FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents Rules, 2003 .
COMPLETE SPECIFICATION
(See Section 10 and Rule 13)
METHOD FOR THE PURIFICATION OF PARATHYROID HORMONE
Intas Biopharmaceuticals Limited
An Indian company having its registered office at:
Plot No.: 423/P/A/GIDC
Sarkhej-Bavla Highway
Moraiya, Tal.: Sanand
Ahmedabad - 382 210
Gujarat, India
The following specification particularly describes the invention and the manner in which it is to be 'performed.

FIELD OF THE INVENTION
The present invention provides a method for the purification of a recombinant teriparatide. BACKGROUND OF THE INVENTION
Parathyroid hormone (PTH) plays an important role in extra-cellular calcium homeostasis and acts on the skeleton to stimulate bone turnover. This hormone is secreted from cells of the parathyroid glands and finds its major target cells in bone and kidney. Like most other protein hormones, PTH is synthesized as a preprohormone. After intra-cellular processing, the mature hormone is packaged within the Golgi into secretory vesicles, and then secreted into blood byexocytosis. Parathyroid hormone is secreted as a linear protein of 84 amino acids.
Recombinant teriparatide or human parathyroid hormone (1-34) (hPTH (1-34)) is the protein with the amino acid sequence 1-34 of the 84 amino acid full-length human parathyroid hormone, expressed by Escherichia coli (E. coli) bacteria. This peptide has a sequence identical to the 34N-terminal amino acids (a biologically active region) of endogenous human PTH. Teriparatide has a molecular weight of 4117.8 Daltons (4.1178 kDa), and the amino acid sequence H-Ser-Val-Ser-Glu-lle-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Phe-Asn-Phe-OH.
Purification of protein is a series of processes intended to isolate a single type of protein from a complex mixture, besides being essential for the characterization of its structure and function. The starting material is usually a biological tissue or a microbial culture expressing PTH (1-34). The various steps in the purification process may free the protein from a matrix that confines it, separate the protein and nonprotein parts of the mixture, and finally separate the desired protein from all other proteins and impurities. Separation of one protein from all others is typically the most laborious aspect of protein purification. Separation steps exploit differences in protein size, physico-chemical properties and binding affinity to variety of chromatographic resins under different conditions.
Because of the importance of recombinant teriparatide in the treatment of osteoporosis, the provision of recombinant teriparatide of high purity and high specific activity is desirable. Recombinant teriparatide requires repeated "injections. Highly purified recombinant teriparatide preparations can be administered subcutaneously, permitting self-administration by the patient and thereby increasing patient convenience and compliance. .

US 5208041 describes essentially pure human parathyroid hormone obtained by the steps of: obtaining a partially purified preparation of E.coli-produced human parathyroid hormone, fractionating the partially purified preparation by reversed-phase high performance liquid chromatography with triethylamine or a salt thereof and collecting, and following the chromatographic step, essentially pure human parathyroid hormone.
US6210925 describes the purification process comprising the following steps: (a) separating host cells from the culture medium; (b) subjecting the medium to cation exchange chromatography and recovering fractions containing said peptide product; (c) subjecting the recovered fraction of step (b) to reverse-phase liquid chromatography and recovering fractions containing peptide product; (d) subjecting the recovered fractions of step (c) to cation exchange chromatography, and (e) thereafter, recovering fractions containing peptide product.
US6103495 describes the purification process comprising the following steps (a) separating host cells from the culture medium; (b) subjecting the medium to reverse-phase liquid chromatography and recovering fractions containing peptide product; (c) subjecting said fractions of step (b) to cation exchange chromatography; and (d) thereafter, recovering fractions containing peptide product.
W02 007112677 describes a method for preparing recombinant human parathyroid hormone 1-34 [rhuPTH(l-34)] comprising the steps of : (1) expressing the expression vector that could express the fusion protein, in which the sequence of the fusion protein is thioredoxin -(His)6- enterokinase recognition site -parathyroid hormone 1-34 peptide from the N-terminal to C-terminal; (2) purifying the fusion protein from the step (1) by Ni-ion chelating affinity chromatography; and (3) digesting the purified fusion protein from the step (2) with enterokinase in order to release the parathyroid hormone 1-34 peptide from the fusion protein.
WO2009019715 describes two-step orthogonal purification process for recombinant hPTH (1-34) comprising cation exchange chromatography, optionally followed by preparative chromatography selected from HIC or RP- HPLC to yield a target protein.
US6962796 describes a process for the production of purified recombinant intact human PTH, comprising the steps of: (a) providing a microorganism that is engineered genetically to produce exogenous and intact hPTH (1-84), wherein said microorganism is selected from the group consisting of Escherichia coli and yeast; (b) expressing said intact hPTH (1-84) within said microorganism which comprises DNA encoding hPTH (1-84): and (c) purifying said intact hPTH (1-84) so as to produce an intact hPTH (1-84) which (1) reacts with antibodies against human PTH in a manner identical to the native rhPTH (1-84) hormone: (2)

has the molecular weight of the native rhPTH (1-84) hormone, and, (3) migrates as a band of approximately 9000 Daltons (9 kDa) when subjected to gel electrophoresis, corresponding to the size of intact rhPTH (1-84).
US5208041 discloses a method for purifying lyophiiized human parathyroid hormone, which comprises the step of fractionating a partially purified human PTH preparation by reverse-phase high performance liquid'chromatography with a cationic ion-pairing agent like tri-ethylamine phosphate.
Hence, there is a need for new methods for purifying recombinant teriparatide that avoid the use of reverse phase chromatography which is costly and uses organic solvent.
SUMMARY OF THE INVENTION
The object of the invention is to-provide a method for purifying a recombinant human parathyroid hormone, preferably recombinant teriparatide.
In a first aspect, the invention provides a method for purifying recombinant teriparatide comprising the following steps:
(1) First Cation exchange chromatography
(2) Enzymatic Digestion of the fusion protein
(3) Second Cation exchange chromatography
(4) Third Cation exchange chromatography
(5) Gel filtration chromatography
The chromatographic steps may be carried out in any order.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 illustrates the present invention, i.e. a process of purification comprising the steps of
(1) First Cation exchange chromatography
(2) Enzymatic Digestion of the fusion protein (3)' Second Cation exchange chromatography

(4) Third Cation exchange chromatography
(5) Gel filtration chromatography
Figure 2 shows flow-chart of a specific embodiment of the present invention, i.e. a process of purification comprising the steps of:

(1) Refolding
(2) Ultrafiltration/ Diafiltration
(3) First Cation exchange chromatographic step
(4) Enzymatic Digestion of the fusion protein
(5) Second Cation exchange chromatographic step
(6) Third Cation exchange chromatographic step
(7) Gel filtration chromatography
Figure 3 shows the comparison of reverse phase high performance liquid chromatogram of Reference Medicinal product (Forteo®) and in-house teriparatide.
DETAILED DESCRIPTION OF THE INVENTION
The invention provides a method for purifying recombinant teriparatide starting from a liquid containing fusion protein of recombinant teriparatide and other impurities comprising the following steps:
(1) First Cation exchange chromatography
(2) Enzymatic Digestion of the fusion protein
(3) Second Cation exchange chromatography
(4) Third Cation exchange chromatography
(5) Gel filtration chromatography
The batch fermentation process for the production of fusion protein of recombinant teriparatide, i.e. 1-34 of the 84 amino acid full-length human parathyroid hormone, is carried out using recombinant E.coli cells in animal source free media. Initially, for inoculum preparation, the cells are cultivated in the shake-flasks (i.e. seed flasks). For production, the inoculum obtained from seed flasks are transferred to 15-25 litre production fermenter and are cultured for 18-22 hours by batch mode fermentation process. During fermentation process, the growing cells are supplemented with air and pH is maintained by controlled addition of acid and base. Cells are induced with Isopropyl-β-D-thio-galactoside (IPTG) at late log phase (12th" 16th hour of batch process) and the fermentation is carried out for another 5-8 hours. The protein teriparatide is produced in intracellular in the form of inclusion bodies. The harvest obtained after the completion of fermentation process is subjected to primary downstream process to isolate the inclusion bodies containing rhuPTH (1 -34) from the cells for further purification.
This inclusion body comprising the fusion protein having teriparatide fused to the chitin binding domain with enterokinase' recognition site in between. Downstream processing begins with solubilization of

inclusion bodies with denaturant, 6 Molar Guanidine Hydrochloride and subsequent, refolding to get biologically active protein. After refolding ultrafiltration and diafiltration is carried out to reduce handling volume and exchange buffer. The obtained mixture comprising the fusion protein is purified further and the purification includes one enzymatic digestion and four chromatographic purification steps in sequence as follows:
(1) First Cation exchange chromatography
(2) Enzymatic Digestion of the fusion protein to cleave chitin binding domain
(3) Second Cation exchange chromatography
(4) Third Cation exchange chromatography
(5) Gel filtration chromatography
Ultrafiltration and Diafiltration
To reduce handling volume and buffer exchange, ultrafiltration and diafiltration is carried out using 10 kDa PES membranes. Buffer exchange is carried out to bring adequate condition for further chromatographic purification steps.
Step 1 - Cation exchange chromatography
For fusion pretein purification, diafiltration output which contains fusion protein and also some host cell proteins are further purified using cation exchange chromatography step I. Fusion protein is captured on cation exchanger resin, and most of the host cell proteins come in flow through. The captured fusion protein is then eluted with high pH.
Step 2 - Enzymatic digestion
The rhuPTH (1-34) comprising chitin binding domain as a tag and a specific site as (AspAspAspAspLys) is digested further by recombinant Enterokinase enzyme to separate teriparatide from the tag.
Step 3 - Cation exchange chromatography.
Digested mixture comprising teriparatide, undigested fusion protein, fusion tag, recombinant enterokinase enzyme and some related impurities formed during digestion is further purified by Cation exchange chromatography Step III. Teriparatide and undigested fusion protein are captured on cation exchange resin and rest impurities come in flow through. Captured undigested fusion protein is removed with high pH wash and the captured teriparatide is eluted with a salt gradient.

Step 4 - Cation exchange chromatography
This step is conducted to concentrate the purified teriparatide protein (as elute of Step III is in diluted form) so as to get desired concentration for Gel filtration chromatography. Diluted teriparatide is capture'' on the cation exchange resin and is eluted with salt in a step gradient.
Step 5 - Gel filtration chromatography
Cation exchange output contains high salt, so in order to remove salt and to get teriparatide in the formulation buffer; buffer exchange is carried out through Gel filtration chromatography.
Example
The present invention will now be illustrated by means of the following example.
Corresponding flow-charts illustrating said examples are presented in Figure 1 and 2. The resulting purified recombinant teriparatide is termed as "teriparatide bulk".
Step 1 - Cation exchange chromatography
The recombinant teriparatide starting material for the purification is prepared from cell harvests comprising fusion protein of recombinant teriparatide, i.e. parathyroid hormone which was produced by recombinant DNA technology .in Escherichia coli cells, in an animal source free medium..The cation exchange chromatography column is first equilibrated with a 20 mM (milli Molar) Sodium acetate buffer at pH 3.5. The liquid comprising the fusion protein recombinant teriparatide is then loaded directly to the resin. After loading, the unbound and bound impurities are washed out using equilibration buffer and wash buffer. The recombinant teriparatide is finally eluted by washing the column with 20 mM Sodium acetate buffer at pH 6.5. The elution pool is processed to the next step. The purity of the eluted fusion protein obtained is more than 85% as determined by RP-HPLC.
Step 2 - Enzymatic digestion
The above purified fusion protein was digested using recombinant Enterokinase enzyme. The matrix of fusion protein was adjusted as per the manufactures instruction to get optimum enzymatic digestion. The said digestion mixture was incubated at room temperature with stirring for about 16 hours. The digestion percentage of fusion protein was analyzed by RP-HPLC.

Ste'p 3 - Cation exchange chromatography
The above said reaction mixture comprising teriparatide, undigested fusion protein, fusion tag and some related impurities was subjected to cation exchange chromatography. The cation exchange resin was equilibrated with 20 mM Sodium "acetate buffer, pH 5.0. The above said reaction mixture was diluted and then loaded onto the resin and was subsequently washed with 20 mM Sodium acetate buffer, pH 6.5. Teriparatide was eluted by using linear gradient of 0-16 % in 24 column volume of elution buffer (20 mM Sodium acetate buffer, 1M NaCl, pH 6.5). The purity of the eluted teriparatide was determined by means of RP-HPLC; it was higher than 98 %.
Step 4 - Cation exchange chromatography
This chromatography step was carried out to concentrate the teriparatide. The cation exchange resin was equilibrated with equilibration buffer 20 mM Sodium acetate buffer, pH 5.0. The output of the above chromatography was diluted and loaded onto the resin and was subsequently washed with equilibration buffer. Teriparatide was eluted by using 60 % step gradient from the resin with elution buffer (20 mM Sodium acetate buffer, 1M NaCl, pH 5.0).
Step 5 - Gel filtration chromatography
This chromatography was carried out for buffer exchange. The Gel filtration resin is equilibrated with equilibration .buffer (Glacial acetic acid 6.8 mM, Sodium acetate 1.2:2 mM, Mannitol 4.54%, pH 4.0). The concentrated teriparatide was loaded onto the column and subsequently flow through was collected. The output was analyzed by means of RP-HPLC and the resulting purify of teriparatide was,more than 98 %.
All the chromatography steps were conducted at room temperature.
Dilution of the output obtained from Step 5
This is to adjust the protein concentration of Gel filtration chromatography output of all batches to an equivalent concentration as decided in specification.
Filtration of bulk & Storage of Drug Substance at -20 °C ± 2 °C
As the size of bacteria is normally more than 0.2μ, the filtration of bulk through Vaccap Supor Membrane
(PES) (0.2 μ) is done in order to reduce bio-burden and stored at -20 °C ± 2 °C if Drug Product has to be
prepared at a later stage. '

We claim
1. A process for the purification of parathyroid hormone that comprises subjecting a liquid
containing parathyroid hormone to
(1) First Cation exchange chromatography
(2) Enzymatic Digestion of the protein fused with the purification tag
(3) Second Cation exchange chromatography
(4) Third Cation exchange chromatography (5) Gel filtration chromatography
which can be carried out in any order.
2. A process as claimed in claim 1, wherein SP Sepharose is used for first and third cation exchange chromatographic step.
3. A process as claimed in claim 1, wherein the enzymatic digestion is carried out with recombinant enterokinase enzyme.

4. A process as claimed in 1, wherein the Source I5S is used for second cation exchange ' chromatographic step.
5. A process as claimed in 1, wherein the Sephadex - G10 is used for gel filtration chromatographic
step.
6. The method of any one of claims 1 to 5, wherein the chromatographic steps are carried out using sodium acetate with sodium chloride as a buffer or as an eluent.
7. The method of any one of claims 1 to 6, wherein the steps are carried out in the following order

(1) First Cation exchange chromatography
(2) Enzymatic Digestion of the fusion protein
(3) Second Cation exchange chromatography
(4) Third Cation exchange chromatography
(5) Gel filtration chromatography
8. A method for purifying recombinant teriparatide comprising the steps of subjecting parathyroid
hormone to
a. Ultrafiltration

b. Cation exchange chromatography on SP Sepharose FF with pH. based elution using
20 mM Sodium acetate buffer with different pH-. Equilibration at pH 3.5, pH 5.0
wash and elution at, pH 6.5
c. Subjecting the eluate of Step 1 to a step, of enzymatic digestion with recombinant
enterokinase enzyme
d. Subjecting the eluate of Step 2 to a step of cation exchange chromatography on
Source 15S with salt based elution using 20 mM Sodium acetate buffer with different
pH. pH 5.0 (equilibration buffer), pH 6.5 (wash buffer) and pH 6.5 with 1 M NaCl as
eluent.
e. Subjecting the eluate of Step 3 to a step of one more cation exchange
chromatography on SP Sepharose FF with 20 mM Sodium acetate buffer at a pH 5.0
and 20 mm Sodium.aceate buffer pH 5.0 with 1 M NaCl as eluent.
f. Subjecting the eluate of Step 4 to a step of Gel filtration chromatography on
Sephadex G 10.
9. A recombinant human teriparatide is obtainable by the process according to the purification process of any preceding claims.
10. A pharmaceutical composition comprising recombinant human teriparatide according to any preceding claims as well as pharmaceutically acceptable excipient.