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Updated: Feb 4, 2026
An Efficient Method for Adenovirus Production
Published on: June 10, 2021
A Flow Cytometry-Based Method to Determine the Titer of Adenoviruses Expressing an Extraneous Gene
Akikazu Asada1, Hideki Hayakawa1, Natsuki Yanase1
1Oncolys BioPharma, Inc.
Abstract:
In recent times, oncolytic viruses expressing an extraneous gene have attracted great interest; in fact, they have been engaged in multiple applications, such as medicine for cancer. Our group made an oncolytic adenovirus, namely, OBP-301, for use in treating solid cancers and press clinical trial to get approval for a pharmaceutical product. In this study, we applied a flow cytometry-based method to determine the titer of adenoviruses expressing an extraneous gene as well as assess their quality. We considered using the green fluorescent protein (GFP)50 titer as a measure of viral quality. The GFP50 titer (GFP50/mL) is the viral load required to render the HeLa S3 cell line 50% GFP-positive by analysing flow cytometry data. We measured the GFP50 titers for three types of recombinant adenoviruses (OBP-401, OBP-1101, and OBP-1106). We compared GFP50/mL and tissue culture infectious dose (TCID50/mL), a conventional titration index, and found that these titers showed a linear correlation, with a correlation coefficient of >0.9. Moreover, GFP50/mL showed high repetitive accuracy. We expect this flow cytometry-based method to be useful in case of clinically relevant viruses expressing an extraneous gene, in particular, to control viral quality.
Insights
A new flow cytometry method accurately measures the quality of oncolytic adenoviruses expressing foreign genes. This GFP50 titer method shows high accuracy and correlates well with traditional titration, aiding clinical applications.
Area of Science:
- Biotechnology
- Virology
- Molecular Biology
Background:
- Oncolytic viruses engineered to express foreign genes are promising cancer therapeutics.
- OBP-301 is an oncolytic adenovirus developed for solid cancer treatment and is undergoing clinical trials.
- Accurate titration and quality control are crucial for clinical-grade viral vectors.
Purpose of the Study:
- To establish and validate a flow cytometry-based method for titering and assessing the quality of recombinant adenoviruses.
- To evaluate the utility of the green fluorescent protein (GFP)50 titer as a measure of viral quality.
Main Methods:
- A flow cytometry assay was developed to determine the GFP50 titer (GFP50/mL).
- This involved measuring the viral dose required to achieve 50% GFP expression in HeLa S3 cells.
- GFP50 titers of three recombinant adenoviruses (OBP-401, OBP-1101, OBP-1106) were measured.
Main Results:
- The GFP50 titer demonstrated a strong linear correlation (R > 0.9) with the conventional tissue culture infectious dose 50 (TCID50/mL).
- The GFP50/mL assay exhibited high repetitive accuracy, indicating reliability.
- The method effectively quantifies viral load and quality for gene-expressing adenoviruses.
Conclusions:
- The flow cytometry-based GFP50 titer assay is a reliable and accurate method for titering and quality control of recombinant adenoviruses.
- This method is expected to be valuable for clinical applications involving oncolytic viruses expressing foreign genes.
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