GFP-tagging of extracellular vesicles for rapid process development

Area of Science:

• Biotechnology
• Cell Biology
• Nanomedicine

Background:

• Extracellular vesicles (EVs) are crucial nano-scale messengers for intercellular communication.
• Therapeutic applications of EVs are rapidly expanding, with significant investment in their development.
• Current limitations in EV production include a lack of rapid, high-throughput analytical methods for process optimization.

Purpose of the Study:

• To develop a quantitative analytical tool for accelerated extracellular vesicle (EV) process development.
• To engineer Chinese hamster ovary (CHO) cells for the production of fluorescently tagged EVs.
• To validate the utility of GFP-tagged EVs in monitoring and quantifying EV purification processes.

Main Methods:

• Engineering CHO cells to express GFP-tagged EVs via fusion to CD81.
• Characterizing parent cells to ensure the absence of non-fused GFP.
• Utilizing the fluorescence of GFP-tagged EVs for rapid quantification of concentration and yield.
• Performing mass balance analysis of ultrafiltration processing to determine product loss.
• Monitoring vesicle elution during chromatography separations and detection via western blotting.

Main Results:

• Successfully generated GFP-tagged EVs from engineered CHO cells.
• Demonstrated rapid quantification of EV concentration and yield throughout the purification process.
• Reconciled up to 97% of initial feed mass during ultrafiltration processing, indicating minimal product loss.
• Facilitated straightforward monitoring of vesicle elution from chromatography using GFP fluorescence and western blotting.

Conclusions:

• GFP-tagged EVs provide a quantitative and accessible tool for accelerating EV process development.
• This approach enables efficient monitoring and optimization of EV production and purification.
• The methodology is crucial for overcoming bottlenecks in the large-scale manufacturing of therapeutic EVs.