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Recombinant protein expression for therapeutic applications

Dana C Andersen* and Lynne Krummen†
In recent years, the number of recombinant proteins used for Mammalian expression systems
therapeutic applications has increased dramatically. Many of Over the past seven to eight years there has been consid-
these applications involve complex glycoproteins and erable progress in fine-tuning mammalian expression
antibodies with relatively high production needs. These systems for high-level recombinant gene expression. CHO
demands have driven the development of a variety of and NS0 murine myeloma cell expression systems have
improvements in protein expression technology, particularly now established themselves as the predominant systems of
involving mammalian and microbial culture systems. choice for mammalian expression. Refinements of vector
construction, choice of selectable markers and advances in
Addresses gene-targeting and high-throughput screening strategies
Cell Culture & Fermentation Research & Development, Genentech, Inc., have made the establishment of recombinant cell lines
1 DNA Way, South San Francisco, CA 94080, USA with high specific productivities (20–60 pg/cell/day for
*e-mail: andersen@gene.com
† e-mail: krummen.lynne@gene.com antibody cell lines) relatively common and have reduced
the time required for cell line development. Recent
Current Opinion in Biotechnology 2002, 13:117–123 advancements in expression technologies using traditional
0958-1669/02/$ — see front matter viral-promoter-based expression vectors include the devel-
© 2002 Elsevier Science Ltd. All rights reserved. opment and refinement of dicistronic expression strategies
using either internal ribosome entry site (IRES) sequences
CHO Chinese hamster ovary
[4–7] or alternative splicing [8,9]. These strategies are
BHK baby hamster kidney 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
Introduction constructs [10••] promise further advances in the augmen-
As the biotechnology industry has rapidly expanded in tation of promoter activity in the near future.
recent years, the expression of a spectrum of recombinant
proteins in different systems for a wide variety of purposes A variety of novel promoter systems are also being devel-
has been a major feature and challenge. In some applica- oped and evaluated both for recombinant drug product
tions, a large array of proteins are needed in relatively expression and for directed metabolic engineering
small quantities for screening applications, whereas in approaches (see [11]). For example, both tetracycline- and
other cases, quantities approaching the metric ton scale are streptogramin-based gene regulation systems have recently
needed for specific therapeutic applications. The majority been demonstrated to be highly useful for regulatable
of therapeutic proteins have been produced in either expression in CHO and other mammalian cell cultures
mammalian cell-culture systems, with Chinese hamster [12••,13]. These systems promise to be useful for multi-
ovary (CHO) cells representing the most common system, component control strategies and for the expression of
or in Escherichia coli [1,2•]. A variety of alternative expres- products which themselves promote cytostasis or cytotoxicity.
sion systems are also being developed and evaluated. It is
not at all clear which of these systems will ultimately be For the alternative application of expressing high numbers of
the most useful for the variety of niches and applications of proteins for screening applications, an alphavirus-based sys-
therapeutic protein production. tem has been reported [14]. Transient systems, in which the
isolation of stable transfectants is bypassed so that protein
Several excellent reviews have provided comprehensive expression is obtained rapidly but only for a limited period of
coverage of various aspects of therapeutic protein produc- time, have also been shown capable of producing reasonable
tion, including specific issues related to large-scale cell protein levels (10–20 mg/L) in reduced times [15]. These
culture production [1] and considerations for the produc- systems are providing an increasing array of tools to enable
tion of specific classes of molecules such as recombinant the manipulation of large panels of genes efficiently.
antibody-related products [3]. This review will focus on
selected results across a range of expression systems One of the most notable advances in recent years has been
(although focusing primarily on mammalian and E. coli the application of genetic engineering approaches to ratio-
cell-culture systems) that illustrate important ways in nally modify specific features of mammalian host cells to
which expression technology is evolving to meet the improve their utility in recombinant protein expression
spectrum of research, development and commercial needs. applications. One such example is in the area of glycosyla-
Rather than provide a comprehensive review of the tion control. Work over the past decade has demonstrated
subject, this review will highlight several specific, recent how various reactions in the glycosylation pathway can be
results across the field. influenced by cell-culture factors, host-cell selection and
118 Biochemical engineering

protein-specific features (reviewed in [16,17,18•]), leading effects and the method of growth control in controlling
to the production of molecules with variable or suboptimal growth-related productivity effects (see [30–32]).
clearance or bioactivity properties. Recent studies have
shown that specific manipulation of oligosaccharide Similarly, work demonstrating the ability of chemical
structures on a recombinant protein by overexpression of inhibitors of apoptosis to extend culture viability has laid
appropriate glycosyltransferases can enhance glycan the foundation for a growing area of work directed towards
quality. Improvements are made either by increasing the genetic engineering of cell death pathways. Overexpression
homogeneity of native structures or by introducing non- of Bcl-2 and BclxL has been shown to inhibit apoptotic
host-cell residues to specialize glycan quality and function. death in several different mammalian culture systems (see
For example, the overexpression of a galactosyltransferase [33,34]). A recent report also showed increased volumetric
and a sialyltransferase in CHO cells led to corresponding antibody expression in butyrate-treated CHO cultures
increases in the galactose and sialic acid content of overexpressing Bcl-2 [35]. Although promising, the effects
expressed recombinant therapeutic proteins [19••]. Other observed in these studies have been relatively modest and
groups have successfully overexpressed N-acetylglu- variable depending on the cell line and the apoptotic stimuli.
cosaminyltransferase III to increase the fraction of bisecting This is perhaps not surprising given the complexity and
N-acetylglucosamine residues on antibodies produced in redundancy of the regulatory pathways involved in apop-
CHO cells [20,21]. Sialic acid has also been introduced in tosis, and multifaceted approaches may be required to
an α2,6-linkage to glycoproteins synthesized by CHO and achieve significant benefits. Researchers are now expand-
baby hamster kidney (BHK) cells that lack the specific ing their approaches to include inhibitors of caspase
sialyltransferase responsible for this transfer [18•,22,23]. activation and mitochondrial cytochrome c release. A recent
genetic engineering study using overexpression of Bcl-2
Another focus of genetic manipulation has been to improve deletion mutants in CHO and BHK cells also suggested
the efficiency of central carbon metabolism and to reduce that the inherent susceptibility of wild-type Bcl-2 to intra-
lactate accumulation. Earlier work showed that overexpres- cellular degradation may explain some of the limited effects
sion of a pyruvate carboxylase gene in BHK cells reduced on productivity that have been reported for Bcl-2-based
lactate accumulation and improved cell yields on glucose, strategies [34,36••].
although it is not clear if this approach would be helpful in
CHO cultures [24]. In an alternative approach, partial In addition to genetic approaches, several recent studies
disruption of the gene encoding lactate dehydrogenase A show that environmental control strategies continue to be
(LDH-A) led to reduced lactate and improved cell-culture useful for manipulating glycosylation as well as carbon
performance in hybridoma cultures [25]. metabolism, cell growth and cell death. For instance,
medium supplementation with the nucleotide sugar pre-
Perhaps the most interesting of the targets for metabolic cursors glucosamine and N-acetylmannosamine enabled
control have been highlighted by recent studies to control some level of glycosylation control of a recombinant glyco-
cell growth and to limit apoptotic cell death. Several protein produced in NS0 and CHO cultures [37,38].
previous studies have shown that recombinant protein Additionally, advancements in fed-batch and batch media
production may be linked to the growth state of the compositions have dramatically improved viable cell den-
culture. These studies have shown both growth-associated sity and the duration of serum-free cell culture through a
and inverse growth-associated production in different general retardation of onset of apoptosis (e.g. [39]).
systems. Recent attempts to exploit this phenomenon Likewise, reduced temperatures have been shown to pro-
have used inducible expression of the product gene in long cell viability and, in some cases, enhance cell-specific
concert with a regulatory protein (such as p27) that induces productivity either through growth arrest phenomenon or
cell-cycle arrest at the G1/S phase border. These studies through the modulation of cold-sensitive gene expression
support the hypothesis that growth arrest, at least in this [27••]. Other studies have identified media additives
case, is associated with as much as a 15-fold increase in directed specifically towards the inhibition of apoptosis
specific productivity [26,27••]. Increased productivity has [40,41]. In a related study, rapomycin was fed to hybridoma
also been observed in response to temperature shift and in cultures in an effort to reduce cell death via cell-cycle
BHK cultures in which cell proliferation was controlled by modulation, yet was found to have a broadly beneficial
expression of the tumor suppressor IRF-1 (interferon effect on growth and productivity in this system [42].
regulatory factor 1) [27••,28,29]. In both instances, however,
direct effects of temperature and IRF-1 on transcriptional Microbial expression systems
and/or translational control may play a role. Whether or not The primary microbial host for producing recombinant
growth control can be balanced with enhancements in therapeutic proteins has been E. coli, and recent reviews
cell-specific productivity in such a way as to significantly have provided excellent summaries of key elements of this
enhance volumetric productivity remains to be seen. topic [2,43].
Although the potential benefits of these approaches are
encouraging, questions remain regarding the general appli- In many cases, the production of soluble, active protein is
cation of the specific promoter systems, cell-line-specific desired (as opposed to inclusion-body formation), and a
Recombinant protein expression for therapeutic applications Andersen and Krummen 119

variety of cases where proteins with chaperone-like activity such as nuclear magnetic resonance (NMR)-based flux
have enhanced folding have been recently reported. DegP analysis to address questions regarding the mechanism of
represents one interesting case as it has been demonstrated the hemoglobin benefit [60,61].
to have chaperone-like activity in at least two cases at
reduced temperature, along with its known protease activity Recombinant protein expression has also been shown to
at higher temperatures [44,45]. Recent work has shown elicit a variety of stress responses in E. coli, some of which
that mutants with modulated activities of the disulfide were recently evaluated using DNA microarrays [62]. These
oxidoreductases DsbA and DsbC can be used to enhance stress responses, in turn, may be highly beneficial for recom-
disulfide isomerization and protein folding in the binant protein expression; for example, the presence of
periplasm [46]. These results complement earlier work stress-induced proteins was recently shown to increase the
showing the dramatic improvements in yields that can be folding and formation of soluble human fibroblast growth
achieved in some cases by overexpressing these proteins factor 2 [63]. A strategy of inducing various stress responses
[47]. Overexpression of the periplasmic peptidylprolyl before recombinant protein expression has been recently
isomerase FkpA has been shown to increase yields of a evaluated and revealed one case where the addition of
single-chain Fv fragment (scFv), although the effect is dithiothreitol 20 min before induction of a green fluorescent
apparently independent of the peptidylprolyl isomerase protein–chloramphenicol acetyltransferase fusion protein
activity [48]. In an alternative approach, Fab antibody led to increased activity of the model protein following
fragments were expressed in the cytoplasm of trxB mutants induction [64]. Another report investigated the role that FIS
in which a variety of chaperones were co-expressed and (a DNA-binding protein implicated in stimulating ribosome
seen to have effects on folding [49••]. ClpG and HtbG synthesis) overexpression might have in increasing heter-
have also been shown to facilitate protein folding in E. coli ologous protein expression [65]. Finally, the possibility of a
[50], a result that highlights the beneficial role that stress role for quorum-sensing mechanisms and the characteriza-
proteins can have in protein folding. Collectively, these tion of complex metabolic oscillations in recombinant E. coli
results demonstrate the utility of co-expressing specific fermentations highlight the complexity and the continuing
factors to modulate the folding environment. potential for surprises [66,67]. In a different study, over-
expression of the rspAB operon (involved in repression of
Although the various promoter systems commonly used for σs-regulated proteins) led to improved recombinant protein
E. coli have been well described [43], several recent reports production, presumably via a homoserine lactone-depen-
have demonstrated new options. In one particularly notable dent mechanism; however, the possibility of cell-to-cell
result, secondary structure was engineered into mRNA tran- signaling effects was also raised in this study [68].
scripts to manipulate message stability and, correspondingly,
expression level [51••]. This provides another technique, With the increasing volume of results from E. coli systems, par-
along with manipulation of translation initiation strength via ticularly as a result of the application of genomic and proteomic
the ribosome-binding site [52], for controlling the relative technologies, the utility of mathematical frameworks to inte-
strength of expression of sets of genes independently. Other grate and analyze this data has become more apparent. Recent
recent reports have developed and demonstrated useful models addressing this issue have been described both at the
protein expression systems for different applications via the whole-cell level [69,70] and for specific elements such as
araE, rhaBAD and nar promoters, which use arabinose, protein folding and aggregation [71] and stress responses [72].
rhamnose and microaerobic conditions, respectively, to
induce recombinant protein expression [53–55]. Other expression systems
Both transgenic plants and animals, as well as plant tissue
As in the case of mammalian systems, much recent work cultures, have been used to produce a variety of different
has focused on metabolic factors that might more broadly recombinant proteins (reviewed in [73–75]). Whereas
improve the effectiveness of recombinant E. coli for limits in glycosylation have been cited as one challenge for
protein production in high cell density fermentors. recombinant protein production in plants, the problem of
Historically, acetate accumulation has provided perhaps proteolysis was also evaluated in a recent report on the
the best example of such an issue for E. coli, and a variety potentially important application of antibody production in
of potential solutions have been reported (see [2•]). Recent plant culture [76••].
strategies to address this issue include the development of
more sophisticated glucose feeding strategies to minimize Insect cells have been used in a variety of protein expression
acetate accumulation and the use of a pyruvate kinase applications, particularly related to high-throughput expres-
mutant [56,57]. The use of E. coli expressing Vitreoscilla sion of sequences for functional screening. One recent major
hemoglobin to improve growth (and product yield) in focus has been on attempts to generate proteins with fully
microaerobic fermentor environments has also been further sialylated, complex oligosaccharides — a topic of some his-
developed. Mutagenesis techniques have been used to torical controversy (see [77]). Overexpression of appropriate
generate improved hemoglobins for this task, and other galactosyltransferases and sialyltransferases has led to some
bacteria have been screened for alternative hemoglobin- success in generating sialylated oligosaccharides on insect-
like proteins [58,59]. Other studies have used techniques derived proteins. It appears, however, that other limitations
120 Biochemical engineering

might also be important for the consistent production of proteins. CHO and NS0 murine myeloma cells are the
highly sialylated proteins [78,79,80••,81,82]. These include predominant mammalian cell lines used, and recent progress
the deficiency of the N-acetylglucosaminyltransferase II has been made in promoter systems and in the manipulation
enzyme responsible for initiation of the second complex- of these cell lines to improve glycoprotein production in
type antennae of the oligosaccharide structure, as well as the fermentors. Rational engineering of E. coli has also contin-
production of the cytidine monophosphate-NeuAc precursor ued, with notable advancements in improving the cellular
and host-specific effects. environment for protein folding. Progress has been made in
addressing the potential limitations of a variety of other
Yeast systems, of which the two major systems are systems including yeast, insect and plant culture systems.
Saccharomyces cerevisiae and Pichia pastoris, are also being One general trend across these platforms has been an expan-
pursued. The use of P. pastoris for the production of recom- sion in the focus of study. Whereas in the past work has
binant human chitinase for clinical studies was recently focused specifically on heterologous product protein expres-
described in a perfusion system, yielding in excess of sion, it has now expanded to include efforts to improve
300 mg/L/day [83]. P. pastoris has also been utilized in a global cellular properties to enable more efficient and effec-
fed-batch system to produce an insulin precursor at yields tive production in industrial environments. Another recent
of 1.5 g/L [84]. Other reports have investigated more trend has been an increased focus on integrated bioprocessing
complex feeding strategies for these systems and include a approaches, such as the development of features in expres-
case for the production of a recombinant scFv antibody sion systems specifically targeted towards improving
fragment [85,86]. Recent studies with S. cerevisiae have recovery and purification of the protein of interest.
explored both the strong cell-cycle dependence and meta-
bolic burden of heterologous protein secretion in these Acknowledgements
cultures. This work provides both interesting points for The authors would like to thank John Joly, Brad Snedecor and Gian Polastri
for discussions and critical review of the manuscript.
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]. References and recommended reading
Papers of particular interest, published within the annual period of review,
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The efficiency of cell-free expression systems also continues
to improve, both for more traditional systems using cell • of special interest
•• of outstanding interest
extracts and for systems reconstituted from purified
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with defining transcriptional control regions to provide enhancement of
In recent years, a variety of different platforms have been stable or regulated gene expression in CHO cells when included on the
investigated for the production of recombinant therapeutic expression plasmid or as a co-transfected element. The authors identify the
Recombinant protein expression for therapeutic applications Andersen and Krummen 121

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