117
Recombinant protein expression for therapeutic applications
Dana C Andersen* and Lynne Krummen†
In recent years, the number of recombinant proteins used for
therapeutic applications has increased dramatically. Many of
these applications involve complex glycoproteins and
antibodies with relatively high production needs. These
demands have driven the development of a variety of
improvements in protein expression technology, particularly
involving mammalian and microbial culture systems.
Addresses
Cell Culture & Fermentation Research & Development, Genentech, Inc.,
1 DNA Way, South San Francisco, CA 94080, USA
*e-mail: andersen@gene.com
† e-mail: krummen.lynne@gene.com
Current Opinion in Biotechnology 2002, 13:117–123
0958-1669/02/$ — see front matter
© 2002 Elsevier Science Ltd. All rights reserved.
Abbreviations
CHO Chinese hamster ovary
BHK baby hamster kidney
Introduction
As the biotechnology industry has rapidly expanded in
recent years, the expression of a spectrum of recombinant
proteins in different systems for a wide variety of purposes
has been a major feature and challenge. In some applica-
tions, a large array of proteins are needed in relatively
small quantities for screening applications, whereas in
other cases, quantities approaching the metric ton scale are
needed for specific therapeutic applications. The majority
of therapeutic proteins have been produced in either
mammalian cell-culture systems, with Chinese hamster
ovary (CHO) cells representing the most common system,
or in Escherichia coli [1,2•]. A variety of alternative expres-
sion systems are also being developed and evaluated. It is
not at all clear which of these systems will ultimately be
the most useful for the variety of niches and applications of
therapeutic protein production.
Several excellent reviews have provided comprehensive
coverage of various aspects of therapeutic protein produc-
tion, including specific issues related to large-scale cell
culture production [1] and considerations for the produc-
tion of specific classes of molecules such as recombinant
antibody-related products [3]. This review will focus on
selected results across a range of expression systems
(although focusing primarily on mammalian and E. coli
cell-culture systems) that illustrate important ways in
which expression technology is evolving to meet the
spectrum of research, development and commercial needs.
Rather than provide a comprehensive review of the
subject, this review will highlight several specific, recent
results across the field.
Mammalian expression systems
Over the past seven to eight years there has been consid-
erable progress in fine-tuning mammalian expression
systems for high-level recombinant gene expression. CHO
and NS0 murine myeloma cell expression systems have
now established themselves as the predominant systems of
choice for mammalian expression. Refinements of vector
construction, choice of selectable markers and advances in
gene-targeting and high-throughput screening strategies
have made the establishment of recombinant cell lines
with high specific productivities (20–60 pg/cell/day for
antibody cell lines) relatively common and have reduced
the time required for cell line development. Recent
advancements in expression technologies using traditional
viral-promoter-based expression vectors include the devel-
opment and refinement of dicistronic expression strategies
using either internal ribosome entry site (IRES) sequences
[4–7] or alternative splicing [8,9]. These strategies are
designed to link expression of the gene of interest to
the selectable marker. In addition, early reports on the
development of host cell lines that express trans-activating
factors or include cis-acting elements on expression
constructs [10••] promise further advances in the augmen-
tation of promoter activity in the near future.
A variety of novel promoter systems are also being devel-
oped and evaluated both for recombinant drug product
expression and for directed metabolic engineering
approaches (see [11]). For example, both tetracycline- and
streptogramin-based gene regulation systems have recently
been demonstrated to be highly useful for regulatable
expression in CHO and other mammalian cell cultures
[12••,13]. These systems promise to be useful for multi-
component control strategies and for the expression of
products which themselves promote cytostasis or cytotoxicity.
For the alternative application of expressing high numbers of
proteins for screening applications, an alphavirus-based sys-
tem has been reported [14]. Transient systems, in which the
isolation of stable transfectants is bypassed so that protein
expression is obtained rapidly but only for a limited period of
time, have also been shown capable of producing reasonable
protein levels (10–20 mg/L) in reduced times [15]. These
systems are providing an increasing array of tools to enable
the manipulation of large panels of genes efficiently.
One of the most notable advances in recent years has been
the application of genetic engineering approaches to ratio-
nally modify specific features of mammalian host cells to
improve their utility in recombinant protein expression
applications. One such example is in the area of glycosyla-
tion control. Work over the past decade has demonstrated
how various reactions in the glycosylation pathway can be
influenced by cell-culture factors, host-cell selection and
118 Biochemical engineering
protein-specific features (reviewed in [16,17,18•]), leading
to the production of molecules with variable or suboptimal
clearance or bioactivity properties. Recent studies have
shown that specific manipulation of oligosaccharide
structures on a recombinant protein by overexpression of
appropriate glycosyltransferases can enhance glycan
quality. Improvements are made either by increasing the
homogeneity of native structures or by introducing non-
host-cell residues to specialize glycan quality and function.
For example, the overexpression of a galactosyltransferase
and a sialyltransferase in CHO cells led to corresponding
increases in the galactose and sialic acid content of
expressed recombinant therapeutic proteins [19••]. Other
groups have successfully overexpressed N-acetylglu-
cosaminyltransferase III to increase the fraction of bisecting
N-acetylglucosamine residues on antibodies produced in
CHO cells [20,21]. Sialic acid has also been introduced in
an α2,6-linkage to glycoproteins synthesized by CHO and
baby hamster kidney (BHK) cells that lack the specific
sialyltransferase responsible for this transfer [18•,22,23].
Another focus of genetic manipulation has been to improve
the efficiency of central carbon metabolism and to reduce
lactate accumulation. Earlier work showed that overexpres-
sion of a pyruvate carboxylase gene in BHK cells reduced
lactate accumulation and improved cell yields on glucose,
although it is not clear if this approach would be helpful in
CHO cultures [24]. In an alternative approach, partial
disruption of the gene encoding lactate dehydrogenase A
(LDH-A) led to reduced lactate and improved cell-culture
performance in hybridoma cultures [25].
Perhaps the most interesting of the targets for metabolic
control have been highlighted by recent studies to control
cell growth and to limit apoptotic cell death. Several
previous studies have shown that recombinant protein
production may be linked to the growth state of the
culture. These studies have shown both growth-associated
and inverse growth-associated production in different
systems. Recent attempts to exploit this phenomenon
have used inducible expression of the product gene in
concert with a regulatory protein (such as p27) that induces
cell-cycle arrest at the G1/S phase border. These studies
support the hypothesis that growth arrest, at least in this
case, is associated with as much as a 15-fold increase in
specific productivity [26,27••]. Increased productivity has
also been observed in response to temperature shift and in
BHK cultures in which cell proliferation was controlled by
expression of the tumor suppressor IRF-1 (interferon
regulatory factor 1) [27••,28,29]. In both instances, however,
direct effects of temperature and IRF-1 on transcriptional
and/or translational control may play a role. Whether or not
growth control can be balanced with enhancements in
cell-specific productivity in such a way as to significantly
enhance volumetric productivity remains to be seen.
Although the potential benefits of these approaches are
encouraging, questions remain regarding the general appli-
cation of the specific promoter systems, cell-line-specific
effects and the method of growth control in controlling
growth-related productivity effects (see [30–32]).
Similarly, work demonstrating the ability of chemical
inhibitors of apoptosis to extend culture viability has laid
the foundation for a growing area of work directed towards
genetic engineering of cell death pathways. Overexpression
of Bcl-2 and BclxL has been shown to inhibit apoptotic
death in several different mammalian culture systems (see
[33,34]). A recent report also showed increased volumetric
antibody expression in butyrate-treated CHO cultures
overexpressing Bcl-2 [35]. Although promising, the effects
observed in these studies have been relatively modest and
variable depending on the cell line and the apoptotic stimuli.
This is perhaps not surprising given the complexity and
redundancy of the regulatory pathways involved in apop-
tosis, and multifaceted approaches may be required to
achieve significant benefits. Researchers are now expand-
ing their approaches to include inhibitors of caspase
activation and mitochondrial cytochrome c release. A recent
genetic engineering study using overexpression of Bcl-2
deletion mutants in CHO and BHK cells also suggested
that the inherent susceptibility of wild-type Bcl-2 to intra-
cellular degradation may explain some of the limited effects
on productivity that have been reported for Bcl-2-based
strategies [34,36••].
In addition to genetic approaches, several recent studies
show that environmental control strategies continue to be
useful for manipulating glycosylation as well as carbon
metabolism, cell growth and cell death. For instance,
medium supplementation with the nucleotide sugar pre-
cursors glucosamine and N-acetylmannosamine enabled
some level of glycosylation control of a recombinant glyco-
protein produced in NS0 and CHO cultures [37,38].
Additionally, advancements in fed-batch and batch media
compositions have dramatically improved viable cell den-
sity and the duration of serum-free cell culture through a
general retardation of onset of apoptosis (e.g. [39]).
Likewise, reduced temperatures have been shown to pro-
long cell viability and, in some cases, enhance cell-specific
productivity either through growth arrest phenomenon or
through the modulation of cold-sensitive gene expression
[27••]. Other studies have identified media additives
directed specifically towards the inhibition of apoptosis
[40,41]. In a related study, rapomycin was fed to hybridoma
cultures in an effort to reduce cell death via cell-cycle
modulation, yet was found to have a broadly beneficial
effect on growth and productivity in this system [42].
Microbial expression systems
The primary microbial host for producing recombinant
therapeutic proteins has been E. coli, and recent reviews
have provided excellent summaries of key elements of this
topic [2,43].
In many cases, the production of soluble, active protein is
desired (as opposed to inclusion-body formation), and a
 Recombinant protein expression for therapeutic applications Andersen and Krummen
variety of cases where proteins with chaperone-like activity
have enhanced folding have been recently reported. DegP
represents one interesting case as it has been demonstrated
to have chaperone-like activity in at least two cases at
reduced temperature, along with its known protease activity
at higher temperatures [44,45]. Recent work has shown
that mutants with modulated activities of the disulfide
oxidoreductases DsbA and DsbC can be used to enhance
disulfide isomerization and protein folding in the
periplasm [46]. These results complement earlier work
showing the dramatic improvements in yields that can be
achieved in some cases by overexpressing these proteins
[47]. Overexpression of the periplasmic peptidylprolyl
isomerase FkpA has been shown to increase yields of a
single-chain Fv fragment (scFv), although the effect is
apparently independent of the peptidylprolyl isomerase
activity [48]. In an alternative approach, Fab antibody
fragments were expressed in the cytoplasm of trxB mutants
in which a variety of chaperones were co-expressed and
seen to have effects on folding [49••]. ClpG and HtbG
have also been shown to facilitate protein folding in E. coli
[50], a result that highlights the beneficial role that stress
proteins can have in protein folding. Collectively, these
results demonstrate the utility of co-expressing specific
factors to modulate the folding environment.
Although the various promoter systems commonly used for
E. coli have been well described [43], several recent reports
have demonstrated new options. In one particularly notable
result, secondary structure was engineered into mRNA tran-
scripts to manipulate message stability and, correspondingly,
expression level [51••]. This provides another technique,
along with manipulation of translation initiation strength via
the ribosome-binding site [52], for controlling the relative
strength of expression of sets of genes independently. Other
recent reports have developed and demonstrated useful
protein expression systems for different applications via the
araE, rhaBAD and nar promoters, which use arabinose,
rhamnose and microaerobic conditions, respectively, to
induce recombinant protein expression [53–55].
As in the case of mammalian systems, much recent work
has focused on metabolic factors that might more broadly
improve the effectiveness of recombinant E. coli for
protein production in high cell density fermentors.
Historically, acetate accumulation has provided perhaps
the best example of such an issue for E. coli, and a variety
of potential solutions have been reported (see [2•]). Recent
strategies to address this issue include the development of
more sophisticated glucose feeding strategies to minimize
acetate accumulation and the use of a pyruvate kinase
mutant [56,57]. The use of E. coli expressing Vitreoscilla
hemoglobin to improve growth (and product yield) in
microaerobic fermentor environments has also been further
developed. Mutagenesis techniques have been used to
generate improved hemoglobins for this task, and other
bacteria have been screened for alternative hemoglobin-
like proteins [58,59]. Other studies have used techniques
119
such as nuclear magnetic resonance (NMR)-based flux
analysis to address questions regarding the mechanism of
the hemoglobin benefit [60,61].
Recombinant protein expression has also been shown to
elicit a variety of stress responses in E. coli, some of which
were recently evaluated using DNA microarrays [62]. These
stress responses, in turn, may be highly beneficial for recom-
binant protein expression; for example, the presence of
stress-induced proteins was recently shown to increase the
folding and formation of soluble human fibroblast growth
factor 2 [63]. A strategy of inducing various stress responses
before recombinant protein expression has been recently
evaluated and revealed one case where the addition of
dithiothreitol 20 min before induction of a green fluorescent
protein–chloramphenicol acetyltransferase fusion protein
led to increased activity of the model protein following
induction [64]. Another report investigated the role that FIS
(a DNA-binding protein implicated in stimulating ribosome
synthesis) overexpression might have in increasing heter-
ologous protein expression [65]. Finally, the possibility of a
role for quorum-sensing mechanisms and the characteriza-
tion of complex metabolic oscillations in recombinant E. coli
fermentations highlight the complexity and the continuing
potential for surprises [66,67]. In a different study, over-
expression of the rspAB operon (involved in repression of
σs-regulated proteins) led to improved recombinant protein
production, presumably via a homoserine lactone-depen-
dent mechanism; however, the possibility of cell-to-cell
signaling effects was also raised in this study [68].
With the increasing volume of results from E. coli systems, par-
ticularly as a result of the application of genomic and proteomic
technologies, the utility of mathematical frameworks to inte-
grate and analyze this data has become more apparent. Recent
models addressing this issue have been described both at the
whole-cell level [69,70] and for specific elements such as
protein folding and aggregation [71] and stress responses [72].
Other expression systems
Both transgenic plants and animals, as well as plant tissue
cultures, have been used to produce a variety of different
recombinant proteins (reviewed in [73–75]). Whereas
limits in glycosylation have been cited as one challenge for
recombinant protein production in plants, the problem of
proteolysis was also evaluated in a recent report on the
potentially important application of antibody production in
plant culture [76••].
Insect cells have been used in a variety of protein expression
applications, particularly related to high-throughput expres-
sion of sequences for functional screening. One recent major
focus has been on attempts to generate proteins with fully
sialylated, complex oligosaccharides — a topic of some his-
torical controversy (see [77]). Overexpression of appropriate
galactosyltransferases and sialyltransferases has led to some
success in generating sialylated oligosaccharides on insect-
derived proteins. It appears, however, that other limitations
120 Biochemical engineering
might also be important for the consistent production of
highly sialylated proteins [78,79,80••,81,82]. These include
the deficiency of the N-acetylglucosaminyltransferase II
enzyme responsible for initiation of the second complex-
type antennae of the oligosaccharide structure, as well as the
production of the cytidine monophosphate-NeuAc precursor
and host-specific effects.
Yeast systems, of which the two major systems are
Saccharomyces cerevisiae and Pichia pastoris, are also being
pursued. The use of P. pastoris for the production of recom-
binant human chitinase for clinical studies was recently
described in a perfusion system, yielding in excess of
300 mg/L/day [83]. P. pastoris has also been utilized in a
fed-batch system to produce an insulin precursor at yields
of 1.5 g/L [84]. Other reports have investigated more
complex feeding strategies for these systems and include a
case for the production of a recombinant scFv antibody
fragment [85,86]. Recent studies with S. cerevisiae have
explored both the strong cell-cycle dependence and meta-
bolic burden of heterologous protein secretion in these
cultures. This work provides both interesting points for
comparison with analyses in microbial and mammalian sys-
tems, as well as an improved fundamental understanding of
the use of yeast for recombinant protein production [87,88].
The efficiency of cell-free expression systems also continues
to improve, both for more traditional systems using cell
extracts and for systems reconstituted from purified
components [89,90••] (reviewed in [2•]). Although currently
useful for high-throughput screening applications, it will
be interesting to see in coming years to what extent these
systems could be useful for larger scale production needs.
Linking expression and purification
Increasing attention has been paid to optimizing expression
systems in the context of the production process to aid in
protein recovery and to improve overall yields and effi-
ciencies. For example, the complementary expression of
holin and lysozyme enzymes in E. coli to facilitate the
release of products has been demonstrated for two differ-
ent cases [91–93] The co-expression of an endonuclease
was also used to improve the properties of the lyzed
fermentation broth for subsequent processing [91]. A very
thorough demonstration of the integration of fermentation
and recovery was reported for human chymotrypsin B pro-
duction in P. pastoris [94•,95]. In this analysis, high-density
fed-batch fermentations and a lower density continuous
process were evaluated with alternative recovery pro-
cedures, including two-phase extraction and expanded-bed
chromatography, to develop an integrated process solution.
In another report, a new affinity tag to provide additional
means for efficient purification of the protein of interest
was demonstrated [96].
Conclusions
In recent years, a variety of different platforms have been
investigated for the production of recombinant therapeutic
proteins. CHO and NS0 murine myeloma cells are the
predominant mammalian cell lines used, and recent progress
has been made in promoter systems and in the manipulation
of these cell lines to improve glycoprotein production in
fermentors. Rational engineering of E. coli has also contin-
ued, with notable advancements in improving the cellular
environment for protein folding. Progress has been made in
addressing the potential limitations of a variety of other
systems including yeast, insect and plant culture systems.
One general trend across these platforms has been an expan-
sion in the focus of study. Whereas in the past work has
focused specifically on heterologous product protein expres-
sion, it has now expanded to include efforts to improve
global cellular properties to enable more efficient and effec-
tive production in industrial environments. Another recent
trend has been an increased focus on integrated bioprocessing
approaches, such as the development of features in expres-
sion systems specifically targeted towards improving
recovery and purification of the protein of interest.
Acknowledgements
The authors would like to thank John Joly, Brad Snedecor and Gian Polastri
for discussions and critical review of the manuscript.
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