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Methods for modulating mannose content of recombinant proteinsMethods for modulating mannose content of recombinant proteins description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070190057, Methods for modulating mannose content of recombinant proteins. Brief Patent Description - Full Patent Description - Patent Application Claims
BACKGROUND OF THE INVENTION [0001]Higher eukaryotes perform a variety of post-translational modifications, including methylation, sulfation, phosphorylation, lipid addition and glycosylation. Such modifications may be of critical importance to the function of a protein. Secreted proteins, membrane proteins, and proteins targeted to vesicles or certain intracellular organelles are likely to be glycosylated. [0002]N-linked glycosylation is a form of glycosylation involving addition of oligosaccharides to an asparagine residue found in recognition sequences (e.g., Asn-X-Ser/Thr) in proteins. N-linked glycoproteins contain standard branched structures, which are composed of mannose (Man), galactose, N-acetylglucosamine (GlcNAc) and neuramic acids. Protein N-glycosylation typically originates in the endoplasmic reticulum (ER), where an N-linked oligosaccharide (e.g., Glc.sub.3 Man.sub.9 GlcNAc.sub.2) assembled on dolichol (a lipid carrier intermediate) is transferred to the appropriate Asparagine (Asn) of a nascent protein. This is an event common to all eukaryotic N-linked glycoproteins. There are two major types of N-linked saccharides: high-mannose oligosaccharides, and complex oligosaccharides. [0003]High-mannose oligosaccharides typically include two N-acetylglucosamines with many mannose residues (e.g., greater than 4). Complex oligosaccharides are so named because they can contain almost any number of the other types of saccharides, including more than the original two N-acetylglucosamines. Proteins can be glycosylated by both types of oligosaccharides on different portions of the protein. Whether an oligosaccharide is high-mannose or complex is thought to depend on its accessibility to saccharide-modifying proteins in the Golgi apparatus. If the saccharide is relatively inaccessible, it will most likely stay in its original high-mannose form. If it is accessible, then it is likely that many of the mannose residues will be cleaved off and the saccharide will be further modified by the addition of other types of group as discussed above. [0004]After an oligosaccharide chain has been added to a protein, the three glucose and one mannose residues are removed by three different enzymes in a fixed order. This event occurs in the ER and is a signal that the protein can be transported to the Golgi for further processing. After the processing in the ER, the high-mannose type oligosaccharide is formed. The three glucose residues and one specific alpha-1,2-linked mannose residue are removed by specific glucosidases and an alpha-1,2-mannosidase in the ER, resulting in the core oligosaccharide structure, Man.sub.8 GlcNAc.sub.2. The protein with this core sugar structure is transported to the Golgi apparatus where the sugar moiety undergoes various modifications. [0005]In mammalian cells, the modification of the sugar chain proceeds via 3 different pathways depending on the protein moiety to which it is added. The three different pathways are: (1) the core sugar chain does not change; (2) the core sugar chain is changed by adding the N-acetylglucosamine-1-phosphate moiety (GlcNAc-1-P) in UDP-N-acetyl glucosamine (UDP-GlcNAc) to the 6-position of mannose in the core sugar chain, followed by removing the GlcNAc moiety to form an acidic sugar chain in the glycoprotein; or (3) the core sugar chain is first converted into Man.sub.5 GlcNAc.sub.2 by removing 3 mannose residues with mannosidase I; Man.sub.5 GlcNAc.sub.2 is further modified by adding GlcNAc and removing 2 more mannose residues, followed by sequentially adding GlcNAc, galactose (Gal), and N-acetylneuraminic acid (also called sialic acid (NeuNAc)) to form various hybrid or complex sugar chains (R. Kornfeld and S. Kornfeld, Ann. Rev. Biochem. 54: 631-664 (1985); Chiba et al., J. Biol. Chem. 273: 26298-26304 (1998)). [0006]The oligosaccharide content of recombinant proteins can affect the safety and efficacy of therapeutic glycoproteins. Accordingly, methods for controlling the oligosaccharide content, particularly the mannose content, of such glycoproteins would be beneficial. [0007]The high mannose content of glycoprotein compositions, particularly therapeutic antibodies, can significantly affect the safety and efficacy of such proteins during therapeutic use. Without being bound by a particular theory, evidence suggests that high-mannose glycoproteins are cleared from circulation faster than their low mannose counterparts due to, for example, mannose receptors on macrophages and dendritic cells. Additionally, high mannose glycoproteins are expected to be more immunogenic. Accordingly, it is desirable to produce therapeutic glycoproteins such as, for example, therapeutic antibodies, having low mannose content. [0008]The present inventors solves this need in the art by providing methods for modulating (e.g., controlling or reducing) the mannose content of recombinantly produced proteins and peptides. SUMMARY OF THE INVENTION [0009]The present invention is based, at least in part, on the discovery of factors that affect mannose content and, in particular, high-mannose content, of recombinantly expressed glycoproteins. [0010]Accordingly, in one aspect, the present invention provides a method of modulating the mannose content (i.e., on an oligosaccharide side chain) of a recombinant glycoprotein produced in a mammalian host cell by manipulating the cell culture conditions such that the glycoprotein produced by the cell has low-mannose content. As used herein, the term "low-mannose content" refers to glycoprotein compositions wherein less than about 10%, or less than about 8%, or less than about 5% (e.g., about 4% or less) of the glycoproteins in the composition have more than 4 mannose residues (i.e., are species of M5 or greater). As used herein, the term "low-mannose content" also refers to glycoprotein compositions wherein less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, or any values between any of these preceding ranges, or even at zero. [0011]In one embodiment of the invention, low-mannose content is achieved by maintaining the cell culture environment at low osmolality (e.g., less than about 600 mOsm/Kg, or less than about 500 mOsm/Kg, or less than about 400 mOsm/Kg, e.g., between about 380 to 250 mOsm/Kg). This enriches the cell culture for glycoproteins having low mannose-content i.e., having 4 or fewer mannose residues on the oligosaccharide side chains of the glycoprotein. Accordingly, in a particular embodiment, the invention provides a method for producing a recombinant glycoprotein having low-mannose content comprising culturing a mammalian host-cell (e.g., in an expansion or production phase of the culture) which expresses the glycoprotein in a medium having an osmolality of about 600 mOsm/Kg or less (e.g., between a range of about 200 and 600 mOsm/Kg, e.g., about 250 and 550 mOsm/Kg, about 250 and 500 mOsm/Kg, about 250 and 450 mOsm/Kg, about 250 and 400 mOsm/Kg, about 250 and 380 mOsm/Kg, or about 250 and 350 mOsm/Kg). [0012]The foregoing osmolality ranges can be achieved by manipulating a number of cell culture parameters including, but not limited to, concentrations of one or more of salts, vitamins, sugars, peptones and amino acids in the cell culture medium. Accordingly, in a particular embodiment, the invention provides a method of producing a recombinant glycoprotein having low-mannose content by culturing a host-cell which expresses the glycoprotein in a medium containing potassium at a concentration of about 70 mM or less (e.g., about 10 mM to about 50 mM); and/or sodium at a concentration of about 200 mM or less (e.g., about 50 mM to about 100 mM) and maintaining the osmolality of the cell culture at about 600 mOsm/Kg or less. [0013]In still another embodiment, the invention provides a method of producing a recombinant glycoprotein having low-mannose content by culturing a host-cell which expresses the glycoprotein in a medium which is substantially free of one or more amino acids selected from the group consisting of alanine, arginine, aspartic acid and glutamic acid, and maintaining the osmolality of the cell culture at about 600 mOsm/Kg or less. [0014]In addition, in still another embodiment, the medium can include one or more vitamins selected from the group consisting of biotin, D-calcium pantothenate, choline chloride, folic acid, i-inositol, niacinamide, pyridoxal HCl, pyridoxine HCl, riboflavin, thamine HCl and cyanocobalamin, at a concentration of about 0.00005 g/L to about 0.9 g/L. In yet another embodiment, the medium includes glucose at a concentration of about 1 mM to about 90 mM. In a further embodiment, the medium includes one or more peptones selected from the group consisting of yeast extract, yeast hydrolysate, soy peptone, soy hydrolysate, wheat peptone and wheat hydrolysate, at a concentration of about 0.5 g/L to about 60 g/L. [0015]In yet a further embodiment of the present invention, the cell culture medium can include one or more osmoprotectants in an amount necessary to maintain the osmolality at a desired level, e.g., about 600 mOsm/Kg or less. Suitable osmoprotectants are known in the art and include, for example, betaine, glycine, L-threonine and L-proline, and derivatives thereof such as, for example, glycine betaine and betaine aldehyde. In a particular embodiment, the osmoprotectant (e.g., betaine) is present at a concentration of about 20 mM or greater in the cell culture medium. In particular embodiments, the osmoprotectant (e.g., betaine) is present at a concentration of about 1 mM to about 100 mM or at about 20 mM to about 30 mM. [0016]Additional cell culture parameters that may be controlled, either alone or in combination with one or more of the parameters described herein include, for example, temperature and duration of time which the cells are cultured for. In certain embodiments, a host-cell expressing a recombinant glycoprotein is cultured at a temperature of about 31.degree. C. to about 38.degree. C. In certain other embodiments, a host cell expressing a recombinant glycoprotein is cultured for a period ranging from about 5 days to about 14 days. [0017]Suitable host cells for expressing recombinant glycoproteins according to the present invention are well known in the art and include any of those described herein, such as CHO cells, lymphocytic cells (e.g., NSO cells) and a variety of other mammalian cells. [0018]The present invention can be employed to product a wide variety of glycoproteins having low-mannose content as described herein. In a particular embodiment, the invention is used to produce a recombinant monoclonal antibody or an antigen-binding fragment thereof having low-mannose content. Suitable antibodies can include, for example, murine, chimeric, humanized and fully human antibodies, as well as other antibody forms known in the art. In another particular embodiment, the antibody binds IL-15, which includes but are not limited to the antibodies disclosed in U.S. Publication No.: 2003-0138421, which is incorporated by reference herein in its entirety. In another particular embodiment, the antibody is a fully human monoclonal antibody that binds IL-15 having a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:4 and/or a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:2, as well as homologous sequences which bind IL-15 (e.g., having amino acid sequences of about 80, 85, 90, 95% or greater identity to SEQ ID NO: 4 or SEQ ID NO: 2, respectively). In a further particular embodiment, the antibody is a human antibody that binds IL-15, or an antigen-binding fragment thereof, having a light chain variable region comprising one or more complementarity determining regions (CDRs) set forth in SEQ ID NOs:8-10, as well as homologous sequences which bind IL-15 (e.g., having amino acid sequences of about 80, 85, 90, 95% or greater identity to any of SEQ ID NOS: 8-10, respectively), and a heavy chain variable region comprising one or more complementarity determining regions (CDRs) set forth in SEQ ID NOs:5-7 as well as homologous sequences which bind IL-15 (e.g., having amino acid sequences of about 80, 85, 90, 95% or greater identity to any of SEQ ID NOS: 5-7, respectively). In a particular embodiment, a human monoclonal antibody that binds IL-15 or an antigen-binding fragment thereof, includes a light chain variable region comprising all three CDRs set forth in SEQ ID NOs:8-10, and a heavy chain variable region comprising all three CDRs set forth in SEQ ID NOs: 5-7, or conservative amino acid substitutions thereof. [0019]In yet another aspect, the present invention provides recombinant glycoproteins having low-mannose content produced by the methods described herein. Accordingly, such glycoproteins may include any of the aforementioned therapeutic glycoproteins, such as antibodies, hormones, enzymes, peptides and other glycoproteins. [0020]Also encompassed by the present invention are compositions comprising any of the aforementioned glycoproteins having low-mannose content. In a particular embodiment, the composition is a pharmaceutical composition that includes an isolated glycoprotein (e.g., an isolated human monoclonal antibody that binds IL-15 or an antigen binding fragment thereof) having low-mannose content and a pharmaceutically acceptable carrier. [0021]Accordingly, in still another aspect, the present invention provides a method of treating or preventing a disorder that is associated with an overexpression of human IL-15 and/or in which a downregulation or inhibition of human IL-15 induced effects is beneficial is provided, by administering to a subject an isolated IL-15 antibody having low-mannose content. Exemplary disorders include, but are not limited to, vasculiitis, psoriasis, multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease (e.g., Crohn's disease or celiac disease), allograft rejection, graft versus host disease, T-cell lymphoma, and T-cell leukemia. [0022]Accordingly, in still another aspect, the present invention provides a method of treating or preventing a disorder that is associated with an overexpression of human IL-15 and/or in which a downregulation or inhibition of human IL-15 induced effects is beneficial is provided, by administering to a subject an isolated IL-15 antibody having low-mannose content. Exemplary disorders include, but are not limited to, arthritides, connective tissue disorders, ophthalmological disorders, neurological disorders, gastrointestinal and hepatic disorders, allergic disorders, hematologic disorders, skin disorders, pulmonary disorders, malignancies, transplantation-derived disorders, endocrinologic disorders, vascular disorders, gynecological disorders and infectious diseases. Continue reading about Methods for modulating mannose content of recombinant proteins... 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