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Toward Translation of Cowpea Mosaic Virus Intratumoral Immunotherapy With a Scalable Production Process
Patrick Opdensteinen1,2,3, Jessica Fernanda Affonso de Oliveira1,2,3, Seongwon Jung2,3,4
1Aiiso Yufeng Li Family Department of Chemical and Nano Engineering, University of California, San Diego, La Jolla, California, USA.
Abstract:
The plant virus cowpea mosaic virus (CPMV) is being developed as a drug candidate for intratumoral immunotherapy for solid tumours. Data from preclinical studies in mice and rats as well as clinical studies in canine cancer patients demonstrate potent efficacy and an excellent safety profile. Toward Investigational New Drug (IND)-enabling studies, a scalable and Good Manufacturing Practice (GMP)-compatible manufacturing process must be developed. Therefore, here, we focused on the downstream process and developed a scalable purification process. We systematically screened the following processes: acidic extraction conditions making use of the virion pH stability, ultrafiltration making use of the nanoparticle character and the virion size, and finally ion exchange chromatography based on the virion's surface charge. The three processes were then combined into a 7-step protocol, providing efficiency compared to the contemporary laboratory procedures involving various centrifugation steps (and a total of 15 steps to yield pure CPMV). Furthermore, we streamlined the removal of endotoxins by combining detergent-based endotoxin removal with ion exchange chromatography. The new ultrafiltration-based process reduced the number of unit operations by more than half and the processing time from ~20 to ~7 h compared to centrifugation-based purification of CPMV. Importantly, toxic organic solvents, ultracentrifugation and isopycnic ultracentrifugation which are difficult to scale were omitted. We used a battery of characterisation methods to validate CPMV's structural integrity and biological activity.
Insights
A new purification process for cowpea mosaic virus (CPMV) has been developed for cancer immunotherapy. This scalable method significantly reduces processing time and complexity, enabling its use in clinical applications.
Area of Science:
- Biotechnology
- Nanomedicine
- Virology
Background:
- Cowpea mosaic virus (CPMV) shows promise as an intratumoral immunotherapy agent for solid tumors.
- Preclinical and clinical studies indicate potent efficacy and a favorable safety profile for CPMV.
- Development of a scalable, Good Manufacturing Practice (GMP)-compatible manufacturing process is crucial for Investigational New Drug (IND)-enabling studies.
Purpose of the Study:
- To develop a scalable and efficient downstream purification process for CPMV.
- To optimize purification by screening acidic extraction, ultrafiltration, and ion exchange chromatography.
- To streamline endotoxin removal and reduce overall processing time and complexity.
Main Methods:
- Screening of acidic extraction conditions based on virion pH stability.
- Utilizing ultrafiltration based on nanoparticle characteristics and virion size.
- Employing ion exchange chromatography leveraging the virion's surface charge.
- Combining optimized steps into a streamlined 7-step protocol, including integrated endotoxin removal.
Main Results:
- A 7-step purification protocol was established, significantly more efficient than traditional 15-step centrifugation methods.
- The ultrafiltration-based process reduced unit operations by over half and processing time from ~20 to ~7 hours.
- Scalable methods replaced toxic organic solvents and ultracentrifugation techniques.
- Characterization methods confirmed the structural integrity and biological activity of the purified CPMV.
Conclusions:
- A scalable, GMP-compatible downstream purification process for CPMV has been successfully developed.
- The new process is more efficient, faster, and amenable to large-scale production compared to existing methods.
- This advancement facilitates the progression of CPMV as a therapeutic agent for cancer immunotherapy.
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