Human Gene Therapy - April 2023 - 262

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OVERTON ET AL.
Additional details of upstream processing, downstream
processing, and analytical methods of the courses are
presented below.
COURSE DESIGN DETAILS
Central to the design of the course, and in particular the
laboratory activities, is a scalable process that has been developed
(Fig. 1) for the production ofAAV serotype 2with a
transgene for green fluorescent protein (AAV2-GFP).
AAV2-GFP is produced by one of two methods: triple
transfection of human embryonic kidney cells (HEK293)
or infection of recombinant Spodoptera frugiperda (Sf9)
cells (F3) with engineered baculovirus. (Note that from an
instructional perspective, insect cells offer the advantage
of being able to execute laboratories in a biosafety level 1
environment vs. biosafety level 2 for HEK293). Because a
significant amount of AAV is intracellular in either production
system, a lysis step is performed by adding Triton
X-100 (1%) and MgCl2 to the bioreactor and holding it for
1 h at 37C and pH 8.0. (Note that Triton X-100 is on the
European Authorisation list (i.e., Annex XIV of the Registration,
Evaluation, Authentication, and Restriction of
Chemicals regulations). This information is covered in the
vector harvest lecture of the course. BTEC has evaluated
lysis with several alternative reagents and plans to use
Tween 80 in the lysis step in the future.) Benzonase is also
added at the end of the 1 h incubation, and the lysate is
incubated for an additional 30 min to digest nucleic
acids for viscosity reduction and to avoid DNA-AAV
complexes.
The resulting lysate is clarified (i.e., cell debris removed)
using a Millistak+ depth filter (there are several
possible options), followed by a 0.22 lm polish filter
(Opticap Durapore XL). Clarification is required
because the lysate contains numerous soluble impurities
(e.g., host cell proteins, host cell DNA, and capsids
without the transgene) that are removed by packed chromatography
beds that would clog if solids are present in
the feed.
The capture (i.e., first) chromatography step mainly
removes process-related impurities such as host cell proteins,
host cell DNA, and surfactant. This is followed by an
anion-exchange step for the enrichment of full capsids.
The affinity step is performed with either of the following
resins: POROS CaptureSelect AAVX affinity resin
from Thermo Fisher Scientific or AVB Sepharose High
Performance resin from Cytiva. Following chromatography,
a tangential flow filtration (TFF) step may be performed
to concentrate AAV2-GFP and to transfer the
product into an appropriate buffer system, typically using
an Ultracel 300 kDa ultrafiltration (UF) membrane from
MilliporeSigma. The filling of drug substance into containers
(from which drug product is produced) is typically
not performed at BTEC.
Upstream processing section
Various materials were used to conduct the upstream
laboratories of the course. The Sf9 recombinant insect cell
line (F3) and shuttle plasmid pTR-Bac-UF26 were developed
in the laboratory of Dr. Sergei Zolotukhin and
obtained under a material transfer agreement with the
University of Florida. In the F3 cell line, rep2 and cap2 are
stably integrated into the Sf9 insect cell genome.7 MultiBac
cells were obtained from Geneva Biotech. Sf9 cells,
Sf900II cells, and fetal bovine serum were obtained from
Thermo Fisher Scientific. AAV293 cells (HEK293), viral
production medium, Optimem, transfection reagent, transfection
booster, transfection enhancer, and 10· lysis buffer
were all components of the Thermo Fisher Scientific AAVMAX
Helper-Free AAV Production System Kit.
However, the data generated used the beta version of the
protocol and kit. Three plasmids, pAAV-RC2, pAAV-GFP,
and pHelper, were obtained fromCell Biolabs. Baculovirus
was generated from the shuttle plasmid pTR-Bac-UF26
based on a protocol from the Thermo Fisher Scientific Bacto-Bac
Baculovirus Expression System, with the exception
of using MultiBac cells instead of DH10Bac.The
virus was titrated using plaque assay. Adherent HEK293
cells (CRL-1573) and Eagle's Minimal Essential Medium
(EMEM) were obtained from ATCC.
The upstream portion of the course consists of lectures
and laboratories (Table 1). Following the introductory
section of the course, the first upstream lecture covers cell
banking, a description of the baculovirus system, methods
to produce AAV using transient transfection into HEK293
cells, and infection of insect cells with recombinant baculovirus.
Other methods to generate AAV are discussed
in the first upstream lecture, however, the instructor
chooses to use the baculovirus expression system and
helper virus-free transient transfection into suspension
HEK293 cells as methods in the laboratory exercises.
These methods are widely used in the literature. In future
course offerings, lecture material will be expanded to
discuss specific differences in product quality between
these two systems. The lecture describes various factors,
such as DNA concentration, molar ratios of the three
plasmids, transfection reagents, cell density, and temperature,
which can be evaluated to optimize AAV titers. The
lecture also covers lentivirus generation and provides an
overview of production processes for ex vivo gene therapy
(CAR-T cells); however, there are no laboratories associated
with these topics.
Following this lecture, students go to the laboratory to
conduct a process design activity that optimizes AAV2GFP
production in AAV293 cells using a design of experiment
(DoE) with a partial factorial design. In this
study, two temperatures (34C and 37C), three DNA
molar ratios of pAAV-GFP:pAAV-RC2:pHelper (ratio 1-
1:1:1, ratio 2-1:1:3, ratio 3-1:1.5:2), and three cell densities
(2.5, 3.0, and 4.0 E+ 6/mL) were evaluated. The
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