Microbial Cell Factories
`
`BioMed Central
`
`Review
`Microbial factories for recombinant pharmaceuticals
`Neus Ferrer-Miralles1,2,3, Joan Domingo-Espín1,2,3, José Luis Corchero3,1,
`Esther Vázquez1,2,3 and Antonio Villaverde*1,2,3
`
`Open Access
`
`Address: 1Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain, 2Department de Genètica i de
`Microbiologia, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain and 3CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-
`BBN), Barcelona, Spain
`
`Email: Neus Ferrer-Miralles - neus.ferrer@uab.cat; Joan Domingo-Espín - Joan.DomingoE@campus.uab.es;
`José Luis Corchero - jlcorchero@ciber-bbn.es; Esther Vázquez - Esther.Vazquez@uab.es; Antonio Villaverde* - antoni.villaverde@uab.cat
`* Corresponding author
`
`Published: 24 March 2009
`
`Microbial Cell Factories 2009, 8:17
`
`doi:10.1186/1475-2859-8-17
`
`Received: 11 February 2009
`Accepted: 24 March 2009
`
`This article is available from: http://www.microbialcellfactories.com/content/8/1/17
`
`© 2009 Ferrer-Miralles et al; licensee BioMed Central Ltd.
`This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
`which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
`
`Abstract
`Most of the hosts used to produce the 151 recombinant pharmaceuticals so far approved for
`human use by the Food and Drug Administration (FDA) and/or by the European Medicines Agency
`(EMEA) are microbial cells, either bacteria or yeast. This fact indicates that despite the diverse
`bottlenecks and obstacles that microbial systems pose to the efficient production of functional
`mammalian proteins, namely lack or unconventional post-translational modifications, proteolytic
`instability, poor solubility and activation of cell stress responses, among others, they represent
`convenient and powerful tools for recombinant protein production. The entering into the market
`of a progressively increasing number of protein drugs produced in non-microbial systems has not
`impaired the development of products obtained in microbial cells, proving the robustness of the
`microbial set of cellular systems (so far Escherichia coli and Saccharomyces cerevisae) developed for
`protein drug production. We summarize here the nature, properties and applications of all those
`pharmaceuticals and the relevant features of the current and potential producing hosts, in a
`comparative way.
`
`Introduction
`Proteins are catalysers of metabolic reactions, structural
`components of biological assemblies, and responsible for
`inter and intracellular interactions and cell signalling
`events that are critical for life. Therefore, deficiencies in
`the production of specific polypeptides or the synthesis of
`mutant, non-functional versions of biologically relevant
`protein usually derive in pathologies that can range from
`mild to severe. In humans, such diseases can be treated by
`the clinical administration of the missing protein from
`external sources, to reach ordinary concentrations at sys-
`temic or tissular levels [1]. Therefore, many human pro-
`teins have an important pharmaceutical value but they are
`difficult to obtain from their natural sources. Recom-
`
`binant DNA (rDNA) technologies, developed in the late
`70's using the bacterium Escherichia coli as a biological
`framework, offer a very potent set of technical platforms
`for the controlled and scalable production of polypep-
`tides of interest by relatively inexpensive procedures. This
`can be done in convenient microbial cells such as bacteria
`and yeasts, whose cultivation can be accomplished by rel-
`atively simple procedures and instrumentation. In early
`80's, the FDA approved the clinical use of recombinant
`human insulin from recombinant E. coli (Humulin-US/
`Humuline-EU) for the treatment of diabetes [2], being the
`first recombinant pharmaceutical to enter the market. The
`versatility and scaling-up possibilities of the recombinant
`protein production opened up new commercial opportu-
`
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`widely used for primarily cloning, genetic modification
`and small-scale production for research purposes. This is
`not surprising as the historical development of microbial
`physiology and molecular genetics was mainly based on
`this species, what has resulted in a steady accumulation
`and worldwide use of both information and molecular
`tools (such as engineered phages, plasmids and gene
`expression cassettes). However, several obstacles to the
`production of quality proteins limit its application as a
`factory for recombinant pharmaceuticals. Recombinant
`proteins obtained in E. coli lack the post-translational
`modifications (PTMs) which are present in most of
`eukaryotic proteins [13]. Glycosylation is the most com-
`mon PTM [14] but many others, such as disulfide bond
`formation, phosphorylation and proteolytic processing
`might be essential for biological activity. PTMs play a cru-
`cial role in protein folding, processing, stability, final bio-
`logical activity, tissue targeting, serum half-life and
`immunogenicity of the protein; therefore PMT deficient
`version might be insoluble, unstable or inactive. Interest-
`ingly, it is possible to attach or bind synthetic PTMs in the
`case of pegylated products [15] such as human growth
`hormone, granulocyte colony stimulating factor, interfer-
`ons alfa-2a and alfa-2b, which renders versions of the pro-
`tein in serum more stable than the naked product. Also,
`the N-linked glycosylation system of Campylobacter jejuni
`has been successfully transferred to E. coli, making this
`approach a promising possibility for the production of
`glycosilated proteins in this species [16]. Furthermore,
`through genetic engineering of the underlying DNA, the
`amino acid sequence of the protein can be changed to
`alter its ADME (absorption, distribution, metabolism,
`and excretion) properties, as it has been observed for insu-
`lin (Table 1) [17].
`
`On the other hand, the frequencies with which the differ-
`ent codons appear in E. coli genes are different from those
`occurring in human genes, and this is directly related to
`the abundance of specific tRNAs. Therefore, genes that
`contain codons rare for E. coli may be inefficiently
`expressed by this organism and cause premature termina-
`tion of protein synthesis or amino acid misincorporation,
`thus reducing the yield of expected protein versions [18].
`This problem can be solved either by site-directed replace-
`ment of rare codons in the target gene by codons that are
`more frequently used in E. coli, or, alternatively, by the co-
`expression of the rare tRNAs (E. coli strains BL21 codon
`plus and Rosetta were designed for this purpose). In addi-
`tion, initial methionine removal depends on the side
`chain of the penultimate amino acid of N-terminal in
`final recombinant proteins produced in E. coli although it
`can be efficiently removed using recombinant methionine
`aminopeptidase [19]. Some mutant E. coli strains have
`been developed to promote disulfide bond formation
`(AD494, Origami, Rosetta-gami) and/or with reduced
`
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`
`nities for pharmaceutical companies. Since the approval
`of recombinant insulin, other recombinant DNA drugs
`have been marketed in parallel with the development and
`improvement of several heterologous protein production
`systems. This has generated specific strains of many
`microbial species adapted to protein production, and has
`allowed the progressive incorporation of yeasts and
`eukaryotic systems for this purpose. Among the 151 pro-
`tein-based recombinant pharmaceuticals licensed up to
`January 2009 by the FDA and EMEA, 45 (29.8%) are
`obtained in Escherichia coli, 28 (18.5%) in Saccharomyces
`cerevisiae, 17 (11.2%) in hybridoma cells, 1 in transgenic
`goat milk, 1 in insect cells and 59 (39%) in mammalian
`cells (Figure 1) [3]. In the next sections, the key properties
`of these expression systems will be analyzed regarding
`both the biological convenience and final quality of the
`products. Alternative promising protein production sys-
`tems such as filamentous fungi, cold-adapted bacteria and
`alternative yeast species among others are under continu-
`ous development but only few biopharmaceutical prod-
`ucts from them have been marketed. Relevant properties
`of such promising systems and their potential as produc-
`ers of therapeutic proteins have been extensively reviewed
`elsewhere [4-12].
`
`Escherichia coli
`The enterobacterium E. coli is the first-choice microorgan-
`ism for the production of recombinant proteins, and
`
`29.8 %
`
`18.5 %
`
`39 %
`
`11.2 %
`
`0.75 %
`
`0.75 %
`
`c e l l s
`c e l l s
`a n i m a l s
`c o l i
`c e r e v i s i a e
`i d o m a s
`Insect cells Mammalian cells
`E. coli
`E .
`I n s e c t
`M a m m a l i a n
`H y b r
`T r a n s g e n i c
`S .
`S. cerevisae
`Hybridomas Transgenic animals
`
`60
`
`50
`
`40
`
`30
`
`20
`
`10
`
`0
`
`Product number
`
`Number (and percentage values siding the bars) of recom-binant proteins approved as biopharmaceuticals in different production systemsFigure 1
`
`
`
`Number (and percentage values siding the bars) of
`recombinant proteins approved as biopharmaceuti-
`cals in different production systems. Data has been
`adapted from Table 1 in [3]. Exubera, an inhalated recom-
`binant human insulin produced in E. coli has been omitted
`since Pfizer stopped its marketing in January 2008. Two
`recently FDA approved products Xyntha and Recothrom
`produced both in CHO cells have also been added.
`
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`Table 1: Recombinant insulins approved for human use.
`
`INN1
`
`Trade name Production system
`
`Modifications from natural
`
`PK2
`
`Insulin human
`
`Insulin human
`
`Humulin
`Insuman
`Exubera3
`Novolin
`
`Insulin lispro
`Insulin glulisine
`Insulin aspart
`
`Humalog
`Apidra
`Novorapid
`
`Insulin glargin
`Insulin detemir
`
`Lantus
`Levemir
`
`E. coli
`
`S. cerevisiae
`
`E. coli
`E. coli
`S. cerevisiae
`
`E. coli
`S. cerevisiae
`
`None
`
`None
`
`PB28K and KB29P
`NB3K and KB29E
`DB28P
`
`NA21G and 2 additional R in B chain
`TB30del and myristic fatty acid attached to KB29 by
`acylation
`
`Short-acting insulin
`
`Short-acting insulin
`
`Rapid-acting insulin analogue
`Rapid-acting insulin analogue
`Rapid-acting insulin analogue
`
`Long-acting insulin analogue
`Long-acting insulin analogue
`
`Insulin is a polypeptide of 51 amino acid, 30 of which constitute A chain, and 21 of which comprise B chain. The two chains are linked by a disulfide
`bond. Mutations in amino acid sequences are noted for each of the chains.
`1INN: International Nonproprietary Names. 2PK:PharmacoKinetics. 3Exubera: Rapid-actin insulin using inhalation route [17], was discontinued in
`2008 by the manufacturer
`
`protease activity (BL21). As an additional technical obsta-
`cle, proteins larger than 60 kDa are inefficiently obtained
`in soluble forms in E. coli [20].
`
`As it has been well documented, bacteria overproducing
`either eukaryotic or prokaryotic recombinant proteins are
`subjected to different stresses (essentially metabolic and
`conformational) [21]. Under this situation, protein
`processing associated to cell stress responses might render
`non useless products, mainly because of lack of solubility,
`and many protein species deposit in high amounts as pro-
`tein aggregates known as inclusion bodies (IBs) [22-25].
`By adjusting media composition, growth temperature,
`inducer concentration, promoter strength and plasmid
`copy number, variable amounts of the target protein can
`be forced to appear in the soluble form [26,27], although
`unfortunately, many eukaryotic proteins are exclusively
`found trapped in IBs and seem to be resistant to process-
`based solubility enhancement. While IBs formed by
`enzymes can be efficient catalysers in enzymatic reactions
`[28-32], pharmaceutical proteins need, in contrast, to be
`dispersed as soluble entities to reach their targets at thera-
`peutic doses. IBs essentially contain the recombinant pro-
`tein in variable proportions (from 60 to more than 90%)
`and some contaminants as chaperones, DNA, RNA and
`lipids [33]. Although stored protein can be released from
`IBs using denaturing conditions, in vitro refolding proc-
`esses are not as effective as expected [34] and other expres-
`sion systems should be tried. In some cases, recombinant
`proteins have been successfully purified from IBs as for
`example Betaferon [35] and insulin [36]. However, for
`non integral membrane proteins, cytosolic and/or soluble
`protein domains, the probability of success is reasonably
`high and E. coli should be then considered as a promising
`expression system [37].
`
`In summary, around 10% of full-length eukaryotic pro-
`teins tested in this system have been successfully pro-
`duced in soluble form in E. coli [38]. Approved
`therapeutic protein-based products from E. coli include
`hormones (human insulin and insulin analogues, calci-
`tonin, parathyroid hormone, human growth hormone,
`glucagons, somatropin and insulin growth factor 1), inter-
`ferons (alfa-1, alfa 2a, alfa-2b and gamma-1b), inter-
`leukins 11 and 2, light and heavy chains raised against
`vascular endothelial growth factor-A, tumor necrosis fac-
`tor alpha, cholera B subunit protein, B-type natriuretic
`peptide, granulocyte colony stimulating factor and plas-
`minogen activator (Additional file 1). Noteworthy, most
`of the recombinant pharmaceuticals produced in E. coli
`are addressed for the treatment of infectious diseases or
`endocrine, nutritional and metabolic disorder disease
`groups (Figure 2).
`
`Saccharomyces cerevisiae
`Production in yeast is usually approached when the target
`protein is not produced in a soluble form in the prokary-
`otic system or a specific PTM, essential for its biological
`activity, cannot be produced artificially on the purified
`product [13]. Yeasts are as cost effective, fast and techni-
`cally feasible as bacteria and high density cell cultures can
`also be reached in bioreactors. Even more, mutant strains
`that produce high amounts of heterologous protein are
`already available. Even though yeasts are able to perform
`many PTMs as O-linked glycosylation, phosphorylation,
`acetylation and acylation, the main pitfall of this expres-
`sion system is related to N-linked glycosylation patterns
`which differ from higher eukaryotes, in which sugar side
`chains of high mannose content affect the serum half-life
`and immunogenicity of the final product. Although less
`studied than in bacteria, the production of recombinant
`
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`Insect cell lines
`Cultured insect cells are used as hosts for recombinant
`baculovirus infections. The production of a recombinant
`viral vector for gene expression is time-consuming, the
`cell growth is slow when compared with former expres-
`sion systems, the cost of growth medium is high and each
`protein batch preparation has to be obtained from fresh
`cells since viral infection is lethal. PTMs are also an impor-
`tant limitation of this expression system because of the
`simple non-syalated N-linked glycosylation which is
`translated in a rapid clearance from human sera [45].
`Although genetic engineering has been used to select
`transgenic insect cell lines (MIMIC™ from Invitrogen and
`SfSWT-3) expressing galactosyltransferase, N-acetylglu-
`cosaminyltransferases, syalic acid synthases and syalil-
`transferases genes [46-48] to obtain humanized complex
`N-linked glycosylation protein patterns, there are still
`unwanted toxicological issues that need to be overcome.
`
`There is only one approved biopharmaceutical product
`containing recombinant proteins from infected insect cell
`line Hi Five, Cervarix, consisting on recombinant papillo-
`mavirus C-terminal truncated major capsid protein L1
`types 16 and 18. Nonetheless, this expression system has
`been extensively used in structural studies since correctly
`folded eukaryotic proteins can be obtained in a secreted
`form in serum free media which enormously simplifies
`protein capture in purification protocols.
`
`Hybridoma cell lines
`Hybridomas are fusion cells of murine origin (B-cells and
`myeloma tumour cells) that are able to express specific
`monoclonal antibodies against a determined antigen,
`thus possessing therapeutic potential [49]. Clone selec-
`tion may account for the progressive enrichment of cells
`displaying a glycosylation profile with reduced potency
`and undesirable immunogenic reaction since the human
`immune system recognizes mouse antibodies as foreign.
`
`Genetic engineering has been applied to obtain human-
`ized monoclonal antibodies using either recombinant
`mammalian cells producing chimeric antibodies or genet-
`ically modified mice to produce human-like antibodies
`[49]. One such product, Remicade, which binds tumour
`necrosis factor-alpha, is a pharmaceutical blockbuster
`used in the treatment of Crohn's disease.
`
`Hamster cell lines
`Most of the therapeutic proteins approved so far have
`been obtained using transgenic hamster cell lines, namely
`49 in chinese hamster ovary cells (CHO) and 1 in baby
`hamster kidney cells (BHK) (Additional file 1). The main
`advantage of this expression system is that cells can be
`adapted to grow in suspension in serum free media
`
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`ACDEGHIJKLMQRT
`
`30
`
`25
`
`20
`
`15
`
`10
`
`5
`
`0
`
`Product number
`
`Insect cells Mammalian cells Microbial cells
`E. coli
`S. cerevisae Hybridomas Transgenic animals
`
`Number of recombinant biopharmaceuticals in different pro-duction systems, grouped by WHO therapeutic indications (see the legend of Additional file for nomenclature)Figure 2
`
`
`
`Number of recombinant biopharmaceuticals in dif-
`ferent production systems, grouped by WHO thera-
`peutic indications (see the legend of Additional file
`for nomenclature). Products from E. coli and S. cerevisae
`are also presented together under the category of microbial
`cells.
`
`proteins also triggers conformational stress responses and
`produced proteins fail sometimes to reach their native
`conformation. Recent insights about conformational
`stress, and in general, to cell responses to protein produc-
`tion in recombinant yeasts have been extensively reviewed
`elsewhere [21,39,40].
`
`The approved protein products produced in yeast are
`obtained exclusively in Saccharomyces cerevisiae [4] and
`correspond to hormones (insulin, insulin analogues, non
`glycosylated human growth hormone somatotropin, glu-
`cagon), vaccines (hepatitis B virus surface antigen -in the
`formulation of 15 out of the 28 yeast derived products-)
`and virus-like particles (VLPs) of the major capsid protein
`L1 of human papillomavirus type 6, 11, 16 and 18, urate
`oxidase from Aspergillus flavus, granulocyte-macrophage
`colony stimulating factor, albumin, hirudin of Hirudo
`medicinalis and human platelets derived growth factor. As
`in the case of E. coli, most of the recombinant pharmaceu-
`ticals from yeast are addressed to either infectious diseases
`or endocrine, nutritional and metabolic disorders (Figure
`2), being these therapeutic areas the most covered by
`microbial products. Interestingly, several yeast species
`other than S. cerevisiae are being explored as sources of
`biopharmaceuticals and other proteins of biomedical
`interest [21,41]. In addition, current metabolic engineer-
`ing approaches [42] and optimization of process proce-
`dures [43,44] are dramatically expanding the potential of
`yeast species for improved production of recombinant
`proteins.
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`(SFM), protein-free and chemically defined media [50].
`This fact increases the biosafety of final products reducing
`risk of introducing prions of bovine spongiform encepha-
`lopathy (BSE) from bovine serum albumin and of infec-
`tious variant Creutzfeldt-Jakob Disease (vCJD) from
`human serum albumin. In addition, recombinant prod-
`ucts can be secreted into the chemical defined media,
`which simplifies both upstream and downstream purifi-
`cation process [51]. PTMs in this expression system are
`almost the same as in human cell lines, although some
`concerns about comparability in the glycosylation pattern
`have arisen when comparing different batches of the same
`manufacturer product and biosimilars [52]. Further devel-
`opment of chemically defined media and fine description
`of growth conditions would help to overcome this issue.
`
`Human cell lines
`In the recent years, three therapeutic proteins produced in
`human cell lines have been approved, namely Dynepo-
`erithropoietin, Elaprase-irudonate-2-sulfatase and Repla-
`gal-alfa-galactosidase A. These products are fully glyco-
`sylated human proteins, so this expression system should
`be addressed when heavily glycosylation is needed. In
`general, recombinant biopharmaceuticals obtained from
`mammalian cells cover a wider spectrum of pathological
`conditions than those obtained from microbes, and the
`distribution of applications is less biased than when
`observing products from E. coli or S. cerevisae (Figure 2).
`
`Transgenic animals
`Transgenic animals (avian and mammals), have been suc-
`cessfully used for the production of recombinant proteins
`secreted into egg white and milk respectively. Protein pro-
`duction using transgenic farm animals supposes a great
`biotechnological challenge in terms of safety concerns
`such as transmission of infectious diseases (including
`viral and prion infections) or adverse allergenic, immuno-
`genic and autoimmune responses. In 2006, ATryn was the
`first and so far single approved rDNA biopharmaceutical
`using transgenic animals and validated manufacturer
`technology platform. It contains human antithrombin
`(432 amino acids) with 15% glycosylated moieties and is
`secreted into the milk of transgenic goats. Another prod-
`uct obtained from the milk of transgenic rabbits (Rhucin)
`has been recently denied for its approval by the EMEA
`although more tests of repeated treatment are underway
`to try again its approval. Despite such limited progress, if
`pharmacovigilance after patient treatment does not reveal
`any adverse side effects, we might envisage, in the next
`years, an increase in the approval rate of recombinant pro-
`tein products from transgenic animal origin.
`
`Alternative, non microbial systems for forthcoming
`products
`As previously discussed, recombinant DNA biopharma-
`ceuticals obtained from bacterial, yeast or mammalian
`
`cell culture bioreactors are quite effective as therapeutic
`agents although production costs are relatively high. One
`way to address the economic-cost benefit hurdle is
`through the use of transgenic organisms to manufacture
`biopharmaceuticals. Biopharming would dramatically
`reduce the cost of recombinant therapeutic proteins not
`only in the initial construction of production facilities but
`also the scale-up process and the final recombinant pro-
`tein yield. Nonetheless, the fact that regulatory guidelines
`are being developed as the same time that the establish-
`ment of protein production processes is creating uncer-
`tainty within biotechnological companies to fulfil drug
`administration requirements.
`
`Transgenic plants have been used as recombinant protein
`producers for research and diagnostic uses due to the
`advantageous low cost of cultivation, high mass produc-
`tion, flexible scale-up, lack of human pathogens and addi-
`tion of eukaryotic PTMs. The first recombinant protein
`product obtained from transgenic tobacco was human
`growth hormone [53] and since then, many other prod-
`ucts have been obtained (including antibodies, the sur-
`face antigen of the Hepatitis-B-Virus, industrial enzymes
`and milk proteins). Again, the main disadvantage is
`related to the plant specific PMTs introduced in recom-
`binant proteins which produce adverse
`immune
`responses. Moreover, the possibility to spread the proteins
`in open fields and the negative public perception of the
`transgenic plants precludes the use of plants as an attrac-
`tive expression system of therapeutic proteins.
`
`Host comparative trends in rDNA biopharmaceutical
`approval
`As mentioned above, human insulin produced in E. coli
`was the first rDNA pharmaceutical approved for use,
`which was followed by a progressively increasing number
`of other protein drugs from bacteria and yeast (Figure 3).
`Since 1995, the progression of products of mammalian
`origin was noticeable and extremely regular, and quanti-
`tatively comparable to that of microbial products. Impor-
`tantly, the incorporation of mammalian cells as factories
`for rDNA pharmaceuticals has neither represented an
`excluding alternative to microbial hosts nor resulted in a
`decrease in the approval rate of microbial products (Fig-
`ure 3). This is probably due to the extremely different bio-
`logically and technologically backgrounds associated to
`protein production, the good quality of microbial prod-
`ucts and the high costs associated to mammalian cell pro-
`duction. In addition, this fact indicates the potential of
`microbial cells in biopharmaceutical industry despite the
`limited PTM performance of their products and other bot-
`tlenecks as discussed above. Also, microbial cell factory
`products cover a spectrum of products and application
`fields that do not necessarily match those addressed by
`mammalian cell factories (Figure 2).
`
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`expected incorporation of unusual mammalian hosts
`such as transgenic animals or plants, microbial cells
`appear as extremely robust and convenient hosts, and
`gaining knowledge about the biological aspects of protein
`production would hopefully enhance the performance of
`such hosts beyond the current apparent limitations. In
`this regard, not only commonly used bacteria and yeasts
`but unconventional strains or species are observed as
`promising cell factories for forthcoming recombinant
`drugs. Their incorporation into productive processes for
`human pharmaceuticals would hopefully push the trend
`of marketed products and fulfil the increasing demands of
`the pharmacological industry.
`
`Abbreviations
`(ADME): absorption, distribution, metabolism, and
`excretion; (BHK): baby hamster kidney cells; (CHO): chi-
`nese hamster ovary cells; (EMEA): European Medicines
`Agency; (FDA): Food and Drug Administration; (IBs):
`inclusion bodies; (PTMs): post-translational modifica-
`tions; (rDNA): Recombinant DNA; (VLPs): virus-like par-
`ticles
`
`Competing interests
`The authors declare that they have no competing interests.
`
`Authors' contributions
`All authors read and approved the manuscript's content.
`
`Additional material
`
`Additional file 1
`Supplemental table
`Recombinant drugs approved for use, grouped by producing host types.
`Click here for file
`[http://www.biomedcentral.com/content/supplementary/1475-
`2859-8-17-S1.doc]
`
`Acknowledgements
`The authors acknowledge the support of MEC, AGAUR and UAB to their
`research on protein drug development and delivery through grants
`BIO2007-61194 to AV (MEC), 2005SGR-00956 to AV (AGAUR) and
`EME2007-08 to NFM (UAB). We also appreciate the support of CIBER de
`Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN, promoted by
`ISCIII), Spain. AV received an ICREA ACADEMIA award from ICREA (Gen-
`eralitat de Catalunya).
`
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`E. coli
`E. coli
`S. cerevisiae
`S. cerevisae
`Hybridomas
`Hybridomas
`Mammalian cells
`Mammalian cells
`Insect cells
`Insect cells
`Transgenic animals
`Transgenic animals
`Microbial cells
`Microbial cells
`
`80
`
`60
`
`40
`
`20
`
`Accumulated product number
`
`0
`1980
`
`1985
`
`1990
`
`1995
`
`2000
`
`2005
`
`2010
`
`Year of approval
`
`Accumulated number of recombinant biopharmaceuticals obtained in different production systems, in front of year of their first time approval (either in US or EU)Figure 3
`
`
`
`Accumulated number of recombinant biopharma-
`ceuticals obtained in different production systems, in
`front of year of their first time approval (either in US
`or EU). Products from E. coli and S. cerevisae are presented
`together under the category of microbial cells.
`
`Interestingly, a plateau in the rate of rDNA drug approval
`during the last 2–3 years is becoming perceivable, irre-
`spective of the production system (Figure 3). Although it
`might be observed as a transient event, this fact seems
`instead to indicate that the current production systems
`could be near to the exhaustion regarding their ability to
`hold the production of complex proteins, protein com-
`plexes or the so-called difficult-to-express proteins. Desir-
`ably,
`recent
`insights about
`system's biology of
`recombinant cells and hosts, and specially, arising novel
`concepts on recombinant protein quality [54-56] and
`host stress responses [21] would enlarge the possibilities
`for metabolic and process engineering aiming to the eco-
`nomically feasible production of new, more complex
`drugs. Indeed, pushed by fast advances in molecular med-
`icine the pharmaceutical industry is urgently demanding
`improved production systems and novel and cheaper
`drugs.
`
`Conclusions and future prospects
`Overcoming the biological and methodological obstacles
`posed by cell factories to the production or rDNA pharma-
`ceuticals is a main challenge in the further development of
`protein-based molecular medicine. Recombinant DNA
`technologies might have exhausted conventional cell fac-
`tories and new production systems need to be deeply
`explored and incorporated into the production pipeline.
`On the other hand, a more profound comprehension of
`host cell physiology and stress responses to protein pro-
`duction would necessary offer improved tools (either at
`genetic, metabolic or system levels) to favour high yield
`and high quality protein production. Apart from the
`
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`Microbial Cell Factories 2009, 8:17
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`http://www.microbialcellfactories.com/content/8/1/17
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