Effect of Source and Time of Collection on rAAV9 Production Yield and Quality: A Comparative Bioprocess Analysis

A comprehensive analysis of Critical Quality Attributes (CQAs) is crucial for extended culture conditions across sources and time points.

The rAAV9-eGFP vectors were produced. Further, collected lysates and culture supernatants (sources) were purified by affinity chromatography (AC) at two time points (72 and 120 hours). Detailed CQA analysis, including vector genome titer, capsid protein abundance, purity, aggregates, and transduction efficiency, was conducted.

The viral titer yield from the 72-h media supernatant was 1.29×1013 ± 7.62×1011 vgc, whereas the lysate yielded 5.28 × 10¹² ± 7.64×1011 vgc. At the 120-h harvest, the supernatant again yielded 7.22 × 10¹² ± 8.97 × 10¹¹ vgc, while the lysate yielded 1.39 × 10¹¹ ± 5.25 × 1010 vgc. ACbased purification showed the absence of degraded capsid proteins, with no detectable host-cell protein contamination across time points. Notably, the SEC revealed pronounced higher-order dimers in 120-h (23% & 55%) lysate-derived vectors purified by AC and CsCl. Transduction efficiency was significantly reduced in the 120-h lysate-purified vectors compared with 72-h lysate (p=0.001) and corresponding 72- and 120-h media supernatants (p=0.004 and p=0.001, respectively).

Substantial variation was observed in total vector yield, with media supernatants consistently yielding higher titers than lysates at both time points. Future studies should focus on process parameters to suppress dimer formation under extended culture conditions and elucidate the impairment of transduction in late-harvest, lysate-derived AAV particles.

The current study established rAAV9 media supernatant harvested at 72-hr and 120-hr as a preferred source selection for improving yield, purity, and potency during scale-up processes.