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Challenges in therapeutic glycoprotein production
Challenges in therapeutic glycoprotein production Natarajan Sethuraman and Terrance A Stadheim Protein-based drugs constitute about a quarter of new approvals with a majority being glycoproteins. Increasing use of glycoproteins, such as monoclonal antibodies, at high therapeutic doses is challenging current production capacity. Mammalian cell culture, which is currently the production system of choice for glycoproteins, has several disadvantages including high cost of goods, long cycle times and, importantly, limited control over glycosylation. In view of this, several expression systems are currently being explored as alternatives to mammalian cell culture, these include yeast, plant and insect expression systems. Each of these has different merits for the production of therapeutic glycoproteins and can lead to enhanced therapeutic efficiency. Addresses GlycoFi, Inc., 21 Lafayette Street, Suite 200, Lebanon, NH 03766, USA Corresponding author: Sethuraman, Natarajan ([email protected])
Current Opinion in Biotechnology 2006, 17:341–346 This review comes from a themed issue on Protein technologies Edited by Deb K Chatterjee and Joshua LaBaer Available online 7th July 2006 0958-1669/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2006.06.010
Introduction The importance of therapeutic proteins has grown rapidly since the emergence of the biotechnology industry more than 30 years ago. Currently, 64 products have been approved by European and US regulatory agencies with some 500 product candidates in clinical and preclinical development. Of these, approximately 70% are glycoproteins. As of 2003, the global market for biopharmaceuticals (recombinant therapeutic protein and nucleic acid based products) was estimated at more than $30 billion [1]. Therapeutic proteins were initially derived from human sources; for example, blood clotting factors and human serum albumin from plasma, insulin from pancreas, and glucocerebrosidase from placenta. However, concerns over product purity and consistency, the potential for viral contamination and, importantly, the emergence of genetic engineering tools has shifted therapeutic protein production into recombinant expression systems. Mammalian cell lines, bacteria, yeast and insect cells have www.sciencedirect.com
evolved as the major recombinant protein expression hosts, although these expression systems vary widely with regard to their ability to incorporate the post-translational modifications found on native human proteins. For example, only mammalian cell lines have the inherent capacity to carry out N-linked glycosylation of proteins during secretion; bacteria lack the N-linked glycosylation machinery and are thus not suitable hosts for glycoprotein production. Because many proteins of therapeutic importance require N-glycosylation for biological activity, expression systems with N-glycosylation capability have become essential for therapeutic glycoprotein production. The process of N-linked glycosylation occurs in a stepwise manner and begins with the transfer of a core oligosaccharide (GlcNAc2Man9Glc3; see Figure 1) to a three amino acid consensus sequence (Asn-X-Ser/Thr where X is any amino acid other than Pro) on the nascent polypeptide in the endoplasmic reticulum (ER). The glycosylated protein then undergoes further glycan remodeling in the ER and Golgi apparatus before it is finally secreted from the cell. As mammalian expression systems produce mainly human glycans, they have become the dominant platform for therapeutic glycoprotein production. However, major advances in the bioengineering of glycosylation pathways in other hosts over the past decade have expanded the options for producing these molecules. Both the function and efficacy of proteins are affected by the presence and composition of N-glycosylation structures. For example, non-human glycoforms can adversely affect pharmacokinetic (PK) properties and raise immunogenicity and safety concerns [2]. Thus, the therapeutic use of glycoproteins derived from expression systems that do not perform human glycosylation could lead to rapid clearance, complement activation, and enhanced immunogenicity by targeting to antigen-presenting cells [2]. Indeed, approximately 1% of circulating immunoglobulin G (IgG) molecules in normal human serum is composed of anti-a-1,3-galactose antibodies, a common modification to N-linked glycans produced by non-primate mammals [3]. Furthermore, non-human glycans such as core a-1,3-fucose (present on proteins expressed in plants and insect cells) and xylose (present in plants) constitute important IgE epitopes. The clinical use of glycoproteins containing such sugars might be accompanied by severe adverse reactions, including life-threatening anaphylaxis, especially in allergic patient populations [4,5]. By contrast, the presence of specific glycoforms can confer significant therapeutic advantages, and expression systems that allow for the control of glycosylation are therefore desirable. Current Opinion in Biotechnology 2006, 17:341–346
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Figure 1
glycans that affect tissue targeting; and glycans that modulate biological activity. Balancing these parameters can dictate the desired glycosylation structure and the choice of protein expression system. The purpose of this review is to highlight the influence of glycosylation on optimal therapeutic efficacy by considering the aforementioned mechanisms, and to evaluate the merits of various expression systems to achieve optimal glycosylation.
The role of glycans for glycoprotein pharmacokinetics PK plays a major role in determining the efficacy of many therapeutic proteins. The circulating half-life of proteins is affected by many factors, including molecular size and net charge, both of which are affected by the presence and composition of glycan structures. Glycans contribute significantly to the hydrodynamic volume and charge of glycoproteins. In particular, the content of sialic acid contributes to the net negative charge, and improves the PK of glycoproteins such as erythropoietin (EPO) [6]. Although glycosylation can improve plasma half-life, the presence of certain carbohydrate moieties can trigger lectin-mediated clearance, thereby reducing plasma halflife. For example, asialylated glycoproteins are subject to receptor-mediated clearance by the asialoglycoprotein receptor (ASGPR) found in the liver [7,8]. Similarly, glycoproteins with terminal mannose or terminal GlcNAc are cleared through the reticulo-endothelial system, which relies on several receptors with high affinity for both mannose and GlcNAc [9]. Human EPO contains three N-linked glycans that can constitute up to 40% of the molecular mass [10]. Although deglycosylated EPO displays threefold higher in vitro potency than its glycosylated counterpart [11], it exhibits no in vivo erythropoietic efficacy [11,12]. A more efficacious form of EPO can be obtained by selecting for multibranched sialylated glycoforms (mostly tetra-antennary; see Figure 1) [13]. The importance of glycans for the in vivo potency of EPO is further illustrated by the development of a more potent erythropoietic factor, which was created by engineering two additional N-linked glycosylation sites into the EPO protein sequence [14]. This modified EPO has weaker in vitro receptor binding but extended serum half-life, resulting in increased efficacy [15].
The role of glycans for tissue targeting Human N-linked glycosylation structures. The structures of the core oligosaccharide, biantennary complex oligosaccharides, triantennary and tetra-antennary fucosylated complex oligosaccharides are illustrated. Note that the fucosylated N-linked glycans depicted here can also be present in an afucosylated form. The key below shows the individual components of the structures.
The different glycoforms can be classified into three categories on the basis of their impact on therapeutic protein efficacy: glycans that affect plasma half-life; Current Opinion in Biotechnology 2006, 17:341–346
Carbohydrate-binding proteins, or lectins, are differentially expressed and thus different tissues have varying affinities for specific glycoforms. Therapeutic proteins with distinct N-glycans can therefore be developed for cell- and tissue-specific targeting. Glucocerebrosidase (GBA), approved for the treatment of Gaucher’s disease, is an example of a therapeutic protein that relies on a glycan-dependent targeting mechanism for therapeutic function. In this case, a specific glycan structure, terminal paucimannose (GlcNAc2Man3), is used to target GBA through mannose-binding lectins to macrophages in the www.sciencedirect.com
Therapeutic glycoprotein production Sethuraman and Stadheim 343
liver, where the recombinant enzyme metabolizes accumulated glucocerebroside [16]. Similarly, ASGPR found on hepatocytes binds to glycoproteins containing terminal galactose or N-acetylgalactosamine; thus, this liver receptor could be used to achieve tissue-specific protein targeting. Interferon exhibits antiviral activity against hepatitis C, yet its therapeutic use is hampered by dose-limiting toxicities [17]. Therefore, the production of interferon with glycoforms that specifically target the site of viral replication (i.e. the liver), might minimize toxicity and thereby increase the therapeutic index.
The role of glycans for modulating biological activity N-Linked glycosylation has been shown to affect the efficacy of some therapeutic glycoproteins by modulating the interaction with specific receptors. For example, immunoglobulin heavy chains contain a single N-linked glycosylation site resulting in two N-glycans per assembled antibody. This glycosylation is essential for efficient interactions with Fc receptors (FcR) and for FcR-mediated effector functions, including antibodydependent cell cytotoxicity (ADCC) and complementdependent cytotoxicity (CDC). Aglycosylated IgG1s exhibit severely impaired ADCC and CDC. IgG-mediated ADCC can be modulated by varying the N-glycan composition. An example of increased ADCC potency was demonstrated by the use of various glycosylation pathway inhibitors during the production of IgG from mammalian cells [18]. The resulting IgGs containing different glycosylation intermediates displayed varying degrees of ADCC and suggested the importance of fucose in modulating ADCC [18].
serum [20]. In addition, glycoproteins produced in CHO cells display inherent glycan heterogeneity, resulting in a mixture of molecules with varying efficacy profiles. Moreover, this heterogeneity is sensitive to culture conditions making batch-to-batch reproducibility a process development challenge [13]. For example, proteins such as EPO, which are believed to require tetra-antennary sialylated glycans for optimal therapeutic efficacy, must be made under culture conditions that favor the formation of such structures (currently involving roller bottles) [13] or could involve the engineering of glycosylation pathways in mammalian cells [21,22]. Although CHO cells are an acceptable expression host for sialylated glycoproteins, proteins requiring non-sialylated glycoforms require in vitro processing to expose the desired glycoforms. Therapeutic GBA is currently produced in CHO cells. However, the N-glycan structure of CHO-produced GBA contains terminal sialic acid and requires a series of enzymatic digests to produce paucimannose through the sequential removal of sialic acid, galactose and GlcNAc [16]. Currently, CHO and the murine myeloma cell line NS0, are the expression systems of choice for the production of monoclonal antibodies. Monoclonal antibodies expressed from CHO cells contain a mixture of fucosylated G0, G1 and G2 complex glycan structures (Figure 1) [23]. Antibody effector function can be increased through the addition of bisecting GlcNAc, which is otherwise absent in proteins derived from CHO cells [24], or through the reduction of fucose [18]. To achieve a higher level of ADCC activity, several groups have engineered fucosedeficient strains [25,26]. These efforts emphasize the importance of glycan engineering in the modulation of antibody efficacy.
Glycoprotein expression systems Mammalian cell culture
Other expression systems
Chinese hamster ovary (CHO) cell lines are currently the preferred host for the production of therapeutic glycoproteins. These cell lines offer several advantages, including extensive know-how of this system and an established infrastructure in the biotechnology industry, process scalability, and the capacity to produce proteins with N-glycans similar to those found on human proteins. However, CHO cell-based expression systems also have several disadvantages: a relatively high cost of goods; the potential for propagating infectious agents, such as viruses and prions; a long development time from gene to production cell line; and the inability to adequately control N-glycosylation. For example, whereas humans lack the pathway for the synthesis of N-glycolylneuraminic acid [19], EPO produced from CHO cells contains both N-glycolylneuraminic acid and N-acetylneuraminic acid [13]. Glycoconjugates that contain N-glycolylneuraminic acid might be subject to clearance by anti-Nglycolylneuraminic acid antibodies present in human
Insect cell lines have been reported to be efficient expression hosts for the production of many glycoproteins [27], including monoclonal antibodies. Insect cells are capable of performing post-translational modifications, such as N-glycosylation [28–30,31]; however, proteins expressed in insect cells contain glycans that are not of the complex type (Figure 1), but instead contain either hybrid, high mannose or paucimannose glycans [32]. Owing to the poor half-life associated with these N-glycans, insect cell lines are not suitable for the expression of glycoproteins for which long serum half-life is desirable. Moreover, depending on the insect cell line used, non-human a1,3-fucose may be present, which raises immunogenicity and safety issues [33]. Efforts to produce sialylated glycans in insect cells have been reported with contradictory results for sialic acid transfer [27,34].
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Transgenic plants are being explored for the production of therapeutic glycoproteins [35]. A major limitation with Current Opinion in Biotechnology 2006, 17:341–346
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plant-based expression systems is the production of nonhuman glycan structures that essentially lack galactose and sialic acid, and which contain the potentially immunogenic sugars xylose and a1,3-fucose [5]. The potential allergic responses to these sugars could limit the development of plants for the production of therapeutic glycoproteins. Efforts to humanize plant N-glycans through the introduction of the enzymatic machinery for the transfer of galactose [36,37] as well as the deletion of xylosyltransferase and a1,3-fucosyltransferase [38] are being pursued. Transgenic animals have gained increased attention as an alternative host, and several glycoproteins including monoclonal antibodies have been expressed [39]. The N-glycans of proteins produced in transgenic animals comprise high-mannose and hybrid type glycans with low sialic acid content compared to human proteins [40]. This could limit their use for the production of proteins requiring a long serum half-life. Monoclonal antibodies with enhanced ADCC have been produced in chicken tubular gland cells and secreted into eggs [41]. The glycosylation of these antibodies shows mostly afucosylated high-mannose type glycans, and thus they exhibit a shorter serum half-life.
glycosylation and the ability to optimize glycosylation for specific therapeutic functions. For example, a panel of glycoengineered P. pastoris strains was used to produce various glycoforms of the monoclonal antibody Rituxan (an anti-CD20 IgG1 antibody) [31]. Although these antibodies share identical amino acid sequences to commercial Rituxan, specific glycoforms displayed 100-fold higher binding affinity to relevant FcgRIII receptors and exhibited improved in vitro human B-cell depletion [31]. The use of glycoengineered yeast to control glycosylation could generate tissue-targeted therapeutic glycoproteins such as interferon, where targeting to the liver might overcome dose-limiting systemic toxicities (as described above). Additionally, the production and secretion of GBA glycosylated with terminal mannose could offer an advantage over current methods, which require downstream in vitro modifications. Another potential use for glycoengineered P. pastoris is the production of recombinant EPO. Engineering yeast to secrete sialylated EPO with high glycan uniformity would increase process robustness resulting in reduced lot-to-lot variability. Alternatively, other physiological functions of glycoproteins can be exploited such as the production of galactosylated EPO, which might be important for ameliorating damage effected by stroke [47].
Yeast expression systems
Yeast-based expression systems have been used for the production of several approved therapeutic proteins including recombinant insulin. Protein production in yeast offers several advantages, such as robust expression, scalable fermentation, and the ability to perform N-glycosylation [42]. The yeast Pichia pastoris has received increasing attention as a protein expression system with several therapeutic proteins produced in this host currently in clinical trials [43]. However, the presence of yeast-specific high mannose sugars has, until recently, limited the use of yeast to proteins that do not require N-glycosylation for therapeutic efficacy (e.g. insulin) [43]. Advances in glycoengineering in P. pastoris have not only eliminated undesirable glycans, but have shown that yeast can be engineered to perform human glycosylation to a high degree of homogeneity [25,44,45]. As discussed, N-glycosylation plays a critical role in the half-life, tissue targeting and potency of therapeutic glycoproteins. Therefore, the availability of an expression system that has the ability to customize the glycosylation of a therapeutic protein is likely to lead to the development of improved therapeutic glycoproteins. To date, only P. pastoris has been glycoengineered to provide a library of individual strains in which each host strain has the ability to produce proteins with a specific human glycoform to a very high degree of uniformity [43,46]. This advance, for the first time, provides an expression system with the capacity to control Current Opinion in Biotechnology 2006, 17:341–346
Conclusions Expression systems used for the commercial production of therapeutic glycoproteins are the result of many years of improvements and process developments. However, an expression system that allows for control over the glycosylation of therapeutic proteins has, until recently, been unavailable. Through the customization of glycan profiles, such as that recently demonstrated in glycoengineered yeast, it is now possible to systematically probe for glycosylation-dependent therapeutic effects. Increased availability of glycoengineered expression systems will enable a greater understanding of structure-function relationships among glycoforms, which should lead to the development of novel therapeutics.
Acknowledgements The authors would like to thank Tillman U Gerngross and Richard A Fisher for stimulating discussions and critical reviews during the preparation of this manuscript.
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35. Giddings G, Allison G, Brooks D, Carter A: Transgenic plants as factories for biopharmaceuticals. Nat Biotechnol 2000, 18:1151-1155. 36. Bakker H, Bardor M, Molthoff JW, Gomord V, Elbers I, Stevens LH, Jordi W, Lommen A, Faye L, Lerouge P et al.: Galactose-extended glycans of antibodies produced by transgenic plants. Proc Natl Acad Sci USA 2001, 98:2899-2904. 37. Misaki R, Fujiyama K, Seki T: Expression of human CMP-N acetylneuraminic acid synthetase and CMP-sialic acid transporter in tobacco suspension-cultured cell. Biochem Biophys Res Commun 2006, 339:1184-1189. This report demonstrates the introduction of a CMP-sialic acid synthesis pathway in tobacco plant cells. Further engineering of this cell line could offer the expression of proteins with mammalian-like N-glycosylation. 38. Strasser R, Altmann F, Mach L, Glossl J, Steinkellner H: Generation of Arabidopsis thaliana plants with complex Nglycans lacking b1,2-linked xylose and core a1,3-linked fucose. FEBS Lett 2004, 561:132-136. 39. Houdebine LM: Antibody manufacture in transgenic animals and comparisons with other systems. Curr Opin Biotechnol 2002, 13:625-629. 40. van Berkel PH, Welling MM, Geerts M, van Veen HA, Ravensbergen B, Salaheddine M, Pauwels EK, Pieper F, Nuijens JH, Nibbering PH: Large scale production of recombinant human lactoferrin in the milk of transgenic cows. Nat Biotechnol 2002, 20:484-487. 41. Zhu L, van de Lavoir MC, Albanese J, Beenhouwer DO, Cardarelli PM, Cuison S, Deng DF, Deshpande S, Diamond JH, Green L et al.: Production of human monoclonal antibody in eggs of chimeric chickens. Nat Biotechnol 2005, 23:1159-1169.
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The expression of human monoclonal antibodies (mAbs) in chicken eggs is reported. This report shows that mAbs produced in chicken eggs display similar antigen binding and antigen-mediated internalization, enhanced ADCC, but increased clearance compared with the CHO-produced mAbs. 42. Cereghino JL, Cregg JM: Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev 2000, 24:45-66. 43. Gerngross TU: Advances in the production of human therapeutic proteins in yeasts and filamentous fungi. Nat Biotechnol 2004, 22:1409-1414. 44. Choi BK, Bobrowicz P, Davidson RC, Hamilton SR, Kung DH, Li H, Miele RG, Nett JH, Wildt S, Gerngross TU: Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris. Proc Natl Acad Sci USA 2003, 100:5022-5027. The production of a glycoprotein with human hybrid N-glycosylation in yeast is described. Also described is a high-throughput screening method for the selection of yeast clones with uniform hybrid N-glycosylation. 45. Bobrowicz P, Davidson RC, Li H, Potgieter TI, Nett JH, Hamilton SR, Stadheim TA, Miele RG, Bobrowicz B, Mitchell T et al.: Engineering of an artificial glycosylation pathway blocked in core oligosaccharide assembly in the yeast Pichia pastoris: production of complex humanized glycoproteins with terminal galactose. Glycobiology 2004, 14:757-766. 46. Wildt S, Gerngross TU: The humanization of N-glycosylation pathways in yeast. Nat Rev Microbiol 2005, 3:119-128. 47. Wang X, Zhu C, Wang X, Gerwien JG, Schrattenholz A, Sandberg M, Leist M, Blomgren K: The nonerythropoietic asialoerythropoietin protects against neonatal hypoxiaischemia as potently as erythropoietin. J Neurochem 2004, 91:900-910.
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Current Opinion in Biotechnology 2006, 17:341–346 This review comes from a themed issue on Protein technologies Edited by Deb K Chatterjee and Joshua LaBaer Available online 7th July 2006 0958-1669/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2006.06.010
Introduction The importance of therapeutic proteins has grown rapidly since the emergence of the biotechnology industry more than 30 years ago. Currently, 64 products have been approved by European and US regulatory agencies with some 500 product candidates in clinical and preclinical development. Of these, approximately 70% are glycoproteins. As of 2003, the global market for biopharmaceuticals (recombinant therapeutic protein and nucleic acid based products) was estimated at more than $30 billion [1]. Therapeutic proteins were initially derived from human sources; for example, blood clotting factors and human serum albumin from plasma, insulin from pancreas, and glucocerebrosidase from placenta. However, concerns over product purity and consistency, the potential for viral contamination and, importantly, the emergence of genetic engineering tools has shifted therapeutic protein production into recombinant expression systems. Mammalian cell lines, bacteria, yeast and insect cells have www.sciencedirect.com
evolved as the major recombinant protein expression hosts, although these expression systems vary widely with regard to their ability to incorporate the post-translational modifications found on native human proteins. For example, only mammalian cell lines have the inherent capacity to carry out N-linked glycosylation of proteins during secretion; bacteria lack the N-linked glycosylation machinery and are thus not suitable hosts for glycoprotein production. Because many proteins of therapeutic importance require N-glycosylation for biological activity, expression systems with N-glycosylation capability have become essential for therapeutic glycoprotein production. The process of N-linked glycosylation occurs in a stepwise manner and begins with the transfer of a core oligosaccharide (GlcNAc2Man9Glc3; see Figure 1) to a three amino acid consensus sequence (Asn-X-Ser/Thr where X is any amino acid other than Pro) on the nascent polypeptide in the endoplasmic reticulum (ER). The glycosylated protein then undergoes further glycan remodeling in the ER and Golgi apparatus before it is finally secreted from the cell. As mammalian expression systems produce mainly human glycans, they have become the dominant platform for therapeutic glycoprotein production. However, major advances in the bioengineering of glycosylation pathways in other hosts over the past decade have expanded the options for producing these molecules. Both the function and efficacy of proteins are affected by the presence and composition of N-glycosylation structures. For example, non-human glycoforms can adversely affect pharmacokinetic (PK) properties and raise immunogenicity and safety concerns [2]. Thus, the therapeutic use of glycoproteins derived from expression systems that do not perform human glycosylation could lead to rapid clearance, complement activation, and enhanced immunogenicity by targeting to antigen-presenting cells [2]. Indeed, approximately 1% of circulating immunoglobulin G (IgG) molecules in normal human serum is composed of anti-a-1,3-galactose antibodies, a common modification to N-linked glycans produced by non-primate mammals [3]. Furthermore, non-human glycans such as core a-1,3-fucose (present on proteins expressed in plants and insect cells) and xylose (present in plants) constitute important IgE epitopes. The clinical use of glycoproteins containing such sugars might be accompanied by severe adverse reactions, including life-threatening anaphylaxis, especially in allergic patient populations [4,5]. By contrast, the presence of specific glycoforms can confer significant therapeutic advantages, and expression systems that allow for the control of glycosylation are therefore desirable. Current Opinion in Biotechnology 2006, 17:341–346
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Figure 1
glycans that affect tissue targeting; and glycans that modulate biological activity. Balancing these parameters can dictate the desired glycosylation structure and the choice of protein expression system. The purpose of this review is to highlight the influence of glycosylation on optimal therapeutic efficacy by considering the aforementioned mechanisms, and to evaluate the merits of various expression systems to achieve optimal glycosylation.
The role of glycans for glycoprotein pharmacokinetics PK plays a major role in determining the efficacy of many therapeutic proteins. The circulating half-life of proteins is affected by many factors, including molecular size and net charge, both of which are affected by the presence and composition of glycan structures. Glycans contribute significantly to the hydrodynamic volume and charge of glycoproteins. In particular, the content of sialic acid contributes to the net negative charge, and improves the PK of glycoproteins such as erythropoietin (EPO) [6]. Although glycosylation can improve plasma half-life, the presence of certain carbohydrate moieties can trigger lectin-mediated clearance, thereby reducing plasma halflife. For example, asialylated glycoproteins are subject to receptor-mediated clearance by the asialoglycoprotein receptor (ASGPR) found in the liver [7,8]. Similarly, glycoproteins with terminal mannose or terminal GlcNAc are cleared through the reticulo-endothelial system, which relies on several receptors with high affinity for both mannose and GlcNAc [9]. Human EPO contains three N-linked glycans that can constitute up to 40% of the molecular mass [10]. Although deglycosylated EPO displays threefold higher in vitro potency than its glycosylated counterpart [11], it exhibits no in vivo erythropoietic efficacy [11,12]. A more efficacious form of EPO can be obtained by selecting for multibranched sialylated glycoforms (mostly tetra-antennary; see Figure 1) [13]. The importance of glycans for the in vivo potency of EPO is further illustrated by the development of a more potent erythropoietic factor, which was created by engineering two additional N-linked glycosylation sites into the EPO protein sequence [14]. This modified EPO has weaker in vitro receptor binding but extended serum half-life, resulting in increased efficacy [15].
The role of glycans for tissue targeting Human N-linked glycosylation structures. The structures of the core oligosaccharide, biantennary complex oligosaccharides, triantennary and tetra-antennary fucosylated complex oligosaccharides are illustrated. Note that the fucosylated N-linked glycans depicted here can also be present in an afucosylated form. The key below shows the individual components of the structures.
The different glycoforms can be classified into three categories on the basis of their impact on therapeutic protein efficacy: glycans that affect plasma half-life; Current Opinion in Biotechnology 2006, 17:341–346
Carbohydrate-binding proteins, or lectins, are differentially expressed and thus different tissues have varying affinities for specific glycoforms. Therapeutic proteins with distinct N-glycans can therefore be developed for cell- and tissue-specific targeting. Glucocerebrosidase (GBA), approved for the treatment of Gaucher’s disease, is an example of a therapeutic protein that relies on a glycan-dependent targeting mechanism for therapeutic function. In this case, a specific glycan structure, terminal paucimannose (GlcNAc2Man3), is used to target GBA through mannose-binding lectins to macrophages in the www.sciencedirect.com
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liver, where the recombinant enzyme metabolizes accumulated glucocerebroside [16]. Similarly, ASGPR found on hepatocytes binds to glycoproteins containing terminal galactose or N-acetylgalactosamine; thus, this liver receptor could be used to achieve tissue-specific protein targeting. Interferon exhibits antiviral activity against hepatitis C, yet its therapeutic use is hampered by dose-limiting toxicities [17]. Therefore, the production of interferon with glycoforms that specifically target the site of viral replication (i.e. the liver), might minimize toxicity and thereby increase the therapeutic index.
The role of glycans for modulating biological activity N-Linked glycosylation has been shown to affect the efficacy of some therapeutic glycoproteins by modulating the interaction with specific receptors. For example, immunoglobulin heavy chains contain a single N-linked glycosylation site resulting in two N-glycans per assembled antibody. This glycosylation is essential for efficient interactions with Fc receptors (FcR) and for FcR-mediated effector functions, including antibodydependent cell cytotoxicity (ADCC) and complementdependent cytotoxicity (CDC). Aglycosylated IgG1s exhibit severely impaired ADCC and CDC. IgG-mediated ADCC can be modulated by varying the N-glycan composition. An example of increased ADCC potency was demonstrated by the use of various glycosylation pathway inhibitors during the production of IgG from mammalian cells [18]. The resulting IgGs containing different glycosylation intermediates displayed varying degrees of ADCC and suggested the importance of fucose in modulating ADCC [18].
serum [20]. In addition, glycoproteins produced in CHO cells display inherent glycan heterogeneity, resulting in a mixture of molecules with varying efficacy profiles. Moreover, this heterogeneity is sensitive to culture conditions making batch-to-batch reproducibility a process development challenge [13]. For example, proteins such as EPO, which are believed to require tetra-antennary sialylated glycans for optimal therapeutic efficacy, must be made under culture conditions that favor the formation of such structures (currently involving roller bottles) [13] or could involve the engineering of glycosylation pathways in mammalian cells [21,22]. Although CHO cells are an acceptable expression host for sialylated glycoproteins, proteins requiring non-sialylated glycoforms require in vitro processing to expose the desired glycoforms. Therapeutic GBA is currently produced in CHO cells. However, the N-glycan structure of CHO-produced GBA contains terminal sialic acid and requires a series of enzymatic digests to produce paucimannose through the sequential removal of sialic acid, galactose and GlcNAc [16]. Currently, CHO and the murine myeloma cell line NS0, are the expression systems of choice for the production of monoclonal antibodies. Monoclonal antibodies expressed from CHO cells contain a mixture of fucosylated G0, G1 and G2 complex glycan structures (Figure 1) [23]. Antibody effector function can be increased through the addition of bisecting GlcNAc, which is otherwise absent in proteins derived from CHO cells [24], or through the reduction of fucose [18]. To achieve a higher level of ADCC activity, several groups have engineered fucosedeficient strains [25,26]. These efforts emphasize the importance of glycan engineering in the modulation of antibody efficacy.
Glycoprotein expression systems Mammalian cell culture
Other expression systems
Chinese hamster ovary (CHO) cell lines are currently the preferred host for the production of therapeutic glycoproteins. These cell lines offer several advantages, including extensive know-how of this system and an established infrastructure in the biotechnology industry, process scalability, and the capacity to produce proteins with N-glycans similar to those found on human proteins. However, CHO cell-based expression systems also have several disadvantages: a relatively high cost of goods; the potential for propagating infectious agents, such as viruses and prions; a long development time from gene to production cell line; and the inability to adequately control N-glycosylation. For example, whereas humans lack the pathway for the synthesis of N-glycolylneuraminic acid [19], EPO produced from CHO cells contains both N-glycolylneuraminic acid and N-acetylneuraminic acid [13]. Glycoconjugates that contain N-glycolylneuraminic acid might be subject to clearance by anti-Nglycolylneuraminic acid antibodies present in human
Insect cell lines have been reported to be efficient expression hosts for the production of many glycoproteins [27], including monoclonal antibodies. Insect cells are capable of performing post-translational modifications, such as N-glycosylation [28–30,31]; however, proteins expressed in insect cells contain glycans that are not of the complex type (Figure 1), but instead contain either hybrid, high mannose or paucimannose glycans [32]. Owing to the poor half-life associated with these N-glycans, insect cell lines are not suitable for the expression of glycoproteins for which long serum half-life is desirable. Moreover, depending on the insect cell line used, non-human a1,3-fucose may be present, which raises immunogenicity and safety issues [33]. Efforts to produce sialylated glycans in insect cells have been reported with contradictory results for sialic acid transfer [27,34].
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Transgenic plants are being explored for the production of therapeutic glycoproteins [35]. A major limitation with Current Opinion in Biotechnology 2006, 17:341–346
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plant-based expression systems is the production of nonhuman glycan structures that essentially lack galactose and sialic acid, and which contain the potentially immunogenic sugars xylose and a1,3-fucose [5]. The potential allergic responses to these sugars could limit the development of plants for the production of therapeutic glycoproteins. Efforts to humanize plant N-glycans through the introduction of the enzymatic machinery for the transfer of galactose [36,37] as well as the deletion of xylosyltransferase and a1,3-fucosyltransferase [38] are being pursued. Transgenic animals have gained increased attention as an alternative host, and several glycoproteins including monoclonal antibodies have been expressed [39]. The N-glycans of proteins produced in transgenic animals comprise high-mannose and hybrid type glycans with low sialic acid content compared to human proteins [40]. This could limit their use for the production of proteins requiring a long serum half-life. Monoclonal antibodies with enhanced ADCC have been produced in chicken tubular gland cells and secreted into eggs [41]. The glycosylation of these antibodies shows mostly afucosylated high-mannose type glycans, and thus they exhibit a shorter serum half-life.
glycosylation and the ability to optimize glycosylation for specific therapeutic functions. For example, a panel of glycoengineered P. pastoris strains was used to produce various glycoforms of the monoclonal antibody Rituxan (an anti-CD20 IgG1 antibody) [31]. Although these antibodies share identical amino acid sequences to commercial Rituxan, specific glycoforms displayed 100-fold higher binding affinity to relevant FcgRIII receptors and exhibited improved in vitro human B-cell depletion [31]. The use of glycoengineered yeast to control glycosylation could generate tissue-targeted therapeutic glycoproteins such as interferon, where targeting to the liver might overcome dose-limiting systemic toxicities (as described above). Additionally, the production and secretion of GBA glycosylated with terminal mannose could offer an advantage over current methods, which require downstream in vitro modifications. Another potential use for glycoengineered P. pastoris is the production of recombinant EPO. Engineering yeast to secrete sialylated EPO with high glycan uniformity would increase process robustness resulting in reduced lot-to-lot variability. Alternatively, other physiological functions of glycoproteins can be exploited such as the production of galactosylated EPO, which might be important for ameliorating damage effected by stroke [47].
Yeast expression systems
Yeast-based expression systems have been used for the production of several approved therapeutic proteins including recombinant insulin. Protein production in yeast offers several advantages, such as robust expression, scalable fermentation, and the ability to perform N-glycosylation [42]. The yeast Pichia pastoris has received increasing attention as a protein expression system with several therapeutic proteins produced in this host currently in clinical trials [43]. However, the presence of yeast-specific high mannose sugars has, until recently, limited the use of yeast to proteins that do not require N-glycosylation for therapeutic efficacy (e.g. insulin) [43]. Advances in glycoengineering in P. pastoris have not only eliminated undesirable glycans, but have shown that yeast can be engineered to perform human glycosylation to a high degree of homogeneity [25,44,45]. As discussed, N-glycosylation plays a critical role in the half-life, tissue targeting and potency of therapeutic glycoproteins. Therefore, the availability of an expression system that has the ability to customize the glycosylation of a therapeutic protein is likely to lead to the development of improved therapeutic glycoproteins. To date, only P. pastoris has been glycoengineered to provide a library of individual strains in which each host strain has the ability to produce proteins with a specific human glycoform to a very high degree of uniformity [43,46]. This advance, for the first time, provides an expression system with the capacity to control Current Opinion in Biotechnology 2006, 17:341–346
Conclusions Expression systems used for the commercial production of therapeutic glycoproteins are the result of many years of improvements and process developments. However, an expression system that allows for control over the glycosylation of therapeutic proteins has, until recently, been unavailable. Through the customization of glycan profiles, such as that recently demonstrated in glycoengineered yeast, it is now possible to systematically probe for glycosylation-dependent therapeutic effects. Increased availability of glycoengineered expression systems will enable a greater understanding of structure-function relationships among glycoforms, which should lead to the development of novel therapeutics.
Acknowledgements The authors would like to thank Tillman U Gerngross and Richard A Fisher for stimulating discussions and critical reviews during the preparation of this manuscript.
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