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.