Downstream processing of MDCK cell-derived equine influenza virus

Area of Science:

• Virology
• Biotechnology
• Bioprocess Engineering

Background:

• Equine influenza virus (EIV) poses a significant threat to equine health.
• Efficient production and purification of EIV are crucial for vaccine development and research.
• Current purification methods require optimization for yield and purity.

Purpose of the Study:

• To develop and optimize a microcarrier-based process for equine influenza virus production.
• To evaluate downstream processing steps for efficient virus purification.
• To assess the recovery of viral hemagglutinin (HA) and neuraminidase (NA) and removal of contaminants.

Main Methods:

• Equine influenza virus (A/Equi 2 (H3N8)) production using Madin Darby Canine Kidney (MDCK) cells on microcarriers.
• Downstream processing including depth filtration, inactivation, ultrafiltration (UF), and size exclusion chromatography (gel filtration).
• Quantification of hemagglutinin (HA) and neuraminidase (NA) activity, host cell proteins, and DNA.

Main Results:

• Ultrafiltration step achieved 100% hemagglutinin recovery and removed over 88% of contaminating proteins and DNA.
• Size exclusion chromatography yielded 37.8% HA and 59.8% NA recovery in the virus peak.
• Non-specific binding to the gel matrix was observed, impacting overall recovery (35.8% HA, 291.6% NA).
• Over 95.7% host cell proteins and 98.7% host cell DNA were removed.

Conclusions:

• A microcarrier-based process coupled with optimized downstream processing can effectively produce and purify equine influenza virus.
• Ultrafiltration is highly effective for HA recovery and initial contaminant removal.
• Further optimization of gel filtration is needed to mitigate non-specific binding and improve overall viral yield.