4.6 High Cell Density-High MOI Production Process: A Case Study
of Three-Bac
When various proteins are co-expressed via multiple baculoviruses, the
kinetic of expression of these proteins have an important effect on the
overall quantitative and qualitative yield of AAV. The optimal MOI (MOI
of 5 of each) of three baculovirus vectors for AAV production was very
high[55]. However, infection of Sf9 cells at such a high MOI in a
high cell density process poses an additional burden on baculovirus
stock preparation. Generally, the baculovirus stock with a concentration
of 1-3x108 pfu/mL requires addition of a large volume
to achieve a high MOI, which often results in a substantial dilution of
the culture. For example, in an AAV production process involving
infection at 3-4 million cells/mL, the baculovirus co-infection at
individual MOI of 5 requires around 17%-20% v/v of each of three
baculoviruses stock (1x108 pfu/mL) which results in an
overall dilution of the culture by around 50%-60% v/v which is not
desirable. The direct solution to this is to use either concentrated
stock of baculovirus, preparations of which has its challenges, or low
MOI process[59], which results in asynchronous infection. An
alternative solution was reported by Cecchini et al., where the
researchers proposed the strategy that mimics low MOI infection culture
behavior and involves baculovirus-infected insect cell (BIIC)[57].
Here, the Sf9 cells were infected at low MOI of each of three
baculoviruses, which results in an asynchronous infection and infection
of the entire cell population within 48 hpi. Following this, the BIIC at
48 hpi were mixed with non-infected insect cells (NIIC) in a ratio of
1:10000 and frozen under liquid nitrogen. When thawed, the cells undergo
a secondary wave of baculovirus infection from the progeny generated
from BIIC and infect the rest of NIIC in the culture within 48-72 hours
post vial thaw. This strategy eliminated the baculovirus infection step
in the actual culture and offered consistency in baculovirus infection
kinetics, and protein expression since the relative proportion of BIIC
and NIIC is kept constant in the frozen cell bank. After the vial thaw,
the culture is volumetrically expanded by the addition of fresh medium
until the desired working volume in a bioreactor is achieved. When
assessed at different scales (shake flask to 200L bioreactor) for
different serotypes (AAV -6 and -9), a comparable yield of AAV serotypes
was reported (Table 1) [57].