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TABLE 9.3
Recombinant Manufacturing System
Industrial Attributes of Expression Systems for Commercial Manufacturing (Fewer Stars
Are Desirable)
Long
High
Cycle
Low
Platform
Transgenic plants **** ** * *** * * *** *
Plant cell ** ** ** *** * * **** **
Plant virus *** *** * ** * ** **** *
Microalgae *** * * *** * * **** **
Yeast ** ** * *** * ** *** **
Bacteria *** *** * *** ** *** ** *
Mammalian cell * * **** * ** * * *
Transgenic animals * * *** ** * * * *
Insect cell ** ** * ** * ** *** **
Filamentous fungi *** * * *** * ** *** ***
9.4 YEAST
Cost
Time
Capacity
Difficult
Propagation Low Yield
Low
Quality
Contamination
Risk
High
Purification
Cost
S. cerevisiae and Pichia pastoris are two yeast species that are increasingly used as an expression
system (Table 9.3). They have high efciency (short doubling time, high cell density, high yield
owing to better mass transfer of nutrients in unicellular growth morphology), and low fermentation costs. In 1991, the Food and Drug Administration (FDA) authorized Novo’s human insulin
as the rst product made from the yeast strain S. cerevisiae. This yeast has since been utilized
to express and synthesize more than 40 distinct recombinant proteins such as insulin peptides,
human serum albumin, and hepatitis vaccines. The full genome sequence of S. cerevisiae is
known, and the FDA has designated it as “Generally Recognized As Safe.” Unlike bacteria, yeast
cells can express or secrete the protein directly into the medium, and the expressed protein can
undergo PTMs.
Yeast cells have the characteristic rigid cell wall. Because yeast cells secrete the expressed protein into the culture medium, unlike bacterial cells, the possibility of host- cell- related impurities is
less with yeast cells but more predominant in bacterial cells. The purication process with the yeast
expression system is also relatively simple compared with that of the bacterial system (owing to
product secretion) and eliminating the need for additional processing such as the recovery of inclusion bodies, their solubilization, and refolding to allow for native protein folding. Despite these
advantages, one of the disadvantages with yeast systems is the lack of PTMs and unexpected mono-
and di- glycosylated forms of the target proteins that may be difcult to remove.
While S. cerevisiae is the most commonly used yeast, Schizosaccharomyces pombe,
Kluyveromyces lactis, and Yarrowia lipolytica have been used. The main reason for the popularity
of using S. cerevisiae is attributed to its high cell densities and fast- growing characteristic that can
be easily achieved in the fermenters without affecting the costs and production timelines. Table 9.3
details all the recombinant proteins that have been produced using S. cerevisiae.
Another yeast strain commonly used for biomanufacturing is Pichia pastoris. Like S. cerevisiae,
the methylotrophic Pichia pastoris can grow to very high cell densities with a tightly regulated
expression system. Like S. cerevisiae, the Pichia pastoris strain has the ability to either secrete the
recombinant protein into the culture medium or express it intracellularly. Pichia, a methylotrophic
microorganism, metabolizes methanol as its primary energy source (carbon), and this characteristic is used in preparation of the expression system. The alcohol oxidase gene 1 (AOX1) promoter
is used for cloning the gene of interest. Methanol is fed during the fermentation process to induce
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protein expression. Additional protective measures must be followed when handling large volumes
of methanol, as it is a volatile solvent. Some of the recombinant proteins produced in Pichia pastoris
include human serum albumin, human insulin, kallikrein inhibitor protein, interferon- alpha 2b, and
microplasmin heparin- binding epidermal growth factor– like growth factor, to name a few. The
N- linked glycosylation pattern for this system is different from that in higher eukaryotes, and this
observation can be generalized for the other yeast strains. One of the major disadvantages with
P. pastoris is proteolytic degradation, which is a major issue particularly with high- cell density
cultures. This results in loss of yield and biological activity. Various strategies, including the addition of protease inhibitors (e.g., phenylmethylsulfonyl uoride, ethylenediaminetetraacetic acid,
benzamidine, and other protease inhibitor cocktails), yeast peptone; optimization of the fermentation culture conditions (e.g., pH); use of certain specic components in the culture medium, feed, or
supplements (e.g., casamino acid); use of other alternative carbon sources; and optimization of the
induction strategy, have been proposed and are being evaluated to overcome this challenge.
Yeast is considered a perfect choice for large- scale production of recombinant proteins that can
mostly be produced in highly complex eukaryotes owing to its highly evolved, yet well- dened,
genetic system, high productivity, rapid growth, ease of scale- up, ability to support certain PTMs,
and decreased manufacturing time and cost. However, management of proteases present in the yeast
is challenging, as these enzymes can degrade the recombinant protein.
The expression vector is the core of the yeast production system. Vectors that can integrate into
and establish themselves in the host cell because of their mitotic stability are commonly used for
expression studies. An alternative to genomic integration is episomal vectors (extrachromosomal
DNA, which can replicate autonomously in the host cell) for some yeast systems. Expression vectors
comprise a yeast promoter/ terminator and a selectable marker cassette, and they enable cloning a
gene insert downstream of a secretion leader. This ability of expression vectors allows the secretion
of heterologous proteins from the cells into the medium. The three most common vectors used for
protein expression in Saccharomyces cerevisiae are
• Yeast integrating plasmid (YIp): This is a single- copy plasmid and can be integrated into the
host genome.
• Yeast centromeric plasmid (YCp): This is a replicating single- copy plasmid and is used for
complementation studies.
• Yeast episomal plasmid (Yep): This is a replicating multicopy plasmid and is used for recombinant protein production.
Figure 9.4 shows the creation of a target protein expression system using S. cerevisiae.
A typical process of creating a target protein expression system using S. cerevisiae starts with
identifying the gene of interest that encodes the protein of interest. The gene of interest is then
used to generate the cDNA cloned into a bacterial system (competent E. coli cells). Positive clones
harboring the modied plasmid are identied using a selection marker, after which the clones are
isolated, transformed into yeast cells for integration of the plasmid into the yeast chromosome,
and screened for positive transformants. Finally, a high expressing clone is selected and scaled up
to create the cell line to further produce the protein of interest. The commonly used methods for
transformation into yeast are spheroplast transformation, whole- cell transformation using lithium
acetate, and electroporation. A combination of lithium acetate and polyethylene glycol is also a
widely used transformation method, mainly for single- stranded DNA. Typically, the transformants
are selected by applying a selectable marker so that they can be detected while screening for positive
clones (Figure 9.4).
• Using dominant selection markers such as resistance to G418, formaldehyde, cupric ions, or
cycloheximide.
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FIGURE 9.4 Yeast protein expression system: Saccharomyces cerevisiae
• By complementation, wherein the plasmid contains the functional gene that is not present
(mutated) in the host strain (e.g., trp1, his3, and ura3).
There is an increase in the number of engineered yeast strains targeted toward either increasing
the yield, altering the composition of N- glycans, and improving the performance of afnity tags.
Despite the several advantages of yeast cells, including quick growth, simple medium
requirements, and PTMs, one of the major drawbacks is the possibility of hyperglycosylation, which
can signicantly affect immunogenicity, as previously discussed; given that the protein is expressed
and secreted into the culture medium, the chances of protein degradation remains a major concern.
Additionally, the low redox potential of the culture medium can also often lead to disulde bond
cleavage.
Overexpression of the recombinant protein, which leads to intracellular accumulation and low
product yield, is another common problem with yeast systems, often leading to increased cellular
stress. Several novel systems are being developed to facilitate easy expression of recombinant
proteins in yeast cells. For improved processing of recombinant biopharmaceuticals, CRISPR/ Cas9
has been successfully used in yeast engineering to insert a site- specic gene or knockout specic
unwanted genes.
9.5 MAMMALIAN CELLS
The impact of cell culture on mankind and the progress achieved in biology have been enormous.
The advancements made in biopharmaceuticals, for example, vaccines, new drug entities, or modalities such as antibody- drug conjugates, bispecic antibodies, cell therapy, and gene therapy, have
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depended mainly on cell culture. There has been an increasing shift toward the use of mammalian
systems (human and nonhuman [animal cell lines]) for production purposes, specically for complex proteins that require PTMs. All cell types vary in their growth prole, optimum conditions, and
nutritional requirements.
A diverse variety of nutrients are required for the dynamic metabolism of animal cells. Hence,
fetal bovine serum, a byproduct of the dairy and cattle industries, is now widely used in cell culture
media. Recent concerns about transmissible spongiform encephalopathies (TSEs) and blood- borne
infections, as well as severe regulatory requirements for TSEs and bovine spongiform encephalopathy (BSEs), have led to a slew of serum- free, animal- product– free, and even protein- free media
formulations. Recently, recombinant insulin, transferrin, and bovine serum albumin (produced by
bacterial fermentation), as well as human serum albumin produced by yeast fermentation, have
replaced animal origin serum in a variety of modern cell culture processes without signicantly
affecting process performance or product quality.
Mammalian cells are extremely sensitive to culture conditions, and they frequently overreact to even small changes in temperature, pH, aeration, and agitation speed. During the log
phase, animal cells proliferate more slowly than bacterial cells, within a doubling time of
15– 48 hours.
Animal cells are naturally more delicate than microbial cells, as their cell wall lacks strength.
Therefore, they are unable to tolerate most of the fermentation conditions that microbial cells tolerate, necessitating the use of specially built fermenters. For a culture- friendly approach of animal
cells, several newer “airlift” and “fermenter” designs have pumps as a substitute for impellers.
During agitation, there is a considerable risk that traditional impellers break fragile cells
suspended in the culture medium. Although impellers with round blades (such as the three- blade
impeller used to drive motorboats) are used for generating reduced shear stresses, the sheer force
with which they come in contact with the cells can be equally harmful.
Forced air sparging or other methods of introducing air into the mixture are used in bioreactors
for the growth and culture of animal cells. In suspension culture, several animal cell lines multiply by
oating around in their liquid media, whereas others require a solid substrate to adhere to the inner
walls of roller bottles, gas- permeable polymer tubes in “hollow- ber” bioreactors, or microcarrier
beads or at disks attached to plastic microcarrier beads or at disks.
For animal cell culture operations, forced air sparging is one way of infusing air into the bioreactor culture medium. A robust substrate bonded to the inner walls of roller bottles, gas- permeable
polymer tubes in “hollow- ber” bioreactors, or plastic microcarrier beads or at disks is required for
culturing some animal cells (anchorage- dependent). Certain animal cell lines multiply by oating in
their liquid medium as a suspension.
Traditionally, anchorage- dependent cells (mainly primary cell lines) have shown high levels of
expression. To achieve high expression levels, streamlined cell lines such as NS0, CHO, human
cervix (HeLa), and HEK- 293 are successfully used in suspension culture. Suspension cell lines, on
the contrary, have proven to be more useful and advantageous, especially for greater volume protein
production procedures.
It is crucial to be aware that some bioreactor cultivation conditions can cause apoptosis (cell
death), and such culture conditions can collect undesired products and cell debris, which will slow
down the downstream processes.
9.5.1 nonhuMan cell lines
9.5.1.1 Chinese Hamster Ovary Cells
Monoclonal antibodies and complex eukaryotic proteins that have undergoing PTMs are commonly produced in mammalian cells such as CHO cells, HeLa cells, African green monkey kidney
cells (COS), BHK cells, and hybridomas. These mammalian cells have been used to produce
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biopharmaceuticals such as monoclonal antibodies and other sophisticated eukaryotic proteins that
have undergone PTMs.
In most cases, the target protein is produced in its original form and secreted directly into the
culture medium. Establishing a small- scale process capable of producing proteins ranging from a
few milligrams per liter to grams per liter may take approximately 4– 5 months, commencing with
gene assembly, clone generation, and selection. Because the protein is expressed in the medium,
the downstream processing is straightforward. The downstream procedures should be robust and
capable of eliminating or minimizing HCPs, cell debris, nucleic acid, and other components that
are commonly produced during the fermentation process. As the possibility of viral contamination
is substantial, steps involving viral inactivation and removal must be included during downstream
processing. To verify viral clearance, extensive control measures (e.g., end- of- production testing,
validation of the presence of virus) must be applied. After the N- 1 or nal purication stage, it is
usual practice to incorporate a viral inactivation step by decreasing pH of the medium and a viral
elimination step by ltration.
Animal cells are more delicate than microbial cells and grow very slowly, which makes them
particularly vulnerable to shear forces; batch or fed- batch cultures are commonly used for antibody
synthesis, while other recombinant proteins can be produced in cultures for 4– 8 weeks constantly.
Animal cells have slower growth and more complications concerning their growth characteristics
than microbial cells. In addition, the fragile nature of these animal cells makes them highly vulnerable to shear forces. The most operated fermentation modes are batch or fed- batch cultures, but
recent advancements have made continuous culture and perfusion equally successful, particularly
for high yields.
The type of nutritional requirements from the culture media makes it extremely expensive relative to those used for microbial and yeast protein expression. This, combined with the low expression levels, makes the overall production process prohibitively costly. However, given that complex
proteins cannot be expressed in microbial or yeast cells, mammalian cells are preferred over transgenic plants or animals.
In cell genetics research, T. Puck created the rst CHO cell line in 1957. Activase, a tissueplasminogen activator created by Genentech, was originally manufactured using CHO cells in
1987. Ever since the CHO cell line has dominated the biopharmaceutical industry as the preferred
system for recombinant protein manufacturing, all the currently approved recombinant products are
produced using CHO cell lines. The initial CHO cell line has been subcloned many times, resulting
in a plethora of variants. The most commonly used cell lines in biomanufacturing are CHO- K1,
CHO DG44, and CHO- S cells, which are derived from a different CHO lineage (Figure 9.5). This
gure shows an illustration from several published papers documenting the history and evolution of
the CHO cell lines widely used today.
CHO- K1 was the rst cell line produced from a single clone of the original CHO cells (ovary
tissue isolate). Later, these cells were modied for industrial use in both suspension and serumfree media. Several expression systems have been developed using this customized CHO- K1 cell
line. They are utilized in association with the glutamine synthetase (GS) selection strategy, wherein
the gene of interest is introduced into the host cell alongside a copy of the GS gene, which allows
stable cells to be selected solely in glutamine- free media. Furthermore, the CHO- K1 cell line was
mutagenized using ethyl methanesulfonate to form the CHO- DXB11 (also known as CHO- DUKX)
cell line, which is decient in dihydrofolate reductase (DHFR) activity.
The original CHO cells were mutagenized by gamma radiation, which resulted in the loss of both
DHFR alleles and the emergence of the DHFR- decient CHO- DG44 cell line. The de novo synthesis of purine, thymidine, and other amino acids, as well as the proliferation of CHO cells, requires
DHFR. Therefore, CHO- DG44 cells must grow on a medium containing glycine, hypoxanthine,
and thymidine (GHT). This feature is used in the same manner as that of the GS selection system
when screening for transformants (selection marker) in a thymidine or GHT- decient medium. If
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FIGURE 9.5 CHO cell lineage
173
the transformed clones (recombinant plasmid DNA expressing the gene of interest and the replacement DHFR gene) have effectively taken up the recombinant plasmid DNA, then they will grow in
a GHT- decient medium.
Methotrexate (MTX) as a selection pressure for screening transformed DHFR- decient cells
encoding the gene of interest is another approach extensively used for screening transformants.
CHO cells lacking DHFR will amplify a transgene when transfected with MTX- treated cells. MTX
binds to the catalytic site of the DHFR enzyme, preventing dihydrofolate from being converted to
the active form tetrahydrofolate, which is required for DNA synthesis. Selection pressure is induced
by increasing MTX concentration of in the culture media of the transformed DHFR- decient CHO
cells (encoding the gene of interest, DHFR, and vector). This causes both DHFR and the gene of
interest to be amplied in the genome, allowing cells to produce a higher amount of recombinant
protein. However, MTX- mediated gene amplication might cause clone instability resulting from
chromosomal abnormalities, which is an important concern in biomanufacturing.
CHO- S is a subset of CHO cells that can grow as suspensions without the need for anchoring or
an adhering surface (originating from the CHO pro- 5 cell line). The ability of CHO cells to grow
in suspension was a game- changer, paving the way for large- scale recombinant synthesis in stirred
tank reactors. Commercially available CHO- S cells with cGMP storage are available.
The diversity of CHO cells dictates different growth conditions, selection pressure, and media
used for protein expression to achieve functional attributes, including PTMs and high cell density,
and specic productivity from the process. Of particular interest with CHO processes is the glycan
distribution, given that variations in glycan patterns can affect product quality, functionality, and
possibly even safety. To a large extent, PTMs are a combination of both the choice of cell line and
the process conditions, including media, feed, supplements, temperature, pH, and dissolved oxygen.
Therefore, optimizing these parameters is key to producing the desired product that meets its critical
quality attributes.
CHO cells are commonly used for expressing recombinant proteins and generating a stable cell
line. A standard approach for this recombinant protein expression using CHO cells lacking the
DHFR enzyme. The target gene and the DHFR gene are both cloned into a single mammalian
expression system. The recombinant DNA plasmid (carrying the two genes) is then transfected into
the host cell (DHFR- decient CHO cells). The DHFR gene serves as a selection marker that allows
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only transfected cells to grow in a thymidine- free medium. Next, using a limiting dilution method,
single- cell stable clone(s) are obtained. The clones are then evaluated further for their expression
levels, product quality, and stability to generate the nal cell line expressing the protein of interest.
9.5.1.2 CHO Expression System
Other mammalian nonhuman cell lines utilized for large- scale pharmaceutical processing include
mouse myeloma cell lines NS0 and Sp2/ 0, BHK cells, Vero cells from the African green monkey,
and Madin- Darby canine kidney (MDCK) cells from a dog. Vero and MDCK cells were utilized
primarily for vaccine research, while the others were employed for the production of recombinant
proteins.
9.5.1.3 Myeloma Cells
Myeloma cells have been used in hybridoma technology since the 1970s and have become a workhorse in antibody development, and only very recently, they have been overtaken by CHO cells
for recombinant protein expression. Some of the advantages of myeloma cells include growing in
suspension culture, using a serum- free medium, and being easily scalable. However, given that myeloma cells have the ability to produce nonhuman glycoforms that cause immunogenicity and adverse
effects, they are viewed as a potential risk. Endogenous retroviruses are abundant in mouse cells,
which make viral clearance very burdensome. The most commonly used myeloma cell lines are NS0
and Sp2/ 0, which have been used successfully for manufacturing biopharmaceuticals. For example,
Sp2/ 0 cell line was used for manufacturing Remicade (iniximab) and Erbitux (cetuximab), and
NS0 was used for Zenapax (daclizumab) and Soliris (eculizumab) in addition to other blockbuster
products. NS0 cells have a decient endogenic expression of GS and require exogenous cholesterol added to the medium for growth and protein production. The risks of using animal- derived
components and the price of synthetic cholesterol have led to the development of cholesterolindependent NS0 clones. The lack of a fully functional GS is used as a selection pressure to identify
transfectants.
To effectively use myeloma cells, it is essential to recognize clones that produce low levels of
these nonhuman glycan structures.
9.5.1.4 Human Cells
The rst human cell line, HeLa, was established in 1951. Human diploid cells were used for vaccine
manufacture in the 1960s, but concerns regarding oncogenic viruses at that time discouraged the
widespread use of human cells. Instead, nonhuman cells, namely CHO and mouse myeloma cell
lines, have been successfully used to manufacture approved recombinant therapeutics. As for
human cell lines, HEK293, brosarcoma HT- 1080 cell line, and Namalwa lymphoma cells have
been used for approved products. More recently, Per.C6 and CAP are being used for products under
development.
HEK293 cell line was established in 1977, and since then, it has been a versatile system for
manufacturing recombinant therapeutics, viral vectors (adenovirus, retrovirus, and lentivirus), and
other proteins for research use. HEK cells can be used as a suspension culture in a serum- free
medium. Four recombinant products (Xigris, Alprolix, Eloctate, and Nuwiq) that are produced in
HEK cells have been approved.
Per.C6 was established in 1998 from nontumorigenic human embryonic retinoblastoma cells and
has been used for adenovirus vector and recombinant protein production. This cell line can also be
grown in suspension culture in a serum- free medium and can reach high cell densities.
The low productivity and low process efciency are commonly encountered challenges with
mammalian cells during recombinant protein production. Both stable and transient expression
processes are being evaluated to improve productivity and ease of scalability.
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9.6 ALGAE
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Green microalgae, such as Chlamydomonas reinhardtii, have been used to produce products such
as full- length human antibodies, signaling molecules such as vascular endothelial growth factor,
and structural proteins such as bronectin, particularly those with strong disulde bonds. Algae
chloroplasts have the same ability to express genes as that of other eukaryotic species such as yeast.
While the algal nuclear genome can also be transformed owing to reduced gene silencing and higher
protein accumulation, the chloroplast genome has been the source in most cases of transgene expression to date.
More than 100 antibodies, subunit vaccines, immunotoxins, subunit oral vaccines, and growth
factors are produced using the algae platform.
While lamentous fungi are widely used to produce enzymes, their effective utilization in the
production of recombinant therapeutic proteins is not widely made because of the low yield and
variability in the protein produced or morphological defects introduced. The common fungi used for
manufacturing recombinant proteins include Aspergillus nidulans, Aspergillus niger, Neurospora
crassa, and Trichoderma reesei.
9.7 INSECT CELLS
In the 1990s, the insect cell baculovirus expression system was created as an alternative to the mammalian expression system. The baculovirus system has quickly gained acceptance, given its potential to convert the lepidopteran insect cells into a high- level expression system. Additionally, these
systems have better glycosylation abilities than yeast and bacterial expression systems. The most
common mechanism of expression for insect cells is transient.
To create expression systems, an insect host cell and a virus vector, such as baculovirus Autographa
californica multiple nuclear polyhedrosis virus, known as AcMNPV, are required. Baculovirus is a
lytic, double- stranded DNA virus frequently amplied in Lepidoptera insects.
It is noninfectious in vertebrates and has inactive promoters in mammalian cells, which makes
it safe for use in humans. Spodoptera frugiperda (Sf9 and Sf21) and Trichoplusia ni are the most
commonly used insect host cell lines. For academic and industrial research and development,
MultiBac, a sophisticated baculovirus/ insect cell system, has been designed and used to generate
multiprotein complexes containing hitherto inaccessible components. Since its launch, many
MultiBac vector derivatives have been produced to express proteins with cleavable N- terminal
signal peptides.
The FDA approved GSK’s groundbreaking insect cell product Cervarix, a human papilloma virus
vaccine, in 2009.
The recombinant DNA plasmid expression (encoding the gene of interest) is co- transfected with
a second plasmid (containing viral genes required for the formation and multiplication of viral
particles) into the host insect cells. The gene of interest is inserted behind a strong promoter, and
transduction of the baculovirus genome into the insect cell leads to the expression of both the viral
gene and the gene of interest. The recombinant viral stock is then puried and amplied. The host
cells are grown to a specic cell density before adding the virus stock at a predetermined time point
(time of infection) with a given number of viruses per cell. The protein production is carried out until
cell lysis occurs, which is typically 48 hours post- infection. Insect cells can be grown in cell suspension in both serum- free and protein- free media as either single cells or as clumps. The optimum
temperature suitable for growth is approximately 27°C– 28°C, but insect cells are more susceptible
to shear stress than mammalian cells. Insect cells are ideal for the production of both cytoplasmic
and secreted proteins.
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9.7.1 insect cell exPRession systeMs: sf9 and sf21
Recombinant Manufacturing System
The entire production cycle is faster in insect cells than in mammalian cells, especially with regard
to the generation of recombinant virus and ending with the purication of the protein of interest.
This cycle takes nearly 4 weeks to complete. Fermentation is carried out only in single or semicontinuous batches owing to the vulnerability of the cells to shear strain. During the fermentation
cycle, the insect cell counts increase by nearly 50- fold within approximately a week but only in
single or semi- continuous batches owing to their sensitivity to shear forces.
Culture media expenses range from moderate (serum- free media) to expensive (bacteria and
yeast media). Because the cells can be cultured in a healthy state before infection, the technique
is well adapted for producing hazardous cell products. Despite this, scaling up is problematic
because of the need for special aeration and the type of plasmid infection required for a high
expression level.
Baculoviruses do not infect vertebrates and hence represent no health danger, while the risk
of adventitious viruses remains unknown, necessitating virus inactivation and active ltration.
Recurrent death and the subsequent lysis of the host cell causes the release of intracellular proteins
and nucleic acids into the media, putting downstream purication processes under extreme pressure
(such as for bacterial cells with inclusion bodies). Insect cells have a major regulatory track record,
with no FDA- approved products yet.
Insect cell culture is advantageous in terms of lower cost and easier cultivation than mammalian
cell culture. The levels of heterologous protein expression achieved with this technique are often
varied, ranging from 1 to 600 mg/ L culture media. At a protein expression level of 10– 40 mg/
L, certain collagens are synthesized in insect cells. One issue with insect cells is that they rarely
produce recombinant proteins, as they prefer to keep the synthesized proteins in the cytoplasm.
This feature is attributed to the transfection method used, and it can make downstream processing
more difcult. Baculovirus expression vectors (noninfectious to humans) are commonly employed
as an expression system for heterologous proteins in cultivated insect cells. Production of multisubunit protein complexes, co- expression of protein- modifying enzymes to increase heterologous
protein production, and new baculovirus display technology applications are some of the recent
advancements.
Insect cell– based expression systems have several drawbacks, including nonmammalian
glycosylation patterns and low productivity. Insect cells are unable to digest proteins that are initially produced as larger inactive precursor proteins (e.g., peptide hormones, neuropeptides, growth
factors, and matrix metalloproteases). Several approaches for circumventing this issue have,
however, been studied. One approach is to co- express these enzymes with the gene of interest
in baculoviruses, while another approach is to introduce mammalian glycosyltransferases into
insect cells.
9.8 COMPARATIVE ANALYSIS
Table 9.3 compares the industrial attributes for the commercial manufacturing of recombinant
products, pointing out the negative attributes (therefore, a low rating is preferred).
The type of target protein, PTMs, expression level, intellectual property rights, and manufacturing cost inuence the choice of the expression system. Each of the current expression systems has
its own set of benets and drawbacks (Table 9.4).
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TABLE 9.4
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Comparison of Various Expression Systems, Advantages, and Disadvantages
Host Advantages Disadvantages
Bacteria, e.g.,
Escherichia coli
Bacteria, e.g.,
Staphylococcus aureus
Mammalian cells, e.g.,
Chinese hamster ovary
cells
Yeasts, e.g.,
Saccharomyces
cerevisiae
A well- characterized system
wide choice of cloning vectors
controlled gene expression
quick doubling times
easy to grow with relatively high yields, the
product constitutes up to 50% of total cell
protein
can be tailored for secretion into growth media
allowing for the elimination of undesirable
N- terminal methionine groups
low cost, short production time, and virus- free
There is no post- translational
modication
Biological activity and immunogenicity
may differ from those of the native
proteins
The gram- negative bacteria have a
highendotoxin level, which can
burden the downstream purication
processes for the removal/ reduction
ofthese endotoxin levels to acceptable
limits
The produced protein product may be
harmful to the cells
Inclusion bodies require additional
processing steps to induce the protein
into its native conformation
There is a high chance of proteolysis
of target protein during cell
disruption, and
additionally, the cells carry the risk of
phage infections
Secretes fusion proteins into the growth media Does not express such high levels as
E. coli; pathogenic
Mammalian cells secrete product
(extracellular)into the media, thus eliminating
cell lysis and other extensive steps to recover
the protein
The proteins expressed have the same biologic
activity as that of native proteins
Mammalian cells can perform advanced post-
translational modications that can closely
mimic even human glycosylation patterns
It can be easily scaled up to signicantly large
volumes
Mammalian cells are more susceptible
to virus contamination, thus requiring
additional processing to demonstrate
viral clearance and removal
Cell lines are expensive
Cells exhibit slow growth with longer
doubling times
Fermentation and seed generation are
time- consuming and lengthen the
production time signicantly
(1– 2 months depending on production
scale)
Yeast has been classied under the generally
regarded as safe (GRAS) category
They are suitable for both intracellular
expression and extracellular expression (i.e.,
secretion into the medium)
Yeast cells do not have any detectable levels of
endotoxin or release any
The fermentation process costs are relatively low
Gene expression is more difcult to
regulate
The glycosylated product is not like that
in the human/ mammalian systems
(N- linked glycan structures are
different from mammalian proteins)
This can have a great impact on both
bioactivity and immunogenicity
Yeasts can support both disulde bond formation
and post- translational modications such as
glycosylation
(continued)
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