Development and optimization of an adenovirus production process

Area of Science:

• Biotechnology
• Gene Therapy
• Virology

Background:

• Adenoviral vectors are valuable tools in gene therapy, particularly for cancer treatment, due to their ability to infect non-dividing cells.
• First-generation adenoviral vectors have limitations, driving the need for improved vectors with enhanced safety and reduced immunogenicity.
• Efficient, scalable, and reproducible production processes are essential to meet the growing demand for adenoviral vectors in clinical gene therapy programs.

Purpose of the Study:

• To optimize the production process of adenoviral vectors using HEK-293 cells.
• To develop scalable and robust manufacturing methods for clinical-grade adenoviral vectors.
• To identify optimal culture conditions and purification strategies for high-titer adenoviral vector production.

Main Methods:

• Evaluation of different culture modes (batch, fed-batch, perfusion) for HEK-293 cells.
• Development of in situ monitoring tools (GFP probe) and quantification techniques (HPLC).
• Implementation of column chromatography for purification, replacing ultracentrifugation.

Main Results:

• An optimized, integrated process for adenoviral vector production in suspension, serum-free medium was developed.
• The process was successfully scaled from 20 L to 100 L, meeting clinical material specifications.
• Perfusion mode with low-shear cell retention devices was identified as key for overcoming metabolic limitations at high cell densities.

Conclusions:

• Scalable adenoviral vector production in suspension and serum-free conditions is achievable.
• The developed process enables robust manufacturing of clinical-grade adenoviral vectors.
• Further improvements require a deeper understanding of viral replication and maturation in host cells.