MXPA99006639A - Practical in vitro - Google Patents

Abstract

This invention provides methods for practical in vitro sialylation of glycoproteins, including recombinantly produced glycoproteins. The methods are useful for large-scale modification of sialylation patterns.

Description

SIALILACION PRACTICA IN VITRO OF RECOMBINANT GLICOPROTEINAS CROSS REFERENCE WITH RELATED REQUESTS This application claims the priority of the Provisional Application of the United States of America 60 / 035,710, filed on January 16, 1997, which is hereby incorporated by reference in its entirety for all purposes. BACKGROUND OF THE INVENTION Field of the Invention This invention pertains to the field of in vivo sialylation of glycoproteins, including recombinant glycoproteins. Background The circulatory lifetime of glycoproteins in the blood depends to a large extent on the structure composition of their N-linked carbohydrate groups. This fact is of direct relevance to therapeutic glycoproteins that are intended to be administered parenterally. In general, the maximum circulating half-life of a glycoprotein requires that its N-linked carbohydrate groups end in the NeuAc-GalNAc sequence. Without terminal sialic acid (NeuAc), the glycoprotein is rapidly evacuated from the blood by means of a mechanism involving recognition of the implied N-acetylgalactosaamine (GalNAc) or galactose residues (Gal) (Goochee et al. (1991)).

Bi o / technology 9: 1347-1355). For this reason, ensuring the presence of terminal sialic acid in the N-linked carbohydrate groups of therapeutic glycoproteins is an important consideration for commercial development. In principle, the mammalian cell culture systems used for the production of most therapeutic glycoproteins have the ability to produce glycoproteiries with fully-sialylated N-linked carbohydrate groups. In practice, however, optimal glycosylation is often difficult to achieve. Under the conditions of a low-scale production, the overproduction of the glycoprotein by the cell may exceed its ability to maintain glycosylation, and this capacity may be influenced positively and negatively by many subtle variables in the culture conditions (Goochee and collaborators, mentioned above). The production of glycoproteins in transgenic animals has some of the same problems as in the culture of mammalian cells. Although the "production" of a glycoprotein can inherently be better controlled, it is also less susceptible to manipulation. If the glycosylation is complete, there is very little to do with the animals to alter the result. With transgenic animals, another problem usually occurs. Although the predominant sialic acid in humans is N-acetyl-neuraminic acid (NeuAc), goats, sheep and cows produce a large fraction of their total sialic acid as N-glycolyl-neuraminic acid (NeuGc). Although the impact of this modification has not been fully explored from a functional or regulatory perspective, it is known that NeuGc substitution is antigenic in humans (Var i (1992) Glycobiology 2: 25-40). As most of the important problems associated with the glycosylation of commercially important recombinant and transgenic glycoproteins involve terminal sialic acid, there is a need for an in vitro procedure to enzymatically "cover" the carbohydrate chains that are needed in a sialic acid terminal. With this procedure, the problem faced with the transgenic glycoproteins could also be approached by resialation with NeuAc once the "non-human" sialic acid NéuGc has been eliminated. The ideal method would employ a sialyltransferase which is capable of efficiently sialylating the N-linked oligosaccharides or recombinant glycoprotein-0 link in a practical scale. The present invention meets these and other needs.

SUMMARY OF THE INVENTION The present invention provides methods for the sialylation of groups of saccharides present in a recombinantly produced glycoprotein. The methods include contacting the saccharide groups with a sialyltrasferase, a half donor of sialic acid and other reagents required for the activity of the sialyltransferase for a sufficient time and under appropriate conditions to transfer the sialic acid from the donor half of the sialic acid to the aforementioned saccharide group. In a preferred embodiment, the methods are carried out using sialyltransferase at a concentration of about 50 mU per mg of glycoprotein or less, preferably between about 5-25 mU per mg of glycoprotein. In general, the concentration of sialyltransferase in the reaction mixture will be between about 10-50 mU / ml, with the glycoprotein concentration of at least about 2 mg / ml of the reaction mixture. In a preferred embodiment, the method results in or results in a sialylation of more than about 80% of the terminal galactose residues present in the aforementioned saccharide groups. In general, the time required to obtain more than about 80% sialylation is less than or equivalent to about 48 hours. The sialyltransferases that are useful in the methods of the invention generally have a sialyl motif that includes about 48-50 amino acids, within which about 40% of the amino acids are identical to the consensus sequence RCAWSSAG DVGSKT (where they indicate a variable number of amino acid residues such that the motif is approximately 48-50 residues in length). Examples of sialyl transferases which are suitable for use in the present invention include S3Gal III (preferably a rat ST3Gal III), ST3Gal IV, ST3GalI, ST6GalI, ST3Gal V, STdGal II, ST6GalNac Y, STdGalNac II, and STdGalNac III ( The nomenclature of the sialyltransferase used here is that described in Tsuj i et al. (1996) Glycobiology 6: v-xiv). The methods of the invention may involve the sialylation of recombinant glycoproteins with more than one sialyltransferase; for example, with an ST3Gal III and an ST3GalI, or an ST3Gal III and a STßGalI, or other combinations of enzymes. The donor half of the sialic acid used in the claimed methods is usually CMP-sialic acid, which can be added to the reaction directly or can be enzymatically generated in your own. The sialic acids used in a preferred embodiment are selected from the groups formed by NeuAc and NeuGc. The invention also provides a glycoprotein having an altered sialylation pattern, where the terminal galactose residues of the aforementioned glycoprotein are sialylated using the methods claimed.

BRIEF DESCRIPTION OF THE ILLUSTRATIONS Figure 1 shows a time course of sialylation mediated with ST3Gal III of the acid glycoprotein that had been treated with neuraminidase. The percentage of the terminal galactose residues that were sialylated are plotted against the reaction time. Figure 2 shows a comparison of the sialylation of the al-glycoprotein acid treated with neuraminidase using two different sialyltransferases, ST3Gal III and ST6GalI.

DETAILED DESCRIPTION Definitions The following abbreviations are used herein: Ara = arabinosil; Fru = fruity; It was = fucosil; Gal = galactosyl; GalNAc = N-acetylgalact; Glc = glucosyl; GlcNAc = N-acetylgluco; Man = manosil; and NeuAc = sialyl (usually N-acetylneuraminyl). The oligosaccharides are considered to have a reducing end and a non-reducing end, whether or not the saccharide at the reducing end is in fact a reducing sugar. According to the accepted nomenclature, the oligosaccharides are shown here with the non-reducing end on the left and the reducing end on the right. All oligosaccharides described herein are described by the name or abbreviation for the non-reducing saccharide (eg, Gal), followed by the configuration of the glycosodic bond (a or β), the ring bond, the position of the ring of the reducing saccharide involved in the binding, and subsequently the name or abbreviation of the reducing saccharide (for example, GlcNAc). The link between two sugars can be expressed, for example, as 2, 3, 2-t > 3 or (2,3). Each sectarian is a pyranosa. The term "sialic acid" refers to any member of a family of nine-carbon carboxylated sugars. The most common member of the sialic acid family is N-acetyl-neuraminic acid (2-keto-5-acetaminido-3,5-dideoxy-D-glycero-D-galactononulopyran-1-ionic acid (often abbreviated as Neu5Ac, NeuAc, or NANA). "A second member of the family is N-glycolyl-neuraminic acid (Neu5Gc or NeuGc), where the N-acetyl group of NeuAc is hydroxylated.A third member of the sialic acid family is 2-keto-3-deoxy-nonulosonic acid (KDN) (Nadano et al. (1986) Bulletin of Chemical Biology 261: 11550-11557; Kanamori et al. (1990) Bulletin of Chemical Biology 265: 21811-21819. include nine substituted sialic acids, such as 9-0-C * -C * acyl-Neu5Ac, such as 9-0-lactyl-Neu5Ac or 9-0-acetyl-Neu5Ac, 9-deoxy-9-fluoro-Neu5Ac and 9- acid-9-deoxy-Neu5Ac For review of the sialic acid family, consult, for example, Varki (1992) Glycobiology 2: 25-40; Sialic Acids: Chemistry, Metabolism and Function , R. Schauer, De. (Springer-Verlag, New York (1992)). The synthesis and use of the sialic acid compounds in a sialylation process is shown in the international application WO 92/16640, published on October 1, 1992. The term "recombinant" when used with reference to a cell indicates that the cell duplicates a heterologous nucleic acid, or expresses a peptide or protein encoded by a heterologous nucleic acid. The recombinant cells may contain genes that are not found within the native (non-recombinant) form of the cell. The recombinant cells can also contain genes found in the native form of the cell, where these genes are modified and reintroduced into the cell by artificial means. The term also includes cells that contain a nucleic acid hendegogenic to the cell that has been modified without removing the nucleic acid from the cell; These modifications include those obtained by the replacement of the gene, specific mutation in the site and related techniques. A "recombinant polypeptide" is one that has been produced by a recombinant cell. A "heterologous sequence" or a "heterologous nucleic acid", as used herein, is one that originates from a source foreign to the particular host cell, or, if it is from the same source, it is modified from its original form. Thus, a heterologous glycoprotein gene in a eukaryotic host cell includes a glycoprotein gene that is hendogenous to the particular host cell that has been modified. Modification of the heterologous sequence may occur, for example, by treating the DNA with a restriction enzyme to generate a DNA fragment that is capable of operably linking to the promoter. Techniques such as site-directed mutagenesis are also useful for modifying the heterologous sequence. A "subsequence" refers to a sequence of nucleic acids or amino acids that includes a portion of a larger sequence of nucleic acids or amino acids (e.g., polypeptides) respectively. A "recombinant expression cartridge" or simply an "expression cartridge" is a nucleic acid construct, synthetically generated, with nucleic acid elements that are capable of affecting the expression of a structural gene in hosts compatible with these sequences . Expression cartridges include at least promoters and optionally, transcription termination signals. In general, the recombinant expression cartridge includes a nucleic acid to be transcribed '(eg, a nucleic acid encoding a desired polypeptide), and a promoter. Additional factors necessary or useful to carry out the expression as described herein may also be used. For example, an expression cartridge can also include nucleotide sequences that encode a sequence of the signals that direct the secretion of a protein expressed from the host cell. Transcription termination signals, enhancers and other nucleic acid sequences that influence gene expression can also be included in the expression cartridge. The term "isolated" is intended to refer to material that is substantially or essentially free of components that generally accompany the enzyme as it is in its native state. In general, the isolated molecules are at least about 80% pure, usually at least about 90%, and preferably at least about 95% pure according to those measured by, for example, the intensity of the band on a silver-stained gel or other method to determine purity. The purity of the protein or homogeneity can be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualization after dyeing. For certain purposes, high resolution and HPLC or a similar means of purification used will be required.

The practice of this invention may involve the construction of recombinant nucleic acids and the expression of transfected host cells. Molecular cloning techniques to achieve these ends are known in the art. A wide variety of in vitro cloning and amplification methods suitable for the construction of recombinant nucleic acids as the expression vectors are well known to the skilled person. Examples of these techniques and sufficient instructions to direct trained people through different cloning exercises are found in Berger and Kimmel, Guide for Molecular Cloning Techniques, Enzymology Methods volume 152 Academic Press, Inc., San Diego, CA (Berger); and Current Protocols in Molecular Biology; Ausubel et al., Eds., Current Protocols, a joint conversion company between Greene Publishing Associates, Inc. and John Wiley $ Sons, Inc., (Supplement 1994) (Ausubel). Suitable host cells for the expression of recombinant polypeptides are known to those skilled in the art and include, for example, eukaryotic cells including insect, mammalian and fungal cells. In a preferred embodiment, Aspergillus niger is used as the host cell.

Examples of protocols sufficient to direct people skilled in the art through in vitro amplification methods, including the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the Qβ replicase amplification and other techniques mediated with RNA polymerase are found in Berger, Sambrook and Ausubel, as well as in Mullis et al. (1987) U.S. Patent No. 4,683,202; PCR Protocols, A Guide for Methods and Applications (Innis et al., Eds) Academic Press Inc. San Diego, CA (1990) (Innis); Arnheim & Levinson (October 1, 1990) C & EN 36-47; The NIH Research Bulletin (1991) 3: 81-94; (Kwoh et al. (1989) Procedures of the US National Academy of Sciences 86: 1173; Guatelli et al. (1990) Procedures of the National Scientific Academy USA: 87: 1874; Lomell et al. (1989) Bulletin of Clinical Chemistry 35: 1826: Landegren et al. (1988) Science 241: 1077-1080, Van Brunt (1990) Biotechnology 8: 291-294, Wu and Wallace (1989) Gene 4: 560, and Barringer et al. (1990) Gene 89: 117. The methods Enhanced for the in vitro cloning of amplified nucleic acids are described in Walace et al., US Patent Number 5,426,039.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention provides methods for efficient sialylation of saccharide groups bound to glycoproteins, in particular recombinantly produced glycoproteins. For example, methods of the invention useful for the sialylation of recombinantly produced therapeutic glycoproteins that are incompletely sialylated during production in mammalian cells or transgenic animals. The methods involve contacting the saccharide groups with a sialyltransferase and a sialic acid donor moiety for a sufficient time and under the appropriate reaction conditions to transfer sialic acid from the donor half of the sialic acid to the saccharide groups. The sialyltransferases include a family of glycosyltransferases that transfer sialic acid from the CMP-sialic acid from the donor substrate to the oligosaccharide substrates of the adsorber. In preferred embodiments, the sialyltransferases used in the methods of the invention are produced recombinantly. The methods of the invention useful for altering the sialylation pattern of glycoproteins. The term "altered" refers to the sialylation pattern of a glycoprotein according to its modifications using the methods of the invention other than those observed in the glycoprotein as they were produced in vivo. For example, the methods of the invention can be used to produce a glycoprotein having a sialylation pattern that is different from that found in the glycoprotein when it is produced by the organism cell of which the glycoprotein is native. Alternatively, methods for altering the sialylation pattern of glycoproteins that are produced recombinantly by expressing a gene encoding the glycoprotein in a host cell, which may be of the species of which the glycoprotein is native, may be used. , or of a different species. Recombinant glycoproteins possessing sialylation patterns that are modified by the methods of the invention can have important advantages over proteins that are in their unaltered, native glycosylation state or that are in a glycosylation state that is less than optimal for a particular application. These non-optimal sialylation patterns may arise when a recombinant glycoprotein is produced in a cell that does not have the proper complement of the glycosylation machinery to produce the desired glycosylation pattern. The optimal or preferred glycosylation pattern may or may not be the native glycosylation pattern of the glycoprotein when it is produced in its native cell. The advantages of optimal sialylation patterns include, for example, the increased therapeutic half-life of a glycoprotein due to a reduced free space percentage. Altering the sialylation pattern can also hide the antigenic determinants in the foreign proteins, thus reducing or eliminating an immune response against the protein. Alteration of the sialylation of a saccharide linked to the glycoprotein can also be used to target a protein to a cell surface receptor that is specific for the altered oligosaccharide, or to block targeting to a receptor that is specific for the unaltered saccharide . Proteins that can be modified by the methods of the invention include, for example, hormones such as insulin, growth hormones (including human growth hormone and bovine growth hormone), tissue-type plasmidgen activator (t-PA), renin, coagulation factors such as factor VIII and factor IX, bombesin, thrombin, hemopoietic growth factor, serum albumin, receptors for hormones or growth factors, interleukins, colony stimulation factors, T cell receptors, MHC polypeptides , viral antigens, glycosyltransferases and the like. Polypeptides of interest for recombinant expression and subsequent modification using the methods of the invention also include al-antitryptin, herithotropin, granulisite-macrophage colony stimulation factor, antitrobin III, interleukin 6, interferon-β, protein C, fibrogen , among many others. This list of polypeptides is exemplary, not exclusive. The methods are also useful for modifying the sialylation patterns for chimeric proteins, including, but not limited to, chimeric proteins that include a half derived from an immunoglobulin, such as IgG. The in vivo sialylation methods provided by the invention are, unlike the previously described sialylation methods, practical for the commercial scale production of modified glycoproteins. In this way, the claimed methods provide a practical means for the large-scale preparation of glycoproteins that possess altered sialylation patterns. The methods are quite suitable for therapeutic glycoproteins that are incompletely sialylated during production in maternal cells or transgenic animals. The processes provide a growing and consistent level of terminal sialylation of a glycoprotein.

One way in which the methods of the invention achieve commercial feasibility is through the use of recombinantly produced sialyltransferases. The recombinant production allows the production of sialyltransferases in large quantities which are required for the large scale modification of the glycoprotein. Removal of the anion domain from the membrane of the sialyltransferases, which makes the sialyltransferases soluble and thus facilitate the production and purification of large amounts of sialyltransferases, can be achieved by the recombinant expression of a modified gene encoding the sialyltransferase. Examples of recombinant sialyltransferases, including those that have eliminated anchor domains, as well as methods for producing recombinant sialyltransferases, are found, for example, in U.S. Patent No. 5,541,083. At least 15 different mammalian sialyltransferases have been documented, and the cDNAs of thirteen of these have been cloned to date (for the systematic nomenclature used in this, consult, Tsuj i et al. (1996) Glycobiology 6: v-xiv). These cDNAs can be used for the recombinant production of sialyltransferases, which can subsequently be used in the methods of the invention.

Commercial viability is also provided by the methods of the invention through the use of bacterial sialyltransferases, either produced recombinantly or produced in native bacterial cells. Two bacterial sisliltransferases have been reported; an ST6Gal II from Photobacterium demsela (Yamamoto et al. (1996) Bulletin of Biochemistry 120: 104-110) and an ST3Gal V from Neisseria meningi tidis (Gilbert et al. (1996) Bulletin of Chemical Biology 271: 28271-28276). The two bacterial enzymes recently described transfer the sialic acid to the Galßl, 4GlcNAc sequence in the oligosaccharide substrates. However, there are no known bacterial proteins that are glycosylated, therefore it was not known whether the Galßl, 4GlcNAc moiety covalently bound to a protein would or would not serve as a substrate adsorb for a bacterial sialyltransferase. Table 1 shows the specificity of the acceptor of these and other sialyltransferases useful in the methods of the invention. In preferred embodiments, the methods of the invention are commercially practical due to the use of sialyltransferases that are capable of sialylating a high percentage of adsorbent groups in a glycoprotein using a low ratio of enzyme units to the glycoprotein. In a preferred embodiment, the desired amount of sialylation will be obtained using approximately 50 mU of sialyltransferase per mg of glycoprotein or less. More preferably, less than about 40 mU of sialyltransferase will be used per mg of glycoprotein, even more preferably, the ratio of sialyltransferase to glycoprotein will be less than or equal to about 45 mU / mg, and still more preferably about 25 mU / mg. mU / mg or less. Most preferably, the desired amount of sialylation will be obtained using less than about 10 mU / mg of sialyltransferase per mg of glycoprotein. Typical reaction conditions will have the sialyltransferase present in a range of about 5-25 mU / mg glycoprotein, or 10-50 mU / ml of the reaction mixture with the glycoprotein present at a concentration of at least about 2 mg / ml. Typically, the chains of saccharides in a glycoprotein possessing sialylation patterns altered by the methods of this invention, such that? Terminal galactose sialylated than the unaltered glycoprotein. Preferably, more than about 80% of the terminal galactose residues present in the saccharide groups linked to the glycoprotein will be sialylated following the use of the methods. More preferably, the methods of the invention will result in more than about 90% sialylation, and even more preferably much more than about 95% of sialylation of the terminal galactose residues. More preferably, essentially 100% of the terminal galactose residues present in the glycoproteins are sialylated following the modification using the methods of the present invention. The methods are generally capable of achieving the desired level of sialylation in about 48 hours or less, and more preferably in about 24 hours or less. Preferably, for the glycosylation of N-linked carbohydrates of glycoproteins, the sialyltransferase will be able to transfer the sialic acid to the sequence Galßl, 4GlcNAc, the penultimate most common sequence implied for the terminal sialic acid for the terminal sialic acid in the carbohydrate structures Fully sialylated Only three of the cloned mammalian sialyltransferases comply with ester specificity requirement of the adsorber, and it has been shown that each of these transfers the sialic acid to the N-linked carbohydrate groups of glycoproteins.

Examples of sialyltransferases using Galßl, 4GlcNAc as the adsorbent are shown in Table 1.

Table 1: Sialyltransferases using the Galßl, 4GlcNAc sequence as the substrate of the adsorber. 1) Goochee et al. (1991) Bio / Technology 9: 1347-1355 2) Yamamoto et al. (1996) Bulletin of Biochemistry 120: 104-110 3) Gilbert et al. (1996) Bulletin of Chemical Biology 271: 28271-28276 The substrate specificity of the sialyltransferases is only the first criterion that an enzyme must satisfy to satisfy a method for the sialylation of commercially important recombinant or transgenic glycoproteins. The sialyltransferase must also be able to carry out sialylation efficiently and completely for a variety of glycoproteins, and support the extension to 1-10 kg of recombinant glycoprotein at a relatively low cost and infrastructure requirements. . There are no published reports documenting that any of these sialyltransferases is adequate to establish a practical process that meets these requirements. An example of a sialyltransferase is useful in the claimed methods is ST3Gal III, which is also referred to as a (2,3) sialyltransferase (EC 2.4.99.6). This enzyme catalyzes the transfer of sialic acid to the glycoside Galßl .3GlcNAc or Galßl .4.4GlcNAc (consult, eg, Wen et al. 81992) Bulletin of Chemical Biology, 261: 21011; Van den Eijnden et al. (1991) Bulletin of Chemical Biology, 256: 3159) and is responsible for the sialylation of oligosaccharides linked with asparagine in glycoproteins. The sialic acid is bound to a Gal with the formation of a bond between the two saccharides. The bond (union) between saccharides is between position 2 of NeuAc and position 3 of Gal. This particular enzyme can be isolated from the liver of the rat (Weintein et al. (1982) Bulletin of Biological Chemistry, 257: 13845); the human cDNA (Sasaki et al. (1993) Bulletin of Chemical Biology 268: 22782-22787; Kitagawa &Paulson (1994) Bulletin of Chemical Biology, 269: 1394-1401) and genomic (Kitagawa et al. (1996) Bulletin of Biology Chemistry, 271: 931-938) DNA sequences are known, facilitating the production of this enzyme by recombinant expression. In a preferred embodiment, the claimed sialylation methods utilize a rat ST3Gal III. Other sialyltransferases, including those listed in Table 1, may also be useful in an economical and efficient large-scale process for the sialylation of commercially important glycoproteins. As a simple test to find the utility of these other enzymes, different amounts of each enzyme (1-100 mU / mg protein) are subjected to reaction with asialo-a * AGP (at 1-10 mg / 1) to compare the ability of the sialyltransferase of interest to sialylate the glycoproteins in relation to either the ST6GalI of bovine, the STT3Gal III or both sialyltransferases. Alternatively, other glycoproteins or glycopeptides, or N-linked oligosaccharides enzymatically released from the backbone of the peptide can be used for a so-called asialo-asialo-a * AGP for this evaluation. Sialyltransferases that show an ability to sialylate glycoprotein-N-linked oligosaccharides more efficiently than ST6GalI may be useful in practical large-scale processes for glycoprotein sialylation (as illustrated for ST3Gal III in this presentation) . The invention also offers methods for altering the sialylation pattern of a glycoprotein by adding sialic acid to an a2, 6Gal bond as well as the a2.3Gal bond, both of which are found in the N-linked oligosaccharides of human plasma glycoproteins . In this embodiment, the ST3Gal III and ST6GalI sialyltransferases are present in the reaction and provide proteins having a reproducible ratio of the two bonds formed in the resialylation reaction. In this way, a mixture of the two enzymes can be of value if both bonds are desired in the final product. An adsorber for the sialyltransferase will be present in the glycoprotein that will be modified by the methods of the present invention. Suitable admisters include, for example, galactosyl admissors such as Galßl, 4GlcNAc, Galßl, GalNAc, Galßl, 3GalNAc, lacto-N-tetraose, Galßl, 3GlcNAc, Galßl, 3Ara, Galßl, 6GlcNAc, Galßl, 4Glc (lactose) , and other receivers known to those skilled in the art (see, for example, Paulson et al. (1978) Bulletin of Chemical Biology 253: 5617-5624). In general, the adsorbers are included in the oligosaccharide chains that are attached to the asparagine, cerina or threonine residues present in a protein. In one embodiment, an adsorber for the sialyltransferase is present in the glycoprotein which will be modified after the in vivo synthesis of the glycoprotein. These glycoproteins can be sialylated using the methods claimed without prior modification of the glycosylation pattern of the glycoprotein. Alternatively, the methods of the invention can be used to alter the sialylation pattern of a glycoprotein that has been modified prior to sialylation. For example, to sialylate a protein that does not include a suitable adsorbent, the protein can be modified to include an adsorber by means of methods known to those skilled in the art. The adsorbent can be synthesized by linking a galactose residue to, for example, a GlcNAc or other appropriate saccharide moiety that is linked to the protein. The oligosaccharides linked to the glycoprotein can first be "compacted" either fully or partially, to expose either an adsorber for the sialyltransferase or a moiety to which one or more suitable residues can be added to obtain an adequate adsorbent. Enzymes such as glycosyltransferases and endoglycosidases are useful for binding and compaction reactions. The claimed methods are also useful for synthesizing a half of saccharide terminated in sialic acid in a protein that is not glycosylated in its native form. A suitable adsorbent for the sialyltransferase is bound to these proteins by methods known to those skilled in the art prior to sialylation using the methods of the present invention. See, for example, US Patent No. 5,272,066 for methods of obtaining polypeptides with suitable receptors for sialylation. Thus, in one embodiment, the invention provides methods for the sialylation of the groups of saccharides present in a glycoprotein which first involves the modification of the glycoprotein to create a suitable adsorbent. A preferred method for synthesizing an acceptor involves the use of a galactosyltransferase. The steps for these methods include: a) galactosylating a compound of the formula GlcNR 'ß (1- ^ 3) Galß-OR with a galactosyltransferase in the presence of a UDP galactose under conditions sufficient to form the compound: Galß (1? 4 ) GlcNR 'ß (1? 3) Galß-O; and b) sialylating the compound formed in (a) with a sialyltransferase in the presence of a CMP derivative of a sialic acid using an a (2,3) sialyltransferase under conditions in which the sialic acid is transferred to the non-reducing sugar for form the compound NeuAca (2? 3) Galß (l? 4) GlcNR '(1? 3) Galß-OR. In this formula, R is an amino acid, a saccharide, an oligosaccharide or an aglycon group that possesses at least one carbon atom. R 'can be either acetyl or allyloxycarbonyl (Alloc) R is linked to or is part of a glycoprotein. The steps of galoctosylar and sialylar are preferably carried out enzymatically, where the galactosilar passage is preferably carried out as part of a galactosyltransferase cycle and the sialylar step is preferably carried out as part of a Sialyltransferase cycle. Preferred conditions and descriptions of other species and enzymes in each of these cycles have also been described. In a preferred embodiment, the galactosylar and sialylation steps are carried out in a simple reaction mixture containing both the sialyltransferase and the galactosyl transferase. In this embodiment, the enzymes and substrates can be combined in an initial reaction mixture, or preferably the enzymes and reagents for a second glycosyltransferase cycle can be added to the reaction medium once the first cycle of the glycosyltransferase approaches at its completion. By carrying out two cycles of glycosyltransferase in sequence in a single vessel, the overall yields are improved over the processes in which an intermediate species is isolated. In addition, the cleaning and disposal of extra solvents and byproducts is reduced. In a preferred embodiment, the sialylation of the glycoprotein is achieved using a sialyltransferase cycle, which includes a CMP-sialic acid recycling system utilizing the CMP-sialic acid synthetase. The CMP-sialic acid is relatively expensive, therefore, the synthesis in si tu of this donor half of the sialic acid increases the economic advantages provided by the methods claimed. Sialyltransferase cycles are described, for example, in U.S. Patent No. 5,347,541. The CMP-sialic acid regeneration system used in this embodiment includes cystidine monophosphate (CMP), a nucleoside triphosphate, a phosphate donor, a kinase capable of transferring phosphate from the phosphate donor to the nucleoside phosphosphates and a nucleoside monophosphate kinase capable of transferring the terminal phosphate of a nucleoside triphosphate to CMP. The regeneration system also employs the CMP-sialic acid synthetase, which transfers sialic acid to CTP. The CMP synthal acid synthetase can be isolated and purified from the cells and tissues containing the synthetase enzyme by methods already known in the art. See, for example, Gross et al. (1987) Bulletin of European Biochemistry 168: 595. Vijay et al. 81975) Bulletin of Biological Chemistry 250: 164; Zapata and collaborators (1989) Bulletin of - Biology. Chemistry 264: 14769; and Higa et al. (1985) Bulletin of Chemical Biology 260: 8838. The gene for this enzyme has also been sequenced. Consultar, Vann et al. (1987) Bulletin of Chemical Biology 262: 17556. Excessive expression of the gene has been reported for use in a gram scale synthesis of CMP-NeuAc. Consultar, Shames and collaborators (1991) Glycobiology. 1: 187 This enzyme is also commercially available. The nucleoside triphosphates suitable for use in accordance with the regeneration system of CMP-sialic acid are adenosine triphosphate (ATP), cystidine triphosphate (CTP), uridine triphosphate (UTP), guanosine triphosphate (GTP), triphosphate deinosin (ITP) and thymidine triphosphate (TTP). A preferred triphosphatide of the nucleoside is ATP. Nucleoside monophosphate kinases are enzymes that catalyze the phosphorylation of nucleoside monophosphates. Nucleoside monophosphate (NMK) kinase or myokinase (MK); EC 2.7.4.3) used according to the regeneration system of the CMP-sialic acid of the present invention are used to catalyze the phosphorylation of the CMP. NMK is commercially available (Sigma Chem. Co., St. Louis, MO; Boehringer Mannheim, Indianapolis, Ind.). A phosphate donor and a catalytic amount of a kinase that catalyzes the transfer of phosphate from the phosphate donor to an activated nucleotide are also part of the regeneration system of the CMP-sialic acid. The phosphate donor of the regeneration system is a phosphorylated compound, whose phosphate group can be used to phosphorylate the phosphate of the nucleoside. The only limitation in the selection of a phosphate donor is that neither the phosphorylated nor the dephosphorylated forms of the phosphate donor can substantially interfere with any of the reactions involved in the formation of the galactosylsiallylated glycoside. Preferred phosphate donors are phosphoenolpyruvate (PEP), creatine phosphate and acetyl phosphate. A particularly preferred phosphate donor is PEP. The selection of a particular kinase for use in a sialic acid cycle depends on the phosphate donor employed. When acetyl phosphate is used as a phosphate donor, the kinase is an acetyl kinase; the creatine kinase is used for a creatine phosphate donor, and when the PEP is used as a phosphate donor, the kinase is pyruvate kinase (PK, EC 2.7.1.40). Other kinases may be used with other phosphate donors since they are well known to those skilled in the art. The kinases are commercially available (Sigma Chem. Co .; St Louis, MO; Boehringer Mannheim, Indianapolis, Ind.). Due to the self-contained and cyclic nature of this glycosylation method, once all reagents and enzymes are present, the reaction continues until the first of the stoichiometric substrates is consumed (eg, Neu5Ac and free PEPs or the adsorber). In the sialylation cycle, the CMP is converted to CDP by the nucleoside monophosphate kinase in the presence of the added ATP. ATP is catalytically regenerated from its secondary product, ADP, by pyruvate kinase (PK) in the presence of the added phosphoenolpyruvate (PEP). The CDP is subsequently converted to CTP, and this conversion is catalyzed by the PK in the presence of PEP. The CTP reacts with the sialic acid to form an inorganic pyrophosphate (Ppi) and CMP-sialic acid, the latter reaction being catalyzed by the CMP synthetase-sialic acid. After sialylation of the galactosyl glycoside, the released CMP re-enters the regeneration system to form the CDP, CTP and the CMP-sialic acid. The Ppi formed is purified as mentioned below, and forms inorganic phosphate (Pi) as a secondary product. Pyruvate is also a secondary product. The secondary product pyruvate can also be used in another reaction in which N-acetyl mannosamine (ManNAc) and pyruvate are reacted in the presence of the AlAdalase NeuAc (EC 4.1.3.3) to form the sialic acid. In this way, the sialic acid can be replaced by the ManNAc and a catalytic amount of Aldolase NeuAc. Although the aldolase NeuAc also catalyses the reverse reaction (NeuAc to ManNAc and pyruvate), the produced NeuAc is irreversibly incorporated into the reaction cycle through the CMP-NeuAc catalyzed by the CMP synthetase-sialic acid. This enzymatic synthesis of sialic acid and its nine substituted derivatives and the use of the resulting sialic acid in a different sialylation reaction scheme is presented in the international application WO 92/16640, published on October 1, 1992. As used in herein, the term "pyrophosphate scavenger" refers to substances that serve to remove the inorganic pyrophosphate from a reaction mixture of the present invention. Inorganic pyrophosphate (PPi) is a by-product of the preparation of CMP-Neu5Ac. The PPi produced can be fed back to inhibit other enzymes in such a way that glycosylation is reduced. Nevertheless, the PPi can be degraded enzymatically or by physical means such as seclusion by a binding substance of the PPi. Preferably the PPi is removed by hydrolysis using the inorganic pyrophosphatase (Ppase; EC 3.6.1.1), a commercially available PPi catabolic enzyme (Sigma Chem. Co., St. Louis, MO, Boehringer Mannheim, Indianapolis, Ind), and this or a similar enzyme serves as the pyrophosphate scavenger. One method for removing the PPi or Pi from the reaction mixture is to maintain a concentration of the divalent metal cation in the medium. In particular, the cations and inorganic phosphate produced form a complex of very low solubility. By supplementing the cations that are lost by precipitation with the pyrophosphate, the reaction ratio can be maintained and the reactions can be carried out to the end (ie, 100% conversion). The complementation can be carried out continuously (for example, by automation) or discontinuously. When the concentration of cations is maintained in this manner, the cycle of the transferase reaction can be carried out to the end. For the glycosyltransferase cycles, the concentrations or amounts of the various reagents used in the processes depend on various factors including the reaction conditions such as temperature and pH value, and the choice and quantity of the adsorber saccharides that will be glycosylated . Because the glycosylation process allows the regeneration of the activated nucleotides the sugars of the activated donors and the clearance of the PPi produced in the presence of catalytic amounts of the enzymes, the process is limited by the concentrations or amounts of the stoichiometric substrates discussed above. The upper limit for the concentrations of the reagents that can be used according to the methods of the present invention is determined by the solubility of these reagents. Preferably, the concentrations of the activation nucleotides, the phosphate donor, the donor sugar and the enzymes are selected in such a way that the glycosylation is carried out until the adsorber is consumed, thus completely isolating the groups of saccharides present in the glycoprotein. The quantities or concentrations of enzymes are expressed in units of activity, which is a measure of the initial relationship of the catalysis. One unit of activity catalyzes the formation of 1 μmol of product per minute at a given temperature (usually 37 ° C) and pH value (usually 7.5). In this way, 10 units of an enzyme is a catalytic amount of this enzyme where 10 μmol of the substrate is converted to 10 μmol of product in one minute at a temperature of 37 ° C and a pH value of 7.5. The above ingredients are combined by mixing in an aqueous reaction medium (solution). This medium has a pH value of about 6 to about 8.5. The medium is free of chelates that bind the co-factors of the enzyme such as Mg * or Mn *. The selection of a medium is based on the ability of the medium to maintain the pH value at the desired level. Thus, in some embodiments, the medium is buffered to a pH value of about 7.5, preferably with HEPES. If a buffer solution is not used, the pH of the medium should be maintained at about 6 to 8.5, preferably about 7.2 to 7.8, by the addition of a base. A suitable base is NaOH, preferably 6 M NaOH. The reaction medium may also include solubilized detergents (e.g., Triton or SDS) and organic solvents such as methanol or ethanol, if necessary. Enzymes can be used free in solution or can be attached to a support such as a polymer. The reaction mixture in this manner is substantially homogeneous at the start, although some precipitate may form during the reaction. The temperature at which a previous process is carried out can vary from just above freezing to the temperature at which the most sensitive enzyme is denatured. This temperature range is preferably from about 0 ° C to about 45 ° C, and more preferably from about 20 ° C to about 37 ° C.

The reaction mixture formed in this manner is maintained for a period of time sufficient for the desired percentage of the terminal galactose residues present in the saccharide groups bound to the glycopretein which has been sialylated. For commercial scale preparations, the reaction will often be allowed to take place for about 8-240 hours, with a time of between about 24 and 48 hours more typically. The following examples are offered to illustrate, but not to limit the present invention.

Example 1 Sialylation of Recombinant Glycoprotins Using ST3Gal III Several glycoproteins were examined for their ability to be sialylated by the recombinant rat ST3Gal III. For each of these glycoproteins, sialylation will be a valuable step in the development of the respective glycoproteins as commercial products. Reaction Conditions The reaction conditions are summarized in Table 2. The reactions of the sialyltransferase were carried out for 24 hours at a temperature between room temperature and 37 °. The degree of sialylation was established by determining the amount of * C-NeuAc incorporated into the oligosaccharides linked to the glycoprotein.

Results and Discussion The results presented in Table 2 show that a remarkable degree of sialylation was achieved in each case, in spite of the low levels of enzyme used (essentially a complete sialylation was obtained based on the calculation of the available terminal galactose). Table 2 shows the amount of enzyme used per mg of protein (mU / mg) as a basis for comparison for different studies. In several of the examples shown, only 7-13 mU ST3Gal III per mg protein was required to provide essentially complete sialylation after 24 hours. These results are in marked contrast to those reported in detailed studies with the STT6GalI of bovine where > 50 mU / mg of protein provided less than 50% of the sialylation, and 1070 mU / mg of protein provided approximately 85-90% sialylation in 24 hours. Paulson et al. (1977) Bulletin of Chemical Biology 252: 2363-2371; Paulson et al. (1978) Bulletin of Chemical Biology 253: 5617-5624. A study of rat a2, 3 and a2, 6 sialyltransferase by another group found that complete sialylation of asialo-a * AGP required enzyme concentrations of 150-250 mU / mg protein. Weinstein et al. (1982) Bulletin of Chemical Biology 257: 13845-13853. These earlier studies jointly suggested that the ST6GalI sialyltransferase requires more than 50 mU / mg and up to 150 mU / mg to achieve complete sialylation. This example demonstrates that silylation of recombinant glycoproteins using the ST3Gal III sialyltransferase requires much fewer enzymes than expected. For a one kilogram reaction, approximately 7,000 units of ST3Gal III silyltransferase would be needed instead of the 100,000-150,000 units indicated in previous studies. The purification of these enzymes from natural sources is prohibitive, with productions of only 1-10 units for a large-scale preparation after 1-2 months of work. Assuming that both the ST6GalI and ST3Gal III sialyltransferase are produced as recombinant sialyltransferases, with equal levels of expression of the two enzymes that are achieved, a fermentation scale 14-21 times greater (or more) would be required for the ST6GalI sialyltransferase relative to the ST3Gal III sialyltransferase. For the ST6GalI sialyltransferase, the expression levels of 0.3 U / 1 in the yeast have been reported. Borsig et al. (1995) Community Research on Biochemistry and Biophysics 210: 14-20. Expression levels of 1000 U / liters of the ST3Gal III sialyltransferase have been achieved in Aspergillus niger. At present levels of expression 300-450 would be required, 000 liters of yeast fermentation to produce enough enzyme for the sialylation of one kg of glycoprotein using the ST3GalI sialyltransferase. In contrast, less than 10 liters of Aspergillus niger fermentation would be required for the sialylation of one kg of glycoprotein using the ST3Gal III sialyltransferase. In this way, the fermentation capacity required to produce the ST3Gal III silyltransferase for a large-scale sialylation reaction would be 10-100 times less than that required to produce the ST6GalI.; the cost of producing the sialyltransferase would be reduced proportionally.

Table 2: Silylation of recombinant glycoproteins using the ST3Gal III sialyltransferase 1. "Cycle" refers to the generation of CMP-NeuAC if you "enzymatically" using standard conditions according to those described in the specification (20 mM NeuAc and 2 mM CMP). The buffer solution was 0.1 M HEPES, pH 7.5. 2. Content of terminal Gal (exposed) in N-linked oligosaccharides determined by the supplier, or from literature values (fetuin, asialo-AAAT). 3. Incorporated NeuAc determined by the incorporation of 14C ~ NeuAc after separation of the free radio-pretreated precursors by gel filtration. 4. The% Rxn refers to the% completion of the reaction based on the Gal terminal content at the theoretical maximum. 5. Antithrombin III 6. Antitrypsin Example 2 Kinetics of Recombinant Glycoprotein Sialation Using ST3Gal III Reaction Conditions The assay mixtures (total volume of 500 μl) consisted of: 25 M of MES pH 6.0, or 5% (v / v) of Triton CF- 54, 2 mg / ml of BSA, 0.04% of sodium azide, 1 mg of al-acid glycoprotein treated with neuraminidase, sialyltransferase (2-100 mUnit / ml (, and 3400 nmoles of CMP-sialic acid with a CMP tracer - [* C [SA added to follow the degree of sialylation] ST3Gal III was produced recombinantly, whereas ST6GalI was purified from bovine colostrum The concentration of al-acid glycoprotein treated with neuraminidase was determined by absorption using a predetermined exintition coefficient (e278 = 0.849 for 1 mg) and by the amount of terminal galactose according to those determined by the galactose dehydrogenase assay (Wallenfels and Kurz, G. (1966) Enzymological Methodology 9: 112-116). At the incubation times indicated at 37 ° C, the degree of sialylation of the glycoprotein to the acid treated with neuraminidase was determined by removing aliquots of 50 μl (10%) of the reaction mixture and the adsorber of glycopretein was precipitated with 1 ml of 1% phosphotungstic acid in 0.5 M HCl to separate it from the CMP-SA donor. The pill was washed twice with phosphotungstic acid followed by dissolving the pill in 400 μl of chloroform / methanol 1: 1 (v / v) at 4 ° C for 20 minutes. After obtaining a final pill by centrifugation, the supernatant was removed and the pill allowed to dry. Subsequently the pill was dissolved in 400 μl of 0.2 M NaCl, 0.5 N NaOH at 37 ° C for 1 hour. Subsequently, the dissolved pill was transferred to scintillation flasks for scintillation counting. The negative controls represented by the omission of the adder were subtracted from each time point. Results and Conditions Figure 1 shows a time course of sialylation using ST3Gal III at a concentration of 20mUhidade / ml (10mUnits / mg admission). These results demonstrate that ST3Gal III efficiently sialylates the open galactose residues on the al-acid glycoprotein treated with neuraminidase. In fact more than 80% of the sialylation achieved in 1 hour. The achievement of more than 80% of the sialylation in 1 hour is significant because the recombinant glycoproteins of therapeutic value can lose bioactivity with extended incubation times at 37 ° C. It should be noted that the al-acid glycoprotein treated with neuraminidase is a particularly difficult glycoprotein to completely sialylate due to the multiple tri-and tetra-antennal N-linked oligosaccharides. In fact, using the glycoprotein al-acid treated with neuraminidase as an ST3Gal III of the adsorbent, is superior to another common sialyltransferase, the STdGalI isolated from bovine colostrum. A comparison of the sialylation capacities of these two enzymes using the glycoprotein al-acid treated with neuraminidase as an adsorber is shown in Figure 2. These results demonstrate that ST3Gal III is superior to ST6GalI at all time points examined., in particular with shorter incubation times. Within one hour, ST3Gal III had sialylated 80% of the open galactose residues of the receptors, whereas only 30% of the sites were saturated by the ST6GalI. When different batches of the al-acid glycoprotein treated with neuraminidase were used as recipients using similar assay conditions, the saturation percentage of the open galactose varied from 75-99% for ST3Gal III and 42-60% for STT6GalI in 24 hours. These results represent experiments in which ST3Gal III and ST6GalI are compared in parallel using identical conditions as defined above. For these experiments the al-acid glycoprotein treated with neuraminidase is separated from the donor by gel filtration as previously described (Weistein et al. (1982) Bulletin of Chemical Biology 257: 13845-13853). In each case examined, ST3Gal III sialylated the admission to a level significantly greater than the degree of sialylation achieved with the STdGalI up to 24 hours. In addition to examining the above mammalian sialyltransferases, two bacterial sialyltransferases were examined for their ability to sialylate the al-acid glycoprotein. An unanticipated finding was that the 2,3 reagent sialyltransferase from Neisseria meningtidis did not transfer the sialic acid to the acid glycoprotein under conditions in which it sialylates the oligosaccharides containing the terminal Galßl, 4 as for example the LNnT lactose. In contrast, a 2,6-sialyltransferase purified from Fotobacterium damasela efficiently incorporated sialic acid into the α-acid glycoprotein treated with neuraminidase as an advisor.

Example III Identification of Sialyltransferases Useful in Methods for Commercially Modified Glycoprotein Modification The members of the mammalian sialyltransferase gene family shown in Table 3 below are recombinantly expressed and examined for their ability to sialylate a variety of glycoproteins in a commercially practical manner. . Table 3. Mammalian Sialyltransferase Sialyltransferases Formated Sequences ST3GalI Neu5Aca2, 3Galßl, 3GalNAc STGal II Neu5Aca2, 3Galßl, 4GalNAc ST3Gal IV Neu5Aca2, 3Galßl, 4GalNAc Neu5Aca2, 3Galßl, 3GalNAc ST5GalNAcI Neu5Ac2, 6GalNAc Galßl, 3GalNAc (Neu5Aca2,6) Galßl, 3GalNAc (Neu5Aca2, 6) Neu5Aca2, 3Galßl, 3GalNAc (Neu5Ac a2 ST6GalNAc II Neu5Ac2,, 6GalNAc Galßl, 3GalNAc (Neu5Aca2,6) ST6GalNAc 111 Neu5Aca2, 3Galßl, 3GalNAc (Neu5Ac a2, 6 The sialyltransferases capable of sialylating the glycoproteins at a level of at least 80% using no more than 50 mUnits / mg of an adsorbent are considered "practical" for use in modifying the glycoprotein on a commercial scale. The assay uses assay conditions that are practical for large-scale use, for example, 1-10 mg / ml of the glycoprotein adsorbent and a concentration of the sialyltransferase of (2-50 mUnity / mg admission). The amount of open galactose is determined by means of the galactose dehydrogenase assay (Wallenfels et al., Supra). After adequate incubation times at 37 ° C, the degree of the glycoprotein is evaluated by removing the aliquots of the reaction mixture and separating the glycoprotein from the donor of the CMP-SA by precipitation or by gel filtration. In addition, the recombinant or purified sialyltransferases of the bacteria presented in Table 4 below can be examined. Once again the concentration of the sialyltransferase did not exceed 50 mUnities / mg of the glycoprotein adsorbent and the glycoprotein concentrations vary from 1-10 mg / ml. Table 4. Sialiltransferases Bacterial Sialyltransferase Organism Structure Sialiltransferase N. meningi tidis Neu5Aca2, 3Galßl, 4G1 N. gonorrheae cNAc ST3Gal VI Campyl obacter Neu5Aca2, 3Galßl, 4G1 j ejuni cNAc ST3Gal VII Haemophilus so nus Neu5Aca2, 3Galßl, 31c H. influenzae NAc STGal VIII ST6Gal II Photobacterium Neu5Aca2, 3Galßl, 4G1 ladies ela cNAc The bacterial and mammalian sialyltransferases listed in Tables 3 and 4 are tested for their ability to completely sialylate the glycoproteins expressed transgenically or recombinantly as shown in Table 5 below. This list is not intended to be exhaustive, however, instead it offers examples of glycoproteins of known therapeutic utility where complete sialylation may favorably alter the pharmacokinetics or biological activity of the glycoprotein. The glycoproteins used in these experiments can be produced in a transgenic animal, or in an aukaryotic cell or cell line. In this experiment, the degree of sialylation and type of glycan that modifies the glycoprotein of interest is examined using standard biochemical techniques such as gel electrophoresis, HPLC and mass spectrometry. This structural information is used to choose the sialyltransferases with the correct specificity characteristics to fully sialylate (or as much as possible) the glycoprotein as judged by gel electrophoresis or the HPLC of the resulting glycans. At this point the pharmacokinetics of the fully sialylated glycoprotein can be compared to the pharmacokinetics of the subsylated glycoprotein by examination in small animals.

It is recognized that certain glycoproteins will require a combination of silayltransferases given the stereochemical and regioselective nature of this class of enzymes. Thus, combinations of sialyltranferases are examined using the conditions defined for their potential large-scale practicality in glycoprotein remodeling. This is of particular importance when examining the glycoproteins of both N-linked and O-linked glycans, as well as those modified by highly branched oligosaccharides. In this regard, sialyltransferases exhibiting multiple specificities such as ST3Gal IV and Campylobacter sialyltransferase may be particularly useful as independent remodeling enzymes when sialylated glycoproteins with N- and N-linked multiple glycans.

Table 5. Candidates for sialylation of the glycoprotein a-lanti-trypsin tissue plasmidgen activator Erythropoietin Gluten-macrophage colony stimulation factor (GMCSF) Antithrombin III Human growth hormone Interleukin human 6 Interferon ß Protein C Fribrinogen Factor IX Factor VII Tumor necrosis factor Tumor necrosis factor receptor protein It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes will be suggested in view of the foregoing to those skilled in the art, and should be included within the scope and scope of this application. and scope of the appended claims. All publications, patents and patent applications cited herein are incorporated herein by reference for all purposes.

Claims (58)

1. CLAIMS 1. A method for sialylating a group of saccharides in a recombinant glycoprotein, the method includes contacting a group of saccharides that includes a galactose or an N-acetylgalactosamine adsorber moiety in a recombinant glycoprotein with a half donor sialic acid and a recombinant sialyltransferase in a reaction mixture that provides the reagents required for the sialyltransferase activity for a sufficient time, and under conditions appropriate for transferring the sialic acid from said donor half of the sialic acid to the aforementioned saccharide group.
2. 2. The method of claim 1, wherein the donor half of the sialic acid is CMP-sialic acid.
3. 3. The method of claim 2, wherein the CMP-sialic acid is generated enzymatically in itself.
4. 4. The method of claim 1, wherein the sialyltransferase is a recombinant eukaryotic sialyltransferase that substantially lacks a membrane extension domain.
5. The method of claim 1, wherein the sialyltransferase includes a sialyl motif having an amino acid sequence that is at least about 40% identical to a sialyl motif of a sialyltransferase selected from the group consisting of ST3GalI, ST6GalI and ST3Gal III.
6. 6. The method of claim 1, wherein the sialyltransferase is a recombinant ST3Gal III.
7. The method of claim 6, wherein the sialyltransferase is a recombinant rat ST3Gal III.
8. The method of claim 1, wherein the sialyltransferase is a recombinant ST3Gal IV.
9. 9. The method of claim 1, wherein the sialyltransferase is a recombinant ST6GalI.
10. The method of claim 1, wherein the sialyltransferase is a recombinant ST3GalI.
11. The method of claim 10, wherein the reaction mixture includes a second recombinant sialyltransferase, wherein the second recombinant sialyltransferase is an ST3Gal III.
12. The method of claim 1, wherein the sialyltransferase is a recombinant bacterial sialyltransferase.
13. The method of claim 12, wherein the bacterial sialyltransferase has an amino acid sequence that is at least 50% identical to an amino acid sequence of a 2,3-sialyltransferase of Neisseria meningi idis.
14. 14. The method of claim 13, wherein the bacterial sialyltransferase is a 2,3-sialyltransferase from Neisseria meningi tidis.
15. The method of claim 12, wherein the bacterial sialyltransferase has an amino acid sequence that is at least 50% identical to an amino acid sequence of a 2,6-sialyltransferase of Photobacterium damsela.
16. 16. The method of claim 15, wherein the bacterial sialyltransferase is a 2,6-sialyltransferase from Photobacterium damsela.
17. The method of claim 12, wherein the bacterial sialyltransferase has an amino acid sequence that is at least 50% identical to an amino acid sequence of a 2, 3-sialyltransferase of Haemophilus.
18. 18. The method of claim 17, wherein the sialyltransferase is a 2,3-sialyltransferase from Haemophi 1 us.
19. The method of claim 12, wherein the bacterial sialyltransferase has an amino acid sequence that is at least 50% identical to an amino acid sequence of a 2, 3-sialyltransferase of Campylobacter j juni.
20. 20. The method of claim 19, wherein the sialyltransferase is a 2,3-sialyltransferase of Campylobacter jejuni.
21. The method of claim 1, wherein the sialyltransferase is produced by the recombinant expression of a sialyltransferase in a host cell selected from the group consisting of an insect cell, a mammalian cell and a fungal cell.
22. 22. The method of claim 21, wherein the host cell is an Aspergillus niger cell.
23. 23. A method for sialylating a group of saccharides in a recombinant glycoprotein, the method includes contacting a group of saccharides including a galactose or an N-acetylgalactosamine adsorber moiety in a recombinant glycoprotein with a sialic acid donor moiety and a bacterial sialyltransferase in a reaction mixture -which offers the required reagents for the activity of the sialyltransferase for a sufficient time, and under appropriate conditions for transferring the sialic acid from the donor half of the sialic acid to the aforementioned saccharide group.
24. The method of claim 23, wherein the bacterial sialyltransferase has an amino acid sequence that is at least 50% identical to an amino acid sequence of a 2,6-sialyltransferase of Photobacterium damsela.
25. The method of claim 24, wherein the bacterial sialyltransferase is a 2,6-sialyltransferase from Photobacterium damsela.
26. 26. The method of claim 23, wherein the bacterial sialyltransferase has an amino acid sequence that is at least 50% identical to an amino acid sequence of a Neisseria meningi tisid 2,3-sialyltransferase.
27. The method of claim 26, wherein the bacterial sialyltransferase is a 2,3-sialyltransferase from Neisseria meningi tidis.
28. The method of claim 23, wherein the bacterial sialyltransferase has an amino acid sequence that is at least 50% identical to an amino acid sequence of a 2, 3-sialyltransferase of Campylobacter jejuni.
29. 29. The method of claim 28, wherein the bacterial sialyltransferase is a 2,3-sialyltransferase of Campylobacter jejuni.
30. 30. The method of claim 23, wherein the bacterial sialyltransferase has an amino acid sequence that is at least 50% identical to an amino acid sequence of a 2, 3-sialyltransferase of Haemophilus.
31. 31. The method of claim 30, wherein the bacterial sialyltransferases is a 2, 3-sialyltransferase of Haemophilus.
32. 32. A method for sialylation in groups of saccharides present in a glycoprotein, this method includes contacting the mentioned saccharide groups with a sialyltransferases, a sialic acid donor half and other reagents required for the sialyltransferase activity for a sufficient time and under the appropriate conditions to transfer the sialic acid from the sialic acid donor moiety to the aforementioned saccharide group, wherein said sialyltransferase is present at a concentration of about 50 mU per mg glycoprotein or less.
33. The method of claim 32, wherein the sialyltransferase is present at a concentration of between about 5-25 mU per ml of glycoprotein.
34. 34. The method of claim 32, wherein the sialyltransferase is present at a concentration of between about 10-50 mU / ml of the reaction mixture and the glycoprotein is present in the reaction mixture at a concentration of less about 2 mg / ml.
35. 35. The method of claim 32, wherein the method produces a glycoprotein possessing a sialylation of at least about 80% of the terminal galactose residues present in the saccharide groups.
36. 36. The method of claim 32, wherein the sialyltransferase is a recombinant sialyltransferase.
37. 37. The method of claim 36, wherein the sialyltransferase substantially lacks a membrane extension domain.
38. 38. The method of claim 32, wherein the sialyltransferase includes a sialyl motif having an amino acid sequence that is at least about 40% identical to a sialyl motif of a sialyltransferase selected from the group consisting of ST3GalI, ST6GalI and ST3Gal III.
39. 39. The method of claim 32, wherein the sialyltransferase is an ST3Gal III.
40. 40. The method of claim 39, wherein the ST3Gal III is a rat ST3Gal III.
41. 41. The method of claim 32, wherein the sialyltransferase is an ST3Gal IV.
42. 42. The method of claim 32, wherein the sialyltransferase is an ST3GalI.
43. 43. The method of claim 42, wherein the reaction mixture includes a second recombinant sialyltransferase, wherein the second recombinant sialyltransferase is an ST3Gal III.
44. 44. The method of claim 32, wherein the sialyltransferase is a bacterial sialyltransferase.
45. 45. The method of claim 44, wherein the bacterial sialyltransferase is a recombinant sialyltransferase.
46. 46. The method of claim 44, wherein the bacterial sialyltransferase has an amino acid sequence that is at least 50% identical to an amino acid sequence of a Neisseria meningi tidis 2,3-sialyltransferase.
47. 47. The method of claim 46, wherein the bacterial sialyltransferase is a 2,3-sialyltransferase from Neisseria meningi tidis.
48. 48. The method of claim 44, wherein the bacterial sialyltransferase has an amino acid sequence that is at least 50% identical to an amino acid sequence of a 2,6-sialyltransferase of Photobacterium damsela.
49. 49. The method of claim 48, wherein the bacterial sialyltransferase is a 2,6-sialyltransferase from Photobacterium damsela.
50. 50. The method of claim 44, wherein the bacterial sialyltransferase has an amino acid sequence that is at least 50% identical to an amino acid sequence of a 2, 3-sialyltransferase of Campylobacter jejuni.
51. 51. The method of claim 50, wherein the sialyltransferase is a 2, 3-sialyltransferase of Campyl obacter jejuni.
52. 52. The method of claim 44, wherein the bacterial sialyltransferase has an amino acid sequence that is at least 50% identical to an amino acid sequence of a 2, 3-sialyltransferase of Haemophilus.
53. 53. The method of claim 52, wherein the sialyltransferase is a 2, 3-sialyltransferase from Haemophi 1 us.
54. 54. The method of claim 32, wherein the donor half of sialic acid is CMP-sialic acid.
55. 55. The method of claim 54, wherein the CMP-sialic acid is enzymatically generated with itself.
56. 56. The method of claim 32, wherein the sialic acid is selected from the group consisting of NeuAc and NeuGc.
57. 57. A method for sialylation in groups of saccharides present in a glycoprotein, the method includes contacting the saccharide groups with an ST3Gal III sialyltransferases, a sialic acid donor moiety and other reagents required for the activity of the sialyltransferase for a sufficient time and under the conditions for transferring the sialic acid from the sialic acid donor moiety to the aforementioned saccharide group, wherein the said ST3Gal III sialyltransferase is present at a concentration of approximately 50 mU per mg of glycoprotein.
58. 58. The method of claim 57, wherein the method further includes contacting the saccharide groups with an ST6GalI sialyltransferase.