BIOTECHNOLOGY
published by
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MANON M.J. COX
Protein Sciences Corporation
1000 Research Parkway
Meriden, CT 06450, USA
Tel +1 203 686 0800 308
Fax +1 203 686 0268
[email protected]
www.proteinsciences.com
Commercial
production of proteins
in insect cells
T
he Baculovirus Expression
Vector System (BEVS) is best
known and used as a research
tool. Thousands of articles
describing the use of the BEVS system
for the production of proteins for
research use have been published over
the past decades. Its reputation is one of
providing quick access to biologically
active proteins. Still today there is no
protein, produced in insect cells
approved human therapeutic or vaccine
use. The case study presented provides
evidence that this technology continues
to have tremendous potential.
BEVS TECHNOLOGY
Parts of this article were presented at the
BioPharmos 2003 meeting, which was held in
March 2003
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JANUARY/FEBRUARY 2004
Baculoviruses are found in nature
commonly on green vegetables, and
therefore, baculoviruses are part of the
menu of healthy individuals. The name
of the virus is derived from the Latin
“Baculum” (rod) describing its scape.
Baculoviruses are characterized by their
narrow host range (1), and cause fatal
disease or death of specific insect
species. A baculovirus infected insect will
transform within days into a glue-like
structure, mostly consisting of the
polyhedrin protein. Polyhedrin protects
the virus from U.V. or day light in its
natural habitat. Summers and Smith
demonstrated in the 1980s that the
DNA encoding this protein was
unnecessary for the survival of the virus
in a laboratory and could be exchanged
for genes encoding proteins of medical
importance, such as β-interferon (2).
Insect cells have the capability to
perform many of the post-translational
modifications such as glycosylation,
disulfide bond formation and
phosphorylation required for the
biological activity of many complex
proteins (3). A new expression system
was born and over the years many
thousands genes have been successfully
cloned and expressed in insect cells.
SELECTION OF AN APPROPRIATE
PROTEIN PRODUCTION SYSTEM
The genomics project has resulted in
the discovery of over 30,000 genes,
all potential disease targets. Identifying
the function of these genes, making
the proteins they encoded, protein
characterization and finding
applications for the resulting products
is generally considered the industry’s
next challenge.
Factors such as time to market, cost
of goods, the characteristics of the
product, regulatory hurdles and patents
can ultimately determine success or
failure of a project. Selecting the
appropriate expression system for your
protein of interest will impact most of
these factors.
The BEVS system offers reliable,
fast production of your protein of
interest. Proteins can usually be
produced in weeks rather than
months or years because the virus
used to infect the insect cells rather
than the cell line is modified. The
baculovirus genome is relatively small
(approximately 160 kb) and can be
easily characterized using genome
digests and Southern blotting
techniques. After the baculovirus
infects the insect cell, the cell is
transformed into a baculovirus protein
production facility prior to cell death.
In case a heterologous protein would
be lethal to the cells, the protein will
still be produced during the short
remainder of the insect cells life span.
The protein of interest is usually
produced under the control of the
polyhedrin promoter, one of the
BIOTECHNOLOGY
Epidermal Growth
strongest promoters
Table I - Strengths and weaknesses of various expression systems.
Factor expressed in
known in nature. Insect
Six different expression systems are qualitatively ranked on various criteria
insect cells using its
cell-produced proteins are from worst to best
natural signal
generally found to be
sequence and the
biologically active because
61 kDa derived
of the capacity of the
signal sequence.
insect cells to perform post
Analyzing the gel by
translational modifications.
using densitometry
Limitations to the BEVS
suggests that
technology include the fact
expression levels
that to date there is no
obtained with the
approved baculovirus
baculovirus derived
product for human vaccine
signal sequence are
or therapeutic use on the
approximately
market, even though a
10-fold higher than
number of products are in
with its natural signal
going advanced clinical
sequence. The right
trials. In addition, the
panel shows an
patent landscape
immunoblot
surrounding the BEVS is
demonstrating the
fairly complex. The
correct identity of the
absence of complex sugars
protein. Protein
in BEVS-produced proteins
Sciences will
raises questions relating to
introduce a protein
the biological activity of the
expression kit that
proteins. The presence of
Spodoptera frugiperda (S.
contains this signal sequence in the
relative “simple” glycosylation can
frugiperda) also referred to as Sf21
fourth quarter of 2003.
be considered an advantage for
and later Sf9 cells. Protein Sciences
some proteins and a disadvantage
has developed a serum free cell line
for others.
S. frugiperda cell line (expresSF+®)
Protease Deficient Baculovirus
For every protein one has to identify
with well defined growth
Vector
the critical parameters and elect the
characteristics (4). The expresSF+®
most optimal expression system. Table I
cell line has been selected to grow to
may provide some guidance for
Some heterologous proteins are very
high density in suspension without
selection of a particular expression
prone to protease degradation. Already
clumping. The doubling time of the
system for a particular protein.
early during the infection process more
cells varies from 18-24 hours
protein is degraded than is being
depending on the selected cultivation
produced. A pragmatic approach to
system (spinners and shakers versus
overcome the degradation of your
OUTSOURCING OF PRODUCTION
bioreactors). Cells can be passaged
protein of interest is to harvest the
OF PROTEINS MADE
for over 50 passages while
protein early in the infection process or,
IN INSECT CELLS
maintaining a high viability. They are
alternatively, add various protease
also highly prone to infection with
inhibitors. We created a baculovirus
Protein Sciences Corporation has
baculovirus resulting in high titer
backbone that lacks the Cathepsin gene
developed technology related to the
baculovirus stock of typically around
and part of the chitinase gene. The use
Baculovirus system that are
108 plaque forming units (pfu) per
of this backbone is advantageous for the
advantageous when considering large
mL and high protein production
production of certain proteins as shown
scale production of proteins in insect
levels. The cells have been used
in Figure 3 but does not in all cases
cells. The Company has tested
successfully in large scale
result in better production levels.
numerous recombinant protein
manufacturing (600 L bioreactors).
vaccines for AIDS, malaria, cancer, and
Figure 1 shows the expresSF+® cells
for viral influenza in phase I and II
during various stages of infections.
human clinical trials, resulting in an
Cells increase approximately fourfold
established track record with the
in size during infection resulting in
regulatory agencies. Large scale (600 L)
increased biomass and protein
production capacity was established in
production. Cells can be purchased
addition to an intellectual property
from Protein Sciences Corporation.
portfolio, providing freedom to operate
to our customers despite the complex
patent landscape.
Signal Sequence
IMPROVEMENTS
TO THE TECHNOLOGY
AND RELATED INTELLECTUAL
PROPERTY
Cell Line
Immortal insect cell lines were
developed from the ovaries of
BIOTECHNOLOGY
Many heterologous proteins are
secreted into the cell media. Protein
Sciences identified a baculovirus signal
sequence derived from the 61 kDa
membrane associated glycoprotein,
which resulted in increased secretion
and expression of heterologous
proteins (5). Figure 2 shows an SDS
PAGE gel compared Vascular
Figure 1 - Infection of SF+ cells at a MOI = 0.2.
SF+ cells can be synchronously infected using
an MOI of 0.2 (as measured in a standard
1 hour plaque assay). By 27 hours post
infection all of the cells show typical
morphological changes associated with
infection. Late in infection (75 hr) the cells
have enlarged to more than twice that of
uninfected cells and the majority of the
infected SF+ cells remain intact
JANUARY/FEBRUARY 2004
45
High Cell Density
Insect cells are
process and
Company
information
derived from six
previously filed
INDs that can be
cross referenced
by our
collaborators.
susceptible to
infection while they
are in their
logarithmic growth
phase, which limits
the possibility of
generating high
concentrations of
Large Scale
biomass prior to
Production
producing the
Capacity
protein of interest.
Under normal
Producing
cultivation conditions
proteins in insect
insect cells can be
cell at scale
successfully infected
requires a high
while at a density of
titer working
2-3 million cells
virus bank and
per mL. We have
an efficient
been able to create
insect cell
fermentation
infection
conditions using a
process. Our
dialysis fermentation
manufacturing
set up under which
Figure 2 - Expression of VEGF.
personnel is
cells can be infected VEGF was expressed either with its natural signal sequence or with PSC’ 61 kDa signal
sequence.
skilled at
at 10 times higher
Left panel represents Coomassie stained SDS-PAGE gel.
producing large
cell densities
Right panel shows Western Blot using VEGF specific antibody for confirmation of identity
quantities insect
(15-20 million cells
cells that are
per mL). The
subjected to infection by the virus bank
principle of the dialysis set up is
To date more than 50,000 doses of
in a batch process. The high cell density
based on the possibility of waste
protein have been administered in over
technology might offer a solution in the
products being removed from the
5,000 subjects and patients without
future but is not yet used in our
media and fresh ingredients be
reportable adverse effects, suggesting
production facility. The expresSF+®
added to the media without going
that BEVS is a safe manufacturing
insect cell line produces high titer virus
through the cumbersome process of
system for the production of proteins.
stocks thereby reducing the amount of
identifying the critical components
We have established a drug master
virus needed to be added to the
(Patent pending).
file that contains general cell line,
bioreactor to approximately 1% of the
total reactor volume. Currently the
production process is performed at a
Upstream Processing
600 L scale and no major hurdles are
envisioned when scaling the process 10
We have successfully developed a
to 50-fold.
clarification method to remove DNA,
lipids and other contaminating agents
from the media. The addition of calcium
CASE STUDY
chloride to a concentration of 10 mM
and Tris base to a concentration of
In 1997 health officials in Hong Kong
28 mM and pH 8.0 allows for the
were alarmed by the death of a child
formation of a calcium gel that batch
following infection with a highly
binds DNA and can be easily separated
pathogenic avian H5N1 influenza strain.
from the supernatant by centrifugation. A
This virus had previously caused the
drawback to this method is certain
death of 70-100% of the chickens in
Ca-dependent proteases may be
infected flocks in Hong Kong. Before
activated during this process, leading to
year-end, six out of eighteen infected
degradation of the protein of interest
people died of the disease (6).
and a reduction in the final overall
Fortunately, the efficiency of
process yield. Adding chelating agents
transmission of this virus between
after centrifugation may be used to
humans was low, but the need for
reduce this effect.
better vaccines became obvious (7).
Particularly alarming was that the usual
Figure 3 - Protease deficient baculovirus.
egg-based influenza vaccine
Clinical and Regulatory Experience
Protein X was cloned in a baculovirus
manufacturing process is incapable of
backbone that lacks the Cathepsin gene and
producing a vaccine for this kind of virus
Protein Sciences has tested many
part of the chitinase gene and in the wildtype
because the chicken embryos used for
different recombinant proteins made in
baculovirus. As shown in this Western Blot
production of the vaccine are killed by
insect cells as vaccine candidates for the
more full length protein X can be detected
this highly pathogenic virus.
prevention of various diseases, such as
intracellular when using protease deficient
backbone
The insect cell based production
HIV, malaria, colon cancer and influenza.
46
JANUARY/FEBRUARY 2004
BIOTECHNOLOGY
technology offered a solution in this
emergency situation. The cDNA
encoding the hemagglutinin gene
from the avian H5N1 strain was used
to produce a sub-unit vaccine. Within
a period of six weeks the Company
was able to produce HA antigen that
was first tested in chickens. These
tests confirmed immunogenicity and
more importantly protection in
chickens from a lethal viral challenge
(8). Four weeks thereafter
1,700 doses were delivered to NIH
for testing in humans. The company
received approval for compassionate
use from the FDA. A clinical study was
conducted using this material. These
studies indeed suggested that the
BEVS expressed H5 HA was able to
induce functional antibodies in
individuals who had no prior exposure
to the H5 viruses (9).
CONCLUDING REMARKS
REFERENCES
The case study demonstrates that the
1) TINSLEY and HARRAP, 1978. “Viruses of
invertebrates” in Comprehensive
Virology; Fraenkel-Conrat, H.; Wagner,
R.R. Editors; Plenum Press: New York,
1978, Vol.12, pp.1-101
2) SMITH et al. Mol. and Cell. Biol. 1983,
3, 2156-2165
3) Reviewed by MILLER “A virus vector for
genetic engineering in invertebrates” in
Genetic Engineering in the Plant
Sciences; Panopaulus, N.J. Ed.; Praeger
Publishers: NY, 1981; pp.203-222
4) US Pat. 6,103,526
5) US Pat. 5,762,939 and US Pat. 6,245,532
6) CLAAS, E.C.J.; et al. Lancet 1998,
351 (9101), 472-476
7) BELSHE, R.B.; Lancet 1998, 351 (9101),
460-461
8) CRAWFORD, J.; et al. Vaccine 1999,
17, 2265-2274
9) TREANOR, J.J. et al. 2001. Vaccine 2001,
19, 1732-1737
BEVS technology is a powerful
manufacturing technology that can
provide healthcare solutions in
pandemic, biodefense and emergency
situations.
We are exploring the potential of
this production technology for the
rapid development of a SARS subunit
vaccine. In addition, a recombinant
trivalent hemagglutinin influenza
vaccine is being tested in a large size
Phase II(b) clinical study in the elderly
population in the US.
For those considering exploring
the BEVS technology for commercial
use or large scale production and
wanting to take advantage of our
experience, we offer process
development services.
Visit our
website:
www.b5srl.com
BIOTECHNOLOGY
JANUARY/FEBRUARY 2004
47