Facing the Challenges in Vaccine Upstream Bioprocessing * Scalable Production of AAV Vectors Table. Method of removal and measurement of AAV vector production impurities AAV Vector Production Impurity Method of Removal Method of Measurement Residual host cell DNA/RNA (nuclease-sensitive) Nuclease treatment (Benzonase) qPCR using amplicons to generic host cell genome (e.g., 18SRNA gene); qPCR using amplicons for sequences of specific concern (e.g., AdE1); qPCR using amplicons for non-vector genome sequences Residual host cell protein Ultracentrifugation; ion exchange chromatography ELISA using polyclonal antibodies detecting representative proteins Residual plasmid DNA (nuclease-sensitive) Ultracentrifugation; ion exchange chromatography qPCR using amplicons for helper virus sequences; infectious titer of helper viruses; ELISA or Western blotting for helper virus proteins Residual helper viruses (nucleic acids and proteins) Nuclease treatment (Benzonase) Various, depending on component AAV empty capsids Ultracentrifugation; ion exchange chromatography Electron microscopy; spectrophotometry Encapsidated host cell nucleic acids (nuclease-resistant) qPCR using amplicons to generic host cell genome sequences Table continued on page 15 12 | GENengnews.com Figure 1. Elements in optimization. Before optimization, the trans-splicing efficiency is about 1-5% when compared to GFP expression from single vector. After optimization, the efficiency reaches about 25-50%.http://www.GENengnews.com