Title of Invention	A PROCESS FOR THE PREPARATION OF ERYTHROPOIETIN
Abstract	The present invention relates to a process for the preparation of a glycoprotein using adherent type substrate in perfusion-mode, wherein in the production phase, the monosaccharide content in the perfusing medium is 7 gm/day to 137.5 gm/day.

Full Text

FORM 2 THE PATENTS ACT, 1970 (39 of 1970) & The Patents Rules, 2003 COMPLETE SPECIFICATIION (See section 10 and rule 13) 1. TITLE OF THE INVENTION : "A PROCESS FOR THE PREPARATION OF GLYCOPROTEIN" 2. APPLICANT (S) (a) NAME (b) NATIONALITY (c) ADDRESS Gennova Biopharmaceuticals Ltd. India T-184, MIDC, Bhosari, Pune 411026, Maharashtra, India 1 FIELD OF THE INVENTION: The present invention relates to a process for the preparation of glycoprotein in high yield. DESCRIPTION OF BACKGROUND AND RELATED ART: A "Glycoprotein" refers generally to peptides and proteins having more than about ten amino acids and at least one glycosylation site that is occupied only in a fraction of the glycoprotein product, i.e., they display variable site-occupancy or variations in N- and O-glycosylation site-occupancy. The glycoproteins may be homologous to the host cell, or preferably, they are heterologous, i.e. foreign to the host cell being utilized, such as a human protein produced by a recombinant cell. Preferably, mammalian glycoproteins (glycoproteins that were originally derived from a mammalian organism) are used, more preferably, those which are directly secreted into the medium. Erythropoietin is an example of a glycoprotein.Erythropoietin (EPO) hormone maintains the physiological levels of circulating red blood cells by regulating the proliferation and differentiation of erythroid progenitor cells and. EPO is an acidic glycoprotein of molecular weight approximately 34,000 dalton. EPO may occur in three forms: alpha, beta and asialo. The alpha and beta forms differ slightly in carbohydrate components, but similar in having the same potency, biological activity and molecular weight. The asialo form is an alpha or beta form with the terminal carbohydrate (sialic acid) removed. When the body is in a healthy state, wherein tissues receive sufficient oxygenation from the existing number of erythrocytes, EPO is present in very low concentrations in plasma. This normal low concentration is enough to stimulate replacement of red blood cells which are lost normally through aging. EPO is primarily produced in the liver in the fetus and about 90% of its production occurs in the kidney after birth. If EPO levels fall due to the reasons such as chronic or acute renal failure, e. g. in cancer patients, EPO must be externally administered to avoid a rising 2 anemia. Since the discovery of the EPO gene and its expression in rodent cells, a therapeutically active human erythropoietin is available. The human EPO gene encodes a 27 amino acid signal peptide and a 166 amino acid protein with a calculated molecular weight of 18399 Dalton. The mature protein has usually 165 amino acids and one amino acid N-terminal deletion. The signal sequence directs the peptide to the cellular compartments leading to a mature protein with three N-and one O-glycosylation site. The sugar moiety, which makes about 40% of the total molecular weight, is essential for the full biological activity of EPO. Several studies have shown that the number of terminal sialic acid residues has a positive effect on the in vivo half-life, although the in vitro activity, i. e. the binding to the receptor, is highest in the non or partly glycosylated form (Takeuchi and Kobata, 1991 (Glycobiology, 1 (4): 337-346). The mean life of erythrocytes in humans is 120 days. A human being loses 1/120 erythrocytes each day and this loss must be continuously restored to maintain an adequate level of red blood cells. The existence of EPO was first postulated by the turn of the century and was definitely proved by Reissman and Erslev early in the'50s, as given by Carnot, et al.,C. R. Acad. Sci. (France), 143,384-6 (1906); Carnot, et al., C. R. Acad. Sci.(France), 143,432-5 (1906); Carnot, et al., C. R. Soc. Biol., 111,344-6 (1906);Carnot, C. R. Soc. Biol, 111,463-5 (1906); Reissman, Blood, 1950,5, 372-80 (1950) and Erslev, Blood, 8,349-57 (1953). Reissman and Erslev's experiments were promptly confirmed by other researchers as described by Hodgson, et al., Blood, 9, 299-309 (1954); Gordon, et al., Proc. Soc. Exp. Biol. Med., 86,255-8 (1954) and Borsook, et al., Blood, 9,734-42 (1954). EPO is produced in the body and is directly proportional to the extent of tissular hypoxia. Hypoxia is a condition when oxygen transport by blood cells is in the circulation is reduced. The amount of erythropoietin in the circulation is increased under these conditions. Hypoxia may be caused by loss of large amounts of blood through hemorrhage, destruction of red blood cells by over-exposure to radiation, reduction in oxygen intake due to high altitudes or prolonged unconsciousness, or various forms of anemia. When 3 tissues undergo hypoxic stress, erythropoietin stimulates the conversion of primitive precursor cells in the bone marrow into proerythroblasts, which subsequently get matured and synthesize hemoglobin and are released into the circulation as red blood cells and thus EPO increases red blood cell production. When the number of red blood cells in circulation is greater than needed for normal tissue oxygen requirements, erythropoietin in circulation is decreased. EPO has shown great efficiency in the treatment of anemia, especially anemia derived from renal failure as given by Eschbach, et al., N. England J. of Med., 316, 2, 73-78 (1987); Krane, Henry Ford Hosp. Med. J., 31, 3,177-181 (1983). Prior attempts to obtain erythropoietin in good yield from plasma or urine have proven relatively unsuccessful. Complicated and sophisticated laboratory techniques are necessary and generally result in the collection of very small amounts of impure and unstable extracts containing erythropoietin. At present, various patents and patent application disclose erythropoietin and the process for preparation of the same. U.S patent 5,756,349 describes production of erythropoietin by vertebrate cells comprising non-human DNA sequences which control transcription of DNA encoding human erythropoietin. Another U.S patent 5,618,698 describes a method for the preparation of active erythropoietin product, where the host cell is transformed with isolated DNA sequences. An international application WO 00/14261 describes the production of erythropoietin in kidney cells transformed with an expression vector containing human erythropoietin gene driven by myeloproliferative sarcoma virus promoter and using tryptophan synthetase gene from E.coli as a selectable marker by utilizing microcarrier and suspension cell culture. Another U.S patent 7,067,279 describes the use of betaine to improve viability of recombinant cells for production of secreted polypeptide such as a soluble TNF receptor, a soluble IL-4 receptor, a soluble IL-1 type II receptor, a soluble flt3 ligand, a soluble CD40 4 ligand, an erythropoietin, an antibody, an Fc-fusion protein, a calcitonin, a growth hormone, an insulin, an insulinotropin, insulin-like growth factors, a parathyroid hormone, an interferon, a nerve growth factor, a glucagons, an interleukins, a colony stimulating factor, a glucocerebrosidase, a super oxide dismutase, a tissue plasminogen activator, a Factor VIII, a Factor IX, an apolipoprotein E, an apolipoprotein A-I, a goblin, an IL-2 receptor, an IL-2 antagonist, alpha-1 antitrypsin, and an alpha-galactosidase A in the culture medium. Another international application WO 03/045995 describes the use of fed batch bioreactor process for producing human erythropoietin by transforming the host cell encoding the recombinant polypeptide by utilizing a medium, which comprised of a plant derived peptone, an energy source, a source of iron, non ferrous metal ions along with a few other supplements. Yet another international application WO 00/27997 describes the use of insulin in the culture medium for massive culture of cells producing erythropoietin by using roller bottle flask. An EP Patent, 0343635 describes process for continuously culturing adherent animal cells in suspension, where (1) a fresh medium is fed into a culture vessel, and a spent medium is withdrawn from the vessel, (2) the adherent animal cells in the serum-free medium in the vessel are maintained in suspension, (3) the adherent animal cells in suspension are caused to exist at a density of at least 3 x 106 cells/ml, and (4) the concentration of a calcium ion in the medium under the culturing conditions is maintained at 0.002 mM to 0.3 mM. One of the disadvantages of suspension cell cultures lies in the increase of metabolic stress on cells due to end product getting contaminated with wastes, which in turn also increases load on the purification process. (Wurm F.M. Nature Biotechnology, Vol. 22, Nov 2004, Page no. 1393-1397). 5 The highest specific productivity in the form of accumulated yield reported for any kind of recombinant protein in general from industrial sources is about 4.7 g/1. (Wurm F.M. Nature Biotechnology, Vol. 22, Nov 2004, Page no. 1393-1397). Current art fails to address the unmet need for large-scale production of EPO and there is a need for more effective, user friendly high yielding process for the preparation of EPO. The present invention addresses the above mentioned needs and provides an improved and a more effective, user friendly high yielding process for the preparation of EPO. SUMMARY OF THE INVENTION Accordingly, the invention provides a process for preparation of a glycoprotein comprising the steps of: (a) growing under nutrient conditions host cells capable of producing/ releasing erythropoietin at its production phase, wherein the host cells are perfused with a nutrient medium having a monosaccharide content of 7gm/day to 137.5 gm/day; and (b) harvesting and recovering the glycoprotein from the medium. In one aspect, the nutrient medium includes a growth medium and a production medium. The production medium is perfused at a rate of 2 to 25 Lit/day, preferably at the rate of 6 to 20 Lit/day. The perfusion may be effected for 45 days. In yet another aspect of the invention, the erythropoietin produced by the process is harvested, isolated and purified by affinity column chromatography, metal chelation chromatography, gel filtration chromatography and/or ion exchange chromatography. BRIEF DESCRIPTION OF THE DRAWINGS: Fig. 1 is a graph that illustrates the cumulative yield (gm/Lit) against process time (hr) for the production of erythropoietin in perfusion batch process, in comparision with prior art. 6 Fig. 2 is a graph that illustrates media flow rate (Lit/day) against process time (hr) for the production of erythropoietin in perfusion batch process, where there is no perfusion till 48 hrs as the glucose consumption rate is very slow initially, then the flow rate is slowly increased based on the glucose consumption rate to a maximum of around 20 Lit/day. DETAILED DESCRIPTION OF THE INVENTION; Definitions: Some of the terms frequently used are defined as given below: "Growth phase" as referred herein includes the complete growth cycle of a cell. "Production phase" of the cell culture refers to the period of time during which the desired glycoprotein, i.e. erythropoietin is produced. The terms "nutrient medium", "growth medium" or "production medium" refer to a nutrient solution used for growing mammalian cells that typically provides at least one component from one or more of the following categories: 1) an energy source, usually in the form of a carbohydrate or a monosaccharide such as glucose; 2) all essential amino acids, and usually the basic set 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 trace elements are defined as inorganic compounds or naturally-occurring elements that are typically required at very low concentrations, usually in the micro molar range. The said medium may be optionally supplemented with a component selected from any of salts, buffers, plasma, nucleosides and bases, proteins and tissue hydrolysates, antibiotics and lipids, including monosaccharide such as glucose. Further, the growth medium may be optionally supplemented with one or more components from any of the following categories: 7 1) Plasma components as defined above and/or growth factors such as, for example, insulin, transferrin, and EGF; 2) Salts and buffers such as, for example, sodium chloride, calcium, magnesium, phosphate, and HEPES; 3) Nucleosides and bases such as, for example, adenosine, thymidine, and hypoxanthine; 4) Protein and tissue hydrolysates; 5) Antibiotics such as GENTAMYCIN.TM. drug; 6) Lipids such as linoleic or other fatty acids and their suitable carriers. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. Preferably, the supplements used are sodium bicarbonate, a monosaccharide, gentamycin, Methotrexate (MTX) and Dialyzed Foetal Bovine Serum (DFBS). Examples of the culture/nutrient medium employed for the purposes of the present invention may be any mammalian cell culture medium suitable for growth of cells producing erythropoietin. Different types of growth mediums may be used, such as a RPMI-1640, Minimum Essential Medium (MEM), Iscove's Modified Dulbecco's Medium (IMDM), Dulbecco's Modified Eagle's Medium (DMEM)/HAM F-l- based formulation such as a DMEM/HAM F-12-based formulation (American Type Culture Collection Catalogue of Cell Lines and Hybridomas, Sixth Edition, 1988, pages 346-349), together with amino acids, sugar, vitamins, glycine, hypoxanthine, thymidine, recombinant human insulin, hydrolyzed peptone such as PRIMATONE HS.TM. or PRIMATONE RL.TM. (Sheffield, England), or its equivalent. The nutrient medium is substantially "serum free". Preferably, no serum may be used in the culture medium. However, traces of serum if present in the medium may range up to 5%, preferably up to 1% or more preferably 0.1%. The serum may be a serum from any mammalian source e.g. fetal bovine serum (FBS) or bovine pituitary extract (BPE). 8 In general, for large scale production of any biological product, the preferred process so far is a fed batch bioreactor but it is limited due to disadvantages of single harvest being isolated, further, the limitation is compounded by accumulation of several byproducts amounting to increase in impurities and there by increasing the load during the down stream process. The cumulative yield of EPO in the bioreactor is generally independent of the quantity of the working volume. Also, the isolated yield if expressed in gm/L also is independent of the volume. For instant invention, the reactor size is 7.5 L with working volume of about 5 L. If the reactor size and working volume is varied, then the isolated yield in the absolute gm quantity would vary proportionately but in terms of gm/L would be almost same. Preferably, the process of the invention may be performed in fed batch mode or perfusion mode. In fed batch mode the medium is supplied to the culruring vessel continuously in discrete increments with or without product harvest before termination of culture. In perfusion mode, the cells are restrained in the culture by anchoring to a carrier or encapsulation or any other method and the nutrient medium is continuously or intermittently introduced into the culruring vessel in discrete increments. Alternatively, the process may be effected in a multi-stage manner, wherein the host cells are cultivated in a first phase and allowed to grow to a viable state and then transferred to another culture medium for production of glycoprotein/erythropoietin. Preferably, the invention employs adherent type of cell culture process, which is advantageous. Further the invention preferably employs packed bed technology, wherein cells are shielded from the turbulence of impeller rotation and sparged gases. The shear forces are also maintained at minimum level in a packed bed, allowing higher rates of agitation which results in increased mass transfer of oxygen and nutrients. Thereby, significantly contributing to cumulative high yield of desired protein. An example is the commercially available FibraCel matrix, which facilitates an easy culturing of large number of cells while concentrating the secreted protein. 9 According to the present invention, cell culture environment during the production phase of the cell culture is controlled. The parameters for obtaining the improved yield are selected during the production phase with respect to the following parameters, a. The monosaccharide content in the perfusing medium is 7 to 137.5 gm/day. b. Perfusion rate of the production medium is in the range of 2 to 25 Lit/day and preferably in the range of 6 to 20 Lit/day. c. Temperature is maintained in the range of 30°C to 40°C, preferably 31°C to 39°C and more preferably in between 32°C to 38°C; d. Agitation speed is maintained in the range of 60 to 140 RPM, preferably in the range of 70 to 130 and more preferably in the range of 80 to 120 RPM; e. Dissolved Oxygen is maintained in the range of 5% to 50%, preferably 8 % to 45 % and more preferably in the range of 10% to 30%; Preferably, the present invention provides with an improved process for the large scale production of erythropoietin using perfusion-based bioreactor, which has the advantages of: a) Multiple harvesting. b) Removal of cellular debris and inhibitory by-products. c) Removal of enzymes (e.g. proteases) released by dead cells that may destroy or damage the product. d) Load mainly is of the desired product in the downstream and yield is more and hence better efficiency of reactor and facilities. e) Shorter exposure time of the final product (EPO) to potentially harsh production conditions (high or low pH). f) High per unit volumetric productivity due to high cell density and metabolism. g) Provides an essentially constant environment. h) Less cumbersome, since production of desired product is in continuous manner. i) Saves time cycle of production batch as the process till upstream is continuous. j) Even if size of the reactor is small, more quantity of production and final product is achieved by perfusion batch culture process due to multiple harvesting. 10 k) Cell bank efficiently used as there is continuous removal of any unwanted side products produced during the batch process in perfusion mode, thereby enhancing yield of the final product by using the same density of cells. "Host cell" as used herein means any cell or cell line capable of producing erythropoietin. Such a cell includes an animal or human cell whose genome contains an active EPO gene which is transcribed and translated during the culture of the cell in a substantially serum free medium. In the alternative, the EPO gene may be introduced into the host cell as an active endogenous gene for production of EPO. Preferably, the CHO (Chinese Hamster Ovary) cell lines are used for production of erythropoietin. The process of the invention may be employed to produce other glycoproteins on large scale. Such glycoproteins include clotting factors such as factor VIIIC, factor IX, tissue factor, and von Willebrands factor; anti-clotting factors such as Protein C; atrial natriuretic factor; lung surfactant; a plasminogen activator; thrombin; hemopoietic growth factor, etc. The glycoprotein of the present invention may be produced by growing cells, which express the desired glycoprotein under cell culture conditions. For instance, cell culture procedures for the large or small-scale production of glycoproteins are potentially useful within the context of the present invention. The present invention is further illustrated by the following, non-limiting examples, which should not be construed to limit the scope of the invention in any manner as they are only intended to illustrate the concept of the invention. Example 1 1) Preparing seed culture using mammalian cells to be used for production phase: Erythropoietin can be produced by making use of CHO (Chinese Hamster Ovary) mammalian cell culture capable of producing EPO, using the technology known in the public domain. 11 According to the preferred aspect of the invention, cell culture conditions are devised to enhance growth of the mammalian cells in the growth phase of the culture. In the growth phase, cells are grown under conditions and for a period of time that is maximized for growth. 2) Inoculation of the seed culture in a bioreactor and the production phase: The seed culture is aseptically transferred to the perfusion mode/batch. The volume of the seed is reached to 2-2.5L and the temperature is reached till 37 °C, with agitation speed in between 60-140 RPM, Dissolved oxygen is in the range of 5-50 % and the pH is in the range of about 7.0 to 7.5, most preferably 7.2 to 7.4. The specific rate of consumption of the monosaccharide is constant throughout the bioreactor operation indicating a consistent metabolic rate of the cells. The perfusion of the IMDM media is continued for 4-7 days and then the initial production is discarded as 10% FBS (foetal bovine serum) may be present in the production. Optionally, IMDM growth medium is replaced by modified CHO-S-SFM II production medium, where modified CHO-S-SFM II medium acts as the production medium and is used for perfusion, where the monosaccharide content is 7 to 137.5 gm/day. Accordingly, herein is a process, describing the preparation of the erythropoietin using adherent type Chinese Hamster Ovary cell in a perfusion batch process, which utilizes packed bed technology comprising of a modified CHO-S-SFM II production medium for production phase. Following the production phase, the glycoprotein of interest can be recovered from the culture medium using techniques, which are well established in the art. The glycoprotein of interest preferably is recovered from the culture medium as a secreted polypeptide, although it may also be recovered from production medium containing the cell culture and secreted protein. 12 3) Harvesting, isolation and purification The harvest containing the glycoprotein in the secreted form is collected in a continuous manner from the bioreactor. Further, the harvest or lysate can be centrifuged to remove particulate cell debris. The glycoprotein thereafter is isolated and purified from contaminant soluble proteins and polypeptides, with either or the combination of following procedures such as, 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.TM.; and protein A SEPHAROSE.TM. columns to remove contaminants such as IgG. In a preferred embodiment, the following chromatographic procedures being exemplary of suitable purification procedures: by affinity chromatography metal chelating chromatography and gel filtration chromatography and ion exchange chromatography. The sequence of the same may be interchangeable. Initially the harvest may be subjected to filtration, wherein, the harvest is optionally treated with a suitable agent, such as beta propiolacton, aldehydes, sodium caprylate, wherein, the preferably used agent is sodium caprylate. The filtrate obtained by filtration may be loaded on an affinity column capable of selective binding of erythropoietin, and erythropoietin may be eluted from the affinity column with elution buffer at a pH around 6.5 to 8.0. The suitable affinity columns are well known in the art and are commercially available. The suitable affinity columns include cibacron blue dye, blue sepharose and lectin sepharose, wherein, the preferably used is a blue sepharose affinity column, which is preferably eluted at a pH, from about 6.5 to about 7.6. The elute was loaded in to the metal chelating column, which can make use of a Poros 20 Resin, saturated with a metal ion solution, in which the metal ion might be a monovalent or 13 divalent ion, preferably is a divalent metal ion, which is selected from Cu, Zn, Fe, Ni. The protein can be passed out in a flow through from the metal chelating column. The flow through can be loaded in gel filtration column, containing sephadex G 25 resin. The eluent now can be loaded into ion exchange column having poros 50 D resin, where the load can be eluted by using elution buffer comprising of Tris solution and NaCl, having a pH range of 7.4 to 8.4 preferably in the range of 7.6 to 8.0. The eluent can be subjected to another ion exchange column containing Poros 50 D resin, where the elution is done by passing elution buffer comprising of citrate buffer solution, having a pH range of 6.0 to 8.0, preferably at 6.5 to 7.6. The erythropoietin recovered and purified following the foregoing protocol typically is more than 95 % pure. The erythropoietin obtained by the process of the invention as set out above is recovered in cumulative yield of 25 to 30 gm/ltr as compared to prior art. The same is illustrated in figure 1. The principles, preferred embodiments, and modes of operation of the present invention have been described in the foregoing specification. The invention which is intended to be protected herein, however, is not to be construed limited to the particular forms disclosed, since these are to be regarded as illustrative rather than restrictive. Variations and changes may be made by those skilled in the art, without departing from the spirit of the invention. Example 2 Preparation of the seed culture Materials and procedure: The recombinant CHO cell line was used The CHO cell line as used may be a known cell line. The growth medium used was IMDM medium (Gibco™, Invitrogen Corporation), which is supplemented with methotrexate, gentamycin, sodium bicarbonate, a monosaccharide and dialyzed foetal bovine serum (DFBS). The different flasks used for the growth were T-25, T-75 and T-175 flask (having 25, 75 and 175 cm2 surface area respectively) and roller 14 bottles (850 cm2). Finally, the cells were fully grown till the required number of cell count was observed. Process: The Erythopoietin producing recombinant CHO cells were taken from manufacture's working cell bank (MWCB) and growth was carried out in a flask/ roller bottle with IMDM growth medium. The IMDM medium included methotrexate, gentamycin, and sodium bicarbonate, a monosaccharide and supplemented with dialyzed foetal bovine serum (DFBS). Here, depending upon the size of the T-flasks, quantity of the IMDM medium varies, e.g., the quantity used in T-175 flask was 50ml. The cell culture amplification was carried out in T-flasks starting from T-25 to T175 flasks, from which the culture was transferred to roller bottles. The cells were grown at a temperature range of 36 °C ± 0.5 °C under atmosphere containing 4-6% (v/v) of C02. When 80-90% of confluence was achieved the monolayer was washed with phosphate buffer saline (PBS) and trypsinized. Then, the cells were seeded and reincubated under similar conditions for a period of about 2.5 days. Cell counting and viability of cells were checked using hemacytometer and trypan blue respectively. Sub culturing in roller bottle is carried out till cell count reaches 10,000-15,000 million cells which issued as a seed culture. Example 3 Production phase Materials and procedure: IMDM was used as a growth medium. CHO-S-SFMII medium as a production medium included sodium bicarbonate, gentamycin, methotrexate (MTX), and supplemented with dialyzed foetal bovine serum (DFBS). The bioreactor used was a perfusion batch process (NEW BRUNSWICK SCIENTIFIC, CELLIGEN PLUS Bioreactor/ fermentor), with capacity of 7.5 L. Process: The seed culture produced as per example 1 was aseptically transferred to the perfusion batch process comprising of IMDM medium, which contains methotrexate (1 uM), gentamycin (50 mg/Lit), sodium bicarbonate (3 gm/Lit), monosaccharide (1.5 gm/Lit) and supplemented with dialyzed foetal bovine serum (DFBS) (10% v/v). The temperature was maintained in the range of 36 °C + 0.5 °C. After 4-7 days of the process in the bioreactor the initial harvest was discarded and the IMDM growth medium was 15 replaced by CHO-S-SFM II medium which contains sodium bicarbonate (2.45 gm/Lit) gentamycin (50 mg/Lit), methotrexate (MTX) (1 uM), dialyzed foetal bovine serum (DFBS) (0.5% v/v). The temperature was decreased at the rate of 0.5 °C per day. The agitation speed was maintained in the range of 80 to 120 RPM. The monosaccharide content was always 7 gm/day to 137.5 gm/day by perfusion of fresh, sterile CHO-S-SFM II medium at a rate of 6 to 20 Lit/day. The pH was maintained in the range of 7.2 to 7.4. After starting the perfusion with CHO-S-SFM II medium, the harvest was collected containing the protein in the secreted form. The total period of production phase was about 45 days. Example 4 Downstream process: Materials and procedure: Sodium caprylate (4.0 mM), For affinity chromatography elution buffer is used with blue sepharose resin. Elution buffer (1L) comprises of Na2HP04.12H20 5.73 gm and NaH2P04.12H20 0.624 gm and NaCl 41.0 gm dissolved in water for injection (WFI), having a pH range of 6.5 to 7.6. In metal chelate chromatography, Poros 20 MC resin was used. Elution buffers I (1L) comprise Na2HP04.12H2 5.8 gm, NaH2P04.12H20 0.62 gm, NaCl 29.23 gm with 5mM imidazole dissolved in water for injection (WFI), having a pH range of6.5to7.6. Elution buffers II (1L) also comprise Na2HP04.12H2 5.8 gm, NaH2P04.12H20 0.62 gm, NaCl 29.23 gm with 110 mM imidazole dissolved in water for injection (WFI), having a pH range of 6.5 to 7.6. In Gel Filtration Chromatography (GFC), sephadex G 25 resin was used. Equilibration buffer (11) used as exchange buffer comprise Tris 2.5 gm dissolved in water for injection (WFI), having a pH range of 7.6 to 8.4. In Ion exchange chromatography, Poros 50 D resin was used. Elution buffer (1L) comprises Tris 2.42 gm, NaCl 10.5 gm dissolved in water for injection (WFI), having a pH range of 7.6 to 8.4. 16 In another Ion Exchange chromatography, Poros 50 D resin was used. Elution buffer (1L) comprised of sodium citrate di hydrate 5.8 gm, citric mono hydrate 0.01 gm, NaCl 29.2 gm dissolved in water for injection (WFI), having a pH range of 6.5 to 7.6. Process: The harvest containing the protein was treated with sodium caprylate followed by filtration through 0.45/0.2 u Millipore filter. The filtrate obtained was subjected to blue sepharose affinity chromatography, wherein the column was eluted using elution buffer having a pH range of 6.5 to 7.6, to elute out the desired protein. The said eluent was subjected to metal chelate chromatography (saturated with 100 mM copper sulphate solution). The protein was passed out in a flow through from the metal chelating column. The column was eluted using elution buffers I and II. The elution buffers I and II (1.5-2.0 CV) containing 20mM PB and 0.5 M NaCl and having a pH range of 6.5 to 7.6 were passed to remove impurities. Further, the flow through was subjected to Gel filtration chromatography containing sephadex G 25 resin, wherein the elution was done by using equilibration buffer comprising of Tris solution. The eluent was loaded into Ion Exchange chromatography having Poros 50 D resin, where elution buffer containing Tris solution with 0.18 M NaCl, having a pH range of 7.6 to 8.0 was used in 0 to 100 % gradient mode. The protein was eluted in a conductivity range of 5 mS to 14 mS. The eluate was diluted and subjected to another Ion Exchange chromatography having Poros 50 D resin. The load was eluted with elution buffer (1.5-2.0 CV) containing citrate buffer solution having a pH range of 6.5 to 7.6. The desired protein thus eluted out was diluted to get the desired protein concentration and filtered through 0.2 uM disposable filter and the bulk was kept as API of EPO and stored at -20 °C. The protein obtained was characterized through in vitro tests as being erythropoietin. The total cumulative yield was found to be 25 to 30gm/ltr and the erythropoietin was recovered in purity >95%. 17 WE CLAIM: 1. A process for preparation of a glycoprotein comprising the steps of: a) growing under nutrient conditions host cells capable of producing/ releasing erythropoietin at its production phase, wherein the host cells are perfused with a nutrient medium having a monosaccharide content of 7gm/day to 137.5 gm/day; and b) harvesting and recovering the glycoprotein from the medium. 2. A process as claimed in claim 1 wherein the nutrient medium includes a medium employed in a growth phase and a medium employed in the production phase. 3. A process as claimed in claim 1, wherein the production medium is perfused at a rate of 2 to 25 Lit/day. 4. A process as claimed in claim 3, wherein the preferred perfusion rate is in a range of 6 to 20 Lit/day. 5. A process as claimed in claim 1, wherein the nutrient medium is supplemented with components selected from a group comprising of salts and buffers, plasma, nucleosides and bases, protein and tissue hydrolysates, antibiotics and lipids, including monosaccharide. 6. A process as claimed in claim 1, wherein the host cells are restrained by anchoring to a carrier. 7. A process as claimed in claim 5, wherein the glycoprotein is recovered by isolating and purifying the glycoprotein from the culture by a chromatographic step selected from affinity column chromatography, metal chelating chromatography, gel filtration chromatography and ion exchange chromatography. 18 8. A process as claimed in claim 1, wherein the cell line is pre-cultured in the growth medium to provide a seed culture. 9. A process for the preparation of erythropoietin, as described herein with respect to the foregoing examples. Dated this 26th day of October, 2006. [RAJESHWARI H.] OF K&S PARTNERS ATTORNEY FOR THE APPLICANT 19 ABSTRACT Title: "A process for the preparation of glycoprotein " The present invention relates to a process for the preparation of a glycoprotein using adherent type substrate in perfusion -mode, wherein in the production phase, the monosaccharide content in the perfusing medium is 7 gm/day to 137.5 gm/day. 21

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1808-mum-2006-drawing.jpg

1808-MUM-2006-FORM 1(12-12-2006).pdf

1808-MUM-2006-FORM 1(16-1-2014).pdf

1808-mum-2006-form 13(1-8-2011).pdf

1808-mum-2006-form 13(30-8-2010).pdf

1808-MUM-2006-FORM 18(30-8-2010).pdf

1808-MUM-2006-FORM 2(TITLE PAGE)-(30-10-2006).pdf

1808-MUM-2006-FORM 26(12-12-2006).pdf

1808-MUM-2006-FORM 3(16-1-2014).pdf

1808-MUM-2006-FORM 5(16-1-2014).pdf

1808-mum-2006-form-1.pdf

1808-mum-2006-form-2.doc

1808-mum-2006-form-2.pdf

1808-mum-2006-form-3.pdf

1808-mum-2006-form-5.pdf

1808-MUM-2006-REPLY TO EXAMINATION REPORT(16-1-2014).pdf