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Development of a robust cell-based potency assay for a coxsackievirus A21 oncolytic virotherapy
Venkateswarlu Chamcha1, Li He2, Jenny Xu1
1Analytical Research and Development, Merck & Co., Inc., Rahway, NJ, USA.
Abstract:
Oncolytic viruses (OV) are part of a burgeoning field of investigational oncolytic therapy (OT), in which lytic viruses dissolve advanced tumors productively and specifically. One such OT is a Coxsackievirus A21 (CVA21) based OV that is currently under clinical evaluation. A tissue culture infectious dose (TCID50) assay was used for CVA21 potency release and stability testing in early clinical development. The titer measured in this method was an extrapolated value from cytopathic effect (CPE) observed during the serial dilution but doesn't represent direct viral killing of cells. Moreover, the assay was not deemed to be optimal to carry into late phase clinical development due to limitations in assay precision, turn-around time, and sample throughput. To address these points, we developed a plaque assay to measure viral plaque forming units to measure the potency value for drug substance (DS), drug product (DP) and virus seed (master and working) stocks. In this manuscript, we describe the steps taken to develop this plaque assay for the late-stage clinical development, which include the assay qualification, validation, and robustness protocols, and describe statistical methods for data analysis. Moreover, the method was validated for linearity, accuracy, precision, and specificity. Furthermore, the plaque assay quantifies OV infectivity with better precision (32% vs 58%), with higher sample throughput (22 samples/week vs 3 samples/week) and shorter assay turnaround time (4 days vs 7 days) than the TCID50 method. This assay development strategy can provide guidance for the development of robust cell-based potency methods for OVs and other infectious viral products.
Insights
A new plaque assay improves oncolytic virus (OV) potency testing for Coxsackievirus A21 (CVA21) therapy. This method offers enhanced precision, throughput, and speed compared to the traditional TCID50 assay for late-stage clinical development.
Area of Science:
- Virology
- Oncolytic Therapy
- Biotechnology
Background:
- Oncolytic viruses (OV) are investigational therapies that selectively destroy tumor cells.
- Coxsackievirus A21 (CVA21) is an OV undergoing clinical evaluation.
- Early clinical development relied on TCID50 assays for CVA21 potency, which had limitations.
Purpose of the Study:
- To develop and validate a plaque assay for CVA21 potency testing suitable for late-stage clinical development.
- To replace the less precise and time-consuming TCID50 assay.
- To establish a robust method for quantifying OV infectivity.
Main Methods:
- Development and qualification of a plaque assay for CVA21.
- Validation of the plaque assay for linearity, accuracy, precision, and specificity.
- Statistical analysis of assay data.
Main Results:
- The plaque assay provides a direct measure of viral plaque-forming units for potency.
- The plaque assay demonstrated improved precision (32% vs. 58% variability) compared to TCID50.
- The plaque assay offers higher sample throughput (22 vs. 3 samples/week) and a shorter turnaround time (4 vs. 7 days).
Conclusions:
- A validated plaque assay is superior to the TCID50 method for CVA21 potency release and stability testing.
- This assay development provides a model for robust cell-based potency assays for OV and other viral products.
- The improved assay supports the progression of CVA21 oncolytic virus therapy into late-stage clinical trials.
