Nocodazole improves rAAV genome titre in two different serotypes
To investigate the effect of nocodazole addition in a system more representative of large scale rAAV production, culture volume was scaled up from the initial microplate culture volume of 0.7 mL to 30 mL culture volume in shake flasks. To rule out a serotype-dependent effect of nocodazole addition, plasmids for both rAAV8 and rAAV9 serotypes were separately transfected in HEK293 cells and cultured with and without the addition of 4 µM nocodazole. rAAV genome titre was measured from 24 – 72 HPT. All measured parameters of nocodazole induced cell cycle arrest (reduced VCD, decreased viability, and increased mean cell volume) and increased rAAV genome titre were consistent with those seen at smaller culture volumes (Fig. 3A & B). Comparable to microplate-based cultures, mean viral genome titre was increased by up to 2.5-fold in nocodazole-treated cultures compared to untreated cultures (Fig. 3C). Importantly there were no observed significant differences in the above-described measurements between cultures producing rAAV8 or rAAV9, suggesting a broad applicability for nocodazole addition in rAAV manufacturing.