FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENT RULES , 2003
PROVISIONAL SPECIFICATION
(See Section 10; rule 13)
" PROCESS FOR PURIFICATION
OF
HUMAN TISSUE TYPE PLASMINOGEN ACTIVATOR"
RELIANCE LIFE SCIENCES PVT.LTD
an Indian Company having its Registered Office at
Dhirubhai Ambani Life Sciences Centre,
R-282, TTC Area of MIDC,
Thane Belapur Road, Rabale,
Navi Mumbai - 400 701 Maharashtra India
The following specification particularly describes and ascertains the nature of this invention and the manner in which it is performed:-

FIELD OF THE INVENTION:
The present invention relates to a simple and efficient process for purification of recombinant protein. The present invention in particular relates to the purification of truncated tissue plasminogen activator produced by optimizing the arginine concentration during chromatography.
BACKGROUND OF THE INVENTION
Thrombosis is an important part of the normal hemostatic response that limits hemorrhage from microscopic or macroscopic vascular injury. Physiologic thrombosis is counterbalanced by intrinsic antithrombotic properties and physiologic fibrinolysis. Under normal conditions, thrombus is confined to the immediate area of injury and does not obstruct flow to critical areas, unless vessel lumen is diminished already such as in atherosclerosis. Under pathological conditions, thrombus can propagate into otherwise normal vessels. Thrombus that has propagated where it is not needed can obstruct flow in critical vessels and can obliterate valves and other structures that are essential to normal hemodynamic function. The principal clinical syndromes that result are acute myocardial infarction (MI), deep vein thrombosis, pulmonary embolism, acute ischemic stroke, acute peripheral arterial occlusion, and occlusion of indwelling catheters.
Both hemostasis and thrombosis depend on the coagulation cascade, vascular wall integrity, and platelets response. Several cellular factors are responsible for thrombus formation. When a vascular insult occurs, immediate local cellular response takes place. Platelets migrate to the area of injury where they secrete several cellular factors and mediators. These mediators promote the clot formation.
During thrombus formation, circulating prothrombin is activated by platelets. In this process, other major step that takes place is fibrinogen getting converted to fibrin, which then creates the fibrin matrix. All this takes place while platelets are being adhered and aggregated. Fibrin-bound plasminogen gets converted by thrombolytic drugs to plasmin, the rate-limiting step in thrombolysis.
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Thrombolytic drug breaks up or dissolves blood clots, which are the main cause of both heart attacks and stroke. By dissolving the clot, the blood is able to start flowing again to that area of the heart. If the blood flow to the heart is started again rapidly, it may prevent long-term damage to the heart muscle and may even stop an event that could be fatal. Thrombolytic agents available today are serine proteases that work by converting plasminogen to the natural fibrinolytic agent plasmin. Plasmin lyses clot by breaking down the fibrinogen and fibrin contained in a clot. Urokinase like plasminogen activators are produced in renal cells. They circulate in blood and are excreted in the urine. Their ability to catalyze the conversion of plasminogen to plasmin is affected slightly by the presence or absence of local fibrin clot.
Not a single plasminogen activator have been approved by the US Food and Drug Administration (FDA) to be labeled for every indication, and new agents and new dosing regimens are under constant investigation. A choice of plasminogen activator is generally based upon the results of ongoing clinical trials and upon the clinician's experience. The most appropriate agent and regimen for each clinical situation changes over time and may differ from patient to patient.
Three groups of thrombolytic agents are available, including enzymes, which act directly upon the fibrin strands within the clot, plasma activator agents, which increase plasma activator activity, and plasminogen activators, such as streptokinase, urokinase, and tissue plasminogen. All these drugs digest clots by increasing the amount of plasmin (plasmin dissolves clots) in the blood. To produce plasmin, the substance plasminogen must first be activated. Plasminogen is converted into plasmin by certain enzymes known as plasminogen activators.
Streptokinase has been used since about 1960. Researchers use streptococci bacteria to produce this drug. Although streptokinase is the least expensive activator, some negative side effects, such as immune responses, have been experienced by patients. Urokinase is found naturally in humans, especially in the urine. Thus, no negative immune response is associated with its use. This therapy is usually administered in small doses and combined with other drugs, because it is difficult to purify, and therefore rather expensive.
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Tissue plasminogen activator (tPA) is currently used most for dissolving blood clots. It is unique because it activates only fibrin-bound plasminogen and thus targets the clot site. tPA in human blood is produced in very small amounts by vascular endothelial cells.
Tissue plasminogen activator (tPA) is a secreted serine protease which converts the proenzyme plasminogen to plasmin, a fibrinolytic enzyme. Plasminogen is synthesized as a single chain which is cleaved by tPA into the two chain disulfide linked plasmin. This enzyme plays a role in cell migration and tissue remodeling. Increased enzymatic activity causes hyperfibrinolysis, which manifests as excessive bleeding; decreased activity leads to hypofibrinolysis which can result in thrombosis or embolism. TPA is an enzyme that helps dissolve clots. tPA is made by the cells lining blood vessels and has also been made in the laboratory. It is systemic thrombolytic (clot-busting) agent and is used in the treatment of heart attack and stroke.
tPA is also known to be secreted naturally from a number of tissue sources including pig heart, fetal kidney, lung, and colon fibroblast cells. Recently, tPA has been produced using recombinant means by a number of groups, initiated by the successful cloning of the cDNA by Pennica, D. et al. Nature (1983) 301:214-221. The protein can be produced in a variety of hosts including E. coli, mouse L cells, CHO cells and yeast. (See, for example, EP Publication No. 174,835 (UpJohn), EP Publication No. 161,935 (Eli Lilly), EP Publication No. 143,081 (Ciba-Geigy), PCT application No. W086/05514 (Chiron).) Native tPA has been produced as disclosed by Snow Brand Milk Products (EP 196,226); Kochi Medical School (EP 194,736); Kowa KK and Asahi (EP 151,996); Meiji Milk Products (GB 2,153,366); Choay, S.A. (EP 133,070) and Asahi and Kowa KK (U.S. 4,505,893); and by Wakamoto Pharmaceutical (Biotechnology, Nov. 1986). Also relevant are two Genentech applications describing the production of tPA in CHO cells (EP 117,059 and EP 117,060).
Qui J, Swartz et.al (Expression of active human tissue-type plasminogen activator in Escherichia coli. Qiu J, Swartz JR, Georgiou G. Appl Environ Microbiol 1998 Dec; 64(12):4891-6) have reported the effect of co-overexpressing native or heterologous cystein oxidoreductases in the bacterial periplasm. The active protein formed was purified
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with an overall yield of 25% by using three affinity steps with, in sequence, lysine-Sepharose, immobilized Erythrina caffra inhibitor, and Zn-Sepharose resins.
Grunfield et.al (Effector-assisted reported the refolding of recombinant tissue-plasminogen activator produced in Escherichia coli. Appl Biochem Biotechnol. 1992 May;33(2):l 17-38.) and has demonstrated the recombinant tissue-plasminogen activator (r-tPA), expression in Escherichia coli cells in an aggregated form, which was solubilized with a strong chaotrope in the absence of any reducing agent. The solubilized molecule was reactivated by a procedure that was developed to mimic the physiological conditions optimal for the functional folding and activity of the native protein. The use of partially purified fibrinogen, as a source of fibrin (the effector), was shown to facilitate the reactivation process and increase its yield by at least a factor of two. The yield of the process was also shown to be particularly dependent on the recombinant protein concentration. Purification of the activated form of r-tPA was achieved with a two-step column-chromatography scheme. This included a gel filtration step on a Sephadex G-50 column followed by an affinity chromatography step on a lysine-sepharose column.
Until recently, tPA have been purified using various types of affinity chromatography. Known examples include concanavalin A-Sepharose (Rijken, D. C. and Collen, D. (1981) J. Biol. Chem. 256, 7035-7041), erythrina trypsin inhibitor (ETI)-Sepharose (Heussen, C, et al. (1984) J. Biol. Chem. 259, 11635-11638), anti-tPA and antibody-Sepharose (Ranby M., et al. (1982) FEBS Lett 146, 289-292) and fibrin-Sepharose (U.S. Pat. No. 4,505,893). Outflow of these immobilized proteins, though in a small amount, is observed during operation. Also, these proteins are undesired heteroantigenic proteins. The ranges of the pi of these proteins are 4.4 to 5.5 for concanavalin, 4.5 to 5.5 for ETI, 5.8 to 7.3 for immunoglobulin G and 5.5 to 5.8 for fibrinogen.
The use of a cation-exchanger for purification of tPA is known (Japanese Patent Laid-Open No. 174727/1985), in which, however, the cation-exchanger is used for recovering tPA fractions for the purpose of partial purification.
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US patent 5,158,882 provides a method of purifying a crude tissue plasminogen activator preparation which was accomplished by dividing a eluting process for selective elution of undesired proteins from the cation exchanger on which tPA and other undesired proteins are adsorbed into two steps by carrying out the steps in a defined order: one step to elute undesired proteins having the pf values equivalent to or lower than that of tPA, and the other step to elute undesired proteins having the pi values equivalent to or higher than that of tPA. The invention teaches appropriate range of the pH for eluents to be used in the two steps. The method of the invention for purifying tPA comprises the steps of:
(a) contacting a crude tPA preparation containing tPA and undesired proteins with a cation exchanger to allow said tPA and undesired proteins to adsorb to said cation-exchanger;
(b) treating said cation-exchanger with an eluent having a pH in the range between 5.2 to 6.5 to elute undesired proteins having the pi values equivalent to or lower than that of said tPA;
(c) treating said cation-exchanger followed by the step (b) with an eluent having a pH in the range between 2.8 to 3.5 and then eluting undesired proteins having the pi values equivalent to or higher than that of said tPA; and
(d) eluting said tPA from the cation-exchanger followed by the steps (b) and (c).
US patent number 5,453,363 provides a process for the activation of tPA after genetic expression in prokaryotes which includes cell digestion, solubilization under denaturing and reducing conditions and activation under oxidizing conditions in the presence of GSH/GSSG. A purification after modification of the thiol groups is carried out by ion exchanger treatment.
US Patent No 4933434 provides a process for the renaturation of denatured proteins in solution in a renaturation buffer, wherein a solution is prepared of the protein to be renatured in the critical concentration in a selected buffer and, after formation of the folding intermediate, further protein to be renatured is added in the amount necessary for the achievement of the critical concentration.
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US Patent No 4898826 providesJPA whether secreted by cells naturally producing it or prepared by recombinant means can be solubilized by providing, in aqueous medium at pH 5-14 and a solubilizing amount of a basic amino acid optionally and preferably, in the presence of a citric acid moiety. The ability to solubilize tPA at concentrations of up to 50 mg is significant as an aid in permitting smaller volumes for purification and permitting single injections of the drug as opposed to intravenous administration.
European Patent No 256836 have demonstrated isolation of Tissue plasminogen activator (tPA) species having a specific molecular weight in a purified form from a crude tPA preparation containing various tPA species having different molecular weights by bringing the crude tPA preparation into contact with hydroxyapatite and then separately eluting the adsorbed tPA species with eluents having different pHs and/or salt concentrations.
European Patent No 245100 have provided a Purification of single-chain and double-chain tissue plasminogen activator. The purification method involved the close contact of the mixture containing a single-chain tissue plasminogen activator (tPA) and/or double-chain tPA with a column carrying an immobilized Erythrina trypsin inhibitor as an affinity agent. Adsorbed proteins are eluted with eluents having different pHs with or without arginine or benzamidine, so that single-chain tPA is obtained in the eluent with a pH at least 4.5 and double-chain tPA is obtained in the eluent with a pH lower than 4.5.
Truncated human tissue plasminogen activator is a complex molecule contain disulfide bonds produced as insoluble inclusion bodies in E.Coli which needs to be solubilised and ideally partially purified by one or two chromatography steps before aiming to refold into biologically active protein followed by purification routinely by affinity (most conventional is lysine affinity chromatography).
A wide variety of purification processes of tPA may be used, but most of these involve, at least in part, chromatographic procedures, gel filtrations, ammonium sulfate precipitations and the like. All of these procedures greatly decrease the total volume of substantially pure form of tPA. The inconvenience of running chromatographic columns, for example,
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increases as the volume of material which needs to be passed over them is increased. The use of arginine hydrochloride alone is workable but quite difficult because of its viscosity. In order for 1 M arginine to be used, for example, high pressure needs to be applied to obtain reasonable flow rates. Since the above conventional methods are elaborative and time consuming and there is a substantial loss of protein due to the involvement of the multiple steps in the process. Further TPA precipitates and/or irreversibly bind to resins and membranes during processing when arginine concentration is less in a buffer system and higher refolding volumes are required
In looking into the need for an efficient purification process of tPA with minimal loss of yield of the pure form of tPA due to conventional multiple chromatographies before refolding, the inventors of the present invention has developed an improved purification process of tPA and have exemplified the problems associated with the conventional purification process of the molecule. The inventors of the present invention have focused on a single step of chromatography for purification by altering the arginine concentration and have managed to achieve an optimum arginine concentration for both ion exchange and lysine affinity chromatography. The present invention has resulted in reducing the refolding volume with out affecting the yield and the purity of the protein. The process developed by the present invention is simple, efficient, commercially viable and a cost effective process. Further the present invention also provides a single step purification for any recombinant proteins forming inclusion bodies like tissue plasminogen activator.
OBJECT OF THE INVENTION
It is the object of the present invention to provide an efficient process of purification of tPA.
It is the object of the present invention to provide a single step ion exchange purification of tPA at high arginine concentration ( upto 0.3 molar)
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It is the object of the present invention to provide a single step lysine affinity chromatography at high salt concentration ( upto 0.2 molar sodium chloride) or cascade of two purification step such as lysine affinity followed by ion exchange.
It is the object of the present invention to provide an optimum arginine concentration in the buffers suitable for ion exchange chromatography.
It is the object of the present invention to reduce the refolding volume.
It is the object of the present invention to provide a single step purification for any recombinant proteins forming inclusion bodies like tissue plasminogen activator.
It is the object of the present invention to minimize loss of proteins due to precipitation in buffer solution during processing.
It is the object of the present invention to minimize the very high dilution of the protein for refolding efficiency.
It is the object of the present invention to perform the refolding reaction at room temperature to reduce the refolding time and avoiding the expenses of cooling.
It is the object of the present invention to minimize the longer batch time, both for refolding and processing time and increase in yield and purity of tPA.
SUMMARY OF THE INVENTION
The present disclosure provides an efficient process for purification of tPA by single affinity step chromatography by altering the arginine concentration. The present invention has also focused on achieving an optimum arginine concentration for ion exchange and sodium chloride concentration for lysine affinity chromatography. The present invention
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has aimed at reducing the refolding volume with out affecting the yield and the purity of the protein resulting in minimal loss of yield.
In one embodiment the present invention provides a process, which comprises steps of direct refolding of the solubilisation of inclusion bodies without any prior chromatography thereby minimizing protein loss, reduction of the batch time and cost of chromatography resin and chemicals. In one preferred embodiment the present invention provides a single step purification for any recombinant proteins forming inclusion bodies like tissue plasminogen activator.
In one embodiment the present invention provides a process with reduction of refolding volume without hampering the refolding efficiency thereby reducing the capital investment for bigger refolding vessel, larger space requirement and handling and processing problems of large volume.
In one embodiment the present invention provides a process wherein the refolding is done at room temperature to reduce the refolding cycle time and also the wholeprocess at room temperature to avoid cost of cooling
In one embodiment the present invention provides a process which gives an industrially feasible process with reduced refolding buffer starting from crude starting material without compromise on the yield and purity.
In one embodiment the present invention provides a process, which utilizes a single step ion exchange chromatography purification without the need of any affinity chromatography directly after refolding.
In one embodiment the present invention provides a process, which utilizes a comparatively higher arginine concentration in the range of 0.1-0.5 molar, ideally 0.3 molar concentration which results in very high conductivity for cation exchange chromatography thus lowering the loss of protein due to precipitation.
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In one embodiment the present invention provides an optimum arginine concentration at the start thus resulting in high start conductivity without affecting the binding of the material to the ion exchange resin.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 Figure 2 Figure 3 Figure 4 Figure 5
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, the inventions of which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
illustrates the effect of temperature on the refolding cycle time of tPA. illustrates the effect of Arginine on Refolding of tPA. illustrates the effect of Urea supplement during refolding of tPA illustrates the effect of Glutathione reduced on refolding of tPA illustrates the effect of Tween 80 concentration on refolding of tPA
DETAILED DESCRIPTION OF THE INVENTION
Definitions:
The term "tPA" as used herein refers to human truncated tissue type plasminogen activator
produced in E. coli
The term "room temperature" indicates a temperature range of 23 to 25 deg. C
The term inclusion bodies refer to foreign protein expressed in bacterial (E. coli) as insoluble protein
The present invention provides a single step of chromatography for purification by altering the arginine concentration. The present invention has also focused on achieving an optimum arginine concentration for both ion exchange and lysine affinity chromatography. The present invention has aimed at reducing the refolding volume with out affecting the yield and the purity of the protein. The present invention mainly focuses on developing an
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industrially viable process of purification of tPA and this process obviates the disadvantages associated with the conventional processes of purification.
The process involves the production of tPA preferably human trunctated tPA in E. Coli by the usual recombinant procedures.
The inclusion bodies of E. coli obtained was harvested after cell lysis. The inclusion bodies were then solubilised in Guanidium hydrochloride and DTT. The solubilised mixture was incubated with stirring at a room temperature for 3 to 5 hours, further the pH was adjusted to the acidic range and kept for dialysis in urea. The dialysed sample was then diluted with urea and slowly supplemented with Tris and oxidized glutathione. Further the pH was adjusted to alkaline and the reaction mixture was incubated at room temperature with stirring.
The reaction mixture was then diluted to 4-8 fold with buffer for refolding. The components of the buffers comprised of Tris, Na- EDTA salt, Tween 80, urea, arginine, reduced glutathione and incubated under suitable temperature preferably Room temperature for 16- 48 hours under stirring. After refolding is complete the reaction mixture was further diluted with sodium citrate buffer and pH adjusted to 4-5.0 with urea and the arginine concentration was finally maintained at 0-0.3M.
The effects of the concentration of the various components of the buffers on the refolding pattern was studied with a aim to provide a process with maximum yield at room temperature and within 16-18 hours of the reaction.
The purification of the reaction mixture was done on a SP Sepharose Column, which comprised 80-160 ml resin for proteins from 0.5-lgm of inclusion bodies and was pre equilibrated with sodium citrate buffer and arginine at a pH of 3-5.0.
After loading of the samples in the column was washed with equilibration buffer and the bound proteins were eluted with a linear gradient with 1 molar sodium chloride in arginine
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(ideally 0.3 molar) and sodium citrate buffer , pH ideally 4 to 4.5 and the purity was achieved as analysed by RP-HPLC (C-18) was not less than 98%. The activity of the protein was determined by Chromozyme tPA activity assay.
The process of the present invention has the following advantageous features:
1. Direct refolding after solubilisation of inclusion bodies without any prior chromatography thereby minimizing protein loss, reduction of the batch time and cost of chromatography resin and chemicals.
2. Reduction of refolding volume to approximately 50% without hampering the refolding efficiency thereby is reducing the capital investment for bigger refolding vessel, larger space requirement and handling and processing problems of large volume.
3. Refolding at room temperature to reduce the refolding cycle time and also the whole process at room temperature to avoid cost of cooling
4. An industrially feasible process with reduced refolding buffer starting from crude starting material without compromise on the yield and purity.
5. Utilization of a single step ion exchange chromatography purification without the need of any affinity chromatography directly after refolding.
6. Utilization of a comparatively higher arginine concentration in the rang of 0.1-0.5 molar, ideally 0.3 molar concentration which results in very high conductivity for cation exchange chromatography thus lowering the loss of protein due to precipitation.
7. To provide a optimum arginine concentration at the start thus resulting in high start conductivity without affecting the binding of the material to the ion exchange resin.
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
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EXAMPLE 1 Preparation of TPA
Human truncated tPA produced in E coli as inclusion bodies was harvested after cell lysis and solubilized in 6M to 8M Guanidium hydrochloride (1: 30 w/v) in 200- 300 mM DTT by incubating with stirring at room temperature for 3 hours and pH was adjusted to 3 with hydrochloric acid (cone) and kept for overnight dialysis against 6M -8 M Urea at 10 to 15 deg. C. The dialysed sample was diluted 10 to 20 fold with 6M -8 M Urea and gradually supplemented with Tris (final concentration 50 mM), Glutathione oxidized (final concentration 25 mM) after adjusting the pH to 9.3 with 3 M sodium hydroxide solution to for 3-5 hours at room temperature with stirring.
EXAMPLE 2: Refolding
The sample was then diluted 4 to 8 fold with buffer for refolding contains Tris (final concentration 150 mM, pH 8.5), Na-EDTA Salt (Final concentration 2mM), Tween 80 (final concentration 0.01 to 0.05%), Urea 0.25M to 1 M , arginine (final concentration 0.3 to 0.5 M) and glutathione reduced (final concentration 0.2 mM to 4mM) and incubated at 10 deg C to room temperature (RT) for 16- 48 hours with stirring. After refolding reaction the sample was diluted with 10 to 50 mM sodium citrate buffer, pH 4.0 to 5.0, ideally pH 4.0 contains 1 M Urea to bring down the arginine concentration to 0.3 M and pH was adjusted to 4.0 to 5.0
EXAMPLE 3: Purification
The sample was then loaded on a SP Sepharose FF (Amershams) column (80 to 160 ml resin used for proteins from 0.5 to 1 gm of IBs) pre equilibrated with 10 to 50 mM sodium citrate buffer, pH 4.0 to 5.0 contains 0.3 M arginine . After sample loading the column was washed with 5 to 10CV of equilibration buffer and then the bound proteins were eluted with a linear gradient (30 CV) or step gradients of equilibration buffer and 10-50 mM sodium citrate buffer, pH 4.0 to 6.0 contains 1 M sodium chloride. ( Process flow diagram 1). The purity achieved as evidenced by RP-HPLC (C-18) is not less than 98%. The protein is fully active as determined by Chromzyme tPA (Roche) activity assay.
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The sample can also be purified by lysine affinity ( CPG Lysine , Millipore) chromatography after supplementing the sample and all the buffers with 0.2 molar sodium chloride and elution with 0.5 molar arginine in 10 mM sodium citrate buffer,0.2 molar sodium chloride, pH 5.0 ( Process flow diagram 2). Purity achived as analysed by RP-HPLC (C-18) is not less than 96% which further can be purified to not less than 98% by ion exchange chromatography as describe above.
EXAMPLE 4: Studies on the various components of the buffer for efficient refolding A) Effect of temperature on the refolding cycle time
The refolding experiments were done at various temperature and the effects of the conventional incubation temperature and at room temperature on the activity has been cited herein in the table below.
Under the same buffer composition the refolding reaction progressed faster and resulted in higher yields at room temperature (23-25 deg C) as compared to 10 deg C.
The effects of the various components of the buffer was studied and the yield in mlU/ml after 16 hours of refolding at room temperature ( 23-25) deg C

Set 1 2
Cone Cone
Arginine (M) 0.5 0.5
Tris buffer (M) 0.15 0.15
Na-EDTA salt (0.2M) 0.002 0.002
Tween 80 ( %, W/V) 0.05 0.05
Glutathione reduced (M) 0.002 0.002
Urea (M) 0.25 0.25
PH 8.5 8.5
Buffer Vol make up (ml) 17.5 17.5
Sample Vol (ml) 2.5 2.5
Incubation (Deg C) 10 RT
Activity (mlU/ml) Abs mlU/ml Abs mlU/ml
0 Hrs. 0.254 0.7 0.249 0.7
18 Hrs. 0.848 2.5 1.241 3.686
24 Hrs. 0.936 2.73 1.311 3.896
42 Hrs. 0.912 2.64 1.137 3.375
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B) Effect of arginine concentration on the yield
The effect of arginine concentration on the yield for refolding of tPA was studied from 0.2-1.0 Molar, the results of which is tabulated below.
The effective concentration of arginine for refolding of tPA was found to be about 0.5 molar.

Argine (M) yield (mlU/ml)
0.2 1.56
0.3 3.228
0.5 3.519
0.8 3.1382
C) Effect of Urea supplement for refolding of tPA.
The effect of urea supplement on the refolding of tPA was studied with concentration ranging from 0 to 1 Molar. The effective concentration was found to be about 0.25 Molar.

Urea (M) Yield (mlU/ml)
0 2.6
0.25 3.686
0.5 3.31
0.75 3.29
1 3.23
D) Effect of Glutathione reduced on the refolding of tPA
The effect of Glutathione reduced on the yield of refolded tPA was studied with concentration of 0.0002 to 0.004 M. The maximum yield was obtained with 0.002 molar concentration of Glutathione.

Glutathione Reduced (M) Yield (mlU/ml)
0.0002 0.5
0.002 3.686
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E) Effect of Tween -80 for refolding of tPA.
The effective concentration of Tween 80 for refolding of tPA was studied with
concentration ranging form 0-0.05 % w/v.
The effective concentration of Tween 80 for refolding of tPA was around 0.05% ( w/v).

Glutathione Reduced (M) Yield (mlU/ml)
0.0002 0.5
0.002 3.686
0.004 2.127
F) Yield calculation
TABLE (1.0 gram IB)

SI.No. Step Vol (ml) Cone (mg/ml) TotalProtein(mg) Activity (IU) Specific activity (IU/mg) StepRecovery (% age) Overall Recovery(% age) RP-HPLC(%) Endotoxin (EU/mg)
1 Sol. IB after dialysis 800 0.359 287.2 ND ND - - ND ND
2 CPG Lysine Load 1500 0.103 154.5 35.91 0.232 53.79 53.79 ND 154.67
3 CPG Lysine Elute 250 0.175 40.75 26.66 0.654 26.37 14.18 74 11.12
4 SP Sepharose Elute 120 0.275 33.0 26.29 0.796 80.98 11.49 98 7.43
G) Analytical results:
RP-HPLC (C18) Purity : 98% (Fig 1)
SEC-HPLC(monomer contents) : 99.2% (Fig 2)
SDS-PAGE: Single band (Fig 3)
Host Cell Protein ( by western blot) : Not detected (Fig 5)
Endotoxin (by Chromogenic LAL) : 7.43 EU/mg (Limit 10EU/mg)
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All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are chemically or physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the invention.

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ABSTRACT
The present invention relates to an efficient process for purification of recombinant protein. The present invention in particular relates to the purification of truncated tissue plasminogen activator produced in E. coli by optimizing an optimum arginine concentration for ion exchange and optimum salt concentration for lysine affinity chromatography. The present invention also provides a process wherein by reducing the refolding volume with out affecting the yield and the purity of the protein.
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