Development of an Efficient Manufacturing Process
for Adenovirus
Mark Fitchmun,1 Mark Snyder, 2 and John Chicca 3
Somatek Inc., 4204 Sorrento Valley Boulevard, Suite G, San Diego, CA
2
Bio-Rad Laboratories, Inc., 6000 James Watson Drive, Hercules, CA
3
Molecular Diagnostic Services, Inc., 4204 Sorrento Valley Boulevard, Suite G, San Diego, CA
1Process Separations

Tech
Note
Bulletin 6719

Abstract
Large-scale downstream processing of viruses for clinical applications poses challenges different from those formany other biotherapeutics. These challenges mostly arise from the size and complexity of the virus. Here, we present
purification results of a process developed for the manufacturing of a recombinant adenovirus. This two-column
process results in an adenovirus preparation comparable to clinical grade products, with high yield and very low hostcell protein (HCP) and DNA contamination. It is readily scalable, simple, rapid, and efficient and thus well-suited for the
production of clinical grade viral vectors.
Introduction
Adenovirus vectors are effective tools for the transfer of genetic
material into mammalian cells. They offer several advantages,
including the capacity to accommodate up to 37 kb of foreign
genetic material, very high infection efficiency, the ability to infecta wide variety of both dividing and nondividing cell types, lack ofintegration into the host chromosome, and production systems
capable of generating high virus titers. These and other qualities
have led to adenoviruses being the most used gene transfer
vectors in experimental therapies, accounting for 25% of all
gene therapy trials; as of 2014, they had been used in almost
500 clinical trials.
Large-scale downstream processing of viruses for clinicalapplications poses challenges that arise, in part, from theviruses’ large size and complexity. In the case of adenovirus,
one intact virus particle (vp) contains more than 2,700 protein
subunits, has a mass of approximately 165 MDa, and has
a diameter of approximately 0.1 µm. The complexity of theparticle gives rise to thousands of charge variants, making it
difficult to establish well-defined binding and elution conditions
on charged separation resin. In addition, adenoviruses tend
to be acid labile, which further increases the complexity ofprocess development.
We developed a two-column efficient purification process fora recombinant adenovirus after screening five chromatography
resins. We show that the final process yields an active,
concentrated virus product with purity, HCP levels, and DNA
contamination comparable to clinical-grade products. The
process is readily scalable and is sufficiently simple, rapid,
and efficient to be used for the production of clinical-grade
viral vectors.

Materials and Methods
Virus

We used a recombinant human adenovirus, Ad5-E1+GFP,
serotype 5, in which the E1a gene was replaced with DNA
coding for Aequorea victoria green fluorescent protein (GFP).
The virus was expanded in HEK 293 cells grown in DMEM +2% fetal bovine serum. The virus was harvested 36 hr after
inoculation by adding a cell permeation agent (Somatek Inc.)to the culture to release the virus from the host cells.
Initial resin screening

Initial resin screening was performed using two 1 ml
prepacked 8 x 20 mm Foresight™ Columns in series.
UNOsphere™ S and Nuvia™ S Cation Exchange Columns(Bio-Rad Laboratories, catalog #732-4730 and #732-4720,
respectively) were equilibrated with 25 mM sodium phosphate,
pH 6.8. The crude harvest was buffer-exchanged into
equilibration buffer prior to column loading and virus was
eluted with 25 mM sodium phosphate, 1 M NaCl, pH 7.4.
A Nuvia™ cPrime™ Hydrophobic Cation Exchange Column(Bio-Rad, catalog #732-4722) was equilibrated with 25 mM
histidine, pH 6.5. The crude harvest was buffer-exchanged into
equilibration buffer prior to column loading and virus was eluted
stepwise with buffers of increasing pH and NaCl concentration.
UNOsphere Q and Nuvia Q Anion Exchange Columns(Bio-Rad, catalog #732-4732 and #732-4721, respectively) were
equilibrated with 25 mM Tris, 250 mM NaCl, pH 8.1. Crude
harvest was loaded directly onto the columns and virus was
eluted stepwise with buffers of increasing NaCl concentration.

Development of an Efficient Manufacturing Process for Adenovirus

Virus quantitation

Results

Focus-forming units (FFU) were determined after tenfold serial
dilutions of virus samples were applied in quadruplicate to
7 x 104 HEK 293 cells grown in 48-well plates. Transgene
expression of GFP was determined by fluorescent spectroscopy
after threefold serial dilutions of virus samples were applied intriplicate to 60–90% confluent HEK 293 cells grown in 96-wellplates. Total virus concentration was determined using thefollowing equation:

Process development

Number of particles =A260 * dilution factor * 1.1 x 1012 particles/A260 unit
These total virus measurements were used only when both
A260/A280 = 1.3 ± 0.1 and A260 = 0.7 ± 0.2 unit.
Purity assessment

SDS-PAGE analysis was performed using 4–20% Criterion™
Tris-HCI Gradient Gels stained with Bio-Safe™ Coomassie
Stain. HCP levels were determined using a HEK 293 HCP
ELISA Kit (Cygnus Technologies). DNA was extracted fromtest samples and subjected to quantitative PCR analysis using
primers and probes directed against a 63 bp region of thehuman 18S ribosomal RNA gene. Readouts were compared
to a quantitative HEK 293 DNA standard curve.
Final process characterization

Data for both the final mass capture and the final anion
exchange operations were generated using 5 ml prepacked
8 x 100 mm Foresight Columns.
The Nuvia cPrime Column (Bio-Rad, catalog #732-4742)
was equilibrated with 25 mM histidine, pH 6.0. The culture
supernatant was diluted 1:3 with equilibration buffer prior
to column loading. The loaded column was washed withequilibration buffer and virus was eluted with 75 mM Tris,
525 mM NaCl, pH 8.5.
The Nuvia Q Column (Bio-Rad, catalog #732-4741) was
equilibrated with 75 mM Tris, 250 mM NaCl, pH 8.0. Eluate
from the Nuvia cPrime Column was diluted 1:1 with 75 mM
Tris, pH 8.0, prior to column loading. The loaded column waswashed with equilibration buffer and then with 75 mM Tris,
440 mM NaCl, pH 8.5. Purified virus was eluted with 75 mM
Tris, 1 M NaCl, pH 7.5.

© 2016 Bio-Rad Laboratories, Inc.

Mass capture

Initial screening showed that of the five resins screened,
three had potential for use in a mass capture process —Nuvia cPrime (hydrophobic cation exchange), UNOsphere Q(anion exchange), and Nuvia Q (anion exchange) — sincevirus was not detected in either the flow-through or wash
fractions (Table 1). Nuvia cPrime was selected for capturing
the adenovirus as it offered the best clearance of feedstream
contaminants. Eluate from this hydrophobic cation exchange
resin could be loaded onto the subsequent column following
a simple pH adjustment. Fouling of anion exchange
chromatography resins in process manufacturing (Close et al.
2013, Drevin et al. 1989) and difficulties in their regeneration(Ng and McLaughlin 2007) have been reported due to thebinding of excessive amount of impurities and ineffective
cleaning. Therefore, the Q resins are more suitable forpolishing purification. Between the two strong anion exchange
resins, Nuvia Q was chosen for its higher binding capacity fortarget virus even in the presence of high NaCl concentrations.
Table 1. Results from the initial resin screening and their implications.

Column Type

Virus in
Flowthrough/
Wash

Virus inEluateNotes/Implications

UNOsphere S
(CEX)

+++

++

Poorly suited for virus
purification in bothbind-and-elute andflowthrough modalities

Nuvia S (CEX)

+++

++

Poorly suited for virus
purification in bothbind-and-elute andflowthrough modalities

Nuvia cPrime
(MM)

—

++++

Partial elution in
125 mM NaCl, pH 6.5;
hence, dilution of crude
harvest required prior
to column loading

UNOsphere Q
(AEX)

—

++++

Could be considered
for direct mass capture

Nuvia Q (AEX)

—

++++

Could be considered
for direct mass capture

CEX, cation exchange; MM, mixed mode; AEX, anion exchange

We developed the mass capture step using Nuvia cPrime by
first replacing the buffer exchange step used in the screening
study with a dilution step. This was carried out since virus
partially eluted from the column when 125 mM NaCl was used.
In addition, a nuclease digestion step was introduced prior tocolumn loading to aid in nucleic acid clearance. Initial masscapture experiments were focused on reducing feedstream
volumes and recovering virus. The chromatograms in Figure 1
illustrate the early progression of these experiments and indicate
when significant transgene expression was detected.

Bulletin 6719

60

1.0

0.4

40

0.8

0.2

20

AU

0
0		 20		 40		 60		 80		100		 120Time, min

0.4

40

0

40

0.2

20
0

60

100
80
60
40
20
0

0

40
20

10

20

30
Time, min

40

50

60

Fig. 2. Behavior of the crude harvest on the UNOsphere Q and Nuvia Q
Columns. OD 260 (—); OD 280 (—); conductivity (—). Blue shading indicatesdetection of significant transgene expression. AU, absorbance units.
mS/cm

AU

50

0

1.6

0

40

0.2

60

0.4

30
Time, min0.4

 eed diluted 1:3 with 75 mM histidine, pH 6.0
F
Wash = 75 mM histidine, pH 6.0
Elution 1 = 75 mM Tris, pH 8.0
Elution 2 = 75 mM Tris + 525 mM NaCl, pH 8.5

0.8

20

0.6

0		 20		 40		 60		 80		100		 120Time, min1.2

10

Nuvia Q

0.8

mAU

0.4

2.0

20

0

mS/cm

AU

60

0

60

0

0.6

0.2

 eed diluted 1:1 with 75 mM histidine, pH 6.0
FWash = 75 mM histidine, pH 6.0
Elution 1 = 75 mM Tris, pH 8.0
Elution 2 = 75 mM Tris + 125 mM NaCl, pH 8.0
0.6

80

mS/cm

0

100

UNOsphere Q

mS/cm

0.6

mS/cm

AU

Development of an Efficient Manufacturing Process for Adenovirus

00		 40		 80		120		160		200		240		280Time, min eed diluted 1:3 with 25 mM histidine, pH 6.0
F
Wash = 25 mM histidine, pH 6.0
Elution = 75 mM Tris + 525 mM NaCl, pH 8.5

Fig. 1. Iterative development of Nuvia cPrime capture. OD 260 (—);
OD 280 (—); conductivity (—). Blue shading indicates detection of significant
transgene expression. AU, absorbance units.

The anion exchange experiments with Nuvia Q were focusedon attaining high product purity (Figure 3). As shown in the
figure, increasing the pre-elution NaCl concentration from 400
to 440 mM prevented a suspected HCP contaminant fromeluting with the product. Therefore, it would be possible tofurther improve the efficiency of this chromatography step bydirectly equilibrating the Nuvia Q Column with 75 mM Tris +440 mM NaCl, pH 8.0.
1.0

Of the two anion exchange resins, UNOsphere Q and Nuvia Q,
Nuvia Q was selected because it could adsorb virus at higher
NaCI concentrations (Figure 2). It was therefore easier to workwith downstream of the Nuvia cPrime capture step, where theeluate had an NaCI concentration of approximately 500 mM
going on to the anion exchange column.

AU

Anion exchange chromatographyc

75 mM histidine
400 mM NaCl
pH 8.0as a b cbs) in-process loada) flowthroughb) pre-elutionc) product elution
suspected
host-cell protein00											55Time, minAU

1.0

s a b cc

75 mM histidine
440 mM NaCl
pH 8.0ab

Increasing [NaCl]
shifted unwanted
protein fromproduct topre-elution peak.

00											55Time, minFig. 3. Chromatograms and gels from anion exchange processdevelopment experiments. OD 260 (—); OD 280 (—); conductivity (—).
* Proteins presumed to be virus capsid components. AU, absorbance units.

© 2016 Bio-Rad Laboratories, Inc.

Bulletin 6719

Development of an Efficient Manufacturing Process for Adenovirus

Mixed-mode chromatography

75 mM Tris, 440 mM NaCl,
pH 8.0 (pre-elution)

0.8

Initial capture was accomplished using Nuvia cPrime
Mixed-Mode Resin (Figure 4). This portion of the processachieved a tenfold reduction in processing volume and asignificant reduction in feedstream contaminants (Figure 6,
lanes 2–4).

80

75 mM Tris, 1,000 mM
NaCl, pH 7.5 (productelution)

100 mM acetate,
1,000 mM NaCl,
pH 4.0 (strip)

AU

75 mM Tris, 250 mM
NaCl, pH 8.0 (equilibration
and wash)Start of load00

Flowthrough

2

100

mS/cm

AU

0

25 mM histidine,
pH 6.0 (wash)

Start of loadmS/cm

Final process10

Pre-elution
20

Product collection

30
Time, min40

50

Fig. 5. Representative anion exchange chromatogram. OD 260 (—);
OD 280 (—); conductivity (—). AU, absorbance units.

Flowthrough0

Analysis of in-process and final product

0
0

60

120

180

240

Time, min

100 mM NaOH
(strip)

60

mS/cm

1.2

AU

75 mM Tris, 525 mM NaCl,
pH 8.5 (elution)00

Product collection
240

250

260

270
Time, min

280

290

Fig. 4. Representative mixed-mode chromatogram. OD 260 (—);
OD 280 (—); conductivity (—). AU, absorbance units.

A notable feature of the chromatograms generated using
this method is a double peak following the application of theelution buffer. The first peak is concurrent with the salt front,
while the second peak presumably corresponds to a pH shift.
Both contained significant amount of virus. Therefore, productcollection spanned both peaks. Increasing just the pH orionic strength alone did not yield an eluate with a satisfactory
amount of virus (data not shown).
Anonion Exchange Chromatography

Final virus purification was accomplished using Nuvia Q Resin(Figure 5). This portion of the process achieved an additional
twofold reduction of product volume along with a significant
improvement in product purity (Figure 6, lanes 4–7). Following
this operation, nonvirus proteins were no longer evident by
SDS-PAGE (Figure 6, lane 7).

We used SDS-PAGE analysis to visualize the progressive
reduction of contaminating proteins at each step of thepurification process (Figure 6). Cell culture media components
such as albumin and transferrin are clearly evident in thevirus culture harvest, that is, the sample loaded onto the
Nuvia cPrime Column (Figure 6, lane 2). These proteins are
effectively separated from the viral protein components, as
they are highly visible in the flow-through fraction from thiscolumn, under the current chromatography conditions(Figure 6, lane 3). Significantly fewer contaminants are seenin the sample loaded onto the Nuvia Q Column (Figure 6,
lane 4). During the anion exchange process, the virus boundto the resin while contaminants either flowed through thecolumn (Figure 6, lane 5) or were pre-eluted (Figure 6, lane 6).
The five most prominent viral proteins — hexon, penton, core
(V), hexon (VI), and core (VII) — are readily visible in the final
purified product (Figure 6, lane 7).
1

2

3

4

5

6

7

kD
250
150
100
75
50

Hexon

PentonCore (V)37
25
20

Hexon (VI)Core (VII)15
10Fig. 6. SDS-PAGE of intermediates and the final product. Lane 1, MW
marker; lane 2, Nuvia cPrime load; lane 3, Nuvia cPrime flowthrough; lane 4,
Nuvia cPrime elution/Nuvia Q load; lane 5, Nuvia Q flowthrough; lane 6, Nuvia
Q pre-elution; lane 7, Nuvia Q product.

© 2016 Bio-Rad Laboratories, Inc.

Bulletin 6719

Development of an Efficient Manufacturing Process for Adenovirus

Virus concentration and DNA levels were evaluated atselect points along the downstream process (Table 2). The
data demonstrate an overall recovery of virus particles ofapproximately 54%, with DNA levels below detection and HCP
at 2 ng/1010 particles. These values are well within current
guidelines for clinical and perhaps commercial use.
Table 2. Viral particle recovery and impurity clearance.
Impurity Levels(ng/1010 Particles)

Conclusions
The final process yields an active, concentrated virus productwith purity, HCP, and DNA levels comparable to clinical grade
products. While the purification methods presented here
were developed using the Ad5-E1+GFP model virus, they
are expected to be applicable to recombinant adenovirusesin general and to constructs derived from serotype 5 virus
in particular. The process is readily scalable and uses
procedures and reagents compatible with cGMPs. Also,
it is sufficiently simple, rapid, and efficient to be used forthe production of clinical grade virus-based gene therapy
products and vaccines.

Total Virus(x1011 Particles)DNA

Bulk harvest

30.6

3,144

n/d

Nuclease-treated harvest

31.8

30

3,022

References

Nuvia cPrime eluate

18.4

n/d

58

Nuvia Q eluate

16.4

<0.02

2

Close EJ et al. (2013). Fouling of an anion exchange chromatography operation
in a monoclonal antibody process: Visualization and kinetic studies. Biotechnol
Bioeng 110, 2425–2435.

Samplen/d, not determined.

HCP

Drevin I et al. (1989). Column performance of Q-Sepharose HP in analyticaland preparative-scale chromatography. J Chromatogr A 477, 337–344.
Ng PK and McLaughlin V (2007). Regeneration studies of anion-exchange
chromatography resins. http://www.bioprocessintl.com/wp-content/uploads/
bpi-content/070505ar06_77602a.pdf, accessed June 9, 2016.

© 2016 Bio-Rad Laboratories, Inc.

Bulletin 6719

Bio-Rad
Laboratories, Inc.
Web site bio-rad.com USA 1 800 424 6723 Australia 61 2 9914 2800 Austria 43 1 877 89 01 177 Belgium 32 (0)3 710 53 00 Brazil 55 11 3065 7550
Canada 1 905 364 3435 China 86 21 6169 8500 Czech Republic 420 241 430 532 Denmark 45 44 52 10 00 Finland 358 09 804 22 00
France 33 01 47 95 69 65 Germany 49 89 31 884 0 Hong Kong 852 2789 3300 Hungary 36 1 459 6100 India 91 124 4029300
Israel 972 03 963 6050 Italy 39 02 216091 Japan 81 3 6361 7000 Korea 82 2 3473 4460 Mexico 52 555 488 7670 The Netherlands 31 (0)318 540 666
New Zealand 64 9 415 2280 Norway 47 23 38 41 30 Poland 48 22 331 99 99 Portugal 351 21 472 7700 Russia 7 495 721 14 04
Singapore 65 6415 3188 South Africa 27 (0) 861 246 723 Spain 34 91 590 5200 Sweden 46 08 555 12700 Switzerland 41 026 674 55 05
Taiwan 886 2 2578 7189 Thailand 66 662 651 8311 United Arab Emirates 971 4 8187300 United Kingdom 44 020 8328 2000

Life ScienceGroup

Bulletin 6719 Ver A

US/EG

16-07090716

Sig 1215