Received: 23 August 2010 Revised: 11 November 2010 Accepted: 12 November 2010 Published online in Wiley Online Library:
(wileyonlinelibrary.com) DOI 10.1002/jctb.2576
Engineering a mammalian super producer
Stefanie Dietmair,∗ Lars K. Nielsen and Nicholas E. Timmins
Mammalian cells are the preferred host for the manufacture of a wide range of biopharmaceuticals, but production costs are high owing to low productivity. A range of rational engineering strategies have been pursued in order to increase volumetric product titres from mammalian cells, such as delaying apoptosis, manipulation of the cell cycle, and improving metabolism and protein processing. Unfortunately, outcomes from these strategies have been mixed, with few instances where signiﬁcant improvements in product yield have been achieved. This article reviews and contrasts many of the engineering strategies attempted to date, highlighting the variability and context speciﬁcity in outcome. The paper argues that this is a reﬂection of the complexity of mammalian cells, and that a deeper understanding of the biology underpinning protein production for biotechnological purposes is required. c 2011 Society of Chemical Industry Keywords: mammalian cell; host cell engineering; review
The market for biologics is one of the fastest growing sectors of the pharmaceutical industry, achieving total sales of over $125 billion in 2009.1 With medical applications ranging from autoimmune, cardiovascular, and infectious diseases through to cancer, this growth is being driven by treatments for both new and existing indications alike. A large proportion of these biopharmaceuticals are complex recombinant proteins (e.g. antibodies) for which correct post-translational modiﬁcations are critical to function and safety.2 Consequently, these products are typically produced in mammalian cells. Compared with bacteria and yeast, mammalian cell culture is time consuming, expensive, and productivities are low. Many biopharmaceuticals must be administered at high doses, and low productivity translates to high treatment costs (e.g. Herceptin at US$ 60 000 per patient per year for treatment of breast cancer;3 Avastin at US$ 40 000 per patient per year for treatment of colon cancer4 ). These costs are unsustainable for health care systems, and constitute a signiﬁcant economic burden to national budgets. Increasing the product titre of mammalian cell cultures could lessen this burden by reducing the cost of production, relieving pressure on limited health resources and improving accessibility to life saving treatments. Three main approaches have been pursued in order to increase productivity of mammalian cell cultures: (1) improvement of expression vectors and transcription of the gene of interest (reviewed by Birch and Racher)5 ; (2) improvement of media composition and cultivation process (reviewed by Birch and Racher5 and Jain and Kumar6 ); and (3) host cell engineering (reviewed by Lim et al.,2 Mohan et al.,7 Schroeder,8 and Sunley and Butler9 ). Optimization of expression vectors has led to substantial improvements in transcription efﬁciency of genes encoding the product of interest. A recent study by Jacob and co-workers10 analysing the transcriptome of Chinese hamster ovary (CHO) cells producing immunoglobulin (IgG), showed that the IgG heavy chain transcripts were the most abundant transcripts in these cells, with light chain transcripts being the third most abundant. Similarly, media and process optimization have led
to signiﬁcant improvements in culture performance. This was dramatically illustrated by Wurm11 through a comparison of typical cell densities and culture durations in 1986 compared with 2004. Process optimizations during this period led to a ﬁve-fold increase in cell densities, a three-fold increase in culture duration, and a 100-fold increase in product titre.11 Product titre is determined by the cell speciﬁc productivity (q) as well as the integral viable cell density. While host cell engineering strategies targeting either or both aspects have proven successful, the successes have been highly context dependent (e.g. host cell, product, culture mode) and overall, outcomes remain mixed. In several instances, positive outcomes for one characteristic (e.g. enhanced q) are coupled to a negative impact on the other (e.g. reduced integral viable cell density), with little or no improvement in ﬁnal product titre as a result. More elegant approaches to identify and modify speciﬁc target mechanisms, or co-ordinate sets of mechanisms, in a predictable manner are required. This review article summarizes and contrasts the outcomes of host cell engineering strategies to date, illustrating that a much deeper understanding of the integrated cellular machine is required if we are to realize the full potential of cellular engineering strategies for generation of a mammalian super producer.
INCREASING INTEGRAL VIABLE CELL DENSITIES THROUGH HOST CELL ENGINEERING
Apoptosis One of the main factors limiting protein production in mammalian cells, is the onset of apoptosis (programmed cell death) when conditions are unfavourable for growth. Depletion of growth
Correspondence to: Stefanie Dietmair, Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, 4072 QLD, Australia. E-mail: firstname.lastname@example.org Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, 4072 QLD, Australia
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baby hamster kidney (BHK).16 – 21 BCL-XL.g. KSBCL2.22 XIAP23 and AVEN24 ) has been shown to increase the survivability of common mammalian production cell lines (e.15 Caspase-8 activates caspase-3 and -7. Alg-2. (2) the death receptor mediated pathway.111
factors or nutrients (e.15 (2) The death receptor pathway is initiated by binding of a ligand (e. glutamine). E1B-19K.15 Cytochrome c binds to apoptotic protease activating factor 1(APAF 1) and caspase-9. XIAP. Eliminating or delaying apoptosis has the potential to both increase viable cell densities and extend culture durations. glucose. ammonia (NH4 )) and high osmolarity. BAX. where it can be taken up by mitochondria through a uniporter.16. FasL) to a death receptor. including up-regulation of anti-apoptotic proteins.12 – 14 The consequent decrease in viable cell density (VCD) limits product yields. but speciﬁc productivity was substantially reduced and no improvement in antibody yield was obtained. including both pro. expression of viral anti-apoptotic proteins (e.g. PUMA. high concentrations of ammonia or lactate. BCL-XL.g. oxygen deprivation. and a variety of strategies to delay its activation have been successfully employed. CRMA).29 Bierau et al. 1). Overexpression of several different anti-apoptotic proteins (e. The underlying biology of apoptosis has been studied extensively15 (Fig.29 Itoh et al.30 reported a reduction in both speciﬁc productivity and ﬁnal titre for BCL-2 overexpressing hybridoma cells.. BCL-2. LK Nielsen and NE Timmins
Figure 1. BHRF. accumulation of toxic by-products (e.
NS0.31 achieved a 2-fold improvement in antibody titre.g.110 (1) The mitochondria mediated pathway is controlled by the B-cell lymphoma-2 (BCL-2) family of proteins.111 High Ca2+ concentration in the mitochondria leads to opening of the permeability transition pore (PTP).15 This activates FADD (Fas-associated death domain protein).g.27.g. improper protein folding or glycosylation.(e.g.com/jctb
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. in turn activating caspase-9. reduced disulﬁde formation).25 showed that overexpression of BCL-2 in hybridoma cells increased the duration of batch cultures by 4 days. MDM2. including overexpression of endogenous anti-apoptotic proteins (e.g.org
S Dietmair. BCL-2 overexpression in NS0 perfusion cultures resulted in a 5-fold increase in cell density. AVEN).112 ER stress can also lead to the release of Ca2+ into the cytosol.15 Alternatively. BCL-2. HSP27. NOXA) and anti-apoptotic proteins (e.26 and fed batch cultures. increased VCD 1. while Fassnacht et al. CHO. HSP70. Figure adapted from Taylor et al. which subsequently activates caspase-8.g. which in turn induces translocation of BAX to the mitochondrial membrane and oligomerization with BAK.soci. and downregulation of pro-apoptotic proteins (e. resulting in increased ﬁnal product titres.15 . lactate.15 Oligomerization of BAX and BAK in the mitochondrial membrane results in the formation of pores releasing cytochrome c. are known to trigger apoptosis in mammalian cell cultures.g. virus infection.www. and resulted in a 4-fold increase in ﬁnal antibody titres.g. BID. serum and nutrient deprivation. initiating the caspase cascade and resulting in apoptosis. cytochrome C) into the cytosol.5-fold.
wileyonlinelibrary. and release of pro-apoptotic factors (e. resulting in apoptosis. BAD. 30KC6.110 Various apoptotic insults can lead to an increase in abundance of pro-apoptotic factors.g.110 Although the exact mechanism of the ER stress mediated pathway is not yet fully understood112 it has been shown that ER stress can activate caspase-12. Three main pathways lead to apoptosis in mammalian cells: (1) the mitochondria mediated pathway. Arden and Betenbaugh110 and Orrenius et al. leading to translocation of BAX to the mitochondrial membrane. BCL-XL). Apoptosis in mammalian cells.g. and (3) the ER stress mediated pathway.111 A range of strategies have been pursued in order to inhibit apoptosis in mammalian cell cultures. BCL-2. down-regulation of pro-apoptotic proteins and disruption of apoptotic pathways.111 Formation of the apoptosome leads to activation of caspase-9. HEK293. requiem)..25 In contrast. hybridoma) when subjected to a variety of apoptotic stimuli (e.17 MCL-1.. Several studies investigating overexpression of anti-apoptotic proteins have also demonstrated improved cell viabilities in the later stages of batch25. chemicals inducing apoptosis).28 and during perfusion culture.15 (3) The ER mediated pathway can be initiated by various perturbations of the protein folding process (e. caspase-8 can activate the mitochondria mediated pathway by truncating BID (t-BID). forming the so called apoptosome.
HSP27 or HSP70 and HSP27.7. extended culture duration by 2 days. translocation of BAX to the mitochondria. A logical target for inhibition of apoptosis is the tumour suppressor protein p53. 1) using zinc-ﬁnger nucleases increased viable cell densities
www.8-fold. p53 upregulates expression of pro-apoptotic proteins such as BAX.33 Similar to overexpression of BCL-2 or BCL-XL.34 or NS0 cells. the frequency of apoptotic cells increased from approximately 6% to 12% on day 9 of culture. Consequently.5-fold increase in product titres respectively.6-fold improvement in product titre. E1B-19K (an adenoviral homolog of BCL-2).49 Enhancing cell proliferation Much as reducing cell death can lead to increases in cell density. nor the effects of dual expression of c-MYC and BCL-2 were reported. successfully increasing viable cell densities of adherent CHO cells in batch culture by 1. Both HSP70 and HSP27 inhibit death receptor mediated apoptosis.35 As for overexpression of mammalian derived anti-apoptotic proteins.Engineering a mammalian super producer Overexpression of BCL-XL in CHO cells producing anti-α1β1 integrin (VLA1) or anti-MCP1 antibodies led to enhanced viabilities over the duration of fed batch cultures. downstream up-regulation of p53 and BAX52 ) a follow-up study investigated the effects of overexpressing anti-apoptotic BCL-2 in addition to c-MYC. O’Connor and colleagues46 found that while expression of epidermal growth factor receptor ErB2 in CHO was barely detectable after 33 days.soci.2-fold increase in VCD for serum containing adherent CHO cultures. HEK293.53 and it has been demonstrated that speciﬁc productivity is linked to cell size.5-fold).32 BHK. HEK293.32 Simultaneous overexpression of HSP70 and HSP27 increased product titre by 2. so can increasing the rate of cell proliferation.43 expression of this protein enabled continued cell growth in serum deprived conditions resulting in 5-fold higher viable cell densities. Kuystermans and Al-Rubeai53 observed a reduction in doubling time by 4. and of HEK293 and CHO cells by 2-fold when cultured in spent medium. 1) has also proven successful. a 2.and 2.
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wileyonlinelibrary. 1). Although their exact roles in apoptosis are unknown. and PUMA (Fig. Majors et al. This can be achieved by manipulating components of the cell cycle regulatory machinery (Fig. suggesting a reduction in cell speciﬁc productivity.50 In serum-free CHO batch cultures overexpressing c-MYC. and its overexpression in adherent CHO cells increased viable cell densities by 1. and XIAP improved some aspects of culture performance.32 In contrast. The transcription factor c-MYC is a key mediator of cell cycle progression.42 Betenbaugh and colleagues utilized mammalian and nonmammalian anti-apoptotic proteins in combination.44 Although overexpression of AVEN. Members of the heat shock protein (HSP) family have also been utilized as a means to inhibit apoptosis.32 HSP overexpression also improved the productivity of BHK cells34 .51 This approach reduced the frequency of apoptotic and necrotic cells in both serum-containing and serum-free cultures by approximately 50% and 66% respectively. Cost et al. resulting in a modest 1.35 increased longevity in batch and fed batch cultures. reducing maximum product titre.42 to CHO.7and 1.to 3. However. in CHO.20 CRMA (cowpox virus)40 and 30KC6 (silkworm haemolyoh)41.1. with no improvement in peak viable cell density.41 Although the mechanism by which 30KC6 inhibits apoptosis is unclear.54 Also targeting cell cycle regulation. and formation of the apoptosome (Fig.8-fold.45 showed that when CHO DG44 cells stably expressing BCL-XL were transiently transfected to produce a therapeutic fusion protein. overexpression of HSP70. although no improvement was observed for NS0. While the majority of studies have investigated the effects of anti-apoptotic proteins in cell lines stably expressing a product of interest. increased cell viabilities in response to induced stress. this increase in VCD did not translate to a higher product yield. This suggests a survival advantage for ErB2 expressing CHO-BCL-XL compared with ErB2 expressing wild-type CHO. A major mediator of apoptosis in response to cell stress. although no signiﬁcant improvement in product titre was observed. much effort has been directed towards engineering mammalian cell metabolism to reduce NH4 and lactate production.com/jctb
. For example. with a modest 1.41 Expression of 30KC6 was also shown to extend culture duration of CHO cells producing IFN-γ in serum free suspension cultures by 3 days. One of the most dramatic examples of this strategy.56 increases in product titre were observed.5-fold in batch cultures with a 5-fold improvement in ﬁnal IgG yield. Overexpression of HSP70 in CHO cells producing IFNγ increased peak viable cell densities almost 2-fold.36 – 39 BHRF (Eppstein Barr virus). C-MYC overexpressing cells were 34% smaller than parental cells.6-fold in fed batch cultures containing serum.32. Improving metabolism Mammalian cells in culture typically exhibit high rates of nutrient consumption (especially glucose and glutamine) and due to metabolic inefﬁciencies.36.50 Neither the impact of this strategy on productivity.38..org of CHO cells 2. Reasoning that this increase in apoptosis is a side effect of c-MYC overexpression51 (e. HSP27 overexpression extended culture duration by 4 days and increased IFN-γ titre 2-fold.5-fold. product yields improved 1. and HeLa cultures. this strategy may also be of value in transient and unstable systems. In a further investigation of c-MYC overexpression. lactate and NH4 ) are generated. 1). NS0 or hybridoma cells. respectively. NOXA. increased growth rates and viable cell densities. correspondingly high concentrations of by-products (e. These byproducts can adversely affect cell growth and viability.6-fold in batch and 2.48 Arden et al. Yet no. overexpression of the transcription factor E2F-1 in suspension adapted CHO.7-fold compared with parental cells. and increased IFN-γ titre 1.20 KSBCL-2 (Kaposi sarcoma associated virus).51 Despite these performance improvements in adherent culture. improvements in ﬁnal product titre were only minor (up to 1.55 and overexpression of CDKL3 (cyclin dependent kinase like 3)56 in adherent CHO. down regulation of pro-apoptotic proteins (Fig. knockdown of apoptosis linked gene 2 (Alg-2) or requiem increased viable cell densities and improved IFN-γ titres by 2.4 h and a 1.55 or only slight.3-fold improvement in VCD was observed.28 Kim and co-workers26 observed that overexpression of BCL-XL in EPO producing CHO cells extended culture longevity. overexpression of c-MYC in suspension culture improved VCD by a modest 24%. is expression of 30KC6 in adherent CHO.47 demonstrated that knockout of BAX and BAK (Fig. As for upregulation of anti-apoptotic proteins.g. introduction of proteins such as E1B-19K (adenovirus). However. and a 10-fold increase in EPO titres.. expression was sustained in a CHO-BCL-XL host.48 inhibited p53 activity by expressing murine double mutant-2 (MDM2).g.5-fold.46 and the strategy may be of beneﬁt for other difﬁcult to express or cytotoxic proteins. 3).
achieving a 48% decrease in glucose uptake and 12% decrease in lactate/glucose yield. and cell densities in continuous culture increased 2. Nevertheless.6-fold in spinner ﬂasks. Kim and Lee61 found that LDH knockdown in CHO reduced speciﬁc lactate production by as much as 79%. CHO cells had a slower growth rate and lower peak VCD in batch culture. Oxaloacetate is subsequently converted to malate and transported into the mitochondria where it participates in the TCA cycle (Fig. culture duration was extended up to 5 days. or ﬂux through glycolysis. Lactate production can also be reduced by expression of pyruvate carboxylase.60 showed that LDH knockdown in a hybridoma line was sufﬁcient to reduce cell-speciﬁc lactate production by 2-fold. Central carbon metabolism in mammalian cells. where GS is used as selection marker and to amplify the copy number of the gene of interest. Similarly. Paredes et al.59 This technology also underpins the highly successful Lonza GS cell lines. overexpression of YPC in HEK293 cells decreased glucose and glutamine consumption. a 2-fold increase in EPO titre was obtained.jp/kegg/).5-fold.2-fold. Down regulation of lactate dehydrogenase (LDH) is a logical strategy by which to reduce lactate production. and 2. α-enolase. and glutamine synthetase (GS).to 1.3-fold. and improved product titre up to 2. and decreased lactate production up to 2.www.5-fold improvement in yield of rhGM-CSF. and improve antibody titre 3-fold. with a reduction in lactate and ammonia production in fed-batch cultures of up to 4-fold and 3-fold respectively.58. Chen et al.soci. and pyruvate carboxylase. resulting in an equal increase in viable cell density. also decreased lactate production. decreased NH4 concentration in CHO cell cultures up to 1. Down regulation
wileyonlinelibrary. 2). resulting in a 2.3-fold in continuous culture. and the glucose transporter GLUT1. 3)). (1) Reductions in NH4 production have been achieved through expression of carbamoyl phosphate synthase and ornithine transcarbamoylase (converting NH4 to citrulline). converting pyruvate to oxaloacetate.genome.com/jctb
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Introduction of carbamoyl phosphate synthetase I and ornithine transcarbamoylase (converting NH4 to citrulline (Fig. is to reduce glucose uptake.org
S Dietmair..64 The clone producing the lowest concentration of lactate and ammonia reached 30% higher cell densities. LK Nielsen and NE Timmins
Figure 2.57 Transfection of hybridoma cells with glutamine synthetase (GS – converts glutamate to glutamine) enabled cell growth in glutamine-free medium and was sufﬁcient to eliminate NH4 production. (http://www.65 Unlike BHK and HEK293 cells.2.65 An alternative strategy by which to decrease lactate concentrations in culture. Introduction of the fructose transporter GLUT5 (allowing cell growth on fructose). Figure modiﬁed from KEGG.63 Similarly. A range of metabolic engineering strategies have been pursued to reduce the concentration of (1) NH4 and (2) lactate. lactate production decreased by 1.62 Culture duration in batch mode was extended by 2–3 days. Expression of yeast pyruvate carboxylase (YPC) in BHK
cells reduced glucose and glutamine consumption.5-fold.64 Although CHO cells overexpressing YPC did not exhibit signiﬁcant decreases in glucose or glutamine uptake rate.62 In glucose-limited perfusion cultures. Mammalian cell lines exhibit an inefﬁcient ‘overﬂow’ metabolism.58 knocked down the glucose transporter GLUT1 in hybridoma cells. with high rates of glycolysis and glutaminolysis leading to the production of high concentrations of NH4 and lactate. (2) Reductions in lactate production have been achieved through knock-down of lactate dehydrogenase (LDH).
Lloyd and colleagues77 showed that for un-arrested producer CHO lines.g.114 The now active E2F-1 initiates transcription of a range of genes related to cell cycle progression (e.68 HeLa. or p53175P (a p53 mutant lacking apoptotic functionality). Whereas induction of growth arrest leads to increases in cell. Progression through the cell cycle is regulated by cyclins and cyclindependent kinases (CDKs).to 15-fold. and two intermediate phases (G1 and G2) during which the cell synthesizes all necessary components for the following S and M phase. P21CIP1 expressing cells continued to grow in size. with a 4-fold increase in cell volume.66 When cultured in fructose based medium. Although.54 showed that over expression of p21CIP1 in CHO cells producing an IgG4 antibody uncoupled cell growth and cell division.68 and BHK69 ) but also reduced q of BHK cells constitutively producing IgG.
Figure 3. binding to and activating CDK4. In stably transfected CHO cells. GLUT5-expressing cells produced less lactate than when cultured in glucose-containing medium.76 Although the underlying mechanisms by which cell cycle arrest enhances cell-speciﬁc productivity are not well understood. Regulation of cell cycle progression in mammalian cells.com/jctb
. and IgG4 productivity increased by approximately 4-fold.soci. follow-up studies investigated the effect of inducible IRF-1 expression on the production of proteins under the control of an IRF-1 inducible promoter. An early example of cellular engineering in this context was inducible expression of interferon regulatory factor-1 (IRF-1).78. respectively. CDK4 then phosphorylates retinoblastoma susceptibility protein (RB). 2). Indeed. C243. growth arrest naturally limits VCD. IRF-1 activation induced cell cycle arrest in a range of cell lines (e. Fussenegger et al.115 Association of Cyclin E with CDK2 enables transition from G1 to S phase. p27KIP1 .71 investigated the productivity of BHK cells producing Factor VII in perfusion culture and observed a decrease in q following activation of IRF-1.113 In response to growth stimuli. p27KIP1 . Speciﬁc productivity increased linearly with cell volume.79 For reasons summarized by Kumar et al.72 achieved a 4-fold increase in productivity on a per vector basis for transiently transfected CHO cells. more rapid proliferation results in higher integral viable cell density. similar to p27KIP1 . p53175P). Bi et al.80 induction of growth arrest by temperature shift is the current method of choice in industrial processes.g. ribosomal protein S6 abundance increased 2fold. and secretion Protein folding is a complex process mediated by a range of different proteins including chaperones (e. Geserick and coworkers70 observed an initial increase in q (up to 6-fold) of BHK cells producing IgG which gradually returned to the level before IRF-1 induction over the course of the culture. expression of p21CIP1 increased cell-speciﬁc productivities up to 4-fold.77 Although these cell cycle engineering strategies have resulted in signiﬁcant improvements in cell-speciﬁc productivity.69 At the same time. The mammalian cell cycle can be divided into four phases: the S phase during which DNA is synthesized. 2).58 Transfection of CHO cells with the fructose transporter GLUT5 enabled growth on fructose. volumetric product titres have shown little or no improvement.Engineering a mammalian super producer
www.g. cyclin E).70.69 Hence.54.81. temperature shifts. Cellular engineering strategies to manipulate the cell cycle have largely focused on induction of growth arrest through overexpression of cell cycle inhibitors (e. Calreticulin) and foldases (protein disulﬁde isomerase (PDI)) (Fig.to 15-fold increase in speciﬁc productivity. Employing a tetracycline inducible dicistronic vector for dual expression of the reporter protein SEAP and one of the cell cycle inhibitors p21CIP1 .66
INCREASING CELL SPECIFIC PRODUCTIVITY THROUGH HOST CELL ENGINEERING
Cell cycle arrest A common approach to enhancing q is to induce cell cycle arrest (Fig. IRF-1 activation increased the concentration of proteins under the control of an IRF-1 inducible promoter by 10fold. and several studies have suggested that protein folding can be a bottleneck in protein production. p27KIP1 improved cell-speciﬁc SEAP productivity by 10.54 Similarly.114 Progression through the remainder of the cell cycle is similarly regulated by the activity of cyclins and CDKs. cyclin D is expressed. co-expression of p21CIP1 with c/EBPα (a stabilizer and inducer of p21CIP1 ) arrested cells in G1.115
of α-enolase (converts 2-phosphoglycerate to phosphoenolpyruvate) decreased glucose uptake by 22% but did not signiﬁcantly reduce lactate/glucose yield.82 For example. On the other hand. causing its dissociation from the transcription factor E2F-1.54 In arrested cells. the major determinant of speciﬁc productivity was cell volume rather than cell cycle state. the clone with the lowest GLUT5 expression had the lowest lactate/fructose yield. and overexpression of c-MYC to enhance cell proliferation.. binding immunoglobulin protein (BiP). transport. while the clone with the highest GLUT5 expression had the highest lactate/fructose yield. or through cellular engineering (recently
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wileyonlinelibrary. This can be achieved through chemical agents. there was no clear correlation between the expression level of GLUT5 and lactate production.75 and expression of p27KIP1 in HEK293 EBNA cells resulted in an up to 6-fold increase in SEAP productivity.g. The clone with the lowest lactate production (and lowest GLUT5 expression). the M phase during which cell division occurs. p21CIP1 . with consequent reductions in ﬁnal product titre. achieved three times higher cell densities in fructose fed batch cultures compared with glucose.org reviewed by Sunley and Butler)67 .66 As for glucose.68 NIH 3T3. this suggests that fructose transporter abundance is important and that limiting sugar uptake may increase metabolic efﬁciency and reduce lactate production.speciﬁc productivity.74 Although stable transfectants for p21CIP1 failed to arrest growth. Calnexin. with a 10. Improving protein folding. mitochondrial mass increased 3-fold. Figure adapted from Dehay and Kennedy113 and Dzau et al.71 Carvalhal et al. glucose regulated protein of 94 kDa (GRP94). although progression through the cell cycle was arrested.73 Expression of the anti-apoptotic gene BCL-XL in addition to p27 (encoded on a tricistronic vector) improved speciﬁc productivity by 30-fold. Quiescent non-dividing cells are said to be in the G0 phase. Co-expression of any one of these proteins resulted in cell cycle arrest exclusively in the G1 phase.74 For NS0 cells producing IgG4 antibody.
and proteins involved in the ER associated degradation (ERAD) process. For example. providing a negative feedback mechanism for the PERK signalling pathway.117 Figure adapted from Hotamisligil. but did increase EPO production in transiently transfected cells whose productivity was signiﬁcantly higher. 4). Overexpression of PDI also failed to improve productivity of IL-15. and was retained intracellularly. Similarly.org
S Dietmair.92 found that XBP-1 overexpression failed to enhance production of antithrombin III.88 TNF receptor : Fc protein was found to complex with PDI.117 Schroeder8 and Ma and Hendershot116 .117 ER stress triggers the release of BiP form these proteins resulting in their activation. PDI overexpression increased cell-speciﬁc productivity up to 1.116 Perturbations of the folding process (e. as well as a number of proteins involved in ER associated protein degradation and attenuation of translation (Fig. mAb. although outcomes have been mixed.8 GADD34 leads to inactivation of EIF2α. PERK (dsRNA-activated protein kinase like ER kinase)96 and IRE1 (inositol-requiring enzyme 1) which are bound to BiP under normal circumstances.86 For antibody-producing CHO cells.8. 4). and proteins involved in ERAD.87 Conversely.g. a mutant form of Factor VIII and von Willebrand factor decreased cell-speciﬁc productivity89.83 A number of cellular engineering approaches aimed at improving productivity have focused on overexpression of proteins known to assist in protein folding (Fig. calnexin and calreticulin.86.8 CHOP downregulates anti-apoptotic proteins and upregulates pro-apoptotic proteins.8 IRE1 activation.com/jctb
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.88 Overexpression of BiP in CHO cells producing mAbs. Ohya et al. although complex formation between PDI and IL-15 was not observed.93 This study supports the logic that targeting protein folding and secretion mechanisms will be of most beneﬁt in those cell lines where these mechanisms are a limiting factor. foldases.93 reported that XBP-1 overexpression did not increase IFNγ .90 while production of M-CSF was not affected.soci.90 As overexpression of a single component of the protein folding machinery may result in bottlenecks elsewhere in the folding process. and its folding capacity. overexpression of X-box binding protein-1 (XBP1) in CHO cells increased SEAP. with the extent of reduction ranging from 20–80% between clones. but not in other cell lines (HEK293.g.117 BiP release from ATF6 initiates translocation of ATF6 to the Golgi apparatus.www. The UPR is a reaction to imbalances in the folding demand imposed on the ER. attempts have been made to simultaneously upregulate all components by activation of the unfolded protein response (UPR. Activation XBP-1 leads to expression of molecular chaperones. XBP-1 overexpression reduced cellspeciﬁc productivity.8 Activation of PERK results in activation eIF2α (eukaryotic translation activation factor eIF2α). protein foldases. leading to apoptosis.91 In CHO cells producing antithrombin.g.
a proteomic comparison of GS-NS0 cells with different antibody productivities revealed that high producers have higher concentrations of chaperones and foldases compared with low producers. Similar to XBP-1. SAMY and VEGF expression.8 To restore equilibrium in the ER. increased speciﬁc productivities of CHO cells producing antithrombin III two-fold.85 while overexpression of PDI had no apparent effect on productivity. PDI) assist in the folding of newly synthesised polypeptide chains in the ER. overexpression of proteins involved in the UPR resulted in mixed outcomes.8 Activated eIF2α increases translation of the transcription factor ATF4. BiP. PDI overexpression in CHO cells producing a TNF receptor : Fc fusion protein decreased productivity. increased q of CHO cells producing TPO approximately two-fold. activates caspase 12. the UPR upregulates expression of a range
of chaperones and foldases. Fig. potentially inducing apoptosis. Overexpression of the UPR associated protein activating transcription factor 4 (ATF4). which is associated with apoptosis (Fig. ATF4 also upregulates C/EBP-homologous protein (CHOP).92 Ku et al. 4).84. calnexin. but slightly reduced growth rates and maximum viable cell densities.92 In addition to enhancing expression of UPR associated genes. which attenuates general protein translation and can cause cell cycle arrest. A range of chaperones (e. calreticulin) and protein foldases (e. HeLa. which in turn activates CHOP (C/EBP-homologous protein) and GADD34 (growth arrest and DNA damage inducible protein 34). GRP94. activation of ATF6 leads to transcription of a range of chaperones. or EPO production in stable CHO or NS0 cells with relatively low productivity.4fold.92 Although CHOP upregulation
wileyonlinelibrary. and that XBP-1 mRNA was already highly expressed in parental cells.. Protein folding in the endoplasmatic reticulum (ER) and the unfolded protein response (UPR). 4). Overexpression of ERp57 (an isoform of PDI). HT1080).8 The main mediators of the UPR are ATF6 (activating transcription factor 6).8 IRE1 activation also leads to the production of active XBP-1. and its folding capacity. accumulation of unfolded proteins) trigger the UPR in an effort to restore the equilibrium between folding demand imposed on the ER. where it is proteolytically processed to yield its active form.8 Similar to overexpression of chaperones. LK Nielsen and NE Timmins
Adv Drug Deliv Rev 58:671–685 (2006).92 ATF4 overexpression was associated with an elevation in growth arrest and DNA damage inducible protein 34 (GADD34) mRNA. Kim Y-G and Lee GM. effects were illustrated by Kuystermans and Al-Rubeai53 for CHO cells engineered to overexpress c-MYC as a way to enhance cell proliferation. HBx (from hepatitis B virus) has been shown to activate the ATF6 and IRE pathways of the UPR. growth inhibitory effects of overexpressing ATF4. 2 Lim Y. outcomes remain mixed. no increase in productivity was observed. Rituximab productivity increased 20-fold in comparison with the parental line (from 2 pcd to 40 pcd).2-fold). Perhaps the most successful approaches to date have been those targeting apoptosis. In stable HBxexpressing clones. Yusuﬁ FN.36. 139 (2010). Such off-target. GlaxoSmithKline cancer drug threatens Herceptin market. Biotechnol Adv 28:385–394 (2010). where accumulation
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wileyonlinelibrary. Retzel EF. and luciferase production by 2. BiP). Though growth arrest has led to dramatic improvements in speciﬁc productivity. HEK293 and HT-1080 cells. Although doubling times decreased.94 Viral activators of the UPR have also been examined for their potential to improve protein productivity in mammalian cells. Nat Biotechnol 23:1453–1454 (2005). and metabolite abundance). despite increases in intracellular TPO concentration. and Sly1. in which the proteins Sly1 and Munc18c play an important role. this may account for the observed reduction in growth.97 have recently targeted protein transport mechanisms as a means to enhance productivity. Anti-apoptotic strategies have consistently improved culture robustness in response to stress.. transport.95 and in a transient system expression of HBx increased antibody production 2. in CHO cells producing TPO. Mulukutla BC and Chuah SH. gene transcription. 10 Jacob NM. respectively.6–4.96 Expression of US11 (from human cytomegalovirus) and NS4B (from hepatitis C virus) were also investigated. Production of recombinant protein therapeutics in cultivated mammalian cells.8-fold.to 2. are at least partially counteracted by slower growth and reduced cell densities.0.to 5-fold compared with parental cells. The concept of systems biology takes a holistic view of the cell.96 As for folding. and integral viable cell densities increased. and has been successful in engineering microbial production systems.98 showed that Munc18b overexpression enhanced protein production in HeLa. Assessment of cell engineering strategies for improved therapeutic protein production in CHO cells.97 In a follow-on study.g. are clearly undesirable. Biotechnol Lett 30:187–196 (2008). 5 Birch JR and Racher AJ. Wong NSC.53 Subsequent proteomic analysis revealed differential expression of over 100 proteins. Biotechnol J 3:624–630 (2008).99 In contrast to the gains made through anti-apoptotic strategies.98 clearly demonstrates that the effects of engineering protein secretion pathways are cell-type speciﬁc.com/jctb
. including down regulation of several that are involved in protein biosynthesis and folding.9–3. signiﬁcant improvements in cell-speciﬁc productivity as a result of engineering protein folding. combining global measurements from different functional levels (e. Engineering eukaryotic protein factories. Similarly. signiﬁcant reductions in peak viable cell densities were also observed.to 2.org of detrimental by-products can be limited by careful control of nutrient feeding.94 q improved approximately 2-fold. Engineering mammalian cells in bioprocessing – current achievements and future perspectives. but signiﬁcant reductions in growth rate and maximum viable cell densities were observed. 9 Sunley K and Butler M. both of these proteins being associated with UPR activation. 3 Waltz E.to 5. the concomitant reduction in peak cell density has so far prevented real gains in terms of ﬁnal product titre. 4 Berenson A. Munc18c.97 Individual overexpression of Sly1 in CHO cells producing Rituximab increased q up to 10-fold. Munc18b overexpression in CHO K1 cells failed to improve productivity.98
www. For example. NY Times (Print) A1:C2 (2006). Kantardjieff A. and improved lentiviral titres from HEK293 derived helper cells. In order to better select targets for cellular engineering and predict the outcomes.5. deeper knowledge of cells as a system is required.7-fold.103 Similarly. Reaching the depth of the Chinese hamster ovary cell transcriptome. In a follow-on study where GADD34 was overexpressed. This approach promises a better understanding of the complex interactions within cells. at a price that many can’t pay. Ku SCY.6. A cancer drug shows promise. Peng et al.5-fold. Although current cellular engineering approaches typically target only single genes/proteins or speciﬁc pathways. Antibody and luciferase productivity increased a further 2.4-fold. and unwanted. Nevertheless. The work of Peng and colleagues97. several studies observed a reduction in productivity. and secretory mechanism. 8 Schroeder M. Upstream processes in antibody production: evaluation of critical parameters.
Despite decades of research and a variety of different strategies to engineer a mammalian super producer. the amount of secreted protein (once above a certain threshold) did not increase with increasing levels of gene copy number or mRNA. and 1.97 With the addition of XBP-1 overexpression to Sly1 and Munc18c. no improvement in productivity was observed for either case. Although direct engineering of cellular metabolism can improve culture performance.Engineering a mammalian super producer in ATF4 expressing cells was only minor (1. This improvement in productivity was further enhanced by transient transfection of stable HBx clones with XBP-1. However. 6 Jain E and Kumar A. Biotechnol Appl Biochem 55:175–189 (2010).39 and alterations in cellular metabolism. Nat Biotechnol 22:1393–1398 (2004). and apparently insufﬁcient to fully induce apoptosis. 11 Wurm FM. cellular complexity and pathway interconnectivity are likely to result in off-target effects. antibody and luciferase productivity increased 2.104 – 109
1 Visiongain. with simultaneous overexpression of both Sly1 and Munc18c increasing q by15-fold.soci.g. These variable outcomes and differences between cell lines are probably a reﬂection of the complexity of mammalian cell biology. Strategies for the enhancement of recombinant protein production from mammalian cells by growth arrest. Biotechnol Bioeng 105:1002–1009 (2010). it has yet to be convincingly demonstrated that increased proliferation results in increased ﬁnal product titres. Wong DCF and Yap MGS. leading to improved productivity in a number of cases. protein synthesis. Antibody production.100 – 102 have not translated directly to mammalian cells. However. transport and secretion of recombinant proteins may also constrain productivity. 7 Mohan C. Lee YY. Protein transport is mediated by a complicated vesicle trafﬁcking system. Biotechnol Adv 26:46–72 (2008). Biosimilars and Follow-On Biologics: Global Market Outlook 2010–2025.7 Peng et al. While such improvements in speciﬁc productivity are impressive. these approaches have not been widely applied in a commercial setting.29. being particularly severe in clones overexpressing XBP-1. while strategies that were effective in yeast (e.
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