WO2001059075A1

WO2001059075A1 - Improved sialylation of glycoproteins

Info

Publication number: WO2001059075A1
Application number: PCT/US2001/003955
Authority: WO
Inventors: Thomas Ryll
Original assignee: Genentech, Inc.
Priority date: 2000-02-08
Filing date: 2001-02-07
Publication date: 2001-08-16
Also published as: AU2001236739A1; WO2001059075A9; WO2001059075A8

Abstract

The present invention provides for cells and processes for producing glycoproteins having enhanced sialyation. The invention also provides for nucleic acid sequences encoding UDP-GlcNac 2-epimerases or modified UDP-GlcNac 2-epimerases. The modified UDP-GlcNac 2-epimerases have reduced binding to sialic acid.

Description

IMPROVED SIALYLATION OF GLYCOPROTEINS

This application is being filed as a PCT application by GENENTECH, INC., a United States national and resident, designating all countries.

Related Application This application claims priority of United States provisional application Serial Number 60/181,074, filed February 8, 2000, and United States provisional application Serial Number 60/191,529 filed March 23, 2000.

Field of the Invention

The invention generally relates to the production of glycoproteins in cell culture. More particularly, the invention relates to processes and cells for improved sialylation of glycoproteins produced in host cells.

Background of the Invention

Differences in glycosylation patterns of recombinantly produced glycoproteins have recently been the focus of attention in the scientific community as recombinant proteins produced as probable prophylactics and therapeutics approach the clinic. Oligosaccharide structures on glycoproteins produced by expression of foreign genes in recombinant host cells in vitro are heterogenous in both their composition and structure. Rademacher et al. (1988) Annu. Rev. Biochem. 57:785-838; Spellman et al., (1989) J. Biol. Chem. 264(24): 14100- 14111; Spellman et al., (1991) Biochemistry 30:2395-2406; Kagawa et al., (1988) J. Biol. Chem. 263(33): 17508-17515.

The oligosaccharide side chains of the glycoproteins affect the protein's function (Wittwer A., and Howard, S.C. (1990) Biochem. 29:4175-4180) and the intramolecular interaction between portions of the glycoprotein resulting in the conformation and presented three dimensional surface of the glycoprotein (Hart, (1992) Curr. Op. Cell Biol. , 4: 1017-1023; Goochee, et al. , (1991)

Bio/Technology, 9:1347-1355; Parekh, R.B., (1991) Curr. Op. Struct. Biol., 1:750-754). In particular, the terminal sialic acid component of the glycoprotein oligosaccharide side chain affects absorption, serum half life, and clearance from the serum, as well as the physical, chemical and immunogenic properties of the glycoprotein (Parekh, R.B., supra; Varki, A., (1993) Glycobiology 3:97-100; Paulson, J. (1989), TIBS, 14:272-276; Goochee, et al., (1991) Biotechnology 9:1347-1355; Kobata, A, (1992) Eur. J. Biochem. 209:483-501). As a result the molar content of sialic acid on a recombinant glycoprotein can be a key feature of the therapeutic glycoprotein 's biological quality.

Several strategies have been proposed to affect the composition of oligosaccharides in recombinant glycoproteins (Minch SL, et al. (1995) Biotechnol Prog 11:348-351; Monaco L, et al. (1996) Cytotechnology 22:197-203; Grabenhorst et al., (1995) Eur. J. Biochem. 232:718-725; U.S. Patent No. 5, 047,335). Expression of human α,6-sialyltransferase in recombinant cell lines such as Chinese hamster ovary (CHO) cells and baby hamster kidney cells (BHK) has been shown to alter the quality of the N-linked oligosaccharide terminal glycoside linkage producing a more human product ( Minch et al., supra; Monaco et al., supra;). The transfected sialyltransferase can compete with endogenous CHO cell enzymes for glycosyl substrate in the attachment of terminal sugar residues (Lee UE, et al. (1989) J Biol Chem 264: 13848-13855). Expression of other glycosyltransferases in recombinant host cell lines has also been reported (Miyoshi E, et al. (1995) J. Biol. Chem. 270(47) :28311-28315; Ernst et al., (1989) J. Biol. Chem. 264(6): 3436-3447). These studies support the potential of genetic strategies directed toward oligosaccharide biosynthesis for altering the glycoprotein production. For glycoproteins whose half-life or biological activity is strongly dependent on the content of sialic acid, insufficient or inconsistent glycosylation is a significant problem for adequate, reproducible dosing of the molecule. From a manufacturing perspective, since the degree of glycosylation can vary as a function of environmental or physiological changes during cell culture, insufficient or inconsistent glycosylation can also be a problem for process consistency. Summary of the Invention

The present invention provides for enhanced activity of a rate-limiting enzyme in the biosynthetic pathway of a specific nucleotide-sugar to induce increased production of glycosylation precursor molecules. In a preferred embodiment, overexpression of the rate limiting enzyme activity in the CMP-sialic acid biosynthesis, UDP-GlcNac 2-epimerase/ManNac kinase (hereinafter "Epimerase") in host cells leads to enhanced intracellular concentration of CMP- sialic acid, the precursor for sialylation reactions. Increased intracellular CMP- sialic acid leads to enhanced sialylation of recombinant glycoproteins produced in the host cells.

Overexpression of Epimerase, e.g., the native enzyme, or preferably of a mutated version having reduced feedback inhibition by the product CMP-sialic acid, is accomplished by expression of a gene encoding the enzyme in the host cell. The host cell may be selected for enhanced enzyme activity, for example, by reducing product feedback inhibition, thereby enhancing intracellular precursor pools for production of the desired oligosaccharides; for example increasing sialylation of glycoproteins in a eukaryotic cell. The invention provides a solution to the problem of inconsistent sialylation of recombinant glycoproteins expressed in varied cell types, under varied cell culture conditions, and the like. The invention also solves inconsistencies in and between glycoprotein production lots in addition to decreasing the heterogeneity of glycoforms in the glycoprotein produced.

The present invention provides host cells and processes for producing a glycoprotein, whereby glycoprotein is produced having enhanced terminal sialylation. According to the invention, host cells and methods direct enhanced sialylation by reduced product feedback inhibition and enhanced activity of the rate-limiting enzyme in the de novo synthetic pathway for the production of CMP- sialic acid.

Feedback inhibition is preferably reduced by use of a mutant enzyme, having altered amino acid residues at the putative binding site for CMP-sialic acid on the Epimerase. For example, the positively charged Arginine (Arg) residues 263 and 266 of the Epimerase amino acid sequence (Seq ID No: 2) may be altered to any other amino acid but Arg residues.

The invention provides host cells expressing mutated Epimerase. Glycoproteins produced in these host cells demonstrate enhanced sialylation, including a shift from terminal galactose to terminal sialyic acid. The method and cells of the invention are useful for the recombinant expression of glycoproteins having sialylic acid terminal residues necessary for a desired reaction, and provide for more consistent sialylation of recombinantly produced glycoproteins.

Brief Description of the Drawings

Figure 1 is a diagramatic representation of the CMP-NANA de novo biosynthetic pathway.

Figure 2 is a HPLC chromatogram showing enhanced intracellular CMP- NANA in cells fed 20 mM N-acetyl Mannosamine. Figure 3 is a graph showing the effect of N-acetyl Mannosamine on the terminal sugar composition of the recombinantly produced protein, TNFr-IgG.

Figure 4 is a scan showing enhanced CMP-NANA in cells overexpressing mutated UDP-GlcNAc 2- epimerase.

Figure 5 is a graph showing enhanced intracellular free sialic acid in cells overexpressing mutated UDP-GlcNac 2- epimerase.

Figures 6A and 6B are MALDI mass spectra of acidic glycans showing an enhanced degree of sialylation on the purified glycoprotein overexpressing mutated UDP-GlcNAc 2- epimerase.

Figure 7 is a graph showing an increased amount of NANA content of protein expressed in cells overexpressing mutated UDP-GlcNAc 2- epimerase.

Detailed Description of the Invention Definitions

The term "eukaryotic cell" or cell line is used to refer to cells established in ex vivo culture. It is a characteristic of the eukaryotic cells of the present invention that it express or is capable of expressing a particular glycoprotein of interest. Eukaryotic cells used in the production of a desired protein product have the means for glycosylating proteins by addition of oligosaccharide side chains. Such cells, in certain embodiments, also have the capability to remove and/or modify enzymatically part or all of the oligosaccharide side chains of glycoproteins.

Examples of suitable eukaryotic host cells within the context of the present invention include insect and mammalian cells. Example host cells include SF9 insect cells (Summers and Smith (1987) Texas Agriculture Experiment Station Bulletin, 1555; and Insect Cell Culture engineering, Goosen Daugulis and Faulkner Eds. Dekker, New York); Chinese hamster ovary (CHO) cells (Puck et al. , (1958) J. Exp. Med. 108:945-955; Puck (1985) Molecular Cell Genetics, Gottersman MM ed. Wiley Intersciences pp37-64) including CHO cells expressing TNFrlgG and aCD20; monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (Graham et al., (1977) J. Gen Virol., 36:59); baby hamster kidney cells (BHK, ATCC CCL 10); and human cervical carcinoma cells (HELA, ATCC CCL 2). The above list is meant only for illustrative purposes and in no way is meant to limit the scope of the present invention.

As used herein, "control cell" refers to a cell that has been cultured in parallel with a cell treated under the specified experimental condition but unlike the treated cell, the host cell has not undergone the specified experimental condition. Control cells represent a baseline from which comparisons are made.

The term "nucleic acid sequence" refers to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide chain. The deoxyribonucleotide sequence thus codes for the amino acid sequence.

The term "expression vector" refers to a first piece of DNA, usually double-stranded, which may have inserted into it a second piece of DNA, for example a piece of foreign DNA. Foreign DNA is defined as heterologous DNA, which is DNA not naturally found in the host cell and includes additional copies of genes naturally present in the host genome. The vector is used to transport the foreign or heterologous DNA into a suitable host cell. Once in the host cell, the vector is capable of integration into the host cell chromosomes. The vector contains the necessary elements to select cells containing the integrated DNA as well as elements to promote transcription of polyadenylated messenger RNA (mRNA) from the transfected DNA. Many molecules of the polypeptide encoded by the foreign DNA can thus be rapidly synthesized.

The terms "expression" or "expresses" are used herein to refer to transcription and translation occurring within a host cell. The level of expression of a product gene in a host cell may be determined on the basis of either the amount of corresponding mRNA that is present in the cell or the amount of the protein encoded by the product gene that is produced by the cell. For example, mRNA transcribed from a product gene is desirably quantitated by northern hybridization. Sambrook et al. , Molecular Cloning: A Laboratory Manual, pp. 7.3-7.57 (Cold Spring Harbor Laboratory Press, 1989). Protein encoded by a product gene can be quantitated either by assaying for the biological activity of the protein or by employing assays that are independent of such activity, such as western blotting or immunoassay using antibodies that are capable of reacting with the protein. Sambrook et al. , Molecular Cloning: A Laboratory Manual, pp. 18.1-18.88 (Cold Spring Harbor Laboratory Press, 1989). As used herein, "glycoprotein" refers generally to peptides and proteins having more than about 10 amino acids and at least one carbohydrate. The glycoproteins may be homologous to the host cell, or preferably, be heterologous, i.e. , foreign, to the host cell. Example glycoproteins that could be expressed using one embodiment of the present invention include molecules such as cytokines and their receptors, chimeric proteins, tumor necrosis factor alpha and beta, tumor necrosis factor receptors, human growth hormone, bovine growth hormone, parathyroid hormone, thyroid stimulating hormone, lipoproteins, alpha- 1- antitrypsin, insulin A-chain, T-cell receptors, surface membrane proteins, monoclonal antibodies, and transport proteins. The list of examples is illustrative only and in no way is meant to limit the scope of the present invention. The term "glycoform" as used within the context of the present invention is meant to denote a glycoprotein containing a particular carbohydrate structure or structures.

The term "glycosylation" as used within the context of the present invention is meant to encompass the process by which a protein is covalently linked with one or more oligosaccharide chains. The term "galactosylation" means the addition of a galactose units to an oligosaccharide chain on a glycoprotein and "sialylation" the addition of a sialic acid to an oligosaccharide chain on a glycoprotein. "Period of time and under such conditions that cell growth in maximized" , and the like, refer to those culture conditions that, for a particular cell line , are determined to be optimal for cell growth and division. Normally, during cell culture cells are cultured in nutrient medium containing the necessary additives generally at about 30-40° C, in a humidified, controlled atmosphere, such that optimal growth is achieved for that particular cell line.

As used herein "cultured for sufficient time to allow selection to occur" refers to the act of physically cul uring the eukaryotic host cells on the selection agent until resistant clones were picked and tested for the relevant characteristic.

As used herein, protein, peptide and polypeptide are used interchangeably to denote an amino acid polymer or a set of two or more interacting or bound amino acid polymers.

As used herein, "Epimerase" means UDP-Glc Nac 2-epimerase/ManNac kinase), the rate limiting enzyme in the de novo synthesis of CMP-NANA.

As used herein, the term "enhance" or "enhancing" or "improved" means increasing the amount, number, pattern, and/or type of oligosaccharide chain which is added to the glycoprotein during cellular production.

The term "mutant" or "modified" Epimerase means the human Epimerase enzyme of Seq. ID. NO: 1 altered by mutation, genetic manipulation, or other means known in the field, to produce Epimerase having an amino acid sequence altered by the change of at least one amino acid, preferably altered so as to reduce CMP-sialic acid bining to the enzyme, or otherwise reducing inhibition of the enzyme's activity. Preferred mutants are those having a substituted amino acid residue at Arg263 or Arg266 or both.

Modes For Carrying Out the Invention Production of glycoproteins in host cell lines does not result in consistent, nonheterogeneous oligosaccharide glycoprotein profiles. According to the present invention, overexpression of the rate-limiting Epimerase enzyme, and particularly of the mutant enzyme permits enhanced enzyme activity in host cells without feedback inhibition, resulting in increased intracellular CMP-sialic acid, and leading to enhanced and improved sialylation of glycoprotein produced in the cell. In a preferred embodiment of the invention, a host cell expressing a first nucleic acid sequence encoding a glycoprotein, and a second nucleic acid sequence encoding Epimerase, preferably mutant Epimerase, produces the glycoprotein having enhanced sialylation. Most preferably, the mutant Epimerase is modified from the amino acid sequence of Seq. ID. NO: 2 by having any other amino acid but Arg substituted for one or both of Arg263 and Arg266. In one embodiment, the mutated Epimerase comprises the following mutation: Arg263Leu, Arg266Trp, Arg266Gln.

Epimerase

In a particular embodiment, a modified Epimerase is introduced into a host cell. The host cell is a eucaryotic cell, for example a CHO cell or insect cell, useful for the production of glycoproteins.

Native, human Epimerase is encoded by the nucleic acid sequence of Seq. ID No: 1 (1), encoding the amino acid sequence shown in Seq. ID No: 2 (2). Patients having the very rare disorder, sialuria, demonstrate an elevated intracellular concentration of free sialic acid. DNA obtained from three sialuria patients each contained a single mutation resulting in the change of the amino acid Arg263Leu, Arg266Trp, or Arg266Gln. Modified amino acid sequences encoding Seq. ID No: 3-7 (3-7) are shown below in comparison to the native, human Epimerase (Seq. ID No: 1 and 2). 263 266

GCA GGG AGC AAA GAG ATG GTT CGA GTG ATG CGG AAG AAG GGC ATT GAG CAT CAT CCC (1) Ala Gly Ser Lys Glu Met Val Arg Val Met Arg Lys Lys Gly lie Glu His His Pro (2)

263 266 Ala Gly Ser Lys Glu Met Val Leu Val Met Arg Lys Lys Gly He Glu His His Pro (3)

Ala Gly Ser Lys Glu Met Val Arg Val Met Trp Lys Lys Gly He Glu His His Pro (4)

Ala Gly Ser Lys Glu Met Val Arg Val Met Gin Lys Lys Gly He Glu His His Pro (5)

Ala Gly Ser Lys Glu Met Val Leu Val Met Trp Lys Lys Gly He Glu His His Pro (6) Ala Gly Ser Lys Glu Met Val Leu Val Met Gin Lys Lys Gly He Glu His His Pro (7)

In the instant invention, nucleic acid sequences encoding Epimerase and having one or more mutations replacing Arg residues of the putative CMP-sialic acid binding site are useful to reduce feedback inhibition and induce enhanced Epimerase acitivity. Host cells transformed with such a gene are provided with enhanced intracellular CMP-silaic acid, the precursor for sialylation of glycoproteins.

Glycoproteins

Nucleic acid encoding the endogenous host cell sequences or the heterologous recombinant glycoprotein gene are available to the skilled artisan and may be obtained by, for example, synthesis by in vitro methods or obtained readily from cDNA libraries. The means for synthetic creation of the DNA, either by hand or with an automated apparatus, are generally known to one of ordinary skill in the art. As but one example of the current techniques available for polynucleotide synthesis, one is directed to Maniatis et al., Molecular Cloning- -A Laboratory Manual, Cold Spring Harbor Laboratory (1984), and Horvath et al., An Automated DNA Synthesizer Employing Deoxynucleotide 3'- Phosphoramidites, Methods in Enzymology 154: 313-326, 1987, hereby specifically incorporated by reference. Alternatively, the gene sequence encoding the recombinant glycoprotein is cloned from cDNA libraries employing techniques available to the skilled artisan. For example polymerase chain reaction techniques may be employed whereby a particular nucleic acid sequence is amplified. Oligonucleotide primers based upon sequences which correspond to the 3' and 5' ends of the segment of the DNA to be amplified are hybridized under appropriate conditions and the enzyme Taq polymerase, or equivalent enzyme, is used to synthesize copies of the DNA located between the primers.

The particular procedure used for the functional expression of the recombinant glycoprotein is not critical to the invention. For example, any procedure for introducing nucleotide sequences into host cells may be used. These include the use of plasmid vectors, viral vectors, and other methods for introducing genetic material into a host cell. It is necessary that the gene or nucleic acid to be expressed be introduced in such a way that the host cell expresses the protein. High level expression is preferred.

For example, expression is typically achieved by introducing into the cells the appropriate glycoprotein along with another gene, commonly referred to as a selectable gene, that encodes a selectable marker. A selectable marker is a protein that is necessary for the growth or survival of a host cell under the particular culture conditions chosen, such as an enzyme that confers resistance to an antibiotic or other drug, or an enzyme that compensates for a metabolic or catabolic defect in the host cell. For example, selectable genes commonly used with eukaryotic cells include the genes for aminoglycoside phosphotransferase (APH), hygromycin phosphotransferase (hyg), dihydrofolate reductase (DHFR), thymidine kinase (tk), neomycin resistance, puromycin resistance, glutamine synthetase, and asparagine synthetase. In selecting an appropriate expression system, a selectable marker for the glycoprotein is chosen to allow for, if necessary, a second transfection with a second suitable amplifiable marker for the expression of a second glycoprotein product or to allow additional functional modifications of the host cell. Such modifications may include augmentation of other activities related to improving cell metabolism of the quantity or quality of recombinant product expression.

The level of expression of a gene introduced into a eukaryotic host cell of the invention depends on multiple factors, including gene copy number, efficiency of transcription, messenger RNA (mRNA) processing, stability, and translation efficiency. Accordingly, high level expression of a desired glycoprotein according to the present invention will typically involve optimizing one or more of those factors.

Further, the level of glycoprotein production may be increased by covalently joining the coding sequence of the gene to a "strong" promoter or enhancer that will give high levels of transcription. Among the eukaryotic promoters that have been identified as strong promoters for high-level expression which are preferred within the context of the present invention are the SV40 early promoter, adenovirus major late promoter, mouse metallothionein-I promoter, Rous sarcoma virus long terminal repeat, and human cytomegalovirus immediate early promoter (CMV). Particularly useful in expression of the desired glycoprotein product are strong viral promoters such as the myeloproliferative sarcoma virus (Artel et al., (1988) Gene 68:213-220), SV40 early promoter (McKnight and Tijian (1986) Cell, 46:795-805).

Enhancers that stimulate transcription from a linked promoter are also useful in the context of the present invention. Unlike promoters, enhancers are active when placed downstream from the transcription initiation site or at considerable distances from the promoter, although in practice enhancers may overlap physically and functionally with promoters. For example, many of the strong promoters listed above also contain strong enhancers (Bendig, (1988) Genetic Engineering, 7:91).

The level of protein production or expression also may be increased by increasing the gene copy number in the host cell. One method for obtaining high gene copy number is to directly introduce into the host cell multiple copies of the gene, for example, by using a large molar excess of the product gene relative to the selectable gene during cotransfectation. Kaufman, (1990) Meth. Enzymol.,

185:537. With this method, however, only a small proportion of the cotransfected cells will contain the product gene at high copy number. Screening methods typically are required to identify the desired high-copy number transfectants.

The use of splice-donor style vectors such as those described by Lucas et al. , (1996) Nuc. Acd Res. 24(9): 1774-1779 and WO/9604391 is also preferred. Yet another method for obtaining high gene copy number involves gene amplification in the host cell. Gene amplification occurs naturally in eukaryotic cells at a relatively low frequency (Schimke,(1988) J. Biol. Chem., 263:5989). However, gene amplification also may be induced, or at least selected for, by exposing host cells to appropriate selective pressure. For example, in many cases it is possible to introduce a glycoprotein gene together with an amplifiable gene into a host cell and subsequently select for amplification of the marker gene by exposing the cotransfected cells to sequentially increasing concentrations of a selective agent. Typically the product gene will be coamplified with the marker gene under such conditions.

The most widely used amplifiable gene for that purpose is a DHFR gene, which encodes a dihydrofolate reductase enzyme. The selection agent used in conjunction with a DHFR gene is methotrexate (Mtx). In this example, an epimerase overexpressing cell line is cotransfected with the glycoprotein gene and a DHFR gene, and transfectants are identified by first culturing the cells in culture medium that contains Mtx. The transfected cells then are exposed to successively higher amounts of Mtx. This leads to the synthesis of multiple copies of the DHFR gene, and concomitantly, multiple copies of the glycoprotein gene (Schimke, (1988) J. Biol. Chem., 263:5989; Axel et al., U.S. Patent No. 4,399,216; Axel et al., U.S. Patent No. 4,634,665; Kaufman in Genetic

Engineering, ed. J. Setlow (Plenum Press, New York), Vol. 9 (1987); Kaufman and Sharp, (182) J. Mol. Biol., 159:601; Ringold et al., J. Mol. Appl. Genet. , 1:165-175; Kaufman et al., Mol. Cell Biol., 5:1750-1759; Kaetzel and Nilson, (1988) J. Biol. Chem., 263:6244-6251; Hung et al., (1986) Proc. Natl. Acad. Sci. USA, 83:261-264; Kaufman et al., (1987) EMBO J., 6:87-93; Johnston and Kucey, (1988) Science, 242:1551-1554; Urlaub et al., (1983) Cell, 33:405-412.

Alternatively, host cells may be co-transfected with a glycoprotein gene, a DHFR gene, and a dominant selectable gene, such as a neo^r gene. Transfectants are identified by first culturing the cells in culture medium containing neomycin (or the related drug G418), and the transfectants so identified then are selected for amplification of the DHFR gene and the product gene by exposure to successively increasing amounts of Mtx.

Another method involves the use of polycistronic mRNA expression vectors containing a product gene at the 5' end of the transcribed region and a selectable gene at the 3' end. Because translation of the selectable gene at the 3' end of the polycistronic mRNA is inefficient, such vectors exhibit preferential translation of the glycoprotein gene and require high levels of polycistronic mRNA to survive selection. Kaufman, (1990) Meth. Enzymol., 185:487; Kaufman, (1990) Meth. Enzymol., 185:537; Kaufman et al., (1987) EMBO J., 6:187. Accordingly, cells expressing high levels of the desired protein product may be obtained in a single step by culturing the initial transfectants in medium containing a selection agent appropriate for use with the particular selectable gene.

A further method suitable within the context of the present invention is integrate the genes encoding the glycoprotein into a transcriptionally active part of the epimerase overexpressing cell genome. Such procedures are described in International Application No. PCT/US/04469.

Other mammalian expression vectors such as those that have single transcription units are also useful in the context of the present invention. Retroviral vectors have been constructed (Cepko et al., (1984) Cell, 37: 1053- 1062) in which a cDNA is inserted between the endogenous Moloney murine Ilekemia virus (M-MuLV) splice donor and splice acceptor sites which are followed by a neomycin resistance gene. This vector has been used to express a variety of gene products following retroviral infection of several cell types.

Introduction of the nucleic acids encoding the glycoprotein is accomplished by methods known to those skilled in the art. For mammalian cells without cell walls, the calcium phosphate precipitation method of Graham and van der Eb, (1978) Virology, 52:456-457 may be used. General aspects of mammalian cell host system transformations have been described by Axel in U.S. 4,399,216 issued 16 August 1983. However, other methods for introducing DNA into cells such as by nuclear injection, by protoplast fusion or by electroporation may also be used (Chisholm et al., (1995) DNA Cloning IV: A Practical Approach, Mammalian Systems., Glover and Hanes, eds., pp. 1-41). In the preferred embodiment the DNA is introduced into the host cells using lipofection. See, Andreason, (1993) J. Tiss. Cult. Meth., 15:56-62, for a review of electroporation techniques useful for practicing the instantly claimed invention. Gene amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA (Thomas, (1980) Proc. Natl. Acad. Sci. USA, 77:5201-5205), dot blotting (DNA or RNA analysis), RT-PCR or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Various labels may be employed in constructing probes, most commonly radioisotopes, particularly ³ P. However, other techniques may also be employed, such as using biotin-modified nucleotides for introduction into a polynucleotide. The biotin then serves as the site for binding to avidin or antibodies, which may be labeled with a wide variety of labels, such as radionuclides, fluorescens, enzymes, or the like. Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.

For the production of glycoproteins, production by growing the host cells of the present invention under a variety of cell culture conditions is typical. For instance, cell culture procedures for the large or small scale production of proteins are potentially useful within the context of the present invention. Procedures including, but not limited to, a fluidized bed bioreactor, hollow fiber bioreactor, roller bottle culture, or stirred tank bioreactor system may be used, in the later two systems, with or without microcarriers, and operated alternatively in a batch, fed-batch, or continuous mode.

Following the glycoprotein production phase, the polypeptide of interest is recovered from the culture medium using techniques which are well established in the art. The polypeptide of interest preferably is recovered from the culture medium as a secreted polypeptide, although it also may be recovered from cell lysates.

As a first step, the culture medium or lysate is centrifuged to remove particulate cell debris. The polypeptide thereafter is purified from contaminant soluble proteins and polypeptides, with the following procedures being exemplary of suitable purification procedures: by fractionation on immunoaffinity or ion- exchange columns; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS- PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; and protein A Sepharose columns to remove contaminants such as IgG. A protease inhibitor such as phenyl methyl sulfonyl fluoride (PMSF) also may be useful to inhibit proteolytic degradation during purification.

The complex carbohydrate portion of the glycoprotein produced by the processes of the present invention may be readily analyzed if desired, by conventional techniques of carbohydrate analysis to confirm the oligosaccharide content of the glycoprotein. Thus, for example, techniques such as lectin blotting or monosaccharide analysis, well-known in the art, reveal proportions of terminal mannose, N-acetylglucosarnine, sialic acid or other sugars such as galactose.

The resulting carbohydrates can be analyzed by any method known in the art including those methods described herein. Several methods are known in the art for glycosylation analysis and are useful in the context of the present invention. Such methods provide information regarding the identity and the composition of the oligosaccharide attached to the peptide. Methods for carbohydrate analysis useful in the present invention include but are not limited to capillary electrophoresis (Rassi and Nashabeh, High Performance Capillary Electrophoresis of Carbohydrates and Glycoconjugates, In, Carbohydrate Analysis (Z. El Rassi, ed.) (1995) 58:267-360), fluorophore-assisted carbohydrate electrophoreses (Starr et al., (1996) J. Chrom. A 720:295-321), high pH anion exchange chromatography pulsed amperometric detection (HPAEC PAD) (Lee (1996) J. Chrom. A 720: 137-151), matrix assisted laser desorption/ionization time of fight mass spectrometry (Harvey (1996) J. Chrom. A 720:429-447; Papac et al., (1996) Anal. Chem. 68:3215-3223; Field et al. (1996) Anal. Biochem. 239:92-98; Hooker et al., (1995) Biotechnology and Bioeng. 48:639-648), high performance liquid chromatogrphy (El Rassi (1995) Reversed phase and hydrophobic interaction chromatogrphy of carbohydrates and glycoconjugates. In, Carbohydrate Analysis (Z. El Rassi, ed.) 58:267-360), and electrospray ionization mass spectrometry Roberts G.D. (1995) Anal. Chem. 67:3613-3625).

Neutral and amino-sugars can be determined by high performance anion- exchange chromatography combined with pulsed amperometric detection (HPAE- PAD Carbohydrate System, Dionex Corp.). For instance, sugars can be released by hydrolysis in 20% (v/v) trifluoroacetic acid at 100EC for 6 h. Hydrolysates - are then dried by lyophilization or with a Speed- Vac (Savant Instruments). Residues are then dissolved in 1 % sodium acetate trihydrate solution and analyzed on a HPLC-AS6 column as described by Anumula et al. (Anal. Biochem. 195:269-280 (1991). Alternatively, immunoblot carbohydrate analysis may be performed.

According to this procedure protein-bound carbohydrates are detected using a commercial glycan detection system (Boehringer) which is based on the oxidative immunoblot procedure described by Haselbeck and Hosel (Haselbeck et al. (1990) Glycoconjugate J., 7:63). The staining protocol recommended by the manufacturer is followed except that the protein is transferred to a polyvinylidene difluoride membrane instead of nitrocellulose membrane and the blocking buffers contained 5% bovine serum albumin in 10 mM tris buffer, pH 7.4 with 0.9% sodium chloride. Detection is made with anti-digoxigenin antibodies linked with an alkaline phosphate conjugate (Boehringer), 1: 1000 dilution in tris buffered saline using the phosphatase substrates, 4-nitroblue tetrazolium chloride, 0.03% (w/v) and 5-bromo-4 chloro-3-indoyl-phosphate 0.03% (w/v) in 100 mM tris buffer, pH 9.5, containing 100 mM sodium chloride and 50 mM magnesium chloride. The protein bands containing carbohydrate are usually visualized in about 10 to 15 rnin. The carbohydrate may also be analyzed by digestion with peptide-N- glycosidase F. According to this procedure the residue is suspended in 14 ml of a buffer containing 0.18% SDS, 18 mM beta-mercaptoethanol, 90 mM phosphate, 3.6 mM EDTA, at pH 8.6, and heated at 100 EC for 3 minutes. After cooling to room temperature, the sample is divided into two equal parts. One aliquot is not treated further and serves as a control. The second fraction is adjusted to about 1 % NP-40 detergent followed by 0.2 units of peptide-N-glycosidase F

(Boehringer). Both samples are warmed at 37° C for 2 hr and then analyzed by SDS-polyacrylamide gel electrophoresis.

The following examples are provided to illustrate the invention only, and should not be construed as limiting the scope of the invention. All literature citations herein are expressly incorporated by reference.

EXAMPLES

Example 1 N-Acetyl Mannosamine (ManNac) increases intracellular CMP-NANA and Glycoprotein Sialylation

Chinese hamster ovary (CHO) cells were incubated in the presence of 20 mM ManNac, and then analyzed for intracellular CMP-NANA. The data are shown in Figures 2 and 3. In Figure 2, the data demonstrate boosting of intracellular CMP-NANA pool by feeding the culture ManNac. Figure 3 demonstrates a shift in the sialylation pattern seen. ManNac treatment resulted in enhanced terminal sialic acid while terminal galactose was diminished.

The data demonstrate that when the intracellular sialic acid pool (CMP- NANA) is increased, for example, by precursor feeding, a desired glycosylation pattern (e.g., terminal sialyation) is achieved. Example 2 Cloning of human and mutant Epimerase

Cloning and Transfection The DNA fragment of 2,169 bp containing the full-length coding sequence of human UDP-GlcNAc 2-epimerase / ManNAc kinase was amplified by PCR using the cDNA library constructed from human small intestine. PfuTurbo DNA polymerase (Stratagene, La Jolla, CA) was used for the polymerization reaction. PCR-amplified DNA fragment was gel-purified and cloned into MPSV vector. DNA sequencing was carried out using ABI Prism BigDye Terminator Cycle Sequencing kit in the Applied Biosystems 310 Genetic Analyzer. The nucleotide sequence has been submitted to the GenBank/NCBI with access number AF155663.

The single mutant (SM, ²⁶⁶R -> W) and double mutant (DM, ²⁶³R - > L and ²⁶⁶R-> W) were introduced using QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jalla, CA). Plasmid DNA (10-50 mg) was transfected into CHO cells by LipofectAMINE PLUS reagent (GIBCO BRL, Rockville, MD). Clones were selected by growth on puromycin (10 mg/ml).

Glycoprotein production

CHO DP12 cells co-expressing TNFrlgG and human UDP-GlcNAc 2- epimerase / ManNAc kinase mutant (SM) was established. TNFrlgG producing cells were seeded at 4xl0⁶ cells per ml in spinner cultures and incubated at 31°C and 6 mM butyrate for 6 days. The cell culture supernatant was harvested and TNFrlgG was recovered by proteinA immunoaffinity chromatography and N- linked glycans were analyzed by MALDI mass spectrometry (Papac et al., 1996). The sialic acid content of TNFrlgG was determined using the method from Anumula, 1995.

Results

Intracelluar concentration of CMP-sialic acid Mock transfected CHO cells and CHO cells overexpressing a mutated form of UDP-GlcNAc 2-epimerase / ManNAc kinase (SM) were extracted using a perchloric extraction method similar to the one described by Ryll and Wagner, 1991. The CMP-sialic acid concentration was measured using a reversed phase HPLC method similar to the one described by Ryll and Wagner, 1991. Chromatograms are shown in Figure 4. The CMP-sialic acid concentration in cell extracts was normalized to the NAD concentration in order to compensate for different numbers of cells extracted. Figure 4 shows a HPLC chromatogram of intracellular nucleotides and nucleotide sugars from a control and a epimerase overexpressing cell line, Figure 5 shows the increase in intracellular free sialic acid. As can be seen from Figures 4 and 5, overexpression of the mutated enzyme does result in an approximately 20 fold increase in intracellular CMP-sialic acid.

Example 3 Overexpression of mutant Epimerase

Increases Intracellular CMP-NANA and glycoprotein sialylation

CHO cells expressing TNFrlgG and transfected with the mutated cDNA coding for human UDP-GlcNAc 2-epimerase / ManNAc kinase (SM) were incubated at 31° C in the presence of 6 mM sodium butyrate for 6 days in spinner flasks. TNFrlgG was purified using ProteinA immunoaffinity chromatography and N-linked gly cans were analyzed by MALDI mass spectrometry (Papac et al., 1996). The sialic acid content of TNFrlgG was determined using the method from Anumula, 1995. As can be seen in Figures 6 and 7, the transfection with UDP- GlcNAc 2-epimerase / ManNAc kinase (SM) resulted in an increase in the sialic acid content of TNFrlgG by 0.5 mol / mol with as corresponding increase of gly cans terminated with sialic acid as compared to the mock transfected control. Figures 6A and 6B demonstrate increased degree of sialylation of acidic glycans on TNFr-IgG produced in CHO cells expressing mutated Epimerase. Figure 7 graphically shows the relative distribution of antennae terminated with GlcNAc, galactose or sialic acid as well as mol per mol content of sialic acid. References

Anumula, K.R. 1995. Rapid Quantitative Determination of Sialic Acids in Glycoproteins by High -Performance Liquid Chromatography with a Sensitive Fluorescence Detection. Anal. Biochem., 230, 24-30

Seppala, R., Lehto, V.-P., Gahl, W.A. 1999. Mutations in the human UDP-N- acetylglucosamine 2-epimerase gene define the disease sialuria and the allosteric site of the enzyme. Am. J. Hum. Genet. 64: 1563-1569

Gu, X., Wang, D.I.C. 1998. Improvement of interferon-g sialylation in Chinese hamster ovary cell culture by feeding of N-acetylmannosamine. Biotechnol. Bioeng., 58:642-648

Papac, D., Wong, A., Jones, A. 1996. Analysis of acidic oligosaccharides and glycopeptides by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Anal. Chem., 68: 3215-3223.

Ryll, T., Wagner, R. 1991. Improved ion-pair high-performance liquid chroma- tographic method for the quantification of a wide variety of nucleotides and sugar- nucleotides in animal cells. J. Chromatography, 570, 77 - 88

Feeding N-acetyl Mannosamine (ManNAc) increases the CMP-sialic acid concentraion and enhanced sialylation of a recombinant glycoprotein.

Cloning of UDP-GlcNAc 2-epimerase / ManNAc kinase - first purification and cloning of rat gene Hinderlich, S., Staesche, R., Zeitler, R., Reutter, W. 1997. A bifunctional enzyme catalyses the first two steps in N-acetylneuraminic acid biosynthesis of rat liver. Purification and characterization of UDP-GlcNAc 2-epimerase / ManNAc kinase. JBC, 272: 24313-24318

Staesche, R., Hinderlich, S., Weise, C, Effertz, K., Lucka, L., Moormann, P., Reutter, W. 1997. A bifunctional enzyme catalyses the first two steps in N- acetylneurarninic acid biosynthesis of rat liver. Molecular cloning and functional expression of UDP-GlcNAc 2-epimerase / ManNAc kinase. JBC, 272: 24319- 24324

- cloning gene of sialuria patients

Other literature around UDP-GlcNAc 2-epimerase I ManNAc kinase

Keppler, O.T., Hinderlich, S., Langer, J., Schwartz- Albiez, R., Reutter, w., Pawlita, M. 1999. UDP-GlcNAc 2-epimerase: A regulator of cell surface sialylation. Science, 284: 1372-1376

Hinderlich, S., Noehring, S., Weise, C, Franke, P., Staesche, R., Reutter, W. 1998. Purification and characterization of N-acetylglucosamine kinase from rat liver Comparison with UDP-N-acetylglucosamone 2-epimerase / N- acetyhnannosamine kinase. Eur. J. Biochem., 252: 133-139

Sialuria related literature

Thomas, G.H., Reynolds, L.W., Miller, C.S. 1985. Overproduction of N- acetymeuraminic acid (sialic acid) by sialuria fribroblasts. Pediatric Research, 19: 451-455 Thomas, G.H., Scocca, J., Miller, C.S., Reynolds, L. 1989. Evidence for non- lysosomal storage of N-acetylneurarniniz acid (sialic acid) in sialuria fibroblasts. Clinical Genetics, 36: 242-249

Krasnewich, D.M., Tietze, F., Krause, W., Prezlaff, R., Wenger, D.A., Diwadkar, V., Gahl, W.A. 1993. Clinical and biochemical studies in an american child with sialuria. Biochemical Medicine and Metabolic Biology, 49: 90-96

Weiss, P., Tietze, F., Gahl, W.A., Seppala, R., Ashwell, G. 1989. Identification of the metabolic defect in sialuria. JBC, 264 : 17635-17636

Seppala, R., Tietze, F., Krasnewich, D., Weiss, P., Ashwell, G., Barsh, G., Thomas, G.H., Packman, S., Gahl, W.A. 1991. Sialic acid metabolism in sialuria fibroblasts. JBC, 266: 7456-7461

Wilken, B., Don, N., Greenaway, R., Hammond, J., Sosula, L. 1987. Sialuria: a second case. J. Inner. Metab. Dis., 10: 97-102

Background literature Gahl, W.A., Krasnewich, D.M., Williams, J.C. 1996. Sialidoses. Handbook of Clinical Neurology, Vol. 22, Chapter 18: 353-375

Thomas, G.H., Scocca, J., Miller, C.S., Reynolds, L.W. 1985. Accumulation of N-acetylneuraminic acid (sialic acid) in human fibroblasts cultured in the presence of N-acetylmamiosmamine. Biochimica and Biophysica Acta, 846: 37-43

Claims

WE CLAIM:

1. A process for enhancing the steady state concentration of CMP-sialic acid in a cell, comprising: introducing into the cell a nucleic acid sequence encoding a modified UDP-

GlcNac 2-epimerase, wherem the epimerase has been modified to reduce binding by sialic acid.

2. A process for the production of glycoprotein in a eucaryotic cell, comprising: expressing in the cell a first nucleic acid sequence encoding a modified UDP-

GlcNac 2-epimerase, wherein the epimerase has been modified to reduce binding by sialic acid; and expressing in the cell a second nucleic acid sequence encoding the glycoprotein.

3. A process for enhancing sialylation of glycoproteins, comprising: expressing in the cell a first nucleic acid sequence encoding a modified UDP- GlcNac 2-epimerase, wherein the epimerase has been modified to reduce binding by sialic acid; and expressing in the cell a second nucleic acid sequence encoding the glycoprotein.

4. The process of claims 1,2, or 3, wherein the epimerase is modified in the region of amino acids 260-270 to replace one or more arginine residues with any amino acid but arginine.

5. The process of claim 3, wherein the epimerase is modified to substitute one or more of Arg263 and Arg266 with any amino acid but arginine.

6. The process of claim 4, wherein said epimerase contains one or more substitution: Arg266Gln; Arg266Trp; or Arg263Leu.

7. The process of claim 4, wherein said epimerase has the amino acid sequence of Seq.ID. No: 3, 4, 5, 6 or 7.

8. A host cell for the production of glycoprotein comprising: a first nucleic acid sequence encoding the glycoprotein; and a second nucleic acid sequence encoding a modified UDP-GlcNac 2- epimerase, wherein the epimerase has been modified to reduce binding by sialic acid.

9. The host cell of claim 8, wherein the second nucleic acid sequence comprises SEQ ID No: 3, 4, 5, 6 or 7.

10. The host cell of claim 8, wherein said epimerase contains one or more mutation at amino acid residues 263 or 266.

11. A process for enhancing the steady state concentration of CMP-sialic acid in a cell, comprising: introducing into the cell a nucleic acid sequence encoding UDP-GlcNac 2- epimerase, wherein the UDP-GlcNac 2-epimerase activity is enhanced compared to host cells.

12. A process for enhancing sialylation of glycoproteins, comprising: expressing in the cell a first nucleic acid sequence encoding a UDP-GlcNac 2- epimerase, wherein the epimerase activity is enhanced compared to a host cell; and expressing in the cell a second nucleic acid sequence encoding the glycoprotein.

13. A host cell for the production of glycoprotein comprising: a first nucleic acid sequence encoding the glycoprotein; and a second nucleic acid sequence encoding a UDP-GlcNac 2-epimerase, wherein the epimerase activity is enhanced compared to a host cell.

14. A process for enhancing the steady state concentration of CMP-sialic acid in a cell, comprising: introducing into the cell a nucleic acid sequence encoding UDP-GlcNac 2- epimerase.