WO2022172284A1 - Purification of a therapeutic protein - Google Patents

Abstract

The disclosed invention relates to a method for purifying a recombinant protein produced in the bacterial protein expression system. Specifically, it relates to the efficient purification of a therapeutic recombinant protein by removing the impurities from a complex liquid mixture containing said recombinant protein and such other impurities coming from the bacterial cell culture fermentation process.

Description

PURIFICATION OF A THERAPEUTIC PROTEIN

DESCRIPTION

FIELD OF THE INVENTION The disclosed invention relates to a method for purifying a recombinant protein produced in the bacterial protein expression system. Specifically, it relates to the efficient purification of a therapeutic recombinant protein by removing the impurities from a complex liquid mixture containing said recombinant protein and such other impurities coming from the bacterial cell culture fermentation process.

BACKGROUND

Over the past three decades a new class of protein-based therapeutics has emerged for the treatment of a variety of the diseases and disorders previously untreatable for the want of medicines. These protein-based therapeutics, also called biopharmaceutical and biosimilar drugs, include as the active pharmaceutical ingredients different types of proteins, which are produced through biochemical processes in fermenters or bioreactors in heterologous expression systems like bacteria, yeasts or animal cell lines. Upon expression in these heterologous systems these proteins require purification to a very high purity that excludes the impurities associates with the expression and manufacturing processes. This process of the purification is difficult and suffers with the technical challenges. In the bio-pharmaceutical industry, the large-scale purification of proteins has become most important manufacturing activity. The pharmaceutical proteins are, generally, produced by cell culture processes using bacterial or animal cell lines. These proteins are over expressed in the cell lines up to about 30 % of the total cellular protein produced during the production stage. Although, the proteins of the interests are produced at a higher level, the recovered cell lysate or the cell culture supernatant is a complex mixture having large amounts of host cell derived components called the process impurities. Before the therapeutic proteins can be used as active pharmaceutical ingredients, they require purification from said process impurities to a level of having no impurities that adversely affect the medicinal preparations of said therapeutic proteins. Further, the product related impurities, which also require removal as they have physiochemical properties very close to the product itself. Hence it is crucial that the purification methods employed are of robust and effective nature as far as the purity of the final medicinal products are concerned. The problem of purification of the therapeutic proteins is complex and requires solutions based on the nature of the protein purified, the conditions under which it is over-expressed and other related matters like the purity of the final product required, etc. The invention disclosed herein relates to a method of purification of a therapeutic protein using a unique combination of unit operations, not disclosed before, for the purification of any protein. It further relates to the use of new steps in said method whereby the purity of the final protein preparation is achieved to 98 % or more, which is suitable for the preparation of medicinal formulations for the human use.

DESCRIPTION OF DRAWINGS

FIGURE lA: Illustrates the determination of pH(I) of anakinra reference product (KINERET) by the capillary isoelectric focusing method.

FIGURE lB: Illustrates the determination of pH(I) of GBL43/ anakinra product by the capillary isoelectric focusing method.

FIGURE 2: Illustrates the capture on and elution from the anion exchange column of GBL43 protein using a step gradient of said first elution solution. FIGURE 3: Illustrates the capture on and elution from the cation exchange column of GBL43 protein using a linear gradient of said second elution buffer.

FIGURE 4A: Illustrates the reducing SDS-PAGE analysis related to different fractions containing GBL43 obtained at the end of anion exchange chromatographic separation. Lane 1: molecular weight marker, lane 2: crude expressed lysate, lane 3: flow-through fraction, lane 5 to 7: eluted factions and lane 9: regeneration fraction.

FIGURE 4B: Illustrates the reducing SDS-PAGE analysis related to different fractions containing GBL43 obtained at the end of cation exchange chromatographic separation. Lane 1: molecular weight marker, lane 2: eluate from anion exchange step, lane 3: flow-through fraction, lane 4 to 7: eluted factions and lane 8: regeneration fraction.

FIGURE 5A: Illustrates the reducing SDS-PAGE analysis of TFF retentate fractions - lane 1: molecular weight marker and lane 3: purified

GBL43 in retentate fraction (visualised by coomassie blue stain).

FIGURE 5B: Illustrates the non-reducing SDS-PAGE analysis of purified fractions [TFF retentates] - lane 1: molecular weight marker and lane 2 to 4: purified GBL43 fractions (visualised by silver stain). FIGURE 5C: Illustrates the reducing SDS-PAGE analysis of purified fractions - lane 1: molecular weight marker and lanes 2 to 4: purified GBL43 fractions (visualised by silver stain).

FIGURE 6A: Illustrates the purity of GBL43 product by the RP-HPLC method.

FIGURE 6B: Illustrates the purity of GBL43 product by the SE-HPLC method.

FIGURE 7: Illustrates the amino acid sequence of the GBL43/ anakinra protein recombinantly expressed in the E. coli host cells.

DESCRIPTION OF INVENTION

The invention disclosed herein relates to a method of purification of a therapeutic protein, called anakinra, to a high level of purity suitable for the medicinal formulations useful in the human medicine. Anakinra is a single peptide protein having 153 amino acids with molecular weight of about 17 kDa and is a derivative of naturally occurring cytokine-type protein present in mammals that inhibits the binding of interleukin -1 cytokines [IL-i] to the IL-i receptor called IL-iRa. It is an inhibitor of IL-ia and IL-ib cytokines binding to its receptors (IL-iR) thereby modulating said cytokine responses in the inflammation and immune reactions. It is non-glycosylated and with one intramolecular disulphide bond between its cysteine residues forming globular protein molecules. By the action of IL-iRa the inflammatory responses in the several disorders like rheumatoid arthritis are controlled or modulated, and this property of IL-iRa is exploited in making anakinra as a therapeutic biopharmaceutical drug molecule. The purity of anakinra to more than 98 % in terms of the peptide molecules is required for medicinal applications. The peptide sequence of anakinra is identical to the IL-iRa with additional methionine residue at the N-terminus due to the requirement of the bacterial expression system.

In one embodiment of the present invention, a synthetic gene expressing the peptide of anakinra (a therapeutic protein) is heterogeneously expressed in the E. coli expression system as a soluble protein in the cytoplasm of cells. An inducible bacterial vector-based expression system is developed expressing the anakinra peptide or protein in the cytoplasm in soluble form in the high density cell culture bioreactors. The artificial gene expressing the anakinra protein is optimised for bacterial over expression such that between 20 and 30 % of total protein consists of said anakinra protein in the cytoplasm of recombinant cells and is designated as GBL43 in this disclosure.

In second embodiment of the present invention, the working volumes for fermentation batches are between 5 and 25 litres, while the fermentation process was carried out in batch, followed by fed-batch modes with induction of the recombinant protein expression happening at the start of the mid-log phase of the fast growing bacterial culture. The fermentation batch sizes may be increased up to 5000 litres capacity with the minimal changes is the running parameters as disclosed therein. The culture is grown at temperature of about 37 °C and pH of about 7 in pre-sterilised chemically defined medium and equipment, under the current GMP conditions throughout the run for all the batches. Further, the rate of agitation, pH management, oxygen and glucose supply rates are maintained to achieve higher density of bacterial cells at about 100 to 160 g/L of wet cell mass at the end of fermentation process. A total time between 18 and 28 h is employed to get high cell numbers at the optical density of about 100 to 140 units at 600 nm (OD600) at the end of fermentation batches, while the recombinant protein production is induced at about 50 to 80 units of optical density at OD600 with addition of about 0.5 to 1.0 mM of IPTG in the fermentation medium.

In another embodiment of the present invention, to recover the recombinant anakinra protein, the bacterial wet cell mass is harvest by centrifugation from the bioreactor stream at the end of the fermentation cycle and is maintained at room temperature for further processing. Then said wet cell mass is further diluted to a level between 10 and 30 times with a lysis buffer. It is stirred at about 22 °C for about 1 h before further procedure. Next, the complete cell lysis is achieved by mechanical shearing of the cells in the lysis buffer under high pressure using a homogeniser operating at a pressure between 700 and 1200 bar, under three repeat cycles to achieve the maximum cell lysis. The homogenised material is maintained at about 22 °C to until used further. The homogenised broken cell mass is then pooled and subjected to the centrifugation at a g-force between 10,000 and 15,000 for up to 30 min to remove the insoluble debris from it, and followed by 0.45 pm membrane filtration to clear it of any visible suspended matter forming a liquid stream. This liquid stream contains most of the anakinra protein in the soluble form. Herein said liquid stream is also called a liquid mixture containing said recombinant protein and contaminants. Said contaminants comprises cellular proteins, lipids, DNA, RNA and other unwanted materials coming from the process and product. The amount of the released recombinant protein is monitored by spectrometry to ensure the effectiveness of the cell lysis protocol.

In further embodiment of the present invention, to determine the quality of the different fractions or streams collected during the purification method disclosed herein, the standard qualitative and quantitative tests used in protein/ peptide chemistry are used. The Bradford’s test to determine protein/ peptide amounts in several fractions is routinely employed. The technique of SDS-PAGE electrophoresis in the reducing or non-reducing conditions is used as per established standard protocols. Further tests using the RP- and SE- HPLC methods are performed as per reported and established protocols. The host cell protein (HCP) amounts are determined by an ELISA test kit, while the bacterial endotoxin amounts are determined using a LAL test kit following the established protocols of manufacturers.

In yet another embodiment of the present invention, the protein anakinra has 153 amino acid residues with a molecular weight of about 17 kDa with 20 negatively and 17 positively charged amino acids making it an acidic peptide with theoretical pH(I) of 5.4, while no empirical pH(I) has been reported. To determine actual pH(I) of anakinra/ GBL43 produced therein, the capillary isoelectric focusing (CIEF) analysis is used. The innovator’s reference drug KINERET (by SOBI) is used as the reference product, however, its empirical pH(I) has not been reported in the prior art. To determine the pH(I) of GBL43 and the reference product, both the products are diluted in water and desalted. The CIEF samples are prepared with internal controls as per the manufacturer’s protocols and the pH(I) values for both the samples determined. Herein recombinant GBL43/ anakinra is also called recombinant protein for convenience.

In yet another embodiment of the present invention, the clarified and filtered lysate of bacteria [herein in called a liquid mixture containing said recombinant protein and contaminants] containing the recombinant GBL43 is subjected to first step of chromatographic purification using a strong anion exchange resin in a Tris buffer system. Herein the capture chromatography column is packed with resin containing quaternary ammonium functional groups [tri-methyl ammonium group attached to resin]. The fraction or eluate [herein called a first stream] obtained in this step containing GBL43 is subjected to the quality control tests along with other fractions to determine the effectiveness of said capture chromatographic step and quality of the GBL43 protein in the eluate [said first stream].

In yet another embodiment of the present invention, the eluate [said first stream] obtained from capture step containing GBL43, is first spiked with citrate buffer and its pH is adjusted to achieve the final pH of about 6.5. This solution is further diluted with acetate buffer to achieve a conductivity between 2 and 3 mS/cm. Then said diluted solution is subjected to a second chromatographic purification using a strong cation exchange resin in acetate buffer system. Herein the separation chromatography column is packed with resin containing sulfoisobutyl functional groups. In yet another embodiment of the present invention, the second eluate containing GBL43 [herein called a second stream] obtained post cation exchange column treatment is subjected to the simultaneous buffer exchange and protein concentration using a tangential flow filtration system. For the effective separation and concentration of GBL43, the filtration cassette with PES membrane with a molecular weight cut-off about 5 kDa is used. Herein, sequential buffer exchange and concentration is achieved using said TFF system and the retentate [herein called concentrated stream] containing concentrated GBL43 protein.

In yet another embodiment of the present invention, to remove the endotoxins from said retentate [said concentrated stream] containing GBL43, it is subjected to a strong anion exchange resin in a citrate buffer system. Herein the chromatography column is packed with resin containing strong quaternary ammonium functional groups [tri-methyl ammonium group attached to resin]. After loading, the column is washed with a mobile phase and the flow through stream [herein called a final stream] containing GBL43 is collected. This stream containing about 170 mg/mL of GBL43 protein is further used for preparing the bulk formulation or drug substance by supplementing it with polysorbate 80 and other buffering components to achieve the final bulk or drug substance product composition of 150 mg/mL of GBL43/ anakinra.

In yet another embodiment of the present invention, the final bulk product containing GBL43 is estimated for various impurities like the aggregation, host cell proteins [HCP] and endotoxins by the analytical methods such as SE-HPLC, RP-HPLC, SDS-PAGE, ELISA, western blotting and LAL test. For RP-HPLC analysis a C-18 reverse phase columns along with an automated HPLC systems are used. For the western blot analyses, the SDS-PAGE gels in reducing or non-reducing conditions are used. To check level of impurities, the protein samples are subjected to the SDS-PAGE gels, followed by protein transfer to NC membranes and then detected with primary (an anti-ILi Ra mouse monoclonal antibody) and secondary (anti-mouse HRP conjugated antibody) antibodies. For visualization, the blots are developed using the

DAB urea method. The percent aggregation is calculated from SEC- HPLC data and the percent impurity from RP-HPLC data. The HCP was estimated using ELISA and the endotoxin was by LAL test.

In yet another embodiment of the present invention, a method of purifying a recombinant protein is disclosed, comprising: providing a liquid mixture containing said recombinant protein and contaminants obtained from lysate of bacteria. Next, contacting said mixture with an anion exchange resin under conditions that allow said recombinant protein to bind or adsorb to it and removing by washing out a first part of said contaminants using an anion exchange wash solution without disrupting said bound recombinant protein. Next, selectively eluting said recombinant protein from said anion exchange resin using a first eluting solution forming a first stream containing said recombinant protein. Next, contacting said first stream with a cation exchange resin under conditions that allow said recombinant protein to bind or adsorb to it and then removing by washing out a second part of said contaminants by using a cation exchange wash solution without disrupting said bound recombinant protein and selectively eluting said recombinant protein from said cation exchange resin using a second eluting solution forming a second stream containing said recombinant protein. Next, concentrating said second stream by molecular filtration under conditions that allow said recombinant protein to get separated from a remaining part of said contaminants forming a concentrated stream containing said recombinant protein and then contacting said concentrated stream with a second anion exchange resin under conditions that allow endotoxins present in said concentrated stream to bind or adsorb to it forming an endotoxins free final stream containing said recombinant protein. Then formulating said final stream by adding pharmaceutical formulation excipients to it.

In yet another embodiment of the present invention, where a first part of said contaminants is removed by washing under conditions that leave said recombinant protein bound to said anion exchange resin. Herein pH of said anion exchange wash solution is between 7.5 and 8.5 with from 15 to 25 mM of Tris buffering agent. Next, herein first elution solution comprises from 500 to 1000 mM of NaCl and from 15 to 25 mM of Tris buffering agent at a pH between 7.5 and 8.5. Next, a second part of said contaminants is removed by washing under conditions that leave said recombinant protein bound to said cation exchange resin. Herein pH of said cation exchange wash solution is between 5.5 and 6.5 with from 10 to 50 mM of acetate buffering agent. Next, a first elution solution comprises from 500 to 1000 mM of NaCl and from 10 to 50 mM of acetate buffering agent at a pH between 5.5 and 6.5. Herein, concentration is performed by means of a tangential flow filtration system along with exchange of buffer solution to formulation buffer comprises from 5 to 25 mM of citrate, from 0.5 to 5 mM of EDTA and from 120 to 160 mM of NaCl at a pH between 5.0 and 7.0.

In yet another embodiment of the present invention, where said recombinant protein is produced in Escherichia coli host cells. And said contaminants comprises one or more of high molecular weight protein aggregates, host cell proteins, DNA, RNA, or endotoxins contributed by said host cells. The E. coli host cells BL21 and/ or BL2i(DE3) and the plasmid pET-24a(+) used herein are imported from EMD Milipore Corporation, Tenecula, California, USA [part Nos. 69449, 69450 and 69749].

In yet another embodiment of the present invention, where said recombinant protein (GBL43/ anakinra) is purified to a purity of at least 90% is having the amino acid sequence of SEQ ID NO: 1 shown in FIGURE 7. Further, said recombinant protein contains less than 1 % high molecular weight protein aggregates. The said recombinant protein is a single chain polypeptide with pH(I) between 5.6 and 6.0. In yet another embodiment of the present invention, where said recombinant protein (GBL/ anakinra) is concentrated to an amount from 120 to 200 g/Lfor the preparation of a pharmaceutical composition comprising pharmaceutically allowed excipients. EXAMPLES

The following examples provide the illustrative data on the various features of the invention disclosed herein, without any limitation to the broad applicability of the invention as may be inferred by the person skilled in the art.

EXAMPLE l: PRODUCTION OF GBL43/ ANAKINRA IN THE HIGH CELL DENSITY BACTERIAL CULTURE SYSTEM

The polypeptide of GBL43/ anakinra was heterologously expressed in the E. coli expression system as a soluble protein in the bacterial cytoplasm. An inducible bacterial plasmid vector-based expression system was developed that expressed the anakinra protein in the cytoplasm in soluble form in the high cell density culture fermenters or bioreactors. The artificial gene expressing the anakinra protein was optimised for bacterial protein over expression such that between 20 and 30 % of total protein consists of said anakinra protein in the cytoplasm of recombinant cells. The working volumes for fermentation batches were between 5 and 25 litres, while the fermentation process was carried out in batch, followed fed-batch modes with induction of the recombinant protein expression happening at the start of the mid-log phase of the fast growing bacterial culture. The culture was grown at temperature of about 37 °C and pH of about 7 in pre-sterilised chemically defined medium and equipment, under the current GMP conditions throughout the run for all the batches. Further, the rate of agitation, pH management, oxygen and glucose supply rates were maintained to achieve higher density of bacterial cells at about 160 g/L of wet cell mass at the end of fermentation process. A total time of about 20 h was employed to get high cell numbers at the optical density of about 140 units at 600 nm (OD600) at the end of fermentation batches, while the recombinant protein production was induced at about 80 units of OD600 with addition of about 0.5 mM of IPTG in the fermentation medium. These steps afforded about 4 g/L of recombinant protein at the end of the fermentation process in each batch. Hereinafter this recombinant anakinra protein is identified as GBL43 in the various steps of the purification method disclosed below.

EXAMPLE 2: RECOVERY OF THE PROTEIN FROM THE BACTERIAL CELLS

To recover the recombinant GBL43, the bacterial wet cell mass was harvested by centrifugation from the fermented stream at the end of the fermentation cycle and maintained at room temperature for further processing. Then said wet cell mass was diluted 10 times with the lysis buffer consisting of about 20 mM of Tris-HCl, 1 mM of disodium EDTA at pH 8.0 in final diluted solution. It was stirred at about 22 °C for about 1 h. Next, the complete cell lysis was achieved by mechanical shearing of the cells in the lysis buffer under high pressure using a homogeniser operating at a pressure of about 1000 bar, under three repeat cycles to achieve the maximum cell lysis. The homogenised material was maintained at room temperature till used further. The homogenised lysed cell mass was pooled and subjected to the centrifugation at a g- force of about 12,000 for up to 30 min to remove the insoluble debris from it, and followed by 0.45 pm membrane filtration to clear it of any visible suspended matter forming a liquid stream. This liquid stream contained most of the anakinra protein in the soluble form. Herein said liquid stream was also called a liquid mixture containing said recombinant protein and contaminants. The amount of the released recombinant protein was monitored by spectrometry to ensure the effectiveness of the cell lysis protocol. The said liquid stream was then subjected to further purification steps to get the API grade anakinra protein/ GBL43, useful for making the pharmaceutical formulation.

EXAMPLE 3: QUALITY CONTROL TESTS

To determine the quality of the different fractions or streams collected during the purification method disclosed herein, the standard qualitative and quantitative tests used in protein/ peptide chemistry have been used. The Bradford’s test to determine protein/ peptide amounts in several fractions was routinely employed. The technique of SDS-PAGE electrophoresis in the reducing or non-reducing conditions was used as per established standard protocols. Further tests using the RP-HPLC and SEC-HPLC methods were performed as per reported and established protocols. The HCP amounts were determined by an ELISA test kit, while the bacterial endotoxin amounts were determined using a LAL test kit following the established protocols. EXAMPLE 4: PHYSIO-CHEMICAL PROPERTIES OF GBL43/ ANAKINRA

Anakinra has 153 amino acid residues with a molecular weight of about 17 kDa with 20 negatively and 17 positively charged amino acids making it an acidic peptide with theoretical pH(I) of 5.4, while no empirical pH(I) has been reported. To determine actual pH(I) of GBL43/ anakinra, the capillary isoelectric focusing [CIEF] analysis was used. The innovator’s reference drug KINERET (by SOBI) was used as the reference product, however, its empirical pH(I) has not been reported in the prior art. To determine the pH(I) of GBL43 and the reference product, both the products were diluted to 5 mg/mL of said protein in water and desalted. The CIEF samples were prepared by mixing 10 pL of desalted GBL43 or reference product separately in a master mix containing urea-CIEF gel, an ampholyte suitable for the pH range from 3 to 10, a cathode stabilizer, an anode stabilizer and markers for pH(I) values of 10.0 and 4.1 as the internal controls. Samples were injected and focused using a capillary of 50 pm id with an effective length of about 20 cm and total length of about 30 cm. The CIEF profiles of the GBL43 and reference product samples are presented in FIGURE lA and lB. The pH(I) of GBL43 was identical to said reference product and was found to be 5.7 (± 0.2) pH unit. EXAMPLE 5: ANION EXCHANGE CHROMATOGRAPHY

In first step of the disclosed purification method, said liquid stream, [herein said liquid stream is also called a liquid mixture containing said recombinant protein and contaminants] obtained in Example 2 containing GBL43, was then subjected to first step of chromatographic purification using a strong anion exchange resin in a Tris buffer system. Herein the capture chromatography column was packed with resin containing quaternary ammonium functional groups [tri-methyl ammonium group attached to resin]. Before loading, the column was sanitized by withholding it in 1 M NaOH solution for about 30 min and then equilibrating with five column volumes (CV) of the Buffer A comprising 20 mM of Tris, pH 8.0. After loading, column was washed with Buffer A until loosely bound process and product related impurities were completely washed away. Next, the impurities were removed using a 6 % step gradient of 1 M NaCl in Buffer A [herein called anion exchange wash solution]. Then the bound GBL43 was eluted using a 12 % step gradient of 1 M NaCl in Buffer A [also called first eluting solution]. It was found that GBL43 is best eluted at a conductivity between 5 and 20 mS/cm, and hence said step gradient was maintained to achieve a conductivity between 7 and 13 mS/cm during the elution of said protein as shown in FIGURE 2 and FIGURE 4A. The fraction [also called first stream] obtained in this step containing GBL43 was subjected to the quality control tests along with other fractions to determine the effectiveness of said capture chromatographic step and quality of the GBL43 protein in the eluate [said first stream]. EXAMPLE 6: CATION EXCHANGE CHROMATOGRAPHY

In second step, the eluate obtained in Example 5 containing GBL43, was first spiked with a solution containing about 10 mM of trisodium citrate of pH 6.4 and further its pH was adjusted with a solution of 1 M sodium acetate, pH 4.0 to achieve the final pH of 6.5. This solution was further diluted to about 10 times with 10 mM of sodium acetate, pH 6.5 buffer to achieve a conductivity between 2 and 3 mS/cm. Next, this diluted solution was subjected to said second step of chromatographic purification using a strong cation exchange resin in an acetate buffer system. Herein the separation chromatography column was packed with resin containing sulfoisobutyl functional groups. Before loading, the column was sanitized by withholding it in 1 M NaOH solution for about 30 min and washed with WFI. Then the column was equilibrated with five column volumes (CV) of Buffer C comprising 20 mM of sodium acetate, pH 6.5. After loading, column was washed with Buffer C [herein called cation exchange wash solution] until loosely bound process and product related impurities were completely washed away. Next, the bound GBL43 was eluted using a linear gradient of 1 M NaCl in Buffer C [herein called second elution solution] using about 10 CV said buffer as shown in FIGURE 3 and FIGURE 4B. The fraction or eluate [herein called second stream] obtained in this step containing GBL43 was subjected to the quality control tests along with other fractions to determine the effectiveness of said separation chromatographic step and quality of the GBL43 protein in the second eluate [said second stream].

EXAMPLE 7: RECOVERY AND CONCENTRATION OF GBL43 In third step, the second eluate obtained in Example 6 containing GBL43, was subjected simultaneous buffer exchange and concentration using a tangential flow filtration system. For the effective separation and concentration of GBL43, the filtration cassette used the PES membrane with a molecular weight cut-off of about 5 kDa. The said cassette was sanitised by 0.5 M NaOH followed by thorough wash with WFI. Then the equilibration of said membrane was carried out with Buffer F, containing about 10 mM of citric acid, about 0.54 mM of disodium EDTA and about 140 mM of NaCl at pH 6.5. Herein, sequential buffer exchange and concentration was achieved using said TFF system. The retentate [herein called concentrated stream] contained GBL43 at about 170 mg/ mL concentration, which was subjected to quality control tests along with other fractions to determine the effectiveness of this purification step as shown in FIGURE 5A to FIGURE 5C.

EXAMPLE 8: REMOVAL OF ENDOTOXINS In fourth step, to remove the endotoxins from said retentate [said concentrated stream] of Example h containing GBL43, it was subjected to a strong anion exchange resin in a citrate buffer system. Herein the chromatography column was packed with resin containing strong quaternary ammonium functional groups [tri-methyl ammonium group attached to resin] . Before loading, the column was sanitized in 1 M NaOH solution and then thoroughly washed with WFI. Next the column was equilibrated with a mobile phase containing about 10 mM of citric acid, about 0.54 mM of disodium EDTA and about 140 mM of NaCl at pH 6.5. After loading, the column was washed with said mobile phase and the flow through stream [herein called final stream] containing GBL43 was collected. This step of the disclosed method led to substantial reduction in the endotoxin level, the amount of the endotoxin in the final bulk product is one of the essential element of said product quality. TABLE 1 below provides reduction in amounts of the endotoxins after each process step of the disclosed method. TABLE l: Endotoxin levels at tend of each step of method of the present invention.

EXAMPLE 9: QUALITY ANALYSIS OF GBL43 BULK PREPARATION

The final bulk product was estimated for various impurities like the aggregation, host cell proteins [HCP] and endotoxins by the analytical methods such as SE-HPLC, RP-HPLC, SDS-PAGE, ELISA, western blotting and LAL test. For RP-HPLC analysis a C-18 reverse phase column 25 cm in length with beads of 5 pm having pores of about 300 A was used with an automated HPLC system. It was equilibrated with Solution A (0.1 % TFA added to 10 % acetonitrile in water) at about 0.8 ml/min flow rate. Then samples containing about 15 pg of GBL43 or the reference product was injected in the column and eluted with an acetonitrile gradient. The amounts of the eluting proteins were determined by UV detector at 214 nm. For the western blot analyses, the SDS-PAGE gels in reducing or non-reducing conditions were used. To check level of impurities, the protein samples of 2.5, 5.0 and 7.5 pg were subjected to the SDS-PAGE gels, followed by protein transfer to NC membranes and then detected with primary (an anti-ILi Ra mouse monoclonal antibody) and secondary (anti-mouse HRP conjugated antibody) antibodies. For visualization, the blots were developed using the DAB urea method. The percent aggregation was calculated from SE- HPLC and the percent impurity from RP-HPLC data as shown in FIGURE 6A and FIGURE 6B. The HCP was estimated using ELISA and the endotoxin was by LAL test.

EXAMPLE 10: FINAL FORMULATION OF GBL43/ ANAKINRA

The said final stream containing about 170 mg/mL of GBL43 protein was supplemented with polysorbate 80 and other buffering components to achieve the final bulk or drug substance product composition of about 150 mg/mL of GBL43/ anakinra with quality attributes as shown in

TABLE 2.

TABLE 2: Quality attributes of the final GBL43 composition

The final formulation made for the use in pre-clinical and clinical trials contained about 150 mg/mL of GBL43/anakinra along with about 1.92 mg/mL of citric acid anhydrous, about 0.18 mg/mL of disodium EDTA, about 8.17 mg/mL of sodium chloride and about 1.0 mg/mL of polysorbate 80. The said final bulk formulation was stored at about 5 °C for up to 90 days.

EXAMPLE 11: PROTEIN PURITY AFTER EACH STEP OF SAID

METHOD

The yield or amount of GBL43/ anakinra recovered after each purification step was determined by Bradford method. The purity of GBL43/ anakinra in eluate after each step was determined by RP-HPLC method. The results are shown in TABLE 3. TABLE 3: Increase in purity of after each step of said method.

Claims

We claim: l. A method of purifying a recombinant protein, comprising:

(a) providing a liquid mixture containing said recombinant protein and contaminants obtained from lysate of bacteria;

(b) contacting said mixture with an anion exchange resin under conditions that allow said recombinant protein to bind or adsorb to it;

(c) removing by washing out a first part of said contaminants using an anion exchange wash solution without disrupting said bound recombinant protein;

(d) selectively eluting said recombinant protein from said anion exchange resin using a first eluting solution forming a first stream containing said recombinant protein;

(e) contacting said first stream with a cation exchange resin under conditions that allow said recombinant protein to bind or adsorb to it; (f) removing by washing out a second part of said contaminants by using a cation exchange wash solution without disrupting said bound recombinant protein;

(g) selectively eluting said recombinant protein from said cation exchange resin using a second eluting solution forming a second stream containing said recombinant protein;

(h) concentrating said second stream by molecular filtration under conditions that allow said recombinant protein to get separated from a remaining part of said contaminants forming a concentrated stream containing said recombinant protein;

(i) contacting said concentrated stream with a second anion exchange resin under conditions that allow endotoxins present in said concentrated stream to bind or adsorb to it forming an endotoxins free final stream containing said recombinant protein; and

(j) formulating said final stream by adding pharmaceutical formulation agents to it.

2. The method as claimed in claim l, wherein removing a first part of said contaminants by washing under conditions that leave said recombinant protein bound to said anion exchange resin.

3. The method as claimed in claim 1, wherein pH of said anion exchange wash solution is between 7.5 and 8.5 with from 15 to 25 mM of Tris buffering agent.

4. The method as claimed in claim 1, wherein said first elution solution comprises 1000 mM of NaCl and from 15 to 25 mM of Tris buffering agent at a pH between 7.5 and 8.5.

5. The method as claimed in claim 1, wherein removing a second part of said contaminants by washing under conditions that leave said recombinant protein bound to said cation exchange resin.

6. The method as claimed in claim 1, wherein pH of said cation exchange wash solution is between 5.5 and 6.5 with from 10 to 50 mM of acetate buffering agent.

7. The method as claimed in claim 1, wherein said first elution solution comprises from 500 to 1000 mM of NaCl and 10 to 50 mM of acetate buffering agent at a pH between 5.5 and 6.5.

8. The method as claimed in claim 1, wherein said concentrating is performed by means of a tangential flow filtration system along with exchange of buffer solution to formulation buffer comprises from 5 to 25 mM of citrate, from 0.5 to 5 mM of EDTA and from 120 to 160 mM of NaCl at a pH between 5.0 and 7.0.

9. The method as claimed in claim 1, wherein said recombinant protein produced in an Escherichia coli host cell.

10. The method as claimed in claim 1, wherein said contaminants comprises one or more of high molecular weight protein aggregates, host cell proteins, DNA, RNA, or endotoxins.

11. The method as claimed in claim 1, wherein said recombinant protein is purified to a purity of at least 90%.

12. The method as claimed in claim 1, wherein said recombinant protein comprises the amino acid sequence of shown in FIGURE 7, or an amino acid sequence that is at least 90 % identical thereto.

13. The method as claimed in claim 1, wherein said recombinant protein is anakinra.

14. The method as claimed in claim 1, wherein said recombinant protein contains less than 1 % high molecular weight protein aggregates.

15. The method as claimed in claim 1, wherein said recombinant protein is a single chain polypeptide of pH(I) between 5.6 and 5.8.

16. The method as claimed in claim 1, further comprises preparing a pharmaceutical composition containing said recombination protein.

17. The method as claimed in claim 1, wherein said recombinant protein is concentrated to amount from 120 to 200 g/L.

18. A recombinant protein purified by the method of any one of claims 1 to 15.

19. A pharmaceutical composition comprising said recombinant protein of claim 17 or 18.