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Vijetha Bhat1*, Ruda Cui1*, Yimeng Zeng1, Bingnan Gu1, Anandita Seth1, and Young Shin1
1. Research & Development, Lonza Houston Inc., Houston, TX, 77047 *Co-first authors
Lentiviral vector is one of the most effective and popular delivery vehicles in cell and gene therapy as the therapeutic gene delivered by LV can be maintained for the lifetime of patients. In addition, LV delivers its
cargo into both dividing and non-dividing cells, due to which LV has broad applications in vivo and ex vivo. The demand for lentiviral vectors is higher than ever with the recent success of many CAR-T therapies as
well as other clinical trials for genetic diseases. Traditionally, the production of lentiviral vectors in a large scale uses transient transfection, where adherent or suspension cells are transfected with multiple
plasmids. However, this approach is constrained by its limited scalability and high cost. Furthermore, a large quantity of plasmid DNA is required in the process, adding more challenges in logistics, quality control,
safety, and process robustness. To overcome these problems associated with transient transfection, Lonza has generated a stably transfected, inducible producer cell line (PCL) for the large-scale production of
lentiviral vectors in suspension culture.
For lentiviral vector production, one needs to provide the Vesicular Stomatitis Virus G protein (VSV-G), the regulator of virion expression (Rev) of human immunodeficiency virus-1 (HIV-1), and group-specific
antigen and polymerase (Gag-Pol) of HIV-1. The sequences of VSV-G and Rev were codon-optimized to allow their higher expression. Each gene was driven from a tetracycline-inducible, cytomegalovirus (CMV)
immediate early promoter. To facilitate efficient, site-specific integration of plasmid DNA into the host cell chromosome, we have adopted PiggyBac transposase technology (exclusively licensed to Lonza) and all
of the genes above were cloned into PiggyBac transposon, making a packaging plasmid. Green fluorescent protein (GFP) was chosen for our gene of interest (GOI) and put into a separate PiggyBac transposon,
making a transfer vector plasmid. These two plasmids were transfected into the 2G7 cell (Lonza’s proprietary cell line for LV production) followed by antibiotic selection to obtain pools of stable producer cell lines.
In this process, we tested different plasmid ratios, transfection agents, and antibiotic concentrations to find the optimal condition for PCL generation. Out of these pools generated from different conditions, Pool-
2 produced one of the highest LV titers (2~4E6 TU/mL by flow cytometry) upon induction. Single-cell cloning was performed by limited dilution using the cells from pool-2. During this process, the growth of cells
from a single cell to patches, was monitored and documented using a CloneSelect Imager. Several single-cell clones were isolated, amplified, and suspension-adapted before banking in this process. The
productivity of each isolated clone was measured. Upon induction with doxycycline, some of the PCL clones produced significantly higher titers (~ 4E7 TU/mL by flow cytometry) than the parental pool-2. The
stability of these PCL clones was assessed by maintaining them through multiple passages, and LV titers were measured across passages post induction. The copy number of packaging and transfer vector
plasmids integrated into the host cell chromosomes will be measured by ddPCR for selected PCL clones. Thus, Lonza has established a procedure to generate a highly productive PCL for the large-scale production
of lentiviral vectors in suspension.
DISCLAIMERAll trademarks belong to Lonza or its affiliates or to their respective third party owners. The information contained herein is believed to be correct and corresponds to the latest state of scientific and technical knowledge. However, no warranty is made, either expressed or implied, regarding its accuracy or the results to be obtained from the use of such information and no warranty is expressed or implied.
Generation of Stable Producer Cell Lines (PCL) of
Lentiviral Vector (LV) Using PiggyBac Transposase
Abstract
Single Cell Cloning and Selection of Top Producer
Process Optimization and Characterization
Summary
Generation of PCL Pools and Evaluation of Productivity
Process Optimization
Harvesting on Day 3 increased the titer by over 3-fold
PiggyBac Transposase-mediated integration
facilitates the efficient integration
pZA01: Packaging Plasmid
pZA02: Transfer VectorTransfection
ReagentA A B B A A A A A
Transfection
Ratio (P1:P2)Ratio 1 Ratio 1 Ratio 1 Ratio 1 Ratio 2 Ratio 2 Ratio 2
pZA01
only
pZA01
only
Transposase No Yes No Yes No Yes Yes No Yes
Clonal Selection
From the PCL Pool-2, 26 single cell clones were isolated and tested through 8 rounds of screening
Two single cell clones – DH4 and ED8 - consistently showed higher LV productivities (> 1E7 TU/mL) and shorter doubling times.
6.0E+05
6.0E+06
6.0E+07
AB1 AF1 AF5 BB1 BB3 CE5 CE7 DE2 DH4 EC5 ED8 Pool-2
Infe
ctio
us
Tite
r (T
U/m
L)
Teaser R1 R2 R3 R4 R5 R6 R7
CloneAverageDoubling Time (hrs)
Average Titer (TU / mL)
DH4 34.0 1.24E07
ED8 31.8 1.58E07
Producer Cell Line Generation
Infection of LV on
target cell
Flow cytometry
orddPCR
VSV-G
HEK293T Stable cell line
Rev Gag/Pol
GOI
Infectious Titers from PCL Pools
1.9E+04
2.1E+06
1.1E+04
1.2E+06
3.4E+04
3.5E+05
1.0E+06
5.6E+04 4.3E+04
1.0E+03
1.0E+04
1.0E+05
1.0E+06
1.0E+07
Pool-1 Pool -2 Pool- 3 Pool- 4 Pool -5 Pool -6 Pool -8 Pool -9 Pool -10
TU/m
L
Transposase enhances LV titer by ~ 2 logs.
Single Cell Cloning
Clonal Expansion
BankingLimited Dilution
Lonza’s 2G7 Cell Line
Clonality and expansion were documented via a CloneSelect imager
Lonza has developed stable producer cell lines (PCL) for production of lentiviral vectors using a unique PiggyBac-based plasmid design developed in-house. Adding Transposase RNA in the transfection increased resulting LV PCL pool titers by ~2 logs. The single cell clones (ED8 and DH4) of LV PCL were generated from Lonza’s suspension-adapted HEK293T clonal line (2G7), the productivity and stability of which were maintained over
20 passages even in the absence of antibiotics. Lonza’s LV-GFP PCL clones produced ~4.5E7 TU/mL at the time of harvest, which is one of the highest titers in the market.
Average infectious LV titer is ~ 4.5E7 TU/mL at time of harvest. Similar titers were observed even in the absence of antibiotics.
Producer Cell Line Productivity and Stability
Single cell clones DH4 and ED8 are stable over 20 passages
1.7E+07 1.8E+07
ULD
5.1E+073.6E+07 4.3E+07
3.2E+07 3.5E+07
31
1
10
100
2.0E
+04
2.0E
+06
2.0E
+08
Uninduced 5 10 15 20
Do
ub
ling
Tim
e (h
rs)
Infe
ctio
us
Tite
r (T
U/m
L)
ED8
Day 2 Day 3 Without antibiotics
1.2E+07 1.5E+07
ULD
5.3E+07 4.5E+074.7E+07
4.0E+07 4.4E+07
33
1
10
100
2.0E
+04
2.0E
+06
2.0E
+08
Uninduced 5 10 15 20
Do
ub
ling
Tim
e (h
rs)
Infe
ctio
us
Tite
r (T
U/m
L)
DH4
Day 2 Day 3 Without antibiotics
Cell seeding densities
Continuous harvest
Supplements
Shaking speeds
Other parameters optimized
1%
100%
354%
0.0
100.0
200.0
300.0
400.0
Day 1 Day 2 Day 3
LV P
rod
uct
ivit
y (%
)
Optimization of Time of Harvest