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Increasing the sialylation of therapeutic glycoproteins: The potential of the sialic acid biosynthetic pathway

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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. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3499–3508, 2009

Section snippets

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 disorders of the central nervous system will grow rapidly. Two very different strategies are used for the development for those novel drugs: (I) High-throughput screens of small molecules libraries to identify new drug candidates1,2 and (II) Development of specific therapeutic proteins using recombinant DNA

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 predominantly N-acetylneuraminic acid, due to a homozygous mutation in the CMP-neuraminic acid hydroxylase gene in the human genome. N-glycolylneuraminic acid is antigenic to humans,14,15 is enriched in tumor cells and is

BIOSYNTHESIS AND ACTIVATION OF SIALIC ACID

The initial reaction in the pathway to form free sialic acid is a conversion of UDP-N-acetylglucosamine (UDP-GlcNAc) to N-acetyl D-mannosamine (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

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.

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 molecules can build polysialic acid.76 In bacteria, polysialic acid is often used as capsular polysaccharide, 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

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 mammalians.86 Especially in humans, glycans of glycoproteins 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. Glycosylation and

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