Figure 3.

Simplified downstream process, with direct loading of clarified lysate on AEX. (a) Schematics of previous and revised DSPs. The dashed box indicates potential execution of depth filter clarification and AEX as a single unit operation. (b) Loading of clarified lysate on 3 ml Sartobind Q anion exchange membrane, followed by elution with a gradient of increasing salt concentration. The peaks labeled 1 and 2 (at 24 and 37 mS/cm) were analyzed by Coomassie‐stained SDS‐PAGE (c), with comparison with virus purified by caesium chloride gradient ultracentrifugation (CsCl) and molecular weight marker (MW, with 80 kDa indicated). Peak 1 contains impurities (notably free hexon protein) while Peak 2 contains predominantly virus. (d) AEX chromatogram obtained using clarified lysate from low MOI upstream process, run at 1000 L scale. Absorbance at 280 nm is shown in blue, conductivity in red. Results are shown from one of two cycles run on a 5000 ml Sartobind Q capsule, each loaded to approximately 1.5 × 10¹³ VP per ml of membrane. Numerals indicate stages: 1 = loading, 2 = wash, 3 = elution, 4 = 1 M NaCl strip and 1 M sodium hydroxide sanitization. (e) Product recovery and quality from the 1000 L scale process shown in (b), after AEX and after final formulation by TFF and 0.2 μm filtration. AEX, anion exchange; DSP, downstream process; MOI, multiplicity of infection; SDS‐PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TFF, tangential flow filtration