The production of recombinant secretory proteinsin mammalian cells is affected by the growthmedia, culture conditions and a large variety of

genetic factors1. The first step of gene expression, tran-scription, has been one of the main focuses in biotech-nological applications using mammalian cells. Theseefforts resulted in efficient and regulated promoter–transactivator systems. The influence of the surround-ing chromosomal DNA on promoter activities has only recently been addressed systematically using virus-based plasmids, transient expression and site-directedrecombination and integration.

Variations in the expression levels of the same con-struct in individual clones initiated the development of sophisticated, multicistronic expression vectors andscreening procedures. In addition to the productivityof individual cells, the final production level dependson the cell number and on the productive lifespan ofthe culture, items addressed by the genetic engineeringof cell growth and the suppression of programmed celldeath.

Apart from achieving high expression levels, productquality is of paramount importance. Post-translationalmodifications can impose significant problems for invivo applications of the product. A major objective ofcurrent research is the use of metabolic engineering to achieve product protein modifications that are compatible with pharmaceutical applications.

High-level, regulated gene expressionMost current applications make use of cell lines with

stably integrated genes under the control of a strongcellular or viral promoter2. Some cellular promoters(e.g. EF1a) or viral promoters, including thecytomegalovirus promoter3, can be used in differentcell lines for high-level protein expression, but theirtranscriptional activity varies depending on the cellu-lar levels of the relevant transcription factors and on thechromatin structure at the integration site. In addition,scaffold- or matrix-attachment regions (S/MAR el-ements) of chromosomal DNA can augment the

expression of heterologous genes and protect themfrom inactivation by the flanking chromatin4. TheS/MAR effects are distinct from those of enhancersbecause, in the absence of chromosomal integrationduring transient assays, S/MARs have no influence onexpression5–7.

Controlled gene expression is of major importancefor functional-genomics research, the production ofgrowth-inhibitory products and complex metabolicengineering. This can be achieved by using regulatablepromoters that respond to medium additives or growthconditions. The first recombinant, regulated gene-expression system developed in mammalian cells isbased on the Escherichia coli lac operator–repressor inter-action: the lac repressor inhibits transcription by bind-ing to lac operator sequences that are inserted betweenthe TATA box and the transcription start site of a mammalian promoter; transcription is activated by the inducer isopropyl-b-D-galactopyranoside, whichinduces the release of the lac repressor from its opera-tor8. Because the regulatability of transgene expressionusing the lac system was relatively poor in most indus-trially relevant cell lines, most currently used regulatedgene-expression systems rely on promoter–transactiva-tor combinations (Fig. 1). For example, efficiently regu-lated, tetracycline-responsive gene expression has beenachieved by the combination of a transactivator basedon the prokaryotic tetracycline repressor and a pro-moter containing multiple binding sites for this transactivator9.

In some cell lines, very high transcription rates havebeen achieved, as well as ranges of expression spanningfive orders of magnitude9. In addition to the tetracycline-dependent transactivator, an inverse system that is tetra-cycline repressible has been developed10. In this system,tetracycline is, in practice, replaced by nontoxic ana-logues such as doxycyclin. The level of the transac-tivator plays a major role in the transcriptional activityof the tetracycline-dependent promoter, but a numberof cell lines expressing tetracycline-regulatable transac-tivators are commercially available. In addition, a bi-directional promoter has been constructed that enablesthe regulated expression of two genes simultaneously11.

Other regulatable transactivators that are widely usedare steroid-hormone receptors. In the absence of hor-mones, these proteins form an inactive complex withheat-shock proteins, but ligand addition induces thetranscriptional activation of the target promoters.

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Genetic optimization of recombinantglycoprotein production by mammalian cellsMartin Fussenegger, James E. Bailey, Hansjörg Hauser and Peter P. Mueller

Genetically modified mammalian cells are the preferred system for the production of recombinant therapeutic glycoproteins.

Other applications include engineering of cell lines for drug screening and cell-based therapies, and the construction of recom-

binant viruses for gene therapy. This article highlights contemporary core genetic technologies and emerging strategies for

genetically engineering mammalian cells for optimal recombinant-protein expression.

M. Fussenegger and J. E. Bailey are at the Institute of Biotechnology,ETH Hönggerberg, HPT, CH-8093 Zurich, Switzerland. H. Hauserand P. P. Mueller (pmu@gbf.de) are at the Department of Gene Regulation and Differentiation, GBF – National Research Centerfor Biotechnology, Mascheroder Weg 1, D-38124 Braunschweig, Germany.

Hormone-dependent transcription activators havebeen constructed by fusing hormone-binding domainsof steroid receptors to DNA-binding domains of

unrelated heterologous proteins12. DNA-bindingdomains from the yeast Saccharomyces cerevisiae Gal4 protein have been used in combination with the hormone-binding domain of the mammalian oestro-gen receptor, the fusion product of which can beinduced by oestradiol13. Analogously, a progesterone-receptor fusion protein has been produced that can beactivated by RU 486 at concentrations much lowerthan those required for antiprogesterone activity14,15, as has a system based on the heterodimeric insectecdysone receptor, which can be induced by the synthetic ecdysteroid compound ponasterone A ormuristerone A (Ref. 16).

The major drawback of isolating stable, inducible orconstitutive high-level producer cell lines is the time-consuming selection procedure. In order to ease the isolation of cell clones with the desired character-istics, a number of sophisticated techniques have beendeveloped.

Controlled, targeted chromosomal-integrationexpression

Low-copy integrated genes are generally expressedmore stably than the multicopy genes usually obtainedwith the calcium coprecipitation method17. To selecttransfectants with low-copy-number integrated genesthat nevertheless are expressed at high levels, aneomycin-phosphotransferase gene with a mutanttranslation-initiation site has been used18. Alternatively,retroviral vectors can be employed to achieve single-copy integration. In principle, if one of the rare siteson the chromosome that allow high gene expressionfrom a single integrated gene copy has been identified,this locus can be used for high-level expression of anydesired transgene.

With the exception of mouse embryonic stem cells,which allow the insertion of DNA fragments byhomologous recombination, the integration of trans-fected DNA into mammalian genomes is unpredictableand occurs almost exclusively by non-homologous(illegitimate) recombination. Therefore, for targetedtransgene integration into the mammalian chromosome,a two-step approach has been developed.

First, a suitable chromosomal location with hightranscriptional activity must be identified by classicaltransfection and subsequent screening. The screeningvectors used for finding these transcriptionally highlyactive sites contain selection markers and reporter genesflanked by artificial recombination sites such as the yeastFlp recombination target (FRT) sites, or the bacterio-phage P1 loxP sites, which are recognized by the Crerecombinase.

The DNA between the recombination sites can thenbe precisely replaced with a second cassette encodingthe expression unit of the gene to be expressed19.Unwanted excision of the insert by recombination ofthe flanking sites can be suppressed by using two FRTsites with different nucleotide sequences20,21. Cassetteexchange is made considerably easier by using a counter-selection procedure (Fig. 2). An extremely effectiveselection procedure for correct cassette replacementuses an initiation-codon-deficient drug-resistancemarker in the first selection construct. The missing in-frame initiation codon is then introduced with thesecond expression cassette. Only cells that have

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A

A A AA

A

TT

A

Preg

Pcon

Transactivator

ProductT

AT

AT

AT

AT

A

Figure 1Artificial transactivator–promoter combinations. These are the most efficient tran-scription systems available and allow highly effective regulation of gene expression.A generic transactivator (T) consists of a heterologous DNA-binding domain (to avoidunwanted activation of endogenous genes), a regulatory-substance (A)-binding domainand a transcriptional-activation domain. The transactivator gene is transcribed froma constitutively active promoter (Pcon) and its activity is regulated by an exogenouslyadded substance. The target promoter (Preg) contains multiple binding sites for thetransactivator upstream of an optimized minimal promoter, from which the productgene is transcribed.

LTR LTRMarker

Product

Product

Flp-drivenintrachromosomalrecombination

Flp-driven recombinationwith plasmidal DNA

Chromosomallyintegratedconstruct

Targetingvector

Recombinantchromosomallocus

Figure 2Heterologous recombination systems. These allow the exchange of expression cassettes at previously marked loci. A gene encoding an easily detectable markerprotein flanked by recombination sites is integrated at random sites into the genomeof the producer cells. To reflect the intrinsic activity of the locus, the integration mustoccur in single copy, which is preferentially obtained by transduction with retroviralvectors. Recombination sites can be inserted into the long terminal repeats (LTRs)of these vectors. Cells with the optimal expression pattern are identified and isolated.Expression of a heterologous recombinase (as an example, the yeast Flp recombi-nase is shown) catalyses the exchange of the chromosomally integrated DNA betweenthe recombination sites (here FRT, a 13-bp inverted-repeat sequence separated byan 8-bp spacer sequence) with a product gene flanked by the same recombinationsites. Improvements of this basic procedure avoid the subsequent deletion of theproduct gene by recombination of the flanking recombination sites with each otherby using two different recombination sites. Deletion of the marker gene is an efficientbut unwanted side reaction that can be selected against by including an incompletedrug-resistance marker gene in the 39 LTR. The missing gene fragment is suppliedby the product cassette, such that only precise recombination events render the celldrug resistant.

recombined precisely become drug resistant22. Thetedious and time-consuming work to improve thisapproach is compensated by the possibility of reusingsuch stable, high-level producer cell lines to express anyother gene of interest.

Multicistronic expressionAlthough the need for screening procedures cannot

be completely removed when generating an ideal pro-ducer cell line, it can be considerably reduced by link-ing the expression of the product to the expression ofa selectable marker. The correlation between selec-table-marker activity and the amount of productsecreted depends on the method used. Cotransfectionof the two genes on different plasmids is the least accu-rate procedure but can be improved by expressing theproduct and the marker gene from the same plasmid.The most stable coupling is obtained when both genesare expressed from the same promoter. Two separatereading frames can be translated from a single mRNAby taking advantage of an internal ribosomal entry site(IRES) to direct translation initiation of the secondcistron. Such artificial genetic configurations havebecome standard for coordinated, dicistronic gene-expression systems in mammalian cells, for example, tolink the gene of interest transcriptionally to a selectionmarker or to express two-component proteins such asimmunoglobulins at defined ratios23–29.

Most of the IRES elements used for dicistronicexpression originate from poliovirus or encephalomyo-carditis virus, but other IRES sequences can be used aswell30,31. The efficiency of IRES-mediated translationinitiation varies greatly between different IRES el-ements and is also dependent on the host cell line used31.

Coordinated expression of two genetic traits can also be achieved by gene fusions32 or by differentialsplicing33, but only IRES-mediated translation can beextended beyond dicistronic configurations (Fig. 3). Anovel family of expression vectors using three and four cistrons have been constructed for coordinated,constitutive or adjustable high-level expression of threeindependent genes in mammalian cells34–36.

Multicistronic expression has enabled the develop-ment of a number of important procedures, including:the coupled expression of multicomponent and multi-subunit proteins; one-step transfection, selection andmaintenance of difficult-to-express genetic configu-rations; the cloning of good producer-cell lines byintroducing a selection marker into the last cistron(auto-selective systems)33; the design of complex regu-latory circuits such as positive-feedback regulatory systems for one-step regulated gene expression in mam-malian cells33; and multigene metabolic engineering ofproduction cell lines using sense, antisense or ribozymetechnology. The applicability of multigene expressioncassettes in genetic immunization is currently underinvestigation.

Advanced selection and screening proceduresEven when using multicistronic expression vectors

for stable transfections, individual clones expressrecombinant proteins at highly variable levels37, andonly a small fraction (generally ,1% of the clones ini-tially obtained) stably express high amounts of thedesired product protein. Improvements in various

methods to isolate these rare high-level producer cellsinclude the use of two selection markers38 instead of asingle selection marker39.

In Chinese hamster ovary (CHO) cells, the standardprocedure for achieving maximal expression is a verytime-consuming procedure that can last for months andincludes a gradual increase in the selection pressure fora cotransfected selection marker such as the dihydro-folate reductase or the glutamine synthetase. A morerapid method to isolate high-producing clones involvesthe use of plasmid vectors that encode green fluorescentprotein (GFP) as a screening and fluorescence-activated-cell-sorting marker. Alternatively, efficientexpression can be enforced by employing selectionmarkers with loss-of-function mutations. It has beenrepeatedly shown that the lower translation efficiencyof such mutated markers is compensated for by theselection of chromosomal integration sites with overall high transcription activity28,40.

Sophisticated screening procedures have been used tomonitor the product level directly (Fig. 4). A relativelysimple and inexpensive method relies on the local trans-fer of a constitutively or inducibly secreted productfrom a colony of producer cells through an agaroseoverlay onto a filter. The bound product can then bedetected immunologically25,41–43. At the single-celllevel, the determination of productivity is not trivialand requires the association of the individual secretingcell with its product. This has been achieved by tethering antibodies to the cell membrane of producercells. In the low-permeability medium, the productaccumulates in the proximity of each producer cell andis captured by the antibody, so that each cell becomesassociated with a certain fraction of its own product.For sorting, the cell-associated product can be labelledwith magnetic beads or with a second antibody44. Analternative method to keep the product associated withindividual producer cells is to embed the cells in geldroplets. The product is caught by antibodies that arelinked to the gel matrix. For quantification, the dropletsare labelled with a second antibody. Beads containingthe cells with the highest expression level can then beselected using flow cytometry45–48.

Controlled-proliferation technologyCell growth and division are of fundamental impor-

tance for multicellular organisms, especially in the first developmental phase, when cell proliferation is

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Figure 3Multicistronic constructs express several reading frames in a defined ratio. The firstcistron is translated similar to most cellular mRNAs in a cap-dependent initiationprocess (ci), while downstream reading frames must be preceded by an internalribosomal-entry site (IRES) to allow cap-independent internal translation initiation (ii).

P

ci ii iiCistron 1 Cistron 2 Cistron 3

AAAAAAAA

DNA

mRNA

Protein 1 Protein 2 Protein 3

Gene 1 Gene 2 Gene 3IRES IRES

essential for growth of the organism. However, whenterminal differentiation and the growth phase are com-pleted, proliferation control becomes the dominantaspect of the genetic stability of higher organisms.Despite the growth-arrested state, the cells produce andsecrete proteins continuously during the lifetime of theorganism. In standard biotechnological productionprocesses, transformed, permanently proliferating ani-mal cells are used, which eventually die of nutrient oroxygen depletion.

The requirement for cell growth to clone and propa-gate producer cell lines to high densities, and reports ofa positive correlation between growth rate and prod-uct formation49–52, stimulated efforts to replace growth-factor-containing animal serum with chemical media

containing defined proteins37,53 and genetically engi-neer the mammalian cell lines to permit growth in low-protein or protein-free medium54–56. In spite ofsuccessful efforts to enhance cell growth, proliferation-based cell-culture technology is hampered by the factthat growth beyond a desired cell density causes nutri-ent limitation, accumulation of toxic compounds and,eventually, cell death accompanied by the release ofhydrolytic enzymes, which can degrade the productand decrease product quality. However, as in the natural situation, an ideal production process wouldinclude proliferation control to allow cells to growrapidly to high cell densities, followed by a prolifer-ation-inhibited production phase in which the cellsdevote their metabolic capability to the formation ofproduct.

Higher productivity of proliferation-inhibited cellswas indeed observed with antibody-producinghybridoma cells57–59. However, as the proliferation-inhibited state was achieved by starving the cells for anessential energy source or the addition of DNA-synthesis inhibitors such as thymidine, hydroxyurea,transforming growth factor b (TGF-b) or geno-toxic agents such as adriamycin, or with temperature-sensitive mutant cells at the non-permissive tem-perature57,59,60, such processes or additives reduce cellviability and are therefore less suitable for long-termproduction systems.

The first attempt to control cell proliferation by the genetic engineering of a baby hamster kidney(BHK) cell line was based on an oestrogen-regulatedfusion between the interferon-responsive factor 1(IRF-1) and the oestrogen receptor61–63. IRF-1 is aDNA-binding transcription activator that accumulatesin cells in response to interferons and has antiviral andantiproliferative activities25,59,61. The induction ofrecombinant protein production can be achieved inIRF-1-arrested cells by placing the transcription of thecorresponding gene under the control of an IRF-1-responsive promoter. In addition, the expression ofoestrogen-responsive IRF-1 in a dicistronic configu-ration with the selection marker stabilizes the growthcontrol in a way that allows several cycles of growth andgrowth-arrested states to be performed with the sameculture63.

In another effort, the cyclin-dependent-kinaseinhibitors p21 and p27, and the tumour-suppressorgene p53 were used to arrest CHO cells reversibly atthe G1 phase of the cell cycle65. p53 functions as a keyregulator at the interconnection of the cell-cycle andapoptosis regulatory networks, and either affects apop-tosis or induces p21, to allow repair and survival66.Overexpressing any one of the three genes leads to arepression of cyclin-E–CDK2-complex phosphorylat-ing activity and prevents entry into S phase66. The G1phase of the cell cycle is expected to be the most suit-able for controlled-proliferation technology as it is thephase in which a cell checks its physiological state andcan arrest to replenish its metabolic supplies and repairgenetic defects66.

CHO cells stably transfected with multicistronic p21-,p27- or p53-containing tetracycline-repressible expres-sion vectors show different responses. The regulatedoverexpression of p27 leads to growth arrest with nosigns of apoptosis and up to 15-times-higher specific

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a Filter immunoassay

(i)

(ii)

(iii)

b Secretion-capture report web

(i) (ii)

c Cell-surface affinity matrix

(i)

P

PP

Figure 4Specialized methods to isolate cells with high expression levels. Inthe simple and inexpensive membrane-immunoassay method (a), cellclones are covered with a thin agar overlay (i) onto which a protein-binding membrane is placed (ii). The product diffuses locally throughthe agar and binds to the membrane, and is detected by labelledantibodies or another appropriate detection method (iii). The ‘secre-tion-capture report web’ (b) is a derivatized agarose bead into whichsingle cells are embedded (i). The secreted product (P) is capturedby biotinylated antibodies (black) that are coupled with avidin (smallcircle) to biotinylated agarose (ii). For detection, the agarose beadis soaked in a solution containing a labelled second antibody (grey).In the cell-surface affinity-matrix method (c), the capture antibody iscoupled to the membranes of biotinylated cells and the product keptin the vicinity of the cell by a special low-permeability medium.

productivity67. The continuous overexpression of p53[even in its mutated, apoptosis-deficient, form(p53175P)] leads to rapid cell death68. No clones couldbe isolated that showed proliferation arrest after induc-tion of p21 expression. Making use of tetracycline-regulated tricistronic expression68, the dicistronicSEAP–p21 expression unit has been extended with acistron encoding the differentiation factor c/ebp a(CAAT-enhancer-binding protein a) that both inducesand stabilizes p21 (Ref. 68). With this construct, sus-tained growth arrest for several weeks and a 10–15-foldhigher specific productivity has been achieved compared with control cells67.

A second example for controlled-proliferation tech-nology is based on a p27–bcl-xL-encoding tricistronicexpression unit. CHO cells stably transfected with thisconstruct showed a 30-times-higher expression levelthan did the controls. Multigene metabolic engineer-ing of the cell cycle has thus proved to be effective inachieving a difficult-to-attain cell-culture state andenhancements in the expression of a heterologoussecreted glycoprotein.

Transient expressionAs an alternative to the cumbersome and time-

consuming selection procedure of stable cell lines, tran-sient transfection strategies can be used for the rapidand efficient production of small quantities of recom-binant product protein. Cell lines that express a particu-lar transcriptional activator can drive high-level expres-sion from specific promoters that contain binding sitesfor the respective transactivator. For example, replicat-ing expression vectors have been developed for thehuman 293 cell line that constitutively expresses theadenoviral E1A transactivator to enhance transcription,concomitant with the EBNA antigen and Epstein–Barrvirus replicon for episomal plasmid maintenance69. Inaddition, COS monkey cell lines are still used for extra-chromosomal plasmid replication and efficient geneexpression from SV40 origin promoters70–72. Many ofthe currently available viral vectors can be used for transient gene expression in mammalian cells. Vectorsderived from Papova viruses, Epstein–Barr virus, adenovirus or alphavirus replicate episomally and oftenreach very high copy numbers, resulting in very high,transient expression levels. However, their productivetime span is limited owing to the loss of the expressionconstruct or the death of the host cell.

Alphavirus-derived vectors have been developed forprotein production73, as they can infect non-dividingcells and have a broad host range. The alphavirusgenome consists of a 12 kb positive-strand RNA mol-ecule, which is translated to produce a replicase imme-diately after infection. Replication signals at both endsof the RNA are required for the amplification of theviral genome that takes place in the cytoplasm until theinfected cell dies. Negative-strand RNA is producedthat serves as a template for the synthesis of both full-length genomic RNA and up to 105 RNA mol-ecules of a subgenomic transcript encoding structuralproteins. These structural genes can be replaced by heterologous genes, and the recombinant viral RNA then packaged with the help of a coexpressedwild-type virus or a packaging cell line expressing complementing structural proteins.

Current alphavirus-based expression vectors allownonviral gene transfer using either in vitro-transcribedRNA or, alternatively, DNA vectors from which theviral RNA is transcribed under the control of a heterologous promoter. The potential risk of a self-replicating infectious virus is reduced by a condition-ally activated Semliki–Forest-virus expression system inwhich a cleavage-deficient spike protein is activated bythe exogenously added protease chymotrypsin74.

Vectors based on vaccinia virus have emerged as themost efficient transient expression system15. However,at present, only a few laboratories have the expertiserequired to handle this system. Among other difficul-ties, the virus might induce changes in the post-translational system of the host cells, which could leadto premature cell death. Therefore, vaccinia-virus andalphavirus vectors have been used mainly in recombi-nant protein production for research purposes and inanimal vaccination75.

Although all of the above-mentioned viral vectorscombine the two prerequisites for strong transientexpression – efficient gene transfer and high episomalcopy number – these properties have also been recentlyachieved by refinements in classical transfection tech-nology. Improvements in DNA preparation and trans-fection protocols now enables the production of grams of recombinant proteins in transient-transfectionbatch-fermentation processes76,77.

Apoptosis engineeringApoptosis is a genetically determined program of

active cell death. Despite its fundamental importancefor multicellular life, apoptosis is an undesirable phe-nomenon in biotechnological production processes,which are often limited by rapid cell death in thedecline phase of a culture. Many commercially impor-tant production cell lines are sensitive to apoptosis,including hybridoma and myeloma cell lines78,79; others, such as HeLa or HL-60 cells, block cellular pro-liferation in response to nutrient limitation or geno-toxic stress rather than initiating apoptosis, and allowcells to replenish their metabolic precursors or repairDNA damage80.

There are three fundamental approaches to suppress-ing apoptosis in cell-culture processes81,82: (1) elimi-nation of nutrient deprivation by feeding strategies; (2)use of chemical medium additives to block apoptosispathways; and (3) metabolic engineering using anti-apoptotic survival genes. Suboptimal concentrations ofamino acids, particularly essential amino acids, can acti-vate apoptotic signalling pathways78,83. The addition ofa single amino acid has recently been shown to rescuehybridoma cultures from starvation-induced apopto-sis84. Medium additives that prevent apoptosis includechemicals that inhibit the activity of the endonucleaseresponsible for DNA cleavage81, antioxidants such asvitamin E (which can prevent apoptosis induced by freeradicals85) and pseudosubstrates for caspases, whichblock the activity of these apoptosis-inducing proteases86.

Apoptosis is induced by a wide variety of stressestypically encountered during normal bioreactor oper-ation87. These include nutrient and serum limitations,accumulation of toxic compounds and metabolites,oxygen deprivation, and hydrodynamic stresses.

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Mammalian cells can be protected against stress-induced apoptosis by genetic engineering to over-express survival genes such as Bcl-2 (Refs 60, 80).Enhanced Bcl-2-mediated survival has also beenreported for Sindbis-virus-based expression systems89.The level of protection varies between different celltypes and cell lines and, in most cases, Bcl-2 over-expression cannot prevent cell death, but it can extendcellular lifetimes and lead to increased produc-tion59,88,90–93. Initial attempts to complement the actionof Bcl-2 using concomitant overexpression of the anti-apoptotic genes bag-1, bcl-xL or the adenoviral E1B-19K gene were encouraging and showed that theprotective effects of individual genes were equal or evenadditive when, for example, Bag-1 and Bcl-2 werecoexpressed94,95. Apoptosis engineering might becomean essential tool for emerging technologies such as genetherapy and tissue engineering.

Glycosylation: a target for post-translationalmetabolic engineering

Although gene dosage, transcription levels and trans-lation rate are key parameters for optimizing heterolo-gous protein production in mammalian cells, post-translational processes have only recently beenperceived as metabolic bottlenecks and potential targetsfor improving the performance of mammalian cell culture. Post-translational modification of a productprotein includes various potentially rate-limiting inter-actions with numerous chaperones and enzymes in thecytoplasm, the endoplasmatic reticulum and the com-partments of the Golgi apparatus. Studies in mam-malian cells96–98, yeast99,100 and insect cells101,102 haveimproved the protein productivity by augmenting thepost-translational capabilities of these cell systems.

One of the main post-translational events is theattachment of complex sugar structures to the major-ity of secreted proteins. Glycoproteins mediate manydiverse functions65,103–106 and the glycosylation patternof secreted proteins influences their activity as well astheir clearance from the body. Certain amino acidresidues serve as specific glycosylation sites as thepolypeptide chain moves from the endoplasmic reticu-lum through the Golgi apparatus. Two major types ofglycosylation are commonly found in eukaryotic cells:N- and O-linked glycosylation, with carbohydratesattached to the amide group of Asn–X–Ser/Thrsequences or the hydroxyl groups of Ser or Thrresidues, respectively.

The final glycosylation pattern of a protein is depend-ent on many parameters including: (1) the polypeptidechain; (2) the host cell and its chromosomal set of glycosyltransferases and glycosidases; and (3) the envi-ronment of the host cell. Different polypeptidesexpressed in the same host under similar conditions canbe glycosylated very differently, showing that thepolypeptide itself exerts a controlling influence on itsown glycosylation107. A technologically relevant exam-ple shows that a single amino acid substitution in tissue-plasminogen activator (tPA) can create a new glycosyl-ation site and even change the glycosylation pattern ofa different native site. Although this tPA glycovariantshowed a ten-times lower plasma clearance than wild-type tPA, its physiological activity decreased toone third of the wild-type level, a phenomenon that

could be alleviated by removing the native glycosylationsite108.

Even with a given protein product and a well-determined host system, the oligosaccharides presenton secreted glycoproteins are built up in a complex cascade of sequential enzyme-catalysed reactions andintracompartmental transport processes, often withmultiple enzymes acting on common substrates to givealternative oligosaccharide products109. A particularglycoprotein produced in a given host system is a popu-lation of different glycoforms. These differ: (1) in thepercentage by which a particular amino acid residue isglycosylated; (2) in high-mannose or complex glyco-forms in N-linked oligosaccharides; (3) the number ofterminal residues (antennae); (4) in bisecting N-acetyl-glucosamine (GlcNAc); (5) in saccharide–saccharidelinkages; and (6) in the percentage of oligosaccharideends that are sialylated.

Glycoforms can significantly differ in their activ-ity107,110, physicochemical properties106,110,111 (solubil-ity, stability, folding and secretion), pharmacokinetics(clearance of a protein pharmaceutical from the bloodstream104,111–114, targeting, immunogenicity, antigen-icity and stability104. Data on the functional impli-cations of attached oligosaccharides are only availablefor a very limited number of glycoproteins. A few general principles of carbohydrate structure–functionrelationships are known. A central concern of bio-pharmaceutical manufacture is the absence of sialic(neuraminic) acid on the termini of complex carbohy-drate structures, which results in more rapid clearance,as does the presence of high-mannose oligosaccharides.

In addition to the protein itself, glycosylation isdependent on the host cell and the culture condi-tions115. Commonly used bacterial hosts do not glyco-sylate their proteins; yeasts glycosylate with high-mannose oligosaccharide structures that are not suitablefor injection into humans. Similarly, baculovirus andplant expression systems synthesize carbohydrates thatare undesirable for pharmaceutical applications. It is thecapability of mammalian cells to accomplish glycosyl-ation in a manner compatible with human applicationsthat has created a special niche for them in the biotech-nology industry. However, even mammalian cells arefar from being perfect in their glycosylation. Currentresearch focuses on strategies to modify targets andoligosaccharides on particular glycoprotein pharma-ceuticals to produce glycoforms with enhanced therapeutic potential.

Methods developed to manipulate glycosylationinclude drugs to inhibit glycosylation as well asinhibitors of glycosylation processing116. Many of thesereagents are toxic and sometimes alter glycosylationpatterns rather inefficiently. Other strategies focus onrandom mutagenesis for the generation of host cellswith altered glycosylation characteristics. Screening isfacilitated by a lectin-based (lectins are toxic carbohy-drate-binding proteins) counterselection of wild-typecells and an enrichment for host cells with altered glycosylation patterns of the surface proteins103. How-ever, such a selection of loss-of-function mutations inthe glycosylation pathways often results in incompleteor truncated carbohydrate structures.

The use of recombinant DNA technology for themetabolic engineering of glycosylation in mammalian

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cells aims to extend the hosts’ oligosaccharide-biosynthesis capabilities by introducing genes encodingheterologous carbohydrate-synthesis enzymes. At pres-ent, most glycosylation-engineering strategies haveconsidered enzymes involved in or near the terminalsteps of complex oligosaccharide biosynthesis117–120.Two main metabolic-engineering strategies are beingfollowed; glycosylation activities can be increased basedeither on: (1) gene activation; or (2) introducing transcriptionally controlled glycosylation genes. Con-versely, antisense and other methods can be used toblock undesired glycosylation. Only a few glycosylationenzymes have been cloned and are available for metabolic engineering120.

CHO cells lack a functional copy of the a2,6-sialyl-transferase gene (2,6 ST)121. This defect of CHO cells,relative to human cells, can be complemented by theexpression of a cloned 2,6 ST, which results in the pro-duction of both a2,3- and a2,6-linked sialic acids on coexpressed tPA (Ref. 117). The glycosylation pat-tern of heterologous pharmaceutical proteins waschanged in BHK cells when the normally absent a2,6-sialyltransferase and a1,3-fucosyltransferase-IIIwere overexpressed116.

The metabolic engineering of glycosylation has sig-nificant potential as a means for the production of glycoproteins and to provide new forms of glyco-proteins with improved therapeutic characteristics. Thispotential is mainly limited by the availability of clonedgenes involved in the glycosylation pathways. As morecomponents involved in glycosylation are discovered,this field will not only have an impact on further development of animal cell technology but will also allow further insights into hidden secrets ofgenome–cell-function relationships.

ConclusionMany promising developments using genetic tools

for the improvement of mammalian cell culture tech-nology could not be reviewed here and many of thebriefly mentioned techniques are still under investi-gation. Some of these recent developments might notbe of any further use, others might become routinemethods in a not-so-distant future. Genetic optimiz-ation of glycoprotein production in mammalian cellshas evolved as a major multidisciplinary field of mod-ern biotechnology in which academia, industry andmedical institutions cooperate closely in order toimprove current therapeutic strategies such as cell andgene therapy, and to ensure a high quality of human life.

AcknowledgmentsResearch in the authors’ laboratories on genetic

engineering to control cell proliferation and to reduceapoptosis is supported by the Swiss Bundesamt für Bildung und Wissenschaft (BBW) and by the Frame-work IV Biotechnology programme of the EuropeanCommission.

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