MXPA97009452A - Process to control the sialilation of proteins produced by cultivation of mamife cells - Google Patents

Abstract

The present invention relates to a novel process for the preparation of glycoproteins by culture of mammalian cells wherein the content of sialic acid of the produced glycoprotein is controlled on a wide scale of values by manipulating the cell culture environment. The invention provides processes in which the content of sialic acid of the glycoprotein is modified by changes in cell culture parameters that affect the specific productivity of cells. Preferred embodiments of the invention include cell culture processes in the osmolality of the cell culture and is controlled as well as the concentration of a transcription enhancer during the reproduction phase of the cell culture. The invention also provides novel preparations of tumor necrosis factor 1 GI and its uses in the treatment of inflammatory or immune related disorders.

Description

Process for Controlling Sialylation of Proteins Produced by Culture of Mammalian Cells Field of the Invention This invention relates to processes for controlling the sialic acid content of glycoproteins produced in mammalian cell cultures. The invention provides processes to increase and decrease the sialic acid content of glycoproteins produced by cultures of mammalian cells. The invention furthermore relates to a process for producing tumor necrosis factor receptor (TNFR) -inmunoglobulin (Ig) chimeras as well as novel preparations of TNFR1-IgG? and its uses in the diagnosis and treatment of various inflammatory and immune disorders. Description of Related Art The differences in glycosylation patterns of recombinantly produced glycoproteins have recently been the subject of much attention in the scientific community as recombinant proteins produced as a probable prophylactic and therapeutic clinical approach. The oligosaccharide side chains of the glycoproteins affect the function of the protein (Witwer A., and Howard, SC (1990) Biochem. 29-4175-4180) and the intramolecular interaction between the portions of the glycoprotein resulting from the conformation and the 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, RB, ( 1991) Curr. Op. Struct. Biol., 1: 750-754). Oligosaccharides may also serve to direct a given polypeptide to certain structures based on specific cellular carbohydrate receptors (Bevilacque, MP and Nelson, R M., (1993) J. Clin. Invest. 91: 379-387; Nelson, RM and others (1993) J. Clin Invest 91: 1157-1166, Norgard, KE et al. (1993) Proc. Nati Acad. Sci. USA 90: 1068-1072; Imai, Y. And others (199) Nature 361,555- 557) The terminal sialic acid component of the glycoprotein oligosaccharide side chain affects absorption, serum half-life, and serum elimination, as well as the physical, chemical and immunogenic properties of the glycoprotein (Parekh, RB, supra, Varki , A, (1993) Glycobiology 397-100 Paulson, J (1989), TIBS, 14: 272-276, Goochee, et al., (1991) Biotechnology 9.1347-1355, Kobata, A (1992) Eur. J. Biochem. 209.483-501) It is therefore important to maintain the sialic acid content of the glycoproteins, particularly those proteins that are intended in using as therapeutics. Much attention has been paid to factors affecting glycosylation during production of recombinant protein such as growth mode (adherent or suspension), fetal bovine serum in medium formulation, culture density, oxygenation, pH, purification schemes and the like (Werner, RY Noe, W (1993), Drug Res 43 1134-1249, Hayter et al. (1992) Biotech And Bioeng 39327-335, Borys et al. (1994) Biotech, and Bioeng 43505-514, Borys et al. (1993) Biotechnology 11 720-724, Heapn et al., (1989) J Cell Biol 108.339353, Goochee et al., In Frontieres in Bioprocessing II, Todd et al., Eds (1992) American Chemical Society pages 199-240; NO 5,096,816; Chotigeat, W., (1994) Citotech., 15: 217-221). Several groups have investigated the process parameters surrounding the production of recombinant proteins and especially the effect of the composition of the medium on the production of recombinant proteins (Park et al., (1992) Biotech, Bioeng. 40686-696: Cox and McCIure, (1993) In Vitro, 19: 1-6, Mizutam et al., (1992) Biochem, Biophys Res Comm 187: 664-669, Le Gros et al. (1985) Lumph Res. 4 (3) .221-227). The addition of alkanoic acids such as butyric acid is known to effect the temporary expression of foreign DNA in recombinant cell culture (Prasad and Sinha, (1976) In Vitro, 12: 125-132, Japanese Patent Application No. 62-48935, Application Japanese Patent No. 55-150440, Klehr et al., (1992) Biochem 31 3322-3329 Gorman and Howard, (1983) Nucleic acid Res 11 7631-7648) However, sodium butyrate has a scale of effects on the expression of genes through several cell lines and compositions of the medium (D'Anna et al., (1980) Biochem 192656-2671, Hagopian, HK, (1977) Cell 12,855- 860) Cell 12855-860) and protein production (Milhard (1980) J Cell Physiol 104 163-170 UK Patent Application No GB 2 122 207 A) suggest that butyrate can modify gene expression (Yuan et al. (1985) J Biol Chem 3778-3783) or inhibit the expression of certain genes (Yuan et al., Supra) European Patent No. 0 239 292 B1, describes a process for the improved production of protein in the presence of an alkanoic acid or a salt thereof such as butyric acid. However, the publication provides little guidance for selecting the appropriate concentrations of the additive and also does not direct the effect of the additive on protein glycosylation. Others have reported that the addition of low levels (0-1.5 mM) of sodium butyrate to the cell culture production medium to increase specific cell productivity leads to a concomitant increase in acid glycoforms (corresponding to the content of sialic acid). increased) of the recombinant protein produced (Chotigeat, et al. (1994) Cytotech., 15: 217-221). Several groups have looked for the effects of osmolarity of cell growth and polypeptide production (Ozturk and Palsson (1991) Biotech, And Bioeng., 37: 989-993; Stubblefield et al., (1960) Cancer Research. 20: 1646-1655; García-Pérez et al. (1989) Journal of biological Chemistry, 264 (28): 16815-16821; Miner and others (1981) Invasión Metastasis. 1: 158-174; GB 2,251,249; EP 481,791; Patent of E.U.A. No. 5,151,359; Patent of E.U.A. No. 4,724,206; Patent of E.U.A. No. 5,122,469; and WO 89/04867). Several osmolarity scales have been proposed for cell development or polypeptide production. Generally, the osmolarity of the cell culture medium is increased via the addition of NaCl or amino acids. Environmental stresses such as increased salt concentrations lead, in some cases, to increased cell product production. The notion that increased expression of mammalian protein products can be achieved in mammalian cell cultures through solute tension, e.g., the addition of salt, lactic acid, ammonia to the culture medium has been reported. (International Publication No. WO 89/04867). These tensions generally inhibit growth but favor the specific productivity of the cells. Others have addressed the effect of glucose concentration on cell development and / or production of polypeptides in recombinant cell culture. See, for example, Park et al., (1992) Biotechnology and Bioengineering, 40: 686-696; Huang et al., (1991) Journal of Biotechnology, 18: 161-162; EP 387,840; Reuveny et al. (1986) Journal of Immunological Methods, 86: 53-59; fine and others (1976) in Vitro; 12 (10): 693-7901; Dircks et al. (1987) Exp. Eye Res., 44: 951-958; Mizutani et al. (1992) Biochemical and Biophysical Research Communications, 187 (2): 664-669; Sugiura, (1992) Biotechnology and Bioengineering, 39: 953-959; WO 88/01643; Graf et al., (1989) DECHEMA Biotechnol. Conf., 3: 615-618; Japanese Patent Application No. JP 1-101882; Patent of E.U.A. No. 3,926,723; WO 87/00195; and Fleishaker, Jr., Ph.D. Thesis, Massachusetts Institute of Technology, pgs. 196-229 (June 1982). However, previous studies have not studied the effect of various process parameters on the sialic acid content of the mature protein, a factor in glycoprotein production that accounts for clinical success. The present invention provides a process for controlling the sialic acid content of glycoproteins produced by mammalian cell cultures.

SUMMARY OF THE INVENTION The present inventors have discovered that certain parameters of the mammalian cell culture process affect cell-specific productivity as well as the degree and type of glycosylation of the proteins produced. More particularly, the present inventors have found that certain factors that increase cell-specific productivity have an inverse effect on the sialic acid content of the protein produced. The present inventors have therefore developed various cell culture processes to enrich the particular glycoforms of glycoproteins produced in mammalian cell cultures. Accordingly, the invention provides a process for controlling the sialic acid content of a glycoprotein produced by mammalian cell culture. According to this aspect of the invention, the variation of the production regimen of the glycoprotein in the production phase of the cell culture leads to variations in the sialic acid content of the mature glycoprotein. More particularly, an increase in cell-specific productivity during the glycoprotein production phase results in a decrease in the sialic acid content of the mature protein. Conversely, a decrease in cell-specific productivity results in an increase in sialic acid content in the mature protein. The present invention provides, in a particular embodiment, for varying the cell-specific productivity of a mammalian host cell during the production phase of mammalian cell culture protein by controlling factors that affect cell-specific productivity. According to one aspect of the invention, the concentration of factors that increase DNA transcription is controlled. In another embodiment, cell-specific productivity is controlled by maintaining the osmolality of the cell culture within certain ranges. According to the invention, any of the above parameters is controlled, alone or in combination, to affect the sialic acid content of mature glycoproteins. In a particular embodiment of the present invention, the factor that increases DNA transcription is an alkanoic acid or salt thereof such as sodium butyrate at a concentration of about 0.1 mM to about 20 mM. According to a second aspect of the invention, the osmolality of the cell culture is maintained between about 250-500 mOsm. In a further aspect, the temperature of the cell culture is controlled between about 30 ° C and 37 ° C. In a preferred embodiment, the invention provides a process for increasing the sialic acid content of the mature glycoprotein produced by the mammalian cell culture comprising maintaining a lower cell-specific productivity by controlling any or all of the parameters of the process identified above, optionally together with other parameters known in the art. In accordance with this aspect of the present invention, culturing the host cell at a concentration of the alkanoic acid or salt thereof from about 0.1mM to about 6mM, and optionally together with maintaining osmolality of the cell culture at about 300 -450 mOsm produces a protein with an increased sialic acid content. In a further preferred embodiment, the invention provides a process for decreasing the sialic acid content of the mature glycoprotein produced by mammalian cell culture comprising increasing the specific productivity of the cell culture. The cell-specific productivity is increased, in a preferred embodiment, by providing a cell culture process which comprises any of, culturing the host cell at a concentration of an alkanoic acid or salt thereof of about 6 mM to about 12 mM; and maintaining the osmolality of the cell culture at about 450-600 mOsm. The invention also provides, in a particular modality, a cell culture process with three phases of cell culture. The invention therefore provides a process for controlling the sialic acid content of a glycoprotein produced by the mammalian cell culture comprising the steps of culturing a host cell that expresses the protein in a growth phase for a period and under said conditions that cell growth is maximized. In accordance with this aspect of the present invention, the growth phase is followed by a transition phase in which cell culture parameters are selected and coupled for the desired sialic acid content of the mature glycoprotein. The transition phase is followed by a production phase of the cell culture in which the parameters selected in the transition phase are maintained and the glycoprotein product is produced and collected. Varying the cell-specific productivity of the cell culture production phase by adding an alkanoic acid or a salt thereof to the cell culture at a concentration of about 0.1 mM to about 20 mM and coupling an osmolality of the cell culture to approximately between 250 and 600 mOsm, optionally in combination with one another during the transition phase produces a protein with different amounts of sialic acid. In a further preferred embodiment, the present invention provides a process for controlling the amount of sialic acid present in a soluble chimeric protein receptor of tumor necrosis factor type 1 (RFNT1) - immunoglobulin G (I gG 1). The present inventors have discovered that, under certain production conditions, the novel glycoform preparations of RFNT1-IgG which exhibit the desirable properties of prolonged blood clearance while retaining significant functional activity can be obtained. A long functional half-life allows for simplified bolus dose administration and contributes to the in vivo potency of the produced glycoprotein allowing lower dosage forms of the glycoprotein.

In accordance with this aspect of the present invention, a glycoprotein molecule of RFNT1-lgd is produced which contains increased sialic acid residues. The cell culture parameters for the production phase of the RFNT1-IgD are selected to obtain the desired sialic acid content. In a preferred embodiment, sodium butyrate is present in the production phase at a concentration of about 0.1 to about 6 mM and the osmolality is maintained at about 300-450 mOsm. In a more preferred aspect, the concentration of sodium butyrate is about 1 mM and the osmolality is maintained at about 350-400 mOsm. In yet another embodiment, the present invention provides a glycoprotein preparation of RFNT1-IgD produced by the process of the present invention. In accordance with this aspect of the invention, a preparation comprising RFNT1-lgd is provided in which the pl scale of the preparation is between about 5.5 and 7.5. In addition, a preparation of RFNT1-lgG is provided? which has a molar ratio of sialic acid to protein of from about 4 to about 7 and especially from about 5 to about 6. In yet another aspect, the RFNT1-lgd preparation has from about 1 to about 2 moles of residues of N-acetylglucosamine exposed per mole of protein. In a further aspect, the preparation has a molar ratio of sialic acid to N-acetylglucosamine from about 0.35 to about 0.5 and more preferably from about 0.4 to about 0.45. The present invention also provides a therapeutic composition comprising the above preparation useful in the treatment of pathological conditions mediated by TNF. Description of the drawings Figure 1A and Figure 1B. Figures 1A and 1B show the method used to calculate the specific productivity of a normal cell culture process in the production phase. The specific productivity can be expressed as a function of viable cell counts (viable cell days) as shown in Figure 1A; or volume of packed cells (VCE) as shown in Figure 1B. The specific production regime is given with the scale for a 90% confidence interval. Figure 2A and Figure 2B. Figures 2A and 2B show the correlation between the specific productivity based on days of viable cells (Figure 2A) and volume of packed cells (VCE) (Figure 2B) during the production phase and the sialic acid content (NANA) of the product collected . The values are shown for 9 different processes (A-1). The data points represent independent production process as described in Table 1. Detailed Description of the Invention 1- Definitions The carbohydrate sentences of the present invention will be described with reference to the nomenclature commonly used for the description of oligosaccharides. A review of carbohydrate chemistry using this nomenclature is found in Hubbard and Ivatt (1981) Ann. Rev. Biochem. 50: 555-583. This nomenclature includes, for example, Man, which represents mannose, GIcNAc, which represents 2-N-acetylglucosamine; Gal representing galactose and Glc, which represents glucose. Sialic acids are described with reference to the handwritten notation NeuNAc, for 5-N-acetylneuraminic acid and NeuNGc for 5-glycolylneuraminic acid (J. Biol. Chem, 1982, 257; 3347; J. Biol. Chem., 1982, 257; 3352). "Osmolalildad" is a measure of the osmotic pressure of solute particles dissolved in an aqueous solution. The solute particles include both ions and non-ionized molecules. The osmolality is expressed as the concentration of osmotically active particles (ie, osmoles) dissolved in 1 kg of solution (1 mOsm / kg H2O at 38 ° C is equivalent for an osmotic pressure of 19 mm Hg). "Osmolarity" in contrast, refers to the number of dissolved solute particles in 1 liter of solution. When used in the present, the abbreviation "oOsm" means a "milliosmole / kg solution". As used herein, "glycoprotein" generally refers to peptides and proteins having more than about ten amino acids and at least one side chain of oligosaccharides. The glycoproteins may be homologous to the host cell, or preferably, they are heterologous, ie, foreign to the host cell being used, such as a human protein produced by a Chinese hamster ovary cell. Preferably, the mammalian glycoproteins (glycoproteins that were originally derived from a mammalian organism) are used, more preferably, those that are secreted directly into the medium. Examples of mammalian glycoproteins include molecules such as cytokines and their receptors, as well as chimeric proteins comprising cytokines or their receptors, including, for example, alpha and beta tumor necrosis factor, their receptors (RFNT-1; EP 417,563 published March 20, 1991; and RFNT-1, EP 417,014 published March 20, 1991) and its derivatives; renin; a growth hormone, including human growth hormone and bovine growth hormone; Growth hormone releasing factor, Parathyroid hormone; thyroid stimulating hormone; lipoproteins, alpha-1-antitrypsin; insulin A chain; B chain of insulin; proinsulin; follicle stimulating hormone, Calcitonin; luteinizing hormone; glucagon; clotting factors such as factor VI II C, factor IX, tissue factor and von Willebrands factor; anticoagulant factors such as Protein C; atrial natriuretic factor; surfactant agent of lungs; a plasminogen activator, such as urokinase or human murine or tissue-type plasminogen activator (t-AP); bombesin; thrombin; hemopoietic growth factor; enkephalinase; RANTES (regulated in activation expressed and normally secreted in T cells); inflammatory protein of human macrophages (PIM-1-alpha); a serum albumin such as human serum albumin; Mulerian inhibitory substance; A chain of relaxin; B chain of relaxin; prorelaxin; peptide associated with mouse gonadotropin; a microbial protein, such as beta-lactamase; DNase; inhibin; ativine; vascular endothelial growth factor (VEGF); receptors for hormones or growth factors; integrin; protein A or D; rheumatoid factors; a neurotrophic factor such as bone-derived neurotrophic factor (FNDH), neurotrophin-3, -4, -5 or -6 (NT-3, NT-4, Nt-5 or NT-6) or a nerve growth factor such as FCN-ß, platelet-derived growth factor (FCDP), fibroblast growth factor such as FCFa and FCFb, epidermal growth factor (EGF), transformation growth factor (FCT) such as FCT-alpha and FCT -beta, including FCT-ß1, FCT-p2, FCT-ß3, FCTß4 or FCTßd, insulin-like growth factor I and II (FCI-I and FCI-II), des (1-3) -FCI-1 ( Brain FCI-1), insulin-like growth factor-binding proteins, CD proteins such as SD-3, CD-4, CD-8 and CD-19, eptropoietin, osteoinductive factors, immunotoxins, an orfogenetic bone protein (PMH), an interferon such as interferon-alpha, -beta, and -game, colony stimulating factors (FEC), v gr, M-FEC, GM-FEC and G-FEC, interleukins (lys), v gr, IL-1 to IL-10, superoxide dismutase, c-receptor T cells, surface membrane proteins, disintegration acceleration factor, viral antigen such as, for example, a portion of AIDS envelope, transport proteins, targeting receptors, adhesives, regulatory proteins, antibodies, chimeric proteins, such as immunoadhesins and fragments of any of the pohpeptides listed above The terms "cell culture medium" and "culture medium" refer to a nutrient solution used for growing mammalian cells that normally provide at least one component of one or more of the following categories 1) a source of energy, usually in the form of a carbohydrate such as glucose, 2) all essential amino acids, and usually the basic group of twenty amino acids plus cysteine; 3) vitamins and / or other organic compounds required at low concentrations, 4) free fatty acids; and 5) trace elements, where the trace elements are defined as inorganic compounds or elements present in nature that are normally required at very low concentrations, usually on the micromolar scale. The nutrient solution can optionally be supplemented with one or more components from any of the following categories: 1) hormones and other growth factors such as, for example, insulin, transferin, and epidermal growth factor; 2) salts and buffer solutions for pH, for example, calcium, magnesium and phosphate; 3) nucleosides and bases such as, for example, adenosine, thymidine and hypoxanthine; and 4) protein and tissue hydrolysates. The term "mammalian host cell", "host cell", "mammalian cell" and the like, refers to cell lines derived from mammals that are capable of growing and surviving when placed in any one layer culture or in suspension culture in a medium containing the appropriate nutrients and growth factors. The growth factors necessary for a particular cell line are easily determined empirically without undue experimentation, as described, for example in Mammalian Cell Culture (Mather, JP ed. Plenum Press, NY [1984]) and Barnes and Sato, (1980). Cell, 22: 649. Normally, the cells are capable of expressing and secreting large amounts of a particular glycoprotein of interest in the culture medium. Examples of suitable mammalian host cells within the context of the present invention may include Chinese hamster ovary cells / DHFR (CHO, Uriaub and Chasin, Proc. Nati, Acad. Sci. EUA, 77: 4216 [1980]); dp12.CHO cells (EP 307,247 published March 15, 1989): monkey kidney CVI line transformed by SV40 (COSA, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 3659 [1977]); baby hamster kidney cells (BHK, ATCC CCL 10); Mouse Bertoli cells (TM4, Mather, Biol. Reprod., 23: 243-251 [1980]); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587): human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mammary tumor of mice (MMT 060562. ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci., 383: 44-68 [1982]); MRC 5 cells; FS4 cells; a human hepatoma line (Hep G2).

Preferred host cells include Chinese hamster ovary cells / DHFR (CHO, Uriaub and Chasin, Proc. Nati, Acad. Sci, USA, 77: 4216 [1980]; dp12.CHO cells (EP 307,247 published on March 15, 1989) It is understood that the term "peptone" within the context of the present invention refers to a supplement of medium that is essentially hydrolyzed animal protein.The source of this protein may be animal by-products of meat houses, purified gelatin or materials for plants The protein is normally hydrolyzed using acid, heat or various enzyme preparations.The preferred peptone mixtures are, for example, "Primatone RL" and "Primatone HS", both of which are commercially available (Sheffield, England) "Cell-specific productivity," "cell-specific regimen," and the like, are used herein to refer to the specific product expression regimen, i.e., per cell or per-cell measure. sa or cellular volume. The cell-specific productivity is measured, for example, in grams of protein produced per cell per day. The specific productivity for cells can be conveniently measured according to the integral method: dP / dt = qpX, or P = qp f: Xdt where qp is the cell-specific productivity constant, X is the number of cells or cell volume, or cell mass equivalents, and dP / dt is the protein production regime. Therefore, qp can be obtained from a plot of product concentration against the time integral of viable cells (J Xdt "days of viable cells") According to this formula, when the amount of glycoprotein product produced is plotted against the days of viable cells, the tilt is equivalent to the cell-specific regimen. Viable cells can be determined by several measures, eg, biomass. O2L lactase dehydrogenase (LKDH) absorption regimen, packed cell volume or turbidity "Growth phase" of the cell culture refers to the period of exponential cell growth (the log phase) where the cells are usually divided rapidly. phase, cells are cultured for a time, usually between 1 and 4 days and under such conditions that cell growth is maximized. Determination of the growth cycle for the host cell can be determined for the particular host cell provided without undue experimentation. time and under said conditions in which cell growth is maximized "and the like, refers to those culture conditions that for a particular cell line were determined to be optimal for cell growth and division. During the growth phase, the cells they are grown in nutrient medium containing the necessary additives generally of about 30-40 ° C in a humidified controlled atmosphere so that optimal growth is achieved for the particular cell line. Cells are maintained in the growth phase for a period between one and four days usually between two to three days The "transition phase" Cell culture refers to the period during which the culture conditions for the production phase are coupled. During the transition phase, environmental factors such as cell culture temperature, osmolality of the medium and the like, are changed from growth conditions to production conditions. The "production phase" of cell culture refers to the period during which cell growth has been planted in boxes. During the production phase, logarithmic cell growth is over and protein production is primary During this period, the medium is generally supplemented to support continuous protein production and to achieve the desired ghcoprotein product. The term "expression" or "expressions" "are used herein to refer to transcription and translation that occur within a host cell. The level of expression of a product gene in a host cell can be determined based on the amount of corresponding mRNA that is present in the host cell. the cell or the amount of the protein encoded by the gene of the product that is produced by the cell. For example, the mRNA transcribed from a gene of the product is conveniently quantified by hybridization of Northern Sambrook et al., Molecular Cloning A laboratory Manual, p. 73 -757 (Cold Spring Harbor Laboratory Press, 1989). The protein encoded by a product gene can be quantified either by analyzing the biological activity of the protein or by employing assays that are independent of said activity, such as Western plot or radioimmunoanalysis using antibodies that are capable of reacting with the protein. Sambrook et al., Molecular Cloning: A laboratory Manual, p. 18.1-18.88 (Cold Spring Harbor Laboratory Press, 1989). For alkaline acid or sa! it is meant an alkanoic acid, saturated or unsaturated, straight or branched chain or salt thereof. The alkanoic acid is generally one to ten carbon atoms and preferably is three to six carbon atoms. An exemplary alkanoic acid is butyric acid and the corresponding salt is sodium butyrate. II. Cell Culture Procedures The present inventors have discovered that the factors that increase the specific cellular productivity during the production of a glycoprotein produced by mammalian cell culture, have an inverse effect on the sialic acid content of the glycoprotein produced. Since proteins expressing one or more sialic acid residues by complex oligosaccharide structure have longer elimination regimes in vivo, the rate of elimination of the produced glycoprotein can be manipulated within wide limits by the overall degree of sialylation of the preparation. . The present invention provides processes for controlling the degree of sialylation of a glycoprotein produced by mammalian cell culture. Following the methodology established herein, one is able to determine the precise parameters of the process that provide the desired sialic acid content of a glycoprotein produced by mammalian cell culture. In accordance with the present invention, a mammalian host cell is cultured to produce a recoverable glycoprotein product. The overall content of sialic acid in the glycoprotein is controlled by the control of cell culture parameters which affect the specific productivity of the cells. Factors that affect the specific productivity of cells are well known in the art and include, but are not limited to, factors that affect the number of DNA / RNA copies. factors that affect RNA, such as factors that stabilize RNA, nutrients from the medium and other supplements, the concentration of transcription enhancers, the osmolality of the culture environment, the temperature and pH of the cell culture, and the like. According to the present invention, the adjustment of these factors, alone or in combination, to increase the specific productivity, generates a protein with a decreased sialic acid content. Adjustment of these factors, alone or in combination, to decrease cell-specific productivity, generates a mature glycoprotein with an increased sialic acid content. The invention will now be described with reference to various cell culture techniques and principles, including those well known. . In accordance with the present invention, mammalian cells are cultured to produce a desired glycoprotein product. By choosing a host cell for the production of the glycoprotein within the context of the present invention it is important to recognize that different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification (v gr, glycosylation, separation) of the expressed proteins Appropriate cell lines should be chosen to ensure that desired post-translational modifications are possible. Alternatively, host cells can be modified to express a desired gene product required for specific post-translational modification. In particular, mammalian cells expressing the protein The desired enzymes should be expressed or manipulated to express particular enzymes so that under the appropriate conditions described herein the appropriate post-translational modification is present. Enzymes include those enzymes necessary for the addition and complexing of N- and O -ligated carbohydrates such as those described in Hubbard and Ivan supra for N-linked okeonates. The enzymes optionally include ohgosacapltransferase, alpha-glucosidase I alpha-glucosidase II. ER alpha (1 2) hands? Dasa. Golgi-alpha-mannosidase I, N-acetyl-glucosaminyltransferase I, Golgi-alpha-anosidase II N-acetylglucosaminyltransferase II, alpha (1 6) fucosyltransferase and β (1 4) galactosyltransferase Additionally, the host cell expresses to bind the terminal sialic acid at the specific position and ligation as part of the host cell genome. Optionally, the host cell can be made to express the appropriate sialyl transferases, for example., by transfection of the host cell with the DNA encoding the sialyltransferase. One would expect the sialyltransferases described above to add the terminal sialic acid residue to the appropriate oligosaccharide matrix structure such as Galβ1-4GlcNAc. Appropriate sialyltransferases within the context of the present invention include, but are not limited to, those sialyltransferases that catalyze the complex sialylation and branching of the N- and O-linked oligosaccharides. For the cultivation of mammalian cells expressing the desired protein and capable of adding the desired carbohydrates in the specific position and ligation, numerous culture conditions can be used paying particular attention to the host cell being cultured. Suitable culture conditions for mammalian cells are well known in the art (J. Immunol.Methods (1983) 56: 221-234) or can be easily determined by the skilled artisan (see, for example, Animal Cell Culture: A Practical Approach 2nd Ed., Rickwood, D. and Hames, BD, eds, Oxford University Press, New York (1992)) and vary according to the particular host cell selected. The mammalian cell culture of the present invention was prepared in a medium suitable for the particular cell being cultured. The commercially available medium such as Ham's f 10 (sigma). Minimum Essential Medium ([MEM], Sigma). RPMI-1640 and Dulbecco's Modified Eagle Medium ([DMEM], Sigma) are illustrative nutrient solutions. In addition, any of the means described in Ham and Wallace, (1979) Meth. Enz .. 58:44: Barnes and Sato. (1980) Anal. Biochem., 102: 255; Patents of E.U.A. Nos. 4,767,704; 4,657,866; 4,927,762; 5,122,469 or 4,560,655; International Publications Nos. WO 90/03430; and WO 87/00195; the descriptions of which are incorporated herein by reference, can be used as a culture medium. Any of these means can be supplemented as necessary with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), solutions pH regulators (such as HEPES). nucleosides (such as adenosine and thymidine). antibiotics (such as the drug Gentamycin ™). trace elements (defined as inorganic compounds usually present at final concentrations on the micromolar scale) lipids (such as linoleic acid or other fatty acids) and their appropriate vehicles and glucose or an equivalent energy source. Any of the other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. In a particular embodiment, the mammalian host cell is an OHC cell. preferably a dp12.0HC cell and a suitable medium contains a basal medium component such as a formulation based on DMEM / HAM F-12 for the composition of DMEM medium and HAM F12. see the formulations of the culture medium in the American Type Culture Collection Catalog of Cell Lines and Hybridomes. Sixth Edition, 1988, pages 346-349) (the formulation of the medium as described in the patent of E.U.A. ,122,469, are particularly appropriate) with modified concentrations of some components such as amino acids, salts, sugar and vitamins, and optionally containing glycine, hypoxanthine and thymidine; recombinant human insulin, hydrolyzed peptone, such as Primatone HS or Primatona RL (Sheffield, England) or the equivalent, a cellular protective agent, such as Pluronic F68 or the equivalent pluronic polyol, Gentamicin and trace elements. The glycoproteins of the present invention can be produced by developing cells that express the desired protein under a variety of cell culture conditions. For example, cell culture methods for large-scale or small-scale production of proteins are potentially useful within the context of the present invention. Processes that include, but are not limited to, a dized bed bioreactor, hollow fiber bioreactor, roller bottle culture or agitated tank bioreactor system can be used in the last two systems, with or without microcarriers and they can operate alternatively in a batch, feed batch, or continuous mode.

In a preferred embodiment, the cell culture of the present invention is performed in a stirred tank bioreactor system and a feed batch culture method is employed. In the preferred batch culture, the mammalian host cells and culture medium are initially supplied to a culture vessel and additional culture nutrients are fed. continuously or in discrete increments, to the crop during the crop, whether or not the cell and / or product is picked up periodically before finishing the crop. The culture of the feedlot may include, for example, a semi-continuous feedlot culture, wherein all the culture is periodically removed and replaced (including cells and medium) by fresh medium. The feedlot culture is distinguished from single-batch culture in which all components for cell culture (including cells and all nutrients of the culture) are cultured. supply to the culture vessel at the beginning of the culture process The culture of the feeding lot can also be distinguished from the perfusion culture in that the supernatant is not removed from the culture vessel during the process (in the perfusion culture, the cells are they restrict the cultivation by, v gr, filtration, encapsulation, union to the microvehicles, etc. and the culture medium is introduced and is continuously or intermittently removed from the culture vessel). In addition, the cells of the culture may be propagated according to any scheme or routine that may be suitable for the particular host cell and the particular production plan contemplated. Therefore, the present invention contemplates a one-step or multi-step culture method In a one-step culture, the host cells are inoculated into a culture environment and the processes of the present invention are employed during a single phase of cell culture production. Alternatively, a multi-stage culture is envisaged. In multi-stage cultures, cells can be grown in a number of steps or phases. For example, the cells can grow in a culture in a first step or growth phase wherein the cells, possibly removed from storage. , are inoculated in a suitable medium to promote growth and high viability. The cells can be maintained in the growth phase for a suitable period by the addition of fresh medium to the host cell culture. In accordance with a preferred aspect of the invention, cell culture conditions in feed or continuous batch are provided to increase the growth of mammalian cells in the growth phase of the cell culture. In the growth phase cells develop under conditions and for a period that are maximized for growth. The conditions of cultivation, such as temperature, pH, dissolved oxygen (dO2) and the like, are those used with the particular host and will be apparent to the ordinary expert. Generally, the pH is adjusted to a level between about 6.6 and 7.5 using either an acid (e.g., CO2), or a base (e.g., Na2CO2 or NaOH). A suitable temperature scale for culturing mammalian cells such as CHO cells is between about 30 to 38 ° C and a suitable dO2 is between 5-90% air saturation. In a particular step, the cells can be used to inoculate a stage or step of production of the cell culture. Alternatively, as described above, the production step or step can be continuous with the inoculation or growth step or step. In accordance with the present invention, the cell culture environment is controlled during the production phase of the cell culture.

In accordance with the process of the present invention, the factors that affect the specific productivity of the cell of the host cell of mammals are manipulated so that the desired sialic acid content is achieved in the resulting glycoprotein. In particular, the factors that increase the cell-specific productivity are controlled during the production phase of the cell culture process so that the resulting glycoprotein product contains the desired sialic acid content. In a preferred aspect, the production phase of the cell culture processes is preceded by a transition phase of the cell culture in which the parameters of the cell culture production phase are coupled. According to one aspect of the present invention, the concentration of a transcription enhancer such as an alkanoic acid is manipulated to affect the cell-specific productivity and hence the sialic acid content resulting from the cell-glycoprotein product of mammals The alkanoic acid may be any of a number of single chain or branched chain alkanoic acids that increase the transcription of mammalian proteins. In a preferred embodiment, the alkanoic acid is butyric acid and especially the salt thereof, butyrate. According to the present invention, the concentration of sodium butyrate is controlled in order to control the specific productivity of the cells. Sodium butyrate concentrations of between 0.1 and 20 mM are used and modified according to the particular host cell being cultured and the desired sialic acid content of the glycoprotein produced. In order to generate a protein with the desired sialic acid content, a concentration of sodium butyrate is chosen which provides the highest cell specific productivity with the most acceptable sialic acid profile. Therefore, in accordance with the concentrations of the present invention a transcriptional enhancer such as sodium butyrate are chosen to obtain the desired sialic acid content. In order to increase the sialic acid content of the mature glycoprotein, lower concentrations of the transcription enhancer are generally used. The lower concentration provides increased transcription but maintains specific productivity for the lower cell while maintaining the viability of the host cell culture. Generally, concentrations of the transcription enhancer such as sodium butyrate between about 0.1 mM and about 8 mM are used. More preferably, concentrations are preferably used between about 1.0 and 6.0 mM. In a particular modality. approximately 6 mM sodium butyrate is used. In another embodiment, about 1 mM sodium butyrate is used. In another embodiment, a glycoprotein with a decreased level of sialic acid is produced. In accordance with this aspect of the present invention, mammalian cells are cultured under conditions such that cell-specific productivity increases. In accordance with this aspect of the present invention, a concentration of alkanoic acid or other appropriate transcription enhancer is chosen such that the increased cell-specific productivity generates a protein with the desired sialic acid profile. In a preferred embodiment, the concentration of the alkanoic acid or salt thereof is between about 5 to 20 mM and more preferably between about 6 mM and 12 mM. In a particular embodiment, the concentration of sodium butyrate is about 12 mM. To determine the appropriate concentration of the transcription enhancer such as an alkanoic acid or salt thereof, reference may be made to Figure 2, as well as Table I infra in Example I. In accordance with the present invention, butyrate concentrations lower generally result in a lower cell-specific productivity. According to the invention, the sodium butyrate concentrations are chosen, they are chosen keeping in mind other parameters of the process such as the osmolality of the production phase. As discussed below, osmolality can affect cell-specific productivity. Butyrate concentrations are chosen keeping in mind the particular osmolality that will be maintained during the production phase. Alternatively, for other mammalian host cells and other glycoproteins. small test cultures and the production regimen of the glycoprotein product can be prepared. that is, the cell-specific productivity can be determined and the resulting sialic acid content can be used to prepare a similar table and figure suitable for the particular host cell being cultured, keeping in mind that decreases in cell-specific productivity leads to to increases in the sialic acid content of the produced glycoprotein The alkanoic acid or salt thereof, such as sodium butyrate and other appropriate transcription enhancer is added to the host cell culture at, or approximately, at the time it is initiated the production phase of the cell culture Conveniently a transition phase is employed during the cell culture process preceding the production phase in which cell culture conditions are coupled as discussed herein for cell-specific productivity wish and therefore the desired glycoform profile Alkalic acid An oic acid or salt thereof is added by any means known in the art. In a preferred embodiment, the sodium batch butyrate is added to the batch culture system with or without other appropriate nutrients as described herein or known to humans. experts in the field of mammalian cell culture According to the present invention the osmolality of the cell culture environment is controlled in addition to the factors observed above to regulate the degree of sialylation of the mature glycoprotein. In one embodiment, the osmolality is controlled by the In order to control the sialic acid content of the mature protein independent of other factors that affect the specific productivity of cells In another modality the osmolality is controlled in addition to controlling other factors that affect the specific productivity for cells. In a preferred embodiment, both the osmolality and the concentration of the alkanoic acid are controlled. The osmolality of the cell culture environment is controlled in order to produce the desired balance between the cell-specific productivity and the sialic acid profile of the resulting glycoprotein. Generally, cell-specific productivity increases when osmolality is increased. An osmolality that produces a protein with the desired sialic acid is chosen keeping in mind that increases in osmolality generally lead to an increase in the production rate of the particular protein. In order to decrease the production rate and increase the sialic acid content of the mature glycoprotein osmolality it is generally maintained at a lower level for the particular cell type being cultured.

For mammalian cell culture, the osmolality of the culture medium is generally about 290-330 mOsm. However, increases in osmolality generally leads to an increase in the protein production rate. An osmolality is chosen so that the production rate corresponds to the desired product profile. In order to increase the sialic acid content, the production rate is decreased and the osmolality is generally kept within a lower range keeping in mind the particular host cell being cultured. Osmolality on the scale from about 250 mOsm to about 450 mOsm is appropriate for an increased content of sialic acid More preferably, the osmolality is maintained at about 300 to 450 mOsm, in accordance with this aspect of the invention and more preferably between about 350 and 400 mOsm and even more preferably about 400 mOsm. For a lower sialic acid content, an osmolality is chosen that provides an increased production rate. According to this aspect of the invention, the osmolality is maintained at about 350-600 mOsm and preferably is between about 450-550 mOsm, according to this aspect of the invention. The skilled practitioner will recognize that the average osmolality depends on the concentration of osmotically active particles in the culture fluid and that a number of variables forming a culture medium of mammalian cells impact the osmolality. The initial osmolality of the culture medium is determined by the composition of the culture medium. The osmolality can be measured using a osmometer such as that sold by Fisher Scientific, Pittsburgh, Pennsylvania under the trade name OSMETTE for the Osmette model 2007. available from Precision Systems, Inc. Natick MA), for example In order to achieve an osmolality on the desired scale, it can be adjusted the concentration of several constituents in the culture medium The solutes that can be added to the medium or culture so as to increase the osmolality thereof include proteins, amino acid peptides, hydrolyzed animal proteins such as peptones, non-metabolized polymers, vitamins, ions, salts, sugars, metabolites, organic acids, liquids and the like. In one embodiment, the osmolality is controlled by the addition of a peptone to the cell culture together with other components of the culture medium during a batch culture procedure. In accordance with the present invention, the osmolality is maintained or adjusted to the desired scale via the addition of, for example, a formulation of basal medium including amino acids, various salts (e.g., NaCl) in addition to a peptone. In a preferred embodiment, the culture medium is supplemented with, for example, a basal culture medium containing excess amino acids (see, e.g., the "Super" medium of U.S. Patent No. 5,122,469), glucose and a peptone. However, it will be appreciated that the concentration (s) of other constituents in the culture medium can be modified in order to achieve an osmolality scale as stated above. By controlling either intermittently or continuously the concentration of glucose (the primary energy source) for example, in the culture medium by cultivation, the osmolality of the medium can be maintained at approximately the desired desirable range. The control of the glucose concentration serves to provide adequate carbon source to the cells and simultaneously control the production of lactic acid by the host cells. This is advantageous since it limits the decrease in pH in the culture medium which necessitates the addition of a neutralizer (e.g., a base such as Na 2 CO 3 or NaOH) which causes the osmolality to rise. The medium can be supplemented to maintain the osmolality within the appropriate margins according to the scheme that is being used to maintain the cell culture. In a preferred embodiment, the culture system is a system of batch culture of feeding and the medium is supplemented in batch in a feeding during the production phase of the cell culture. Additionally, the medium can be supplemented during the production phase as described above. Alternatively, intermittent off-line sampling of the culture medium can be carried out. The osmolality of the culture medium can be modified by the modulation of a feeding solution as required. It will be understood by the skilled person that the cell culture methods of the present invention are selected to achieve the desired level of sialylation of the protein produced. Process parameters in addition to those described herein that influence the degree of sialylation, include oxygen level and glucose level. The density, culture, time and storage conditions such as temperature also influence sialylation. It is understood that the present invention includes those process parameters that are also more suitable for increased sialylation. lll. Recovery of the Glycoprotein Following the production phase of the polypeptide, the polypeptide of interest is recovered from the culture medium using techniques that are well established in the art. The polypeptide of interest is preferably recovered from the culture medium as a secreted polypeptide, although it can also be recovered from the host cell lysates. As a first step, the culture medium or lysate is centrifuged to remove cell debris into particles. Therefore, the polypeptide is purified from contaminating soluble proteins and polypeptides, the following procedures being illustrative of suitable purification procedures by immunoaffinity fractionation or ion exchange columns.; Ethanol precipitation, reverse phase HPLC, chromatography on silica or in a cathodic exchange resin such as DEAE; chromatofocusing; SDS-PAGE; precipitation of ammonium sulfate; 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 (FFMS) may also be useful for inhibiting proteolytic degradation during purification. One skilled in the art will appreciate that suitable purification methods for the polypeptide of interest may require modification to account for changes in the character of the polypeptide when expressed in the recombinant cell culture. Especially within the context of the present invention, the purification techniques and processes which select the carbohydrates of the invention are preferred. The desired glycoforms of the present invention can be enriched for acid-containing molecules eg, ion exchange or soft-gel chromatography. using cationic or anionic exchange resins, where the most acidic fraction is collected. IV Glycoprotein Analysis The complex carbohydrate portion of the ghcoprotein produced by the process of the present invention can be easily analyzed, if desired, by conventional techniques. carbohydrate analysis Thus, for example, techniques such as lectin agglutination, well known in the art, reveal proportions of terminal sugar or other sugars such as galactose. The termination of mono, bi, tp or tetra-antenapo oligosaccharide by sialic acids can be confirmed by the release of sugars from the protein using anhydrous hydrazine or enzymatic methods and fractionation of ohgosacducts by ion exchange or size exclusion chromatography and other well-known methods in the subject The glycoprotein pf can also be measured, before or after neuraminase treatment to remove sialic acids An increase in pl after neuraminidase treatment indicates the presence of sialic acids in the glycoprotein The carbohydrate structures of the present invention They occur on the protein expressed as N-linked or olized carbohydrates N-linked or olized carbohydrates differ mainly in their core structures N-linked glycosylation refers to the binding of the carbohydrate portion via GIcNAc to a residue of asparagine in the peptide chain. The N-linked carbohydrates contain a core structure Man1-6 (Man1-3) Manß1-4GlcNAcß1-4GlcNAcß-R. Therefore, in the described core structure, R represents an asparagine residue of the protein produced. The peptide sequence of the produced protein will contain an asparagine-X-serine, asparagine-X-threonine and asparagine-X-cysteine, where X is any amino acid except proline. O-linked carbohydrates, in contrast, are characterized by a common core structure, which is the GalNAc bound to the hydroxyl group of a threonine or serine. Of the N-linked and O-linked carbohydrates, the most important are the N- and O-linked complex carbohydrates. These complex carbohydrates will contain several antennal structures. The mono-, bi-, tri- and tetra-exterior structures are important for the addition of terminal sialic acids. Such chain structures provide additional sites for the specific sugars and ligatures comprising the carbohydrates of the present invention. The resulting carbohydrates can be analyzed by any method known in the art including those methods described herein. Various 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 composition of the oligosaccharide attached to the peptide. Methods for analyzing carbohydrates useful in the present invention include but are not limited to lectin chromatography. HPAEC-DAP, which uses high pH anion exchange chromatography to separate oligosaccharides based on charge; NMR; mass spectrometry; CLAR; CFG; composition analysis of monosaccharides; Sequential enzymatic digestion. Additionally, methods for releasing the oligosaccharides are known. These methods include 1) enzymatic, which is commonly carried out using peptide-N-glycosidase F / endo-β-galactosidase; 2) ß elimination using unpleasant alkaline environment to release mainly O-linked structures and 3) chemical methods using anhydrous hydrazine to release both N- and O-linked oligosaccharides. The analysis can be carried out using the following steps: 1. Dialysis of the sample against deionized water, to remove all regulatory salts, followed by lyophilization. 2. Release the intact chains of oligosaccharides with anhydrous hydrazine. 3. Treatment of intact oligosaccharide chains with methanic anhydride HCl to release individual monosaccharides as O-methyl derivatives. 4. N-acetylation of any primary amino groups. 5. Derivatization to give per-O-trimethylsilylmethyl glycosides 6. Separation of this derivative, by capillary CGL (gas-liquid chromatography) on a CP-SIL column. 7. Identification of individual glycoside derivatives by CGL retention time and mass spectroscopy, compared to known normal. 8. Quantification of individual derivatives by FID with an internal standard (13-O-methyl-D-glucose). Neutral and amino sugars can be determined by high performance anion exchange chromatography combined with pulsed amperometric detection (Carbohydrate System IAAR-DAP, Dionex Corp.). For example, sugars can be released by hydrolysis in 20% (v / v) trifluoroacetic acid at 100 ° C for 6 h. The hydrolysates are then dried by lyophilization or with a Speed-Vac (Savant Instruments). The residues are then dissolved in 1% sodium acetate trihydrate solution and analyzed on a column of CLAR-AS6 as described by Anumula et al. (Anal. Biochem, 195: 269-280 (1991). determined separately by the direct colorimetric method of Yao et al. (Anal. Biochem. 179; 2332-335 (1989)) in triplicate samples In a preferred embodiment, thiobarbituric acid (ATB) is used from Warren, LJ Biol. Chem. 238: (8) (1959) Alternatively, immuno carbohydrate analysis can be carried out In accordance with this procedure, protein-bound carbohydrates are detected using a commercial glycan detection system (Boehringer) which is based on in the oxidative immunoassay procedure described by Haselbeck and Hosel [Haselbeck et al., Glycoconjugate J., 7:63 (1990)] The staining protocol recommended by the manufacturer is followed in that the protein is transferred to a membrane of difluoride of polyvinylidene in place of a nitrocellulose membrane and blocking buffer solutions containing 5% bovine serum albumin in tris 10 mM pH buffer, pH 7.4 with 0.9% sodium chloride. Detection was performed with anti-digoxigenin antibodies bound with an alkaline phosphate conjugate (Boehringer), dilution 1: 1000 in tris saline with regulated pH using phosphatase substrates, tetrazolium 4-nitro blue chloride, 0.03% (p. / v) and 5-bromo-4-chloro-3-indoyl-phosphate 0.03% (w / v) in tris 100 mM pH buffer solution, pH 9.5, containing 100 mM sodium chloride and 50 mM magnesium chloride. Protein bands containing carbohydrate are usually visualized in approximately 10 to 15 min. The carbohydrate can also be analyzed by digestion with peptido-N-glycosidase F. According to this procedure, the residue is suspended in 14 μl of a buffer solution containing 0.18% SDS, 18 mM beta-mercaptoethanol, 90 mM phosphate, 3.6 mM EDTA at pH 8.6 and heated at 100 ° C for 3 minutes. After cooling to room temperature, the sample is divided into two equal parts. An aliquot is not treated additionally and serves as a control. The second fraction is adjusted to approximately 1% detergent NP-40 followed by 0.2 units of peptido-N-glycosidase F (Boehringer). Both samples are heated at 37 ° C for 2 hours and then analyzed by SDS poiacrylamide gel electrophoresis. V. Tumor Necrosis Factor-Immunoglobulin Receptor Chimeras In a preferred embodiment, the processes of the present invention are used to produce tumor necrosis factor receptor (TNFR) -immunoglobulin (Ig) chimeras. Especially among this class of chimeric proteins, the RFNT-IgGi type 1 is preferred. The RFNT1-IgD chimeras produced are useful in the treatment or diagnosis of many diseases and disorders mediated by TNF or related to TNF. The term "treatment" in this context, it includes both prophylactic (prevention), suppression (eg, of a symptom) and treatment of an existing condition. Pathological conditions associated with TNF include, but are not limited to, large negative and gram positive bacteremia, endotoxic shock, graft rejection phenomenon, rheumatoid arthritis, systemic lupus, Crohn's disease and other autoimmune and inflammatory diseases associated with TNF. The RFNT1-IgG preparations of the present invention are generally useful in those indications in which monoclonal antibodies to TNF have been found useful. For example, in animal models, monoclonal antibodies to TNF-alpha were found to have protective effect when used prophylactically (Tracey, K.JK. et al. (1987) Nature 330: 662). In a phase I clinical study reported by Exley, A.R., et al. (1990) Lancet 335: 1275, a murine monoclonal antibody to recombinant human TNF-alpha was found safe when administered to human patients with severe septic shock. RFNT1-IgG1 is used appropriately in the treatment of rheumatoid arthritis as well as septic shock. In a method for treating a disease or disorder associated with TNF, a therapeutically active amount of preparation of RFNT1-IgG is administered? to a subject who needs such treatment. The preferred subject is a human being. An effective amount of a glycophorus preparation of RFNT1-IgD of the present invention for the treatment of a disease or disorder is in the dose scale of 0.01-100 mg / patient, preferably 1 mg-75 mg / patient and more preferably between approximately 10 and around 50 mg / patient. For administration, the preparation of RFNTI-IgGi must be formulated in an appropriate pharmaceutical or therapeutic composition. Said composition normally contains a therapeutically active amount of the preparation of RFNT1-IgD and a pharmaceutically acceptable excipient or carrier such as saline, saline with regulated pH, dextrose or water. The compositions may also comprise specific stabilizing agents such as sugars, including mannitol and mannitol and local anesthetics for injectable compositions, including, for example, lidocaine. The present invention provides compositions that further comprise a therapeutically active amount of an additional active ingredient such as monoclonal antibodies, (e.g., anti-FNT antibodies, antibodies to Mac 1 or LFA 1) or other receptors associated with the production of TNF. , eg, IL-1 or IL-2 receptors, etc. A preferred therapeutic composition for single or combined therapy, as above, comprises an RFNT1-IgD preparation of this invention, which exhibits prolonged blood clearance while retaining significant functional activity. Said prolonged functional half-life, allows the administration of simplified bolus dose and contributes to the potency in vivo. The chimeras of RFNT1-lgG? preferred in the therapeutic composition includes RFNT1-IgD and preparations described herein, for example: (1) RFNT1-IgD preparations comprising a complex oligosaccharide terminated by one or more residues of a sialic acid; (2) Preparations of RFNT1-lgG? where the scale of isoelectric point, pl of the preparation is between 5.5 and 7. 5 determined by chromatofocusing, in which the pl is sensitive to the neuraminidase treatment; (3) Preparations of RFNT1-lgd having approximately 1-2 moles of N-acetylglucosamine residues per mole of protein. (4) RFNT1-lgd preparations having a molar ratio of sialic acid to protein of about 4-7, especially about 5-6. (5) RFNTI-lgd preparations having a molar ratio of sialic acid to N-acetylglucosamine from about 035 to about 05 and more preferably from about 04 to about 045 Administration routes for the individual or combination therapeutic compositions of the present invention include normal routes, such as, for example, intravenous infusion or bolus injection. The use of an RFNT1-lgd preparation of this invention in the manufacture is also provided. of a medicament for the treatment of a human or animal having been generally described, the same will be more easily understood by reference to the following examples which are provided by way of illustration and are not intended to limit the present invention, unless specified Examples Biological effects of TNF-alpha and FNT-beta are mediated through specific receptors (Dembic et al. (1990) Cytokines, 2231) Molecular cloning has demonstrated the existence of two distinct types of TNF receptors (RFNT) with evident molecular masses of 55 kD (type 1) (Schall et al. (1990) Cell 61 361) and 75 kD (type 2) (Smith and others (1990) Science 248 1019), each of which naturally binds both to TNF-alpha and FNT-beta (Loetscher et al. (1990) Cell, 61 351, Shall et al. (1990) Cell, 61 361, Kohno et al. (1990) Proc Nati Acad Sci USA 878331) The extracellular portions of both receptors naturally occur as TNF binding proteins (Kohno et al., Supra). TNF agonists have been created, which block the detrimental effect of TNF in various immune and inflammatory events (Peppel et al. (1991) J. Exp. Med., 174: 1483-1489. Ulich (1993) Am. ., 142: 1335-1338, Howard, OMZ (1993) Proc. NMtl Acad Sci USA 90: 2335-2339: Wooley PH, (1993) J. Immunol. 151: 6602-6607). One such agonist (Werner et al. (1991) J. Cell. Biochem. Abstracts, 20th annual meeting, p. 115) combines the extracellular domain of human type 1 RFNT 55 kD with a portion of the joint and chain Fe regions. thick G1 of human immunoglobulin. In this Example, the cells of mammals transfected with a vector containing the cDNA encoding the chimera of RFNT1-IgD were cultured. Methods A. Cell Line The Chinese hamster ovary (OHC) cell line used as the mammalian host cell line was derived from OHC-K1 (ATCC No. CCL61 OHC-K1). A deficient cell line dihydroftlatoreductase (DHFR) mutant of OHC-K1 named (DHFR) OHC-K1. DUX-B11 (obtained from Dr. L. Chasin of Columbia University, Simonsen, CC, and Levinson, AD, (1983) Proc. Nati, Acad. Sci. USA 80: 2495-2499, Uriaub G. and Chasin. (1980 = Proc. Nati, Acad. Sci. USA 77: 4216-4220) was then used to obtain a cell line with a reduced requirement for insulin by transfection with the vector containing the cDNA for proinsulin (Sures et al. ) Science, 108: 57-59.) The selected clone designated dp12.OHC requires glycine, hypoxanthine and thymidine for growth, thus verifying its DHFR genotype). B. Construction of the Chimera of RFNT-lgG! Type 1 Soluble A chimera of soluble type 1 RFNT-lgGi was constructed by fusion of genes from the extracellular domain of human type 1 RFNT with the region of articulation and heavy chain CH2 and CH3 domains of Igd (also known as RFNTI-lgd) . The human type 1 RFNT that encodes DNA sequence (see Loetscher and others, Supra) was obtained from the pRK-FNT-R plasmid (Schall et al., Cell 61: 361 (1990).] To construct this starting plasmid, a 2.1 kb placental cDNA clone was inserted into the mammalian expression vector. pRK5, the construction of which is described in EP Pub. No. 307,247, published March 15, 1989. This cDNA starts at a 64 position of the nucleotide of the frequency reported by Loetscher et al., with the starting methionine 118 pb downstream The source of the sequence encoding Igd was the CD4-lgG expression plasmid pRKCD42Fc1 [Capón, DJ Y otros, Nature 337, 525 (1989); Byrn et al., Nature 344. 667 (1990)], containing a cDNA sequence encoding a hybrid polypeptide consisting of residues 1-180 of the mature human CD4 protein (two N-terminal variable domains CD4) fused with a sequence of Human IgG 1 starting from an aspartic acid 216 (taking amino acid 114 as the first heavy chain constant region residue [Kabat et al., Sequences of Proteins of Immunological Interest 4th Edition (1987)] which is the first residue of the Igd after the cysteine residue involved in the thick-light chain linkage) and ending with residue 441 to include the CH2 and CH3 Fe domains of Igd. Was RFNT1-lgG built? generating restriction fragments of plasmids pRK-FNT-R and pRKCD42Fc1 and binding them, using deletion mutagenesis, so that the threonine residue 171 of mature FRNT is juxtaposed to the IgGi heavy chain aspartic acid residue 216 [Kabat et al. supral The resulting plasmid of pRKTNFR-IgG contained the full length coding sequence for RFNT! Igd. C. Cell Culture The gene encoding the soluble type 1 RFNT-lgd was introduced into the dp12.OHC cells by transfection. This was achieved using the calcium phosphate technique to introduce DNA into mammalian cells. Two days after cell transfection, trypsinization was performed and replated in plaques in selective medium (glycine-hypoxanthine and Ham's F-12 DMEM free thymidine, 1: 1 v / v with 2% dialyzed serum). Subsequent isolates were screened for secretion of RFNTI-IgGt. The clones expressing RFNT1-lgd were amplified in methotrexate producing high expression clones and subsequently adapted to serum free medium. These cells were under continuous selective pressure until they were transferred to non-selective medium for growth and expansion of the inoculum. To provide cells for the production crops of RFNTI-lgd, the cell population described above was expanded from methotrexate-containing medium by subcultures in ERIE in containers to increase volumes of growth medium that do not contain methotrexate. For these steps of the process, the non-selective growth medium was the formulation based on DMEM / HAM F-12 (see US Patent 5: 122,469, for example) with modified concentrations of some components, such as glucose, amino acids, salts, sugar, vitamins, glycine, hypoxanthine and thymidine; recombinant human insulin, hydrolyzed peptone (Primatone HS or Primatone RL) a cellular protective agent such as Pluronic F68 (pluronic polyol) or the equivalent; Gentamicin; Lipids and trace elements. The cultures were controlled at pH 7.2 +/- 0.4 by the use of CO2 gas (acid) and / or Na2CO3 (base). The temperature was controlled near 37 ° C during the growth period. Dissolved oxygen was maintained above 5% air saturation by direct spraying with air and / or oxygen gas. Osmolality during the inoculum expansion phase was maintained between approximately 250 mOsm and 350 mOsm. The growth phase for each crop was followed by a second phase or transition phase where the parameters of the optimum growth culture were changed to production conditions. During this transition phase, the temperature of the culture system decreased, generally to approximately between 30 and 35 ° C. Butyrate was added and a certain scale of osmolality was coupled. The product accumulated during this production phase was analyzed for sialic acid content.

In a normal production form approximately 1.2x106 cells derived from the inoculum expansion of the selective stage were grown in a growth phase with a starting osmolality of 300 mOsm. The growth medium is supplemented with trace elements, recombinant human insulin and hydrolysed peptone. The cells developed under these conditions for 2 days. At the beginning of the third day, the cell culture temperature decreased. Simultaneous to or after the change in temperature, sodium butyrate was added to the cell culture and the desired production osmolality was coupled by the addition of various components of the medium. The cells were developed under these conditions with feeding for 9-10 days. The cells were fed with several components of the medium. Table I describes the production conditions for various production processes.

Table I Productivity Content of Specific Osmolality of Sialic Acid of Conversion Version of cell phase day 5 and RFNT1-lgG? in Butirate process production day 10 day 10 (mml / 1) (mOsmol / kg) (pg /) (mol / mol) A 1 360-420 0.6-1.5 6.4-7.2 N = 4 B 1 480-510 1.7-2.2 6.0-6.3 N = 3 C 6 460 4.8 4.7 N = 1 D 6 370-420 2.4-2.8 5.5-5.6 N = 3 E 6 350-370 1.4-2.3 5.4-6.3 N = 3 F 12 390 4.0 5.3 N = 1 G 12 490-510 2.9-5.2 4.0-5.2 N = 4 H 12 400-430 2.0-2.8 5.8-6.0 N = 3 I 12 370-380 2.0-2.2 5.5-5.9 N = 3 D. Recovery of the RFNT-lgG The chimera of RFNT1-lgG? more than 95% homogeneity was purified by affinity chromatography on Staphylococcus aureus Protein A immobilized as described by Capon et al., supra. E. Analysis of Carbohydrates The content of sialic acid was analyzed by the method of Warren, L. (1959) J. Biol. Chem. 234: 1971-1975. Results The cell-specific productivity for each of the production cultures described in Table I is plotted against the sialic acid content of the cultivated product. The results are presented in Figure 1. The higher sialic acid content was observed when the parameters of the process were maintained at a production osmolality between approximately 360 and 420 mOsm and a butyrate concentration of approximately 1 mM. The sialic acid content could be controlled over a wide range of values by adjusting the process parameters Example II Plasma pharmacokinetic half-life regimens and / or elimination of different preparations were determined. Preparations having a higher sialic acid content in the regimens of increased plasma half-life and / or lower total elimination compared to preparations having a decreased sialic acid content Methods Seventeen male Sprague-Sawly rats were randomly assigned weighing 272-315 g for one of three groups of fusion protein treatments of RFNT1-IgD (N = 5 or 6 per group) Animals were injected intravenously with a nominal dose of 5 mg / kg of test material via a femoral vein cannula. The test materials chosen included two preparations of RFNT1-IgG? of a process in which the concentration of butyrate during the production phase was 6 mM and the osmolality during the production phase was maintained at approximately 400 mOsm The process I and a third preparation of RFNT1-lgd of a process in which the concentration of butyrate was 12 mM and the osmolality during the production phase was 12 mM and the osmolality during the production phase was approximately 500 mOsm, Process II. Blood samples of two ml were collected in EDTA (8.5%) before the dose and at 5 minutes and 2, 6, 10, 24, 34, 48, 56, 72, 96 and 120 hours after the dose. The blood samples were centrifuged, the plasma was collected and the samples were analyzed for fusion protein concentrations. An enzyme-linked biological and immunological binding assay (AIBUE) was used to quantify RFNT1-IgD in rat plasma. This analysis is based on the ability of FNT-alpha coupled to horseradish peroxidase (FNT-alpha-HRP) to bind to the receptor portion of the fusion protein of RFNT1-IgG ?. In this analysis, the goat antihuman IgGFc Fab fragments reversed in wells of microtitre plates were used to capture RFNT1-IgG? by interaction with the Fe portion of the molecule. FNT-alpha-HRP was added to the wells and the receptor portion of the RFNT1-IgG was allowed to bind? captured. The quantification was determined by measuring a color produced by peroxidase reaction with peroxide and an ortho-phenylenediamine (OFD) substrate. The scale of the analysis is from 0.003 to 0.02 mg / ml. It was shown that the presence of EDTA in the plasma samples has no effect on the assay performance. The dose was normalized to count the concentration differences of dose solution. All calculations were based on the concentration versus time data through the 120 hr time point. The area truncated under the curve (AUC0-? 2o) was calculated using the trapezoidal rule and truncated elimination normalized by weight (CL0-? 2o / P) was calculated as dose / AUC0-? 2o- Result Elimination regimens varied depending on of the version of the process used (Process I compared to Process II) and the amount of sialic acid present in the product. The elimination regimes for individual animals and the resulting mean and normal deviations from this study are presented in the following Table II. Process I exhibits a slower and more favorable elimination regime. Table II ELIMINATION 1-120 (mL / hr / kg) Process I Process I Process II 2.12 2.08 2.144 2.03 2.31 2.99 1.63 2.20 2.61 1.89 2.22 3.27 1.85 1.99 2.81 1.66 3.42 STDEV 1.91 2.08 2.88 average 0.02 0.23 0.45 Example lll COMPOSITION OF MONOSACARID OF RFNT1-laG? The determination of oligosaccharide carbohydrate composition and structure of RFNT1-IgG? prepared as described in Example I, showed that the sialic acid content of the different versions of the process varied. The rapid elimination of the plasma with highly exposed GIcNAc, lower sialic acid in the oligosaccharide chains and (by inference) ability of the protein access to mannose or galactose receptors was associated. Slow elimination of the plasma was associated with more terminal sialic acid residues. A. Methods RFNT1-IgD was produced according to the methods described in Example 1 above. Process I material was obtained from cell culture using 6mM butyrate and an osmolality of about 400 mOsm during the production phase. Process material II was obtained from the cell culture using 12 mM butyrate and an osmolality of about 500 mOsm during the production phase. B. Methods The release of neutral sugars and intact amino sugars was determined by high pH anion exchange chromatography combined with pulsed amperometric detection. The analysis was performed using the following steps: 1. The exchange of pH buffer solution was performed with RFNT1-IgD (approximately 50 μg / ml) and the appropriate reference samples so that the final sample was contained in the acetic acid at 1%. 2. Approximately 90 μg of RFNT1-lgd as well as samples of the reference materials were frozen in a dry ice and alcohol bath and the frozen sample was lyophilized overnight. 3. The dried frozen samples were reconstituted in 500 μl of trifluoroacetic acid and incubated at 120 ° C for 1 hour. 4. After acid hydrolysis, the RFNT1-lgd and the reference samples were cooled and evaporated to dryness. 5. Samples were reconstituted with water at a final concentration of approximately 0.6 mg / ml. 6. Separation of the monosaccharides was carried out at room temperature by anion exchange chromatography at high pH with pulsed amperometric detection using a Dionex CarboPac PA l (4x250 mm) column (Dionex Corp., Sunnyvale, CA). 7. The quantification of individual monosaccharides was by comparison to the reference monosaccharides. C. Results The relative molar content of each monosaccharide in the two preparations is shown in the following Table l l l: Table III Relative MOLAR CONTENT OF MONOSACCHARIDES IN TWO PREPARATIONS OF RFNT1-lgd Monosaccharide Process I Process II Fucose 43 ± 02 44 Galactose 6 5 + 00 4 7 Mañosa - | 2 2 + 06 12 5 N-acetylglucosamine 14 1 + Q 143 Sialic acid 3 7 ± 03 49 + 0 3 N-acetylgalactosamine 0 3 0 5 + 0 1 Sia co / GIcNAc 026 035 acid ratio The above results show that the parameters of the process selected for the production phase of the co-protein affects the carbohydrate composition of the mature glycoprotein. Preparations having a higher sialic acid content generally exhibited prolonged serum half-life Tables IV and V presented below demonstrate the carbohydrate composition of Oligosaccharide side chains of chimeric preparations of RFNTI-IgD produced under the conditions of the type of process I and process II Table V presents carbohydrate data in the receptor portion of the chimeric molecule, subtracting the glycosylation site Table IV Pi EL £ i Pll Pll Sialic Acid 5.8 5.8 5.7 5.8 3.7 3.5 Fucosa 4.0 4.0 3.6 4.1 4.4 4.3 Gal NAc 0.3 0.2 0.2 0.4 0.3 0.4 Glc NAc 12.9 15.0 14.8 14.3 14.3 14.4 Gal 7.4 7.6 7.1 7.0 4.7 4.1 Man 12.0 12.0 10.0 12.0 12.5 11.9 I laughed SA / G1cNAc 0.45 0.39 0.39 0.41 0.26 0.24 Table V Pll Pll P - L P-I Moles exposed from 3.18 3.2 1.11 1.32 GIcNAc per mol Moles exposed from Gal 0.97 0.08 1.45 -0.26 per mole Gal antenna 4.47 4.72 6.45 6.24 Sialic acid per mole 3.5 4.8 5.00 6.5 The results presented in Tables IV and V demonstrate that the composition of RFNT1-lgd is normal for oligosaccharides complexes of sialic acid terminated in mono-, bi-, and triantenas. It can be concluded that the RFNT1-lgG? prepared by Process I contains a significantly higher proportion of sialic acid and a significantly lower proportion of exposed GIcNAc. Sialic acid per mole of Indica protein that this material could have a lower isoelectric point than the material produced by Process II. Comparing with the results in Table II, these results also indicate that the slower plasma removal of Process I material correlates with exposed lower GIcNAc and generally with a higher sialic acid content. Tables IV and V show that the preparations of RFNT1-lgG? comprising a complex oligosaccharide terminated by one or more sialic acid residues. The preparations of RFNTI-lgG! Preferred comprise RFNT1-IgD molecules having approximately 1-2 moles of exposed N-acetylglucosamine residues per mole of protein. The molar ratio of sialic acid to protein is about 4-7. The RFNT1-IgD preparations have a molar ratio of sialic acid to N-acetylglucosamine from about 0.4 to about 0.45. Example IV The pl of the highly sialylated preparation is lower than the pl of the slightly sialylated preparation. Methods The isoelectric focus was carried out by the various preparations described in Example II. The isoelectric focusing gels separate the glycoproteins from the preparation according to their isoelectric point, pl, using a pH gradient created with ampholytes of different pH. In this study, the analysis of the preparations was carried out using a pH gradient of 10 to 4. Results The RFNT1-lgd preparations exhibit an isoelectric point scale of about 5.5 to about 7.5 determined by chromatofocusing in which the pl is sensitive to neuroaminase treatment. The references cited above are incorporated herein by reference, whether or not they are specifically incorporated. While this invention has been described in relation to specific embodiments thereof it will be understood that it is capable of further modifications. It is intended that this application covers any variations, uses or adaptations of the following inventions, in general, the principles of the invention and including said deviations from the present description given that they fall within the known or common practice within the art to which The invention pertains and may be applied to the essential aspects exhibited before in the following manner in the scope of the appended claims.

Claims (23)

1. CLAIMS 1. A process for controlling the amount of sialic acid present in an oligosaccharide side chain of a glycoprotein produced by mammalian cell culture which comprises: i) adding an alkanoic acid or a salt thereof to the cell culture at a concentration from about 0.1 mM to about 20 M; and ii) maintaining the osmolality of the cell culture from about 250 to about 600 mOsm.
2. 2. The process according to claim 1, wherein the amount of sialic acid present in the oligosaccharide side chain of the glycoprotein is increased and wherein: the specific productivity of cell culture cells decreases by culturing the host cell at a concentration of alkanoic acid or salt thereof from about 0.1 to about 6 mM and maintaining the osmolality at about 300-450 mOsm.
3. 3. The process of claim 2, wherein the host cell is an OHC cell.
4. 4. The process according to claim 3, wherein the alkanoic acid or salt thereof is sodium butyrate.
5. 5. The process according to claim 4, wherein the glycoprotein produced is a mammalian glycoprotein.
6. 6. The process according to claim 5, wherein the glycoprotein is a tumor necrosis factor-immunoglobulin receptor chimera.
7. 7. The process of claim 6, wherein the host cell is a dp12.OHC cell line transfected with the vector carrying the cDNA encoding a tumor necrosis factor-immunoglobulin receptor chimera. The process according to claim 1, wherein the amount of sialic acid present in the oligosaccharide side chain of the glycoprotein decreases and wherein: the cell-cell specific productivity is increased by culturing the host cell at a concentration from about 6 mM to about 12 mM of the alkanoic acid or salt thereof and maintains the osmolality at about 450-600 mOsm. 9. The process of claim 8, wherein the host cell is an OHC cell. 10. The process according to claim 9, wherein the alkanoic acid or salt thereof is sodium butyrate. 11. The process according to claim 10, wherein the glycoprotein produced is a mammalian glycoprotein. The process according to claim 11, wherein the glycoprotein is a tumor necrosis factor-immunoglobulin receptor chimera. The process of claim 14, wherein the host cell is a dp12.OHC cell line transfected with the vector carrying chimeric cDNA of RFNT1-IgD. 14 A process to produce a chimeric tumor necrosis factor-immunoglobulin receptor protein RFNT1-IgG? which comprises a) culturing a mammalian host cell expressing a chimera of RFNT-IG in a growth phase under said conditions and for such a period that maximum cell growth is achieved, b) culturing the host cell in a production phase in the presence of sodium butyrate at a concentration of about 6 mM to about 12 mM, c) maintaining the osmolality of the production phase of about 450-600 mOsm 15 A process to produce a chimeric protein of receptor tumor necrosis factor-immunoglobulin RFNT1-lgd comprising a) culturing a mammalian host cell expressing a chimera of RFNT-IG in a growth phase under said conditions and for a period such that maximum cell growth is achieved , b) culturing the host cell in a production phase in the presence of sodium butyrate at a concentration of about 1 mM to about 6 mM, c) maintaining the mole of the production phase of about 300-450 mOsm 16 The process of claim 15, wherein the host cell is an OHC cell 17. The process of claim 16, in which the host cell is a line of dp12.OHC. 18. A preparation that comprises the RFNT1-lgG? produced by the process of claim 17. 19. A therapeutic composition comprising the RFNT1-Igd produced by the process of claim 15, and a pharmaceutically acceptable excipient thereof. 20. A preparation of RFNT1-lgG? which comprises molecules of RFNT1-lgd wherein the RFNTI-lgd molecules have a molar ratio of sialic acid to protein of about 4-7. 21. An RFNTI-IgG-preparation, comprising RFNTI-IgD molecules wherein the RFNT1-IgD molecules have approximately 1-2 moles of N-acetylglucosamine residues per mole of RFNT1-IgG protein. 22. A preparation of RFNTI-IgG, comprising RFNT1-IgG molecules wherein the RFNT1-IgG molecules have a molar ratio of sialic acid to N-acetylglucosamine from about 0.35 to about 0.5. 23. The preparation of RFNT1-IgG "of claim 22, wherein the molar ratio of sialic acid to N-acetylglucosamine is from about 0.39 to about 0.45.