(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2016/005903 A2 14 January 2016 (14.01.2016) P O P C T
(51) International Patent Classification: (IN). SRINIVASAN, Sankaranarayanan; E-504, E-504, C07K 14/605 (2006. X) Jalvayu Heights, Opp. Jal Soudha, Jalahalli, HMT Main Road, Bangalore 560013 (IN). HORA, Anuradha; H. No. (21) International Application Number: 706, Church Road, Civil Lines, Uttar Pradesh, Sitapur PCT/IB20 15/055 133 261001 (IN). N, Bairavabalakumar; 175A, Velan Nagar, (22) International Filing Date: Velasaravakkam, Tamil Nadu, Chennai 600087 (IN). 7 July 2015 (07.07.2015) NAIR, Karthika; P-6, Senior Officers Enclave, Head Quarters Training Command, Indian Air Force J.C Nagar (25) Filing Language: English Post, Karnataka, Bangalore 560006 (IN). THANIGAFV- (26) Publication Language: English EL, Aswini; 29, Wellala Street, 2nd Floor, Aminbikarai, Tamil Nadu, Chennai 600029 (IN). MALIWALAVE, (30) Priority Data: Amol; 239, 2nd floor, 5th main, 1st Block, Valagerahalli, 3042/CHE/2014 8 July 2014 (08.07.2014) IN nanabharathi PO, Kengeriupanagar, Karnataka, Bangalore (71) Applicant: THERAMYT NOVOBIOLOGICS 560056 (IN). SHENOY, Bharath R ; # 74/94, 9th Cross, PRIVATE LIMITED [IN/IN]; 118/1 1 Prasad Enclave, 4th main, Bapujinagar, Karnataka, Bangalore 560026 (IN). Yeshwantpur Industrial Suburb, 2nd Stage, 5th Main, KURUP, Anisha; 51-E, Pocket A-3, Mayur Vihar, Phase - Yeshwantpur, Bangalore 560022 (IN). 3, Delhi 110096 (IN). (72) Inventors: CHATTERJEE, Sohang; 45, 1st A Cross, (74) Agents: THACKER, Ankita et al; K&S Partners | Intel AECS Layout, RMV 2nd Stage, Sanjay Nagar, Bangalore lectual Property Attorneys, 4121/B, 6th Cross, 19A Main, 560094 (IN). RODRIGUES, Kavitha Iyer; Apartment HAL II Stage (Extension), Bangalore 560038 (IN). No. 121B, Sobha Magnolia, Bannerghatta, Road, Ban (81) Designated States (unless otherwise indicated, for every galore 560029 (IN). GHOSH, Maloy; D 903, NNC Urban kind of national protection available): AE, AG, AL, AM, Aster Park, 5/2 Mandala Kunte, Yelahanka New Town, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, Bangalore 560064 (IN). MAITY, Sunit; 8A 25, Nandi BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, Garden, Phase 1, JP Nagar, 9th Phase, Anjanapura, Ban DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, galore 560062 (IN). PENDSE, Rajeshwari; # 688/13, HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, Narasimhaswamy building, RK Layout Hebbagodi, Ban KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, galore 560100 (IN). UNNIKRISHNAN, Divya; No. 44, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, 4th Cross, 4th Block, Nandini Layout, Bangalore 560096 PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, (IN). B. M., Yogendra Manjunath; #85, 1st ' Cross SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, Road, RMV 2nd Stage, 3rd Block, Devasandra, Bangalore TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. 560054 (IN). HAZARIKA, Jahnabi; # 302, Golf Manor Apartments, Murgeshpalya, Wind Tunnel Road, Bangalore (84) Designated States (unless otherwise indicated, for every 560017 (IN). M, Sathyabalan; No. 52 4th cross, Dug- kind of regional protection available): ARIPO (BW, GH, galamma Layout, Rajagoplal Nagar, Laggre, Peenya III GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, phase, 560058 Bangalore (IN). M, Pavithra; 1-171-1A, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, Mathra Smurthi, Near Prabhakar Tiles, Ferry Road, Kund- TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, apur 576201 (IN). PRASAD, Bhargav; New No. 6, 1st DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, street 5/55, 3rd Main Road, Kannadasan Nagar, Kodun- LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, gaiyur, Tamil Nadu, Chennai 6001 18 (IN). K, Veeresha; SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, No. 682, 2nd Block, Kanteeravanagar, Nandini Layout GW, KM, ML, MR, NE, SN, TD, TG). Post, Bangalore 560096 (IN). PATHAK, Prabhat Ku¬ Declarations under Rule 4.17 : mar; S-8/226,A-l, Sudhakar Road, Khajuri, Pandeypur, Uttar Pradesh, Varanasi 221002 (IN). BHATTACHAR- — of inventorship (Rule 4.17(iv)) JEE, Sanghamitra; G-07, Bloomingdale apartments, 1st Published: Main, 11th cross, Near Corporation Bank, Pai layout old Madras Road, Doorvaninagar, Bangalore North, Bangalore — without international search report and to be republished 560016 (IN). D, Pravin Kumar; No.32, MGR Nagar, 1ST upon receipt of that report (Rule 48.2(g)) Street Velachery, Tamil Nadu, Chennai 600042 (IN). — with sequence listing part of description (Rule 5.2(a)) < HALAN, Vivek; # 619, Jakkanarai, Thumboor village, jackanarai, The Nilgiris, Tamil Nadu, Aravenu 643201 o
© © v o (54) Title: A PROCESS FOR OBTAINING EXENDIN-4 (57) Abstract: The present disclosure provides a process for preparing Exendin-4 from high cell density employing specific steps to obtain purified Exendin-4. The process produces Exendin-4 in higher purity and yield, employing chromatographic and non-chroma - tographic techniques in the process. The present disclosure also provides for a process for purification of Exendin-4. A PROCESS FOR OBTAINING EXENDIN-4
TECHNICAL FIELD The present disclosure relates to the field of biotechnology, particularly, to the field of Biopharmaceuticals. The present disclosure provides a process for preparing Exendin-4 from high cell density employing specific steps to obtain purified Exendin-4.
The process produces Exendin-4 in higher purity and yield using chromatographic and non- chromatographic techniques in the process. The present disclosure also provides for a process for purification of Exendin-4.
BACKGROUND OF THE DISCLOSURE The production of large quantities of relatively pure, biologically active polypeptides and proteins is important economically for the manufacture of bio-pharmaceuticals. The advent of recombinant DNA technology has extended the range of potential microbial fermentation products. It is possible to introduce genes from higher organisms into microbial cells such that the recipient cells are capable of synthesizing foreign proteins by recombinant DNA technology. Examples of the hosts for such foreign genes include Escherichia coli, Saccharomyces cerevisiae etc. Use of E.coli has advantages like low cost, less time, robustness, easy transformation and fermentation, protein yield etc. Fed-batch processes have most often been used to obtain high cell productivity.
Products produced in such genetically manipulated organisms include interferon, insulin, human serum albumin, factor VIII and factor IX, epidermal growth factor, bovine somatostatin and bovine chymosin. Chemically defined media are used during the fermentation of microorganisms. Main advantages of a chemically defined medium are more consistent titres, easier process control, high yield and cost effectiveness. The composition of the medium is identical in every process in contrast to complex media. Therefore, the downstream process is simplified, because only a few substances have to be removed.
It is important to maintain the integrity and biological property/activity of the proteins. Numerous purification techniques have been employed to purify the product of interest with maximum purity without affecting the product quality. One example in this area is production of large quantities of the protein 'insulin' which is used in treatment of Diabetes mellitus (DM). The hallmark of diabetes mellitus is hyperglycaemia resulting from impaired carbohydrate metabolism. Type 2 diabetes has a complex pathophysiology characterised by deficient insulin activity arising from decreased insulin secretion secondary to beta-cell failure, compromised insulin action in peripheral target tissues (insulin resistance), or a combination of the two abnormalities. This abnormal metabolic state is exacerbated by excess hepatic glucose production and altered metabolism of proteins and lipids, which along with hyperglycaemia, contribute to microvascular and macrovascular complications.
Type 2 diabetes accounts for approximately 85% to 95% of diabetes cases in developed regions like the European Union. Age and weight are established risk factors for type 2 diabetes. The majority of patients with type 2 diabetes are overweight or obese.
Glucagon-like peptide- 1 agonist (GLP-1 agonist) is one of the most sought after drug category in current diabetes treatment options. Byetta®, a GLP1 analog is prescribed for twice a day injection for diabetics. The product is manufactured through chemical synthesis route.
Byetta (Exenatide or Exendin-4) contains exenatide which is an incretin mimetic. Endogenous incretins, such as glucagon like peptide 1 (GLP-1), facilitate insulin secretion following their release from the gut into the circulation in response to food intake. Exenatide is licensed for the treatment of type 2 diabetes mellitus in combination with metformin and/or a sulfonylurea, or pioglitazone in patients who have not achieved adequate glycaemic control with these drugs alone or in combination.
As, there is no single biosimilar approved for Byetta®, the current disclosure reports a method for developing the TMB01 a biosimilar to Byetta® using microbiological platform. The present disclosure outlines the program/method/protocol which has been followed (aligning to all regulatory guidelines) in developing Exendin-4.
Byetta is a synthetic version of Exendin-4, a hormone found in the saliva of Gila monster. Exenatide is a 39 amino acid peptide amide with gluco-regulatory effects and is a glucagon- like peptide- 1 (GLP-1) receptor agonist that exhibits several anti-hyperglycaemic actions of GLP-1. It stimulates glucose dependent insulin secretion, suppresses inappropriately elevated glucagon secretion, which is known to be elevated in type 2 diabetes, and slows gastric emptying. However, Exenatide does not impair the normal glucagon response and other hormone responses to hypoglycaemia and is resistant to breakdown by dipeptidyl peptidase-4 (DPP) and therefore has a much longer half-life than endogenous GLP-1. Exenatide is intended for use in combination with basal insulin with or without metformin and/or a thiazolidinediones for the treatment of type 2 diabetes mellitus. Exendin-4 is administered as sub-cutaneous injection for the treatment of Type 2 Diabetes mellitus, a metabolic disorder that is characterized by hyperglycemia due to insulin resistance or insufficient levels of insulin secretion by the pancreatic cells.
Byetta was approved in the U.S. in April 2005 as adjunctive therapy to improve glycemic control in patients with type 2 diabetes taking certain oral medications but who have been unsuccessful at controlling their blood sugar levels. In 2009, the FDA approved an expanded use for Byetta as a stand-alone therapy (monotherapy) along with diet and exercise. It was approved as an add-on therapy to insulin glargine in 2011, expanding the indication for the use of Byetta across the continuum of type 2 diabetes care.
Exendin-4 facilitates glucose control in following ways:
1. It enhances insulin secretion by the pancreatic beta cells upon ingestion of food (glucose dependent) to control the blood glucose levels. Once the blood sugar level reaches to normal values, the pancreas response to produce insulin is reduced which prevents hypoglycemia. 2. It also suppresses pancreatic release of glucagon in response to eating, which helps stop the liver from overproducing sugar when it is unneeded, which prevents hyperglycemia. 3. It helps slow down gastric emptying and thus decreases the rate at which meal-derived glucose appears in the bloodstream. 4. It has a prolonged effect to reduce food intake resulting in weight loss. 5. It offers an increased half-life when injected to a patient due to its resistance against DPP.
Also, the generally known process for purification in the art is by subjecting the protein containing sample to three to four chromatography steps, which makes the process costly and time consuming. Thus, the present disclosure provides a solution to the problems faced in the prior art by providing a cost effective and more efficient process of producing Exenatide in microorganisms. Also, the process disclosed in the present disclosure gives maximum purity and yield of the product.
STATEMENT OF THE DISCLOSURE Accordingly, the present disclosure to a process for obtaining Exendin-4, said process comprising acts of a) obtaining vector comprising gene for Exendin-4 set forth in SEQ ID No. 1 and transforming host cell with the vector to obtain transformed cell b) culturing the transformed cell in culture medium and subjecting to fermentation c) lysing the cell to obtain crude Exendin-4 and d) optionally processing the crude Exendin-4 to obtain purified Exendin-4; a process of purifying Exendin - 4, said method comprising acts of a) lysing cell comprising Exendin-4 protein and subjecting the cell lysate to clarification to obtain clarified sample b) subjecting the clarified sample to affinity chromatography and tangential flow filtration c) digestion of protein; and subjecting the digested protein to chromatography and filtration to obtain purified Exendin - 4 and Exendin-4 as set forth in SEQ ID No. 1, wherein the Exendin - 4 is formulated into dosage form in formulation buffer.
BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES In order that the disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figures together with a detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, in accordance with the present disclosure where:
Figure 1 depicts the cellular mechanism of binding of the Exenatide to the GLP- 1receptor and how the binding brings about the increase of glucose uptake by the cells.
Figure 2 depicts the pET-32 expression vector having Exendin-4.
Figure 3 depicts the pairwise alignment of amino acid sequence between Exendin-4 of the present disclosure and Byetta®. Figure 4 depicts the coinciding Tryptic peptide map profile between Exendin-4 of the present disclosure and Byetta®.
Figure 5 depicts the identical mass profile between Exendin-4 of the present disclosure and Byetta®.
Figure 6 depicts the similar UV spectroscopy between Exendin-4 of the present disclosure and Byetta®.
Figure 7 depicts the CD spectroscopy profile of Exendin-4 of the present disclosure suggested as alpha helical.
Figure 8 depicts the test principle and the results obtained with the Exendin-4 bioassay.
Figure 9 depicts the SDS profile of both Byetta® and Exendin-4 of the present disclosure.
Figure 10 depicts the size exclusion HPLC profile of Exendin-4 of the present disclosure with that of Byetta®. The peak observed at 15 minutes is that of the monomer whereas the peak at 25 minutes is that of meta-cresol.
Figure 11 depicts the results of the oral glucose tolerance test, wherein the Exendin-4 and Byetta® show comparable reduction in blood glucose.
Figure 12 depicts the level of blood glucose in the form of total area under the curve (AUC) for Exendin-4 and Byetta® during the OGTT test. The results depict similar reduction of blood glucose for both Exendin-4 and Byetta®.
Figure 13 depicts the change in plasma insulin with respect to time for Exendin-4 and Byetta®.
Figure 14 depicts the level of insulin release in the form of total area under the curve (AUC) using Exendin-4 and Byetta® in bar graph format. DETAILED DESCRIPTION OF THE DISCLOSURE The present disclosure to a process for obtaining Exendin-4, said process comprising acts of: a) obtaining vector comprising gene for Exendin-4 set forth in SEQ ID No. 1 and transforming host cell with the vector to obtain transformed cell; b) culturing the transformed cell in culture medium and subjecting to fermentation; c) lysing the cell to obtain crude Exendin-4; d) optionally processing the crude Exendin-4 to obtain purified Exendin-4.
In an embodiment of the present disclosure, the gene sequence for the Exendin-4 includes solubility or affinity tag or combination thereof; and the Exendin-4 is expressed as a fusion protein.
In another embodiment of the present disclosure, the host cell is E.coli; and the vector is selected from group comprising pET-32a and pET24.
In another embodiment of the present disclosure, the transforming is carried out by technique selected from group comprising Heat shock method, electroporation and chemical method or any combinations thereof; and the culturing is carried out at temperature ranging from about 25°C to about 40°C, preferably about 37°C, for time duration ranging from about 1 hour to about 20 hours, preferably about 17 hours, at pH ranging from about 6.8 to about 7.4, preferably about 7.2.
In another embodiment of the present disclosure, the fermentation is carried out at temperature ranging from about 35°C to about 40°C, preferably about 37°C, for time duration ranging from about 11 to 13 hours, at pH ranging from about 6.5 to 7.5.
In another embodiment of the present disclosure, the lysing of cell is carried out by subjecting the cell to high pressure Homogenizer or Microfluidizer or French Press, or any combinations thereof; and wherein said lysing of cell is followed by clarification of the cell lysate.
In another embodiment of the present disclosure, the processing of step d) is carried out by subjecting the crude Exendin-4 to technique selected from group comprising chromatography including affinity chromatography or reverse phase chromatography; filtration including tangential flow filtration, ultrafiltration, diafiltration, sterile filtration; and digestion of protein; or any combinations thereof.
In another embodiment of the present disclosure, the Exendin - 4 obtained by the process has a purity of at least 97%, preferably about 99%; and the Exendin-4 is formulated into dosage form in formulation buffer comprising component selected from group of sodium acetate, acetic acid, D-Mannitol and Meta-cresol or any combinations thereof.
The present disclosure also relates to a process of purifying Exendin - 4, said method comprising acts of: a) lysing cell comprising Exendin-4 protein and subjecting the cell lysate to clarification to obtain clarified sample; b) subjecting the clarified sample to affinity chromatography and tangential flow filtration; c) digestion of protein; and subjecting the digested protein to chromatography and filtration to obtain purified Exendin - 4.
In an embodiment of the present disclosure, the lysing of cell is carried out by subjecting the cell to high pressure Homogenizer or French Press or Microfluidizer, or any combinations thereof; and the clarification of the cell lysate is carried out by techniques selected from group comprising centrifugation, tangential flow filtration and microfiltration; or any combinations thereof.
In another embodiment of the present disclosure, the digestion of protein is with enterokinase enzyme; and the chromatography of step c) is selected from group comprising affinity chromatography and reverse phase chromatography, or combination thereof; and the filtration is selected from group comprising ultrafiltration, diafiltration and sterile filtration, or any combinations thereof.
In another embodiment of the present disclosure, the protein is fusion protein; and the filtration of step c) is followed by sterile filtration.
The present disclosure relates to Exendin-4 as set forth in SEQ ID No. 1, wherein the Exendin - 4 is formulated into dosage form in formulation buffer. In an embodiment of the present disclosure, the Exendin-4 has purity of at least 97%, preferably about 99%; and the formulation buffer comprises component selected from group of sodium acetate, acetic acid, D-Mannitol and Meta-cresol or any combinations thereof.The instant disclosure overcomes the drawbacks observed in the prior art and arrives at process of producing Exendin-4 in E.coli using different Chromatographic & Non-Chromatographic methods sequentially in combination which results in purified Exendin-4 with higher purity.
In an embodiment, the terms "Exendin-4", "Exenatide", instant/final product are interchangeably used and have the same meaning within the scope of the present disclosure.
In another embodiment, "Byetta" and "reference product" are interchangeably used and have the same meaning within the scope of the present disclosure.
The present disclosure relates to a process for obtaining Exendin-4.
In another embodiment, the fusion Exendin-4 contains affinity and solubility tag, specifically the solubility tag is thioredoxin protein and the affinity tag is poly (His) tag. In another embodiment, the solubility tag helps in expressing the product in soluble form in E.coli and to ease the purification process.
In another embodiment of the present disclosure, the culture medium is a chemically defined medium.
In a specific embodiment, the purification step comprises the following steps:
Step 1) Subjecting the cells to lysis by high pressure homogenizer to extract the intracellular protein after suspending the cells in lysis buffer; Step 2) Subjecting the cell lysate to clarification by centrifugation process; Step 3) Subjecting the clarified sample to first Affinity chromatography and removal of the affinity tag and solubility tag from the fusion Exendin-4; Step 4) Subjecting the Exendin-4 to second affinity chromatography having negative binding phase to remove affinity tag and solubility tag. Step 5) Subjecting the Exendin-4 to the final chromatography step or the 'polishing step' which is reverse phase chromatography to obtain pure Exendin-4. Step 6) Subjecting the Exendin-4 to ultrafiltration/diafiltration to formulate. Step 7) Subjecting the Exendin-4 product into sterile filtration using 0.22 micron filter.
In an embodiment of the present disclosure, the purification carried out by chromatography and filtration comprises the following sequence of steps:
Step 1) Subjecting the crude Exendin-4 to first affinity chromatography step;
Step 2) Subjecting the sample obtained in step 1) to tangential flow filtration system; Step 3) Removal of the affinity tag and solubility tag from the fusion Exendin-4; Step 4) Subjecting the Exendin-4 to second affinity chromatography having negative binding phase; Step 5) Subjecting the Exendin-4 to the final chromatography step which is the reverse phase chromatography or 'polishing step' to obtain pure Exendin-4.
In an embodiment of the present disclosure, Exendin-4 is produced using a high cell density fed-batch fermentation process using completely chemically defined media (CDM).
In an embodiment, the upstream fermentation parameters are optimized for production of Exendin-4, wherein the optimum temperature is about37.0 + 0.5 °C and the optimum pH is about 7.00 + 0.1.
In another embodiment of the present disclosure, Exendin-4 is purified from high cell density E.coli cells.
In an embodiment, the cells are harvested by centrifugation and are lysed by High Pressure Homogenizer after re-suspending in appropriate lysis buffer. The cell lysate contains cell debris, cell wall materials along with fusion protein. The crude lysate is subjected to clarification by centrifugation.
In another embodiment, the clarified supernatant sample is subjected to affinity Chromatography. The affinity chromatography eluted sample is buffer exchanged to appropriate buffer by tangential flow filtration. The solubility and affinity tags are cleaved using respective enzyme. The sample is passed onto affinity chromatography, where target protein is eluted in the flow through. This is followed by Reverse Phase chromatography. In another embodiment, in order to achieve maximum purity of the protein, various resins, buffers and other conditions are screened and evaluated.
The present disclosure relates to a process of producing Exendin-4, wherein the process uses only two chromatography techniques to purify the Exenatide from E.coli.
In an embodiment, the downstream process is designed considering the product size and process and product related impurities.
In an embodiment of the present disclosure, Exendin is a 4.18 kDa peptide with a pi of 4.70, which is expressed as a fusion protein with a molecular weight of 21.1 kDa.
In another embodiment, the fusion protein is expressed as a soluble protein in E.coli, which contains thioredoxin solubility tag to express in soluble form and His-tag as an affinity tag to ease the purification process and to reduce proteolytic effects.
In another embodiment, after cell lysis, the clarified sample is loaded on an affinity chromatography column. The elute is subjected to Tangential Flow Filtration (TFF) to reduce imidazole and NaCl for Enterokinase (EK) digestion of fusion proteins. In a specific embodiment, EK digestion step cleaves Thioredoxin-Histidine tag from exenatide molecule. The sample is loaded on Affinity chromatography column, which is in negative binding mode where the exenatide molecule elutes in the Flow through and fusion tags and other impurities binds to the column.
The FT sample is passed onto Reverse Phase Chromatography (RPC) and the bound exenatide is eluted in acetonitrile containing elution buffer in gradient. The RPC eluted sample is subjected to Ultrafiltration/Diafiltration for Drug substance (DS) preparation. The Drug Product (DP) is prepared by adding 2X D-Mannitol and meta-Cresol in DS buffer to a final concentration of about 225 to about 275 µg/ml.
In an embodiment, the cost of the instant process is very less because only two chromatography columns used with buffers, throughout the process. In another embodiment, the downstream processing of Exenatide consists of nine steps (refer flow chart in subsequent sections) which includes from cell lysis to final drug product (DP) preparation. The process flow chart shows the steps involved in the product development.
The present disclosure relates to the use of a high cell density fermentation process for higher expression and various chromatography techniques to purify Exendin-4 in E.coli.
The present disclosure primarily relates to an approach of producing Exendin-4 in E.coli using different Chromatographic & Non-Chromatographic methods sequentially in combination which result in Exendin-4 with higher purity and yield.
In another embodiment, MFCS DA software is used for powerful supervisory fermentation process control through extended visualization, data acquisition and trend display.
In an embodiment, the process of the present disclosure has two parts, wherein one part is upstream processing (fermentation) and the second part is downstream purification.
In another embodiment, the process of the present disclosure yields around -180 to 200 mg of Exenatide with the purity of at least 97%, preferably about 99%.
In another embodiment of the present disclosure, the Exendin-4 is formulated with formulation buffer in cartridges. In another embodiment, the formulation buffer contains sodium acetate, acetic acid, D-Mannitol and Meta-cresol or any combinations thereof.
The present disclosure is further described with reference to the following examples, which are only illustrative in nature and should not be construed to limit the scope of the present disclosure in any manner.
The biological material used in the present application is obtained/sourced from Harlan Laboratories, United States of America.
EXAMPLES EXAMPLE 1: DEVELOPMENT OF CELL LINE In an embodiment of the present disclosure, Exenatide is obtained from microbial source, specifically E.coli.
The E. coli cells are transformed with a plasmid (Figure 2) carrying an optimized, partially synthetic, structural gene for Exendin-4 as set forth in SEQ ID No. 1. The peptide sequence is obtained and codon optimized for ideal expression in the current microbial system i.e. E.coli. As soluble form of expression is preferred, the pET-32 series of vector (Novagen®) is used for cloning and high-level expression of peptide sequence. This vector system contains the Trx Tag™ (thioredoxin protein solubilization tag). These tags are used especially for recombinant proteins expressed in chaperone-deficient species such as E. coli, to assist in proper folding of proteins and keep them from precipitating. The poly (His) tag is used (appended to the peptide) so that the expressed peptide is purified from their crude biological source using an affinity technique enabling higher recovery.
A large number of clones resulting from this transformation are then screened to identify the production clone i.e. a clone which combines high quality and robust productivity. The final clone selected for full development is characterized for clonal stability. Several research cell banks of final clone are created and stored in -80°C freezer.
EXAMPLE 2 : DEVELOPMENT OF THE FERMENTATION PROCESS To initiate the fermentation process, a vial of the Research Cell Bank is thawed under controlled conditions and its contents are transferred into a shake flask containing a sterile fully chemically defined medium.
The cells are grown in this shake flask till mid log phase and then transferred to bioreactor (fermenter). All of the steps in the fermentation process are optimized to yield a product quality that is highly similar to Byetta®. At all stages, nutrient media without any animal- derived component are used to avoid the risk of contamination and lot to lot variation with such components.
The cultivation conditions are systematically optimized in an iterative manner with many fermentation runs, manipulating the culture conditions until it is able to achieve the required product quality highly similar to Byetta®. This includes the optimization of cultivation conditions such as temperature, stirring speed, air input, and pH. The feeding parameters such as composition of feed, frequency and amount of feed to be added are also optimized. EXAMPLE 2.1: PREPARATION OF MEDIA The E.coli cells transformed with the protein i.e. Exendin-4/Exenatide is grown in a chemically defined/optimized media, which helps the transformed cells to grow uniformly and produce biosimilar of Exendin-4 in large quantities. The optimization of this chemically defined media is such that it helps to produce Exendin-4. The media employed in the instant disclosure are media employed for culturing cells such as E.coli.
EXAMPLE 2.2: INOCULUM DEVELOPMENT (SHAKE FLASK) • 400 ml of seed media is re-constituted by adding required amount of glucose solution, sulphate solutions to a sterile conical flask containing phosphates and citric acid. Vitamins and trace elements are added such that the final concentration of each is 0.1%. Ampicillin is added such that the final concentration is 0.05%. • A vial(s) from the research cell bank of the clone which produces exenatide is taken from - 80°C freezer and rapidly thawed on ice at 4°C. • The flask containing aforementioned seed media is inoculated with 0.1% of inoculum and then incubated at 37°C and 220 rpm in a shaker incubator. • The duration of incubation is such that an actively growing culture (OD600 of 3 +0.3) is obtained prior to seeding of fermenter i.e. about 10 + 1 hours incubation. • The flask(s) is sampled at the end of incubation to check the OD@600 nm, which is about 3.0 + 0.3. • The seed is thereafter transferred to the fermenter.
EXAMPLE 2.3: FERMENTATION RUN The following parameters as set forth in the table below are set on the control unit for the fermenter run. As can be observed the below table depicts the parameters and set points maintained during fermentation.
TABLE NO. 5 Gas flow rate 3 1pm
Protocol: • The calculated volume (as shown below) of the seed culture is aseptically transferred into vessel using a transfer flask. • The batch is inoculated with 10% of the inoculum. Volume of seed inoculum needed = Total fermenter culture volume/10. • To maintain the dissolved oxygen (DO) level, the saturation level is set at '30% saturation' throughout the fermentation run, the DO cascading option is activated to cascade the dissolved oxygen to gas flow rate limits ( 0 1pm to 3 1pm). • After about 5 hours of growth, the addition bottle with the feed media is connected to the substrate pump of the fermenter. • The OD@600nm and residual glucose is monitored and the substrate pump is activated when the OD@600nm value reaches approximately 6.0+1 OD@600nm and residual glucose reaches 11.0+1 g/L. • The culture is grown for about 8.5 + 0.5 hours, time duration till the OD@600nm reaches 45.0 + 4. • Volume corresponding to 10 OD@600nm of the sample is pelleted & named as "BP denoting before induction with IPTG and stored at - 20°C. • The culture is induced with 35 mL of Isopropyl β-D-l-thiogalactopyranoside (IPTG) solution. The following formula is used to calculate the amount of IPTG to be added. (IPTG in grams = 0.0238 X (OD@600nm before induction) X batch volume in L).
• Volume corresponding to 10 OD@600nm of the sample is collected every hour starting from induction time point till harvest time (4 hours after Induction). • The collected samples are pelleted at about 10,000 rpm for about 5 minutes using micro centrifuge. • Cell pellet samples before and after induction are subjected to SDS PAGE analysis. Final OD600 after harvest should not be less than 49.0 • The culture is harvested at the end of the induction period by unclamping the harvest tube and closing the exhaust vent filter, until all the culture is drained out. • The harvested culture is pelleted by centrifugation at 5180 relative centrifugal force (rcf), 4°C for 30 min using refrigerated centrifuge. • The pellet is subjected to downstream processing. The instant examples relate to Exenatide manufacturing process, which broadly consists of following steps: • Cell line development • Upstream process and fermentation • Downstream process and purification • Formulation
In an embodiment, the Exenatide has been processed and developed to be similar to the reference product Byetta® with respect to active pharmaceutical ingredient (API) and formulation.
EXAMPLE 3 : DOWNSTREAM PROCESSING AND PURIFICATION As the product accumulates inside of the cells in soluble form, a process for lysing the cells is developed. This is achieved by exerting the cells to high pressure and then rapidly releasing this pressure using a high pressure homogenizer. This process is effective, yet gentle, and is optimized to ensure the Exenatide is not damaged in any way. The Exenatide is then isolated from the cell lysate using a centrifuge (separator) to remove cell debris and components of the E. coli cell. Purification of Exenatide is a sequential chromatography process, which exploits the physical and chemical difference between Exenatide and other biomolecules. Purification steps using various types of chromatography are evaluated, modified and refined in an iterative manner to yield product without detectable limit of product related as well as process related impurities as per regulatory standard. The selected process contains a series of sequential chromatography and tangential flow filtration (TFF) steps that use complementary purification methods.
The process broadly consists of three chromatography steps with TFF process after first affinity chromatography step and final UF/DF step after RPC stage. The first step is to capture the Exenatide/Exendin-4 from large number of E.coli proteins using an Affinity Chromatography and this yields relatively pure Exendin-4/Exenatide. The sample obtained from capture chromatography is buffer exchanged using tangential flow filtration to bring optimum buffer conditions for enzyme assay. To remove the affinity tag and solubility tag from Exendin-4, an enzyme is added and incubated at appropriate temperature. After the cleavage, the protein sample containing Exendin-4 is subjected to the second chromatography step. The Exendin-4 sample with residual impurities is loaded on the third chromatography column, which is called as a 'polishing step'.
In the end, the resulting Exendin-4 has high purity. The optimized purification process yields around -180 to 200 mg of Exenatide with the purity of around 99%. Moreover, the purification requires only two chromatography columns and buffers are employed in the process to purify the product. The instant process proves to be efficient and cost-effective process.
The purified Exendin-4 is frozen in special sterile plastic bottles until needed for the manufacturing of finished product. Various buffers are used in the present disclosure which are prepared as per standard parameters.
The broad sequential steps followed in the instant process to obtain the product is provided in the below flow chart.
FLOW CHART NO. 1 3.1 CELL LYSIS The transformed cells having the protein of interest i.e. Exendin-4 are subjected to cell lysis in order to recover the protein as it accumulates within the transformed E.coli cells. The cells are lysed using a technique which involves subjecting cells to high pressure and then rapidly releasing the pressure using a High pressure homogenizer which has been optimized to ensure that the protein of interest is not damaged. The OD 600 of the cell suspension typically is decreased after cell lysis.
3.2 CELL LYSATE CLARIFICATION The cell lysate is subjected to centrifugation to separate the crude molecules. Instrument Used: Centrifuge, Weighing balance
The supernatant collected carefully for capture chromatography step.
3.3 AFFINITY CHROMATOGRAPHY The centrifuged sample is subjected to chromatographic techniques in order to further purify the product.
Immobilized metal affinity chromatography (IMAC) is a type of affinity chromatography which is widely used as capture step in the purification of His tagged proteins.
3.4 TANGENTIAL FLOW FILTRATION (TFF) The chromatographic techniques are intermittently separated by tangential flow filtration/cross-flow filtration.
Tangential flow filtration is also called as cross flow filtration and is used for diafiltration and concentration. Hence, elute from IMAC is subjected to buffer exchange by Tangential flow filtration.
3.5 ENTEROKINASE EK DIGESTION The fusion protein has a DDDDK (Asp-Asp-Asp-Asp-Lys) site before the 1st amino acid of exenatide molecule. Enterokinase is known for site specific cleavage of the product and it cleaves the protein at specific site and releases the final product. • After incubation, the reaction is arrested by adding 0.5 n M phenylmethanesulfonylfluoride (PMSF).
3.6 AFFINITY CHROMATOGRAPHY (NEGATIVE BINDING) IMAC is used as a negative binding, where the Exendin-4/exenatide molecule free of fusion tag protein (His tag and Thioredoxin protein) will elute in the Flow through and His- Thioredoxin fusion protein along with some impurities will bind to the column. This chromatography is used as an intermediate purification step. The purity of IMAC flow through is achieved in the range between 80 to 90 %.
3.7 REVERSE PHASE CHROMATOGRAPHY (RPC) Reverse phase chromatography is used as a polishing step and the protein molecules separated based on hydrophobicity. RPC has become increasingly important for high resolution separation of protein molecules. The protein binds to resin in aqueous phase and eluted using organic solvent. This is the final chromatography step, where 99% of purity is achieved.
3.8 DRUG SUBSTANCE(DS) PREPARATION BY ULTRAFILTRATION (UFVDIAFILTRATION (DF) UF/DF is used for preparation of DS, where the RPC eluted fractions with highest purity is diluted to reduce the acetonitrile concentration and the fractions are buffer exchanged to DS buffer.
Instrument Used: AKTA Purifier 100, Biophotometer
Protocol: • Connect the Omega 50CM2 TFF Cassette of 1 kDa NMWCO to Lab Scale TFF system. The cassette is primed with purified water followed by DS buffer (30 mM Sodium acetate buffer pH 4.50+0.05) • After buffer priming, the sample is added to the reservoir and re-circulated for approximately 5 mins. The sample is concentrated to a final volume of ~ 100 ml and buffer exchanged to 5 diavolume with EK Digestion buffer. • After diafiltration, measure the protein concentration of DS sample and diluted to approximately 1 to 1.5mg/ml final concentration. 3.9 DRUG PRODUCT (DP) PREPARATION & STERILE FILTRATION Add required volume of DS buffer to bring the concentration of the protein to ~0.5mg/ ml. Add equal volume of DS buffer containing 2X D-Mannitol and meta-Cresol to bring the concentration of DP in the range between 225 µ g to 275 g/ml; filter using 0.2µ filter and fill in 3 ml cartridges under aseptic conditions.
EXAMPLE 4 : DEVELOPMENT OF THE FORMULATION The product obtained by DP preparation and sterile filtration is thereafter developed into a formulation. The instant Exendin-4 formulation is identical to that used for Byetta®. The formulation components used for Exendin-4/Exenatide is as mentioned in the table below.
TABLE NO. 7
The Exenatide is formulated as a clear, colourless solution at concentration of about 250µ / η1and each pen contains 2.4ml with a total concentration of 0.6 mg.
EXAMPLE 5 : DEMONSTRATION OF BIOSIMILARITY Analytical demonstration of Biosimilarity In order to determine biosimilarity of the instant product to Byetta®, orthogonal analytical tools are employed which determine the higher order structure of the instant product and also biological functions of it.
EXAMPLE 5.1; DETERMINE LINK BETWEEN STRUCTURE, FUNCTION, AND PHYSIOLOGICAL RESPONSE The mechanism of action of Exendin-4 is mediated through its binding to its receptor
(GLP1R) which has been depicted in Figure 1. The GLP1R is characterized as a seven- transmembrane domain with confirmed expression in pancreatic periductal and b-cells, kidney, heart, blood vessels, stomach, and brain. Both GLP-1 and Exendin-4 are a-helical peptides that interact with the GLP- 1 receptor by binding multiple extracellular contact points to induce receptor signalling. The actions of GLP-1 are mediated, at least in part, through adenylate cyclase. Moreover, GPCR recruitment of GPCR kinases (GRKs) and β-arrestins is characterized as inducing desensitization of G-protein mediated signal transduction.
The structure and function of Exendin-4 determines the in-vivo physiological response, and must therefore be controlled. Critical molecular attributes (commonly termed as "quality attributes") that are responsible for the in-vivo physiological response to Exendin-4 are the following: • the amino acid sequence (identity) for correct folding required for Exendin-4/GLPlR interaction • high-molecular weight variants/aggregates (purity) • the overall higher order structure which serves as the scaffold for correct positioning of the binding sites • receptor binding reflecting the integrity of the overall structure and the binding sites • in-vitro proliferation assay (potency) for demonstrating activation of downstream signalling cascades as a result of full structural integrity of the molecule.
In designing an analytical analysis program, emphasis is placed on evaluating the quality attributes that are known to have clinical relevance, as outlined in the table below, which depicts quality attributes responsible for in-vivo physiological response to Exendin-4. TABLE NO. 8
The final head-to-head analytical comparison is carried out comparing several batches of Exenatide of the present disclosure with four batches of Byetta®.
EXAMPLE 5.2: ANALYTICAL COMPARISON. The analytical comparison of Exenatide of the present disclosure to the reference product i.e. Byetta® is carried out in a stepwise manner that assesses structure, function, potential variants and stability. The analysis is performed with the drug product. As a first step, comparison of the structure of the biosimilar Exenatide with Byetta® is carried out first, as the amino acid sequence and protein folding have to match.
The structures are seen to be highly similar, thereafter functional assays are conducted to establish that receptor binding properties and potency are also similar. For Exendin 4, in vitro potency assay is analysed by several orthogonal methods. Moreover, in order to further justify the in vitro data, efficacy is also analysed using both Oral Glucose Tolerance Test OGTT and streptozotocin treated rat model. Once high similarity is established with instant product - Exendin -4 and Byetta®, further analyses are conducted to establish that Exendin -4 exhibits the same product stability profile as that of Byetta®.
EXAMPLE 5.3: STRUCTURAL ASSAYS A. The primary structure of Exendin - 4 and Byetta® are identical It is absolutely essential to have same amino acid sequence for safety, efficacy and immunogenicity. This aspect is confirmed by several different orthogonal methods, including N Terminal Edman sequencing and sequencing by MS/MS. Figure 3 shows the pairwise alignment between instant product - Exendin-4 and Byetta® confirming the sequence similarity. Moreover, peptide mapping analysis with UV and mass spectroscopic detection is carried out to confirm that the sequence and molecular weight between - Exendin - 4 and Byetta® are identical. Peptide mapping analysis is carried out after performing digestion with Trypsin. Figure 4 shows an overlay of the RP-LC chromatogram of a Tryptic digest map between Exendin - 4 and Byetta®. In addition, mass spectroscopy confirms the identity of individual peak by comparing mass of them (Figure 5).
B. The higher order structure does not differ between Exendin-4 of the present disclosure and Byetta® The structure of Exendin-4 drives its function by interacting with its receptor GLP1R. The overall structure defines the efficacy of the molecule. To determine the similarity with respect to both primary and secondary structure, UV and CD spectroscopic analysis is carried out. UV analysis suggests the primary structure of a protein, whereas CD analyses folding at the level of the secondary structure, i.e. the coiling of the strand of amino acids into alpha helices or its organization into beta sheets. UV spec data confirms the similar primary structure as observed from the lambda max value of 282.4 nm as shown in Figure 6.
For all tested Exenatide/Exendin - 4, the far UV-CD spectra indicates a folded protein with high amounts of a-helical secondary structure composition as for the reference product Byetta® confirming the similar profile as seen in Figure 7. EXAMPLE 5.4: FUNCTIONAL ASSAYS A. Target binding of Exendin - 4 and Byetta® with GLP1R is similar Binding affinities between Exendin-4 and its receptor reflect the structural integrity of the molecule as well as the correct three dimensional conformation generated by the amino acid residues within the target binding regions.
Binding affinity to GLP1R of both products is tested by Bio-Layer Interferometry (BLI) which is a label free, biosensor technology that allows the real-time measurement of molecular interaction. This assay is carried out using Octet® instrument from Pall. Calculated affinity is observed in micro molar range.
Comparable binding of Exendin - 4 with that of Byetta® is further confirmed by performing competitive ELISA with Exendin-4. This assay is carried out using available commercial kit from Phoenix Pharmaceutical.
B. In vitro potency by activated GPCR also confirms similar potency Exendin-4 Bioassay monitors GPCR activity by detecting the interaction of b-Arrestin with the activated GPCR using b-galactosidase (b-gal) enzyme fragment complementation. It is carried out by Path Hunter® Exendin-4 Bioassay kit from DiscoverX. The results for this assay are depicted in Figure 8. It can be interpreted from the results that relative potency is comparable to that of Byetta®.
EXAMPLE 5.5: SIZE VARIANTS The overall size of a protein and its size variants are assessed by use of multiple complementary techniques, including gel electrophoresis and size exclusion chromatography. Examples of size variants are dimers, oligomers, aggregates and lower molecular weight fragments generated during synthesis in the cell. Aggregates are known to have caused immunogenicity and are therefore tightly controlled in protein-based therapeutics.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a gel-based technique that measures the size of the protein on the based on movement through a gel pores following application of a directed electrical field. Figure 9 shows a representative Coomassie Blue stained SDS-PAGE gel from the head-to-head biosimilarity exercise. The highly similar migration confirms the size similarity and absence of size variants in Exendin - 4 and Byetta®.
Size exclusion chromatography is another orthogonal technique for detection of multimer formation of proteins. All Exendin -4 and Byetta® batches evaluated using size exclusion chromatography (SEC) show prominent monomer peak of around 96%, free of any aggregates as depicted in Figure 10.
EXAMPLE 5.6: IN VIVO ASSAY Type 2 diabetes mellitus (T2DM) is characterized by glucose intolerance, which is contributed by peripheral insulin resistance as well as islet β-cell dysfunction. The glucose tolerance test is a standard procedure to evaluate the rate of exogenous glucose clearance from blood. The efficacy of Exendin-4 is assessed in comparison with that of Byetta® in oral glucose tolerance test (OGTT) in male C57BL/6J mice. Mean blood glucose levels, change in blood glucose levels as well as total and delta area under the curve (AUC) is calculated. Plasma insulin analysis is also carried out. Both Exendin-4 and Byetta treated groups showed a significant reduction in total and delta AUC when compared to the vehicle control group (Figure 11-12). A significant increase in insulin release at all the tested time points is also observed (Figure 13-14).
Multiple analytical orthogonal characterization experiments confirm that Exendin-4 of the present disclosure and Byetta are highly similar.
Exendin -4 is developed to be analytically similar in major quality attributes to its reference product Byetta®. The comprehensive analytical program that has been conducted confirms that Exendin -4 is similar to Byetta® with regard to sequence and folding structure. By different orthogonal analysis, it has also confirmed that Exendin -4 binds to its receptor GLP1R in a similar fashion as like Byetta®. Exendin -4 also remains highly similar during product storage, as demonstrated by comparative stability studies. Thus, in view of the above studies, it is evident that the biosimilar Exendin -4/Exenatide of the present disclosure is highly similar to the reference molecule i.e. Byetta® with respect to its efficacy, binding, genetic sequence, size etc.
The advantages of the present disclosure are as follows: • The instant process produces Exendin-4 having higher yield and purity. • The purification step uses only two chromatography columns and three chromatography totally which helps in reduction of cost and time required for the process. • Optimization of the fermentation parameters during upstream processing yields Exendin-4 having biosimilaity as that of Byetta. • The purification of crude Exendin-4 carried out by the combination of techniques like affinity chromatography and Tangential flow filtration (TFF) and their specific sequence during the purification process helps in achieving higher purity of the product i.e. Exendin-4. • Overall reduction in cost, owing to the use of two columns in the chromatography etc.
Overall, it is evident from the present disclosure that the instant process provides for a highly cost effective, pure and equally efficacious biosimilar of Byetta®. SEQUENCE LISTING
<110> Theramyt Novobiologics Private Limited <120> A PROCESS FOR OBTAINING EXENDIN-4 <130> IP30790/MT/js <140> 3042/CHE/2014 <141> 2014-07-08
<160> 1
<170> Patentln version 3.5
<210> 1 <211> 117 <212> DNA <213> Escherichia coli
<220> <221> misc_recomb <222> (1)..(117)
<400> 1 catggcgaag gcacctttac cagcgatctg agcaaacaga tggaagaaga agcggtgcgc 60 ctgtttattg aatggctgaa aaacggcggc ccgagcagcg gcgcgccgcc gccgagc 117
<210> 2 <211> 39 <212> PROTEIN <213> Escherichia coli
<220> <221> misc_recomb <222> (1)..(39)
<400> 1 HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS 39 We Claim:
1. A process for obtaining Exendin-4, said process comprising acts of: a) obtaining vector comprising gene for Exendin-4 set forth in SEQ ID No. 1 and transforming host cell with the vector to obtain transformed cell; b) culturing the transformed cell in culture medium and subjecting to fermentation; c) lysing the cell to obtain crude Exendin-4; d) optionally processing the crude Exendin-4 to obtain purified Exendin-4.
2. The process as claimed in claim 1, wherein the gene sequence for the Exendin-4 includes solubility or affinity tag or combination thereof; and the Exendin-4 is expressed as a fusion protein.
3. The process as claimed in claim 1, wherein the host cell is E.coli; and the vector is selected from group comprising pET-32a and pET24.
4. The process as claimed in claim 1, wherein the transforming is carried out by technique selected from group comprising Heat shock method, electroporation and chemical method or any combinations thereof; and the culturing is carried out at temperature ranging from about 25°C to about 40°C, preferably about 37°C, for time duration ranging from about 1 hour to about 20 hours, preferably about 17 hours, at pH ranging from about 6.8 to about 7.4, preferably about 7.2.
5. The process as claimed in claim 1, wherein the fermentation is carried out at temperature ranging from about 35°C to about 40°C, preferably about 37°C, for time
duration ranging from about 11 to 13 hours, at pH ranging from about 6.5 to 7.5.
6. The process as claimed in claim 1, wherein the lysing of cell is carried out by subjecting the cell to high pressure Homogenizer or Microfluidizer or French Press, or any combinations thereof; and wherein said lysing of cell is followed by clarification of the cell lysate. 7. The process as claimed in claim 1, wherein the processing of step d) is carried out by subjecting the crude Exendin-4 to technique selected from group comprising chromatography including affinity chromatography or reverse phase chromatography; filtration including tangential flow filtration, ultrafiltration, diafiltration, sterile filtration; and digestion of protein; or any combinations thereof.
8. The process as claimed in claim 1, wherein the Exendin - 4 obtained by the process has a purity of at least 97%, preferably about 99%; and the Exendin-4 is formulated into dosage form in formulation buffer comprising component selected from group of sodium acetate, acetic acid, D-Mannitol and Meta-cresol or any combinations thereof.
9. A process of purifying Exendin-4, said method comprising acts of: a. lysing cell comprising Exendin-4 protein and subjecting the cell lysate to clarification to obtain clarified sample; b. subjecting the clarified sample to affinity chromatography and tangential flow filtration; c. digestion of protein; and subjecting the digested protein to chromatography and filtration to obtain purified Exendin-4.
10. The process as claimed in claim 9, wherein the lysing of cell is carried out by subjecting the cell to high pressure Homogenizer or French Press or Microfluidizer, or any combinations thereof; and the clarification of the cell lysate is carried out by techniques selected from group comprising centrifugation, tangential flow filtration and microfiltration; or any combinations thereof.
11. The process as claimed in claim 9, wherein the digestion of protein is with enterokinase enzyme; and the chromatography of step c) is selected from group comprising affinity chromatography and reverse phase chromatography, or combination thereof; and the filtration is selected from group comprising ultrafiltration, diafiltration and sterile filtration, or any combinations thereof.
12. The process as claimed in claim 9, wherein the protein is fusion protein; and the filtration of step c) is followed by sterile filtration. 13. Exendin-4 as set forth in SEQ ID No. 1, wherein the Exendin-4 is formulated into dosage form in formulation buffer.
14. Exendin-4 as claimed in claim 13, wherein the Exendin-4 is obtained by the process
as claimed in claim 1; the Exendin-4 has purity of at least 97%, preferably about 99%; and the formulation buffer comprises component selected from group of sodium acetate, acetic acid, D-Mannitol and Meta-cresol or any combinations thereof.