The present invention relates to novel process for the preparation of glycoproteins by mammalian-cell culture wherein the sialic acid content of the glycoprotein' produced is controlled over a broad range of values by manipulating the cell culture environment. The invention provides for processes in which the sialic acid content of the glycoprotein is modified by changes in cell culture parameters which affect cell specific productivity. Preferred embodiments of the invention include cell culture processes in the osmolality of the cell culture is controlled as well as the concentration of a transcription enhancer during the production phase of the cell culture. The invention further provides for novel preparations of-soluble type 1 tumor necrosis factor immunoglobulin G1 and their uses in the treatment of inflammatory or immune related disorders.

Description of the Invention

SUMMARY OF THE INVENTION

The present inventors have discovered that certain mammalian cell culture process parameters affect cell specific productivity as well as the extent and type of glycosylation of the proteins produced. More particularly, the present inventors have found that certain factors which enhance cell specific productivity have an inverse effect on the-sialic acid content of the produced protein. The present inventors have therefore devised various cell culture processes to enrich particular glycoforms of glycoproteins produced in mammalian cell culture.

Accordingly, the invention provides for a process for controlling the sialic acid content of a glycoprotein produced by mammalian cell culture. According to this aspect of the invention, varying the production rate 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 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 protein production phase of mammalian cell culture by controlling factors which affect cell specific productivity. According to one aspect of the invention, the concentration of factors which enhance DNA transcription are controlled. In another embodiment cell specific productivity is controlled by maintaining the osmolality of the cell culture within certain margins. According to the invention, any of the foregoing parameters are controlled, alone or in combination, to affect the mature glycoprotein sialic acid content.

In a particular embodiment of the present invention, the factor which enhances 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 250-600 mOsm. In a further aspect, the temperature of the cell culture is controlled between about 30.degree. C. and 37.degree. C.

In a preferred embodiment, the invention provides for a process for increasing the sialic acid content of the mature glycoprotein produced by mammalian cell culture comprising maintaining a lower cell specific productivity by controlling any or all of the above identified process parameters, optionally together with other parameters known in the art. According to this aspect of the present invention, culturing the host cell at a concentration of the alkanoic acid or salt thereof of about 0.1 mM to about 6 mM, and optionally together with maintaining the 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 for a process for decreasing the sialic acid content of the mature glycoprotein produced by mammalian cell culture comprising increasing cell 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 further provides, in a particular embodiment, for 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 mammalian cell culture comprising the steps of culturing a host cell which expresses the protein in a growth phase for a period of time and under such conditions that cell growth is maximized. According to this aspect of the present invention, the growth phase is followed by a transition phase in which cell culture parameters for the desired sialic acid content of the mature glycoprotein are selected and engaged. The transition phase is followed by a production phase of the cell culture wherein parameters selected in the transition phase are maintained and glycoprotein product is produced and harvested. Varying the cell specific productivity of the production phase of the cell culture 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 engaging an osmolality of the cell culture at about between 250 and 600 mOsm, optionally in combination with one another during the transition phase produces a protein with differing 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 type 1 tumor necrosis factor receptor (TNFR1)-immunoglobulin G.sub.1 (IgG.sub.1) chimeric protein. The present inventors have discovered that, under certain conditions of production, novel TNFR1-IgG.sub.1 glycoform preparations may be obtained which exhibit the desirable properties of prolonged clearance from the blood while retaining significant functional activity. A long functional half-life permits simplified, bolus-dose administration and contributes to in vivo potency of the glycoprotein produced allowing for lower dose forms of the glycoprotein.

According to this aspect of the present invention, a TNFR1-IgG.sub.1 glycoprotein molecule is produced that contains increased sialic acid residues. The cell culture parameters for the production phase of the TNFR1-IgG.sub.1 are selected to obtain the desired sialic acid content. In a preferred embodiment, the 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 sodium butyrate concentration is about 1 mM and the osmolality is maintained at about 350-400 mOsm.

In yet another embodiment, the present invention provides for a preparation of TNFR1-IgG.sub.1 glycoprotein produced by the process of the present invention. According to this aspect of the invention a preparation is provided comprising TNFR1-IgG.sub.1, in which the range of pI of the preparation is between about 5.5 and 7.5. Further provided is a TNFR1-IgG.sub.1 preparation having a molar ratio of sialic acid to protein of about 4 to about 7 and especially about 5 to about 6. In yet another aspect, the TNFR1-IgG.sub.1 preparation has about 1 to about 2 moles of exposed N-acetylglucosamine residues per mole of protein. In a further aspect, the preparation has a molar ratio of sialic acid to N-acetylglucosamine of about 0.35 to about 0.5 and more preferably about 0.4 to about 0.45.

The present invention also provides a therapeutic composition comprising the above preparation useful in the treatment of TNF-mediated pathologic conditions.

DETAILED DESCRIPTION OF THE INVENTION

II. Cell Culture Procedures

The present inventors have discovered that factors which increase cell specific productivity during the production of a glycoprotein produced by mammalian cell culture have an inverse effect on sialic acid content of the glycoprotein produced. Since proteins expressing one or more sialic acid residues per complex oligosaccharide structure have longer clearance rates in vivo the clearance rate of the glycoprotein produced may be manipulated within broad limits by the overall degree of sialylation of the preparation. The present invention provides for processes for controlling the extent of sialylation of a glycoprotein produced by mammalian cell culture. Following the methodology set forth herein, one is able to determine the precise process parameters that provide for the desired sialic acid content of a glycoprotein produced by mammalian cell culture.

According to 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 controlling cell culture parameters which affect cell specific productivity. Factors which affect cell specific productivity are well known in the art and include but are not limited to, factors which affect DNA/RNA copy number, factors which affect RNA, such as factors which stabilize RNA, media nutrients 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 adjustment of these factors, alone or in combination, to increase cell specific productivity generates a protein with a decreased sialic acid content. The adjustment" of these factors, alone or in combination, to decrease cell specific productivity, generates a mature glyccprotein with an increased sialic acid content.

The invention will now be described with reference to various, including well known, cell culture techniques and principles.

According to the present invention mammalian cells are cultured to produce a desired glycoprotein product. In 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 (e.g., glycosylation, cleavage) of the proteins expressed. Appropriate cell lines should be chosen to ensure that the desired post translational modifications are possible. Alternatively, host cells can be modified to express a desired gene product required for the specific post-translational modification.

In particular, the mammalian cells which express the desired protein should express or be manipulated to express the particular enzymes such that under the appropriate conditions; described herein, the appropriate post translational modification occurs in vivo. The enzymes include those enzymes necessary for the addition and completion of N- and O- linked carbohydrates such as those described in Hubbard and Ivan supra for N-linked oligosaccharides. The enzymes optionally include oligosaccharyltransferase, alpha-glucosidase I, alpha-glucosidase II, ER alpha(1,2) mannosidase, Golgi alpha-mannodase I, N-acetylyglucosaminyltransferase I, Golgi alpha-mannodase II, N-acetylyglucosaminyl-transferase II, alpha(1,6) fucosyltransferase, and .beta.(1,4) galactosyltranferase. Additionally, the host cell expresses the appropriate sialyl transferase that can be expected to attach the terminal sialic s acid in specific position and linkage as part of the host cell genome. optionally, the host cell can be made to express the appropriate sialyl transferases by, for instance, transfection of the host cell with the DNA encoding the sialyl tranferase.

The sialyl transferases described above would be expected to add the terminal sialic acid residue to the appropriate, oligosaccharide core structure such as Gal.beta.1-4GlcNAc. Appropriate sialyl transferases within the context of the present invention include, but are not limited to, those sialyl transferases which catalyze the complex sialylation and branching of the N- and O-linked oligosaccharides.

For the culture of the mammalian cells expressing the desired protein and capable of adding the desired carbohydrates in specific position and linkage, 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, 2.sup.nd Ed., Rickwood, D. and Hames, B. D., eds. Oxford University Press, New York (1992)), and vary according to the particular host cell selected.

The mammalian cell culture of the present invention is prepared in a medium suitable for the particular cell being cultured. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ([MEM], Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ([DMEM], Sigma) are exemplary nutrient solutions. In addition, any of the media described in Ham and Wallace (1979) Meth. Enz., 58:44; Barnes and Sato (1980) Anal. Biochem., 102:255; U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 5,122,469 or 4,560,655; International Publication Nos. WO 90/03430; and WO 87/00195; the disclosures of all of which are incorporated herein by reference, may be used as culture media. Any of these media may 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), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics (such as gentamycin (gentamicin) drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range) lipids (such as linoleic or other fatty acids) and their suitable carriers, and glucose or an equivalent energy source. Any 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 a CHO cell, preferably a dp12.CHO cell and a suitable medium contains a basal medium component such as a DMEM/HAM F-12 based formulation (for composition of DMEM and HAM F12 media, see culture media formulations in American Type Culture Collection Catalogue of Cell Lines and Hybridomas, Sixth Edition, 1988, pages 346-349) (the formulation of medium as described in U.S. Pat. No. 5,122,469 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 Primatone RL (Sheffield, England), or the equivalent; a cell protective agent, such as Pluronic F68 or the equivalent pluronic polyol; gentamycin; and trace elements.

The glycoproteins of the present invention may be produced by growing cells which express the desired protein under a variety of cell culture conditions. 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 s system may be used, in the later two systems, with or without microcarriers, and operated alternatively in a batch, fed-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 fed batch culture procedure is employed. In the preferred fed batch culture the mammalian host cells and culture medium are supplied to a culturing vessel initially and additional culture nutrients are fed, continuously or in discrete increments, to the culture during culturing, with or without periodic cell and/or product harvest before termination of culture. The fed batch culture can include, for example, a semi-continuous fed batch culture, wherein periodically whole culture (including cells and medium) is removed, and replaced by fresh medium. Fed batch culture is distinguished from simple batch culture in which all components for cell culturing (including the cells and all culture nutrients) are supplied to the culturing vessel at the start of the culturing process. Fed batch culture can be further distinguished from perfusion culturing insofar as the supernate is not removed from the culturing vessel during the process (in perfusion culturing, the cells are restrained in the culture by, e.g., filtration, encapsulation, anchoring to microcarriers, etc. and the culture medium is continuously or intermittently introduced and removed from the culturing vessel).

Further, 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 single step or multiple step culture procedure. In a single step culture the host cells are inoculated into a culture environment and the processes of the instant invention are employed during a single production phase of the cell culture. Alternatively, a multi-stage culture is envisioned. In the multi-stage culture cells may be cultivated in a number of s steps or phases. For instance, cells may be grown in a first step or growth phase culture wherein cells, possibly removed from storage, are inoculated into a medium suitable for promoting growth and high viability. The cells may be maintained in the growth phase for a suitable period of time by the addition of fresh medium to the host cell culture.

According to a preferred aspect of the invention, fed batch or continuous cell culture conditions are devised to enhance growth of the mammalian cells in the growth phase of the cell culture. In the growth phase cells are grown under conditions and for a period of time that is maximized for growth. Culture conditions, such as temperature, pH, dissolved oxygen (dO.sub.2) and the like, are those used with the particular host and will be apparent to the ordinarily skilled artisan. Generally, the pH is adjusted to a level between about 6.5 and 7.5 using either an acid (e.g., CO.sub.2) or a base (e.g., Na.sub.2CO.sub.3 or NaOH). A suitable temperature range for culturing mammalian cells such as CHO cells is between about 30 to 38.degree. C. and a suitable dO.sub.2 is between 5-90% of air saturation. At a particular stage the cells may be used to inoculate a production phase or step of the cell culture. Alternatively, as described above the production phase or step may be continuous with the inoculation or growth phase or step.

According to the present invention, the cell culture environment during the production phase of the cell culture is controlled. According to the process of the present invention, factors affecting cell specific productivity of the mammalian host cell are manipulated such that the desired sialic acid content is achieved in the resulting glycoprotein. In particular, factors which increase cell specific productivity are controlled during the production phase of the cell culture process such that the resulting glycoprotein product contains the desired sialic acid content.

In a preferred aspect, the production phase of the cell culture process is preceded by a transition phase of the cell culture in which parameters for the production phase of the cell culture are engaged.

According to one aspect of the present invention the concentration of a transcription enhancer such as an alkanoic acid, is manipulated to affect cell specific productivity and therefore the resulting sialic acid content of the mammalian cell glycoprotein product. The alkanoic acid may be any one of a number of single or branched chain alkanoic acids that enhance transcription of mammalian proteins. In a preferred embodiment the alkanoic acid is butyric acid and especially the salt thereof, sodium butyrate.

According to the present invention the concentration of sodium butyrate is controlled to control the cell specific productivity. Concentrations of sodium butyrate 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 for the highest cell specific productivity with the most acceptable sialic acid profile. Therefore, according to the present invention concentrations of a transcription enhancer such as sodium butyrate are chosen to obtain the desired sialic acid content.

To increase sialic acid content of the mature glycoprotein, generally, lower concentrations of the transcription enhancer are used. The lower concentration provides for enhanced transcription, but maintains lower cell specific productivity 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 between about 1.0 and 6.0 mM are used. In a particular embodiment about 6 MM sodium butyrate is used. In another embodiment about 1 mM sodium butyrate is used.

In another embodiment a glycoprotein is produced with a decreased level of sialic acid. According to this aspect of the present invention, mammalian cells are cultured under conditions such that cell specific productivity is increased. According to this aspect of the present invention a concentration of alkanoic acid or other appropriate transcription enhancer is chosen such that 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 and 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.

In determining the appropriate concentration of the transcription enhancer such as an alkanoic acid or salt thereof, reference can be made to FIG. 2 (see Original Patent) as well as Table I (see Original Patent) infra at Example I (see Original Patent). According to the present invention lower butyrate concentrations generally result in lower cell specific productivity. According to the invention concentrations of sodium butyrate are chosen, keeping in mind other process parameters such as the osmolality of the production phase. As discussed below the osmolality can affect the cell specific productivity. Concentrations of butyrate are chosen keeping in mind the particular osmolality to be maintained during the production phase.

Alternatively, for other mammalian host cells and other glycoproteins, small test cultures can be prepared and rate of glycoprotein product production, i.e. the cell specific productivity can be determined and the resulting sialic acid content can be used to prepare a similar table and figure appropriate for the particular host cell being cultured, keeping in mind that decreases in cell specific productivity lead to increases in the sialic acid content of the glycoprotein produced. The alkanoic acid or salt thereof, such as sodium butyrate, or other appropriate transcription enhancer is added to the host cell culture at or about the time the production phase of the cell culture is initiated. Conveniently, a transition phase is employed during the cell culture process preceding the production phase in which the cell culture conditions as discussed herein are engaged for the desired cell specific productivity and hence the desired glycoform profile.

The alkanoic acid or salt thereof is added by any means known in the art. In a preferred embodiment the sodium butyrate is added in batch to the fed batch culture system with or without other appropriate nutrients as described herein or known to those skilled in the art of mammalian cell culture.

According to the instant invention the osmolality of the cell culture environment is controlled in addition to the factors noted above to regulate the extent of sialylation of the mature glycoprotein. In one embodiment, osmolality is controlled to control the sialic acid content of the mature protein independent of other factors which affect cell specific productivity. In another embodiment osmolality is controlled in addition to controlling other factors which affect cell specific productivity. In a preferred embodiment both osmolality and the concentration of alkanoic acid are controlled.

The osmolality of the cell culture environment is controlled to produce the desired balance between the cell specific productivity and the sialic acid profile of the resulting glycoprotein. Generally, cell specific productivity is increased when osmolality is increased. An osmolality which produces a protein with the desired sialic acid is chosen keeping in mind that increases in the osmolality generally lead to increase in production rate of the particular protein. In order to decrease the production rate and increase the sialic acid content of the mature glycoprotein osmolality 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 the osmolality generally lead to increase in production rate of proteins. An osmolality is chosen such that the production rate corresponds with the desired product profile. To increase the sialic acid content, production rate is decreased and osmolality is generally maintained within a lower margin is keeping in mind the particular host cell being cultured. Osmolality in the range from about 250 mOsm to about 450 mOsm is appropriate for an increased sialic acid content. More preferably, the osmolality is maintained at about, between 300 and 450 mOsm, according to this aspect of the invention, and more preferably between about 350 and 400 mOsm, and most preferably about 400 mOsm.

For a lower sialic acid content, an osmolality which provides an increased production rate is chosen. According to this aspect of the invention, the osmolality is maintained at about between 350-600 mOsm, and is preferably between about 450-550 mOsm, according to this aspect of the invention.

The skilled practitioner will recognize that media osmolality is dependent upon the concentration of osmotically active particles in the culture fluid and that a number of variables that make up a complex mammalian cell culture medium impact osmolality. The initial osmolality of the culture medium is determined by the composition of the culture medium. The osmolality can be measured using an osmometer such as that sold by Fisher Scientific, Pittsburgh, Pa., under the brand name OSMETTE (or the Osmette model 2007, available from Precision Systems, Inc. Natick Mass.), for example. In order to achieve an osmolality in the desired range, the concentration of various constituents in the culture medium can be adjusted.

Solutes which can be added to the culture medium so as to increase the osmolality thereof include proteins, peptides, amino acids, hydrolyzed animal proteins such as peptones, non-metabolized polymers, vitamins, ions, salts, sugars, metabolites, organic acids, lipids, and the like. In one embodiment, the osmolality is controlled by the addition of a peptone to the cell culture along with other components of the culture medium during a fed batch culture procedure.

According to the present invention the osmolality is maintained or adjusted to the desired range via the addition of for instance, a basal medium formulation including amino acids, various salts (e.g. NaCl), in addition to a peptone. In a preferred embodiment, the culture medium is supplemented with, for instance, a basal culture medium containing excess amino acids (see, e.g., the "Super" medium of U.S. Pat. No. 5,122,469), glucose, and a peptone.

It will be appreciated however, that the concentration(s) of other constituents in the culture medium can be modified in order to achieve an osmolality range as set forth above. By controlling either intermittently or continuously the concentration of glucose (the primary energy source), for instance, in the culture medium throughout the culturing, the osmolality of the medium can be maintained at about the desirable range specified. Controlling 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 in that it limits the pH decrease in the culture medium which necessitates the addition of a neutralizer (e.g., a base such as Na.sub.2CO.sub.3 or NaOH), which causes the osmolality to rise.

The medium can be supplemented to maintain the osmolality within the appropriate margins according to whatever scheme is being used to maintain the cell culture. In a preferred embodiment, the culture system is a fed batch culture system and the medium is supplemented in batch in a feed during the production phase of the cell culture. Additionally the medium can be supplemented during the production phase as described infra.

Alternatively, intermittent off-line sampling of the culture medium can be carried out. The osmolality of the culture medium can then be modified by the modulation of a feed solution as required.

It will be understood by the skilled artisan that the cell culture procedures of the present invention are selected to achieve the desired level of sialylation of the produced protein. Process parameters in addition to those described herein which influence the degree of sialylation include oxygen level, and glucose level. Culture density, time and storage conditions such as temperature also influence sialylation. The present invention is meant to include those process parameters which are additionally most suitable for enhanced sialylation.

III. Recovery of the Glycoprotein

Following the polypeptide 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 host 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. One skilled in the art will appreciate that purification methods suitable for the polypeptide of interest may require modification to account for changes in the character of the polypeptide upon expression in recombinant cell culture.

Especially preferred within the context of the present invention are purification techniques and processes which select for the carbohydrates of the invention. The desired glycoforms of the present invention may be enriched for sialic acid-containing molecules by, for example, ion-exchange soft gel chromatography or HPLC using cation- or anion-exchange resins, wherein the more acidic fraction is collected.

IV. Analysis of the Glycoprotein

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. Thus, for example, techniques such as lectin blotting, well-known in the art, reveal proportions of terminal mannose or other sugars such as galactose. Termination of mono-, bi-, tri-, or tetra-antennary oligosaccharide by sialic acids can be confirmed by release of sugars from the protein using anhydrous hydrazine or enzymatic methods and fractionation of oligosaccharides by ion-exchange or size exclusion chromatography or other methods well-known in the art. The pI of the glycoprotein can also be measured, before and after treatment with neuraminidase to remove sialic acids. An increase in pI following neuraminidase treatment indicates the presence of sialic acids on the glycoprotein.

The carbohydrate structures of the present invention occur on the protein expressed as N-linked or O-linked carbohydrates. The N-linked and O-linked carbohydrates differ primarily in their core structures. N-linked glycosylation refers to the attachment of the carbohydrate moiety via GlcNAc to an asparagine residue in the peptide chain. The N-linked carbohydrates all contain a common Man1-6 (Man1-3) Ma.n.beta.1-4GlcNAc.beta.1-4GlcNAc.beta.g-R core structure. Therefore, in the core structure described, R represents an asparagine residue of the produced protein. The peptide sequence of the protein produced will contain an asparagine-X-serine, asparagine-X-threonine, and asparagine-X-cysteine, wherein X is any amino acid except proline. O-linked carbohydrates, by contrast are characterized by a common core structure, which is the GalNAc attached to the hydroxyl group of a threonine or serine. Of the N-linked, and O-linked carbohydrates, the most important are the complex N- and O-linked carbohydrates. Such complex carbohydrates will contain several antennary structures. The mono-, bi-, tri, -, and. tetra-, outer. structures are important for the addition of terminal sialic acids. Such outer chain structures provide for additional sites for thee specific sugars and linkages that comprise the-carbohydrates of the instant invention.

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 lectin chromatography; HPAEC-PAD, which uses high pH anion exchange chromatography to separate oligosaccharides based on charge; NMR; Mass spectrometry; HPLC; GPC; monosaccharide compositional analysis; sequential enzymatic digestion.

Additionally, methods for releasing oligosaccharides are known. These methods include 1) enzymatic, which is commonly performed using peptide-N-glycosidase F/endo-.beta.-galactosidase; 2) .beta. elimination using harsh alkaline environment to release mainly O-linked structures; and 3) chemical methods using anhydrous hydrazine to release both N-and O-linked oligosaccharides.

Analysis can be performed using the following steps: 1. Dialysis of the sample against deionized water, to remove all buffer salts, followed by lyophilization. 2. Release of intact oligosaccharide chains with anhydrous hydrazine. 3. Treatment of the intact oligosaccharide chains with anhydrous methanolic HC1 to liberate individual monosaccharides as O-methyl derivative. 4. N-acetylation of any primary amino groups. 5. Derivatization to give per-O-trimethylsilyl methyl glycosides. 6. Separation of these derivative, by capillary GLC (gas--liquid chromatography) on a CP-SIL8 column. 7. Identification of individual glycoside derivatives by retention time from the GLC and mass spectroscopy, compared to known standards. 9. Quantitation 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 (HPAE-PAD Carbohydrate System, Dionex Corp.). For instance, sugars can be released by hydrolysis in 20% (v/v) trifluoroacetic acid at 100.degree. C. 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).

Sialic acid can be determined separately by the direct colorimetric method of Yao et al. (Anal Biochem. 179:332-335 (1989)) in triplicate samples. In a preferred embodiment the thiobarbaturic acid (TBA) of Warren, L. J. Biol Chem 238: (8) (1959) is used.

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. Glycoconjugate J., 7:63 (1990)]. 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 trio 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 min.

The carbohydrate may also be analyzed by digestion with peptide-N-glycosidase F. According to this procedure the residue is suspended in 14 .mu.l 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.degree. C. for 3 min. 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 370.degree. C. for 2 hr. and then analyzed by SDS-polyacrylamide gel electrophoresis.

V. Tumor Necrosis Factor Receptor-Immunoglobulin Chimeras

In a preferred embodiment the processes of the present invention are used to produce tumor necrosis factor receptor (TNFR)-Immunoglobulin (Ig) chimeras. Especially preferred among this class of chimeric proteins is the soluble type 1 TNFR IgG.sub.1. The TNFR1-IgG.sub.1 chimeras produced are useful in the treatment or diagnosis of many TNF-mediated or TNF-related diseases and disorders. The term "treatment" in this context includes both prophylactic (prevention), suppression (e.g., of a symptom) and treatment of an existing condition. The pathologic conditions associated with TNF include but are not limited to gram negative and gram positive bacteremia, endotoxic shock, graft rejection phenomena, rheumatoid arthritis, systemic lupus, Crohn's disease and other is autoimmune and inflammatory diseases associated with TNF.

The TNFR1-IgG.sub.1 preparations of the instant invention are useful generally in those indications where monoclonal antibodies to TNF have been found to be useful. For instance, in animal models, monoclonal antibodies to TNF-alpha were found to have protective effect when employed prophylactically (Tracey, K. J. 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 to be safe when administered to human patients with severe septic shock. TNPRI-IgG.sub.1 is suitably used in the treatment of rheumatoid arthritis as well as septic shock.

In a method of treating a disease or disorder associated with TNF a therapeutically active amount of TNFR1-IgG.sub.1 preparation is administered to a subject in need of such treatment. The preferred subject is a human.

An effective amount of a TNFR1-IgG.sub.1 glycoform preparation of the instant invention for the treatment of a disease or disorder is in the dose range of 0.01-100 mg/patient; preferably 1 mg-75 mg/patient and most preferably between about 10 and about 50 mg/patient.

For administration, the TNFR1-IgG.sub.1 preparation should be formulated into an appropriate pharmaceutical or therapeutic composition. Such a composition typically contains a therapeutically active amount of the TNFR1-IgG.sub.1 preparation and a pharmaceutically acceptable excipient or carrier such as saline, buffered saline, dextrose, or water. Compositions may also comprise specific stabilizing agents such as sugars, including mannose and mannitol, and local anesthetics for injectable compositions, including, for example, lidocaine.

The present invention provides compositions which further comprise a therapeutically active amount of an additional active ingredient such as monoclonal antibodies (e.g. anti-TNF antibodies, antibodies to Mac1 or LFA 1) or other receptors associated with TNF production, e.g. IL-1 or IL-2 receptors, etc.

A preferred therapeutic composition for single or combined therapy, as above, comprises a novel TNFR1-IgG.sub.1 preparation of this invention which exhibits prolonged clearance from the blood while retaining significant functional activity. Such a prolonged functional half-life permits simplified, bolus-dose administration and contributes to potency in vivo. Preferred TNFR1-IgG.sub.1 chimeras in the therapeutic composition include the TNFR1-IgG.sub.1 and preparations described herein, for example: (1) TNFR1-IgG.sub.1 preparations comprising a complex oligosaccharide terminated-by one or more residues of a sialic acid; (2) TNFR1-IgG.sub.1 preparations wherein the range of isoelectric point, pI, of the preparation is between 5.5 and 7.5 as determined by chromatofocusing, in which the pI is sensitive to neuraminidase treatment; (3) TNFR1-IgG, preparations having about 1-2 moles of exposed N-acetylglucosamine residues per mole of protein. (4) TNFR1-IgG.sub.1 preparations having a molar ratio of sialic acid to protein of about 4-7, especially about 5-6. (5) TNFR1-IgG.sub.1 preparations having a molar ratio of sialic acid to N-acetylglucosamine of about 0.35 to about 0.5, and more preferably of about 0.4 to about 0.45.

Routes of administration for the individual or combined therapeutic compositions of the present invention include standard routes, such as, for example, intravenous infusion or bolus injection.

Also provided is the use of a TNFR1-IgG.sub.1 preparation of this invention in the manufacture of a medicament for the treatment of a human or animal.
 

Claim 1 of 23 Claims

1. A process for controlling the amount of sialic acid present on an oligosaccharide side chain of a glycoprotein produced by mammalian cell culture which comprises: culturing the mammalian host cell in a production phase of the culture which is characterized by; i) 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 ii) maintaining the osmolality of the cell culture at about 250 to about 600 mOsm.

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