Optimization of Protein Expression in

Mammalian Cells
Molly Hunter,1 Ping Yuan,1 Divya Vavilala,1 and Mark Fox1,2
1
ATUM, Newark, California
2
Corresponding author: mfox@atum.bio

Recombinant proteins, such as monoclonal antibodies, are produced in mam-

malian cell lines to introduce proper protein folding and post-translational
modifications, which are essential for full biological activity. In both the indus-
trial and academic environments, the use of recombinant proteins varies widely
and, with it, the method of production. The amount of an antibody needed for
a toxicity study is far greater than that needed by a research lab performing
cellular assays, and the amount of effort put into the development of the protein
will vary accordingly. There is no universal strategy for mammalian expression
systems, and scientists often struggle to develop a suitable process from the
myriad of choices at each step. Here, we elaborate on the various obstacles en-
countered when planning high-yield experiments to produce the recombinant
proteins of interest. 
C 2018 by John Wiley & Sons, Inc.

Keywords: expression vector r gene optimization r mammalian cell culture

r protein expression r recombinant protein r stable cell lines r transient
expression

How to cite this article:

Hunter, M., Yuan, P., Vavilala, D., & Fox, M. (2018). Optimization of
protein expression in mammalian cells. Current Protocols in Protein
Science, e77. doi: 10.1002/cpps.77

INTRODUCTION scale (De Jesus & Wurm, 2011). Many of these

Mammalian protein production has become
standard for the manufacture of state-of-the-art
biotherapeutics, as well as becoming routine in
the production of protein samples for research.
Protein production in mammalian cell lines
offers the unique advantage of generating as
close to wild-type environments for transcrip-
tion and translation, coupled with the relevant
chaperone, secretory, and redox environments
and post-translational modifications that lead
to functionally relevant and active proteins.
While it is possible to produce human proteins
in prokaryotic expression systems, the result-
ing proteins often have undesirable character-
istics that impact efficacy, such as a lack of
glycosylation. Currently, prokaryotic systems
cannot match the advantages of mammalian
systems. Much of the technological advance-
ment in mammalian systems has been focused
on industrial processes, especially the produc-
tion of biotherapeutics at the multi-kilogram
developments are also available to scientists.
Despite these advancements, selecting a pro-
cess for recombinant protein expression is not
necessarily simple. Variables such as cell line,
culture media, and culture method all impact
the yield, physical characteristics, and biologic
activity of the proteins expressed, as depicted
in Figure 1. This review focuses on the prac-
tical aspects of developing a strategy for the
rapid, economical production of proteins in
mammalian hosts that will be of use to new
scientists.
CELL LINES AS EXPRESSION
HOSTS
The most commonly used mammalian cell
lines found in research and industrial set-
tings for protein production are Chinese ham-
ster ovary cells (CHO) and human embry-
onic kidney 293 cells (HEK-293) (S. Estes &
Melville, 2014; Hacker et al., 2013; Swiech,

Hunter et al.

Current Protocols in Protein Science e77 1 of 28

Published in Wiley Online Library (wileyonlinelibrary.com).
doi: 10.1002/cpps.77

C 2018 John Wiley & Sons, Inc.
 Figure 1 A summary of the many elements that need to be considered for the development of
a rapid and economical mammalian expression system. Optimization of these factors cannot be
made in isolation; gene optimization, vector selection, type of selection pressure, molecule type
to be expressed, transient or stable expression, cell type, media selection, production scale, and
post-translational modifications all affect the final protein yield and biological activity, and they are
interdependent.

Picanco-Castro, & Covas, 2012). Hundreds
of other cell lines exist, and a considerable
amount of effort has been employed in the en-
gineering of the next generation of high pro-
ducing cell lines, but HEK-293 and CHO are
the mainstays. The CHO cell line was culti-
vated from a biopsy of the ovary of the Chi-
nese hamster. The HEK-293 cell line was cre-
ated when human embryonic kidney cells were
transformed with fragments of adenovirus type
5 DNA, resulting in a cell line that replicated
rapidly, tolerated reduced serum concentra-
tions in the media, and was easily transformed
(Graham, Smiley, Russell, & Nairn, 1977).
Both HEK-293 and CHO cells have since been
genetically modified to improve responsive-
ness to different expression vector elements.
For example, the HEK-293T derivative en-
codes the SV40 Large T antigen, allowing
for episomal replication of plasmids contain-
ing the SV40 origin of replication (DuBridge
et al., 1987; Rio, Clark, & Tjian, 1985). The
CHO-K1 derivative was cultured from a clone
of the CHO line and contains fewer chromo-
somes than the original (Kao & Puck, 1968).
HEK-293 and CHO cells are maintained
as either suspension or adherent cultures, with
suspension culture being particularly favored
Hunter et al. for their ease of handling and scale up. Both
2 of 28
cell lines can produce and post-translationally
modify eukaryotic proteins in functional
forms at high levels. HEK-293 and CHO cells
in suspension typically have a doubling time
of less than 24 hours and can grow to densities
higher than 5 million cells per ml, depending
on culture conditions. Both cell types are
readily transfected or virally transduced to
introduce foreign DNA that codes for target
proteins. Productivities of grams of protein per
liter of culture, generated by transient trans-
fection, have been reported for both HEK-293
and CHO cells. Higher yields are possible
from stable pools, and clonally selected stable
lines have been reported expressing tens of
grams per liter, with cellular productivities of
more than 50 pg/cell/day (Huang et al., 2010;
C. Liu, Dalby, Chen, Kilzer, & Chiou, 2008).
HEK-293 and CHO lines are available
from cell line repositories, such as the
American Type Culture Collection (ATCC;
Rockville, MD), or through commercial
suppliers. Genetically modified cell lines, in
which key steps of a metabolic pathway are
mutated or removed, are also available, and
they are vital for selection procedures used in
the generation of stable cell lines. Suspension
cell kit-based systems that package optimized
cell lines, media, transfection reagents, and
Current Protocols in Protein Science
their corresponding protocols are convenient,
work well, and are accessible to anyone (Taki
et al., 2015). Examples include the Expi
systems from ThermoFisher Scientific, the
MEXi system from IBA Lifesciences, and
the Epi-CHO system from Acyte Biotech
(C. Liu et al., 2008; Prevato et al., 2015; Taki
et al., 2015). Even though HEK-293 and CHO
are most frequently used, other cell lines are
available for protein production. The NS0
and Sp2/0 lines were derived from mice and
used to generate drugs such as Synagis and
Erbitux (Butler & Spearman, 2014; Chartrain
& Chu, 2008; Johnson et al., 1997; Peakman
et al., 1994). New lines such as the CAP and
CAP-T, from human amniocyte (Fischer et al.,
2012), and PER.C6 R
(Jones et al., 2003),
from human retina, are reported to have
very high cellular productivities and generate
human-type post-translational modifications.
Scientists are continually researching and
engineering new cell lines with desired at-
tributes, such as higher yields, specific post-
translational modifications, or improved se-
lection pressure for stable cell lines, among
others. The availability of omics data has
dramatically improved our understanding of
production cell lines, allowing cell line im-
provements to be made via cell line engineer-
ing instead of random mutagenesis (Xu et al.,
2011). The discoveries of the CRISPR/Cas9
(Cong et al., 2013; Jinek et al., 2013; Mali
et al., 2013), zinc finger nucleases (Carroll,
2011), and transposases (Mates et al., 2009)
systems have accelerated the development of
cell line engineering technologies. Of partic-
ular interest are the glycosylation profiles of
secreted proteins and their effect on func-
tion. For antibodies, glycosylation can dic-
tate properties such as antibody-dependent
cellular cytotoxicity (ADCC), complement -
dependent cytotoxicity (CDC), and serum
half-life. Changes to the glycosylation pro-
file of a protein can be accomplished either
by modifying the genome of the cell or by
co-expression of plasmids encoding other fac-
tors (Le Fourn, Girod, Buceta, Regamey, &
Mermod, 2014; Yin et al., 2015). The corre-
lation between low levels of core-fucosylation
and increased ADCC activity (Shinkawa et al.,
2003) has led to the creation of cell lines
with reduced or eliminated core-fucosylation
of secreted proteins. The FUT8 knockout
CHO cell line is unable to synthesize α(1,6)-
fucosyltransferase, resulting in the expres-
sion of completely defucosylated antibodies
(Yamane-Ohnuki et al., 2004). Methods for re-
moving the ability of existing stable cell lines
Current Protocols in Protein Science
to attach core fucose to nascent N-glycan moi-
eties include suppression of the three key genes
involved in fucose modification using RNAi
(Imai-Nishiya et al., 2007), overexpression
of b(1,4)-N-acetylglucosaminyltransferase III
(GnTIII) (Umana, Jean-Mairet, Moudry, Am-
stutz, & Bailey, 1999), and bacterial oxidore-
ductase GDP-6-deoxy-D-lyxo-4-hexulosa re-
ductase (RMD) (von Horsten et al., 2010). The
challenge with these processes is to prevent
significant changes in cell character beyond
fucosylation. High-mannose cell lines have
been introduced in recent years, created ei-
ther through mutagenesis and selection pres-
sure (Stanley, 1983) or by site-directed mu-
tagenesis of the Mgati gene (Sealover et al.,
2013). Enhanced ADCC activity of antibod-
ies with high mannose glycans is well docu-
mented, but they are also known to decrease
serum half-life (Reusch & Tejada, 2015). The
sialylation of a glycoprotein has also been
found to play a significant role in serum half-
life. Cell engineering efforts have identified
various gene targets for enhancing sialic acid
biosynthesis, transfer, and preservation with
the goal of improving serum half-life. Current
approaches include the overexpression of ei-
ther UDP-GlcNAc2-epimerase or CMP-sialic
acid transporter, RNA interference of siali-
dases, and the overexpression of sialyltrans-
ferases such as St6gal (Lin et al., 2015). In all
of these cases, glycosylation modulation has
been shown to have a significant impact on
clinical efficacy and pharmacokinetics.
Despite the variety of mammalian cell lines
available for protein production, CHO cells
remain the workhorse of biopharmaceutical
production since the approval of the first CHO-
derived recombinant protein, tissue plasmino-
gen activator, tPA, in 1986. The process of
biomanufacturing in CHO cells involves cell
line development and screening to select the
stable pool or clone that has the highest cell vi-
ability, growth, expression level, and product
quality. CHO cells are involved in the produc-
tion of over 70% of therapeutic recombinant
proteins, most of which are monoclonal anti-
bodies (Jayapal, Wlaschin, Hu, & Yap, 2007).
In 2017, the global revenue from monoclonal
antibodies was over $90 billion, providing a
strong incentive for the continued develop-
ment of cell line engineering technologies.
CELL CULTURE MEDIA FOR
PROTEIN PRODUCTION
Modern cell culture media was developed Hunter et al.
over 60 years ago starting with 199 (M199;
3 of 28
 Morgan, Morton, & Parker, 1950) and Harry
Eagle’s minimum essential medium (MEM;
Eagle, 1959). While media formulations are
tailored for specific applications, they all have
the same goal: to supply the nutrients re-
quired for healthy cell division and propaga-
tion. Due to the ease of use, complex media
components such as fetal bovine serum, serv-
ing as cellular protectants against shear, pH
change, and nutrient stress were favored in
the past for protein production. However, con-
cerns of undefined raw materials potentially
containing adventitious agents, in conjunction
with serious ethical issues regarding the wel-
fare of the donor fetus during harvest (Asher,
1999) pushed the industry to replace serum
with defined raw materials. For example, in
the 1990s, bovine spongiform encephalopa-
thy/transmissible spongiform encephalopathy
(BSE/TSE) spread in beef cattle in the United
Kingdom, resulting in the deaths of over 200
people. As a result, the United States Food and
Drug Administration restricted the importa-
tion of bovine serum from countries that were
affected by the BSE epidemic (Asher, 1999;
Keen & Rapson, 1995). Cell culture media
for mammalian cell production has now pro-
gressed to formulations that are serum-free or
chemically defined, typically containing 50 to
100 components grouped as energy substrates,
chelators, amino acids, surfactants, pH buffers,
vitamins, nucleic acid derivatives, trace el-
ements, salts, fatty acids, and lipids (Keen
& Rapson, 1995; McCoy, Costa, & Morris,
2015). The added benefit of serum-free cell
culture media is that it simplifies the down-
stream purification process.
Published literature is sparse as to the
exact composition of many of the latest media
formulations, as it is proprietary information
for commercial products readily available
from suppliers such as HycloneTM (GE Life
Sciences), GibcoTM (Thermo Fisher Scien-
tific), Lonza and Irvine Scientific. Serum-free
medium consists of nutritionally complete
basal medium supplemented with an empiri-
cally determined mixture of hormones, growth
factors, attachments factors, and binding pro-
teins. These mixtures are commercially avail-
able and referred to as serum replacements
(Keen & Rapson, 1995). Vegetable-derived
protein hydrolysates have been shown to be
a capable replacement for serum and animal-
derived components (Pazos et al., 2004). How-
ever, hydrolysates by their nature are complex
and not fully defined. The latest step in media
technology has been to develop culture media
Hunter et al.
4 of 28
that are not only free of animal-derived compo-
nents but are also chemically defined, meaning
that the quantity of each component is known.
Development of a sophisticated defined media
is not without significant challenges. Compo-
nents in culture media frequently interact due
to the complexity of cellular metabolic path-
ways. Furthermore, nutritional requirements
are defined by the system in which the cell
is used. It cannot not be expected that media
designed for transient transfection in CHO
cells to be the best media for clonal selection
of a CHO stable line. Transient expression
media must contain the appropriate nutrients
to support the growth of high density cultures
expressing recombinant proteins, while media
for clonal cell line development must promote
the growth of cells at extremely low densities.
Selecting a media to produce recombinant
proteins can be as simple or complicated as
the process requires. For a commercially avail-
able expression system like those offered by
HycloneTM (GE Life Sciences) and GibcoTM
(ThermoFisher), the choice is simple: cell lines
can be easily adapted to these commercial me-
dia, or cells can be bought in a kit with the
media and transfection reagents. However, for
more sophisticated processes, the decision of
which media to use will significantly affect
the amount and quality of the biotherapeutics
made. High cell densities and cell viability lead
to higher protein productivities. However, the
nutritional requirements for cell growth can
conflict with those for production of proteins.
Media composition is divided between these
two tasks. An initial approach would be to
assess the qualities of serum-free and chemi-
cally defined media sold by a variety of man-
ufacturers. Cells may require a series of adap-
tation steps to new growth media, typically
performed by weaning, in which the new and
old media are mixed, gradually increasing the
proportion of new media as the cells adapt.
Changing the media may lead to improved cell
growth and productivity but must not come at
the expense of an unacceptable change in pro-
tein quality, such as glycosylation, proteolytic
cleavage, or protein aggregation.
Component titration is the classic approach
to media development, involving a series of
experiments to determine the cell line’s dose
response to various media components, adding
each one at a time. For example, there have
been several reports on the specific effects of
copper in cell culture application. In one study
of CHO cells expressing the IgG-fusion pro-
tein B0, the addition of 50 μM copper sulfate
Current Protocols in Protein Science
to the media reduced lactate and increased cell
viability and protein yield. However, reducing
sulfate concentration to 5 μM had the same
positive effects, as well as reduced protein
aggregation (Qian et al., 2011). Copper sul-
fate has also been shown to increase the pro-
ductivity of recombinant factor VIII expressed
in both baby hamster kidney cells (BHK) and
CHO cells (Chan & Harris, 1997). Component
titration is labor-intensive (a typical cell cul-
ture media contains 50 to 100 ingredients) and
does not always reveal interactions between
components. Media blending is an alternative
method that rapidly generates new media by
mixing existing formulations. An excellent ex-
ample of such an approach is the blending of
the classical formulations DMEM and Ham’s
F12 media, resulting in DMEM/F12, a ubiq-
uitous formulation that is frequently used as
a basal media (D. Barnes & Sato, 1979). Al-
though blending is an effective and faster pro-
cess, the performance of such mixtures can-
not be attributed to a single component within
the media, making troubleshooting difficult.
Nevertheless, this approach can be very suc-
cessful when mixing media that differ only
by a few elements. It is possible to increase
growth and productivity by following a statis-
tical approach to media mixing using a design
of experiments (DOE) methodology (Mande-
nius & Brundin, 2008). Spent media analy-
sis provides a stoichiometric approach to me-
dia design. Analysis of nutrient consumption
can deliver information regarding nutritional
requirements and culture chemistry changes
over the course of a process, providing insights
into the balance of amino acids and other nu-
trients. However, assays for every media com-
ponent are not available. Most assays are lim-
ited to glucose, lactate, ammonia, and amino
acids, delivering a simplistic overview of a far
more complex biological problem. Ultimately,
rational culture media design requires that all
approaches are considered and used when ap-
propriate (Fletcher, 2005).
Nutritional considerations for stable cell
fed-batch systems differ from transient sys-
tems in that the media for fed-batch cultures
must support cell growth and production of
the product for much longer time periods than
transient cultures. For secreted proteins and
cytoplasmic proteins, titers are proportional to
cell density, so in batch culture, generating and
maintaining high viability, high-density cul-
ture is crucial. At high cell densities, nutrients
are rapidly depleted. Glucose feeding is rou-
tinely employed to support cell growth, and a
Current Protocols in Protein Science
variety of cell feeds and hydrolysates are avail-
able that contain glucose, amino acids, lipids
and other factors to maintain the culture (Kyr-
iakopoulos & Kontoravdi, 2014; McCoy et al.,
2015; Mosser et al., 2013). DOE can be used to
screen and optimize feed types, amounts, and
timing. Cell culture metabolite data that are
measured by a Nova system, or more simply
with glucose testing kits, can also be applied
to the DOE. It must be noted that glucose feed-
ing can lead to harmful lactate buildup, which
may need to be monitored along with increas-
ing ammonium levels, to avoid inhibiting pro-
tein production and altering glycosylation (Y.
Fan et al., 2015; B. Liu et al., 2014). Feeding
and media additions must not change the cell
culture osmolality significantly, as this may
also affect productivities and protein quality.
Commercial cell culture media typically have
buffering systems, such as HEPES, that work
alongside bicarbonate buffering provided in
conjunction with CO2 from the cell culture in-
cubator (Zigler, Lepe-Zuniga, Vistica, & Gery,
1985). pH is also actively controlled in biore-
actors if this equipment is available (Howorth,
1975).
One of the most critical parameters to
consider when developing cell culture me-
dia is the method of transfection to be used.
There are several commercially available for-
mulations that can be used with both HEK-
293 and CHO cells that allow levels of re-
combinant protein expression in the range of
many hundreds of milligrams per liter. How-
ever, several contain ingredients that can in-
hibit transfection. Iron (III) citrate (Eberhardy,
Radzniak, & Liu, 2009), and dextran sulfate
both inhibit polyethylenimine-mediated trans-
fection but can improve protein production
if added later in the process. Other compo-
nents may increase protein production lev-
els by aiding transient transfection efficien-
cies, such as N,N dimethylacetamide (DMA;
Rajendra et al., 2015), lithium acetate (LiAc;
Ye et al., 2009) and dimethyl sulfoxide
(DMSO; Ye et al., 2009). Further enhancement
of protein expression for both transient and
stable line producers is achieved by supple-
menting the expression phase of a process with
small molecule additions. In particular, the
use of histone deacetylase inhibitors, such as
sodium butyrate (Mimura et al., 2001) and val-
proic acid, have been shown to increase protein
titers in both HEK-293 and CHO cells (Backli-
wal et al., 2008). Most of these inhibitors mod-
ify host histone acetylation or methylation pat-
terns, thereby increasing cellular transcription
Hunter et al.
5 of 28
 levels and boosting protein production many
folds. The concentration and timing of addi-
tions must be determined empirically as they
may result in cellular toxicity and arrest of cell
growth (Backliwal et al., 2008). Other small
molecules have been shown to have positive
impacts on protein production, such as LiAc
(Cervera et al., 2015) and caffeine (Cervera
et al., 2015; Dell, Mcgrew, Trentalange, &
Van, 2004).
Finally, media composition may also af-
fect post-translational modifications of pro-
teins. Of these modifications, glycosylation
has been the most extensively studied due to its
ability to influence drug clearance rates, spe-
cific activity, solubility, immunogenicity, and
further processing of the protein. Various nu-
trients, sugar precursors, amino acids, trace
metals, and hormones have been shown to in-
fluence glycan distribution. Glucose starvation
has been shown to reduce the incidence of gly-
cosylation at asparagine residues, while the
supplementation of the sugar galactose in the
presence of manganese chloride (MnCl2 ) can
alter the types and abundance of the glycosy-
lation (St Amand, Radhakrishnan, Robinson,
& Ogunnaike, 2014). Controlling osmolality
of the cell culture has been shown to provide
a means to control fucosylation during per-
fusion and fed-batch cultures. Hypoosmolal-
ity of the culture has been shown to reduce
levels of fucosylation of monoclonal antibod-
ies (Konno et al., 2012). The impact of me-
dia composition changes on post-translational
modifications must be taken into consideration
during development, as it can have a direct
impact on the biological activity of the target
protein.
CULTURE METHODS
Traditional laboratory-scale tissue culture
using culture dishes seldom produces more
than a few micrograms of purified recombinant
proteins, antibodies, or viruses from cultures
of adherent cells. With burgeoning needs for
milligrams of protein for crystallography, drug
discovery, or feasibility tests of a novel drug,
the static tissue culture dish has been replaced
by large-scale systems. Cell culture scale-up
of adherent cells has become routine since the
development of the round glass culture dish
by Richard Petri in 1877, cell culture flasks
developed by Alex Carrel in 1923, and roller
bottles, the concept of growing cells as rotat-
ing cultures, designed by George Gey in 1933.
Some of these systems remain relevant today.
Hunter et al.
Erythropoeitin, the first recombinant protein
6 of 28
made from mammalian cells, is still produced
in adherent CHO cells cultivated in roller bot-
tles (F. M. Wurm, 2004). Although much of
the process has been roboticized, the classic
roller bottle remains the pillar of the process
(F. M. Wurm, 2004).
Traditional Methods
The challenge of scaling traditional ad-
herent flask-based processes is addressed by
maximizing the total surface area in a planar
vessel containing multiple layers. Multilayer
vessels have been available for over 30 years
(Stacey & Davis, 2007) and are commercially
available as stacked-plate systems called
Cell FactoriesTM (NuncTM ) and CellStacks R
R
(Corning ). Stacked-plate systems have
allowed a traditional T-25 laboratory flask,
with a surface area of 25 cm2 , to be scaled to
6,360 cm2 in a 10-layered vessel, 25,440
cm2 in a 40-layer vessel, and 60,000 cm2
in a 120-layer hyperstack R
vessel (Stacey &
Davis, 2007). Increased surface area brings
its own set of challenges, including even
distribution of nutrients, gas exchange, and
the manual manipulation of the stacks. Equip-
ment manufacturers have addressed these
issues by adding bioreactor control to planar
culture systems and creating more compact
systems. For example, the xPansion R
mul-
R
tilayer bioreactor (PALL ) provides a cell
growth surface area up to 122,400 cm2 with
temperature monitoring, agitation control, and
disposable pH and dissolved oxygen sensors.
Researchers needing to scale up adher-
ent expression systems for secreted proteins
should also consider a process based on mi-
crocarriers, which can be applied to stir tanks
(Hu et al., 2000) and wave bioreactors (Singh,
1999). This approach allows for optimization,
flexibility in feeding parameters, scalability
up to 10,000 liters, and a higher surface area
to volume ratio. A less common combina-
tion of bioreactors and adherent cultures is the
packed-bed bioreactor, in which the cells are
immobilized on a substrate that is enclosed
in a packed bed within a bioreactor. A wide
array of substrates and configurations exist,
but the most widely used are beads, porous
structures, fibers and hollow fibers (Meuwly,
Ruffieux, Kadouri, & von Stockar, 2007; Park
& Stephanopoulos, 1993; Wang et al., 1992).
The primary advantage of a packed-bed cul-
ture is the lower shear-stress forces on the
cells, compared with microcarriers that are ag-
itated in a stir tank (Brindley et al., 2011).
Researchers who need more than a few
milligrams of a product are encouraged to
Current Protocols in Protein Science
switch to a suspension-adapted cell line when
possible. Suspension culture of mammalian
cells is operationally more facile than adherent
culture and is adaptable to automated liquid
handling and scale up to bioreactor culture
(Muller, Girard, Hacker, Jordan, & Wurm,
2005). Switching to a suspension-adapted
cell line can be as simple as placing a small
platform shaker and a tray of deionized H2 O,
for humidity, inside an existing CO2 incubator.
Tubes containing just a few milliliters of cul-
ture can be scaled up to large flasks containing
multiple liters. If the goal is to test many prod-
ucts or expression conditions, investment in a
CO2 shaker incubator, available from suppli-
ers such as Infors AG and Kuhner Shaker Inc,
provides a unified platform from screening
to small-scale biomanufacturing. Variable or-
bital shaking diameters of 3, 12.5, 25, and 50
mm provide the tools to handle a wide range
of cell culture volumes and containers. For
high throughput testing, 96- or 24-well blocks
are particularly useful for screening both
transiently transfected HEK-293 cells during
protein engineering projects (Bos et al., 2015;
Davies, Greene, Lullau, & Abbott, 2005) and
stable CHO cells for clone selection. Also, 96-
and 24-deep well plates significantly increase
throughput as liquid handling robots are suit-
able for incorporation at all stages of the pro-
cess, including purification (Chaturvedi, Sun,
O’Brien, Liu, & Brooks, 2014). As more pro-
tein is required, convenient 50 ml Tubespin
bioreactors are available for growth at larger
scales (10 to 30 ml), and these vessels are also
liquid handling compatible (Gomez et al.,
2017; Legmann et al., 2009). Shake flasks are
typically used for volumes between 30 ml and
5 liters (Stettler, Zhang, Hacker, De Jesus, &
Wurm, 2007). It may be necessary to test the
scalability of the product from the small to
large scale, as differences in cell growth, shear
factor, oxygenation, and other factors may
affect protein titer and quality. It is essential to
use the correct shaker orbit relative to culture
vessel size, fill volume, and shake speed.
For transient transfection, these parameters
must also be considered for maintaining
the integrity of the transfection complex in
culture and its ability to enter a cell during
transfection.
For further scale-up, wave-mixed bioreac-
tors can be used. In wave-mixed bioreactors,
a one- or two-dimensional movement of the
bioreactor platform induces a wave in a ster-
ile bag, providing oxygen transfer and media
mixing throughout the culture. These bioreac-
tors are easy to operate, minimize shear stress,
Current Protocols in Protein Science
and offer control of temperature, oxygenation,
gas exchange, pH, and other factors (Singh,
1999). Cell growth and product generation are
highly dependent on wave development and
propagation, which is mainly influenced by
rock rate, rock angle, fill level, bag geom-
etry, and culture media (Eibl, Loffelholz, &
Eibl, 2014). Wave-mixed bioreactors are scal-
able up to 500 liters and enable rapid gener-
ation of material for early drug development
either by large-scale transient expression, or
expansion of stable pools or clones. Despite
the same working principle of wave bioreac-
tor systems, commercial reactors differ in their
control unit, platform movement, bag design,
number of sensors, and the PID loops present
in each unit. Wave Rocker (GE Life Sciences),
BIOSTAT R
RM (Sartorius), CELL-tainer R
TM
(Biotech BV) and Allegro XRS 25 biore-
actor System (PALL) are commercially avail-
able wave-mixed bioreactors. As a less expen-
sive option, Kuhner Shaker, Inc and Infors AG
supply trays that can be inserted into a CO2
shaking incubator to provide an alternative to
an independent wave unit.
Bioreactors
Stir tank bioreactors are the most com-
monly used bioreactor type for cultivating
suspension cells. Their general design cri-
teria was derived from E.coli fermentation.
They have been successfully used for the cul-
R
tivation of a variety of suspension cells in-
cluding CHO, baby hamster kidney (BHK),
HEK-293, and PER.C6 R
, and are available
for culture volumes from 10 ml (Ambr R
sys-
tem) to many thousands of liters (Janakiraman,
Kwiatkowski, Kshirsagar, Ryll, & Huang,
2015; Smelko et al., 2011). Stainless steel
tanks are used at laboratory and pilot scales
for research grade material but require sterility,
cleaning, and maintenance. Single-use biore-
actors enable fast setup and breakdown and
reduce contamination risks (Irfan, Rees, Bar-
rett, Markicevic, & Segarra, 2015). Specific
challenges should be taken into consideration
when moving to an impeller driven bioreac-
tor. An ideal bioreactor should impart low
shear stress on the cells, minimizing me-
chanical damage from the eddies formed by
the impeller motion or bursting gas bubbles,
while ensuring uniform mixing of the nutri-
ents in cell culture media and reducing gradi-
ents across the tank. Once optimized, stir tank
bioreactors typically enable consistent scala-
bility and control of all culture conditions, re-
sulting in higher yields and more controllable
Hunter et al.
product qualities.
7 of 28
 Bioreactors may be operated in batch,
extended batch (also known as fed-batch),
or perfusion mode. Fed-batch cultivation of
mammalian cells is the most common, and
it involves the gradual addition of selected
nutrients during the growth-culture cycle to
improve productivity and growth, typically
lasting 10 to 14 days (Pollock, Ho, & Farid,
2013). The development of an extended
batch process requires an understanding of
the nutritional requirements of the cell line.
Perfusion mode enables long production runs
of stable cell lines (for 30 to 60 days or
longer) as product and waste are removed and
replaced with fresh growth media (Pollock
et al., 2013). This culture method is particu-
larly amenable to the production of secreted
proteins but less so for that of cytoplasmic
proteins. Perfusion growth may utilize micro-
or macro-carriers that immobilize the cells,
protecting the cells from shear and allowing
easy media exchange (Butler, Imamura,
Thomas, & Thilly, 1983). Other perfusion
techniques include the use of cell separators,
Alternating Tangential Flow (ATF)/Tangential
Flow Filtration (TFF) devices, or hollow fiber
systems. These methods enable extremely
high live cell densities, exceeding 200 million
cells per ml (Clincke et al., 2013). The highest
productivity cultures reported, such as the
27 g/L antibody titer from the PER.C6 R
cells,
typically have utilized perfusion bioreactor
culture (Tchoudakova et al., 2009).
Transient vs Stable Systems
The choice between transient and stable
expression systems depends upon throughput,
the quantity and quality of material required,
and the turnaround time. Both methods
involve getting target DNA into cells, but in
transient protein production, the expression
vector never integrates into the genome of the
cell. For large amounts of protein production,
stable cells are preferred, as they provide high
yields with consistent quality. However, at
the early stage of a project, where milligrams
to grams of protein is required, transient
transfection is preferred, due to speed and
lower cost (Gutierrez-Granados, Cervera,
Kamen, & Godia, 2018). Furthermore, it
provides an opportunity to focus on protein
engineering experiments, construct design,
growth parameter improvements, and product
feasibility assessments. Transient systems
offer a product with the quality suitable
for preclinical evaluation, thus speeding the
“Proof of Concept” stage when screening
Hunter et al.
multiple candidates. This can be achieved
8 of 28
before committing significant amounts of time
and resources into stable cell line generation
(Gutierrez-Granados et al., 2018).
Several different cell lines have been shown
to be capable of being transiently transfected
for the expression of recombinant proteins, in-
cluding HEK-293 (Jordan, Kohne, & Wurm,
1998), CHO (Preuss, Connor, & Vogel, 2000),
COS (African green monkey kidney) (Blasey,
Aubry, Mazzei, & Bernard, 1995) and BHK
(baby hamster kidney) cells (F. Wurm &
Bernard, 1999). The HEK-293 cell line ex-
hibits very high transfection efficiency and
reliable translation and processing of pro-
teins, resulting in higher titers compared to
other mammalian cells. Significant develop-
ment work has been performed in suspen-
sion HEK-293 cells, providing many options
for reagents and scales for transfection (Jager
et al., 2013). Many variables, including the
health and density of cells at transfection,
growth conditions, culture vessel, expression
construct, duration of expression, and trans-
fection method, must be optimized to achieve
a high level of transient expression, as protein
production diminishes over time due to dilu-
tion of the expression vector by cell division
(Hopkins, Wall, & Esposito, 2012) (Fig. 1).
The HEK-293T (Lebkowski, Clancy, & Calos,
1985) and HEK-293EBNA (Cachianes et al.,
1993; Sun, Goh, Wong, Mori, & Yap, 2006)
cell lines are integrated with a gene encoding
simian virus 40 (SV40) large T and Epstein-
Barr nuclear antigen-1 (EBNA), respectively.
Cells producing these proteins allow for epi-
somal gene replication and segregation when
an SV40 ori or oriP element is present in the
expression plasmid. This prevents dilution of
the plasmid as the cells replicate and often re-
sults in improved production yield (Daramola
et al., 2014; Magistrelli et al., 2010).
One drawback of using HEK-293 expres-
sion systems is the differeing glycosylation
patterns from those proteins made in CHO
cells, the ultimate host for biopharmaceuti-
cals production. It is therefore beneficial to
keep the host cell type consistent throughout
product development, reducing the likelihood
of changes in activity or biophysical proper-
ties. Traditionally, CHO transient expression
systems have been limited by transfection ef-
ficiencies and titers that are insufficient for in
vivo pharmacokinetic/pharmacodynamic stud-
ies (Derouazi et al., 2004; Galbraith, Tait,
Racher, Birch, & James, 2006). Both CHO
cell lines and expression vectors have been
engineered for improved productivity. Engi-
neering cells for increased transcription rates
Current Protocols in Protein Science
and transgene expression, regulating cellu-
lar metabolism, reducing the induction of
apoptosis, and over-expressing of XBP1 (Cain
et al., 2013), ERO-La (Cain et al., 2013) or
EBNA-1 and GS (Daramola et al., 2014) have
all been shown to increase the final titer pro-
duced in CHO. Engineered CHO lines such
as these are generally not commercially avail-
able, and patents may limit their use. Com-
mercially available transient CHO expression
systems have improved significantly, and pro-
tein titers observed with the ExpiCHO sys-
tem (ThermoFisher) have been reported to be
higher than those seen in both the FreeStyle-
MAX CHO and Expi293 systems.
Stable cell lines, in particular clonally se-
lected stable cell lines, have not been sur-
passed for high levels of protein production.
Stable cultures can show consistent produc-
tivities for weeks, if not months, in both the
batch and continuous culture. The traditional
strategy for making a cell line involves nu-
merous labor-intensive steps, from transfec-
tion to the isolation of the final clonal line (Lai,
Yang, & Ng, 2013). The development spans
months and consists of screening hundreds of
clones for productivity and quality attributes
before a smaller set are selected as candidate
production cell clones. Development of a re-
combinant CHO cell line relies on the ran-
dom integration of a plasmid into the genome
by non-homologous recombination, an ineffi-
cient process occurring in a small portion of
cells that are transfected. The cells are sub-
jected to increasing levels of selective pressure
to select for transfectants that have integrated
the gene of interest into their genome. Repeat-
ing this selection process with increased se-
lection pressure can result in cell clones with a
high number of copies of the marker gene, co-
amplification of the gene of interest, and high
levels of protein expression. However, lack of
control of gene insertion, as well as the effects
of gene amplification, can lead to unwanted
phenotypic heterogeneity due to the varying
accessibility of integration sites. These issues
often result in unstable cell lines that show re-
duced production over time (Lai et al., 2013).
For long-term, consistent protein produc-
tion, it is necessary to stably integrate genes
of interest into the genome of the host cell
at sites that are transcriptionally active, that
do not experience gene silencing, (Wipper-
mann, Rupp, Brinkrolf, Hoffrogge, & Noll,
2015) and are not susceptible to genetic re-
arrangement (Dorai et al., 2012). Viral vec-
tors target transcriptionally active regions of
Current Protocols in Protein Science
the genome to integrate their DNA, and they
have inbuilt mechanisms to divert the cellu-
lar machinery in their favor. Lentiviral vec-
tors can transduce CHO cells with high vector
copy, resulting in high levels of product that
is appropriately glycosylated. Integration via
lentiviral vectors has been shown to remain
stable for many months (Baranyi et al., 2013;
Gaillet et al., 2010). Any virus-based protein
production system raises safety issues which
need to be addressed if the stable cell line will
be moved into biotherapeutic production for
humans. An alternative to a complete viral sys-
tem is non-viral adeno-associated virus (AAV)
expression. This approach takes advantage of
the AAV machinery, which integrates into spe-
cific sites in the genome, to bias integration to-
wards open chromatin that favors high expres-
sion and genomic stability (Dorai et al., 2012).
Another novel viral vector system developed
from alphaviruses, Semliki Forest virus (SFV),
uses an RNA vector system to generate stable
cell lines (Casales et al., 2010).
Plasmids bearing genetic regulator
elements can also be used to confer a tran-
scriptionally active state during the random
integration of a transgene into a heterochro-
matic environment. Some of these elements,
such as scaffold/matrix attachment regions
(S/MARs) and locus control regions (LCRs),
function through actively switching regions of
the genome to an active transcriptional state
(Saunders, Sweeney, Antoniou, Stephens,
& Cain, 2015). One of the most significant
challenges to reliable expression from stable
cell lines is gradual epigenetic silencing via
methylation. Another category of regulatory
elements works to prevent this silencing
by restricting heterochromatin expansion,
maintaining a region of euchromatin under
active transcription. This category includes
insulators, ubiquitous chromatin opening ele-
ments (UCOEs), and stabilizing anti-repressor
elements (STAR) (Neville, Orlando, Mann,
McCloskey, & Antoniou, 2017). Use of these
plasmid elements in the creation of stable
CHO cells has been shown to promote protein
yields and sustain transgene expression
(Kwaks & Otte, 2006).
Site-specific recombination can generate
high yielding stable clones reproducibly.
Different gene recombination systems have
been used, including the bacteriophage P1-
derived Cre recombinase, the yeast-derived
FLP-FRT, and the C31 integrase, derived
from Streptomyces phage f31(Ahmadi,
Damavandi, Akbari Eidgahi, & Davami,
Hunter et al.
9 of 28
 2016). However, these systems do require the
establishment of a platform cell line whose
genome is integrated either randomly or at a
limited number of sites, with reporter cassettes
flanked by short cis-acting DNA target se-
quences recognized by specific recombinases.
The Cre recombinase-mediated integration
process recognizes the short 34 bp cis-acting
DNA target sequence, loxP, and was the first
system to be used for human monoclonal
antibody production in CHO (Kawabe et al.,
2017). Similarly, the Flp recombinase requires
a 48 bp Flp Recombination Target (FRT)
sequence for recombination. Cells integrated
with the FRT sequence are available from
ThermoFisher, with the Flp-InTM System, and
have been used successfully to generate sta-
bles lines that consistently produce polyclonal
antibodies (Cacciatore, Leonard, & Chasin,
2012; Zhou, Liu, Sun, Huang, & Yu, 2010).
The downside of both approaches is that the
site-specific recombination is reversible, as
the target sequence remains after integration.
λ integrase and φC31 integrase target 2 dif-
ferent sequences, attP, and attB and catalyzes
irreversible recombination by altering the
attB and attP sites upon cassette exchange
(Cacciatore et al., 2012). With the genome
sequence of several production cell lines now
available, it is possible to efficiently engineer
these genomic sequences with nucleases.
Customized nucleases such as zinc finger
nucleases (ZNFs), transcription activator-
like effector nucleases (TALENs), and
clustered regularly interspaced short palin-
dromic repeats (CRISPR)-associated (CAS)
could be an advantageous choice for the future.
The CRISPR/Cas system has been success-
fully used for genome editing in CHO during
cell line development (Lee, Grav, Lewis, &
Faustrup Kildegaard, 2015), and recently it
has been reportedly used for site-specific
integration of a therapeutic gene in CHO (Lee,
Kallehauge, Pedersen, & Kildegaard, 2015).
Transposase enzymes can integrate recom-
binant genes into the genomes of mammalian
cell lines. When transposases recognize short
inverted DNA sequences, they excise DNA
that is flanked by these repeats and inte-
grate it at a new location in the cell genome
(Fig. 2A). Transposases can be used to cre-
ate stable cell lines by co-transfecting cells
with a donor plasmid and some form of the
transposase. The donor plasmid contains the
gene of interest and a selection marker flanked
by the repeats, called a transposon. The trans-
posase can be transfected as a helper plas-
Hunter et al.
mid coding for the transposase, or the purified
10 of 28
transposase enzyme, or the mRNA coding for
the transposase. Using the purified transposase
enzyme or its mRNA avoids overexpression
of the transposase in the cell and prevents
the integration of the transposase DNA into
the genome. Using transposases to integrate
the gene of interest into the genome results
in much higher integration rates than random
integration, with fewer instances of transpo-
son truncation or scrambling (Fig. 2B). Sev-
eral approaches are commercially available,
including the PiggyBac Transposon system
(SBI System Biosciences), and Leap-In Trans-
posase system (ATUM). Both approaches have
been successfully used to rapidly generate sta-
ble pools with volumetric productivities higher
than those produced by traditional random in-
tegration methods (Balasubramanian, Rajen-
dra, Baldi, Hacker, & Wurm, 2016; Matasci
et al., 2011; Matasci, Baldi, Hacker, & Wurm,
2011) (Fig. 2C). The timeline for generating
stable pools can be as little as 2 to 4 weeks
(Balasubramanian et al., 2015; Balasubrama-
nian et al., 2016) from transfection to protein
recovery, establishing cell pools suitable for
protein production and the selection of clonal
cell lines (Fig. 2D). The speed with which sta-
ble pools are made makes this approach a vi-
able option for researchers making the switch
from transient expression to stable lines.
TRANSFECTING CELLS
The gene encoding the protein of interest
must be inserted into a cell before the
protein can be produced. Many transfection
procedures are available, broadly classified
as chemical, viral, and physical methods
(Kim & Eberwine, 2010). The approach
offers high transfection efficiency, low cell
toxicity, and reproducibility. The choice of
method depends on the cell type, the amount
of protein required, and the cost involved.
In choosing an approach, the downstream
application must also be considered: it may
be possible to use a technique successfully
at the small scale that is not cost-effective at
large scale. All transfection approaches have
their advantages and drawbacks, and once a
method is selected, critical parameters need to
be optimized at each stage of the development
process (Kim & Eberwine, 2010).
Chemical Methods
Chemical transfection methods, in which
a chemical is added to the DNA to facili-
tate entry into the cell, have been the most
widely used since their introduction in 1973.
Current Protocols in Protein Science
Figure 2 Transposase enzymes allow rapid integration of recombinant genes into genomes of
cultivated mammalian cells. (A). Transposases can excise DNA flanked by short inverted DNA
sequences recognized by the transposase and integrate them at a new location in the cell genome,
keeping the entire region (expression cassette) between the short-inverted DNA sequences intact.
Without the transposase, the expression cassette can be truncated or scrambled, resulting in
transgene-transgene fusion (TG/TG fusion) sequences. This approach has been successfully
used to rapidly generate stable pools with volumetric productivities higher than those produced by
traditional random integration methods. (B). Targeted Locus Amplification (TLA) sequencing data
of transgenes and their integration sites in a stable cell line. The table shows the proportions of
identified integration sites that were accomplished via transposition and via random integration,
as well as the number of TG/TG fusion sequences that result from concatemerization. The data
shows that the number of TG/TG fusions is far greater in the cells without transposase. (C). Rapid
selection of clonal lines with consistent productivity from stable pools. The highest expressing
clones are selected from pools giving high protein productivities of approximately 4 g/L (y-axis).
(D). The timeline for generating stable pools can be as little as 2 to 4 weeks from transfection to
protein recovery, establishing cell pools suitable for protein production and selection of clonal cell
lines for research cell banking (RCB).

CaCl2 (Graham & van der Eb, 1973) or DEAE- ability to transfect a variety of cell types at
dextran (McCutchan & Pagano, 1968; Vaheri high efficiency. A wide range of cationic lipid
& Pagano, 1965) are the two oldest approaches products has become commercially available,
and are very cost-effective but may suffer from such as TransFectinTM (Biorad), FuGENE R

low transfection efficiency. Transfection effi- HD (Promega), FectoPro (Polyplus transfec-

ciency is the percentage of cells transformed
and is typically assessed using a report plas-
mid. DEAE-dextran is not suitable to generate
stable lines, and both approaches cause tox-
icity to the cells (Gluzman, 1981). Cationic
lipids are the most popular chemical transfec-
tion method and can be scaled up to thou-
sands of liters. Their main advantage is their
tion) and Lipofectamine (ThermoFisher). Al-
ternatively, polyplexes like Polyethelyimine
(PEI) are an economical method of transfec-
tion (Rajendra, Kiseljak, Baldi, Hacker, &
Wurm, 2011). Different chemical forms of
PEI are available from a variety of suppli-
ers, and many can generate transfection ef-
ficiencies of 100% with little cellular toxicity. Hunter et al.

11 of 28
Current Protocols in Protein Science
 However, some growth media are not com-
patible with PEI transfection as they contain
components that interfere with PEI activity.
This can be mitigated by a media change or
cell wash before transfection, or by select-
ing a suitable growth media initially. PEI is
scalable from 1 ml or less to many thousands
of liters (Ye et al., 2009). The processes by
which each of these approaches introduces
DNA into a cell are similar. Nucleic acids
are negatively charged due to their polyphos-
phate backbone, and these positively charged
chemicals interact with the DNA forming
complexes. Presumably, these positively
charged complexes attach to the negatively
charged cell surface (sialic acid residues), trig-
gering cell encapsulation by endocytosis and
phagocytosis. The DNA is transported to the
nucleus of the cells, where the cells transcrip-
tion and translation machinery produces the
protein of interest (Gutierrez-Granados et al.,
2018).
PHYSICAL METHODS
Physical transfection methods include elec-
troporation, direct injection, biolistic particle
delivery, and laser-based transfection. Electro-
poration is the most widely used approach for
delivering DNA to cells for protein production.
It uses pulsed electric fields to introduce DNA
into the cells (Longo, Kavran, Kim, & Leahy,
2013). Cells and the plasmid containing the
gene of interest are suspended in a conduc-
tive solution enclosed in an electrical circuit.
A short electrical pulse is discharged through
the suspension, disturbing the phospholipid bi-
layer of the cell membrane, creating tempo-
rary pores through which the DNA can pass
(Shigekawa & Dower, 1988). Electroporation
is applicable to both stable and transient trans-
fection and transfects many cells in a short pe-
riod once optimal electroporation conditions
have been determined. The primary disadvan-
tages are a lack of scalability and high levels
of cell death caused by the high voltage pulses.
Equipment to electroporate cells are available
from many vendors, including ThermoFisher,
Biorad, and Eppendorf. MaxCyte has devel-
oped a scalable transfection system using Flow
ElectroporationTM . However, this technology
is effective only up to 200 liters, significantly
short of the thousands of liters achieved by the
chemical approach.
Viral Methods
Viral vector systems consist of a defective
Hunter et al.
virus that encapsulates the gene of interest for
12 of 28
transfer into cells and are the most efficient
vehicle for DNA transfer. However, there are
significant safety issues regarding their use,
and they are not permitted for the develop-
ment of recombinant proteins that may en-
ter the clinic. Lentiviral vectors are capable
of transducing a broad range of cell types and
have been shown to generate stable cell lines in
both HEK-293 and CHO cells. One significant
drawback to the lentiviral system is that the
capsid is limited to a packaging size of approx-
imately 10 kb, restricting the size of the protein
that can be made (Kumar, Keller, Makalou, &
Sutton, 2001). The BacMam System uses a
baculovirus, a virus that infects insect cells,
that has been modified to swap promoter el-
ements that are active in insect cells to ones
that are active in mammalian cells, such as the
cytomegalovirus (CMV) promoter (Fornwald,
Lu, Wang, & Ames, 2007). The viruses are
generated and amplified in insect cells and are
then used to infect mammalian cells, where the
target protein will be produced. Improved Bac-
Mam vectors have been generated, express-
ing vesicular stomatitis virus glycoprotein G
in the viral envelope to make the infection of
mammalian cells more efficient. These sys-
tems have proved popular as the baculovirus
can deliver its payload to a large variety of
cell types. However, the amount of virus re-
quired to infect mammalian cells effectively
often needs large baculovirus production runs,
which take a few weeks and have their inher-
ent costs (Barsoum, Brown, McKee, & Boyce,
1997). BacMam is widely used in the mem-
brane protein structural biology field, possi-
bly because insect cell culture has been heav-
ily utilized by this group, making it a smooth
transition between systems (Dukkipati, Park,
Waghray, Fischer, & Garcia, 2008).
SELECTION METHODS
Selection techniques for generating stable
cell lines are broadly grouped into drug re-
sistance, metabolic rescue, or physical meth-
ods, and these techniques can be used alone
or in combination. A typical cell line develop-
ment program starts with a transgenic plasmid
containing the gene of interest and a selection
marker. After integration into the cell genome,
a selection agent is used to kill off the cell pop-
ulation that has little to no expression of the
transgene. In some instances, selection pres-
sure is increased to enable gene amplification
and elevate protein expression. When creating
clonal cell lines, cells that survive the selec-
tion process are diluted to single cells and ex-
Current Protocols in Protein Science
Figure 3 Selection drug kill curves are shown for three commonly used antibiotics (A). Blasticidin,
(B). Puromycin and (C). Zeocin on HEK-293 cells grown for 6 days and viability measurements taken
at day 0, 1, 2, 3 and 6. Percent viability is shown for each time-point along the y-axis. Untreated HEK
293 cells are used as positive controls. In order to generate a stable line expressing a transgene it is
important to generate selection-drug kill curve to determine the minimum amount of drug required to
kill cells that were not transfected or transduced with a drug resistance marker.

panded as clonal populations. Timescales for
the generation of a stable cell line, in which
the gene of interest becomes integrated into
the host cell genome, can also vary widely be-
tween selection methods.
Positive selection markers that rely on toxic
agents were the first approach to be devel-
oped. These drug resistance strategies are
highly efficient, with selection times of only
a few days. HEK-293 and CHO cells are sen-
sitive to drugs such as Zeocin, Puromycin,
Hygromycin B, Ouabain, and G418 (Lanza,
Kim, & Alper, 2013; Treschow et al., 2007).
Some cell strains, such as HEK-293T, are
resistant to certain drugs, so optimal antibi-
otic selection must be determined for a given
cell and media combination to generate a
kill curve. For any drug resistance selection
method, the generation of a selection-drug kill
curve will determine the minimum amount of
drug required to kill cells that were not trans-
fected or transduced (Fig. 3). The modes of
action of these drugs are entirely different.
Zeocin is a bleomycin analog that binds and
cleaves DNA (Oliva-Trastoy, Defais, & Larmi-
nat, 2005). G418 and Hygromycin are amino-
glycosides that kill eukaryotic cells by binding
ribosomal components and inhibiting transla-
tion (Bernard, Krammer, & Rowekamp, 1985).
Puromycin is an aminonucleoside that also dis-
rupts translation (Azzam & Algranati, 1973),
and Ouabain is a clinically used cardiac gly-
coside and selective Na+ /K+ -ATPase inhibitor
(Treschow et al., 2007).
Metabolic selection markers, utilizing cell
strains that are deficient in critical biosynthetic
pathways, are commonly used in high-level
expression systems. Cell line development
technologies used by most biopharmaceutical
companies are usually based off either the glu-
tamine synthetase (GS) system (Bebbington
et al., 1992) or the methotrexate (MTX) ampli-
fication technology (Kaufman & Sharp, 1982).
Glutamine synthetase knockout cell lines are
available for CHO (L. Fan et al., 2012) in
which the GS gene is deleted, requiring the cell
culture media to be supplemented with glu-
tamine for the cells to grow. Transformation of
the genes of interest into a cassette with genes
for the expression of exogenous glutamine
synthetase allows for the rescue of transformed
cells in glutamine-deficient media (Knox et al.,
2013). Selective pressure can be increased
by using the drug methionine sulphoximine
(MSX), which inhibits the GS enzyme, or
by weak promoters driving GS production, or
even by destabilizing the GS protein by the ad-
dition of PEST sequences, which act as a signal
for protein degradation (L. Fan et al., 2013).
Increasing selective pressure can lead to gene
amplification and even higher expression lev-
els of the target protein. These processes force
the cell to make more of the protein of inter-
est, but some of these amplification techniques
can lead to genomic instability. Cell line gen-
eration using GS is reported to be faster than
the related dihydrofolate reductase selection
system.
The CHO-DG44 line is a dihydrofolate re-
ductase (DHFR) deficient cell line, in which
selection is based on media that does not con-
tain thymidine, glycine or hypoxanthine, as
DHFR is critical for the synthesis of these
molecules (Cacciatore, Chasin, & Leonard,
2010). Transformation of cells with the gene
of interest and the gene for the DHFR enzyme
allows for growth in thymidine, glycine or hy-
poxanthine deficient media which is then cou-
pled with methotrexate (MTX) drug selection
and complementary gene amplification (Vish-
wanathan et al., 2014). Other metabolic selec-
tion pathways have been investigated such as
folate and purine synthesis, and given the rel-
ative ease of CRISPR/CAS technologies and Hunter et al.

13 of 28
Current Protocols in Protein Science
 genome sequencing, it seems likely that many
new selection methods will soon become avail-
able (Costa et al., 2012; Rothem, Berman,
Stark, Jansen, & Assaraf, 2005).
Each selection method will result in a pop-
ulation of cells producing target proteins at
different levels. If a stable pool is not suffi-
cient for production (stable pools can gener-
ate grams per liter of antibodies), then further
selection techniques can be used to create a
clonal cell line. Limited dilution is the process
of making serial dilutions of a cell stock and
then dispensing a cell suspension into individ-
ual wells of multi-well plates to isolate a single
cell in each well. This cell will proliferate, cre-
ating a group of cells that are clonally identi-
cal. Limited dilution is tedious and inefficient
but does not require sophisticated equipment.
Fluorescence-activated cell sorting (FACS) is
utilized to sort a cell suspension into single
cells (Misaghi et al., 2016). FACS can be used
to sort a population of cells, selecting cells
with high target protein production levels by
linking the gene of interest to a fluorescent
marker, such as in the IRES-GFP system, or
by cell surface display, or by ‘cold’ sorting to
capture protein secretion in the membrane of
the cell, which can then be probed by a fluo-
rescent secondary antibody (Brezinsky et al.,
2003). In each of these techniques, the level of
fluorescence associated with a cell correlates
to the level of production of the target pro-
tein, so that only highly producing cells will
be captured. Cells may also be sorted in ad-
herent culture using lasers to induce cell death
of non-producers (Koller et al., 2004) . Fluo-
rescent selection is also utilized in semi-solid
media, where target proteins being secreted
from the cell remain in close proximity (Caron
et al., 2009; Mangalampalli et al., 2015). Prob-
ing with a fluorescent antibody against the se-
creted protein reveals high-producing clones
that are automatically picked and transferred
into multi-well plates for further growth (Dhar-
shanan, Chong, Swee Hung, Zamrod, & Ka-
mal, 2011).
EXPRESSION VECTORS
Expression vectors, also known as expres-
sion constructs, are critical to the production
of proteins, as they are the vehicle by which
the gene that codes for the protein of interest
is carried into the target cell. These vectors
must contain the essential elements by which
the cells’ machinery recognizes the DNA and
begins transcription and translation of the
Hunter et al.
protein of interest. These elements include
14 of 28
a promoter, a multi-cloning site (MCS),
and a 3’ untranslated region (3’UTR) con-
taining a polyadenylation (polyA) sequence
(Kaufman, 2000). Other elements often
include enhancers, introns, viral amplifiers,
IRES elements, selection markers, origins
of replication, and chromatin remodeling
factors (Fig. 4A). Accordingly, different
vector design strategies mix and match these
elements to produce large amounts of stable
messenger RNA and, by extension, proteins.
Enhancers and promoters are upstream of
the MCS and cooperate to determine the
rate of transcription. A promoter is an ar-
rangement of short regulatory sequences that
attract sequence-specific transcription factors
that drive transcription initiation (Dalton &
Barton, 2014). Enhancers are sequences that
bind activator proteins that increase the rate
of transcription and can be adjacent or far
from the promoter they influence. Commonly
used promoters are the CMV, Ef1α, CAG, and
SV40 (Dalton & Barton, 2014). Many viral
promoter regions contain proximal enhancers,
such as CMV and SV40, frequently incorpo-
rated into vector cassettes that are in some
cases cell-specific (Brown, Sweeney, Main-
waring, & James, 2014; Mariati, Ho, Yap, &
Yang, 2010).
Simultaneous expression of the light and
heavy chain of a monoclonal antibody can
be achieved using two independent plasmids.
However, this approach is heavily influenced
by transfection efficiency, the copy number of
each plasmid that enters the cell, and, in the
case of stable cell lines, genomic integration
sites. An alternative approach is to use a single
vector with two independent promoters. How-
ever, transcription interference can occur, in
which the influence of a strong transcriptional
process reduces the expression of the sensitive
promoter. Transcriptional interference can be
solved with the use of an internal ribosome
entry sites (IRES), either of cellular or viral
origin. The IRES approach involves the design
of a bicistronic construct where the two genes
are separated by an IRES sequence, allowing
two different proteins to be translated from
a single mRNA. This intervening IRES
sequence functions as a ribosome-binding site
for efficient cap-independent internal initia-
tion of translation. The IRES system offers the
advantage of using a single promoter for the
expression of both polypeptides of the mon-
oclonal antibody, and it allows the expression
of the gene of interest in the presence of a
selection marker for stable line generation
(Li et al., 2007).
Current Protocols in Protein Science
Figure 4 (A). Expression vectors, or expression constructs, are critical to the production of
proteins. These vectors must contain essential elements, including a promoter, multiple cloning
site (MCS), and polyadenylation sequence by which the cells’ machinery recognizes the DNA and
begins transcription and translation of the protein of interest. They may also include additional
elements such as enhancers, introns, viral amplifiers, IRES elements, selection markers, origins
of replication and chromatin remodeling factors, which can increase expression levels. Modular
vector designs utilizing common cloning sites allow different vector design strategies to mix and
match these elements to optimize protein expression in the cell line of choice. (B). Expression of
trastuzumab in different vector configurations is shown. Vector configurations tested are shown
along the x-axis and protein productivity in mg/L is shown along the y-axis. The features of each
vector are listed in Table 1. Selection of a vector is complex: no single expression vector works
for all systems, therefore a systematic approach that optimizes each element in combination with
others needs to be undertaken. Design of Experiments (DOE) was used by ATUM to design a
set of vectors containing elements like those in Figure 4A to be highly productive in a particular
mammalian host compared to standard off the shelf vectors.

Just as the promoter initiates transcription,
the 3’ untranslated region is imperative for
transcript termination. The 3’ end of the RNA
terminates with a polyA sequence that pro-
motes nuclear export, stability, and efficient
translation (Edmonds, 2002; Wickens, Ander-
son, & Jackson, 1997). Commonly used mam-
malian terminators are the SV40, hGH, BGH
and rbGlob polyA sequences that provide
both polyadenylation and termination (Gil &
Proudfoot, 1987; Goodwin & Rottman, 1992;
Lanoix & Acheson, 1988; Schek, Cooke, &
Alwine, 1992). Termination and polyadeny-
lation of an mRNA are a coordinated process.
The terminator, a sequence-based element, de-
fines the end of the transcript, creating a free
3’ end and initiates release of the mRNA from
the transcriptional machinery. The free 3’ end
is then available for the addition of the polyA
tail. Hunter et al.

15 of 28
Current Protocols in Protein Science
 The addition of an intron or an untranslated
exon sequence upstream from the initiation
codon of the gene of interest has been shown
to induce higher levels of protein expression
than plasmids without these elements (Buch-
man & Berg, 1988; Lacy-Hulbert et al., 2001;
Melcher, Grosch, & Hasilik, 2002). These
untranslated exons contain splicing elements
that are thought to improve mRNA transport
from the nucleus to the cytoplasm, or enhance
RNA stability and or half-life, thus provid-
ing a greater abundance of the transcript. As
a result, introns between the promoter and
MCS of commercially available mammalian
expression vectors are often included. For ex-
ample, Intron A in combination with hCMV
IE1 enhancer-promoter can radically increase
levels of translatable mRNA, leading to higher
cellular productivity.
The effects of these additional elements in
expression vectors depend on whether the gene
of interest is integrated into the genome or ex-
pressed transiently. Human CMV promoters
may be more active than the mouse promot-
ers in stable cell lines, as they are less prone
to epigenetic silencing. Elements such as uni-
versal chromatin opening elements (UCOE)
may inhibit chromatin condensation around
target integrated genes, inhibiting transcrip-
tional silencing (Betts, Croxford, & Dickson,
2015; Rocha-Pizana, Ascencio-Favela, Soto-
Garcia, Martinez-Fierro, & Alvarez, 2017).
Woodchuck hepatitis post-transcriptional reg-
ulatory element (WPRE) and Hepatitis B virus
regulatory element (HPRE) may enhance post-
transcriptional processing or export of mRNA
leading to increased protein production (Klein
et al., 2006).
It should be noted that all of these high pro-
ductivity expression vectors are not without
their drawbacks (Halff, Versteeg, Brondijk, &
Huizinga, 2014). The quality of protein pro-
duced must be assessed as well as the quan-
tity. Decreasing effective promotor strength
has been demonstrated to lead to reduced pro-
tein aggregation. Intron sequences may have
cryptic donor or acceptor sites in the target
gene leading to mRNA splice variants that do
not code for the target protein. High rates of
production may swamp the post-translational
modification apparatus leading to heteroge-
neous product formation or in cellulo aggre-
gation, like Russell bodies (Hasegawa, Woods,
Kinderman, He, & Lim, 2014). Some of these
problems may be ameliorated by codon op-
timization, by stuffer DNA, by choosing a
Hunter et al.
16 of 28
‘weaker’ promoter, or by engineering cells to
have enhanced properties (B. Estes et al., 2015;
Rajendra et al., 2012).
Inducible vectors are used in the produc-
tion of target proteins that may be toxic to cell
growth, such as integral membrane proteins.
These systems allow transfected cells to grow
uninduced at a similar rate to the parental cell
line, and then, upon induction, synthesize the
desired protein. Inducible transient expression
systems typically rely on the co-transfection of
a plasmid that expresses a regulatory protein
which binds to operator sequences upstream
of the gene of interest. Alternatively, stable
cells lines can be developed that constitutively
express the regulatory protein. Commercially
available inducible systems include the Tet
operon (from Clontech) which can be used
to turn on expression in the presence of the
small molecule drug doxycycline (Dox; Do-
rai et al., 2012; Gaillet et al., 2010), although
there are many more systems available. The
Cumate inducible system can be used sim-
ilarly (Baranyi et al., 2013). Other systems
have utilized temperature, isopropyl β-D-1-
thiogalactopyranoside (IPTG), metals, light,
and insect hormones such as Ecdysone (Ah-
madi et al., 2016; Casales et al., 2010; Kwaks
& Otte, 2006; Neville et al., 2017; Saunders
et al., 2015).
No single expression plasmid works for all
systems and selecting one will be dependent
upon the cell line, the type of expression, and
perhaps other factors not considered in this
review. Traditional protein expression vectors
employed by many are an assembly of nat-
urally occurring parts chosen from a limited
pool of available enhancers, promoters, termi-
nators, translation initiation signals, selection
markers, etc. Many of these vectors are merely
functional constructions used either because of
historical precedent or because an academic
lab or company have identified a new regula-
tory element. To know which combination of
features will provide the best productivity for
the gene of interest in the chosen expression
host, a panel of vectors needs to be tested in
parallel to empirically determine the best solu-
tion, which can be accomplished using suites
of vectors with common cloning sites. While
the number of potentially useful elements con-
tinues to expand, a systematic approach needs
to be undertaken to optimize each element
in combination with the others to maximize
vector properties. Optimization tools such as
DOE can be used to provide a framework for
Current Protocols in Protein Science
Table 1 Features for Each Vector Displayed in Figure 4B

Viral Viral
Origin of replication
Vector ID Enhancer Promoter Intron WPRE replication protein
pTT5 CMV CMV synthetic N/A OriP N/A
pCDNA3.4 CMV CMV N/A yes N/A N/A
1 CMV CMV CMVa N/A OriP EBNA
2 CMV CMV CMVa N/A OriP EBNA
3 CMV CMV CMVa N/A OriP EBNA
4 CMV CMV CMVa N/A OriP EBNA
5 CMV CMV CMVc N/A N/A N/A
6 CMV CMV CMVa N/A SV40 SV40 T
antigen
7 CMV CMV CMVa N/A N/A N/A
8 CMV CMV CMVa N/A OriP N/A
9 CMV CMV CMVa N/A OriP EBNA
10 CMV CMV synthetic N/A N/A N/A
11 synthetic synthetic synthetic N/A N/A N/A
12 synthetic synthetic synthetic N/A N/A N/A
13 N/A synthetic synthetic N/A OriP EBNA
14 synthetic synthetic N/A N/A OriP EBNA
15 synthetic CMV N/A N/A N/A N/A
16 synthetic CMV synthetic N/A N/A N/A
17 synthetic CMV synthetic N/A N/A N/A
18 N/A synthetic synthetic N/A N/A N/A
19 synthetic synthetic N/A N/A N/A N/A
20 CMV CMV N/A N/A N/A N/A
21 synthetic CMV CMVc N/A N/A N/A
22 CMV CMV CMVa N/A OriP EBNA
23 SV40 SV40 N/A N/A N/A N/A

CMV stands for cytomegalovirus, as the promoter and enhancer are originally from the genome of the human cy-
tomegalovirus. The SV40 promoter and enhancer were taken from the genome of simian virus 40. The synthetic
enhancers, promoters, and introns are of rodent origin. The CMVa and CMVc introns are two different regions from the
CMV enhancer/promoter that have been shown to increase expression in mammalian cells. WPRE stands for Woodchuck
Hepatitis virus posttranscriptional regulatory element, which has been shown to increase protein yield when present
in the 3’ untranslated region. Viral origins of replication and their viral replication proteins allow episomal replication
of plasmids. The OriP/EBNA pair is from the Epstein-Barr virus, and the SV40/SV40 T-antigen pair is from Simian
virus 40.

experimentation in which several element GENE OF INTEREST

combinations can be simultaneously tested. In
conjunction with de novo DNA synthesis, it is
possible to rapidly screen independently de-
signed constructs based on their performance
(Fig. 4B, Table 1). The outcome of these ex-
periments allows the researcher to purchase,
with confidence, a vector that will be highly
productive in a defined mammalian host.
OPTIMIZATION
Considerable effort has been made to
improve the expression of the gene of in-
terest (GOI), including the development of
mammalian expression hosts, cell culture
media improvements, construction of con-
trolled bioreactors, development of rapid and
extended expression methods, and vector
Hunter et al.

17 of 28
Current Protocols in Protein Science
 optimization. Despite these advances, one
underlying subject remains to be discussed,
the GOI itself. The open reading frame used
to express the protein of interest goes far
beyond merely stating the order of amino
acids. In its native context, it may contain sec-
ondary structures, mRNA that might inhibit
ribosome processivity, alternative splicing
sites, sequence elements signaling mRNA
degradation, or codons that are rarely used in
an expression host. Therefore, the variables
that affect the open reading frame of the GOI
and its ability to produce large amounts of
protein in mammalian cells can be numerous
(Gustafsson et al., 2012; Welch, Villalobos,
Gustafsson, & Minshull, 2009). De novo DNA
synthesis provides the researcher with the
ability to create a full-length gene considering
these parameters and controlling for the
presence or absence of specific restriction
sites or motifs.
De novo gene synthesis allows fast and ef-
ficient construction of genes tailored for a spe-
cific application, as well as the design of an
“optimal” gene. One interpretation of “opti-
mal” could mean designing a DNA sequence
that increases the cell’s ability to express a
recombinant protein at high levels by opti-
mizing codon choices. Many examples ex-
ist demonstrating the impact of codon usage
on gene expression. Specifically, research has
demonstrated that gene expression levels cor-
relate with the use of optimal codons, codons
that have the highest tRNA gene copy num-
ber. They demonstrate that there is a high fre-
quency of optimal codons in highly expressed
genes, so a simple strategy for increasing ex-
pression of the protein of interest would be
to construct the GOI with optimal codons
(Lavner & Kotlar, 2005). Another optimiza-
tion strategy would be to utilize codon usage
bias and assign the most highly used codons
to all instances of the amino acid sequence.
This line of reasoning means that an ideal
codon is defined by its frequency, which is
often measured by the Codon Adaption Index
(CAI) score (Sharp & Li, 1987). Such an ap-
proach has many drawbacks, including gener-
ating a high codon concentration subset of the
tRNA population, resulting in an imbalanced
tRNA pool, skewing codon usage, and in-
creasing translational error (Plotkin & Kudla,
2011). This approach has been superseded by
the development of advanced algorithms that
not only take these issues into account, but
can also avoid repetitive elements and mRNA
structure (Villalobos, Ness, Gustafsson, Min-
Hunter et al.
18 of 28
shull, & Govindarajan, 2006). Furthermore,
they provide the researcher with the oppor-
tunity to manipulate their sequence to include
or remove sequence elements such as restric-
tion sites. A well-designed optimization of
a gene is a big challenge; however, a large
variety of software is available in the pub-
lic domain or as commercial sequence pack-
ages. The software for optimization must take
into consideration many parameters spanning
the entire gene of interest, including short se-
quence motifs, splice site recognition patterns,
GC-content and codon usage (Villalobos et al.,
2006).
In addition to the optimization of the gene
that will produce the recombinant protein of
interest, we must consider where the protein
will be present once made. The widespread
use of mammalian cells has been dominated
by the production of secreted proteins that re-
quire a signal sequence that directs the pro-
tein of interest outside of the cell. These are
typically short 20-30 hydrophobic amino acid
helices which are recognized by cellular ma-
chinery and used to target the protein of in-
terest down the correct pathway. Signal se-
quences may also be present on proteins that
are not secreted from the cell but embedded
into the cell membrane, such as integral mem-
brane proteins or cell surface receptors. It has
been reported that the expression of a recom-
binant protein does not always correlate with
its mRNA levels (L. M. Barnes, Bentley, &
Dickson, 2004) and a rate-limiting step can
be the secretory pathway. Intuitively, the best
choice of a signal sequence may be the pro-
teins’ native signal. However, testing a panel
of commonly utilized signal sequences is de-
sirable (Fig. 5). Not surprisingly, the signal se-
quence can have a dramatic effect on protein
productivity, with fourfold enhancement of ex-
pression levels being reported (Kober, Zehe, &
Bode, 2013; Stern, L.C., Trobe, Ravneberg, &
Pryme, 2007).
Recombinant proteins are commonly ex-
pressed in eukaryotic expression systems to
ensure the formation of disulfide bridges and
proper glycosylation. Antibody and antibody-
like molecules are examples of proteins that
are relatively easily made in mammalian cells
and are easily purified by Protein A or Protein
G resins. However, some recombinant pro-
teins, sub-domains, and mutant protein ver-
sions can be plagued with issues regarding
misfolding, aggregation, or lack of expression
entirely. Fusion tags are frequently used to ad-
dress these problems and provide a convenient
Current Protocols in Protein Science
Figure 5 Effect of different secretion signals on the expression of antibody light chain (LC)
and heavy chain (HC) is shown. Secretion signals tested are shown on the x-axis and model
regression weights shown on the y-axis. A total of 23 different secretion signals were tested on
the light and heavy chains of 5 different antibodies. ATUM utilizes DOE and machine learning
algorithms that assign model regression weights based off the mean protein titer of each antibody
(y-axis) to different secretion signals (x-axis), predicted for their effect on (A). light (LC) and (B).
heavy chains (HC) of an antibody. The model regression weights of each signal sequence are
represented as a cluster of the 5 antibodies on which it has been tested. Positive contributions
have positive regression weights, and the magnitude of the weight corresponds to the magnitude
of the effect of the secretion sequence. As the data shows, some secretion signals can have
positive effects on one antibody and negative effects on another, while others have net positive
effects on all 5 antibodies tested.

means for single step purification. This review
will not extensively cover these tags. Com-
monly used fusion partners include the con-
stant domain of IgG (the Fc region), maltose
binding protein (MBP), small ubiquitin-like
modifier (SUMO), and human serum albumin
(HSA) tag (Bokhove et al., 2016; Carter et al.,
2010). All have been shown to improve yield,
solubility, or both for the protein that they were
fused to, but the extent of their influence does
appear to be protein specific. The Fc tag does
cause artificial dimerization of fused proteins,
which may induce aggregation with certain
partners. As many of these tags are large, it
is often necessary to remove them after the fu-
sion protein has been purified. The most com-
mon solution is to engineer a protease cleavage
site between the solubility tag and the partner
recombinant protein.
CONCLUSIONS
With the continuing growth in demand for
recombinant proteins, researchers are seeking
rapid methods to produce their protein of in-
terest. To meet these needs there are several
mammalian expression platforms that can be
bought off the shelf from commercial suppli-
ers. However, as most of the information re-
garding the composition of commercial kits is
proprietary, these platforms do not lend them-
selves to easy troubleshooting. In order to
fully understand the outcome of a mammalian
expression system, all of the inputs must be
Current Protocols in Protein Science
known. But beyond deciding on the protein of
interest, it can be difficult to choose a starting
point for process development.
The numerous variables that are involved
in mammalian protein expression are interde-
pendent, so changing one can have unexpected
downstream effects. A common starting point
is selection of the expression host, which has
a direct connection to the culture media, trans-
fection method, and expression vector used.
HEK-293 cells are often used for transient ex-
pression due to its ease of transfectability and
high titers. CHO cells are more commonly
used for stable cell line expression, as most
FDA-approved biotherapeutics are produced
in CHO. It is not unusual for a biotherapeutic
development process to start with HEK-293
expression and move into CHO once screening
has been done on several protein versions. This
switch requires further development of the me-
dia, expression vector, and transfection and se-
lection methods. Scaling up the culture size as
development progresses affects protein char-
acteristics like glycosylation, and these must
be accounted for and optimized as well.
No choice in the development of a mam-
malian expression system is made in isolation,
as they tend to have both upstream and down-
stream effects (Fig. 1). Despite the increasing
number of approaches for expressing the gene
of interest in a mammalian platform, there is
no system capable of meeting all challenges.
The scale and expectations for those requiring Hunter et al.
19 of 28
 material for biological research greatly differ
from the requirements for those needing ma-
terial for drug discovery or development. De-
veloping a system that results in the protein of
interest at the correct scale, with correct char-
acteristics and biologic activities, at the right
price, can take months, or even years and is
outside the scope of this review. This process
can be accelerated with the help of a contract
research organization (CRO) or contract man-
ufacturing organization (CMO).
ACKNOWLEDGEMENTS
The authors would like to thank Miles
Scotcher, Ferenc Boldog, Alan Villalobos, and
Medini Gore for proof reading and intellectual
input.
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