Title of Invention	A NOVEL PROCESS FOR PRODUCING THERAPEUTIC GRADE STREPTOKINASE
Abstract	The present invention relates to a novel process for producing therapeutic grade Streptokinase from genetically engineered E. coli. The yields of streptokinase produced by r-DNA technology, though better than those obtained from natural resources, have been low. In therapeutic proteins the presence of endotoxins is not desirable but removal of endotoxins poses problems particularly for streptokinase produced from E. coli. This invention progressively reduces DNA and endotoxin content in different steps of the procedure, top get extremely low levels of these contaminants in the final purified pr-oduct. There is a drastic reduction in quantity of chemicals due to reduction in the volume of ,. buffers at every step by employing suitable high capacity chromatographic matrix. B .. The activity of streptokinase is not affected. The process can be easily scaled up ~ for large scale purification. The therapeutic grade streptokinase obtained ~from this invention can be used in thrombolytic therapy used in significant .. medical problem of thrombosis which is potentially fatal, often affecting organs such as heart or lungs. --

Title of Invention

A NOVEL PROCESS FOR PRODUCING THERAPEUTIC GRADE STREPTOKINASE

Abstract

The present invention relates to a novel process for producing therapeutic grade Streptokinase from genetically engineered E. coli. The yields of streptokinase produced by r-DNA technology, though better than those obtained from natural resources, have been low. In therapeutic proteins the presence of endotoxins is not desirable but removal of endotoxins poses problems particularly for streptokinase produced from E. coli. This invention progressively reduces DNA and endotoxin content in different steps of the procedure, top get extremely low levels of these contaminants in the final purified pr-oduct. There is a drastic reduction in quantity of chemicals due to reduction in the volume of ,. buffers at every step by employing suitable high capacity chromatographic matrix. B .. The activity of streptokinase is not affected. The process can be easily scaled up ~ for large scale purification. The therapeutic grade streptokinase obtained ~from this invention can be used in thrombolytic therapy used in significant .. medical problem of thrombosis which is potentially fatal, often affecting organs such as heart or lungs. --

Full Text	A NOVEL PROCESS FOR PRODUCING THERAPEUTIC GRADE STREPTOKINASE Field of the invention The present invention generally relates to a novel process for producing therapeutic grade streptokinase. More particularly it relates to a novel process for producing therapeutic grade streptokinase from genetically engineered E. coli. Background of the invention A significant medical problem, namely thrombosis, is presented by the occlusion of blood vessels due to the presence of thrombi (blood clots). Such thrombosis is potentially fatal, often affecting organs such as heart or lungs. Over the last few years, intravenous thrombolysis (blood clot dissolution) has become the standard therapeutic approach for patients with myocardial infarction. The reduction in short and long term mortality rates caused by thrombolytic therapy has been demonstrated in several large-scale clinical trials (1-6). One of the agents commonly used in thrombolytic therapy is the bacterial protein streptokinase (7), which is already in clinical use in many countries, treating patients with lung thrombus and acute myocardial infarction and has proved very effective. Its capacity to cause lysis of blood clots was first described in 1933 (8). Clinical studies over the last 10 years have shown that streptokinase decreases the morbidity and mortality. Streptokinases, secretory proteins elaborated by many strains of hemolytic streptococci, belong to a widely distributed and heterogeneous class of proteins known as plasminogen activators, which includes Urokinase and Tissue type plasminogen activator (9-11). These plasminogen activators are at present used as thrombolytic agents in the treatment of disorders which collectively represent one of the greatest causes of death in the world, such as myocardial infarction, pulmonary, arterial or venous thromboembolism, and thrombosis due to surgical complications and other causes. Interestingly streptokinase (SK) differs from most other plasminogen activators in that it is not a proteolytic enzyme. The primary structure of streptokinase (12) has revealed that streptokinase does have sequence homology to serine proteases. It is not known, however, whether this structural homology contributes to the plasminogen activation. Although devoid of intrinsic protease activity, streptokinase avidly binds to plasminogen (from many mammalian species) and generate streptokinase-plasminogen activator complex (SK-PAC) that has protease activity (13, 14). This SK-PAC catalyses the conversion of the zymogen-plasminogen into the active enzyme plasmin. Plasmin then cleaves fibrin, the protein backbone of thrombus, resulting in thrombolysis. Streptokinase's properties have been reviewed in detail (15). It functions in the species-specific conversion of plasminogen to plasmin. It complexes with human plasminogen or plasmin, forming an enzyme activator complex (which can directly convert human or bovine plasminogen to plasmin), a property which neither plasmin nor streptokinase alone possess (16). Bovine plasminogen, however, is essentially unreactive towards streptokinase with respect to the formation of plasminogen activator. As of now plasminogen and plasmin are the only substrates with which streptokinase is known to react. Streptokinase is a bacterial protein derived from pathogenic strains of the streptococcus family (17). The function of streptokinase in the pathogenicity of streptococci is not known exactly, although potentially it may contribute to elimination or avoidance of formation of fibrin barriers around the infection. It is an extra cellular secretory protein produced in group C strains of beta-hemolytic streptococci with a molecular mass of 47,000 Daltons and is devoid of carbohydrate, cyteine and cystine (18,19). Streptokinase has been purified for clinical usage and studied by several investigators. The primary source of this streptokinase has been the culture fluid resulting from the growth of the beta-hemolytic group C organism streptococcus equisimilis. Method of purifying streptokinase from culture medium of streptococci has been described by several investigators. In US Patent No. 5840315, the streptokinase is prepared by affinity chromatography of culture medium by using monoclonal antibodies matrix. In US Patent No. 3855065, purification is attempted by using gel filtration chromatography but purity level of the protein has not been described. In US Patent No. 3186920, purification is achieved by mixing the culture supernatant with Carboxyl Methyl (CM) polysaccharide at higher temperature and eluting the bound protein with salt gradient. In US Patent Nos. 299742 and 3042586 is described a procedure for purification of streptokinase using Diethyl Aminoethyl (DEAE) cellulose chromatography. In KR Patent No. 9512897, a combination of ammonium sulphate precipitation and gel filtration chromatography has been used for purification of streptokinase. In KR Patent No. 9702903, a procedure involving cation exchange chromatography for purification of streptokinase has been described. / In KR Patent No. 136613, the method describes a procedure which consists of treating the culture medium with reducing agent and incubating with a reagent R-X where R is reactive group capable of reacting with free thiols and effect impurity removal. In GB Patent No. 894151, the purification procedure comprises precipitation by sodium chloride and anion exchange chromatography. In GB Patent No. 966672, the method describes a combination of methanol and ammonium sulphate precipitations for purification of streptokinase. In EP Patent No.0150424, a combination of ammonium sulphate precipitation and gel filtration chromatography has been used for purification of streptokinase. In Indian Patent No. 137890, the method described involves CM cellulose chromatography and CaCk precipitation to obtain streptokinase. The drawbacks of producing streptokinase from natural sources by these methods are: 1. The presence of toxic and undesirable substances makes the purification process more complex. 2. Low yields of streptokinase (0.1 to 0.2 g/L) and consequent handling of huge volumes of cultures medium makes the process inefficient and costly. 3. As streptokinase is produced by culturing hemolytic streptococci, which is a pathogenic organism, handling during production may cause inconvenience and hazards to workers. 4. Extreme care has to be taken to avoid hazards to the environment in case of leakage of streptococci. 5. The streptococcus strains used for industrial production of streptokinase secrete other products into the culture medium such as deoxyribonucleases, streptolysin or hyaluranidase and proteases, which makes the process of purifying the desired protein difficult. 6. Since many of these secretory contaminants are toxic or potentially toxic to humans, the preparation of streptokinase from culture fluid for clinical usage involves multi-steps which must be performed with great care to ensure a clinically acceptable product. On the other hand it has not yet been possible to obtain genetically improved strains from these hosts due to the lack of a developed methodology for the gene transfer. It is due to these drawbacks that the cloning of different isolated genes which code for these proteins has been attempted using prokaryotic and eukaryotic hosts (20). The recombinant DNA technology has opened up entirely new and important ways to use living cells to produce biologically active material such as enzymes and hormones in large amounts. Several methods of streptokinase purification have been published (21, 22). Besides chromatography, aqueous two-phase system (ATPS) composed of polyethylene glycol/dextran and polyethylene glycol/salt have been employed for separation and purification of streptokinase from culture supernatant (23). The streptokinase gene from streptococcus equisimilus has been cloned (24) and expressed in streptococcus sanguis, an organism that does not normally produce streptokinase (25). As an alternative to the above bacterial systems, streptokinase has been expressed in methylotropic yeast Pichia pastoris. Several methods of purification of streptokinase from recombinant host have been published (26). Recently a gene for streptokinase has been cloned from the chromosomal DNA of the group C streptococcal strain H46A (27). The gene product has the same substrate specificity and antigenicity as streptokinase from natural source. The complete primary sequence of streptokinase has been published both from protein sequencing (28) and from elucidation of the nucleotide sequence of the entire cloned streptokinase gene from streptococcus equisimilis (29). The gene for streptokinase was expressed in E. coli with deduced amino acid sequence agreeing well with the known sequence of commercial streptokinase. In US Patent No. 5296366, streptokinase has been expressed in E. coli as well as in Pichia systems with expression levels of 350 mg/L and 1 g/L respectively. In US Patent application No. 2003/059921, is described a method which consists of solubilising the recombinant streptokinase from E. coli cells with GuCl and subjecting the extract to hydrophobic and anion exchange chromatography. In Indian Patent No. 183828, the method consists of adsorption on silica and gel ■ filtration for purifying streptokinase and has employed affinity chromatography using acylated plasminogen for purifying streptokinase analogues. In Indian Patent No. 185350, the protein is expressed as a periplasmic protein and an expression yield of 400 mg/L culture has been reported. Streptokinase has also been expressed as fusion protein with maltose binding protein(US 5854049) or with histidine tag (US Patent No. 6087332) to enable affinity purification. Streptokinase mutant proteins with desired characteristics, have also been expressed in bacterial system (US Patent Nos. 6309873, 5876999, 5240845, IN Patent 183828). The purification strategies include gel filtration, ion exchange, hydrophobic high performance liquid chromatography or isoelectric focusing techniques or combination of these techniques. Although higher specific activity and yield have been reported in US Patent application No 2003/059921 which described a process using ion exchange and hydrophobic chromatographic procedure to produce r-streptokinase from E. coli, their method is devoid of special focus to bring down the endotoxin to the prescribed level for therapeutic use. Furthermore, solubilization of cells with 6M Guanidine hydrochloride (GuCl) as used in their procedure would completely solubilise the lysed membrane structures and consequently result in high endotoxin content in the extract. This high level of endotoxin cannot be handled by ion exchange and hydrophobic column alone as their method has no other efficient steps to bring down the endotoxins level as required for therapeutic purpose. Thus although good yields are reported in this method, it is not suitable for obtaining therapeutic grade protein. The advantages in producing streptokinase by recombinant technology are as follows. 1) The pathogenic organisms and toxic substances are absent thus making the development and purification process simpler and cost effective. 2) Manufacturing process is absolutely safe and worker-friendly. 3) The process is safe and environment friendly. 4) Higher safety may be exhibited for patients as it is free from streptolysin and streptodornase. 5) Higher yields of streptokinase are possible (Ig/L). Although above mentioned expression and purification of streptokinase by recombinant method overcomes some of the problems and potential risk associated with the production from natural sources, it has its own drawbacks, which are as follows: 1) The yields though better than those from natural sources, have been low. 2) Presence of endotoxins is not desirable and removal of endotoxins poses a serious problem, particularly for streptokinase produced in E. coll Endotoxins are an integral part of the cell membrane of gram negative bacteria and constitute approximately three quarters of the bacterial surface which consists of endotoxins. They are responsible for the organization and stability and consequently the cell lysate obtained from E.coli contains extremely high amount (~ >108 EU/ml) of endotoxins. Bacterial endotoxins show strong biological effects even at very low concentration in human beings and in many animals when entering the blood stream, for example, during a bacterial infection or via intravenous application of a contaminated medicament. Because of their toxicity in vivo and in vitro, endotoxins removal is extremely important for a safe parental administration and therefore the endotoxin level has to be extremely low for therapeutics proteins. Streptokinase is a high volume product with dose level of 15 mg/dose, which means stringency levels are high as far as endotoxin contamination is concerned. This requirement dictates that the process employed should be very efficient to bring down the endotoxin to the prescribed level. Several methods of endotoxin removal from protein solutions involving chromatographic or detergent extraction have been described in literature (30,31). Although ion exchange and hydrophobic interaction chromatography columns do reduce endotoxins, the reduction efficiency are 2-3 log only, which means it is extremely difficult to obtain the endotoxin level which is required for therapeutic application with these column chromatography steps alone. In view of the above mentioned disadvantages there is a need for a cost effective, simple and efficient purification process for producing therapeutic grade r-streptokinase from genetically engineered E.coli. Objects and Advantages of the invention Accordingly the present invention has several objects of achieving different advantages. One object of the present invention is to provide a purification process for production of therapeutic grade streptokinase from genetically engineered E.Coli, which is simple, efficient and results in high recovery of the expressed protein. Still another object of the present invention is to provide a process which can be scaled up easily for large scale purification of streptokinase Yet another object of the present invention is to provide a process giving removal of both free and protein bound endotoxins without affecting the activity of streptokinase thereby yielding streptokinase which is virtually devoid of endotoxins. Further object of the present invention is to provide a process that yields recombinant streptokinase with specific activity in close agreement with that of streptokinase from natural source. Summary of the invention: The present invention describes a novel process of purification for producing therapeutic grade streptokinase from genetically engineered bacteria. High yielding genetically engineered E. coli containing streptokinase gene derived from streptococci equisimilis strain H46A are grown, harvested and lysed. The lysate obtained is subjected to a series of purification steps such as ATPS, different types of chromatography and filtration to obtain highly purified r-streptokinase which has extremely low levels of host cell proteins, DNA, endotoxins and other contaminants. The final streptokinase product has high level of purity and is of therapeutic grade. Detailed description This invention provides a purification process for producing therapeutic grade streptokinase from genetically engineered E. coli. High yielding genetically engineered E. coli cells containing streptokinase gene derived from streptococci equisimilis strain H46A are grown in suitable growth media, harvested and lysed. In a preferred embodiment of the present invention, the lysate obtained is subjected to a series of sequential purification steps such as the ATPS, anion exchange chromatography, hydrophobic chromatography, cross-flow filtration, ATPS with detergent, adsorption chromatography, dye-ligand chromatography, and ultrafiltration/diafiltration in order to obtain highly purified r-streptokinase which has extremely low levels of host cell proteins, DNA, endotoxins and other contaminants. These steps are described below in detail. First ATPS: ATPS is an important emerging technique for separation, concentration, and purification of proteins, cell organelles, and other biological products. Initial clarification of lysate is achieved by using the first ATPS which is created by mixing the lysate with a phase forming salt such as ammonium sulphate to a final concentration of about 10% w/w to about 30% w/w and a phase forming polymer such as polyethylene glycol (PEG) with about 400 MW to about 2000 MW to a final concentration of about 5% w/w to about 30% w/w and a buffer comprising Tris HC1 of concentration of about lOmM to about lOOmM and pH of about 6.5 to about 8.5, to form a first mixture yielding a first ATPS. Polyethylene glycol is nontoxic and has a protective effect on proteins. The mixture is stirred vigorously for about 30 minutes to about 2 hours at about 200 rpm to about 300 rpm, at about 4°C to about 25°C. This first mixture is left to stand for a duration of about 1 hour to about 24 hours at temperature of about 5°C to about 35°C. This leads to formation of phases that are then separated by centrifugation for about 30 minutes to about 2 hours at about 5000 rpm to about 15000 rpm at about 4°C to about 25°C. Three distinct phases are formed as a result of centrifugation. The top polymer phase, referred to as PEG phase primarily contains PEG and the r-streptokinase along with E. coli proteins. The unlysed cells, cell debris, lipoproteins, lipopolysaccahrides, endotoxins, and other contaminants form the interphase or the middle phase, whereas the bottom phase, referred to as the salt phase, consists essentially of nucleic acids and highly hydrophilic proteins along with the salt. The PEG phase is then carefully collected using peristaltic pump aspiration which is a method commonly known to person skilled in the art and subjected to further purification steps described below to obtain the desired level of purity. This step results in the removal of the cell debris, DNA, some pigments, lipoproteins and substantial reduction (about 3 log to about 4 log reduction) of endotoxins. The reduction in level of endotoxin at this early stage of purification is a major advantage of the present invention leading to increase in efficiency of endotoxin reduction of the subsequent steps of this invention. This step results in enrichment of r-streptokinase in the PEG phase by adjusting the extreme volume ratios of the ATPS. It is only the PEG phase that is subjected to further purification steps. This significant reduction in the treatable volume is another major advantage of the present invention, which makes industrial scale production of r-streptokinase from E. coli lysate feasible. Anion exchange chromatography: The PEG phase collected as above is mixed and diluted with a first loading buffer in order to prepare the phase for the anion exchange chromatography. The dilution is carried out so that the dilution effect is multiplied to about 2 to about 10 times. The first loading buffer used for dilution is Tris HC1 with a pH of about 6.5 to about 8.5, preferably from about 7.0 to about 8.5, and has a concentration of about 10 mM to about 100 mM. The desired concentration of the mixture of the first loading buffer and the PEG phase is about 20mM, and is arrived at by adding required volume of the first loading buffer to the PEG phase. The diluted sample is clarified using a 0.45 micron filter and applied onto a chromatographic matrix which is of anion exchange nature and which has been pre-equilibrated with the first loading buffer. The unbound material passing through the column is collected and the column is washed with the first loading buffer till all loosely bound proteins are removed as monitored by measuring absorbance at 280 nm. The r-streptokinase, which is adsorbed on the stationary phase, is then eluted from the ion-exchange column by increasing the conductivity of the first loading buffer with salt such as sodium chloride applied in a step gradient mode. The streptokinase elutes with the first loading buffer of concentration of about 100 mM to about 600 mM NaCl. The r-streptokinase fraction is collected separately and processed for further chromatography. The UnosphereQ, a macroporous matrix, is used as the stationary phase in the anion exchange chromatography mentioned above. This matrix exhibits at least 20-30 times higher binding capacity compared to other conventional anion. exchange matrices. The present invention has a major advantage that the use of this matrix results in consequent reduction of volume of buffers, quantity of chromatography matrix, operation time and cost of production. With the improved thermodynamic properties, the matrix allows the use of high flow rates while still giving high resolution, thus resulting in shorter process time. In any scale-up operation, where time is one of the major factors that influences the effectiveness of such operation, this is of great advantage. The anion exchange chromatography results in further reduction of DNA, lipids, lipoproteins, endotoxins (2-log reduction), and host-cell proteins. Hydrophobic interaction chromatography: The r-streptokinase fraction obtained from anion exchange chromatography is further prepared for processing with hydrophobic interaction chromatography. A second loading buffer containing salt such ammonium sulphate is added so as to arrive at a salt concentration of about lOOmM to about 2000mM, preferably about 400mM to about 800mM. The pH of the solution is then adjusted to about 6.5 to about 8.5, preferably about 7.0 to about 8.0, with dilute sodium hydroxide. The resultant second mixture of the salt and r-streptokinase fraction is applied onto a hydrophobic column made of phenyl sepharose (low substitute) matrix that has been pre-equilibrated with the same second loading buffer. The r-streptokinase component is adsorbed on the stationary phase. Analysis by SDS gel electrophoresis reveals elimination of most of the host cell protein contaminants in the unadsorbed fraction. Next, a Tris HC1 buffer containing lower salt concentration of about 100 mM to about 300 mM is used to elute the bound proteins, including r-streptokinase, from the column. This chromatography results in further reduction in DNA and endotoxins (about 1 log reduction). Streptokinase obtained is about 98% pure, however traces of high molecular weight aggregates are present and which need to be removed with further purification measures. Also, although progressive endotoxin reduction is achieved at all foregoing purification steps, the resultant level of endotoxin content is still too high for therapeutic applications on humans. Additional purification measures are applied to further reduce the endotoxin levels. Ultra filtration: The concentration of eluted fraction obtained from the hydrophobic chromatography stage is then increased by about ten times by cross-flow ultrafiltration with membrane with a molecular weight cut-off value of lOkD. This filtration removes contaminants with small molecular weight from the protein solution. At the end of this step, there are still protein-bound endotoxins present. ATPS technique with use of detergents is applied next to remove protein-bound endotoxins. Second ATPS with detergent: A second ATPS technique using detergent such as Triton X 114 at a concentration of about 0.5% to about 5% effectively removes the traces of both free and protein-bound endotoxin. This detergent has a cloud point of about 25°C. When protein solution is mixed with a mixing buffer comprising Tris HC1 of concentration of about lOmM to about lOOmM and pH of about 6.5 to about 8.5 and with the detergent below the cloud point, it dissolves in water and dissociates the endotoxin from the protein. The detergent containing endotoxin separates as a distinct phase when the temperature is increased above the cloud point in the range of about 25°C to about 40°C, preferably about 30°C to about 37°C. Centrifugation is then applied to achieve more rapid separation of the phases. It is important to note that the second ATPS technique with detergent removes both free and protein-bound endotoxin without affecting the activity of r-streptokinase. The detergent treatment causes little loss as well as no dilution of protein. Adsorption chromatography: After Triton XI14 treatment, the r-streptokinase fraction is subjected to adsorption chromatography on Amberlite XAD-2 chromatography which removes the traces of Triton XI14. This removal of Tritoi XI14 is required because non-anionic detergents such as Triton are more toxic and less biodegradable than anionic detergents and are not adsorbed to ion exchange columns. Ambertile XAD-2 is used for removal of detergents from larg volumes of protein solutions such as those used on an industrial scale (32). This i particularly so as regeneration is a simple, inexpensive and quick process. After the Amberlite XAD-2 adsorption, the Triton XI14 content in the r-streptokinase fraction is reduced to Dye-Ligand Chromatography: The r-streptokinase fraction which is now most! devoid of endotoxin is then subjected to dye-ligand chromatography on Blue sepharose fast flow matrix. Blue sepharose fast flow chromatography works on negative chromatographic mode by trapping all the high molecular weight impurities which co-elute with r-streptokinase both in ion exchange and in hydrophobic interaction chromatography and allowing the r-streptokinase to pas; through without binding. An advantage of the present invention is that negative chromatography on blue sepharose fast-flow matrix yields purified r-streptokinase in the same buffer, without any volume increase. Diafiltration: The r-streptokinase after the blue sepharose fast-flow matrix chromatography is diafiltered using about lOkD to about 50kD membrane, preferably about 30kD membrane, to remove small molecular weight contaminants and to transfer the sample in formulation buffer. It is then sterile filtered using 0.22 micron filter, formulated and Iyophilized for therapeutic application. The product obtained at the end of the purification process described herein is extremely pure with very negligible host cell protein ( The method of this invention comprises steps that remove endotoxin and DNA right from clarification step. The aqueous two-phase extraction used for clarification gives 3-4 log reduction of endotoxins. Subsequent chromatography steps results in 1-2 log reduction per step and the final polishing step is designed to bring the level of endotoxin very much lower than the prescribed limits. Also by using two different ATPSs, one at the clarification and the other at the final polishing step the present invention achieves extremely low endotoxin content. The product obtained is virtually free of endotoxins and DNA and thus suitable for therapeutic application. A further advantage of this invention is that it does not denature the protein as r-streptokinase activity is fully retained after the treatment. It has been observed that streptokinase purification from E. coli gives high molecular weight contaminants which are difficult to remove by ion exchange/hydrophobic chromatography and can be removed only by gel filtration which involves use of long column which is inconvenient in scale up operation. This invention achieves removal of these impurities by using affinity binding on blue sepharose chromatography which avoids use of long column and hence easily scalable. A major advantage of the process described in the foregoing embodiment is that it does not use chaotropic agents and consequently maintains the streptokinase in active form throughout the process. This results in elimination of the inconsistency in activity usually associated with procedures involving denaturation and renaturation of proteins. As a further advantage, the procedure of this example uses an ATPS step which drastically reduces the endotoxins in streptokinase prior to chromatographic methods. Also the use of two different ATPSs, one at the clarification and the other at the final polishing step, leads to achieve extremely low endotoxin content. The product obtained is virtually free of endotoxins and DNA and thus suitable for therapeutic application. Thus the subject process for the production of recombinant streptokinase from the genetically engineered E. coli is simple, efficient and easily scalable for large-scale production. The yield is higher than those reported from other systems. The method yields highly purified product with extremely low endotoxin content. The specific activity obtained is in line with that reported for streptokinase from natural source. In another embodiment of the invention, the purification is carried out using all of the steps used in the above mentioned embodiment. However, the difference lies in the sequence in which these steps are executed* Here, the hydrophobic chromatography is carried out before anion exchange chromatography. All details of these two steps are same as that for the preferred embodiment except in the hydrophobic interaction chromatography where the sample preparation is made by adding about 400mM to about 800mM ammonium sulphate to the diluted PEG extract. In yet another embodiment of the invention, the PEG- ATPS system has been left at about 4°C to about 25°C for about 10 hours to 24 hours, before collecting the PEG phase. The salt phase formed by gravity is siphoned out and the remaining solution is centrifuged to recover the PEG phase. This reduces the centrifugation volume and consequently the centrifugation time by >50%. Except for this change, details of all the subsequent steps are same as that for the preferred embodiment. Examples: Example 1: Referring to the steps for downstream processing and purification of streptokinase from lysate, from fermentation onwards, to the broken cell extract obtained from 301itres culture, solid ammonium sulphate was added to a final concentration of 20% w/w followed by PEG 1000MW to a final concentration of 8% w/w. The suspension was stirred at 200 rpm to 300 rpm for 30 minutes at 25°C. It was then centrifuged at 7000 rpm for 60 minutes at 4°C and the upper PEG phase containing streptokinase was separated by centrifugation. The extract of the above step was diluted by 5 times with a buffer of 20 mM Tris HC1 pH 7.5 and was made 600mM with respect to ammonium sulphate. The pH of the solution was then adjusted to 7.5 with sodium hydroxide, clarified by filtration through 0.45 micron membrane and applied onto a hydrophobic interaction column made up of phenyl sepharose (low substitute) matrix of 2000 ml, pre-equilibrated with same buffer. Decreasing ammonium sulphate gradient was then used to elute the bound proteins from the column. Bound streptokinase was eluted with 300 mM ammonium sulphate concentration. This chromatography resulted in further reduction in DNA and endotoxins. The enriched streptokinase fraction from hydrophobic interaction chromatography was subjected to anion exchange chromatography on Unosphere Q column of 1200 ml volume and eluted with a step-gradient of NaCl. The streptokinase was eluted with 300mM NaCl. This fraction was collected separately and processed for subsequent chromatography step. Streptokinase obtained was 98% pure with only traces of high molecular weight aggregates. Although progressive endotoxin reduction was achieved at all the above purification steps, endotoxin content was still too high for therapeutic applications. The concentration of eluted fraction was then increased by ten times by cross flow filtration with membrane with a cut-off value of 10 kD to reduce the fraction volume to approximately 1 litre for further operations. The concentrated streptokinase fraction was subjected to ATPS containing 1% Triton X 114 at 4°C to remove the free as well as the bound endotoxins from the streptokinase fraction. When the temperature of the ATPS was increased to 37 degree C phase separation occurred. The triton along with the separated endotoxin formed the bottom phase and was discarded, and streptokinase was fully retained in the aqueous phase and quantitatively recovered. After Triton XI14 treatment the Streptokinase fraction was subjected to adsorption chromatography on Ambertile XAD-2 column of 300ml, to remove the traces of Triton XI14. After the Amberlite XAD-2 adsorption, the Triton XI14 content in the Streptokinase sample was reduced to 0.002% as adjudged by HPLC. The flow-through which is free of triton XI14 was subjected to dye-ligand chromatography on Blue sepharose fast flow column of 300ml, to remove high molecular weight contaminants. Blue sepharose chromatography worked in the negative chromatographic mode by trapping all the high molecular weight impurities and allowing the streptokinase to pass through without binding. The product obtained at the end of the chromatographic procedure was extremely pure with very negligible host cell protein ( The streptokinase after the Blue sepharose chromatography was diafiltered using 30kD membrane to remove small molecular weight contaminants and to transfer the sample in formulation buffer. It was then sterile filtered using 0.22 micron filter, formulated and lyophilized with excipients for therapeutic application. Table 1 details the specific activity and the purity of purified recombinant streptokinase. Biological activity of streptokinase was determined by Clot-Lysis assay in which Fibronogen, plasminogen and thrombin were used in the assay. The assay was calibrated with a reference standard obtained from National Institute of Biological Standards and Control, U.K. Data are presented for 5 batches of purified streptokinase. By clot lysis procedure the natural streptokinase from streptococcus equisimilus strain H46A was found to have a specific activity of l.lx 105 IU/mg of streptokinase (US Patent application no 2003/059921) Thus the specific activity of recombinant streptokinase made in this example (Table 1) is in close agreement with the protein derived from the natural source. Table 2 illustrates endotoxin content in streptokinase bulk. Endotoxin limit as per European Pharmacopoiea is 23.3 EU/lxlO5 IU of streptokinase. Example 2: Referring to the steps for downstream processing and purification of streptokinase from lysate, from fermentation onwards, to the broken cell extract obtained from 301itres culture, solid ammonium sulphate was added to a final concentration of 20% w/w followed by PEG 1000MW to a final concentration of 8% w/w. The suspension was stirred at 200 rpm to 300 rpm for 30 minutes at 25°C. It was then centrifuged at 7000 rpm for 60 minutes at 4°C and the upper PEG phase containing streptokinase was separated by centrifugation. The extract of the above step was diluted by 5 times with a buffer 20 mM Tris HC1 pH 7.5 and clarified by filtration through 0.45 micron membrane. The above extract was subjected to anion exchange chromatography on Unosphere Q column of 1200 ml volume and eluted with a step-gradient of NaCl. The streptokinase was eluted with 300mM NaCl. This fraction was collected separately and processed for subsequent chromatography step. The enriched streptokinase fraction from anion exchange chromatography was made 600mM with respect to ammonium sulphate, pH adjusted to 7.5 with sodium hydroxide and applied onto a hydrophobic interaction column made up of phenyl sepharose (low substitute) matrix of 2000 ml, pre-equilibrated with same buffer. Decreasing ammonium sulphate gradient was then used to elute the bound proteins from the column. Bound streptokinase was eluted with 300 mM ammonium sulphate concentration. This chromatography resulted in further reduction in DNA and endotoxins. Streptokinase obtained was 98% pure with only traces of high molecular weight aggregates. Although progressive endotoxin reduction was achieved at all the above purification steps, endotoxin content was still too high for therapeutic applications. The concentration of eluted fraction was then increased by ten times by cross flow filtration with membrane with a cut-off value of 10 kD to reduce the fraction volume to approximately 1 litre for further operations. The concentrated streptokinase fraction was subjected to ATPS containing 1% Triton X 114 at 4°C to remove the free as well as the bound endotoxins from the streptokinase fraction. When the temperature of the ATPS was increased to 37 degree C phase separation occurred. The triton along with the separated endotoxin formed the bottom phase and was discarded, and streptokinase was fully retained in the aqueous phase and quantitatively recovered. After Triton XI14 treatment the Streptokinase fraction was subjected to adsorption chromatography on Ambertile XAD-2 column of 300ml, to remove the traces of Triton XI14. After the Amberlite XAD-2 adsorption, the Triton XI14 content in the Streptokinase sample was reduced to The flow-through which was free of triton XI14 was subjected to dye-ligand chromatography on Blue sepharose fast flow column of 300ml5 to remove high molecular weight contaminants. Blue sepharose chromatography worked in the negative chromatographic mode by trapping all the high molecular weight impurities and allowing the streptokinase to pass through without binding. The product obtained at the end of the chromatographic procedure was extremely pure with very negligible host cell protein ( The streptokinase after the Blue sepharose chromatography was diafiltered using 30kD membrane to remove small molecular weight contaminants and to transfer the sample in formulation buffer. It was then sterile filtered using 0.22 micron filter, formulated and lyophilized with excipients for therapeutic application. Other references 1. Gruppo Italiano per lo studio della streptochinai nell' Infarto miocardico (G1SS1). Effectiveness of intravenous thrombolytic treatment in acute myocardial infarction. Lancet 1986:1: 397-401. 2. AIMS trial study group. Effect of intravenous APSAC on mortality after acute myocardial infarction. Preliminary report of a placebo-controlled clinical trials. Lancet 1988 -1 - 545-549. 3. Simoons, M.L., Serruys, P.W., Van. Den. Brand. M, et.al. Early thrombosis in acute myocardial infarction. Limitation of infarct size and improved survival. J. Am. Coll. Cardiol. 1986: 7: 717-28. 4. Van de. Werf. F., Arnold AER and the European Co-operative study group for recombinant tissue type plasminogen activator (rtPA). Intravenous tissue plasminogen activator and size of infarct, left ventricular function and survival in acute myocardial infarction Br. Med. Journal 1988: 297: 2374-9. 5. Wilcox, R.G., Vonder, Lippe, G, Olsen C.G., Jenson G, Skeine, A.M., Hampton, J.R., Trial of tissue plasminogen activator for mortality reduction in acute myocardial infarction; the Anglo-scandinavian study of early thrombolysis (ASSET). Lancete 1988: 2. 525-30. 6. ISIS-2, (second International Study of infarct survival) Collaborative group. Randamised trial of intravenous streptokinase, oral aspirin, both or neither among 17187, cases of suspected acute myocardial infarction. Lancet 1988: 2, 349-60. 7. Ambler, R.P. (1972) Methods in Enzymology 25,143-154. 8. W.S. Tillet and R.L. Garner. J. ExpH. Med. 58, 485 (1933) J. ExpH. Med. 58,485(1933). 9. Martin, M., Streptokinase in chronic arterial diseases CRC, Boca Raton, Fl, 1982. 10. Collen. D. and Verstracte. M., Systemic thrombolytic therapy of active myocardial infarction? Circulation 68 (1983) 462-465. 11. Van de Werf F.? Eudbrook P.A., Bergmann S.R., Tiefenbrun, A.J., Fox, K.A.A., DeGeest H., Verstracte M., Collen D. and Sobel B.E. Coronary thrombolysis with tissue plasminogen activator in patients with evolving myocardial infarction. New England J. Med. 310 (1984) 609-613. 12. Jackson, K.W., and Tang, J., (1982). Biochemistry 21, 6620-6625. 13. Schick L. A., and F.J. Castellino 1973. Interaction of streptokinase and rabbit plasminogen Biochemistry 12,4315. 14. Bajaj, A.P., and F.J. Castellino 1977. Activation of human plasminogen by equimolar levels of streptokinase. J. Biol. Chem 252; 492. 15. Brogden, R.N., Speight, T.M. and Avery G.S. (1973) Drugs 5, 357-445. 16. Davies. M.C., Englert, M.E., and De. Renzo., E.G., (1964). J. Biol. Chem. 239,2651-2656. 17. Reddy K.N., 1988 - Streptokinase, Biochemistry and clinical application. Enzyme 40: 79. 18. Taylor, F.B. and Botts. J. (1968) Biochemistry 7, 232-238. 19. Brockway, W.J., and Castellino F.J. (1974) Biochemistry 13, 2063-2070. 20. United States Patent No. 5296366. Garcia et.al. March 22,1994. 21. E.C. De Renzo, P.K. Siiteri, B.L., Hutching and P.H. Bell. J. Biol. Chem 242, 533 (1967) 22. R.H. Tomar, Proc. Soc. ExpH. Biol. Med. 127,239 (1968). 23. A.Otto,G.Lorenz &G.Kopperschlager (1995) Bioseparation 51, 35-40 24. Malke, H, and Ferretti. JJ. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3551-3561. 25. Malke H, Gerlach. D., Kohler, W., and Ferretti. J.J. 1984 MMG, Mol. Gen. Genet 1996,360-365. 26. Ferretti, et. al., United States Patent No. 4,764,469. August 16,1988. 27. Malke, H., and Ferretti. J.J. Streptokinase: Cloning expression and excretion by Escherichia coli. Proc. Natl. Acad. Sci. USA. 81 (1984) 3557-3561. 28. Jackson, K.W., and Tang, J. (1982) Biochemistry 21,6620-6623. 29. Malke, H, and Ferretti. JJ. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3551-3561. 30. Dagmar Petsch and Friedrich Birger Anspach (2000) Journal of biotechnology ,76:97 - 119 31. Yoshitomi Aida and Michael J.Pabst (1990)Journal of Immunological Methods,132,191-195 32. Peter S.J. Cheetham (1979) Analytical Biochemistry 92,447-452. While the above description and examples contains many specificities, these are intended for the purpose of illustration and are not to be construed as limitations on the scope of the invention, but rather as an exemplification of one preferred embodiment thereof. Accordingly, the scope of the invention should be determined not by the embodiments illustrated, but by the appended claims and their legal equivalents. We Claim: 1. A novel process for producing therapeutic grade streptokinase, which process is simple, cost effective, efficient, easily scalable and results in high recovery of r-streptokinase which is virtually devoid of endotoxins, with specific activity of said r-streptokinase being in close agreement with native streptokinase, comprising the steps of: (a) mixing a phase forming polymer, a phase forming salt, and a buffer with a high r-streptokinase yielding bacteria lysate thereby forming a first mixture; wherein, said phase forming polymer comprises polyethylene glycol, with molecular weight of about 400 to about 2000 and final concentration of about 5% w/w to about 30% w/w; wherein, said phase forming salt comprises ammonium sulphate, with final concentration of about 10% w/w to about 30% w/w; wherein, said buffer comprises Tris HC1, of concentration of about lOmM to about 100 mM, with pH about 6.5 to about 8.5; and wherein, said bacteria comprises genetically engineered E.coli; (b) stirring said first mixture at about 200 rpm to about 300 rpm, for about 30 minutes to about 120 minutes, at a temperature of about 4° C to about 25° C and allowing the stirred mixture to stand for about 1 hour to about 24 hours at a temperature of about 5° C to about 35° C; thereby forming three phases; (c) separating said three phases of step(b) by centrifugation, thereby forming a top polymer phase, a middle phase, and a bottom phase; wherein said centrifugation is carried out at a speed of about 5000 rpm to about 15000 rpm, for about 30 minutes to about 120 minutes, and a temperature of about 4° C to about 25° C; (d) removing said bottom phase of step(c) by siphoning; (e) collecting said top polymer phase of step(c) by using peristaltic pump aspiration, thereby forming a polymer phase extract; (f) diluting said polymer phase extract of step(e) by mixing said extract with a first loading buffer, wherein said first loading buffer comprises Tris HC1 of concentration of about lOmM to lOOmM with pH of about 6.5 to about 8.5, preferably with pH of about 7.0 to about 8.5, with said dilution being carried out to about 2 times dilution to about 10 times dilution, thereby making concentration of mixture of said polymer phase extract and said first loading buffer about 20 mM; (g) clarifying the diluted mixture of step(f) by centrifugation or filtration, preferably by said filtration by using a 0.45 micron filter; (h) applying the clarified diluted mixture of step(g) onto anion exchange chromatography column, said column being pre-equilibrated with said first loading buffer, and adsorbing r-streptokinase on the stationary phase of said anion exchange chromatography column, with matrix of said column being of high binding capacity such as Unosphere Q; (i) collecting separately unbound material passing through said column of step(h); (j) washing said column of step(h) with said first loading buffer till all loosely bound proteins are removed; (k) eluting the adsorbed r-streptokinase of step(h) by using said first loading buffer while adding to it a salt such as sodium chloride applied in step gradient mode, capably increasing conductivity of said first loading buffer and maintaining the buffer concentration at about 100 mM to about 600mM sodium chloride; (1) mixing a second loading buffer with the eluted r-streptokinase fraction of step(k), thereby forming a second mixture; wherein said second loading buffer comprises solution of ammonium sulphate with salt concentration of about lOOmM to about 2000mM, preferably about 400mM to 800mM, with pH of said solution of step(l) being adjusted to about 6.5 to about 8.5, preferably about 7.0 to about 8.0, by addition of dilute sodium hydroxide; (m) applying said second mixture of step(l) onto a hydrophobic interaction chromatography column which is pre-equilibrated with said second loading buffer of step(l), wherein the stationary phase of said hydrophobic interaction chromatography column comprises phenyl sepharose low substitution; (n) eluting the bound r-steptokeinase and other proteins by using Tris HC1 buffer comprising a lower concentration salt, wherein said salt of step(n) is a kosmotropic salt such as ammonium sulphate with concentration of about lOOmM to about 300mM; (o) concentrating the eluted fraction of step(n), by using cross-flow ultra filtration which uses a membrane with molecular weight cutoff value of lOkD; (p) mixing the concentrated eluted fraction of step(o) with a neutral detergent, such as Triton X 114 at a concentration of about 0.5% to about 5%, which neutral detergent is at a temperature below 25° C, and with a mixing buffer which comprises Tris HC1 of about lOmM to about lOOmM concentration and of pH of about 6.5 to about 8.5; (q) increasing temperature of the mixture formed during step(p) to 40° C, capably forming two phases; (r) rapidly separating said two phases of step(q) by applying centrifugation to said mixture of step(q) (s) applying the separated phase of step (q) containing r-streptokinase fraction onto Amberlite XAD-2 chromatography column, capably removing traces of said Triton X 114; (t) applying the flow-through fraction of step (s) to blue sepharose fast flow chromatography column and collecting unbound eluted fraction; (u) diafiltering the resultant eluted fraction of step (t) through diafiltration membrane of 10 kD to 50 kD size, preferably 30kD size, capably transferring the r-streptokinase into a formulation buffer. (v) sterile filtering, said r-streptokinase contained in said formulation buffer of step(u), said sterile filtering being effected through 0.22 micron filter; and subsequently formulating with required excipient. 2. A novel process for producing therapeutic grade streptokinase, substantially as herein described with reference to examples and accompanying drawing.

Full Text

A NOVEL PROCESS FOR PRODUCING THERAPEUTIC GRADE STREPTOKINASE
Field of the invention
The present invention generally relates to a novel process for producing therapeutic grade streptokinase. More particularly it relates to a novel process for producing therapeutic grade streptokinase from genetically engineered E. coli.
Background of the invention
A significant medical problem, namely thrombosis, is presented by the occlusion of blood vessels due to the presence of thrombi (blood clots). Such thrombosis is potentially fatal, often affecting organs such as heart or lungs.
Over the last few years, intravenous thrombolysis (blood clot dissolution) has become the standard therapeutic approach for patients with myocardial infarction. The reduction in short and long term mortality rates caused by thrombolytic therapy has been demonstrated in several large-scale clinical trials (1-6).
One of the agents commonly used in thrombolytic therapy is the bacterial protein streptokinase (7), which is already in clinical use in many countries, treating patients with lung thrombus and acute myocardial infarction and has proved very effective. Its capacity to cause lysis of blood clots was first described in 1933 (8). Clinical studies over the last 10 years have shown that streptokinase decreases the morbidity and mortality.

Streptokinases, secretory proteins elaborated by many strains of hemolytic streptococci, belong to a widely distributed and heterogeneous class of proteins known as plasminogen activators, which includes Urokinase and Tissue type plasminogen activator (9-11). These plasminogen activators are at present used as thrombolytic agents in the treatment of disorders which collectively represent one of the greatest causes of death in the world, such as myocardial infarction, pulmonary, arterial or venous thromboembolism, and thrombosis due to surgical complications and other causes.
Interestingly streptokinase (SK) differs from most other plasminogen activators in that it is not a proteolytic enzyme. The primary structure of streptokinase (12) has revealed that streptokinase does have sequence homology to serine proteases. It is not known, however, whether this structural homology contributes to the plasminogen activation.
Although devoid of intrinsic protease activity, streptokinase avidly binds to plasminogen (from many mammalian species) and generate streptokinase-plasminogen activator complex (SK-PAC) that has protease activity (13, 14). This SK-PAC catalyses the conversion of the zymogen-plasminogen into the active enzyme plasmin. Plasmin then cleaves fibrin, the protein backbone of thrombus, resulting in thrombolysis.
Streptokinase's properties have been reviewed in detail (15). It functions in the species-specific conversion of plasminogen to plasmin. It complexes with human plasminogen or plasmin, forming an enzyme activator complex (which can directly convert human or bovine plasminogen to plasmin), a property which neither plasmin nor streptokinase alone possess (16). Bovine plasminogen, however, is essentially unreactive towards streptokinase with respect to the formation of plasminogen activator. As of now plasminogen and plasmin are the only substrates with which streptokinase is known to react.

Streptokinase is a bacterial protein derived from pathogenic strains of the streptococcus family (17). The function of streptokinase in the pathogenicity of streptococci is not known exactly, although potentially it may contribute to elimination or avoidance of formation of fibrin barriers around the infection. It is an extra cellular secretory protein produced in group C strains of beta-hemolytic streptococci with a molecular mass of 47,000 Daltons and is devoid of carbohydrate, cyteine and cystine (18,19).
Streptokinase has been purified for clinical usage and studied by several investigators. The primary source of this streptokinase has been the culture fluid resulting from the growth of the beta-hemolytic group C organism streptococcus equisimilis. Method of purifying streptokinase from culture medium of streptococci has been described by several investigators.
In US Patent No. 5840315, the streptokinase is prepared by affinity chromatography of culture medium by using monoclonal antibodies matrix.
In US Patent No. 3855065, purification is attempted by using gel filtration chromatography but purity level of the protein has not been described.
In US Patent No. 3186920, purification is achieved by mixing the culture supernatant with Carboxyl Methyl (CM) polysaccharide at higher temperature and eluting the bound protein with salt gradient.
In US Patent Nos. 299742 and 3042586 is described a procedure for purification of streptokinase using Diethyl Aminoethyl (DEAE) cellulose chromatography.
In KR Patent No. 9512897, a combination of ammonium sulphate precipitation and gel filtration chromatography has been used for purification of streptokinase.

In KR Patent No. 9702903, a procedure involving cation exchange chromatography for purification of streptokinase has been described.
/ In KR Patent No. 136613, the method describes a procedure which consists of treating the culture medium with reducing agent and incubating with a reagent R-X where R is reactive group capable of reacting with free thiols and effect impurity removal.
In GB Patent No. 894151, the purification procedure comprises precipitation by sodium chloride and anion exchange chromatography.
In GB Patent No. 966672, the method describes a combination of methanol and ammonium sulphate precipitations for purification of streptokinase.
In EP Patent No.0150424, a combination of ammonium sulphate precipitation and gel filtration chromatography has been used for purification of streptokinase.
In Indian Patent No. 137890, the method described involves CM cellulose chromatography and CaCk precipitation to obtain streptokinase.
The drawbacks of producing streptokinase from natural sources by these methods are:
1. The presence of toxic and undesirable substances makes the purification process more complex.
2. Low yields of streptokinase (0.1 to 0.2 g/L) and consequent handling of huge volumes of cultures medium makes the process inefficient and costly.
3. As streptokinase is produced by culturing hemolytic streptococci, which is a pathogenic organism, handling during production may cause inconvenience and hazards to workers.

4. Extreme care has to be taken to avoid hazards to the environment in case of leakage of streptococci.
5. The streptococcus strains used for industrial production of streptokinase secrete other products into the culture medium such as deoxyribonucleases, streptolysin or hyaluranidase and proteases, which makes the process of purifying the desired protein difficult.
6. Since many of these secretory contaminants are toxic or potentially toxic to humans, the preparation of streptokinase from culture fluid for clinical usage involves multi-steps which must be performed with great care to ensure a clinically acceptable product.
On the other hand it has not yet been possible to obtain genetically improved strains from these hosts due to the lack of a developed methodology for the gene transfer.
It is due to these drawbacks that the cloning of different isolated genes which code for these proteins has been attempted using prokaryotic and eukaryotic hosts (20). The recombinant DNA technology has opened up entirely new and important ways to use living cells to produce biologically active material such as enzymes and hormones in large amounts. Several methods of streptokinase purification have been published (21, 22). Besides chromatography, aqueous two-phase system (ATPS) composed of polyethylene glycol/dextran and polyethylene glycol/salt have been employed for separation and purification of streptokinase from culture supernatant (23).
The streptokinase gene from streptococcus equisimilus has been cloned (24) and expressed in streptococcus sanguis, an organism that does not normally produce streptokinase (25). As an alternative to the above bacterial systems, streptokinase has been expressed in methylotropic yeast Pichia pastoris. Several methods of purification of streptokinase from recombinant host have been published (26).

Recently a gene for streptokinase has been cloned from the chromosomal DNA of the group C streptococcal strain H46A (27).
The gene product has the same substrate specificity and antigenicity as streptokinase from natural source. The complete primary sequence of streptokinase has been published both from protein sequencing (28) and from elucidation of the nucleotide sequence of the entire cloned streptokinase gene from streptococcus equisimilis (29). The gene for streptokinase was expressed in E. coli with deduced amino acid sequence agreeing well with the known sequence of commercial streptokinase.
In US Patent No. 5296366, streptokinase has been expressed in E. coli as well as in Pichia systems with expression levels of 350 mg/L and 1 g/L respectively.
In US Patent application No. 2003/059921, is described a method which consists of solubilising the recombinant streptokinase from E. coli cells with GuCl and subjecting the extract to hydrophobic and anion exchange chromatography.
In Indian Patent No. 183828, the method consists of adsorption on silica and gel
■
filtration for purifying streptokinase and has employed affinity chromatography using acylated plasminogen for purifying streptokinase analogues.
In Indian Patent No. 185350, the protein is expressed as a periplasmic protein and an expression yield of 400 mg/L culture has been reported.
Streptokinase has also been expressed as fusion protein with maltose binding protein(US 5854049) or with histidine tag (US Patent No. 6087332) to enable affinity purification. Streptokinase mutant proteins with desired characteristics, have also been expressed in bacterial system (US Patent Nos. 6309873, 5876999, 5240845, IN Patent 183828). The purification strategies include gel filtration, ion

exchange, hydrophobic high performance liquid chromatography or isoelectric focusing techniques or combination of these techniques.
Although higher specific activity and yield have been reported in US Patent application No 2003/059921 which described a process using ion exchange and hydrophobic chromatographic procedure to produce r-streptokinase from E. coli, their method is devoid of special focus to bring down the endotoxin to the prescribed level for therapeutic use. Furthermore, solubilization of cells with 6M Guanidine hydrochloride (GuCl) as used in their procedure would completely solubilise the lysed membrane structures and consequently result in high endotoxin content in the extract. This high level of endotoxin cannot be handled by ion exchange and hydrophobic column alone as their method has no other efficient steps to bring down the endotoxins level as required for therapeutic purpose. Thus although good yields are reported in this method, it is not suitable for obtaining therapeutic grade protein.
The advantages in producing streptokinase by recombinant technology are as follows.
1) The pathogenic organisms and toxic substances are absent thus making
the development and purification process simpler and cost effective.
2) Manufacturing process is absolutely safe and worker-friendly.
3) The process is safe and environment friendly.
4) Higher safety may be exhibited for patients as it is free from streptolysin and streptodornase.
5) Higher yields of streptokinase are possible (Ig/L).
Although above mentioned expression and purification of streptokinase by recombinant method overcomes some of the problems and potential risk associated with the production from natural sources, it has its own drawbacks, which are as follows:

1) The yields though better than those from natural sources, have been low.
2) Presence of endotoxins is not desirable and removal of endotoxins poses a serious problem, particularly for streptokinase produced in E. coll
Endotoxins are an integral part of the cell membrane of gram negative bacteria and constitute approximately three quarters of the bacterial surface which consists of endotoxins. They are responsible for the organization and stability and consequently the cell lysate obtained from E.coli contains extremely high amount (~ >108 EU/ml) of endotoxins.
Bacterial endotoxins show strong biological effects even at very low concentration in human beings and in many animals when entering the blood stream, for example, during a bacterial infection or via intravenous application of a contaminated medicament. Because of their toxicity in vivo and in vitro, endotoxins removal is extremely important for a safe parental administration and therefore the endotoxin level has to be extremely low for therapeutics proteins.
Streptokinase is a high volume product with dose level of 15 mg/dose, which means stringency levels are high as far as endotoxin contamination is concerned. This requirement dictates that the process employed should be very efficient to bring down the endotoxin to the prescribed level. Several methods of endotoxin removal from protein solutions involving chromatographic or detergent extraction have been described in literature (30,31).
Although ion exchange and hydrophobic interaction chromatography columns do reduce endotoxins, the reduction efficiency are 2-3 log only, which means it is extremely difficult to obtain the endotoxin level which is required for therapeutic application with these column chromatography steps alone.

In view of the above mentioned disadvantages there is a need for a cost effective, simple and efficient purification process for producing therapeutic grade r-streptokinase from genetically engineered E.coli.
Objects and Advantages of the invention
Accordingly the present invention has several objects of achieving different advantages.
One object of the present invention is to provide a purification process for production of therapeutic grade streptokinase from genetically engineered E.Coli, which is simple, efficient and results in high recovery of the expressed protein.
Still another object of the present invention is to provide a process which can be scaled up easily for large scale purification of streptokinase
Yet another object of the present invention is to provide a process giving removal of both free and protein bound endotoxins without affecting the activity of streptokinase thereby yielding streptokinase which is virtually devoid of endotoxins.

Further object of the present invention is to provide a process that yields recombinant streptokinase with specific activity in close agreement with that of streptokinase from natural source.
Summary of the invention:
The present invention describes a novel process of purification for producing therapeutic grade streptokinase from genetically engineered bacteria. High yielding genetically engineered E. coli containing streptokinase gene derived from streptococci equisimilis strain H46A are grown, harvested and lysed. The lysate obtained is subjected to a series of purification steps such as ATPS, different types of chromatography and filtration to obtain highly purified r-streptokinase which has extremely low levels of host cell proteins, DNA, endotoxins and other contaminants. The final streptokinase product has high level of purity and is of therapeutic grade.
Detailed description
This invention provides a purification process for producing therapeutic grade streptokinase from genetically engineered E. coli. High yielding genetically engineered E. coli cells containing streptokinase gene derived from streptococci equisimilis strain H46A are grown in suitable growth media, harvested and lysed.
In a preferred embodiment of the present invention, the lysate obtained is subjected to a series of sequential purification steps such as the ATPS, anion exchange chromatography, hydrophobic chromatography, cross-flow filtration, ATPS with detergent, adsorption chromatography, dye-ligand chromatography, and ultrafiltration/diafiltration in order to obtain highly purified r-streptokinase which has extremely low levels of host cell proteins, DNA, endotoxins and other contaminants. These steps are described below in detail.

First ATPS: ATPS is an important emerging technique for separation, concentration, and purification of proteins, cell organelles, and other biological products. Initial clarification of lysate is achieved by using the first ATPS which is created by mixing the lysate with a phase forming salt such as ammonium sulphate to a final concentration of about 10% w/w to about 30% w/w and a phase forming polymer such as polyethylene glycol (PEG) with about 400 MW to about 2000 MW to a final concentration of about 5% w/w to about 30% w/w and a buffer comprising Tris HC1 of concentration of about lOmM to about lOOmM and pH of about 6.5 to about 8.5, to form a first mixture yielding a first ATPS. Polyethylene glycol is nontoxic and has a protective effect on proteins. The mixture is stirred vigorously for about 30 minutes to about 2 hours at about 200 rpm to about 300 rpm, at about 4°C to about 25°C. This first mixture is left to stand for a duration of about 1 hour to about 24 hours at temperature of about 5°C to about 35°C. This leads to formation of phases that are then separated by centrifugation for about 30 minutes to about 2 hours at about 5000 rpm to about 15000 rpm at about 4°C to about 25°C.
Three distinct phases are formed as a result of centrifugation. The top polymer phase, referred to as PEG phase primarily contains PEG and the r-streptokinase along with E. coli proteins. The unlysed cells, cell debris, lipoproteins, lipopolysaccahrides, endotoxins, and other contaminants form the interphase or the middle phase, whereas the bottom phase, referred to as the salt phase, consists essentially of nucleic acids and highly hydrophilic proteins along with the salt. The PEG phase is then carefully collected using peristaltic pump aspiration which is a method commonly known to person skilled in the art and subjected to further purification steps described below to obtain the desired level of purity.
This step results in the removal of the cell debris, DNA, some pigments, lipoproteins and substantial reduction (about 3 log to about 4 log reduction) of endotoxins. The reduction in level of endotoxin at this early stage of purification

is a major advantage of the present invention leading to increase in efficiency of endotoxin reduction of the subsequent steps of this invention.
This step results in enrichment of r-streptokinase in the PEG phase by adjusting the extreme volume ratios of the ATPS. It is only the PEG phase that is subjected to further purification steps. This significant reduction in the treatable volume is another major advantage of the present invention, which makes industrial scale production of r-streptokinase from E. coli lysate feasible.
Anion exchange chromatography: The PEG phase collected as above is mixed and diluted with a first loading buffer in order to prepare the phase for the anion exchange chromatography. The dilution is carried out so that the dilution effect is multiplied to about 2 to about 10 times. The first loading buffer used for dilution is Tris HC1 with a pH of about 6.5 to about 8.5, preferably from about 7.0 to about 8.5, and has a concentration of about 10 mM to about 100 mM. The desired concentration of the mixture of the first loading buffer and the PEG phase is about 20mM, and is arrived at by adding required volume of the first loading buffer to the PEG phase. The diluted sample is clarified using a 0.45 micron filter and applied onto a chromatographic matrix which is of anion exchange nature and which has been pre-equilibrated with the first loading buffer.
The unbound material passing through the column is collected and the column is washed with the first loading buffer till all loosely bound proteins are removed as monitored by measuring absorbance at 280 nm. The r-streptokinase, which is adsorbed on the stationary phase, is then eluted from the ion-exchange column by increasing the conductivity of the first loading buffer with salt such as sodium chloride applied in a step gradient mode. The streptokinase elutes with the first loading buffer of concentration of about 100 mM to about 600 mM NaCl. The r-streptokinase fraction is collected separately and processed for further chromatography.

The UnosphereQ, a macroporous matrix, is used as the stationary phase in the anion exchange chromatography mentioned above. This matrix exhibits at least 20-30 times higher binding capacity compared to other conventional anion. exchange matrices. The present invention has a major advantage that the use of this matrix results in consequent reduction of volume of buffers, quantity of chromatography matrix, operation time and cost of production. With the improved thermodynamic properties, the matrix allows the use of high flow rates while still giving high resolution, thus resulting in shorter process time. In any scale-up operation, where time is one of the major factors that influences the effectiveness of such operation, this is of great advantage.
The anion exchange chromatography results in further reduction of DNA, lipids, lipoproteins, endotoxins (2-log reduction), and host-cell proteins.
Hydrophobic interaction chromatography: The r-streptokinase fraction obtained from anion exchange chromatography is further prepared for processing with hydrophobic interaction chromatography. A second loading buffer containing salt such ammonium sulphate is added so as to arrive at a salt concentration of about lOOmM to about 2000mM, preferably about 400mM to about 800mM. The pH of the solution is then adjusted to about 6.5 to about 8.5, preferably about 7.0 to about 8.0, with dilute sodium hydroxide. The resultant second mixture of the salt and r-streptokinase fraction is applied onto a hydrophobic column made of phenyl sepharose (low substitute) matrix that has been pre-equilibrated with the same second loading buffer. The r-streptokinase component is adsorbed on the stationary phase.
Analysis by SDS gel electrophoresis reveals elimination of most of the host cell protein contaminants in the unadsorbed fraction. Next, a Tris HC1 buffer containing lower salt concentration of about 100 mM to about 300 mM is used to elute the bound proteins, including r-streptokinase, from the column.

This chromatography results in further reduction in DNA and endotoxins (about 1 log reduction). Streptokinase obtained is about 98% pure, however traces of high molecular weight aggregates are present and which need to be removed with further purification measures. Also, although progressive endotoxin reduction is achieved at all foregoing purification steps, the resultant level of endotoxin content is still too high for therapeutic applications on humans. Additional purification measures are applied to further reduce the endotoxin levels.
Ultra filtration: The concentration of eluted fraction obtained from the hydrophobic chromatography stage is then increased by about ten times by cross-flow ultrafiltration with membrane with a molecular weight cut-off value of lOkD. This filtration removes contaminants with small molecular weight from the protein solution.
At the end of this step, there are still protein-bound endotoxins present. ATPS technique with use of detergents is applied next to remove protein-bound endotoxins.
Second ATPS with detergent: A second ATPS technique using detergent such as Triton X 114 at a concentration of about 0.5% to about 5% effectively removes the traces of both free and protein-bound endotoxin. This detergent has a cloud point of about 25°C. When protein solution is mixed with a mixing buffer comprising Tris HC1 of concentration of about lOmM to about lOOmM and pH of about 6.5 to about 8.5 and with the detergent below the cloud point, it dissolves in water and dissociates the endotoxin from the protein. The detergent containing endotoxin separates as a distinct phase when the temperature is increased above the cloud point in the range of about 25°C to about 40°C, preferably about 30°C to about 37°C. Centrifugation is then applied to achieve more rapid separation of the phases.

It is important to note that the second ATPS technique with detergent removes both free and protein-bound endotoxin without affecting the activity of r-streptokinase. The detergent treatment causes little loss as well as no dilution of protein.
Adsorption chromatography: After Triton XI14 treatment, the r-streptokinase fraction is subjected to adsorption chromatography on Amberlite XAD-2 chromatography which removes the traces of Triton XI14. This removal of Tritoi XI14 is required because non-anionic detergents such as Triton are more toxic and less biodegradable than anionic detergents and are not adsorbed to ion exchange columns. Ambertile XAD-2 is used for removal of detergents from larg volumes of protein solutions such as those used on an industrial scale (32). This i particularly so as regeneration is a simple, inexpensive and quick process.
After the Amberlite XAD-2 adsorption, the Triton XI14 content in the r-streptokinase fraction is reduced to Dye-Ligand Chromatography: The r-streptokinase fraction which is now most! devoid of endotoxin is then subjected to dye-ligand chromatography on Blue sepharose fast flow matrix. Blue sepharose fast flow chromatography works on negative chromatographic mode by trapping all the high molecular weight impurities which co-elute with r-streptokinase both in ion exchange and in hydrophobic interaction chromatography and allowing the r-streptokinase to pas; through without binding.
An advantage of the present invention is that negative chromatography on blue sepharose fast-flow matrix yields purified r-streptokinase in the same buffer, without any volume increase.

Diafiltration: The r-streptokinase after the blue sepharose fast-flow matrix chromatography is diafiltered using about lOkD to about 50kD membrane, preferably about 30kD membrane, to remove small molecular weight contaminants and to transfer the sample in formulation buffer. It is then sterile filtered using 0.22 micron filter, formulated and Iyophilized for therapeutic application.
The product obtained at the end of the purification process described herein is extremely pure with very negligible host cell protein ( The method of this invention comprises steps that remove endotoxin and DNA right from clarification step. The aqueous two-phase extraction used for clarification gives 3-4 log reduction of endotoxins. Subsequent chromatography steps results in 1-2 log reduction per step and the final polishing step is designed to bring the level of endotoxin very much lower than the prescribed limits. Also by using two different ATPSs, one at the clarification and the other at the final polishing step the present invention achieves extremely low endotoxin content. The product obtained is virtually free of endotoxins and DNA and thus suitable for therapeutic application. A further advantage of this invention is that it does not denature the protein as r-streptokinase activity is fully retained after the treatment.
It has been observed that streptokinase purification from E. coli gives high molecular weight contaminants which are difficult to remove by ion exchange/hydrophobic chromatography and can be removed only by gel filtration which involves use of long column which is inconvenient in scale up operation. This invention achieves removal of these impurities by using affinity binding on blue sepharose chromatography which avoids use of long column and hence easily scalable.

A major advantage of the process described in the foregoing embodiment is that it does not use chaotropic agents and consequently maintains the streptokinase in active form throughout the process. This results in elimination of the inconsistency in activity usually associated with procedures involving denaturation and renaturation of proteins.
As a further advantage, the procedure of this example uses an ATPS step which drastically reduces the endotoxins in streptokinase prior to chromatographic methods. Also the use of two different ATPSs, one at the clarification and the other at the final polishing step, leads to achieve extremely low endotoxin content. The product obtained is virtually free of endotoxins and DNA and thus suitable for therapeutic application.
Thus the subject process for the production of recombinant streptokinase from the genetically engineered E. coli is simple, efficient and easily scalable for large-scale production. The yield is higher than those reported from other systems. The method yields highly purified product with extremely low endotoxin content. The specific activity obtained is in line with that reported for streptokinase from natural source.
In another embodiment of the invention, the purification is carried out using all of the steps used in the above mentioned embodiment. However, the difference lies in the sequence in which these steps are executed* Here, the hydrophobic chromatography is carried out before anion exchange chromatography. All details of these two steps are same as that for the preferred embodiment except in the hydrophobic interaction chromatography where the sample preparation is made by adding about 400mM to about 800mM ammonium sulphate to the diluted PEG extract.
In yet another embodiment of the invention, the PEG- ATPS system has been left at about 4°C to about 25°C for about 10 hours to 24 hours, before collecting the

PEG phase. The salt phase formed by gravity is siphoned out and the remaining solution is centrifuged to recover the PEG phase. This reduces the centrifugation volume and consequently the centrifugation time by >50%. Except for this change, details of all the subsequent steps are same as that for the preferred embodiment.
Examples:
Example 1: Referring to the steps for downstream processing and purification of streptokinase from lysate, from fermentation onwards, to the broken cell extract obtained from 301itres culture, solid ammonium sulphate was added to a final concentration of 20% w/w followed by PEG 1000MW to a final concentration of 8% w/w. The suspension was stirred at 200 rpm to 300 rpm for 30 minutes at 25°C. It was then centrifuged at 7000 rpm for 60 minutes at 4°C and the upper PEG phase containing streptokinase was separated by centrifugation.
The extract of the above step was diluted by 5 times with a buffer of 20 mM Tris HC1 pH 7.5 and was made 600mM with respect to ammonium sulphate. The pH of the solution was then adjusted to 7.5 with sodium hydroxide, clarified by filtration through 0.45 micron membrane and applied onto a hydrophobic interaction column made up of phenyl sepharose (low substitute) matrix of 2000 ml, pre-equilibrated with same buffer. Decreasing ammonium sulphate gradient was then used to elute the bound proteins from the column. Bound streptokinase was eluted with 300 mM ammonium sulphate concentration. This chromatography resulted in further reduction in DNA and endotoxins.
The enriched streptokinase fraction from hydrophobic interaction chromatography was subjected to anion exchange chromatography on Unosphere Q column of 1200 ml volume and eluted with a step-gradient of NaCl. The streptokinase was eluted with 300mM NaCl. This fraction was collected separately and processed for subsequent chromatography step.

Streptokinase obtained was 98% pure with only traces of high molecular weight aggregates. Although progressive endotoxin reduction was achieved at all the above purification steps, endotoxin content was still too high for therapeutic applications.
The concentration of eluted fraction was then increased by ten times by cross flow filtration with membrane with a cut-off value of 10 kD to reduce the fraction volume to approximately 1 litre for further operations.
The concentrated streptokinase fraction was subjected to ATPS containing 1% Triton X 114 at 4°C to remove the free as well as the bound endotoxins from the streptokinase fraction. When the temperature of the ATPS was increased to 37 degree C phase separation occurred. The triton along with the separated endotoxin formed the bottom phase and was discarded, and streptokinase was fully retained in the aqueous phase and quantitatively recovered.
After Triton XI14 treatment the Streptokinase fraction was subjected to adsorption chromatography on Ambertile XAD-2 column of 300ml, to remove the traces of Triton XI14. After the Amberlite XAD-2 adsorption, the Triton XI14 content in the Streptokinase sample was reduced to 0.002% as adjudged by HPLC.
The flow-through which is free of triton XI14 was subjected to dye-ligand chromatography on Blue sepharose fast flow column of 300ml, to remove high molecular weight contaminants. Blue sepharose chromatography worked in the negative chromatographic mode by trapping all the high molecular weight impurities and allowing the streptokinase to pass through without binding.

The product obtained at the end of the chromatographic procedure was extremely pure with very negligible host cell protein ( The streptokinase after the Blue sepharose chromatography was diafiltered using 30kD membrane to remove small molecular weight contaminants and to transfer the sample in formulation buffer. It was then sterile filtered using 0.22 micron filter, formulated and lyophilized with excipients for therapeutic application.
Table 1 details the specific activity and the purity of purified recombinant streptokinase. Biological activity of streptokinase was determined by Clot-Lysis assay in which Fibronogen, plasminogen and thrombin were used in the assay. The assay was calibrated with a reference standard obtained from National Institute of Biological Standards and Control, U.K. Data are presented for 5 batches of purified streptokinase.

By clot lysis procedure the natural streptokinase from streptococcus equisimilus strain H46A was found to have a specific activity of l.lx 105 IU/mg of streptokinase (US Patent application no 2003/059921) Thus the specific activity

of recombinant streptokinase made in this example (Table 1) is in close agreement
with the protein derived from the natural source.
Table 2 illustrates endotoxin content in streptokinase bulk.

Endotoxin limit as per European Pharmacopoiea is 23.3 EU/lxlO5 IU of streptokinase.
Example 2: Referring to the steps for downstream processing and purification of streptokinase from lysate, from fermentation onwards, to the broken cell extract obtained from 301itres culture, solid ammonium sulphate was added to a final concentration of 20% w/w followed by PEG 1000MW to a final concentration of 8% w/w. The suspension was stirred at 200 rpm to 300 rpm for 30 minutes at 25°C. It was then centrifuged at 7000 rpm for 60 minutes at 4°C and the upper PEG phase containing streptokinase was separated by centrifugation.
The extract of the above step was diluted by 5 times with a buffer 20 mM Tris HC1 pH 7.5 and clarified by filtration through 0.45 micron membrane.

The above extract was subjected to anion exchange chromatography on Unosphere Q column of 1200 ml volume and eluted with a step-gradient of NaCl. The streptokinase was eluted with 300mM NaCl. This fraction was collected separately and processed for subsequent chromatography step.
The enriched streptokinase fraction from anion exchange chromatography was made 600mM with respect to ammonium sulphate, pH adjusted to 7.5 with sodium hydroxide and applied onto a hydrophobic interaction column made up of phenyl sepharose (low substitute) matrix of 2000 ml, pre-equilibrated with same buffer. Decreasing ammonium sulphate gradient was then used to elute the bound proteins from the column. Bound streptokinase was eluted with 300 mM ammonium sulphate concentration. This chromatography resulted in further reduction in DNA and endotoxins. Streptokinase obtained was 98% pure with only traces of high molecular weight aggregates. Although progressive endotoxin reduction was achieved at all the above purification steps, endotoxin content was still too high for therapeutic applications.
The concentration of eluted fraction was then increased by ten times by cross flow filtration with membrane with a cut-off value of 10 kD to reduce the fraction volume to approximately 1 litre for further operations.
The concentrated streptokinase fraction was subjected to ATPS containing 1% Triton X 114 at 4°C to remove the free as well as the bound endotoxins from the streptokinase fraction. When the temperature of the ATPS was increased to 37 degree C phase separation occurred. The triton along with the separated endotoxin formed the bottom phase and was discarded, and streptokinase was fully retained in the aqueous phase and quantitatively recovered.
After Triton XI14 treatment the Streptokinase fraction was subjected to adsorption chromatography on Ambertile XAD-2 column of 300ml, to remove the traces of Triton XI14. After the Amberlite XAD-2 adsorption, the Triton XI14

content in the Streptokinase sample was reduced to The flow-through which was free of triton XI14 was subjected to dye-ligand chromatography on Blue sepharose fast flow column of 300ml5 to remove high molecular weight contaminants. Blue sepharose chromatography worked in the negative chromatographic mode by trapping all the high molecular weight impurities and allowing the streptokinase to pass through without binding.
The product obtained at the end of the chromatographic procedure was extremely pure with very negligible host cell protein ( The streptokinase after the Blue sepharose chromatography was diafiltered using 30kD membrane to remove small molecular weight contaminants and to transfer the sample in formulation buffer. It was then sterile filtered using 0.22 micron filter, formulated and lyophilized with excipients for therapeutic application.

Other references
1. Gruppo Italiano per lo studio della streptochinai nell' Infarto miocardico
(G1SS1). Effectiveness of intravenous thrombolytic treatment in acute
myocardial infarction. Lancet 1986:1: 397-401.
2. AIMS trial study group. Effect of intravenous APSAC on mortality after
acute myocardial infarction. Preliminary report of a placebo-controlled
clinical trials. Lancet 1988 -1 - 545-549.
3. Simoons, M.L., Serruys, P.W., Van. Den. Brand. M, et.al. Early
thrombosis in acute myocardial infarction. Limitation of infarct size and
improved survival. J. Am. Coll. Cardiol. 1986: 7: 717-28.
4. Van de. Werf. F., Arnold AER and the European Co-operative study group
for recombinant tissue type plasminogen activator (rtPA). Intravenous
tissue plasminogen activator and size of infarct, left ventricular function
and survival in acute myocardial infarction Br. Med. Journal 1988: 297:
2374-9.

5. Wilcox, R.G., Vonder, Lippe, G, Olsen C.G., Jenson G, Skeine, A.M., Hampton, J.R., Trial of tissue plasminogen activator for mortality reduction in acute myocardial infarction; the Anglo-scandinavian study of early thrombolysis (ASSET). Lancete 1988: 2. 525-30.
6. ISIS-2, (second International Study of infarct survival) Collaborative group. Randamised trial of intravenous streptokinase, oral aspirin, both or neither among 17187, cases of suspected acute myocardial infarction. Lancet 1988: 2, 349-60.
7. Ambler, R.P. (1972) Methods in Enzymology 25,143-154.
8. W.S. Tillet and R.L. Garner. J. ExpH. Med. 58, 485 (1933) J. ExpH. Med. 58,485(1933).
9. Martin, M., Streptokinase in chronic arterial diseases CRC, Boca Raton, Fl, 1982.
10. Collen. D. and Verstracte. M., Systemic thrombolytic therapy of active myocardial infarction? Circulation 68 (1983) 462-465.
11. Van de Werf F.? Eudbrook P.A., Bergmann S.R., Tiefenbrun, A.J., Fox, K.A.A., DeGeest H., Verstracte M., Collen D. and Sobel B.E. Coronary thrombolysis with tissue plasminogen activator in patients with evolving myocardial infarction. New England J. Med. 310 (1984) 609-613.
12. Jackson, K.W., and Tang, J., (1982). Biochemistry 21, 6620-6625.
13. Schick L. A., and F.J. Castellino 1973. Interaction of streptokinase and rabbit plasminogen Biochemistry 12,4315.
14. Bajaj, A.P., and F.J. Castellino 1977. Activation of human plasminogen by equimolar levels of streptokinase. J. Biol. Chem 252; 492.
15. Brogden, R.N., Speight, T.M. and Avery G.S. (1973) Drugs 5, 357-445.
16. Davies. M.C., Englert, M.E., and De. Renzo., E.G., (1964). J. Biol. Chem. 239,2651-2656.
17. Reddy K.N., 1988 - Streptokinase, Biochemistry and clinical application. Enzyme 40: 79.
18. Taylor, F.B. and Botts. J. (1968) Biochemistry 7, 232-238.
19. Brockway, W.J., and Castellino F.J. (1974) Biochemistry 13, 2063-2070.

20. United States Patent No. 5296366. Garcia et.al. March 22,1994.
21. E.C. De Renzo, P.K. Siiteri, B.L., Hutching and P.H. Bell. J. Biol. Chem 242, 533 (1967)
22. R.H. Tomar, Proc. Soc. ExpH. Biol. Med. 127,239 (1968).
23. A.Otto,G.Lorenz &G.Kopperschlager (1995) Bioseparation 51, 35-40
24. Malke, H, and Ferretti. JJ. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3551-3561.
25. Malke H, Gerlach. D., Kohler, W., and Ferretti. J.J. 1984 MMG, Mol. Gen. Genet 1996,360-365.
26. Ferretti, et. al., United States Patent No. 4,764,469. August 16,1988.
27. Malke, H., and Ferretti. J.J. Streptokinase: Cloning expression and excretion by Escherichia coli. Proc. Natl. Acad. Sci. USA. 81 (1984) 3557-3561.
28. Jackson, K.W., and Tang, J. (1982) Biochemistry 21,6620-6623.
29. Malke, H, and Ferretti. JJ. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3551-3561.
30. Dagmar Petsch and Friedrich Birger Anspach (2000) Journal of biotechnology ,76:97 - 119
31. Yoshitomi Aida and Michael J.Pabst (1990)Journal of Immunological Methods,132,191-195
32. Peter S.J. Cheetham (1979) Analytical Biochemistry 92,447-452.
While the above description and examples contains many specificities, these are intended for the purpose of illustration and are not to be construed as limitations on the scope of the invention, but rather as an exemplification of one preferred embodiment thereof. Accordingly, the scope of the invention should be determined not by the embodiments illustrated, but by the appended claims and their legal equivalents.

We Claim:
1. A novel process for producing therapeutic grade streptokinase, which process is simple, cost effective, efficient, easily scalable and results in high recovery of r-streptokinase which is virtually devoid of endotoxins, with specific activity of said r-streptokinase being in close agreement with native streptokinase, comprising the steps of:
(a) mixing a phase forming polymer, a phase forming salt, and a buffer with a
high r-streptokinase yielding bacteria lysate thereby forming a first
mixture;
wherein, said phase forming polymer comprises polyethylene glycol, with
molecular weight of about 400 to about 2000 and final concentration of
about 5% w/w to about 30% w/w;
wherein, said phase forming salt comprises ammonium sulphate, with
final concentration of about 10% w/w to about 30% w/w; wherein, said
buffer comprises Tris HC1, of concentration of about lOmM to about 100
mM, with pH about 6.5 to about 8.5; and
wherein, said bacteria comprises genetically engineered E.coli;
(b) stirring said first mixture at about 200 rpm to about 300 rpm, for about 30 minutes to about 120 minutes, at a temperature of about 4° C to about 25° C and allowing the stirred mixture to stand for about 1 hour to about 24 hours at a temperature of about 5° C to about 35° C; thereby forming three phases;
(c) separating said three phases of step(b) by centrifugation, thereby forming a top polymer phase, a middle phase, and a bottom phase;
wherein said centrifugation is carried out at a speed of about 5000 rpm to about 15000 rpm, for about 30 minutes to about 120 minutes, and a temperature of about 4° C to about 25° C;

(d) removing said bottom phase of step(c) by siphoning;
(e) collecting said top polymer phase of step(c) by using peristaltic pump aspiration, thereby forming a polymer phase extract;
(f) diluting said polymer phase extract of step(e) by mixing said extract with a first loading buffer, wherein said first loading buffer comprises Tris HC1 of concentration of about lOmM to lOOmM with pH of about 6.5 to about 8.5, preferably with pH of about 7.0 to about 8.5, with said dilution being carried out to about 2 times dilution to about 10 times dilution, thereby making concentration of mixture of said polymer phase extract and said first loading buffer about 20 mM;
(g) clarifying the diluted mixture of step(f) by centrifugation or filtration, preferably by said filtration by using a 0.45 micron filter;
(h) applying the clarified diluted mixture of step(g) onto anion exchange chromatography column, said column being pre-equilibrated with said first loading buffer, and adsorbing r-streptokinase on the stationary phase of said anion exchange chromatography column, with matrix of said column being of high binding capacity such as Unosphere Q;
(i) collecting separately unbound material passing through said column of step(h);
(j) washing said column of step(h) with said first loading buffer till all loosely bound proteins are removed;
(k) eluting the adsorbed r-streptokinase of step(h) by using said first loading buffer while adding to it a salt such as sodium chloride applied in step

gradient mode, capably increasing conductivity of said first loading buffer and maintaining the buffer concentration at about 100 mM to about 600mM sodium chloride;
(1) mixing a second loading buffer with the eluted r-streptokinase fraction of step(k), thereby forming a second mixture; wherein said second loading buffer comprises solution of ammonium sulphate with salt concentration of about lOOmM to about 2000mM, preferably about 400mM to 800mM, with pH of said solution of step(l) being adjusted to about 6.5 to about 8.5, preferably about 7.0 to about 8.0, by addition of dilute sodium hydroxide;
(m) applying said second mixture of step(l) onto a hydrophobic interaction chromatography column which is pre-equilibrated with said second loading buffer of step(l), wherein the stationary phase of said hydrophobic interaction chromatography column comprises phenyl sepharose low
substitution;
(n) eluting the bound r-steptokeinase and other proteins by using Tris HC1 buffer comprising a lower concentration salt, wherein said salt of step(n) is a kosmotropic salt such as ammonium sulphate with concentration of about lOOmM to about 300mM;
(o) concentrating the eluted fraction of step(n), by using cross-flow ultra filtration which uses a membrane with molecular weight cutoff value of lOkD;
(p) mixing the concentrated eluted fraction of step(o) with a neutral detergent, such as Triton X 114 at a concentration of about 0.5% to about 5%, which neutral detergent is at a temperature below 25° C, and with a mixing buffer which comprises Tris HC1 of about lOmM to about lOOmM concentration and of pH of about 6.5 to about 8.5;

(q) increasing temperature of the mixture formed during step(p) to 40° C, capably forming two phases;
(r) rapidly separating said two phases of step(q) by applying centrifugation to said mixture of step(q)
(s) applying the separated phase of step (q) containing r-streptokinase fraction onto Amberlite XAD-2 chromatography column, capably removing traces of said Triton X 114;
(t) applying the flow-through fraction of step (s) to blue sepharose fast flow chromatography column and collecting unbound eluted fraction;
(u) diafiltering the resultant eluted fraction of step (t) through diafiltration membrane of 10 kD to 50 kD size, preferably 30kD size, capably transferring the r-streptokinase into a formulation buffer.
(v) sterile filtering, said r-streptokinase contained in said formulation buffer of step(u), said sterile filtering being effected through 0.22 micron filter; and subsequently formulating with required excipient.
2. A novel process for producing therapeutic grade streptokinase, substantially as herein described with reference to examples and accompanying drawing.

Documents:

1103-che-2004 abstract duplicate.pdf

1103-che-2004 claims duplicate.pdf

1103-che-2004 description (complete) duplicate.pdf

1103-che-2004-abstract.pdf

1103-che-2004-claims.pdf

1103-che-2004-correspondnece-others.pdf

1103-che-2004-correspondnece-po.pdf

1103-che-2004-description(complete).pdf

1103-che-2004-form 1.pdf

1103-che-2004-form 19.pdf

1103-che-2004-form 26.pdf

1103-che-2004-form 3.pdf

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Patent Number

220936

Indian Patent Application Number

1103/CHE/2004

PG Journal Number

31/2008

Publication Date

01-Aug-2008

Grant Date

11-Jun-2008

Date of Filing

20-Oct-2004

Name of Patentee

SHANTHA BIOTECHNICS PRIVATE LIMITED

Applicant Address

Inventors:

#	Inventor's Name	Inventor's Address
1	DAMOTHARAN VIJAYARANGAM

PCT International Classification Number

C12N 9/70

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA