High-cell-density cultivations to increase MVA virus production

Area of Science:

• Biotechnology and Bioprocessing
• Vaccine Manufacturing
• Cell Culture Technology

Background:

• High-cell-density (HCD) cultivations present challenges in balancing medium consumption, nutrient limitation, and toxic metabolite accumulation for economical vaccine production.
• Optimizing HCD conditions is crucial to prevent apoptosis or autophagy during early viral infection phases, ensuring efficient vaccine yield.
• Rational analysis of host cell line cultivation and viral vaccine strain infection conditions is essential for each specific manufacturing process.

Purpose of the Study:

• To develop and illustrate a strategy for producing modified vaccinia Ankara (MVA) virus isolate MVA-CR19 in avian suspension cells (AGE1.CR.pIX) using HCD cultivations.
• To optimize perfusion rates and feeding strategies for enhanced cell growth and virus propagation in HCD bioreactor systems.
• To evaluate the impact of different feeding strategies on MVA-CR19 yield and productivity at HCD compared to conventional-cell-density (CCD) processes.

Main Methods:

• Perfusion rate adjustment based on measured cell concentration and glucose consumption in a 0.8 L bioreactor with an ATF2 system.
• Analysis of various feeding strategies in small-scale shake flask cultivations to optimize virus propagation phase at HCD.
• Implementation of discontinuous perfusion (semi-perfusion), medium exchange at infection, and fed-batch feeding strategies.

Main Results:

• Achieved cell concentrations up to 57 x 10^6 cells/mL with >95% viability in the bioreactor, and up to 63 x 10^6 cells/mL in shake flasks.
• Identified the necessity of medium exchange at infection and optimized feeding strategies for improved virus yields at HCD.
• Demonstrated a combination of fed-batch and medium exchange strategies yielding 10-fold higher MVA-CR19 titers and four times higher volumetric productivity compared to CCD.

Conclusions:

• HCD cultivation of AGE1.CR.pIX cells is feasible and can be optimized for MVA-CR19 production.
• Strategic implementation of perfusion control, medium exchange, and fed-batch feeding is critical for maximizing virus yield and productivity in HCD processes.
• The developed strategy offers a significant improvement in MVA vaccine manufacturing efficiency, achieving high titers and productivity.