell passage number, affected assay variability at both high and low viral titers. While no significant interactions were found among parameters, individual main effects such as cell passage and overlay volume were optimized to minimize variability.
  2. Qualification Phase: The plaque assay was then qualified using representative CVA21 drug substance (DS) preparations across a five-point dilution range. Key performance indicators such as linearity (R² = 0.98), accuracy (% recovery from -0.43% to 23.79%), and intermediate precision (RSD = 26.4%) were achieved, and assay system suitability criteria were established using a consistent system suitability sample (SSS).
  3. Validation Phase: With pre-defined acceptance criteria derived from the qualification data, the assay was validated over an expanded titer range (3.31E+06 to 8.11E+09 PFU/mL). It met all criteria for linearity (slope = 1.011), accuracy (87.6%–99.8%), precision (repeatability RSD = 22%, intermediate precision RSD = 32%), specificity (no plaques in negative controls), and range.
  4. Robustness Testing: The validated assay was further subjected to robustness testing using a D-optimal design that varied critical parameters at three levels. Despite significant main effects (e.g., higher cell passage improving titer at high concentrations), the assay consistently met all acceptance criteria, demonstrating resilience to routine procedural variability. It is important to note that robustness testing is typically incorporated as a critical component of the overall validation process, ensuring the assay’s reliability under routine operational variations.

Optimization of Critical Variables

During the initial development of the CVA21 plaque assay, key parameters were systematically evaluated to ensure assay robustness and reproducibility. SK-MEL-28 cells were selected as the host cell line due to their high susceptibility to CVA21, driven by ICAM-1 and DAF receptor expression. Critical variables, including plate format, overlay composition and concentration, cell seeding density, virus input, adsorption time, and plaque development duration, were optimized to establish baseline assay conditions.

An initial One Factor At a Time (OFAT) approach guided the stepwise refinement of individual parameters. Following this, a Design of Experiment (DOE) analysis was conducted to assess the combined effects of cell density, adsorption time, overlay volume, and cell passage number on assay variability. Although no significant main or interaction effects were identified, the I-optimal response surface model determined the optimal settings that minimized variability across both high and low viral loads. These conditions were incorporated into the final validated protocol.

Qualification Criteria for Oncolytic Virus Potency Assays

Qualification Protocol

The plaque assay qualification was conducted after finalizing assay parameters through prior optimization. Representative CVA21 drug substance (DS) was diluted to generate five target concentrations, alongside a system suitability sample (SSS) and a negative control. Two analysts each performed six runs, with each run including six plates, five for test articles (TAs) and one for controls.

The study assessed linearity, accuracy, specificity, and intermediate precision using USP Chapter 1024 guidelines. The resulting data were used to define the SSS titer range and acceptance criteria for assay performance, forming the foundation for the subsequent validation phase.

Method Qualification

Following optimization, the CVA21 plaque assay was qualified to evaluate performance characteristics including linearity, accuracy, repeatability, intermediate precision, and system suitability. Key acceptance criteria were established, such as a system suitability titer range of 2.6E+09 to 8.2E+09 PFU/mL, and reportable titers required to be within 0.3 log of their geometric mean. Recommended qualification criteria for Oncolytic Virus Potency Assays are shown in Table 1.

 

Table 1: Recommended Qualification Criteria for oncolytic virus potency assay are shown in Table 1.
(With CVA21 Assay Results in Brackets)

Qualification ParameterGeneral Recommendation for Oncolytic Virus Potency Assays (CVA21 Result)
LinearityThe assay should demonstrate strong linear correlation (R² ≥ 0.98) with minimal dilution bias (≤ ±5%).(R² = 0.98; Dilution bias = –1.05%)
AccuracyThe average relative bias across test samples should remain within an acceptable range (e.g., –10% to +25%).(–0.43% to +23.79%)
RepeatabilityIntra-assay %RSD should not exceed 25%.(21.6%)
Intermediate PrecisionInter-assay %RSD should remain within 30%.(26.4%)
System SuitabilityThe system suitability sample (SSS) should have a defined acceptable titer range, and %RSD across runs should be ≤ 62%.(Titer range: 2.6E+09–8.2E+09 PFU/mL; %RSD ≤ 62%)

Validation Criteria for Oncolytic Virus Potency Assays

Validation Protocol

The validation protocol followed the qualified plaque assay procedure using representative CVA21 drug substance (DS) diluted across a broader concentration range than in qualification, covering expected drug product (DP) levels. Due to high assay dilutions, differences between DS and DP matrices were considered negligible.

Twelve runs were conducted by two analysts, mirroring the qualification design, to assess linearity, accuracy, and precision. All acceptance criteria—including those for system suitability sample (SSS) and assay variability—were pre-defined based on qualification data. The SSS had to fall within the validated range of 2.6E+09 to 8.11E+09 PFU/mL, ensuring assay consistency and reliability for clinical testing.

 Method Validation

Subsequently, the assay was validated using pre-defined acceptance criteria derived from the qualification phase. Validation confirmed the assay met all criteria for specificity, accuracy (87.6%–99.8%), linearity, precision, and range (3.31E+06 to 8.11E+09 PFU/mL), supporting its reliability for release and stability testing of clinical samples.

A robustness study was then conducted to determine the assay’s resilience to minor procedural variations. A total of 100 data points were analyzed using a mixed-effects response surface model. While cell passage and overlay volume had some impact on titers at specific concentrations, all observed differences remained within acceptable limits. The assay demonstrated robustness across expected operational ranges, confirming its suitability for routine GMP testing.

 

Table 2: Recommended Validation Criteria for Oncolytic Virus Potency Assays

Assay Performance ParameterGeneral RecommendationCVA21 Validation Result
SpecificityNo plaques should be observed in negative control wells, confirming no assay interference from sample matrix.No plaques detected in negative controls; assay specific to CVA21.
AccuracyRecovery for each sample should fall within 55%–175% of the expected value.Recovery range: 87.6% to 99.8%
Intermediate PrecisionInter-assay variability (%RSD) should be ≤40% across different runs and analysts.%RSD: 32%
RepeatabilityIntra-assay variability (%RSD) should be ≤30% within a single run.%RSD: 22%
LinearityDilution bias across reportable range should be ≤5% per two-fold dilution. R² should be ≥0.98.Dilution bias: 0.768%; R² = 0.995
RangeAssay should demonstrate acceptable accuracy, precision, and linearity across a suitable dynamic range.Validated range: 3.31E+06 to 8.11E+09 PFU/mL

 

Validation Outcomes of the CVA21 Plaque Potency Assay

The CVA21 plaque assay demonstrated consistent and reliable performance across qualification, validation, and robustness studies. High slope and R² values in all protocols confirmed the assay’s strong linearity and quantitative capability. Specificity was also maintained, as no plaques were observed in negative control samples. Importantly, each protocol progressively broadened the tested concentration range, enhancing the assay’s applicability for both high- and low-titer samples. The validated dynamic ranges were 1.58E+07 to 1.58E+09 PFU/mL during qualification, 3.31E+06 to 8.11E+09 PFU/mL during validation, and 3.30E+05 to 8.11E+09 PFU/mL in the robustness study.

Robustness

The final step in the assay development was a robustness study, which is normally a part of validation studies, designed to confirm that the plaque assay could withstand small, intentional variations in key parameters during routine use. Conducted by two analysts over five days, the study evaluated 100 data points using a mixed-effects response surface model. Key findings included a significant impact of cell passage at high titer (8.11E+09 PFU/mL), and significant effects of both cell passage and overlay volume at low titer (3.30E+05 PFU/mL). Two interaction effects—between cell passage and density, and between overlay volume and adsorption time—were also noted. However, all observed variations remained within the predefined acceptance criteria. Additionally, the assay maintained acceptable linearity, accuracy, and precision under these perturbations, confirming consistent performance comparable to that observed in the qualification and validation studies.

Phase-Appropriate Potency Assays for Oncolytic Viruses

Research from Merck has outlined a blueprint for developing potency assays for oncolytic viruses (OVs), beginning with the selection of a method that aligns with the virus’s mechanism of action (MOA), followed by systematic parameter optimization, assay qualification, validation, and assessment of robustness. This structured approach enables the development of phase-appropriate potency assays—bioanalytical methods tailored to meet the evolving scientific and regulatory requirements across the drug development continuum, from preclinical studies to commercial licensure.

These assays typically progress through three key stages. First is fit-for-purpose demonstration, used in preclinical and Phase 1 trials to show that the assay can reliably measure viral potency in a biologically relevant system. Second is assay qualification, conducted in Phase 2, to confirm robustness and suitability for process development, dose optimization, and broader clinical evaluation. Finally, full assay validation is required during Phase 3 and for commercial release, ensuring the assay meets regulatory expectations for accuracy, precision, specificity, linearity, and robustness.

For OVs, where infectivity and tumor cell lysis are central to therapeutic activity, potency assays must accurately reflect the virus’s ability to bind, infect, replicate within, and lyse cancer cells. Assays such as the plaque assay must therefore be mechanistically relevant and fully aligned with FDA and ICH guidelines for Quality Control (QC) and Quality Assurance (QA). By developing phase-appropriate assays, developers ensure that each stage of OV clinical advancement—including IND-enabling studies and pivotal efficacy trials—is supported by a scientifically sound and regulatory-compliant testing strategy. This not only protects data integrity and streamlines regulatory submissions but also supports the successful translation of OV candidates from the research bench to the clinic.

 

Table 3: Stages of Potency Assay Development for Oncolytic Viruses Across Clinical Phases*

Clinical PhaseAssay StagePurpose in Oncolytic Virus Development
PreclinicalStage 1 – Fit-for-Purpose: A method that reliably measures virus infectivity and supports early go/no-go decisions.Used in exploratory studies for screening virus candidates, evaluating basic oncolytic activity, and mechanism of action.
Phase 1 ClinicalStage 1 – Fit-for-Purpose: Assay demonstrates sufficient accuracy, reproducibility, and biological relevance to support early-phase studies.Supports initial safety and pharmacokinetic assessments and guides early manufacturing process development.
Phase 2 ClinicalStage 2 – Qualified Assay: Assay shows intermediate precision, accuracy, specificity, linearity, and range. Aligned with ICH Q2(R2) guidelines.Used for dose optimization, expanded safety evaluation, and refining OV production processes.
Phase 3 ClinicalStage 3 – Validated Assay: Fully validated under FDA/EMA/ICH guidance, compliant with GMP/GLP, and supported by SOPs and QC/QA systems.Enables confirmatory efficacy and safety studies, lot release, and real-time stability monitoring of OV products.
CommercialStage 3 – Validated Assay: Same as above, but with strict adherence to validation protocols, comprehensive documentation, and full regulatory compliance.Required for commercial lot release, post-marketing surveillance, and global assay transfer for import testing.

*Read our white paper “Selecting Bioanalytical Assays for FDA Approved Clinical Trials and Commercialization” for detailed information.

Conclusion

The CVA21 plaque assay sets a benchmark for OV potency testing, combining precision, efficiency, and regulatory compliance. By replacing TCID50 with a method that directly measures infectivity, this approach enhances reliability in dose determination and quality control for late-stage clinical development. Its success underscores the importance of systematic assay optimization and validation in accelerating OV-based therapies to market.

Disclaimer: This blog post is intended solely for educational and scientific informational purposes. Any mention of therapeutic drug names, including FDA-approved medications, is for the purpose of accurate reporting and discussion of biomedical research and does not constitute medical advice, endorsement, or promotion. Readers should not interpret the content as a recommendation for any specific treatment. Always consult a qualified healthcare professional for medical advice or treatment decisions.

 

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