Commercial Production in Insect Cells by linzhengnd


									C      H A P T E R                         THREE

Commercial Production
in Insect Cells
One Company’s Perspective

by Manon M.J. Cox

           he human genome project
                                            Figure 1: The author’s qualitative ranking showing strengths and weaknesses of various
           sequenced more than              protein expression systems.
           30,000 genes, all of them
           potential disease targets.
           Identifying the function of
those genes, making the proteins
they encoded, characterizing those
proteins, and finding applications
for them is generally considered the
biotechnology industry’s next
challenge. Factors such as time to
market, cost of goods, product
characteristics, regulatory hurdles,
and intellectual property can
ultimately determine the success or
failure of a given project. Selecting
an appropriate expression system for
the protein of interest will affect
most of those factors. For any
protein of interest, the critical
parameters must be identified before
the most optimal expression system
can be found. Figure 1 provides
some basic guidance for selecting an       this technology continues to have               structure mostly consisting of
expression system.                         tremendous potential.                           polyhedrin protein. In the virus’s
    The baculovirus expression vector                                                      natural habitat, polyhedrin protects
system (BEVS) is best known and            SCIENCE                                         it from ultraviolet light. Scientists
used as a research tool. Thousands of      Baculoviruses are commonly found                demonstrated in the 1980s that the
articles describing the use of the         on green vegetables, and are                    DNA encoding the protein was
BEVS system for the production of          therefore part of the diet of healthy           unnecessary for the survival of the
proteins for research use have been        individuals. The name is derived                virus in a laboratory, so it could be
published during the past few              from the Latin baculum (rod),                   replaced by genes encoding proteins
decades. Its reputation is one of          which describes the shape of the                of medical importance, such as
providing quick access to biologically     virus. Baculoviruses are                        interferon (2). Insect cells can
active proteins. But today no protein      characterized by a narrow host                  perform many of the
produced in insect cells has been          range (1), mainly lepidopteran insect           posttranslational modifications
approved for human therapeutic or          species (moths and butterflies), in             required for the biological activity of
vaccine use. The case study presented      which they cause fatal disease.                 many complex proteins: e.g.,
in this chapter provides evidence that     Within days, a baculovirus-infected             glycosylation, disulfide bond
                                           insect will transform into a glue-like
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formation, and phosphorylation (3).        envisoned before embarking on the               They are also highly susceptible to
Over the years, many thousands of          path of this expression system.                 baculovirus infection, typically
genes have been successfully cloned           An absence of complex sugars in              resulting in high-titer virus stock
and expressed in insect cells.             BEVS-produced proteins raises                   around 108 plaque forming units
    With the BEVS system, proteins         questions relating to their biological          (pfu) per mL and accordingly high
can be produced in weeks rather            activity. But the presence of                   protein production levels. Figure 2
than in months or years because the        relatively simple glycosylation can be          shows the various stages of infection
virus (rather than the cell line itself)   an advantage for some proteins and              in the serum-free cell line. The cells
is genetically modified to infect          a disadvantage for others.                      increase about fourfold in size
insect cells.                                 Protein Sciences Corporation                 during infection, increasing biomass
    The baculovirus genome is              (PSC) has developed technology for              and protein production.
relatively small (about 160 kb), and       large-scale (600 L) production of                  Signal Sequence: Many
it can be easily characterized using       proteins in insect cells using the              heterologous proteins are secreted
genome digesting and Southern              BEVS. The company has tested                    into the growth medium. We
blotting techniques. After the virus       recombinant protein vaccines for                identified a baculovirus signal
infects an insect cell, the cell is        AIDS, malaria, cancer, and for viral            sequence (derived from a 61-kDa
transformed into a baculovirus             influenza in phase I and II human               membrane-associated glycoprotein),
protein production facility before it      clinical trials, establishing a                 which increased secretion and
dies. Heterologous proteins that           regulatory track record.                        expression of heterologous proteins
would be lethal to the cells could be                                                      (5, 6). The SDS-PAGE gel in Figure
produced during the short                  TECHNOLOGY DEVELOPMENT                          3 compares vascular endothelial
remainder of their lifespan. The           PSC developed immortal insect cell              growth factor (VEGF) expressed in
protein of interest is usually made        lines from the ovaries of Spodoptera            insect cells using its natural signal
under control of the polyhedrin            frugiperda, referred to as Sf 21 and            sequence with the 61-kDa–derived
promoter, which is one of the              Sf 9 cells, and a serum-free cell               signal sequence. Analyzing the gel
strongest known promoters. Insect-         culture line with defined growth                by densitometry suggested that
cell–produced proteins are generally       characteristics (4). The serum-free             expression levels obtained with the
bioactive because of the cells’ ability    cell line was selected to grow at               baculovirus-derived signal sequence
to perform complicated                     high densities in suspension without            are about tenfold higher than with
posttranslational modifications.           clumping. Cell doubling time varies             the natural signal sequence. The
    To date no approved BEVS-made          from 18 to 24 hours, depending on               right panel shows an immunoblot
product is on the market for human         the selected cultivation system                 demonstrating the correct identity
vaccine or therapeutic use, even           (spinner and shaker flasks,                     of the protein.
though a number of products are in         bioreactors of varying size). Cells               Protease-Deficient Baculovirus
advanced clinical trials. For example,     can be passaged more than 50 times              Vector: Some heterologous proteins
Dendreon Corporation (www.                 while maintaining a high viability.             are prone to protease degradation. is conducting phase
III clinical studies of its prostate
cancer vaccine candidate produced          Figure 2: Infection of SF+ cells at MOI = 0.2 (as measured in a standard 1-hour plaque
                                           assay). The cells can be synchronously infected, and by 27 hours postinfection, all show
by insect cells. GlaxoSmithKline
                                           typical morphological changes. Late in infection (75 hours), the cells have enlarged to more
( is performing clinical       than twice normal size, but the majority of them remain intact.
studies with a human papilloma virus
vaccine candidate, and Protein
Sciences Corporation plans to initiate
a phase III trial for its influenza
vaccine candidate in the fall of 2004.
    A major challenge is presented by
the fairly complex patent landscape
surrounding the expression system.
Over 5000 patents refer to the use
of insect cells or baculovirus,
varying among multiple cell lines,
signal sequences, promoters, dual-
promoter systems, and the
production of specific types of
proteins. It is therefore
recommended to conduct a directed
patent study toward any application

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Figure 3: Expression of VEGF showing advantages of the PSC signal sequence. VEGF was
expressed either with its natural signal sequence or with the 61-kDa signal sequence. Left   high–cell-density technology might
panel shows a Coomassie-stained SDS-PAGE gel; right panel shows a Western blot using         offer a future solution, but it is not
VEGF-specific antibody for confirmation of identity.                                         yet used in our production facility.
                                                                                             The serum-free insect cell line
                                                                                             produces high-titer virus stocks,
                                                                                             thereby reducing the amount of virus
                                                                                             that needs to be added to the
                                                                                             bioreactor to about 1% of the total
                                                                                             reactor volume. Currently the process
                                                                                             is performed at a 600-L scale, and no
                                                                                             major hurdles are envisioned when
                                                                                             scaling the process 10 to 50-fold.

                                                                                             CLINICAL AND
                                                                                             REGULATORY HISTORY
                                                                                             We have tested a number of
                                                                                             recombinant proteins made in insect
                                                                                             cells as vaccine candidates for the
                                                                                             prevention of various diseases,
                                                                                             including AIDS, malaria, colon
                                                                                             cancer, and influenza. To date more
                                                                                             than 50,000 doses of protein have
                                                                                             been administered in more than 5000
                                                                                             subjects and patients without
Early during the BEVS infection                 oxygen, nutrient supplies, and               reportable adverse effects. The data
process, more protein may be                    reduced shear forces necessary for           suggest that BEVS is a safe
degraded than is being produced. A              high-density cell propagation. These         manufacturing system for protein
pragmatic approach to overcome this             include continuous circulation of            production. Our drug master file
degradation would be to harvest the             cells from the bioreactor, through a         contains general cell line, process, and
protein of interest early in the                semipermeable hollow-fiber filter,           company information derived from
infection process — or to add                   then back to the bioreactor in a             six previously filed INDs that can be
protease inhibitors. PSC created a              manner analogous to the circulation          cross referenced by its collaborators.
baculovirus backbone that lacks the             of blood through the kidneys (with
cathepsin gene and has an incomplete            inline oxygenation as in the lungs).         ONE APPLICATION
chitinase gene. Figure 4 illustrates its           Upstream Processing: The                  In 1997, health officials in Hong
use for the production of certain               clarification step needs to remove           Kong were alarmed by the death of a
proteins. However, it does not result           DNA, lipids, and other                       child following infection with a
in better production levels in all cases.       contaminating agents from the cell           highly pathogenic avian H5N1
   High Cell Density: Insect cells are          culture medium. Addition of calcium          influenza strain. The virus had
susceptible to infection while they are         chloride to a concentration of 10            previously caused the death of
in their logarithmic growth phase,              mM and Tris base to a concentration          70–100% of the chickens in infected
which limits the potential for                  of 28 mM at pH 8.0 allows the                flocks there. Before year’s end, six of
generating high concentrations of               formation of a calcium gel that binds        18 infected people died of the disease
biomass before producing the                    DNA and can be separated easily              (7). Fortunately, the efficiency of
protein of interest. Under normal               from the supernatant by                      virus transmission between humans
cultivation conditions, insect cells can        centrifugation. A drawback to this           was low, but the need for better
be successfully infected at a density of        method is that certain calcium-              vaccines became obvious (8).
2–3 million cells/mL. Our                       dependent proteases may be                   Particularly alarming was that the
fermentation conditions use a dialysis          activated during the process, which          usual egg-based influenza vaccine
fermentation set-up under which                 can lead to degradation of the               manufacturing process cannot
cells can be infected at higher cell            protein of interest and a reduction of       produce a vaccine for this kind of
densities (15–20 million cells/mL).             the overall process yield. Adding            virus because the chicken embryos
   The dialysis set-up is based on              chelating agents after centrifugation        used for vaccine production are killed
waste products being removed from               may reduce this effect.                      by this highly pathogenic virus.
the media while fresh ingredients                   Large-Scale Production Capacity:            The insect-cell–based production
are added without the cumbersome                Producing proteins in insect cells at        technology offered a solution. The
process of identifying critical                 larger scales requires a high-titer          cDNA encoding the hemagglutinin
components. It provides favorable               working virus bank and an efficient          (HA) gene from the avian H5N1
                                                insect cell infection process. The
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                                                     7 Claas ECJ; et al. Human Influenza A
                                                                                               Figure 4: Protease-deficient baculovirus.
strain was used to produce a subunit             H5N1 Virus Related to a Highly Pathogenic
                                                                                               Protein X was cloned in a baculovirus
vaccine. Within six weeks, PSC made              Avian Influenza Virus. Lancet 1998, 351:
                                                                                               backbone that lacks the cathepsin gene and
HA antigen for testing in chickens.                                                            part of the chitinase gene found in the wild
                                                     8 Belshe, R.B. Influenza as a Zoonosis:   type baculovirus. As shown in this Western
Tests confirmed immunogenicity —                 How Likely is a Pandemic? Lancet 1998,        blot, more intracellular full-length protein
and more important, protection                   351: 460–461.                                 can be detected when using the protease-
from a lethal viral challenge — in                   9 Crawford, J; et al. Baculovirus-        deficient backbone.
chickens (9). Four weeks thereafter,             Derived rHA Vaccines Protect Against Lethal
1700 doses were delivered to NIH                 Influenza Infections by Avian H5 and H7
for testing in humans. The company               Subtypes. Vaccine 1999, 17: 2265–2274.
received FDA approval for                           10 Treanor; et al. Safety and
                                                 Immunogenicity of rHA H5 Vaccine in
compassionate use. A clinical study              Humans. Vaccine 2001, 19: 1732–1737.
was conducted using the material
produced. And the studies suggested
that the BEVS-expressed H5 HA                    Manon M.J. Cox is chief operating
induced functional antibodies in                 officer at Protein Sciences Corporation,
individuals who had no prior                     Meriden, CT 06450. Previously she was
exposure to the H5 viruses (10).                 with Gist-brocades (now part of DSM),
    The BEVS technology can be a                 a Dutch company specializing in
powerful manufacturing technology                fermentation, where she held various
to provide health care solutions to              management positions, most recently in
pandemic, biodefense, and emergency              new business development, and before
scenarios. We are exploring the                  that in research and development (R&D)
potential of this production                     and production. Before joining Gist-
technology for rapid development of              brocades, she was a molecular biologist       Rochester, NY, and a PhD in molecular
a SARS subunit vaccine. A                        on the development of a PCR screening         biology from the University of
recombinant trivalent hemagglutinin              test for cervical cancer at the University    Nijmegen, The Netherlands. Parts of this
influenza vaccine is being tested in a           of Amsterdam. She received her MBA            article were presented at the
large US phase II(b) clinical study in           with distinction from the University of       BioPharmos 2003 meeting, which was
the elderly. For those exploring the             Nijenrode and the University of               held 26–28 March 2003 in Monte
BEVS technology as a commercial                                                                Carlo, France.
large scale production system, the
company offers process development                FOR FURTHER READING
                                                  Ikonomou, L; Schneider YJ; Agathos           engineering issues related to high-
REFERENCES                                        SN. Insect Cell Culture for Industrial       density culture (new bioreactor types,
    1 Tinsley and Harrap. Viruses of              Production of Recombinant Proteins.          gas exchange, feeding strategies) are
Invertebrates. In Comprehensive Virology 12,      Appl. Microbiol. Biotechnol. 2003,           addressed in view of their relevance to
Fraenkel-Conrat H, Wagner RR, Eds.                62(1): 1–20.                                 large-scale culture.
(Plenum Press, New York, NY, 1977); pp.
1–101.                                            From the Abstract: Our current               SEE ALSO:
    2 Smith, GE; et al., Production of            knowledge of insect cell metabolism is       Griffiths CM; Page MJ. Production of
Human Beta Interferon in Insect Cells             summarized, and emphasis is placed           Heterologous Proteins Using the
Infected with a Baculovirus Expression            on elements useful in the rational           Baculovirus/Insect Expression System.
Vector. Mol. and Cell. Biol. 1983, 3:
                                                  design of serum-free media. The culture      Methods Mol. Biol. 1997, 75:
    3 Miller. A Virus Vector for Genetic
                                                  of insect cells in the absence of serum      427–440.
Engineering in Invertebrates. In Genetic          is reaching maturity, and promising
                                                                                               Wang, M; Cox, M. Baculovirus Protein
Engineering in the Plant Sciences, Panopaulus     serum substitutes (hydrolysates, new
Ed. (Praeger: New York, NY, 1981); pp.
                                                                                               Expression Vector System Tutorial:
                                                  growth and production-enhancing
203–222                                                                                        Optimized Sf+ Cell Line, Meda,
                                                  factors) are being evaluated. Proteolysis
    4 Smith, GE; et al. Spodoptera                                                             Baculovirus-Derived Signal Peptide,
                                                  is a problem of the BEVS system due to
Frugiperda Single Cell Suspension Cell Line in                                                 and Promoter Efficiently Provide High
Serum-Free Media, Methods of Producing and
                                                  its lytic nature, and can, therefore, be a
                                                                                               Yields. Gen. Eng. News 23(20), 15
Using. US Patent No. 6,103,526; 8 October         critical issue in insect cell
                                                                                               November 2003.
1998.                                             bioprocessing. Several cell- or
    5 Smith, GE; et al. Method for Producing      baculovirus proteases are involved in        Wilkinson, BE; Cox, M. BioProcess
Influenza Hemagglutinin Multivalent               degradation events during protein            Tutorial: Baculovirus Expression System
Vaccines Using Baculovirus. USP 5,762,939;        production by insect cells. Methods for      — The Production of Proteins for
13 September 1993.
                                                  control, the optimal inhibitors and          Diagnostic, Human Therapeutic, or
    6 Smith, GE; et al. Method for Producing
Influenza Hemagglutinin Multivalent
                                                  culture and storage conditions which         Vaccine Use. Gen. Eng. News 18(19),
Vaccines. USP 6,245,532; 9 October 1998.          affect proteolysis are discussed. Finally,   1 November 1998.

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