Simplified production and concentration of HIV-1-based lentiviral vectors using HYPERFlask vessels and anion exchange membrane chromatography.

Abstract

During the past twelve years, lentiviral (LV) vectors have emerged as valuable tools for transgene delivery because of their ability to transduce nondividing cells and their capacity to sustain long-term transgene expression in target cells in vitro and in vivo. However, despite significant progress, the production and concentration of high-titer, high-quality LV vector stocks is still cumbersome and costly.
Here we present a simplified protocol for LV vector production on a laboratory scale using HYPERFlask vessels. HYPERFlask vessels are high-yield, high-performance flasks that utilize a multilayered gas permeable growth surface for efficient gas exchange, allowing convenient production of high-titer LV vectors. For subsequent concentration of LV vector stocks produced in this way, we describe a facile protocol involving Mustang Q anion exchange membrane chromatography.
Our results show that unconcentrated LV vector stocks with titers in excess of 108 transduction units (TU) per ml were obtained using HYPERFlasks and that these titers were higher than those produced in parallel using regular 150-cm2 tissue culture dishes. We also show that up to 500 ml of an unconcentrated LV vector stock prepared using a HYPERFlask vessel could be concentrated using a single Mustang Q Acrodisc with a membrane volume of 0.18 ml. Up to 5.3 x 1010 TU were recovered from a single HYPERFlask vessel.
The protocol described here is easy to implement and should facilitate high-titer LV vector production for preclinical studies in animal models without the need for multiple tissue culture dishes and ultracentrifugation-based concentration protocols.

Full text

Page 1

BioMed Central
Page 1 of 7
(page number not for citation purposes)
BMC Biotechnology
Open Access
Methodology article
Simplified production and concentration of HIV-1-based lentiviral
vectors using HYPERFlask vessels and anion exchange membrane
chromatography
Robert H Kutner*1, Sharon Puthli1, Michael P Marino1,2 and Jakob Reiser1,2
Address: 1Gene Therapy Program, Vector Core, Louisiana State University Health Sciences Center, New Orleans, LA, USA and 2Division of Cellular
and Gene Therapies, Office of Cellular, Tissue and Gene Therapies, US Food and Drug Administration, Center for Biologics Evaluation and
Research, Bethesda, MD, USA
Email: Robert H Kutner* - rkutne@lsuhsc.edu; Sharon Puthli - sputhli@yahoo.com; Michael P Marino - Michael.Marino@fda.hhs.gov;
Jakob Reiser - Jakob.Reiser@fda.hhs.gov
* Corresponding author
Abstract
Background: During the past twelve years, lentiviral (LV) vectors have emerged as valuable tools
for transgene delivery because of their ability to transduce nondividing cells and their capacity to
sustain long-term transgene expression in target cells in vitro and in vivo. However, despite
significant progress, the production and concentration of high-titer, high-quality LV vector stocks
is still cumbersome and costly.
Methods: Here we present a simplified protocol for LV vector production on a laboratory scale
using HYPERFlask vessels. HYPERFlask vessels are high-yield, high-performance flasks that utilize a
multilayered gas permeable growth surface for efficient gas exchange, allowing convenient
production of high-titer LV vectors. For subsequent concentration of LV vector stocks produced
in this way, we describe a facile protocol involving Mustang Q anion exchange membrane
chromatography.
Results: Our results show that unconcentrated LV vector stocks with titers in excess of 108
transduction units (TU) per ml were obtained using HYPERFlasks and that these titers were higher
than those produced in parallel using regular 150-cm2 tissue culture dishes. We also show that up
to 500 ml of an unconcentrated LV vector stock prepared using a HYPERFlask vessel could be
concentrated using a single Mustang Q Acrodisc with a membrane volume of 0.18 ml. Up to 5.3 ×
1010 TU were recovered from a single HYPERFlask vessel.
Conclusion: The protocol described here is easy to implement and should facilitate high-titer LV
vector production for preclinical studies in animal models without the need for multiple tissue
culture dishes and ultracentrifugation-based concentration protocols.
Background
LV vectors provide powerful tools for transgene delivery
into dividing as well as nondividing cells and for long-
term transgene expression in target cells in vitro and in
vivo. Currently, the use of LV vectors is commonplace and
applications in the fields of neuroscience, hematology,
Published: 16 February 2009
BMC Biotechnology 2009, 9:10doi:10.1186/1472-6750-9-10
Received: 17 September 2008
Accepted: 16 February 2009
This article is available from: http://www.biomedcentral.com/1472-6750/9/10
© 2009 Kutner et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Page 2

BMC Biotechnology 2009, 9:10http://www.biomedcentral.com/1472-6750/9/10
Page 2 of 7
(page number not for citation purposes)
developmental biology, stem cell biology and transgene-
sis have emerged [1]. LV vectors are also being pursued in
a number of clinical trials (see http://www.gemc
ris.od.nih.gov/). Despite significant progress, the produc-
tion and concentration of high-titer, high-quality LV vec-
tors for preclinical studies in animal models is still
cumbersome and costly [2].
The production of LV vectors is typically carried out using
transient transfection approaches involving tissue culture
dishes or flasks [3], cell factories [4-6], or stirred-tank bio-
reactors [7]. These protocols are cumbersome to scale up
(dishes, flasks) or technically challenging (cell factories,
bioreactors), preventing their routine use in a standard
laboratory setting.
Typical LV vector titers involving the vesicular stomatitis
virus (VSV) G glycoprotein range from 106 to 108 TU/ml
[8]. Higher titers can be achieved by physical concentra-
tion [2], including ultracentrifugation [3,9-11], or filtra-
tion approaches such as ultrafiltration [3,10,12-14], and
diafiltration [4,6]. Vector production for large-scale in vivo
applications in animal models requiring high-titer LV vec-
tor stocks is challenging due to the lack of simple proce-
dures allowing rapid processing of large volumes of LV
vector-containing cell culture supernatants. The tradi-
tional ultracentrifugation-based methods are limited in
terms of their capacity to handle large volumes, thus mak-
ing this procedure extremely
approaches such as diafiltration are well suited for
processing large volumes of vector supernatants. How-
ever, they are difficult to implement in a standard labora-
tory setting. Thus, there is an emerging need for simple
and less laborious procedures that result in a rapid reduc-
tion of the volume of the cell culture supernatant to be
processed without the need for a centrifugation step.
tedious. Filtration
One problem with the centrifugation and filtration-based
methods outlined above is that cell-derived components
are concentrated along with the vector particles. These
have the potential to cause immune and inflammatory
responses [15]. For example, concentrated VSV-G-pseudo-
typed LV vector preparations were shown to be contami-
nated with tubovesicular structures of cellular origin
which carried nucleic acids, including the plasmid DNAs
that were used to generate the LV vector stocks. DNA car-
ried by these tubovesicular structures acted as a stimulus
for innate antiviral responses, triggering Toll-like receptor
9 and inducing alpha/beta interferon production [16].
Thus, additional steps including chromatography-based
methods such as anion exchange chromatography are
needed in order to reduce host cell and cell culture-
derived contaminants from LV vector preparations. Meth-
ods based on anion exchange column chromatography of
LV vectors pseudotyped with VSV-G [17,18] or the bacu-
lovirus GP64 glycoprotein were previously described [19].
However, the yields and purity of the LV vector stocks
obtained in this way were not reported.
In an attempt to simplify the production and concentra-
tion of LV vectors and to make this approach more repro-
ducible and cost-effective for preclinical studies in
animals, we have worked out a facile LV vector production
system based on HYPERFlasks. We also implemented a
straightforward concentration procedure based on Mus-
tang Q anion exchange membrane chromatography. Mus-
tang Q anion exchange-based chromatography protocols
for concentrating/purifying LV vectors were previously
reported [4,20]. Such vector preparations displayed
reduced toxicity compared to vectors concentrated using
ultracentrifugation [20], as well as enhanced purity [4].
Results and discussion
Simplified production of lentiviral vectors using
HYPERFlasks
With a view toward improving high-titer LV vector pro-
duction for preclinical studies in animals, we tested the
usefulness of HYPERFlask vessels that have a total growth
area of 1720 cm2, corresponding to ten standard T175
flasks. HYPERFlasks consist of ten interconnected growth
surfaces each containing a membrane pretreated to allow
improved cell adherence. The membrane is gas permea-
ble, allowing exchange of oxygen and carbon dioxide
through the base of the membrane, resulting in gas expo-
sure to a large surface area within the flask [21].
To test the usefulness of HYPERFlasks for LV vector pro-
duction involving calcium phosphate-mediated transfec-
tion [10], we compared the titers of LV vector stocks
prepared side-by-side using either ten 150-cm2 dishes or a
single HYPERFlask. The data shown in Table 1 represent
the results of three independent productions each for the
150-cm2 dish system and the HYPERFlask system. These
data indicate that the titers of unconcentrated LV vectors
produced using HYPERFlasks were up to 2.3 × 108 TU/ml
while the titers of LV vectors produced in 150-cm2 dishes
were lower, up to 6.9 × 107 TU/ml. The total yields were
up to 1.2 × 1010 TU from ten 150-cm2 dishes and up to 1.3
Table 1: Lentiviral vector production using 150-cm2 dishes or
HYPERFlasks
Production vessels TU/mlTotal TU
150-cm2 dishes
HYPERFlask
5.6 ± 1.3 × 107
2.0 ± 0.3 × 108
9.5 ± 2.1 × 109
1.1 ± 0.16 × 1011
Ten 150-cm2 dishes (total volume: 170 ml) and one HYPERFlask
vessel (volume: 550 ml) were used for LV vector production.
Productions were performed side-by-side (n = 3). Vector titers were
determined using HOS cells. Data are expressed as the mean ±
standard deviation (SD).

Page 3

BMC Biotechnology 2009, 9:10http://www.biomedcentral.com/1472-6750/9/10
Page 3 of 7
(page number not for citation purposes)
× 1011 TU for the HYPERFlask vessels. This corresponds to
a productivity of up to 8 × 106 TU per cm2 for the 150-cm2
dishes. For the HYPERFlask, the productivity was about
10-fold higher, up to 0.75 × 108 TU per cm2. The higher
productivity observed with HYPERFlasks may be related
to better gas exchange during LV vector production. Over-
all, the 293T cells used for production appeared healthier
and there was less cell debris from transfections carried
out in HYPERFlaks compared to transfections carried out
in 150-cm2 dishes (data not shown). We expect the
HYPERFlask production protocol described here to be
compatible with other DNA transfection formats such as
polyethylenimine-mediated transfection [22].
Simplified concentration of lentiviral vectors using
Mustang Q Acrodiscs
The concentration and purification of LV vector stocks on
a large scale presents a significant bottleneck and methods
allowing rapid processing of large volumes of LV vector-
containing supernatants are needed. Chromatography-
based methods appear to be particularly attractive in this
regard [2]. For example, methods to concentrate/purify LV
vectors based on anion exchange or affinity chromatogra-
phy have been established [17-19]. Recently, Segura et al.
[7] described an approach involving heparin affinity chro-
matography to concentrate LV vector particles directly
from clarified supernatants. During this step, a recovery of
53% of infectious LV particles was achieved while 94% of
the impurities were removed. This strategy may ultimately
result in vector stocks of improved purity, increased infec-
tivity and reduced toxicity. However, these approaches are
cumbersome and difficult to implement in a standard lab-
oratory setting.
We have recently described a facile membrane-based
chromatography approach involving Mustang Q anion
exchange chromatography cartridges (Acrodiscs) to purify
adenoviral vectors [23]. Mustang Q membranes resulted
in high recoveries of infectious viral particles and allowed
the processing of adenoviral vector particles from lysates
in a fraction of the time required using the traditional
cesium chloride-based ultracentrifugation method. Mus-
tang Q membranes include a matrix that has a high
dynamic binding capacity for adenoviral vectors and is
capable of withstanding high flow rates. Other benefits of
Mustang Q membrane chromatography cartridges include
higher peak resolution at faster flow rates as compared to
traditional ion exchange resins. Also, they can easily be
adapted to bench-scale work and they are syringe-adapta-
ble. Finally, there is no need for an HPLC setup or a col-
umn packing step. In work with LV vectors carried out by
us [20,24] and by others [4], Mustang Q-based cartridges
were used to concentrate/purify LV vectors. While these
initial studies were encouraging, the capacity of Mustang
Q membranes for crude LV vector supernatants was rather
low [24], possibly caused by cell-derived proteins, serum
proteins, or contaminating nucleic acids that bound to
Mustang Q membranes during vector capturing.
To determine the binding capacity of Mustang Q mem-
branes for LV vectors produced in HYPERFlask vessels
compared to 150-cm2 dishes, a capture study was con-
ducted. Figure 1 shows a typical elution profile of HYPER-
Flask-produced LV vectors following capturing onto a
0.18-ml Mustang Q Acrodisc and elution using a gradient
ranging from 0.3 to 1.5 M NaCl. TU were determined by
FACS analysis of transduced HOS cells. The results pre-
sented in Table 2 show that the capacity of Mustang Q
Acrodiscs at 10% breakthrough for LV vector supernatants
produced in 150-cm2 dishes was up to 3.9 × 109 TU per ml
of membrane while the capacity of Mustang Q mem-
branes at 10% breakthrough for HYPERFlask vessel-
derived LV vector samples was up to 9.6 × 1010 TU per ml
of membrane. This is consistent with the observation that
LV vector supernatants produced using HYPERFlasks con-
tained fewer cellular proteins and nucleic acids compared
to supernatants produced in 150-cm2 dishes (Table 3) that
may have interfered with the binding of the LV vector par-
ticles. To remove excess NaCl, pooled Mustang Q frac-
tions were subjected to a buffer exchange using Sepharose
CL-4B. Table 4 summarizes the recovery of LV vectors fol-
lowing processing of 500-ml aliquots of HYPERFlask ves-
sel-derived supernatants. Up to 4.7 × 1010 TU were
obtained after Mustang Q chromatography and up to 5.3
Table 2: Capacity of Mustang Q Acrodiscs for crude lentiviral vector stocks
Production vesselsInput (TU)Unbound (TU)Bound (TU)Recovered (TU)YieldCapacity of Acrodisc (TU/ml)
150-cm2 dishes
HYPERFlask
1.2 ± 0.5 × 109
1.6 ± 0.1 × 1010
6.4 ± 5.1 × 108
2.8 ± 6.4 × 108
5.5 ± 1.6 × 108
1.6 ± 1.6 × 1010
3.2 ± 0.8 × 108
1.2 ± 0.1 × 1010
28.9 ± 5.4%
76.0 ± 0.70%
3.0 ± 0.9 × 109
≥8.7 ± 0.9 × 1010
Mustang Q Acrodiscs were challenged with 75-ml aliquots of vector-containing supernatants. The crude vector supernatants were filtered using a
0.45 μm filter prior to anion exchange chromatography on Mustang Q Acrodiscs. Input corresponds to the total number of TU loaded. Unbound
corresponds the total number of TU found in the flow-through. Bound represents input TU minus unbound TU. Recovered corresponds to the
total number of TU following elution. Capacity corresponds to the total number of TU bound per ml of the Mustang Q membrane. Since the
HYPERFlask-derived samples did not result in greater than 10% breakthrough, the upper limit of the capacity for these samples is not known. Titers
were determined by FACS using HOS cells. Data from three independent experiments involving 150-cm2 dish-derived and HYPERFlask vessel-
derived vector supernatants are shown. The data are expressed as mean ± SD.

Page 4

BMC Biotechnology 2009, 9:10 http://www.biomedcentral.com/1472-6750/9/10
Page 4 of 7
(page number not for citation purposes)
× 1010 TU after subsequent buffer exchange using Sepha-
rose CL-4B (Table 4).
Finally, we wanted to compare the titers and total yields
of our concentrated LV vectors with those reported by
other groups. To do this, we carried out a side-by-side titer
comparison of a HYPERFlask-derived LV vector stock con-
centrated by Mustang Q anion exchange chromatography
using HOS cells, 293T cells and HeLa cells. Table 5 shows
that the titers of fresh (unfrozen) vector stocks were simi-
lar in HOS cells and 293T cells and slightly lower in Hela
cells. Upon freezing and thawing of Mustang Q-concen-
trated vector stocks there was a 40% drop in vector titers
on average (data not shown).
Overall, the titers obtained using the HYPERFlask system
were as high or higher compared to those obtained using
150-cm2 dishes and ultracentrifugation-based concentra-
tion approaches [3]. The yield (total TU) from a single
HYPERFlask (cell growth area: 1720 cm2) was above 1011
TU (Table 1). This is about two orders of magnitude
higher than the yields reported by Karolewski et al. [5]
who used a cell factory system (total cell growth area:
6320 cm2), and at least one order of magnitude higher
than the yields reported by Geraerts et al. 2005 [6] who
also used Cell Factories.
Conclusion
In conclusion, we present here a simple protocol for LV
vector production and concentration involving HYPER-
Flasks in conjunction with Mustang Q anion exchange
membrane cartridges. These protocols are easy to imple-
ment in a standard laboratory setting and allow high-titer
LV vector production without the need for multiple tissue
culture dishes or flasks or ultracentrifugation-based con-
centration procedures.
Methods
Cell lines
293T cells (CRL-11268), human osteosarcoma (HOS)
cells (CRL-1543) and HeLa cells (CCL-2) were obtained
from the American Type Culture Collection (ATCC) and
propagated in DMEM (high glucose), 10% fetal bovine
serum (FBS, Invitrogen-GIBCO), 1% GlutaMAX (Invitro-
gen), 1% Pen/Strep (Invitrogen).
Production of lentiviral vectors using 150-cm2 dishes
LV vectors were generated by calcium phosphate-medi-
ated transfection of 293T cells as described [10,25], with
modifications. 293T cells were plated in 150-cm2 dishes at
a density of 8 × 106 cells per dish in 25 ml DMEM medium
supplemented with 10% FBS, 1% Pen/Strep, 0.3% HyQ
CelPro-LPS (HyClone). Twenty four h later, chloroquine
(Sigma-Aldrich) was added to the medium at a final con-
centration of 25 μM. LV vector, packaging (helper), and
envelope plasmid DNAs were combined in 3 ml of 0.25 M
CaCl2 and then mixed with 3 ml of 2 × HEPES-buffered
saline (2 × HBS) [26] using gentle vortexing. The DNA/
CaCl2/HBS mixture was then added to medium. The
amount of plasmid DNA used per dish was 21 μg of the
pNL-EGFP/CMV/WPREΔU3 LV vector plasmid DNA [20]
(Addgene plasmid 17579), 14 μg of the pCD/NL-BH*ΔΔΔ
packaging plasmid DNA [27] (Addgene plasmid 17531)
and 7 μg of the VSV-G-encoding pLTR-G plasmid DNA
[12] (Addgene plasmid 17532). The medium was
removed 16 h later and replaced with 17 ml of fresh
DMEM/10% FBS/1% GlutaMAX per plate. Forty eight h
later, the vector-containing medium was collected and
spun at 500 × g for 5 min, filtered through a 0.45-μm pore
size filter (Corning) and stored at -80°C.
Production of lentiviral vectors using HYPERFlask vessels
293T cells (2 × 108 cells) were seeded into a HYPERFlask
Cell Culture Vessel (Corning) in 550 ml of DMEM
medium supplemented with 10% FBS, 1% GlutaMAX, 1%
Pen/Strep, 0.3% HyQ CelPro-LPS. Twenty four h later, the
medium was removed from the HYPERFlask vessel. Sixty
ml of the medium were discarded, and chloroquine was
added to the remaining medium at a final concentration
of 25 μM. LV vector, packaging, and VSV-G plasmid DNAs
Table 3: Protein and DNA concentrations for crude lentiviral
vector stocks
Production vesselsProtein (mg/ml)DNA (μg/ml)
150-cm2 dishes
HYPERFlask
4.35 ± 0.25
3.03 ± 0.07
11.17 ± 1.6
7.22 ± 0.32
Protein and DNA concentrations of 150-cm2 dish-derived and
HYPERFlask vessel-derived vector supernatants were determined
using a Qubit assay kit (Invitrogen). Productions were performed
side-by-side (n = 3). Data are expressed as mean ± SD.
Table 4: Recovery of lentiviral vectors following Mustang Q anion exchange chromatography and buffer exchange
Input (TU)Unbound (TU) Bound (TU)Recovered following Mustang Q
chromatography (TU)
Recovered following buffer exchange (TU)
7.3 ± 2.0 × 1010
7.4 ± 2.3 × 109
6.5 ± 1.8 × 1010
4.1 ± 0.6 × 1010
4.5 ± 0.8 × 1010
Mustang Q Acrodiscs were challenged with 500-ml aliquots of HYPERFlask vessel-derived vector supernatants. Buffer exchange was carried out by
Sepharose CL-4B size exclusion chromatography as outlined under Materials and Methods. Titers were determined by FACS using HOS cells. Data
from three independent productions are shown. Data are expressed as mean ± SD.

Page 5

BMC Biotechnology 2009, 9:10http://www.biomedcentral.com/1472-6750/9/10
Page 5 of 7
(page number not for citation purposes)
Concentration of LV vectors by ion exchange chromatography using Mustang Q Acrodiscs
Figure 1
Concentration of LV vectors by ion exchange chromatography using Mustang Q Acrodiscs. A HYPERFlask vessel-
derived LV vector-containing supernatant (500 ml) was adjusted to 25 mM Tris-HCl, pH 8.0, 0.3 M NaCl and loaded onto a
Mustang Q Acrodisc (bed volume 0.18 ml) at a flow rate of 10 ml/min for 8 min using an AKTA purifier HPLC (Amersham
Pharmacia) and Unicorn 4.0 Software (Amersham Pharmacia). The Acrodisc's membrane was washed with 2 ml of 25 mM Tris-
HCl, pH 8.0, 0.3 M NaCl and LV vectors were eluted with a 10-ml gradient ranging from 0.3 to 1.5 M NaCl in 25 mM Tris-HCl,
pH 8.0 and 1-ml fractions were collected. Flow through, wash, and eluate fractions were collected using a FRAC 950 collector
(Amersham Pharmacia) and the OD at 280 nm was recorded (top panel); mAU = milli-absorbance units. Eluate fractions were
immediately processed to analyze vector TU (bottom panel). The total TU for each fraction are presented.
mAU OD 280nm
0
1000
2000
3000
4000
5000
Fraction number
246810 12 1416
Transduction Units
0.0
5.0e+9
1.0e+10
1.5e+10
2.0e+10
2.5e+10
3.0e+10
3.5e+10
Eluate