Human Gene Therapy - April 2023 - 263

SHORT COURSE IN AAV MANUFACTURING
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effects of the addition of enhancer were also evaluated.
Students counted the cultures prepared by staff, set up
cultures at different cell densities, made up the DNA/
transfection reagent complex, and transfected the cultures
according to the conditions assigned to each group.
Seventy-two hours post-transfection, the staff counted
the cultures, confirmed GFP expression, and lysed the
culture using 10 · lysis buffer. The samples were then
analyzed by quantitative polymerase chain reaction
(qPCR) to determine the vector genomes/mL. Staff analyze
the data using statistical analysis software, specifically
JMP Pro software (v15.1.0). Using ap-value cutoff
of 0.05, it was shown that only temperature and cell density
had a statistically significant impact on the vector titer.
This finding is illustrated (Fig. 2), which shows the desirability
profile from the statistical analysis performed by
the JMP software. L1 refers to the absence of enhancer.
This is a complicated experiment to run in class, as each
group executes one set of conditions in the DoE. If one
group makes an error (such as forgetting to add one of the
DNAs), the entire experiment is in jeopardy.
With students at varying levels of general laboratory
technique and cell culture skills, it was decided to simplify
the design of the laboratory after running the experiment as
described in six offerings. Students are now given data from
theDoE described above and then set up flasks to confirm the
effect of temperature and evaluate the addition of enhancer.
In the next upstream processing lecture, students are
introduced to scale up in single-use bioreactors (SUBs)-
both rocking and stirred tank. The scale-up of adherent and
suspension cells is discussed. Topics include bioreactor
bag films and the effect of leachables, setting up a 50L
reactor (video), as well as various tools (welders, sealers,
aseptic connectors, and disconnecting devices) that are
needed to connect and disconnect addition vessels to the
bioreactor.
To reinforce the topics presented in the lecture, students
return to the laboratory (day 1) and observe, under the microscope,
adherent HEK293 cells growing in a 10-stack cell
stack and manipulated the cell stack. A short video of harvesting
cells from T-flasks and seeding the 10-stack cell
stack is shown. The term scale out instead of scale-up is
introduced, and students are able to fully understand why it
is preferable, due to the time involved, chance for contamination,
and vessel-to-vessel variability, to scale up with
suspension cells where possible. Next, students open up a
rocker bag (which would be used for small-scale production
of AAV or to prepare inoculum for a larger production reactor),
familiarize themselves with the purpose of the parts
on the bag, and position the bag on the bioreactor platform.
The instructor reviews the hardware and software for
the rocker bag, and students, following the laboratory
protocol, inflate the bag with air. Once the bag is inflated,
the students weld the bag of viral production medium to
the inflated bag and add 4.0 L of viral production medium
by weight. The students also familiarize themselves with
the parts of the 50L SUB, in which an AAV2-GFP production
run using baculovirus is ongoing.
In this run, the F3 cell line, which has a stably integrated
Rep2 and Cap2,isused. Rep and Cap are not expressed
until infection with the baculovirus.7 The cells are scaled up
to 50L at *1.5E+6 cells/mL and infected with GFP-ITR
baculovirus infected insect cells (BIICs) at a ratio of 7mL
BIIC to 50 L.8 Students are given the batch record, filled out
Figure 2. Data from a DoE study looking at the effect of temperature, DNA molar ratio, cell density, and addition of enhancer during transfection of AAV293
cells on AAV2-GFP titer. The data, analyzed with the statistical analysis software JMP, show that a lower temperature and lower cell density yielded optimal
titers. DoE, design of experiment.

Human Gene Therapy - April 2023

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