NOVEL PROCESS FOR THE EXPRESSION AND PURIFICATION OF PARATHYROID HORMONE (1-34) WITH CHITIN BINDING DOMAIN (CBD) AS FUSION PARTNER

Field of invention:

The present invention relates to a process for the production and purification of recombinant human parathyroid hormone, rhu (PTH 1-34).To accomplish this, a DNA fragment coding for parathyroid hormone (1-34) is fused to a DNA fragment coding for amino acid sequence recognized by a proteolytic enzyme and a DNA sequence coding for protein chitin binding domain(CBD);inserted into a cloning vector and an appropriate host is transformed; upon expression, a fused protein (CB0-(ASP)4-Lys-PTH) is produced; the fusion proteiKn is purified through specific affinity chromatography and the purified fusion protein is separated into PTH (1-34) and chitin binding domain employing recombinant bovine enterokinase; PTH (1-34) thus obtained is purified employing ion-exchange chromatography techniques.

BACKGROUND OF THE INVENTION

It is recognized that osteoporosis is a major public heafth problem associated with substantial morbidity and socio¬economic burden. As a result, one in two women and one in five men over the age of 50years sustain fractures. A recent report stated that 29% prevalence of osteoporosis is in 30-60 year age group Indian women. Osteoporosis is a condition characterized by a decrease in bone density, decreasing its strength and resulting in fragile bones which lead to abnormally porous bone. Recombinant human parathyroid hormone (PTH) (1-34] is a new class of anabolic therapies for management of severe osteoporosis both in men and women.

Recently developed techniques have made it possible to employ microorganisms capable of rapid and abundant growth for the synthesis of commercially useful proteins and peptides. These techniques make it possible to genetically endow a suitable microorganism with the ability to synthesize a protein or peptide normally made by another organism. In brief. DNA fragments coding for the protein are inserted into a cloning vector such as a plasmid. An appropriate host is transformed with the cloning vector and is cultivated to promote expression of the desired protein. Proteins so produced are then isolated from the culture medium for purification,

Many purification techniques have been employed to harvest proteins produced by recombinant DNA techniques. Such techniques generally include segregation of the desired protein based on its distinguishing molecular properties, e.g. by dialysis, density-gradient centrifugation and chromatography. Such techniques are not universally applicable and often result in consumption of the purification materials which may have considerably more value than the protein being purified particularly, where substantial quantities of highly purified protein are desired.

Other procedures have been developed to purify proteins based on solubility characteristics of the protein. For example, isoelectric precipitation has been employed to purify proteins since the solubility of proteins varies as a function of pH.

Similarly, solvent fractionation of proteins is a technique whereby the solubility of a protein varies as a function of the dielectric constant of the medium. Solvent fractionation, while giving good yields often causes denaturation of the protein molecule. Neither isoelectric precipitation nor solvent fractionation is useful in obtaining highly purified protein. Such techniques are typically employed in tandem with other procedures.

Affinity chromatography has also been employed in the purification of proteins. Affinity chromatography for protein purification involves a selective adsorption of the proteins to be purified onto substrate. The non-binding species are removed by washing and the desired protein recovered by eluting with a specific desorbing agent Identifying such a substrate having specificity to a particular protein for adsorption, desorption and also do not cause any damage to the protein of interest during the process is of great interest and needs a lot of research which will be valuable for further usage universally. Thus, there is a continuing need for techniques which enable large scale purification of proteins produced through recombinant DNA processes without the above described problems. It would be particularly advantageous to provide an affinity purification process which utilizes an abundant and inexpensive substrate to which the fused protein would bind and can be eluted with an equally abundant and inexpensive eluent.

Therefore it is an object of the present invention to provide processes for the production and purification of recombinant human parathyroid hormone, rhu PTH (1-34).

Chrtin binding domain (CBD) of a fusion protein binds to chitin. This property is exploited for isolating the fusion protein containing CBD from a mixture of host cell proteins. In the present case this method is employed for isolation of CBD-PTH (1-34).Fusion protein CBD-PTH(1-34) thus obtained is cleaved using recombinant bovine enterokinase yielding crude PTH (1-34).This is further separated and purified by cation exchange chromatography from other host cell proteins.

Alternatively, CBD-PTH (1-34) fusion protein is isolated from a mixture of host cell proteins employing CaptoMMC, a cation exchange resin.

Use of either chitin or CaptoMMC for the capture of fusion protein CBD-PTH (1-34) enables the elution with inorganic salt free eluent which facilitates further purification avoiding a step of salt removal in further process.

Further object of the present invention is to provide a cost effective, simple and industrially viable method for the production and purification of PTH (1-34).

Summary

The first aspect of the present invention is a process for the production and purification of the fusion protein comprising of:

1. Production of a fusion protein CBD-PTH (1-34),by fermentation employing recombinant Ecoli having the plasmid for expression of fusion protein wherein a DNA fragment coding for parathyroid hormone (1-34) is fused to a DNA fragment coding for amino acid sequence recognized by a proteolytic enzyme and a DNA sequence coding for protein chitin binding domain(CBD). The fused DNA is inserted into a cloning vector and an appropriate host is transformed.

2. Homogenization involving lysis of the bacteria at an appropriate pressure.

3. Clarifying the lysate obtained in step (2) to reduce the concentration of host cell proteins of Ecoli generated during homogenization.

4 Capturing the fusion protein CBD-PTH (1-34) obtained in step (3) using chitin as an affinity matrix or CaptoMMC resin.

5. Cleaving fusion protein using endopeptidase (recombinant bovine enterokinase) to yield crude PTH (1-34).

6. Purifying the PTH (1-34) obtained employing Ion-Exchange Chromatography.

Another aspect of the present invention is to provide an appropriate resin for capturing fusion protein containing chitin binding domain from the lysate, wherein one such substrate is naturally occurring Chitin and the other is a cation exchange resin namely CaptoMMC.

Another aspect of the invention is a process of purification of PTH (1-34) comprising of:

a) Capturing fusion protein onto affinity matrix or cation exchanger resin

b) Digesting the fusion protein of step (a) using endopeptidase to yield crude PTH(1-34)

c) Further purification of crude PTH (1-34) employing cation exchange chromatography to a purity of £ 98%.

Another embodiment of the present invention is to provide a process for producing and purifying polypeptide or fused protein molecule employing recombinant DNA technique. More specifically, a DNA fragment coding for a protein molecule, e.g. a polypeptide, parathyroid hormone (1-34) is fused to a DNA fragment coding for a binding protein such as the DNA coding for the chitin binding domain. The fused DNA is inserted into a cloning vector and an appropriate host is transformed. Upon expression, a fused protein is produced which can be purified by binding it to the substrate for which it has specific affinity i.e. by affinity chromatography. The fusion protein so purified may be cleaved to obtain the protein of interest. This can be done by inserting a DNA sequence coding for an amino acid that can be recognized by a particular proteolytic enzyme. The present invention also relates to certain methods for the purification of the fusion protein containing the CBD as a tag and a protease recognition site in between chitin binding domain and parathyroid hormone (1-34).

Further aspect of the present invention is to provide a method for the capture of fusion protein containing chitin binding domain as fusion partner to PTH (1-34) by affinity chromatography using cheap and naturally occurring substrate having specificity for chitin binding domain obtained from Bacillus circulans. Otherwise fusion protein having chitin binding domain from Bacillus circulans as fusion partner can also be captured by binding it on to a cation exchange resin CaptoMMC Elution of the captured fusion protein from the above said two resins allows elution in eluent with low inorganic salt concentrations thus avoiding the salt removal step in further purification of material.

Another aspect of the present invention is to provide the process for cleaving the PTH (1-34) from its fusion partner chitin binding domain using proteolytic enzyme in it's lowest possible concentration. Thus making the process more economical.

Detailed description of the invention

The invention therefore provides a process for producing fusion protein, purifying the fusion protein, cleavage of the fusion protein to give chitin binding domain and PTH(1-34) and purification to yield high purity PTH (1-34). A DNA fragment coding for a protein, e.g. a polypeptide parathyroid hormone (1-34) is fused to a DNA fragment coding for a binding protein such as chitin binding domain. The fused DNA is inserted into a cJoning vector and an appropriate host is transformed, Upon expression, a fusion protein is produced which can be purified by affinity chromatography or ion exchange chromatography

The present invention also relates to certain methods for the purification of the fusion protein containing the CBD as a fusion partner and a protease recognition site fused to parathyroid hormone (1-34).

An important feature of the invention is to use a naturally occurring polymer chitin as affinity substrate to capture the fusion protein. The alternative method of capturing the chitin binding domain to a cation exchange resin Capto MMC which offers high recoveries Both the methods offer a common advantage of facilitating the elution with eluent containing least salt concentration thus removing the salt removal step further. The present invention also highlights the process of separating the PTH (1-34) from it's fusion partner chitin binding domain using recombinant bovine enterokinase (a endopeptidase) in its lowest concentration. All together, the process becomes economically viable through less input costs, less number of steps in downstream process with high recoveries.

A process for the production and purification of the fusion protein comprises of:

I. Producing a protein by fermentation of recombinant E.coli having the plasmid for expression of fusion protein wherein a DNA fragment coding for a protein or polypeptide is fused to a DNA fragment coding for a fusion partner such as chitin binding domain.

II. Lysis of the bacteria at an appropriate pressure.

III. Clarifying the lysate obtained in step (ii) to reduce the concentration of host cell proteins

IV. Capturing the fusion protein obtained in step (iii) using affinity substrate namely, naturally occurring chitin or cation exchange resin

V. Separating PTH (1-34) from its fusion partner (chitin binding domain) using recombinant bovine enterokinase (an endopeptidase).

VI. Purifying the PTH (1-34) thus obtained utilizing ion-exchange chromatography.

Feeds used during the fermentation of the recombinant E.Coli are of two types; Feed 1 and Feed II.

Feed I is growth media with a glucose concentration ranging from 3-8%.

Feed II is a growth media with a glucose concentration ranging from 7-14%

Initially feed I is used followed by Feed II.

The cell pellet obtained in fermentation broth was suspended in citrate buffer, pH 6.0 to 8.0 and the homogenate is passed twice through high pressure homogenizer set at 1000bar.

The lysate containing CBD-PTH(1-34) obtained by passing the E.coli culture through high pressure homogenizer at a pressure of 1000bar was clarified by acid precipitation using an acid such as acetic acid or hydrochloric acid preferably 25% aqueous acetic acid. Most of the host cell proteins are separated from fusion protein CBD-PTH (1-34) by centrifugation. The clarified supernatant with a pH between 5.0 and 9.0 is loaded on to the column packed with chitin.

Alternatively, pH of the clarified supernatant is adjusted between 5.0 and 7.0 with glacial acetic acid and is loaded onto column packed with cation exchange resin, Capto MMC equilibrated at pH 5.0-7.0

The substrate used for capturing fusion protein from the lysate is a natural polymer such as chitin or a cation exchange resin, CaptoMMC and the like. More particularly, the resin used for performing affinity chromatography for a fusion protein with chitin binding domain as tag obtained from Bacillus ciroulans is chitin.

The chitin substrate to which the fusion protein is bound is washed with the buffer whose pH is in between 5.0 and 9.0 and at a concentration of 20 -100mM for two column volumes and later the buffer whose pH is between 2.0 and 5.0 is passed onto the chitin column until all the protein is eluted to get a final purity of £ 60%.

Alternatively, the fusion protein bound to CaptoMMC resin is eluted with Tris-HCI whose pH between 8.0 -10.0.

The soluble fusion protein is digested with recombinant bovine enterokinase whose concentration ranges from 0.05 IU to 2 lU/mg of fusion protein and magnesium sulphate concentration ranging from 1mM to 10mM is added and incubated for a period of 15-20hrs, the digested target protein is purified to a purity of £ 90% using cation exchange resin Tosho 650S and optionally to a purity of s 98% using cation exchanger SP-FF (SP Sepharose Fast Flow)

The process for the production and purification of the PTH (1-34) as claimed in claim 1, wherein the lysate is centrifuged at 10800g for 30 minutes and the supernatant thus obtained is collected and the pellet containing the cefl debris is discarded. The supernatant is collected, subjected to acid precipitation under cold conditions by addition of 25% acetic acid slowly to bring the pH to 4.0-6.0. The homogenate is centrifuged at 10800g for 30 min. to sediment precipitated proteins. Supernatant is collected and the pH is again adjusted to 6.0-8.0 by slow addition of 4N NaOH and the resultant homogenate is again centrifuged at 10800g for 30minutes to separate the precipitated host cell proteins. The clarified supernatant thus obtained is loaded on to chitin column between pH 5.0 and 9.0 and eluted with citrate buffer pH between 2.0- 5.0 to yield a fusion protein of purity ≥ 60%

The pH of elute from the above step is adjusted to 7.0-9.0 and Enterokinase enzyme is added to it at the concentration of 0 05 to 2.0IU per mg of the fusion protein. Magnesium sulphate is added to the reaction mixture at the concentration of 1 0-50mM to enhance the activity of the enzyme and stirred for 15 to 20hrs at 22-25 ° C. The pH of the digestion mixture is adjusted to pH 3.0-6,0 and is bound to cation exchanger resin and the target protein is eluted using sodium chloride gradient whose concentration ranges from 02 to1.0M.The eluted protein is desalted using tangential flow filtration or direct dilution with water for injection to a conductivity of 1 -3ms/cm2 .

The pH of the desalted elute containing PTH (1*34) is adjusted to 3.0^6.0 with sodium hydroxide and is bound to another cation exchanger resin. The bound protein is again eluted with a salt (NaCI) gradient ranging from 0.2-1.0M.The resultant protein is £98% pure, desalted and is stored at -20°C.
PTH is a single chain 84-amino acid peptide in which the structural requirements for biological activity are satisfied by the first 34 NH2-teminal amino acids. Deletion of the few amino acids from either the amino or carboxy terminal of the active fragment PTH (1-34) results in the progressive decline tn the biological activity, Hence there is a necessity to develop a process to obtain rhPTH (1-34) to maximum purity (≥98%) for full activity. Often the use of aqueous solvents is recommended for the purification of peptide by RP-HPLC so as to avoid the oxidation of peptide when exposed to air. But the cost involved in the purification by RP-HPLC and the removal of traces of organic solvents is huge and the generation of impurities during the removal of the solvents is also a problem. Hence the cost effective process is developed eliminating the use of organic solvents.

Examples:
Example 1: Preparation of seed culture:
Escherichia coli strain BL21 transformed to express rhPTH (1-34) is maintained as glycerol stock. An aliquot of the glycerol stock was inoculated in 100ml of growth media (Terrific Broth with kanamycin, 25ug/ml) and incubated at 37°C at 180 rpm for 6-8hrs. The culture (80ml each), is inoculated into 4 flasks containing growth media (Yeast Extract-1%, Tryptone-1%, and kanamycin-25ug/ml and glocose-2%-5% and incubated in rotary shaker incubator at 37°C for 3-5hrs at 1 BOrpm.

Example 2: Fed batch fermentation
The above mentioned flask culture was used to inoculate 14 liter fermentor containing 4500ml of basal medium, wherein basal media composition per liter of fermentation media vide Table-1. Fermentation is carried out at 37°C and pH is maintained at 7.0 by the addition of 12.5% of ammonia solution or 25% hydrochloric acid whenever necessary. Stirring is set at 650-750 rpm and air supply set at 1.0LPM is maintained throughout the fermentation. Addition of feed medium -I (feed-l media composition per liter given Table-2) is started when the pH of the fermentation broth rises from 7.0 to 7,2 and continued for a total of 24hrs starting from inoculation. The culture was induced with 1.0-5.0mM of fPTG and continued the fermentation for 12hrs. Addition of Feed media-ll (feed-ll media composition per liter given in Table-3) to the fermentor is started after the entire feed -I is consumed. Samples were taken before induction and every 2hrs after induction for checking the expression of the fusion protein (Fig. 1).

Table 1:

Table 2:

Table 3:

Trace element solution composition / liter Table 4:

Example 3: Cell Lysis and clarification:
The harvested fermented broth is centrifuged at 10800 rpm for 20minutes to remove the media from cells. The pellet thus obtained is resuspended in lysis buffer (Tris-20mM and EDTA 5mM. pH-8.0) and passed through high pressure homogenizer set at 800-1200 bar pressure. The solution containing lysed cells is centrifuged at 10800g for 30min and the resultant pellet is discarded. The supernatant is adjusted to pH 4.0-6.0 with 25% aqueous acetic acid and the solution is centrifuged at 10800g for 30minutes.Pellet is discarded and the supernatant is adjusted to pH 8.0-10.0 with sodium hydroxide. The solution is again centrifuged at 10800g for 30minutes to separate the precipitate. The resultant supernatant contains soluble fusion protein.

Example 4: Capturing of fusion protein:
The clarified supernatant containing fusion protein is loaded to a column of chitin which was pre-equiiibrated with 20-100mM citrate buffer of pH 5.0-9.0. The fusion protein is eluted with 20-1 OOmM citrate buffer or pH 2.0-5.0 to get a protein which is £60% pure. Fig. 2 exhibits the chromatogram of CBD-PTH fusion protein elution from chitin column. Alternatively, the pH of the clarified supernatant containing fusion protein containing fusion is adjusted to 5.0-7.0 with 25% aqueous acetic acid and loaded to column with cation exchanger resin, namely Capto MMC. The bound protein is eluted from the resin using Tris-HCI buffer at pH 8.0-10.0 to get a purity of £60%. Fig.3 exhibits the chromatogram of CBD-PTH fusion protein elution from CaptoMMC column.

Example 5: Digestion of fusion protein with Enterckinase
The pH of elute from the above step is adjusted to 7.0-9.0 and enterokinase enzyme, 0.05-2IU/mg of protein is added to it with stirring. Aqueous magnesium sulphate (1-50mM) is added as co-factor to the reaction mixture to enhance the efficiency of enzyme. The reaction is continued for 15-20 hrs at 23-27°C to complete the cleavage yielding crude PTH (1-34). Figure 4: exhibits the chromatogram of cleavage of fusion protein separating PTH (1-34) and CBD.

Example 6: Cation Exchange chromatography -1
The pH of the solution containing PTH (1-34) and CBD is adjusted to 3.0-6.0 with 25% aqueous acetic acid. The resultant protein after cleavage is passes through cationic exchanger resin, namely Toyopeart SP-650S pre-equiiibrated with 20-40mM citrate buffer at pH 3.0-6.0 and is eluted with the same buffer with salt gradient concentration of 0.5-1.0M sodium chloride to get a purity of (PTH 1 -34) to 90-95%. Fig.5 exhibits the chromatogram of purified PTH (1-34) eluted from Toyopearl SP-650S resin having purity ≥94%.

Example 7: Cation Exchange chromatography - II
The above elute is either desalted using 1kd pore si2e PVDF membrane by tangential flow or directly diluted to get a conductivity of <10ms/cm2,pH is adjusted between 3.0-6.0 and is loaded to cationic exchanger resin (SP Sepharose Fast Flow) column pre-equiiibrated with 10-50mM citrate buffer (pH 3.0-6.0) and eluted with the same buffer with salt gradient of concentration 0.5-1.OM sodium chloride to get a purity of target protein (PTH 1-34) to £98-% as analyzed by RP-HPLC method. Fig. 6 exhibits the chromatogram of PTH (1-34) eluted from SP Sepharose FF having purity ≥98%.

Example 8: Final Filtration
The eluted protein is desalted by diafiltration using tangential flow filtration equipment, concentrated, sterile filtered and is stored at -20°C.

PTH (1-34) purified by the above methods was analyzed by the following methods

1. Determination of the sequence of first five amino acids from N-terminal which confirmed the sequence as Ser Val Ser GU Iso.

2. Bioassay using cAMP assay with UMR106 cell line proved our PTH (1-34) to be bioactive.
Fig. 7: SDS-PAGE showing single band of recombinant human PTH (1-34). The separated recombinant human PTH (1-34) of present invention compared with commercially available recombinant human PTH (1-34) along with various molecular weight proteins. It is found that the Rf value of Forteo and recombinant human PTH (1-34) of the present invention is similar in nature. There are no other bands observed in the sample of the present invention.

Brief description of the accompanying drawings:

Figure 1: SDS -PAGE exhibits the expression of CBD-PTH after induction of E.Coli culture

Figure 2: Exhibits the chromatogram of CBD-PTH fusion protein elutton from chitin column

Figure 3: Exhibits the chromatogram of CBD-PTH fusion protein elution from CaptoMMC column

Figure 4: Exhibits the chromatogram of cleavage of fusion protein separating PTH (1-34) and CBD

Figure 5; Exhibits the chromatogram of purified PTH (1-34) eluted from Toyopearl SP-650S resin having purity ≥94%

Figure 6: Exhibits the chromatogram of PTH (1-34) eluted from SP Sepharose FF having purity ≥98%

Figure 7; SDS-PAGE exhibiting single band of recombinant human PTH (1-34)

claim:

1. A process for the production and purification of PTH (1-34) comprising of:

a. Production of fusion protein CBD-PTH {1-34) by fermentation employing recombinant E.coli having the plasmid for expression of the fusion protein wherein a DNA fragment coding for a polypeptide PTH (1-34) is fused to a DNA fragment coding for a binding protein such as the gene coding for the chitin
binding domain (CBD) and the fused DNA is inserted into a cloning vector and an appropriate host is
transformed.

b. Lysis of the bacteria obtained in step (a) at an appropriate pressure.

c. Clarifying the lysate obtained in step (b) to reduce the concentration of host cell proteins of E.coli.

d. Separation of fusion protein obtained in step (c) using cation exchange chromatography or naturally occurring polymer, namely, chitin which has affinity for chitin binding domain obtained by the expression of gene obtained from Bacillus circulans.

e. Cleaving the fusion protein using a proteolytic enzyme to yield crude PTH(1-34) f. Purification of the crude PTH (1-34) thus obtained utilizing ion-exchange chromatography to a purity of ≥98%.

2. The process for the production and purification of PTH(1-34) as in claim 1a, wherein the feeds used for recombinant E.coli fermentation expressing fusion protein CBD-PTH are of two types, Feed-I and Feed-ll. The concentration of glucose in the feed -1 is 3-8% and the concentration of glucose in the feed - II is 7-14%.

3. The process for the production and purification of PTH (1-34) as in claim 1a, wherein the DNA sequence coding for the expression of chitin binding domain used as fusion partner to PTH (1-34) is obtained from Bacillus circulans.

4. The process for the production and purification of the PTH(1-34) as in claim 1c, wherein the lysate was clarified by acid precipitation using acid such as acetic acid or hydrochloric acid preferably 25% aqueous acetic acid.

5. The process for the production and purification of the PTH (1-34) as in claim 1d, wherein affinity substrate used for capturing fusion protein from the clarified supernatant is a natural polymer.

6. The process for the production and purification of the fusion protein as in claim 1d, wherein natural polymer is chitin.

7. The process for the production and purification of the PTH (1-34) as in claim 1d, wherein the clarified supernatant is adjusted to pH to 5.0 - 9.0 and is loaded to chitin substrate column at pH 5.0-9.0 to bind the fusion protein.

8. The process for the production and purification of the PTH{1-34) as in claim 1d, wherein to elute the fusion protein bound to chitin. the buffer used is 20-100mM citrate buffer (20-100mM citric acid and 10-50mM sodium phosphate) pH between 2.0 and 5.0,preferably 20-100mM citrate buffer to get a final purity of ≥ 60%

9. The process for the production and purification of PTH (1-34) as in claim 1d, wherein the resin used for performing cation exchange chromatography to capture fusion protein from clarified supernatant is CaptoMMC.

10. The process for the production and purification of PTH (1-34) as in claim 1d, wherein the pH of the clarified supernatant is adjusted from 5.0 -7.0 with aqueous acetic acid and is loaded onto Capto MMC column at pH 5.0-7.0

11. The process for the production and purification of PTH(1-34) as in claim 1d, wherein to elute the fusion protein from CaptoMMC .the buffer used is10-40mM Tris-HCI buffer at pH 8.0 -10 to get a purity of ≥60%

12. The process for the production and purification of PTH(1-34) as in claim 1e, wherein the proteolytic enzyme used to cleave and separate PTH(1-34) from chitin binding domain is endopeptidase, more particularly recombinant bovine Enterokinase

13. The process for the production and purification of PTH (1-34) as in claim 1e. wherein soluble fusion protein is digested with recombinant bovine enterokinase whose concentration ranges from 0.05IU to 2IU per milligram of fusion protein.

14. The process for the production and purification of PTH (1-34) as in claim 1e, wherein aqueous magnesium sulphate is added to fusion protein as a co-factor with concentrations ranging from 1mM to 50mM and incubated for a period of 15 -20hrs with stirring at 23-27°C.

15. The process for the production and purification of PTH (1-34) as in claim 1f, wherein cationic exchange resin used to separate and purify PTH (1-34) is SP Sepharose or Toyopearl SP650S, preferably Toyopeari SP650S.

16. The process for the production, separation and further purification of PTH(1-34) as in claim 1f,where in the pH of the solution containing PTH(1-34) and chitin binding domain is adjusted to 3.0 -6.0 with aqueous acetic acid and is loaded to cation exchange column, namely, Toyopearl SP650S.

17. The process for the production separation and final purification of PTH (1-34) as in claim If, wherein the pH of the solution containing PTH(1-34) to be loaded onto cation exchange column, namely, SP(FF) is adjusted to 3.0-6.0 with 4NNaOH.

18. The process for the production and purification of PTH (1-34) as in claim 1f, wherein the process to purify rhPTH (1-34) to a purity of £98% having the required biological activity.

19. The process for the production and purification of PTH (1-34) substantially such as herein described and exemplified.