THE FULLY SCALABLE PRODUCTION SYSTEM FORGENE THERAPY VECTORS

CAP®GT

CAP®GT The Fully Scalable Production System for Gene Therapy Vectors Switch to the expression system best suited to address industrial needs:

• High titer production of lentiviral, adenoviral, and AAV vectors

• Stable helper virus-free AAV packaging cell lines

• Stable LV packaging cell line

• Generation of custom-made producer cell lines

• Serum-free suspension culture with high cell densities

• Easy scale-up in stirred tank bioreactors from 15 mL to industrial scale

• Cost-efficient production by increased cGMP batch sizes

• Compliance with regulatory needs by using one of the best documented cell lines available

• Referenceable biologics master file filed with the US FDA

Suspension cell platform for

commercially scalable cGMP production of

gene therapy vectors

Stable AAV ProductionUnique stable helper virus-free AAV production system, addressing the pro-duction gap in AAV-based gene therapies.

AAV manufacturing using stable cell lines is the best option for scaling up the production process. Stable AAV producer cell lines avoid the challenges of tran- sient transfections.

Screening of 2 different batches of single cell clones of the stable AAV pack-aging platform identifies clones with comparable productivity to the transient approach, even before process optimization.

CAP®GT

Stable production of AAV vectors in CAP-GT packaging and producer cells

CAP-GT cells CAP-GT AAV packaging cell line

+ inducible helper, AAV Rep and AAV Cap genes

+ viral genome with GOI

scale-up+ induction

CAP-GT AAV producer cell line

AAV production

SCC 1 SCC 2 transient SCC 4 SCC 5 backgroundSCC 3

Prod

uctiv

ity

per c

ell c

ompa

red

to tr

ansi

ent p

rodu

ctio

n 3.0

2.5

2.0

1.5

1.0

0.5

0.0

SCC 1 transient SCC 2 SCC 4 SCC 7SCC 5 SCC 6 SCC 8 SCC 9SCC 3

vg/m

L re

lativ

e to

tran

sien

t 1.2

1.0

0.8

0.6

0.4

0.2

0.0

Transient AAV ProductionScalable suspension system for high titer transient AAV production in stirred tank bioreactors.

AAV-titers for different serotypes from crude harvests and before process op-timization, obtained by standard transient, PEI-mediated transfection of CAP-GT cells with corresponding 2-plasmid system (PlasmidFactory) and electron microscopic image of AAV2 particles produced with CAP-GT cells (courtesy of Paragon Bioservices).

Development of a fully scalable protocol for transient transfection without medium exchange yielding high-titer AAV8 production in stirred tank bioreac-tor (STBR) using PlasmidFactory‘s 2-plasmid system and minicircle approach.

CAP®GT

Thaw cells Expand CAP cells in shake flasks (125 mL – 5 L). Culture in medium supporting cell growth and transfection

PEI transfection in conditioned medium in stirred tank bioreactor, 3 days post-seeding

Transfect helper/packagingplasmids + gene of interest

Mix+ 15 min incubation at RT

Transfectionmedium

Transfectionmedium

PEI

Highly scalable process for transient AAV vector production

100 mm

1E+09

10 L STBR2 L STBR 2 L STBRminicircle

vg/L

1E+13

1E+12

1E+11

1E+10

1E+14

1E+15

control rAAV2-GFP rAAV5-GFP rAAV8-GFP

vg/L

1E+13

1E+12

1E+11

1E+10

1E+09

1E+14

Transient Lentivirus ProductionScalable suspension system for high titer transient lentivirus production in stirred tank bioreactors.

PEI-mediated transfection efficiencies and corresponding relative lentiviral vector titers in CAP-GT and adherent HEK293T cells.

Development of a fully scalable protocol for transient transfection without medium exchange yielding high-titer lentiviral production in stirred tank bioreactor.

CAP®GT

Thaw cells Expand CAP cells in shake flasks (125 mL – 5 L). Culture in medium supporting cell growth and transfection

PEI transfection in conditioned medium in stirred tank bioreactor, 3 days post-seeding

Transfect helper/packagingplasmids + gene of interest

Mix+ 15 min incubation at RT

Transfectionmedium

Transfectionmedium

PEI

Highly scalable process for transient LV vector production

200 mL15 mL 2 Lbioreactor

TU/m

L 1.0E+08

1.0E+07

1.0E+06

1.0E+05

8.0E+05Tran

sfec

tion

effici

ency

% G

FP p

osit

ive

mea

n flu

ores

cens

e in

tens

ity

HEK293T cellsadherent

blank CAP-GT cellssuspension

140 2.0E+06

1201.6E+06

100

1.2E+0680

60

4.0E+0540

20

0 0.0E+00

HEK293T cellsadherent

blank

Rel

ativ

e vg

/mL 7.0

6.0

5.0

4.0

3.0

2.0

1.0

0.0

CAP-GT cellssuspension

Adenovirus ProductionScalable suspension system for high titer production of replication deficient adenoviral vectors.

The CAP cell line produces 5000 -10 000 infectious adenovirus particles per cell. No undesired replication competent adenovirus (RCA) can be detected in 5x1010 viral particles (VP) from CAP cells, whereas 5-500 RCAs can be detected in 5x1010 VP produced in HEK293 cells.

CAP cells show high levels of coxsackie and adenovirus receptor (CAR) expression

CAP cells produce high levels of adenovirus

Infectious units/cell(E1-deleted ad-vector)

Conditions non-optimized non-optimized optimized

2500-6000 500-3000 4000-7000

CAP Per.C6 293

Nucleus+ adenovirus E1 genes

RCA-free production of adenovirus in CAP cells

Replication deficientadenovirus Producer cell

Viral genetherapy product

RCA:HEK: 5-500 in 5×1010 VPCAP: None in 5×1010 VP

+E1

P2

102 104

Coun

t

CAR

CHO

P2 P2P3 P3 P3

CAR

293A

CAR

CAP

104 105 102 102103 103104 104105 105

Coun

t

Coun

t

CAP®GT

CEVEC is a center of expertise for the production of biopharmaceuticals using a unique human cell-based expression system.

CAP-GT is a fully scalable manufacturing platform for viral vector production. CEVEC has successfully de-veloped CAP-GT suspension cell-derived viral packaging cell lines which enable better scale-up and com- petitive production costs when compared to adherent cell culture systems. CAP-GT suspension cell lines grow to high cell densities and show excellent productivity for a broad range of viruses. Gene therapy vec-tors such as lentivirus (LV), adenovirus (AV) and adeno-associated virus (AAV) can be produced at industrial scale.

For further information please contact: CEVEC Pharmaceuticals GmbHGottfried-Hagen-Str. 60-62 | 51105 Köln | GermanyPhone +49 (0)221 46020800 | E-Mail info@cevec.com

For business development please contact: bizdev@cevec.com | www.cevec.com

All information provided is for the purpose of presenting CEVEC and its technology. All information provided is given to CEVEC‘s best knowledge and shall not create any guarantee, representation, warranty or liability of any kind, express or implied. Any claims concerning this information shall be governed and construed in accordance with the laws of Germany, excluding its conflicts of law principles.

CAP® is a registered international trademark of CEVEC Pharmaceuticals GmbH, all rights reserved. All trade-marks are owned by their respective companies.