VACCIN E S
G E N E T HE R AP Y
VIR OT HE R AP Y
CASE STUDY: iCELLis Fixed-bed Bioreactor
System for the Production of Oncolytic Adenovirus
(CGTG-102) with A549 Cells
www.vibalogics.com | Zeppelinstr. 2 | 27472 Cuxhaven | Germany
The vaccine manufacturer.
RESULTS
Recombinant Adenovirus (Ads) have a reputation of safe clinical
use. After several replication-deficient Ads disappointed regarding
clinical efficacy, more potent replication-competent Ads are now
entering the clinical arena. Oncos Therapeutics is a clinical stage
Biotech Company and it‘s recombinant Ad comprises three major
modifications (Fig. 2). Δ24 E1a deletion for tumour-selective replication, hGM-CSF expression to induce strong anti-tumoral immune
responses and chimeric Ad3/Ad5 fiber to exploit more abundant
Ad3 receptor expression on tumour cells compared to Ad5 receptor.
The cell line of choice for the production of Replication-Competent
Ads is the human A549 cell line. Unfortunately, it is more or less
impossible to adapt this cell line to suspension growth and therefore
a scalable adherent system is required to enable viable commercialization. Here we describe the evaluation of a scalable, disposable,
fixed-bed bioreactor system (iCELLis, ATMI) for Ads production using
adherent A549 cells.
Growth studies without virus infection were performed to determine the confluence density of the system. Peak densities of
around 1.8E+05 cells/cm2 at the top of the fixed-bed were observed.
In the middle/bottom of the fixed bed, even 2.7E+05 cells/cm2 was
measured. This is about twice the peak densities measured from
T-flasks (data not shown). The minimum cell inoculation density
required to achieve confluent outgrowth was determined to be
~1E+04 cells/cm2. (Fig. 3) shows stained A549 cells on micro-fibers
several hours post-inoculation. This density (1E+04 cells/cm2) was
used for the virus production runs. For all iCELLis fermentations
the following settings were used: DO: 50%, pH: 7.2, Temp.: 37 °C,
stirring: 500 rpm. We targeted to infect the culture at ~70-80% of
the density reached at confluence using the count from the top bed
area. The cultures were infected with MOI of 10 (virus run-1) or 5
(virus run-2). (Fig. 4) illustrates the process flow applied to produce
Adenovirus and to generate a crude virus lysate by chemical lysis
of the fixed carrier bed. Cells and virus present in the medium loop
was also monitored to make sure that no significant cell detachment
and lysis had occurred until the medium loop was uncoupled from
the bioreactor volume. (Fig. 5) shows a typical iCELLis fermentation
documentation chart (Temp, DO, and pH control). The fermentation
data of virus run-1 are summarized in (Fig. 6). Consistently, lactate
levels peaked around 18 mM. iCELLis virus productivity data (Fig. 7)
from both runs were very high and cell productivities of 7.4E+04
TCID50 /cell (run-1) and 2E+04 TCID50 /cell (run-2) were achieved. Productivities in T-flasks were also very good. Lower productivity from
run-2 is very likely related to the lower MOI used here. This indicates
that the MOI used in run-1 (10) allowed more or less for optimal
CGTG-102 production.
iCELLis™ BIOREACTOR (Fig. 1)
The iCELLis™ system combines the advantages of single-use technologies with the benefits of a fixed-bed, small volume system. The
compact, fixed-bed matrix is composed of macrocarriers (each 11.2
cm2 surface) made of medical-grade polyester microfibers (PET) and
provides a growth surface between 0.8-500 m2. For all system scales,
the bed height is fixed to 10 cm. Instead of containing a donutshaped basket as is ATMI's fully single use, commercial scale iCELLis
500, the nano system has a cylindrical fixed bed. Linear scalability
between the systems is given due to the fixed bed height. Culture
medium passes the carrier bed from bottom to top. Here, the medium falls as a thin film down the outer wall like a “waterfall” enabling oxygenation/CO2- stripping. An integrated medium-loop allows
sufficient supply with medium. A full perfusion system is optional.
All commercial systems are fully disposable, a fully-disposable nano
system is under development.
VIRUS PRODUCTION CASE STUDY SUMMARY
Adherent A549 cells in OptiPro SFM / 1% FBS / 4mM Glutamax
iCELLis Nano system, 10 cm bed height, 2.7 m2 growth surface
Optimal inoculation of iCELLis at 1E+04 cells /cm2
NucleoCounter (ChemoMetec; Denmark) cell counting
Daily analysis of glucose and lactate levels
Fed–batch fermentation with glucose
Ad virus production in iCELLis compared to T-flask
Analysis of virus productivity by TCID50 on HEK293 cells
SUMMARY & CONCLUSIONS
The iCELLis system is an optimal solution for production and
harvest of Adenovirus through chemical lysis of cell containing fixed
bed, thereby generating a concentrated virus lysate for downstream
operation.
Fig. 1. iCELLis technology. (Left) Proprietary principle of
waterfall oxygenation / CO2 stripping. (Right) Commercial
scale iCELLis 500 unit with 25 L fixed-bed mounted on process
control skid. The system offers ease of handling and closed
system operation mode.
Fig. 3. Attachment of A549 cells to microfibers. Crystal
violet stained A549 cells. iCELLis nano was inoculated at
1E+04 cells/cm2. Picture taken about 3h post-inoculation
(100x magnification).
Fig. 2. Recombinant Oncolytic Adenovirus CGTG-102.
The following modifications apply: Δ24-deletion in E1A
gene, expression of hGM-CSF in E3 from Major Late Promoter,
Chimeric Ad3/Ad5 fiber protein with knob domain from human
Ad3 serotype.
1,8E+05
Cell inoculation at 1E+04 viable cells/cm2 (rpm=500;37 °C)
20
1,6E+05
1,4E+05
Expansion for ~ 5 days and Infection of culture at ~ 70-80%
of confluence density
Virus production for ~ 72 h at 37 °C
Decoupling of media loop and induction of virus lysis by
addition of 2mM MgCl2, Benzonase (15U/mL)
and 0.1% v/v Triton-X-100 (37 °C, 1000 rpm, 1 h)
18
infec1on with CGTG-­‐102 virus (MOI=10) 16
14
1,2E+05
12
1,0E+05
harvest 8,0E+04
10
start of medium loop 8
6,0E+04
6
addi1on of glucose 4,0E+04
4
2,0E+04
2
0,0E+00
glucose [g/L], lactate [mM] concentration
INTRODUCTION
K. Lehmann1, M. Paradies1, E. Haavisto2, S. Beyer1, A. Vuolanto2 and K. Lipinski1. 1Vibalogics GmbH,
Zeppelinstr. 2, 27472 Cuxhaven, Germany, kai.lipinski@vibalogics.com; 2Oncos Therapeutics, Saukonpaadenranta,
FI-00180 Helsinki, Finland.
cell density [cells/cm²]
iCELLis Fixed-bed Bioreactor System for the Production of
Oncolytic Adenovirus (CGTG-102) with A549 Cells
0
0
24
48
72
96
120
run time [h]
total cell density (carrier)
glucose
144
168
192
216
lactate
Recovery of crude virus lysate (CVL) from bioreactor
Clarifying Centrifugation of CVL
Fig. 6. Cell growth profile and infection. Shown are total cell count (carrier), lactate and
glucose levels. Batch was fed if glucose dropped below ~2 g / L. Infection was on day-5 of
fermentation at 1.38E+05 cells / cm2 with and MOI=10 (in case of 1st virus run).
Supernatant CVL stored and analysed
Fig. 4. Process flow chart summary. For the first time, an Oncolytic Adenovirus was recovered
from the fixed carrier bed by chemical-induced cell lysis (Triton-X-100) in the presence of Benzonase to reduce viscosity and achieve downstream conditioning.
Virus Run-1
(MOI=10)
Peak densities of 2.7E+05 cells/cm were observed (middle and
bottom of bed)
Peak productivity of 7.4E+04TCID50 /cell were observed for the
iCELLis system
The availability of the fully disposable Nano system will allow
cost- and time-efficient generation of IMP (Phase I/II) clinical
material
2
Fig. 5. Typical fermentation controller data chart. Set points were: DO: 50%, pH: 7.2, Temp:
37°C. pH was controlled with CO2 gasing and NaOH. DO was controlled through headspace air/O2
cascade; blue: temperature, purple: pH and orange: DO%.
TCID50
Titer
Harvest Volume (mL)
Total produced TCID50
particles
Productivity
TCID50 / cell
Relative
surface productivity
iCELLSis bulk
harvest
2,90E+11
900
2,61E+14
7,40E+04
1,02
T-flask lysate
control
4,90E+10
34
1,67E+12
1,23E+05
1
Virus Run-2
(MOI=5)
Titer
TCID50 / mL
Harvest Volume (mL)
Total produced TCID50
particles
Productivity
TCID50 / cell
Relative
surface productivity
iCELLSis bulk
harvest
8,90E+10
900
8,01E+13
1,97E+04
0,77
T-flask lysate
control
2,00E+10
34
6,80E+11
4,75E+04
1
Fig. 7. Summary virus production in iCELLis and T-flask. Virus productivity was analysed
by TCID50 on HEK293 cells. Two independent virus production runs were performed. To compare
T-flask and iCELLis, virus production was normalized either against growth surface or cell
number as indicated.