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Vaccine xxx (2016) xxxxxx

Contents lists available at ScienceDirect

Vaccine
journal homepage: www.elsevier.com/locate/vaccine

Optimization and scale-up of cell culture and purication processes

for production of an adenovirus-vectored tuberculosis vaccine
candidate
Chun Fang Shen a, , Danielle Jacob a , Tao Zhu c , Alice Bernier a , Zhongqi Shao b ,
Xuefeng Yu b , Mehul Patel a , Stephane Lanthier a , Amine Kamen a,d
a
Human Health Therapeutics Portfolio, National Research Council of Canada, Montreal, Quebec, Canada
b
Tianjin CanSino Biotechnology Inc., Tianjin, China
c
Tianjin University of Science and Technology, Tianjin, China
d
Department of Bioengineering, McGill University, Montreal, Quebec, Canada

a r t i c l e i n f o a b s t r a c t

Article history: Tuberculosis (TB) is the second leading cause of death by infectious disease worldwide. The only available
Available online xxx TB vaccine is the Bacille CalmetteGuerin (BCG). However, parenterally administered Mycobacterium
bovis BCG vaccine confers only limited immune protection from pulmonary tuberculosis in humans.
Keywords: There is a need for developing effective boosting vaccination strategies. AdAg85A, an adenoviral vector
Adenovirus-vectored vaccine expressing the mycobacterial protein Ag85A, is a new tuberculosis vaccine candidate, and has shown
Tuberculosis vaccine
promising results in pre-clinical studies and phase I trial. This adenovirus vectored vaccine is produced
Cell culture
using HEK 293 cell culture.
Process development
Purication
Here we report on the optimization of cell culture conditions, scale-up of production and purication
of the AdAg85A at different scales. Four commercial serum-free media were evaluated under various
conditions for supporting the growth of HEK293 cell and production of AdAg85A. A culturing strategy
was employed to take advantages of two culture media with respective strengths in supporting the cell
growth and virus production, which enabled to maintain virus productivity at higher cell densities and
resulted in more than two folds of increases in culture titer. The production of AdAg85A was successfully
scaled up and validated at 60 L bioreactor under the optimal conditions.
The AdAg85A generated from the 3 L and 60 L bioreactor runs was puried through several purication
steps. More than 98% of total cellular proteins was removed, over 60% of viral particles was recovered after
the purication process, and purity of AdAg85A was similar to that of the ATCC VR-1516 Ad5 standard.
Vaccination of mice with the puried AdAg85A demonstrated a very good level of Ag85A-specic antibody
responses. The optimized production and purication conditions were transferred to a GMP facility for
manufacturing of AdAg85A for generation of clinical grade material to support clinical trials.
Crown Copyright 2016 Published by Elsevier Ltd. All rights reserved.

1. Introduction million TB deaths among HIV positive people (http://www.who.int/

Tuberculosis (TB) remains a global health concern. Globally in
2014, there were an estimated 1.5 million deaths from TB (1.1
million HIV-negative and 0.4 million HIV-positive). TB now ranks
alongside HIV as a leading cause of death worldwide. HIVs death
toll in 2014 was estimated at 1.2 million, which included the 0.4
Corresponding author at: Human Health Therapeutics Portfolio, National
Research Council of Canada, 6100 Royalmount Ave, Montreal, Quebec H4P 2R2,
Canada. Tel.: +1 514 496 7881.
E-mail address: chunfang.shen@nrc-cnrc.gc.ca (C.F. Shen).
tb/publications/global report/en/). TB is caused by Mycobacterium
tuberculosis (M.tb) that most often affect the lung. This leading
infectious cause of death remains highly prevalent with one third
of the worlds population latently infected with M.tb in spite of rou-
tine vaccination against TB in endemic areas. The only approved TB
vaccine is the Bacille CalmetteGuerin (BCG), however the BCG has
variable efcacy in providing protection against pulmonary TB [1].
There is a need for developing effective vaccines.
Human adenovirus (AdV) type 5 is the most characterized
among the wide variety of human and non-human adenoviral
vectors, and its biology is well described [2]. AdV often induces
humoral, mucosal and cellular immune responses to antigens

http://dx.doi.org/10.1016/j.vaccine.2016.04.090
0264-410X/Crown Copyright 2016 Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Shen CF, et al. Optimization and scale-up of cell culture and purication processes for production of
an adenovirus-vectored tuberculosis vaccine candidate. Vaccine (2016), http://dx.doi.org/10.1016/j.vaccine.2016.04.090
G Model
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encoded by the inserted foreign genes, and are still considered
among the most effective vaccine vectors [37]. Numerous clin-
ical trials have been initiated with adenovirus vectors to control
infectious diseases and treat different types of cancer [8]. Recom-
binant AdV vectors have the ability to replicate at high titers in
complementing cell lines.
AdAg85A, which consists of a nonreplicating human adenovirus
type 5-expressing mycobacterial antigen 85A and is a new promis-
ing tuberculosis vaccine candidate, has been studied with mouse,
guinea pig, goat and cow animal models, and was shown to be effec-
tive against M.tb infection [9]. In previous studies, the AdAg85A
vaccine was shown to elicit strong T cell responses in various ani-
mal models as well as in human [10]. A phase I trial on AdAg85A
demonstrated that AdAg85A was safe and highly immunogenic
[11]. AdAg85A is produced in HEK 293 cell culture. To further evalu-
ate the efcacy of this vaccine, by assessing the associated humoral
response which has been suggested to be important for the protec-
tion against TB [12], and to make it available to large populations, a
robust and cost-effective cell culture production process had to be
developed for manufacturing large quantities of AdAg85A required
for further clinical trials.
The production of AdV is affected by a number of factors includ-
ing cell line physiology, cell culture conditions and virus construct,
and has been reviewed in several publications [1315]. In general,
nutrient limitation and/or accumulation of inhibitory metabolites
are still the main factors limiting the yield of AdV production [16].
Scale-up of production process and improvement of product yield
while maintaining product quality attributes through upstream
and downstream processing steps are key success factors for man-
ufacturing large quantity of Ad-vectored vaccines. Here we report
our study on optimization of cell culture conditions, successful
scale up to 60 L bioreactor and purication of AdAg85A at different
scales. Overall, this work contributed to signicantly improve the
nal product titer and recovery, and provide highly puried mate-
rials for preclinical study. The optimized conditions for production
and purication were transferred to a GMP facility for AdAg85A
manufacturing for clinical trials.
2. Materials and methods
2.1. AdAg85A construct
Construction and characterization of recombinant replication-
decient adenoviral vaccine (AdAg85A) are described by Wang
et al. [17]. Briey, Ag85a complementary DNA (cDNA) without
its own signal sequence gene region were amplied by PCR from
M.tb genomic DNA. A shuttle plasmid vector pJW24 was designed
and constructed through multiple subcloning steps. The presence
of adenoviral sequences in pJW24 allows homologous recombina-
tion with adenoviral rescuing vector pBHG10 to form recombinant
replication-decient adenovirus AdAg85A. An AdAg85A seed, pro-
vided by Dr. Zhou Xing (McMaster University, Ontario), was
amplied as a virus stock for this study.
2.2. HEK 293SF-3F6 cell lines, cell maintenance and culture in
serum-free media
The HEK 293SF-3F6 cell line was derived from HEK 293S cells
which constitutively express Ad5 E1A + E1B genes that support the
replication of the E1A + E1B-defective adenoviral particle. This cell
line was adapted to grow in suspension and serum-free media
through multi-steps of adaptation and clone selection [18]. The HEK
293SF-3F6 cell culture was maintained at 37 C and 5% CO2 and sub-
cultured in 125 mL plastic shake asks (Corning, NY) three times a
week at 22.5 105 cells/mL in serum-free SFM4HEK-293 medium
Please cite this article in press as: Shen CF, et al. Optimization and scale-up of cell culture and purication processes for production of
an adenovirus-vectored tuberculosis vaccine candidate. Vaccine (2016), http://dx.doi.org/10.1016/j.vaccine.2016.04.090
(HyClone, Logan, UT). These cells will be referred to as HEK293 for
conciseness.
2.3. Optimization of culture conditions
Small-scale experiments were all performed in 125 mL shake
asks with a working volume of 25 mL. Four serum-free media,
SFM4Transfx-293, SFM4HEK293 (both from HyClone), Adenovirus
Expression Medium (AEM) and CD 293 (both from Life Technolo-
gies) were evaluated for growth of HEK293 cells and production
of AdAg85A. Cultures were infected with AdAg85A at MOI of
10 (throughout the study) with or without a complete medium
exchange before infection, depending on the experimental design.
Samples taken at different times during the infection were kept at
80 C for subsequent analyses.
2.4. Scale-up and validation of AdAg85A production in 3 L and
60 L bioreactors
The production of AdAg85A was operated in a Chemap 3.5 L
bioreactor under controlled conditions. The bioreactor was inocu-
lated with HEK 293 cells and SFM4HEK293 medium at a volume of
1.5 L and at a cell density of 0.55 106 cells/mL. Temperature, agi-
tation and dissolved oxygen (DO) in the bioreactor were controlled
at 37 C, 90 rpm and 40% of air saturation, respectively. The pH was
controlled at 7.2 0.1 by using CO2 in the gas inlet and base con-
sisting of 5% NaOH and 7.5% NaHCO3 . The culture was grown up to
2.5 106 cells/mL, then diluted with 1.5 L of CD293 medium before
infecting with the AdAg85A. The infected culture was harvested at
42 hours post infection (hpi) for downstream processing.
The production of AdAg85A was further scaled-up and validated
in a 60 L Applikon bioreactor with a culture volume of 45 L. Similar
to the operation in 3 L bioreactor, the 60 L bioreactor was inoculated
at a culture volume of 22 L. The culture was grown to a cell density
of 2 106 cells/mL and then diluted with 22 L of CD293 medium
before the viral infection. All operating parameters were the same
as in the 3 L bioreactor runs, whereas the agitation was controlled
at 70 rpm. The infected culture was harvested at 43 hpi for down-
stream processing according to a standard adenovirus purication
process [13,19].
2.5. Harvest, primary separation and chromatography steps in
AdAg85A purication
The purication steps were briey described as follows. The cul-
ture from 3 L production was centrifuged at 375 g for 10 min at
4 C. The pelleted cells were resuspended in the Lysis Buffer (10 mM
Hepes, 2 mM MgCl2 , pH 7.5) to approximately 1/10th the original
volume. The 10 concentrated lysate from the rst 3 L run was
frozen at 80 C, then thawed in a water bath set at 37 C on the day
of purication. The lysate from the second 3 L run was proceeded
immediately. The lysate was treated with Benzonase (DNase I,
EMD Millipore) for 1 h at room temperature and centrifuged at
4560 g at 4 C for 10 min. The supernatant was conditioned with
a concentrated NaCl buffer to 300 mM NaCl and was ltered
through a 0.45 m dead end lter before being loaded onto a
Q-Sepharose HP (56 mL) anion exchange chromatography (IEX) col-
umn.
The IEX column was pre-equilibrated with 300 mM NaCl in
Buffer A (50 mM HEPES, 2 mM MgCl2 , 2% sucrose, pH 7.5), and then
was washed with the equilibrating buffer followed by consecu-
tive steps of 450 mM NaCl in Buffer A, 600 mM NaCl in Buffer A
and 1 M NaCl in Buffer A. The IEX puried adenovirus eluate was
collected, diluted with Buffer A to reach 0.5 M NaCl in Buffer A,
ltered through a 0.45 m CA membrane and loaded onto a 74 mL
Capto Core 700 multimodal chromatography column previously
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Fig. 1. Productivity of AdAg85A in batch cultures with different culture media infected at a cell density of 1 106 cells/mL with or without medium exchange before the
virus infection. (a) Concentration of total viral particles; (b) titer of infectious viruses; (c) productivity of AdAg85A in cultures grown with SFM4HEK293 medium, and then
diluted with CD293 before the virus infection at cell densities of 1 106 cells/mL and 2 106 cells/mL.

equilibrated with 0.5 M NaCl in Buffer A for polishing. The ow
through pool was collected.
Same strategies and purication steps were employed to purify
the 45 L culture produced in the 60 L bioreactor except large-scale
equipment was employed at various steps. Briey, the infected cul-
ture was harvested by using a Centritech Cell II system (Sorvall). IEX
was carried out in a Q-Sepharose HP column with a size of 2.6 L. The
polishing step was performed using Capto Core 700 column with a
volume of 1.06 L.
2.6. Quantication methods
Quantication of viable and total cells, total viral particles have
been described previously [16]. The infectious titer of AdAg85A vec-
tors was assayed by the Clontech Adeno-X Rapid Titer Kit according
to the User Manual.
Samples from critical purication steps were also analyzed for
total protein concentration (RC/DC Protein Determination Assay,
Bio-Rad, Hercules, CA), SDS page (415% gradient precast gel,
Bio-Rad) and Western Blot. The nal product was further quan-
tied using absorbance reading at 260, 280 nm in presence of
SDS.
2.7. Immunogenicity with AdAg85A in mice and serum IgG titer
determination
Six- to 8-week-old female BALB/c mice (Vital River Labora-
tory Animal Technology Co. Ltd., Beijing, China) were divided into
groups of 10 each and immunized intramuscularly with AdAg85A at
1 1010 , 1 108 or 1 106 pfu/mouse. Two weeks after prime vac-
cination, animals were boosted by the injection of the same amount
of the antigen. Serum samples were collected 14 days after prime
and boost vaccination respectively. The Ad5Ag85 materials which
were manufactured at both 3 L and 60 L scales were tested.
The Ag85A-specic IgG titers in mouse sera were determined
by ELISA with plates coated with the recombinant Ag85A protein
(CanSino, Tianjin, China).
3. Results
3.1. Screening of culture media and optimization of culture
conditions for the production of AdAg85A
SFM4HEK-293, SFM4Transfx-293 and AEM media supported the
growth of HEK293SF-3F6 cell to 45 106 cells/mL in the batch cul-
tures, with a cell doubling time of 24 h in the log growth phase. The

Please cite this article in press as: Shen CF, et al. Optimization and scale-up of cell culture and purication processes for production of
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2.0 16E+10 30 4.0

a Glucose b

Conc. of total and infecous viral parcles

25 Lactate

Conc of glucose and lactate (mM)

Cell density (x 106 cells/mL)

1.5 12E+10 NH3 3.0

20

Conc of NH3 (mM)

1.0 8E+10 15 2.0

10
Total cell density
0.5 Viable cell density 4E+10 1.0
Virus conc. (vp/mL) 5
Virus conc. (pfu/mL)

0.0 0 0 0.0
0 19 25 43 49 67 19 hpi 25 hpi 43 hpi 49 hpi 67 hpi
Hours post-infecon (h) Hours of post infecon

Fig. 2. (a) Total () and viable () cell density, total viral particles () and infectious virus titer () concentration over the time course of post culture infection in a shake
ask culture. (b) Concentrations of residual glucose and accumulated inhibitory metabolites, lactate and ammonia.

HEK293 cell however did not grow in CD293 medium, but remained 2 106 cells/mL, reaching 4.8 1010 vp/mL, and 5.3 109 pfu/mL,
alive in this medium. respectively (Fig. 1c).
SFM4HEK-293 and SFM4Transfx-293 supported similar produc-
tions of AdAg85A total viral particles at 1.8 1010 vp/mL in the 3.2. Kinetic analysis of AdAg85A production process
batch cultures without medium exchange (Fig. 1a). The produc-
tivity of AdAg85A in the cultures grown with AEM medium was The production of AdAg85A over the course of post-culture
much lower, at 3.1 109 vp/mL. A medium exchange before the infection (Fig. 2a) revealed that a low concentration of virus
virus infection improved the production of total virus particles (4.4 107 pfu/mL) was already detected in the culture at 19 hpi, the
by more than 50%, reaching 2.73.1 1010 vp/mL in the cultures production of viruses increased over time, and reached a maximum
grown in SFM4HEK-293 or SFM4Transfx-293, and 4.9 109 vp/mL at 43 hpi. The virus titer however decreased with the increased
in the culture grown in AEM. duration of culture. A similar trend was also observed in the con-
Titer of infectious viruses in the cultures grown in SFM4HEK-293 centration of total viral particles.
and SFM4Transfx-293 was similar, at 2.0 109 pfu/mL (Fig. 1b), Concentrations of glucose and amino acids, and also concen-
which is about 10% of the total viral particles concentration. How- trations of inhibitory metabolites (lactate and ammonia) in the
ever, the positive effect of medium exchange on the production samples taken over the time course (Fig. 2b) revealed there was
of infectious viruses was less conclusive, mainly due to a large sufcient residual glucose in the culture and none of the amino
variability inherent to the biological assay. The titer of infectious acids was depleted. The maximum concentration of generated lac-
viruses in the cultures grown in AEM was below the detection limit tate and ammonia was 25 and 1.8 mM, respectively, which is within
(1 108 pfu/mL). a range usually observed in healthy cultures.
Although CD293 medium did not support the growth of
HEK293 cell, the production of AdAg85A (both vp/mL and pfu/mL) 3.3. Scale-up and validation of AdAg85A production in 3 L and
was dramatically improved when the batch cultures grown in 60 L bioreactors
SFM4HEK-293 was diluted with an equal amount of CD293 medium
before the virus infection (Fig. 1c), reaching 3.9 1010 vp/mL Cell densities, viability and virus productivity listed in Table 1
and 4.0 109 pfu/mL, respectively in the cultures infected at showed that the two 3 L bioreactor runs were very reproducible.
1 106 cells/mL. These values are one fold more than those The validation of AdAg85A production in the 60 L bioreactor was
(1.8 1010 vp/mL and 2.0 109 pfu/mL) achieved in the culture performed under the same conditions used in the 3 L bioreac-
grown in SFM4HEK-293 and infected without medium exchange. tor. However, the data in Table 1 revealed that the post-infection
The volumetric productivity of AdAg85A was also increased when cell growth and cell viability (95%) at the harvest in the 60 L run
the cell density at infection was increased from 1 106 cells/mL to were much higher. The virus productivity in the 60 L bioreactor

Table 1
Summary of key process data and virus productivity in two 3 L and one 60 L bioreactor runs.

Key process data 1st 3 L run 2nd 3 L run 60 L run

Total cell density and viability before dilution 2.5 10 cells/mL;

6
2.9 10 cells/mL;
6
2.03 106 cells/mL;
97% 98% 99%
Total cell density and viability before virus infection 1.4 106 cells/mL; 1.5 106 cells/mL; 1.04 106 cells/mL;
99% 99% 99%
Total cell density and viability at harvest (43 hpi) 1.4 106 cells/mL; 1.7 106 cells/mL; 1.76 106 cells/mL;
81% 83% 95%
Total viral particle (vp) conc. of harvest broth 2.5 1010 vp/mL 2.7 1010 vp/mL 9.8 1010 vp/mL
Infectious virus (pfu) conc. of harvest broth 2.3 109 pfu/mL 2.6 109 pfu/mL 9.7 109 pfu/mL
pfu/vp (%) 9.1% 9.6% 10%

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production was nearly 4 folds of those obtained in the 3 L bioreactor

pfu/vp ratio
runs or 2 times of that achieved in the shake ask cultures.

16%
17%

13%
10%

N/A
3.4. Purication of AdAg85A

Key performance indicators in the critical purication steps in

two 3 L runs and one 60 L production are presented in Table 2.

Viral particles
Both vp and pfu in the samples from the purication steps were

recovery
quantied, however, the concentration of vp (more precise) was

100%
75%
79%
75%
67%
used to estimate the recovery of viruses (or viral particles) in each
purication step.
The recovery of viral particles in the rst 3 L run was more than
60% after the Capto Core 700 polishing. The data in Table 2 also

Total viral particle

showed that the recovery of viral particles in the purication steps

conc. (vp/mL)
in the rst 3 L bioreactor run was systemically higher than those in

9.8 1010
8.3 1011
8.4 1011
2.2 1012
1.9 1012
the second run. This difference was more likely due to the additional
freeze-thaws step applied to the 10 concentrated lysate from the
rst run. It has been known that freeze-thaw cycle of adenovirus

60 L bioreactor run
culture or lysate helps to release viruses from the cells. The pfu/vp
ratio in the samples from the purication steps varied from 7% to

Volume (mL)
13% in the rst run, and from 6% to 21% in the second run. There is
no trend in the pfu/vp ratio associated with the samples from dif-

45,000
3965
4169
1500
1553
ferent purication steps. The difference in the pfu/vp ratio is more
likely due to the large variability inherent to the biological assay of
infectious virus particles.

2nd run
The same sequential purication steps were employed to purify
the 45 L culture produced in the 60 L bioreactor. Data in Table 2

10%
21%
6%
14%
6%
showed the recovery of total viral particles was 67% which is very

pfu/vp ratio
similar to that achieved in the rst 3 L run. The ratio of pfu/vp was

Concentrations and recoveries of total viral particles, and pfu/vp ratios in critical purication steps in two 3 L and one 60 L runs.
slightly higher.

1st run

9%
12%
7%
7%
7%
More than 99% of total cellular proteins was removed during the
purication process in both 3 L purication runs (Table 3). Most of
the total proteins removal was achieved during the IEX step, and

Viral particles recovery

2nd run
followed by Capto Core 700 polishing. Total protein concentration

100%
49%
64%
60%
37%
in the puried AdAg85A product was about 60 g/mL. The removals
of proteins were also conrmed by the result from SDS-Page, sliver
stain analysis of samples from the purication steps (Fig. 3a). Result
1st run
from the SDS-Page and Western Analyses also revealed that purity
100%
72%
101%
73%
61%
of AdAg85A was identical to that of the ATCC VR-1516 Ad5 standard
(Fig. 3b). The removal of total proteins in the 60 L run was 98% after
the Capto Core 700 polishing step (Table 3), which is similar to what
2.7 1010
1.3 1011
1.7 1011
3.2 1011
2.1 1011
2nd run

was achieved in the purication of rst 3 L run.

Total viral particle

3.5. Animal studies with the AdAg85A vaccine

conc. (vp/mL)

2.5 1010
1.7 1011
2.3 1011
2.8 1011
2.3 1011
1st run

No Ag85A-specic antibody response was detected when ani-

mals were injected with Ad5Ag85A at the dose of 1 106 pfu.
Vaccination at 1 108 pfu/mouse resulted in a good antibody level,
which could be boosted by the second injection of AdAg85A. A
2nd run

very high antibody titer, >105 , was detected in mice which only
3 L bioreactor runs

2932
304
300
146
140

received one injection of the vaccine at 1 1010 pfu/mouse. No sig-

nicant difference was observed in the Ag85A-specic antibody
Volume (mL)

responses generated by AdAg85A produced at 3 L and 60 L scales

1st run

(Fig. 4), demonstrating that the immunogenicity of the vaccine was

2852
300
315
185
188

not affected by scaling up the manufacturing process.

4. Discussion
Capto Core 700 owthrough
Q-Seph HP 1 M NaCl peak

Improvement of product yield and scale up of the production

process were the most critical and pressing issues for manufac-
turing large quantity of AdAg85A required for clinical trials, and
10 conc. lysate
Q-Seph HP feed

commercial manufacturing. Although the production of adenovirus

Harvest broth

has been investigated and reviewed in a number of publications

Samples

[13,15,20], it still remains challenging to signicantly improve the

Table 2

yield of production to reduce the cost of goods and maintain eco-

nomical viability. The virus productivity in Fig. 1a showed that

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Table 3
Concentrations and recoveries of total proteins in critical purication steps in two 3 L and one 60 L runs.

Samples 3 L bioreactor runs 60 L bioreactor run

Volume (mL) Total protein conc. Protein recovery Volume (mL) Total protein Protein
(mg/mL) conc. (mg/mL) recovery

1st run 2nd run 1st run 2nd run 1st run 2nd run

Harvest broth 2852 2932 0.39 0.53 100% 100% 45,000 0.66 100%
10 conc. lysate 300 304 3.31 3.54 89% 69% 3965 4.29 57.7%
Q-Seph HP feed 315 300 2.80 1.76 79% 34% 4169 4.15 58.3%
Q-Seph HP 1 M NaCl peak 185 146 0.37 0.37 6% 3% 1500 0.93 4.7%
Capto Core 700 owthrough 188 140 0.06 0.05 1% 0.5% 1553 0.36 2.0%

Fig. 3. (a) SDS-Page, silvered-stained and (b) western blots of ATCC Ad5 standard and samples from critical steps in the purication process of culture harvested from 3 L
bioreactor run. (1) BMW Std, Bio-Rad; (2) Harvest Broth; (3) Q-Seph HP Feed; (4) Q-Seph HP Flowthrough Pool; (5) Q-Seph HP 450 mM NaCl Peak; (6) Q-Seph HP 600 mM
NaCl Peak; (7) Q-Seph HP 1 M NaCl; Peak; (8) CaptoCore 700 Flowthrough Pool; (9) sterilely ltered product; (10) VR-1516 ATCC Ad5 Standard.

production yield was further increased by only 30% instead of

Fig. 4. Immunogenicity of AdAg85A produced at 3 L and 60 L scales.
although SFM4HEK-293, SFM4Transfx-293 and AEM supported the
growth of HEK293 cell to a density of 4 106 cells/mL, their capa-
bilities varied in supporting the virus production. This result clearly
indicates that the formulations of these media were not optimal
for supporting both HEK293 cell growth and AdAg85A production.
This statement is further supported by the improvement of virus
productivity in the cultures initially grown in SFM4HEK-293, then
diluted with CD293 before the viral infection, as CD293 did not even
support the growth of HEK293. These results illustrate the differ-
ence in metabolic requirements of HEK293 during the phase of cell
growth and the phase of virus production.
Although the dilution of SFM4HEK-293 culture with CD293
medium signicantly improved the virus productivity, the
100% when the cell density at infection was increased from
1 106 cells/mL to 2 106 cells/mL (Fig. 1c). This might indicate
that nutrient(s) became limiting again in the SFM4HEK293 culture
diluted with CD293 when the culture was infected at a cell den-
sity of 2 106 cells/mL. All of these results suggest the medium
exchange or dilution of culture with CD293 medium before the
virus infection provided additional nutrient(s) required for infected
HEK293 cells during the phase of virus production to further
increase the virus productivity. Nutrient limitation could be a crit-
ical factor hindering the virus productivity in the batch culture
process when cultures are infected at higher cell densities.
Infecting cultures at higher cell densities while maintaining
the specic productivity obtained at low cell density is one of
the goals to improve the virus productivity. Achievable cell den-
sity at infection depends on cell intrinsic characteristics and also
cell culture conditions. The result from this experiment showed
that the dilution of culture with CD293 was not able to maintain
the specic virus productivity when the culture was infected at
2 106 cells/mL. Other production modes, such as fed-batch or per-
fusion culture, could be explored for further improvement of the
virus productivity.
Parameters governing the production yield of adenovirus have
been extensively reviewed by Dormond et al. [15]. Among the
parameters, cell environment is a key factor affecting the prod-
uct yield in various production processes. The cell environment
is characterized by availability of nutrients (medium formulation
and feeding strategies), physicochemical conditions and various
stresses induced by the different culture modes. Medium exchange,
fed-batch and perfusion processes have been used to improve
the supply of nutrients, thus improving the production yield of

Please cite this article in press as: Shen CF, et al. Optimization and scale-up of cell culture and purication processes for production of
an adenovirus-vectored tuberculosis vaccine candidate. Vaccine (2016), http://dx.doi.org/10.1016/j.vaccine.2016.04.090
G Model
JVAC-17631; No. of Pages 7 ARTICLE IN PRESS
C.F. Shen et al. / Vaccine xxx (2016) xxxxxx
adenovirus. Medium exchange and perfusion process are also able
to minimize the accumulation of inhibitory metabolites, and to
prevent potential inhibitory effects on the cell growth and virus
production. However, operation complexity and cost of feeding
processes have to be taken into consideration when perfusion or
medium exchange process would be applied to improve the pro-
duction yield.
Cellular shear stresses are common problems associated with
the cell culture in bioreactors, especially at large-scale. The virus
productivities, which achieved in the shake ask cultures, 3 and
60 L bioreactor runs (at 4.8 1010 vp/mL, 2.6 1010 vp/mL and
9.8 1010 vp/mL, respectively), indicate that the production of
AdAg85A is scalable; however, the virus productivity could be sig-
nicantly affected by the conguration of bioreactors. The lower
virus productivity in the 3 L bioreactor runs and higher yield in the
60 L bioreactor tted the trend in previous results from various pro-
ductions of different viruses and viral vectors carried out in 3 L and
60 L bioreactors. Based on the design of 3 L and 60 L bioreactors, it is
believed that the lower virus productivity was due to higher shear
stress in the 3 L bioreactor.
The AdAg85A cultures produced in the 3 L and 60 L bioreactors
were successfully puried with over 98% removal of total cellu-
lar proteins and more than 60% recovery of total viral particles. The
ratio of pfu/vp in the samples from the critical steps was in the range
of 10%, indicating the purication steps did not signicantly impact
the infectivity of AdAg85A. This result was further conrmed by
high Ag85A-specic antibody responses generated by AdAg85A
produced at both 3 L and 60 L scales. The high purity (the same
as ATCC standard) and immunogenicity of the puried AdAg85A
demonstrated that the purication process employed in this study
was feasible and transferable to sites for generation of puried
materials for pre-clinical or clinical applications. The experimental
results demonstrate that the purication process reported in this
study is scalable from 3 L to 45 L scale. Our future study includes
evaluation of scalable extraction step such as cell disintegration by
Triton X-100 and ltration to replace the centrifugation and the
freeze-thaw lysis steps while reducing labor cost associated with
the manufacturing process.
In conclusion, this study demonstrates a successful produc-
tion process improvement and scale-up for manufacturing and
purication of an adenovirus-vectored tuberculosis vaccine can-
didate. Optimization of culture conditions resulted in over one
fold improvement in virus yield. The developed production and
purication processes were able to generate AdAg85A materials
with purity equal to that of the ATCC VR-1516 Ad5 standard. The
Please cite this article in press as: Shen CF, et al. Optimization and scale-up of cell culture and purication processes for production of
an adenovirus-vectored tuberculosis vaccine candidate. Vaccine (2016), http://dx.doi.org/10.1016/j.vaccine.2016.04.090
7
puried AdAg85A material was bioactive and generated the tar-
geted Ag85A-specic antibody response.
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