MINI-REVIEW
`
`Increasing the Sialylation of Therapeutic Glycoproteins:
`The Potential of the Sialic Acid Biosynthetic Pathway
`
`KAYA BORK, RU¨ DIGER HORSTKORTE, WENKE WEIDEMANN
`
`Institut fu¨ r Physiologische Chemie, Martin-Luther-Universita¨t Halle-Wittenberg, Hollystr.1 D-06114 Halle, Germany
`
`Received 29 August 2008; accepted 10 December 2008
`
`Published online 6 February 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21684
`
`ABSTRACT: The number of therapeutic proteins has increased dramatically over the
`past years and most of the therapeutic proteins in the market today are glycoproteins.
`Usually, recombinant glycoproteins are produced in mammalian cell lines, such as
`Chinese-hamster-ovary-cells to obtain mammalian-type of glycosylation. The terminal
`monosaccharide of N-linked complex glycans is typically occupied by sialic acid.
`Presence of this sialic acid affects absorption, serum half-life, and clearance from the
`serum, as well as the physical, chemical and immunogenic properties of the respective
`glycoprotein. From a manufacturing perspective, the degree of sialylation is crucial since
`sialylation varies the function of the product. In addition, insufficient or inconsistent
`sialylation is also a major problem for the process consistency. Sialylation of over-
`expressed glycoproteins in all mammalian cell lines commonly used in biotechnology
`for the production of therapeutic glycoproteins is incomplete and there is a need for
`strategies leading to homogenous, naturally sialylated glycoproteins. This review will
`shortly summarize the biosynthesis of sialic acids and describe some recent strategies to
`increase or modify sialylation of specific therapeutic glycoproteins. ß 2009 Wiley-Liss, Inc.
`and the American Pharmacists Association J Pharm Sci 98:3499–3508, 2009
`Keywords:
`sialic acid; recombinant glycoproteins; therapeutic proteins; protein
`stability
`
`RECOMBINANT THERAPEUTIC PROTEINS
`AND GLYCOSYLATION
`
`Major demographic shifts in the industrial
`nations will prompt numerous changes in social
`and health systems. The need for novel drugs
`fighting serious diseases such as cancer or dis-
`orders of the central nervous system will grow
`rapidly. Two very different strategies are used
`
`Correspondence to: Ru¨ diger Horstkorte (Telephone: 49-345-
`5573873; Fax: 49-345-5573804;
`E-mail: ruediger.horstkorte@medizin.uni-halle.de)
`
`Journal of Pharmaceutical Sciences, Vol. 98, 3499–3508 (2009)
`ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association
`
`for those novel drugs:
`for the development
`(I) High-throughput screens of small molecules
`libraries to identify new drug candidates1,2 and
`(II) Development of specific therapeutic proteins
`using recombinant DNA technologies.3,4 Both
`strategies have advantages and disadvantages.
`Here we will focus on therapeutic glycoproteins.
`Due to their outstanding specificity, therapeutic
`glycoproteins will revolutionize our possibilities
`to treat serious diseases. However, there are
`several restrictions and problems during the
`production process, purification and application.
`In nature, nearly all proteins outside the cell are
`glycoproteins and glycosylation represents the
`most common posttranslational modification of
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`BORK, HORSTKORTE, AND WEIDEMANN
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`proteins.5–7 Glycosylation has dramatic impact on
`the function, stability, solubility and immuno-
`genicity of recombinant glycoproteins.8,9 This
`explains the general interest for novel strategies
`to engineer glycosylation and to increase the value
`of therapeutic proteins. However, glycosylation
`is not directly encoded by the genome and by far
`not understood.10 Glycans of glycoproteins are
`synthesized in the Golgi apparatus by specific
`glycosyltransferases, which attach nucleotide-
`activated monosaccharides to specific sugar resi-
`dues of glycoproteins.11 Although these glycans
`have a common core structure (Fig. 1A) the
`combination of different monosaccharides and
`different linkage-type allows the cell to create an
`enormous number of different structures. The
`terminal and most exposed monosaccharide of
`most glycoproteins is sialic acid,12 which will be in
`the focus of this review (Fig. 1B).
`
`SIALYLATION OF (THERAPEUTIC)
`GLYCOPROTEINS
`
`Sialic acids represent a family of aminosugars
`with 9-carbons with over 50 members derived
`from N-acetylneuraminic acid12,13 (Fig. 2A). Most
`mammals express N-glycolylneuraminic acid, the
`hydroxylated form of N-acetylneuraminic acid
`at position C5. However, humans express pre-
`dominantly N-acetylneuraminic acid, due to a
`
`Figure 1. Diversity of glycosylation. (A) Schematic
`structure of three major types of N-glycans. The arrows
`indicate further diverse glycosylation. A red box indi-
`cates the common core structure. (B) Branching of
`typical N-glycans. The arrows indicate further diverse
`glycosylation. The potential linkages are shown in the
`left bi-antennary structure.
`
`Figure 2.
`(A) Structure of sialic acids. (B) Biosynth-
`esis pathway of sialic acid and feedback inhibition of the
`GNE. Enzymes: (1) GNE ¼ UDP-N-acetylglucosamine
`2-epimerase/N-acetylmannosamine kinase, (2) N-acetyl
`neuraminic acid-9-phosphate synthase,
`(3) N-acetyl
`neuraminic acid-9-phosphatase, (4) CMP-N-acetyl neura-
`minic acid synthase, (5) CMP-N-acetyl neuraminic acid
`hydroxylase, (6) CMP-N-acetyl neuraminic acid Golgi
`transporter, (7) Several specific sialic acid transferases.
`Figure modified and printed with permission from Bork
`et al.49
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`homozygous mutation in the CMP-neuraminic
`acid hydroxylase gene in the human genome.
`acid
`is
`antigenic
`to
`N-glycolylneuraminic
`humans,14,15 is enriched in tumor cells and
`is originated most probably from the diet.16 This
`is an important issue since this is one of the
`reasons why the nonhuman N-glycolylneuraminic
`acid has to be avoided in any production process
`of recombinant therapeutic glycoproteins. This
`problem has been overcome recently by using
`antisense strategies to reduce the activity of
`the CMP-neuraminic acid hydroxylase in CHO-
`cells.17 The respective sialic acids possess differ-
`ent highly specific recognition and binding proper-
`ties for a variety of cellular receptors.18 This
`structural and functional diversity of sialic acid
`is exploited by viruses, bacteria and toxins, and
`by the sialoglycoproteins and sialoglycolipids
`involved in cell-cell, cell-matrix or molecular
`recognition.19
`Sialic acid is only one component out of several
`monosaccharides building glycans of glycopro-
`teins, but has an outstanding impact on the
`quality and stability of any therapeutic glycopro-
`teins for several reasons: (I) terminal galactose
`residues are one of the major factors determining
`the serum half-life of glycoproteins. The serum
`half-life is regulated by the expression of liver
`asialo-glycoprotein receptors. These receptors
`bind nonsialylated glycoproteins (on free galac-
`tose residues, see Fig. 1) and bound asialo-
`glycoproteins are removed from the serum by
`endocytosis.20 As a consequence, expression of
`terminal sialic acid on galactose residues prevents
`serum glycoproteins from degradation.21
`(II)
`Sialic acids are important for masking antigenic
`determinants or epitopes.12 It is known that the
`receptors of the immune system (T- and B-cell
`receptors) often prefer nonsialylated structures.
`Therefore, the possibility of the generation of
`antibodies (neutralizing antibodies) against the
`therapeutic glycoproteins22 correlates with the
`degree of its sialylation. (III) Negatively charged
`sialic acids influence protein-specific parameters
`such as the thermal stability,23 the resistance to
`proteolytic degradation24 or its solubility.25
`For these reasons, sialic acids are crucial for the
`production process and especially for the approval
`of therapeutic glycoproteins. Manufactures have
`to ensure the homogeneity of each batch of
`therapeutic glycoproteins. This includes
`the
`degree of sialylation. Therefore, great efforts
`have been done to standardize sialylation during
`the fermentation process since in many cases only
`
`one third of the primary glycoprotein after the
`fermentation fulfils the standard of homogenous
`gylcosylation/sialylation.
`In the following, we will introduce the biosynth-
`esis of sialic acid and consequently present some
`aspects and possibilities to improve and/or engi-
`neer higher levels of sialylation.
`
`BIOSYNTHESIS AND ACTIVATION OF
`SIALIC ACID
`
`The initial reaction in the pathway to form free
`sialic acid is a conversion of UDP-N-acetylgluco-
`samine (UDP-GlcNAc) to N-acetyl D-mannosa-
`mine (ManNAc) since the physiological precursor
`of all sialic acids is ManNAc (Fig. 2B). ManNAc
`is formed from UDP-N-acetylglucosamine (UDP-
`GlcNAc) by epimerization of the hydroxyl-group
`in position 2 and cleavage of UDP by the UDP-N-
`acetylglucosamine 2-epimerase.26 Cardini and
`Leloir originally discovered this enzyme in rat
`liver.27 All ManNAc produced by the UDP-N-
`acetylglucosamine 2-epimerase is metabolized to
`sialic acid. The biosynthesis of sialic acid is
`regulated by the feedback inhibition of the key
`enzyme of sialic acid biosynthesis, the UDP-N-
`acetylglucosamine 2-epimerase/ManNAc kinase
`(GNE). GNE is a bifunctional enzyme, which
`catalyzes the conversion of UDP-GlcNAc to
`ManNAc and the phosphorylation of ManNAc to
`ManNAc-6-phosphate26,28 (see Fig. 2B). The next
`step is a condensation of ManNAc-6-P and pyru-
`vat resulting in sialic acid-9-phosphate by the
`N-acetyl-D-neuraminyl-9-phosphate synthase.29,30
`The formation of N-acetyl neuraminic acid is
`completed by specific phosphatase acting on sialic
`acid-9-phosphate.31 Then, the resulting primary
`sialic acid (N-acetyl neuraminic acid) is either
`modified to the other members of the sialic acid
`family or is activated in the nucleus to CMP-sialic
`acid.32,33 It is very important to mention, that
`CMP-sialic acid inhibits the epimerase activity of
`GNE in a feedback dependent manner.34 CMP-
`sialic acids are than transported to the Golgi
`apparatus by a specific CMP-sialic acid trans-
`porter35 and attached to free galactose residues by
`specific sialyltransferases.36 Since not only mam-
`malian, but also plant cells, which are not able to
`generate mammalian type of glycosylation are
`used for the production of glycoproteins, these
`cells have to be engineered with the proper
`glycosylation machinery.37 However, plant cell
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`glycosylation and sialylation is not the focus of
`this review.
`
`INCREASING SIALYLATION OF
`RECOMBINANT GLYCOPROTEINS
`
`Several approaches have been made to enhance
`sialylation of glycoproteins and to maximize the
`yield of high quality glycoproteins for therapeutic
`use in mammalian cell lines.
`
`Application of ManNAc
`
`As described above, N-acetylmannosamine (Man-
`NAc)
`is the specific precursor for increasing
`intracellular sialic acid pools.38 To bypass the
`GNE-feedback mechanism (the rate-limiting step
`in the biosynthesis of sialic acids), cells can be
`supplemented with ManNAc, which intercept the
`pathway beyond the feedback mechanism of GNE
`(see Fig. 2B), since only the epimerase activity of
`the GNE is feedback-controlled. Any cellular
`ManNAc is phosphorylated and subsequently
`converted to sialic acid. Supplementation with
`ManNAc therefore leads to an increased intra-
`cellular concentration of sialic acids. However,
`aminosugars such as ManNAc does not easily pass
`the cell membrane39 and there is no detailed
`cellular uptake mechanism known (most prob-
`ably, ManNAc is taken up during endo- or
`pinocytosis). This is one reason, why very high
`concentrations (mM range) have to be used to
`increase intracellular ManNAc levels and sialyla-
`tion.40 Recent studies revealed that application
`of peracetylated ManNAc, which can cross the
`plasma membrane, helps to reduce the concentra-
`tion needed to increase sialylation.41
`It was reported that ManNAc feeding to
`Chinese-hamster-ovary-cells
`(CHO-cells), pro-
`ducing recombinant human interferon-gamma
`(CHO IFN-g) increased intracellular sialic acid
`concentration.42 Interferons (IFNs) are proteins
`produced by a wide variety of cells of the immune
`system of many vertebrates in response to
`challenges by foreign pathogens such as viruses,
`parasites or tumor cells.43 IFNs belong to the large
`class of glycoproteins known as cytokines. They
`assist the immune system to response by (I)
`inhibiting viral replication within the host cells,
`(II) Activating natural killer cells and macro-
`phages, (III) Increasing antigen presentation to
`lymphocytes, and (IV) Inducing the resistance of
`
`host cells to viral infection. Interferon-g exists in
`three sialylated glycoforms: double-glycosylated
`(at Asn25 and Asn97), single glycosylated (at
`Asn25) or nonglycosylated.44 Without further
`efforts, 25% of the total IFN-g produced in
`CHO-cells is nonsialylated after 140 h,45 which
`is a nontolerated reproducibly for a production
`process. Application of ManNAc led to a 15%
`increasing in the sialylation of IFN-g and satura-
`tion was reached with the addition of 40 mM
`ManNAc.42
`This approach was also successfully adopted to
`increase the sialylation of erythropoietin (EPO).
`EPO is a glycoprotein hormone that is a cytokine
`for erythrocyte precursors cells.46 Of all clinical
`approved recombinant growth factors, EPO has
`the broadest indication spectrum and economic
`potential. EPO is available as a therapeutic agent
`produced by recombinant DNA technology in
`mammalian CHO-cells and is used in treating
`anemia resulting from chronic kidney disease,
`from the treatment of cancer (chemotherapy and
`radiation), and from other critical illnesses (heart
`failure or chronic infections). EPO contains three
`complex type N-glycans located on Asn residues at
`position 24, 38, and 83, and an O-glycosylation site
`at position 126 and has a maximum of 14 sialic
`acids per molecule.47 These terminal sialic acids
`determine the biological activity of EPO.47 Less
`sialylated or nonsialylated EPO has dramatically
`decreased in vivo activity compared to sialylated
`EPO. Interestingly, mutated and nonsialylated
`EPO has strong in vitro activity, indicating the
`importance to the asialo glycoprotein receptor
`system.48 Application of 10 mM ManNAc to EPO-
`producing CHO-cells led to increased and more
`homogenous sialylation as demonstrated by two-
`dimensional gel electrophoresis.49
`However, although ManNAc supplementation
`does increase the intracellular sialic acid pool
`dramatically,49 ManNAc-application does not
`automatically lead to an increase of sialylation
`of the product. This was shown for NS0 cells
`producing a recombinant humanized IgG150 and
`to CHO- or NS0-cells producing TIMP-I.51 In
`addition, there exist first reports in the literature
`that ManNAc application influences cell prolife-
`ration and differentiation.52 Finally, from a manu-
`facturer point of view, ManNAc is an expensive
`supplement, which has to be used in very high
`concentration during the fermentation process.
`Furthermore, the distribution of ManNAc and the
`process lead-through is difficult to control. There-
`fore, ManNAc application is not first choice to
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`increase intracellular sialic acid concentrations in
`a large-scale production process.
`
`Overexpression of Enzymes Involved in the
`Biosynthesis of Glycans
`
`CHO-cells are widely employed to produce glyco-
`sylated recombinant glycoproteins. However,
`CHO-cells do not express alpha-2,6-sialyltrans-
`ferase53 and therefore cannot produce glycopro-
`teins similar to human glycoproteins that are
`characterized by both a2,6- and a2,3-linked
`terminal sialic acid residues.54 Several appro-
`aches have been performed to enhance the
`sialylation machinery of CHO-cells. Coexpression
`of 2,3-sialyltransferase together with CMP-sialic
`acid synthase did not further increase sialylation,
`although a further increase in the intracellular
`pool of CMP-sialic acid was measured. As
`explanation, it was postulated that the transport
`capacity of CMP-sialic acid into the Golgi lumen
`was limited, thereby causing the reduced avail-
`ability of CMP-sialic acid substrate for sialyla-
`tion55 and be responsible for the lack of (further)
`increase in sialylation of the recombinant protein.
`As CMP-sialic acid transport affects the avail-
`ability of CMP-sialic acid substrate in the Golgi, in
`addition the CMP-sialic acid transporter was
`over-expressed. The result was increased sialyla-
`tion of EPO.55 Similar results were obtained in
`CHO-cells producing recombinant human inter-
`feron gamma (CHO IFN-g).56 Using this approach
`it could be demonstrated that over-expression of
`the CMP-sialic acid transporter represents a
`novel and powerful approach to improve sialy-
`lation during recombinant glycoprotein produc-
`tion.55,56 As described before, terminal sialylation
`of human proteins is characteristically in alpha-
`2,3 and alpha-2,6 linkage to galactose. Therefore,
`CHO-cells were stably transfected with human
`alpha-2,6-sialyltransferase cDNA to increase
`the sialylation of recombinant glycoproteins.57,58
`Using this approach it could be demonstrated that
`sialylation and clearance of IFN-g was improved
`compared to untransfected CHO-cells.57 However,
`in one study analyzing the EPO-bioactivity after
`engineering CHO-cells to express alpha-2,6-sia-
`lyltransferase, no significant differences could be
`detected.58
`
`Over-Expression of GNE
`
`The intention of this approach is trying to enhance
`the intracellular precursor pools for the biosynth-
`
`esis of the sialic aids, ManNAc, and thereby
`increasing sialylation of glycoproteins in the
`respective eukaryotic cell by the endogenous
`cellular metabolism. In first line experiments,
`GNE was over-expressed and it was possible to
`increase GNE activity in in vitro assays.59 But
`unfortunately the pool of intracellular sialic aids
`was not increased.60 This could be explained by
`the feedback inhibition of the GNE by activated
`CMP-sialic acid. To avoid or bypass the feedback
`inhibition of the epimerase activity of the GNE, a
`human sialic acid biosynthesis disease could be
`one way out. Sialuria is a rare human inborn error
`of the sialic acid metabolism.34 A defect of the
`feedback inhibition of the GNE is caused by point
`mutations within the epimerase domain of GNE
`(263L, 266Q, 266W) leads to excessive synthesis of
`sialic acid34 (see Fig. 1). In several experiments it
`could be shown that transfection of CHO-cells
`with sialuria-mutated GNE increases the intra-
`cellular pool a sialic acids and consequently
`increases the sialylation of some model glycopro-
`teins, such as EPO.49,60
`
`Resialylation
`
`Coagulation factor IX produced by CHO-cells
`exhibited complex-type glycosylation with carbo-
`hydrate chains capped with sialic acid in alpha 2–
`3 linkage.61 Human plasma-derived coagulation
`factor IX contains terminal sialic acid in alpha
`2–6-linkage. Using a strategy of desialylation
`followed by resialylation with specific sialyltrans-
`ferases it was possible to convert of CHO-cell-
`derived sialylation pattern into human-like
`sialylation pattern in vitro.62 To increase the
`serum half-life of Etanercept1,63 a recombinant
`fusion protein, which is used to treat patients
`suffering from ankylosing spondylitis,
`juvenile
`rheumatoid arthritis, psoriasis, psoriatic arthritis
`or rheumatoid arthritis, a resialylation strategy
`after expression was developed. Etanercept1 is a
`disulfide-linked dimer of a polypeptide composed
`of the extracellular portion of the human type 2
`(p75)
`tumor necrosis factor receptor (TNFR)
`fused to the hinge and Fc regions of the human
`IgG1 heavy chain.64 This bivalent antibody-like
`molecule contains two N-glycosylation sites per
`polypeptide in the receptor domain.60 The hetero-
`geneous N-linked oligosaccharides of TNFR-IgG
`fusion protein contain sialic acid, galactose, and
`N-acetylglucosamine as terminal sugar residues.
`To increase the level of terminal sialylation,
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`BORK, HORSTKORTE, AND WEIDEMANN
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`TNFR-IgG was re-galactosylated and/or re-sialy-
`lated using beta-1,4-galactosyltransferase and/or
`alpha-2,3-sialyltransferase. Treatment of TNFR-
`IgG with beta-1,4-galactosyl-transferase and
`UDP-galactose showed that the number of oligo-
`saccharides with terminal N-acetylglucosamine
`residues was significantly decreased with a
`concomitant increase in the number of terminal
`galactose residues.64 Similar treatment of TNFR-
`IgG with alpha-2,3-sialyltransferase and CMP-
`sialic acid produced a molecule with an approxi-
`mately 11% increase in the level of terminal
`sialylation but still contained oligosaccharides
`with terminal N-acetylglucosamine residues.
`When TNFR-IgG was treated with a combination
`of beta-1,4-galactosyltransferase and alpha-2,3-
`sialyltransferase (either in a single step or in a
`stepwise fashion), the level of terminal sialylation
`was increased by approximately 20–23%. These
`results suggest that in vitro galactosylation and
`sialylation of
`therapeutic glycoproteins with
`terminal N-acetylglucosamine and galactose resi-
`dues can be achieved in a single step, and the
`results are similar to those for the stepwise
`reaction.65 This type of in vitro glycosylation is
`suggested to be applicable to other glycoproteins
`containing terminal N-acetylglucosamine and
`galactose residues and could prove to be useful
`in increasing the serum half-life and homogeneity
`of therapeutic glycoproteins. Recently, Hayashi
`et al.66 reported the chemical sialylation of TNF-
`alpha using an acyl azide method and proofed that
`also chemical methods can be used to sialylated
`glycoproteins.
`
`Inhibition of Sialidases
`
`This important issue was interestingly very rarely
`investigated during the last years. Many cell types
`including CHO-cells are known to express several
`sialidases and under standard cell culture con-
`ditions, many recombinant glycoproteins lose
`sialic acid during the course of the fermentat-
`ion process.67,68 Rising numbers of dead cells
`correlates with increasing sialidase activity of
`CHO-cell culture supernatant, resulting in oligo-
`saccharide desialylation and reduced biological
`activity of recombinant glycoproteins. Inhibition
`of sialidase-activity in the supernatant by addi-
`tion of a substrate analogue to the culture
`medium69 or genetic manipulation of sialidase
`synthesis via antisense-RNA70,71 have already
`been applied to overcome the desialylation pro-
`blem in IFN-g expressing CHO-cells.
`
`Novel Unnatural Sialylation of Glycoproteins
`
`It has been shown that cells take up and efficiently
`metabolize synthetic N-acyl-modified D-manno-
`samines to the respective N-acyl-modified sialic
`acids in vitro and in vivo.72,73 N-acyl-modified D-
`mannosamines successfully employed in this way
`include N-propanoyl- (ManNProp), N-butanoyl-
`(ManNBut)-, N-pentanoyl- (ManNPent), N-hex-
`anoyl-
`(ManNCrot),
`(ManNHex), N-crotonoyl-
`N-levulinoyl- (ManNLev), N-glycolyl- (ManNGc),
`and N-azidoacetyl D-mannosamine (ManNAc-
`azido) (Fig. 3). All these synthetic, unnatural
`sialic acid precursors are metabolized by the
`promiscuous sialic acid biosynthetic pathway in
`the cytosol and are incorporated into sialoglyco-
`conjugates, where, depending on the cell type,
`they replace 10–85% of normal, physiological
`sialic acids (for review: see Ref. 40). This method
`of introducing novel, nonphysiological sialic acids
`into glycoconjugates has been termed biochemical
`engineering of the side chain of sialic acid. As
`mentioned before the biological half-life time of
`many glycoproteins is regulated via terminal
`sialic acids. Therefore, it was not unexpected that
`we found that the half-life of the highly sialylated
`CEACAM1, a member of the immunoglobulin
`superfamily, was increased by more than 50%
`after replacement of the N-acetylneuraminic acid
`by the novel, engineered N-propanoylneuraminic
`acid. This demonstrates that biochemical engi-
`neering not only modulates the structure of cell
`surface sialic acids, but that biochemical engi-
`neering also influences biological stability of
`defined glycoproteins.74
`
`POLYSIALIC ACID IS A PROMISING
`ANTI-IMMUNOGENIC POLYMER
`
`Polysialic acid represents a homopolymer of alpha
`2-8 linked sialic acids.75 Up to 150 sialic acid
`
`Figure 3. Structure of a modified N-acyl mannosa-
`mine with an elongated N-acyl side chain (N-propanoyl
`mannosamine). Note that for example N-propanoyl
`mannosamine contains one CH2-group more compared
`to the physiological human sialic acid precursor
`ManNAc.
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`molecules can build polysialic acid.76 In bacteria,
`polysialic acid is often used as capsular poly-
`saccharide, including neuroinvasive Escherichia
`coli K1 or Neisseria meningitidis group B.77
`Polysialic acid is also uniquely expressed on the
`neural cell adhesion molecule of mammals.78 As a
`consequence, polysialic acid alone, as a complex or
`a conjugate, is poorly immunogenic.79 Chemical
`coupling of a variety of polymers to therapeutic
`proteins has been studied as a way of improving
`their pharmacokinetics and pharmacodynamics
`in vivo. Conjugates have been shown to possess
`greater stability, lower immunogenicity, and a
`longer blood circulation time due to the chemico-
`physical properties of these hydrophilic long chain
`molecules.80–82 Naturally occurring polysialic
`acid has been investigated as an alternative to
`synthetic polymers such as poly(ethylene glycol)83
`due to its lower toxicity and natural metabolism.
`It has been demonstrated that polysialic acid
`conjugates to antibodies or their fragments
`increased biological stability and availability
`compared to the nonconjugated Fab.84
`
`SUMMARY
`
`Nearly each naturally eukaryotic secreted protein
`is a glycoprotein85 and therefore most therapeutic
`proteins in development are glycoproteins. Sialic
`acid is a crucial monosaccharide in mamma-
`lians.86 Especially in humans, glycans of glyco-
`proteins determine functional properties of the
`respective protein. This review concentrates on
`the role of sialic acid and possibilities to increase
`the content of sialic acid during the production
`process of recombinant glycoproteins. Glycosyla-
`tion and sialylation (as one part of the glycosyla-
`tion) have different consequences on (therapeutic)
`glycoproteins and therefore one has to distinguish
`between glycosylation and sialylation. Glycosy-
`lation per se has major impact on solubility and
`resistance to proteolysis of glycoproteins.87,88
`Glycosylation can be achieved by expressing the
`respective protein in a cell system, which is
`capable to glycosylate proteins, such as insect cells
`or mammalian cells like CHO.89 However, in none
`of major cell line used for production has the
`product a human-type of sialylation.90 However,
`the biological effect of therapeutics depends often
`on sialic acids. Terminal sialic acids in the proper
`linkage on glycoproteins reduce their immuno-
`genicity12,22 and increase the biological half-life of
`
`respective glycoproteins20,21 and are therefore
`not only important, but also a necessity for most
`therapeutic glycoproteins.
`It
`is notable that
`during the fermentation process of recombinant
`glycoproteins, sialylation is difficult to control and
`there is still a need for further improvement
`homogenous sialylation. In addition the degree of
`sialylation is important for the batch-to-batch
`consistency during the fermentation process and
`approval.
`Taken together: The best strategy to generate
`native sialylation would be producing the respec-
`tive sialylated glycoprotein in cell lines, which
`are derived from the original tissue, where the
`glycoprotein is expressed in vivo. Since this is not
`applicable, there are several strategies to increase
`and humanize sialylation during the production
`process of recombinant therapeutic glycoproteins.
`However, it is difficult to predict a method to yield
`maximal sialylation. In most cases a combination
`of more than one strategy seems to be advisable.
`
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