Title of Invention

A PROCESS FOR THE PREPARATION OF BIOLOGICALLY ACTIVE PRODUCT

Abstract

The present invention relates to a process for the preparation of TNK-tPA by inoculating said seed culture for production phase in a perfusion mode/batch using adherent type Chinese Hamster Ovary cell with packed bed technology comprising of a modified CHO-S-SFM II production medium; wherein the residual glucose level is maintained in the range of 0.15 g/L to 0.75 g/L, during the production phase.

Full Text

FORM 2 THE PATENTS ACT, 1970 (39 of 1970) & The Patents Rules, 2003 COMPLETE SPECIFICATION (See section 10 and rule 13) 1. TITLE OF THE INVENTION "A NOVEL PROCESS FOR THE PREPARATION OF BIOLOGICALLY ACTIVE PRODUCT" 2. APPLICANT (S) (a) NAME Gennova Biopharmaceuticals Ltd. (b) NATIONALITY : India (c) ADDRESS : T-184, MIDC, Bhosari, Pune 411026, Maharashtra, India 1 FIELD OF THE INVENTION The present invention is directed to an improved process for preparing a tissue plasminogen activator (tPA), a particular variant of plasminogen activators. In particular the present invention relates to a process for preparation of Tenecteplase, a modified tissue plasminogen activator (tPA) in high yield. DESCRIPTION OF BACKGROUND AND RELATED ART Plasminogen activators are enzymes that activate the zymogen plasminogen to generate the serine proteinase plasmin (by cleavage at Arg560-Val561) that degrades various proteins, including fibrin. Among the plasminogen activators studied are a) streptokinase, a bacterial protein b) urokinase, an enzyme synthesized in the kidney and elsewhere and originally extracted from urine, and c) human tissue plasminogen activator (tPA), an enzyme produced by the cell lining blood vessel walls. The mechanism of action of each of these plasminogen activators differs: Streptokinase forms a complex with plasminogen, generating plasmin activity, urokinase cleaves plasminogen directly, and tPA forms a ternary complex with fibrin and plasminogen, leading to plasminogen activation in the locality of the clot, hereby providing the desired effect dissolving the clot. tPA has been identified and described as a particularly important and potent new biological pharmaceutical agent that has shown extraordinary results in the treatment of vascular diseases, such as myocardial infarction, due to its high fibrin specificity and potent ability to dissolve blood clots in-vivo. (As described by Anderson etal U.S Patent No. 5,385,732). tPA has been the subject of numerous scientific and patent application disclosures. Although its existence prompted numerous investigations by several scientific groups, it was first identified as a substantially pure isolate from a natural source, and tested for 2 requisite plasminogen activator activity in-vivo, by a) Collen et al., European Patent Application Publn. No. 41,766, published Dec. 16, 1981, based upon a first filing of Jun. 11,1980. b) Rijken et al., J. Biol. Chem., 256: 7035 (1981). Natural tPA has a plasma half-life of typically about six minutes or less, when administered to patients in therapeutically effective amounts. Such a half-life was desirable under certain situations, for example, when acute aggressive therapy of a life-threatening disease such as myocardial infarction or pulmonary embolism is undertaken. In this high-risk situation, patients may be treated who have significant or unrecognized potential for uncontrolled bleeding. If such bleeding occurred, drug administration could be stopped and the causative tPA levels would be rapidly depleted by high clearance rates. However, in other circumstances, for example, in the treatment of myocardial infarction following reperfusion, the desired therapeutic regimen using naturally occurring tPA proved to be less aggressive and an extended duration of 4 to 12 hours was envisaged which resulted in the discovery of a modified tPA having a longer half-life (or slower clearance rate) termed as TNK-tPA. TNK-tPA was first disclosed by Anderson et.al. in U.S Patent Application 5,385,732, this is generically know as Tenecteplase and is available as a marketed preparation under the name of Tnkase®. Tenecteplase is produced by recombinant DNA technology using an established mammalian cell line (Chinese Hamster Ovary cells), which is a 527 amino acid glycoprotein, and has been developed through modifications of the complementary DNA (cDNA) for natural human tPA: wherein a substitution of threonine 103 with asparagine and a substitution of asparagine 117 with glutamine, has been made both within the kringle 1 domain, and a tetra-alanine substitution at amino acids 296-299 has been done in the protease domain. In addition to this, several variants of tPA have been reported for example, U.S Patent No. 5407819 issued on April 18, 1995 discloses a method of preparation of a tPA variant by displacement of a particular amino acid group in the amino acid sequence. Another 3 U.S Patent 5,612,029 issued on March 18, 1997 discloses a variant of tPA , which is glycosylated at any positions between 103-105 and devoid of functional carbohydrate structure at position 117 of wild type human tPA amino acid sequence. U.S Patent 5,520,911 issued on May 28th, 1996 discloses preparation of DNA sequences to encode the tPA variant. U.S. Patent No. 5,424,198 issued on June 13, 1995, discloses a method for the production of tPA, by transforming the cells with mutant or wild-type DHFR using methotrexate (MTX). U.S Patent No. 6586206 issued on July 1, 2003, discloses a method for production of various recombinant proteins including tPA, using apoptosis inhibitor. At present, various patents disclose natural tPA or TNK-tPA and the process for preparation of the same. U.S Patent Nos. 5,728,567, 5,714,145, 5,366,886, 5,094,953 and 5,407,819 disclose a method of preparation of t-PA and t-PA variant. One U.S Patent No. 6,506,598, issued on January 14, 2003 discloses a process for preparation of an increased amount of % of type-1 tPA in the presence of butyrate salt along with the reduction in temperature during the production phase using fed-batch process. In yet another U.S Patent No. 6,610,516 issued on August 26, 2003, to Genentech, Inc. (South San Francisco, CA), butyrate is replaced by bivalent metal ions using fed-batch process. Both the U.S. Patents 6,506,598 and 6,610,516, mentioned herein uses CHO cell lines along with basal 20 medium as a growth and production media. The process uses fed batch type bioreactor to yield a harvest which is centrifuged to remove particulate cell debris and the glycoprotein thereafter is purified from contaminant soluble proteins and polypeptides, using conventional purification techniques. 4 There is growing demand of TNK-tPA (Tenecteplase), for the treatment of cardiovascular complications, arising out of current life style. The demand supply ratio for TNK- tPA is presently not balanced; hence, there is an urgency to comply with the market requirements of Tenecteplase. Hence, biotech industries are aiming for higher yields of tenecteplase. However, the prior art literatures do not address the issue of production of TNK- tPA on large scale, wherein the yield of TNK- tPA is acceptable to biotech industries at large. 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). The prior art processes which are disclosed in U.S Patents 6,610,516 and 6,506,598, mostly showed utilization of fed batch processes with suspension cell culture. Though suspension cell cultures have the advantages of scale up and can handle large quantity volumes with all physiological parameters in precise control, yet the disadvantages of suspension cell cultures lies in the increase of the metabolic stress on the cells due to the end product getting contaminated with wastes, which in turn also increases the load in the purification process. (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 TNK-tPA and there is a need for more effective, user friendly high yielding process for the preparation of TNK-tPA. The present invention addresses the above needs and provides an improved and a more effective, user friendly high yielding process for the preparation of TNk-tPA. SUMMARY OF THE INVENTION Accordingly the present invention provides an improved process for production of TNK-tPA comprising the steps of (a) culturing a cell line capable of producing TNK-tPA in a 5 medium while maintaining level of residual glucose at a range of 0.15gms/ltr to 0.75gms/ltr through the production phase; (b) harvesting and recovering TNK-tPA. In one aspect the cell lines employed may be any host cell adapted to produce TNK-tPA. Such a line may be such as a recombinant Chinese Hamster Ovary (CHO) cell line or any other suitable line. Preferably, the process may be conducted in perfusion mode to obtain enhanced yield of the final product. In another aspect, residual glucose at the production phase is maintained at a range of 0.15 to 0.75 gms/ltr. The additional glucose added may be of a concentration of lgm/ltr to 3 gms/ltr. The medium may be any medium suitable and adapted to promote the growth of cells; such a medium may be selected from the group comprising of Iscove's Modified Dulbecco's Medium (IMDM), Dulbecco's Modified Eagle's Medium (DMEM), PRIMATONE HS.™ and PRIMATONE RL.™ (Kerry Biosciences). The medium employed is not necessarily limited to the above-exemplified media and other commercially available media may also be used. In addition, the mdium may be supplemented with components selected from the group comprising of a plasma, salts and buffers, nucleosides and bases, protein and tissue hydrolysates, antibiotics and Lipids and apoptosis inhibitors, Preferably, the host cell lines capable of producing TNK-tpa may first be grown in a flask as a seed culture and then when the cells have reached a sustainable stage, the culture may be preserved and used as a stock for inoculation. In yet another aspect of the invention the TNK-tPA is harvested, isolated and purified by affinity column chromatography, metal chelating chromatography and gel filtration chromatography. 6 DETAILED DESCRIPTION: Accordingly the present invention provides an improved process for production of TNK-tPA comprising the step of culturing a cell line capable of producing TNK-tPA in a medium while maintaining level of residual glucose at a range of 0.15gms/ltr to 0.75gms/ltr through the production phase; and harvesting and recovering TNK-tPA. Some of the terms frequently used are defined herebelow: "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. TNK -tpA is produced. The terms "cell culture medium", "culture medium", refer to a nutrient solution used for growing mammalian cells. The culture medium may be any commercially available medium such as Iscove's modified Dulbecco medium, Eagle Medium, Primatone etc. The medium may be supplemented with any of: 1) an energy source, usually in the form of a carbohydrate 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 cell culture production 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 upto 5%, preferably upto 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). 7 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 thereby increasing the load during the down stream process. The cumulative yield of TNK-tPA 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. In contrast, the present invention provides with an improved process for the large scale production of TNK- tPA, using perfusion mode, which has an advantage of: a) Multiple harvesting. b) Potential 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 the reactor and facilities. e) Shorter exposure time of the product to potentially harsh production conditions (high or low pH). f) High per unit volumetric productivity due to high cell density and metabolism. g) Provides essentially the constant environment. h) Less cumbersome, since the production of the desired product is in continuous manner, i) Saves the time cycle of the production batch as the process till upstream is continuous, j) Even if the size of the reactor is small, more quantity of the production is achieved in perfusion batch culture process due to multiple harvesting. k) There is efficient use of manufacture's working cell bank as there is continuous removal of any unwanted side products produced during the batch process in perfusion 8 mode, hereby enhancing the yield of the product by using the same density of cells Ifl perfusion batch culture. Correspondingly the present invention also makes use of 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. Modes for Carrying out the Invention Cell Culture Procedures: According to the present invention mammalian cells are cultured to produce a desired glycoprotein product. The mammalian cell culture of the present invention can be prepared in a medium suitable for the particular cell being cultured. Different types of growth mediums can be used, suitable for culturing the cells, which are described in the art, such as a 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 Ceil Lines and Hybridomas, Sixth Edition, 1988, pages 346-349), 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.TM. or PRIMATONE RL.TM. (Sheffield, England), or the equivalent. Preferably, the medium used is Iscove's Modified Dulbecco's (IMDM) Medium. The growth medium can be optionally supplemented with one or more components from any of the following categories: 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, glucose, gentamycin, Methotrexate (MTX) and Dialyzed Foetal Bovine Serum (DFBS). In a preferred embodiment the process for preparation of TNK-tPA is performed using a perfusion mode with adherent cell culture along with packed bed technology to provide a higher yield of the recombinant protein. In a particularly preferred embodiment, the mammalian host cell is a CHO cell. 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. 10 1) Preparing seed culture using mammalian cells to be used for production phase: The modified tPA is termed as TNK-tPA, which can be produced by making use of CHO (Chinese Hamster Ovary) mammalian cell culture using the technology known in the public domain. The CHO cell line as referred herein may be any known cell line. 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 modebatch. 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 glucose 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 to maintain the required residual glucose level in the bioreactor. Accordingly, herein is a process, describing the production of the TNk-tPA, wherein the process includes culturing the CHO cell lines in the growth medium to provide a seed culture followed by inoculation of seed culture in a perfusion type of perfusion mode/batch using adherent type of CHO cells with packed bed technology comprising of a modified CHO-S-SFM II production medium. [Gibco Laboratories, USA] 11 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. Residual glucose concentration during the production phase is maintained in the rangeof0.15g/lto0.75g/l. b. Additional glucose concentration in modified CHO-S-SFM II medium is in the range of 1.0 g/L to 3.0 g/L, preferably in the range of 1.5 g/L to 2.5 g/L. c. Perfusion rate of the production medium is in the range of 10 to 25 1/day and preferably in the range of 12.5 to 23.5 1/day. d. 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; e. 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; f. 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 %; In a further preferred embodiment provided herewith is a process to produce tPA in substantially in a single-chain form, comprising the use of a protease inhibitor, such as aprotinin, pepstatin A, E-64, betastatin, leuoeotin and phenylmethylsulfonyl fluoride (PMSF), wherein the preferably used protease inhibitor is aprotinin. 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. 3) Harvesting, isolation and purification The harvest containing the glycoprotein in the secreted form is collected in a continuous manner from the bioreactor. 12 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. A protease inhibitor such as phenyl methyl sulfonyl fluoride (PMSF) also may be useful to inhibit proteolytic degradation of the desired glycoprotein during purification. One skilled in the art will appreciate that purification methods suitable for the glycoprotein of interest may require modification to account for changes in the character of the glycoprotein upon expression in recombinant cell culture. Also of utility within the context of the present invention are purification techniques and processes that select for the carbohydrates of the invention. Such techniques include, for example, ion-exchange soft gel chromatography or HPLC using cation- or anion-exchange resins, wherein the more acidic or more basic fraction is collected, depending on which carbohydrate is being selected for. Several techniques may be used for recovery of the t-PA. For example, at the end of the culture, tangential flow filtration, including high-pressure-tangential flow filtration, can be used to remove the medium containing t-PA from the cells. The cell culture supernatants may be concentrated, diafiltered, and loaded onto an affinity column capable of specific binding of t-PA, typically a lysine affinity column. Under the chromatography conditions employed, t-PA adheres selectively to the affinity column from which it can be recovered and subjected to further purification. In a preferred embodiment, the following chromatographic procedures being exemplary of suitable purification procedures: by affinity chromatography, metal chelating 13 chromatography and gel filtration 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 t-PA, and t-PA 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. To further reduce the load of the impurities, metal chelating chromatography can be introduced, 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 divalent ion, preferably is a divalent metal ion, which is selected from Cu, Zn, Fe, Ni. The load in the column is eluted in the second elution by buffer comprising of agents, such as imidazole or EDTA, in high concentration, wherein, the preferably used agent is imidazole. The sample can then be loaded on another affinity chromatography column for more specific binding of the protein, which makes use of a lysine affinity column. Lysine affinity columns are well known in the art and are commercially available. Suitable columns include Lysine CPG.TM. (Bioprocessing), ECH Lysine CL.TM. (Pharmacia), and Lysine Hyper D.TM. (Biosepra). The preferably used column is a lysine Hyper D column. The desired protein is eluted out from the lysine affinity column by the elution buffer comprising arginine and 6-Amino hexanoic acid at pH range of 6.0 to 8.0, preferably at 6.5 to 7.6. The purity of TNK-Tpa was found to be more than 95% after this step, which was then confirmed by SDS-PAGE. The pool can be concentrated by using TFF (tangential flow filtration). The concentrate is loaded in Gel Filtration Chromatography (GFC) column and eluted with GFC buffer and the protein is collected. 14 The purification protocol further optionally includes additional steps that inactivate and/or remove retroviruses that might potentially be present in the cell culture fluid of continuous mammalian cell lines. A significant number of viral clearance steps are available, including additional ultrafiltration/diafiltration steps, treatment with chaotropes such as urea or guanidine, pH extremes, detergents, heat, chemical derivatization, such as formaldehyde, proteases, conventional separation, such as ion-exchange or size exclusion chromatography, organic solvents, etc. The recombinant human t-PA recovered and purified following the foregoing protocol typically is more than 95 % pure (depending on the lysine resin). 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 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. The present invention is further illustrated by the following, non-limiting examples. Example 1 Preparation of the seed culture Materials and procedure: The recombinant CHO cell line was used. The growth medium used was IMDM medium (Gibco™, Invitrogen Corporation), which contains methotrexate, gentamycin, sodium bicarbonate, glucose and supplemented with 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 bottles (850 cm2). Finally, the cell culture was fully grown till the required number of cell count was observed. Process: The modified tPA producing recombinant CHO cells were taken from manufacture's working cell bank (MWCB) and growth was carried out in a flask/ roller 15 bottle with IMDM growth medium. The IMDM medium included methotrexate, gentamycin, and sodium bicarbonate, glucose 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 cell cultures 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 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. This provided around 10,000-15,000 million cells as a seed for next steps. Example 2 Production phase Materials and procedure: IMDM was used as a growth medium. CHO-S-SFM II medium as a production medium included sodium bicarbonate, gentamycin, methotrexate (MTX), aprotinin and supplemented with dialyzed foetal bovine serum (DFBS). The bioreactor used was a perfusion type bioreactor (NEW BRUNSWICK SCIENTIFIC, CELLIGEN PLUS Bioreactor/ fermentor), with capacity of 7.5 L suitable for perfusion batch process. Process: The seed culture produced from example 1 was aseptically transferred to the perfusion type bioreactor comprising of IMDM medium, which contains methotrexate (1 uM), gentamycin (50 mg/L), sodium bicarbonate (3 g/L), glucose (1.5 g/L) 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 replaced by CHO-S-SFM II medium which contains sodium bicarbonate (2.45 g/L) gentamycin (50 mg/L), methotrexate (MTX) (1 uM), aprotinin (7 mg/L), dialyzed foetal bovine serum (DFBS) (0.5% v/v). The temperature was decreased at the rate of 0.5 °C per day. The residual glucose level in the bioreactor was always maintained in the range of 0.15 g/1 to 0.75 g/1 16 by perfusion of fresh, sterile modified CHO-S-SFM II production medium, having additional glucose concentration of 2g/L. Perfusion rate of the production medium in the bioreactor was maintained in the range of 12.5 to 23.5 1/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 rate of harvest and perfusion is dependant on the residual glucose concentration as mentioned above. The total period of production phase was about 25 days. Example 3 Downstream process: Materials and procedure: Sodium caprylate (4.0 mM), For 1ST affinity chromatography elution buffers I and II used with blue sepharose resin was used. Elution buffer I (1 ltr)comprised of Na2HP04.12H20 4.45 g and NaH2P04.12H20 0.41 g and NaCl 117.0 g dissolved in water for injection (WFI). Elution buffer II (1 ltr) comprised of Na2HP04.12H2 3.8 g, NaH2P04.12H20 0.41 g and NaCl 58.5 g, Urea 180.15 g dissolved in water for injection (WFI). In metal chelate chromatography, POROS 50 MC resin was used.. Elution buffer I (1 ltr) comprised of Na2HP04.12H2 3.8 g, NaH2P04.12H20 0.41 g, NaCl 58.3 g dissolved in water for injection (WFI), with 5mM imidazole. Elution buffer II (1 ltr) also comprised of same ingredients with same concentration except for imidazole having 110mM concentration. In second affinity chromatography, Lysine Hyper D resin was used. Elution buffer I (1 ltr) comprised of Na2HP04.12H23.8 g, NaH2P04.12H20 0.41 g, NaCl 58.5 g. dissolved in water for injection (WFI). Elution buffer II (1 ltr) comprised of arginine 87.1 g, 6-amino hexanoic acid 26.9 g, and phosphoric acid 19 g. dissolved in water for injection (WFI). In Gel Filtration Chromatography (GFC), sephadex G 25 resin was used. Elution buffer (1 ltr) comprised of 55 g L-Arginine, 430 uL polysorbate 20 and 70 g phosphoric acid dissolved in water for injection (WFI). The elution buffers referred above have a pH in the range of 6-8. 17 Process: The harvest containing the protein in the secreted form was treated with sodium caprylate followed by filtration through 0.45/0.2 \i Millipore filter. The filtrate obtained was subjected to blue sepharose affinity chromatography, wherein the column was eluted using elution buffers I and II. The elution buffer I (1.5-2.0 Column Volume i.e., CV) was passed to remove impurities, which was followed by the elution buffer II (1.5-2.0 Column Volume i.e., CV) having a pH range of 6.5 to 7.6, to elute out the desired protein. The said eluent was now subjected to metal chelate chromatography. The resin was initially saturated with 0.1 M Zinc chloride solution. The column was eluted using elution buffers I and II. The elution buffer I (1.5-2.0 CV) was passed to remove impurities, which was followed by elution buffer II (1.5-2.0 CV). The protein was eluted out in elution buffer II, having a pH range of 6.5 to 7.6. Further, the elute was diluted and loaded into second affinity column, containing lysine Hyper D resin. The column was eluted by passing elution buffer I, to remove the impurities, which was discarded and elution buffer II, comprising of 6-Amino hexanoic acid and arginine was used to elute out the desired protein. The pool was concentrated by using tangential flow filtration (TFF). The concentrate obtained was then loaded in a Gel Filtration Chromatography (GFC) column, containing sephadex G 25 resin having a pH range of 6.5 to 7.6 to yield the required protein, which if required could be diluted with arginine and was analyzed for "% of protein content". The final solution so obtained having desired % of protein was lyophilized to yield the final formulation. The cumulative yield of the TNK-tpa produced as a result of the process of the invention is about 8gm/ltr and its purity may be more than 95%. 18 WE CLAIM: 1. A process for the preparation of TNK-tPA comprising the steps of: (a) culturing a cell line capable of producing TNK-tPA in a medium while maintaining level of residual glucose at a range of 0.15gms/ltr to 0.75gms/ltr through the production phase; (b) harvesting and recovering TNK-tPA.. 2. A process as claimed in claim 1, wherein the medium includes a medium employed in a growth phase and a medium employed in the production phase. 3. A process as claimed claim 1, wherein the level of residual glucose is maintained in the range of 0.15 g/L to 0.75 g/L. 4. A process as claimed in claim 3, wherein the concentration of glucose added is within a range of 1 g/L to 3 g/L. 5. A process as claimed in claim 4, wherein the concentration of glucose added is preferably within a range of 1.5 g/L to 2.5 g/L. 6. A process as claimed as in claim 1, wherein the medium is perfused at a rate of 10 to 25 L/day. 7. A process as claimed in claim 6, wherein the preferred perfusion rate is within a range of 12.5 to 23.5 L/day. 8. A process as claimed in claims 1 and 2, wherein the medium used for growth phase is selected from the group comprising of Iscove's Modified Dulbecco's Medium (IMDM), Dulbecco's Modified Eagle's Medium (DMEM), PRIMATONE HS.™ and PRIMATONE RL.™ 19 9. A process as claimed in claim 8, wherein the medium for growth phase is Iscove's Modified Dulbecco's Medium (IMDM). 10. A process as claimed in claims 1 and 2 wherein the medium employed for production phase is CHO-S-SFMII. 11. A process as claimed in claim 1 , wherein the medium is supplemented with components selected from a group comprising of a plasma, salts and buffers, nucleosides and bases, protein and tissue hydrolysates, antibiotics,lipids and apoptosis inhibitors. 12. A process according to claim 1 wherein the TNK-tpa is isolated and purified by a chromatographic technique selected from the group comprising of affinity column chromatography, metal chelating chromatography and gel filtration chromatography. 13. A process as claimed in claim 1, wherein the cell line is pre-cultured in the growth medium to provide a seed culture. 14. A process for preparation of TNK-tPA, substantially as described herein with respect to accompanying examples. Dated this 26th day of October, 2006. [RAJESHWARI H.] OF K&S PARTNERS ATTORNEY FOR THE APPLICANT 20 ABSTRACT: Title: "A novel process for the preparation of biologically active product" The present invention relates to a process for the preparation of TNK-tPA by inoculating said seed culture for production phase in a perfusion mode/batch using adherent type Chinese Hamster Ovary cell with packed bed technology comprising of a modified CHO-S-SFM II production medium; wherein the residual glucose level is maintained in the range of 0.15 g/L to 0.75 g/L, during the production phase. 21

Documents:

1807-mum-2006-abstract.pdf

1807-MUM-2006-CLAIMS(AMENDED)-(29-8-2013).pdf

1807-MUM-2006-CLAIMS(AMENDED)-(9-7-2014).pdf

1807-MUM-2006-CLAIMS(MARKED COPY)- (29-8-2013).pdf

1807-MUM-2006-CLAIMS(MARKED COPY)-(9-7-2014).pdf

1807-mum-2006-claims.doc

1807-mum-2006-claims.pdf

1807-mum-2006-correspondance-received.pdf

1807-MUM-2006-CORRESPONDENCE(12-12-2006).pdf

1807-MUM-2006-CORRESPONDENCE(2-9-2011).pdf

1807-MUM-2006-CORRESPONDENCE(23-4-2012).pdf

1807-MUM-2006-CORRESPONDENCE(3-8-2011).pdf

1807-MUM-2006-CORRESPONDENCE(30-8-2010).pdf

1807-mum-2006-description (complete).pdf

1807-MUM-2006-FORM 1(12-12-2006).pdf

1807-MUM-2006-FORM 1(29-8-2013).pdf

1807-MUM-2006-FORM 1(9-7-2014).pdf

1807-mum-2006-form 13(30-8-2010).pdf

1807-MUM-2006-FORM 18(30-8-2010).pdf

1807-MUM-2006-FORM 2(TITLE PAGE)-(30-10-2006).pdf

1807-MUM-2006-FORM 2(TITLE PAGE)-(9-7-2014).pdf

1807-MUM-2006-FORM 26(12-12-2006).pdf

1807-MUM-2006-FORM 3(29-8-2013).pdf

1807-MUM-2006-FORM 5(29-8-2013).pdf

1807-mum-2006-form-1.pdf

1807-mum-2006-form-2.doc

1807-mum-2006-form-2.pdf

1807-mum-2006-form-3.pdf

1807-mum-2006-form-5.pdf

1807-MUM-2006-REPLY TO EXAMINATION REPORT(29-8-2013).pdf

1807-MUM-2006-REPLY TO HEARING(9-7-2014).pdf