A Direct Comparison of rAAV5 Variants Derived from the Baculovirus Expression System Using LC-MS Workflows...

Area of Science:

• Molecular Biology
• Biotechnology
• Gene Therapy

Background:

• Adeno-associated virus (AAV) vectors are promising for gene therapy but face challenges in production, immunogenicity, and cost.
• Improving production yield and understanding critical quality attributes are essential for commercial viability.
• Capsid protein stoichiometry and the ratio of empty to full capsids are key parameters affecting AAV product quality and safety.

Purpose of the Study:

• To investigate the impact of different start codons (ATG, CTG, ACG) on the cap gene during recombinant AAV serotype 5 (rAAV5) production using the baculovirus expression vector system (BEVS).
• To characterize rAAV5 variants using advanced liquid chromatography-mass spectrometry (LC-MS) techniques.
• To correlate translational start codon modifications with capsid packaging, protein stoichiometry, and in vitro potency.

Main Methods:

• Production of rAAV5 vectors in Sf9 insect cells utilizing the BEVS with modified cap gene start codons (ATG, CTG, ACG).
• Comprehensive characterization of produced AAV5 variants using novel LC-MS techniques.
• Analysis of virus yield, capsid protein ratios (VP1/VP2), and percentage of empty capsids.

Main Results:

• The ACG start codon yielded the highest virus production but resulted in lower transduction efficiency in mammalian cells.
• Lower transduction efficiency correlated with a reduced VP1/VP2 ratio and an increased proportion of empty capsids.
• LC-MS analysis provided detailed insights into capsid composition and packaging efficiency.

Conclusions:

• Translational start codon choice significantly influences rAAV5 production yield, capsid composition, and functional potency.
• The ACG start codon, while increasing yield, compromises capsid quality and therapeutic efficacy.
• The developed LC-MS workflow is a robust and transferable method for comprehensive AAV characterization, aiding process optimization and commercial manufacturing.