Next-Generation Transfection Reagent for Large Scale AAV
Manufacturing
Mathieu Porte, Mégane Denu, Marine Ricordel, Jonathan Havard, Coralie Stritt, Yann Philipson, Malik Hellal, Patrick Erbacher
Polyplus-transfection®, Vectura, 75 rue Marguerite Perey, 67400 Illkirch, FranceAbstract
The number of ATMP therapeutic-based medicines for inherited genetic disorders is in constant growth, with a global 32% increase in new clinical trials in the last 4 years. ATMPs have demonstrated their success withalready more than ten approved for commercialization. The success of AAV as the most promising viral vector for gene therapy is due to low immunogenicity, broad tropism and non-integrating properties. One major
challenge for translation of promising research to clinical development is the manufacture of sufficient quantities of AAV. Transient transfection of suspension cells is the most commonly used production platform, as
it offers significant flexibility for cell and gene therapy development. However, this method presents some limitations in large scale bioreactors: inadequate transfection protocol, reduced transfection efficiency andlower productivity. To address this concern, we present data on a novel transfection reagent showing: i) increased AAV titers, ii) improved transfection protocol for large scale bioreactors and iii) reproducibility of viral
titers at different production scale. The aforementioned optimized parameters make this novel transfection reagent ideal for cell and gene therapy developers by combining the flexibility of transient transfection withscalability and speed to market.

Improved transfection protocol
for large scale suspension bioreactors

Materials & Methods
pAAV-GFP

D4

D1

D7

Workflow chart of the rAAV2 production and analysis. (D0) Suspension-adapted HEK-293T cells are seeded at 1x106 cells/mL in 30mL Freestyle™ F17 culture medium in a 125 ml shaker flask. The cells are incubated at 37°C and 8% CO2 at 130 rpm. (D1) Afterincubation, cells are in exponential growth phase at around 2 million cells/mL. After cell counting, triple-transfection of rAAV2 coding
plasmids with GFP as gene of interest is performed. (D4) rAAV2 are harvested after cell lysis by 3 thaw/freeze cycles. Aftercentrifugation, the virus physical titer is assayed in the supernatant either by capsid ELISA (Viral Particles or VP) or by qPCR (ViralGenomes or VG) analysis. The functional titer (Transduction Units or TU) is measured with an infectivity assay : adherent HEK-293Tcells are infected with serial dilutions of harvested rAAV2. (D7) three days post-infection, cells are analyzed by flow cytometry todetermine the number of GFP-positive transduced cells.

B.

Screening

HITS
Structure-activity
relationship study

Leadcompounds

9080706050403020101 2 3 4 5 6 7 8 9 101112131415161718192021222324252627282930313233
Hit-to-lead molecules

Discovery process and validation of abreakthrough transfection reagent forsuspension rAAV production.
A. Description of the discovery process
followed to identify and optimize theperfect reagent for suspension rAAVproduction.
B. Suspension HEK-293T cells weretransfected with a GFP plasmid using
molecules from the hit-to-lead screening.
Transfection efficiency was assayed at 48h
post-transfection using flow cytometry.
C. rAAV were produced in suspension HEK293T cells either with PEIpro® or with thelead compounds. The graph shows theresults of 12 different molecules in 12
different experiments. Functional titer wasmeasured with an infectivity assay

Optimization

Functional titer (TU/mL)

1E+08

Formulation

1E+07

FectoVIR®-AAV

1E+00

6h

Viral genomes

Leads

C.

Complexation medium
Functional titers (TU/mL)

1E+12
1E+10
1E+08
1E+06
1E+04
1E+02
1E+00

Freestyle®
F17

Functional rAAV2 production
1E+11

1E+10

1.5 µg DNA/millioncells
1.5 µg
DNA:reagent ratio: 1µg:4µL
PEI MAX reagent
Other PEI-basedDMEM

BalanCD®
HEK-293

PBS

Opti-MEM™

Volume increase

6050
4033

1E+0430
201E+02
103130 mL

10100 mL

300 mL

1L1,5L

0Stirred Tank Reactor

FectoVIR®-AAV shows a great scalability with perfectly consistent results between shake flasks and stirred tank reactor in a 50-foldvolume increase study. HEK-293T cells in suspension were transfected with FectoVIR®-AAV with 1 µg DNA/million cells and a DNA :reagent ratio of 1 µg : 1 µL. Complexes were prepared in DMEM in 1% of the final culture volume and incubated for 30 min before being
added to the cells. Functional titer was measured with an infectivity assay
Lot-to-lot reproducibility

OtherPEIPEI MAX
based reagent
1.5 µg DNA/million cells
1.5 µg
DNA:reagent ratio: 1µg:4µL
OtherPEI-based
PEI-based
Otherreagent
PEI MAXreagent

Viral genomes (VG/mL)

1E+14

Shake flasks

Packaging efficiency

B.

10%

FectoVIR®-AAV

As a robust and reliable product, FectoVIR®-AAV
demonstrates
anexcellent
lot-to-lot
reproducibility. HEK-293T cells in suspension weretransfected with FectoVIR®-AAV with 1 µgDNA/million cells and a DNA : reagent ratio of 1 µg :1 µL. Complexes were prepared in DMEM in 5% of
the final culture volume and incubated for 30 min
before being added to the cells. Functional titer wasmeasured with an infectivity assay.

1E+08

1 µg DNA/millioncells
1 µg
DNA:reagent ratio: 1µg:1µL
FectoVIR-AAV
FectoVIR®-AAV

5%

1E+06

1E+00

1E+11

1 µg DNA/million
cells
1 µg
DNA:reagent ratio: 1µg:1µL
FectoVIR®-AAV
FectoVIR®-AAV

1%50

FectoVIR-AAV
FectoVIR®-AAV

1E+09

1E+01

1E+0805101520

Full capsid ratio (%)

With 33% less DNA, an improvement in bothviral genome production and packaging
efficiency with FectoVIR®-AAV results in a 10fold increase in functional rAAV2 production incomparison with competitor.
Suspension-HEK-293T cells were transfectedwith the optimal conditions for the other PEIbased reagent (1.5 µg/million cells, ratio DNA :PEI of 1 µg : 4 µL) and FectoVIR®-AAV (1µg/million cells, ratio DNA : reagent of 1 µg : 1µL) following the recommended protocol foreach reagent.
A. The viral genome production was measured
by qPCR analysis.
B. Capsid titer was determined by ELISA. The full
capsid ratio was calculated by dividing the VGtiter (VG/mL) by the capsid titer (VP/mL).
C. Functional titer was measured with aninfectivity assay.

PEIpro® and FectoVIR® are registered trademarks of Polyplus-transfection®.
Freestyle™ and Opti-MEM™ are trademarks of Thermo Fisher Scientific®.
BalanCD® is a registered trademark of Irvine Scientific®.

Functional titer (TU/mL)

Viral Genomes (VG/mL)
Total TU (30 mL)C.

3hFunctional titer

Increased AAV titers using less DNA

1E+10

2h

1E+02

Scalability and reproducibility

1E+06

PEIpro®

A. 1E+121h30

By offering great flexibility in the transfection mix
preparation, FectoVIR®-AAV allows to overcome themajor limitations encountered in large-scalebioreactors transfection.
For each experiment, HEK-293T cells in suspension
were transfected with FectoVIR®-AAV using 1 µg
DNA/million cells and a DNA : reagent ratio of 1 µg : 1µL. If not mentioned otherwise, transfection complexes
were prepared in DMEM in 5% of the final volume ofculture and incubated 30 min before being added tothe cells.
A. After preparation, the transfection mix wereincubated at room temperature at rest from 15 min to6h. Functional titer was measured with an infectivity
assay.
B. Transfection complexes were prepared in a volume
ranging from 1 to 10% of the final volume of culture (30mL). Viral genomes were measured using qPCR analysis.
C. Transfection complexes were prepared in different
complexation media. Viral genomes were assayed usingqPCR analysis and functional titer was determined in aninfectivity assay.

100

1E+09C.

Upscale1h

1E+03

Transfection efficiency of hit-to-lead molecules0

Chemicallydefinedmolecule

15 min 30 min 45 min

1E+04

FectoVIR®-AAV
FectoVIR-AAV

Transfection efficiency (% GFP-positive cells)Internalchemicallibrary

Functional titer (TU/mL)

A.

1E+08

1E+07

Volume of complexation

1E+05

1E+09

Functional titer (TU/mL)
Viral genomes (VG/mL)

D0

Infection

HarvestB.

Transfection mix incubation time

Volume fold increase

Transfection

Cell seeding

A.

Analysiso Flow cytometry

Viral genomes per cell (VG/cell)

pHelperpAAV-RepCap

Functional titer (TU/mL)

Analysiso qPCRo ELISA

1E+06
1E+04
1E+02
1E+00

Lot A

Lot B

Lot C

Lot D

FectoVIR®-AAVConclusion

High performance: superior yields for rAAV production in suspension
Ease of use: reduces complexation volume, stable transfection mix
Cost-saver: reduces DNA amount
Scalable: from small to large scale industrial production
Flexible: compatible with different culture medium
GMP compliance: GMP grade available

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