Purification process for pth

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The present invention relates to improved method for purification of a recombinant parathyroid hormone (rhPTH1-34 or teriparatide), said process for purification of parathyroid hormone comprising following essential steps: (a) Enzymatic cleavage; (b) anion exchange chromatography, followed by other suitable purification steps; wherein step (a) and (b) can be carried out in any order.

The present invention provides improved method for purification of a recombinant parathyroid hormone (rhPTH1™34 or teriparatide). The process of purification of PTH according to the present invention comprises use of an anion exchange chromatography in the first ste i prior to use of any cation exchange chromatography. Such process of purification results in highly purified rhPTH1-34, with more than 99% purity, without employing any HPLC column step in the process of purification.

BACKGROUND OF THE INVENTION Recombinant human parathyroid hormone (rhPTH1 -34) or teriparatide is a biologically active N-terminal fragment of endogenous human parathyroid hormone (PTH). Therapeutically, teriparatide is used for the treatment of men and postmenopausal women with osteoporosis who are at high risk of fracture. It increases bone mineral density and reduces the risk of vertebral and noil-vertebral fractures.

The inventors of the present invention have indigenously developed teriparatide by recombinant DNA technology using genetically engineered E. coli cells as host system. Teriparatide comprising 34 natural amino acids has a theoretical molecular weight of 41 17.8 Da. Teriparatide is a cysteine-free polypeptide chain. In human body, PTH is mainly synthesized and secreted by the chief cells of the parathyroid glands, as a 84 amino acids (9.5 kDa) containing single polypeptide chain. Upon release in to the blood stream, PTH binds to the specific membrane receptor mainly present in bone and kidney to maintain serum Ca2+ level. The hormone-receptor interaction leads to activation of both the cAMP-dependent protein kinase A and the calcium-dependent protein kinase C signaling pathways with a typical cascade system. In circulation, the endogenous native PTH has a half-life of 2 to 5 min and more than 90% of its clearance is mediated by liver and kidney.

It has been observed through several biochemical and structural studies that the N- terminal 1 - 34 amino acids fragment of PTH produced recombinantly or synthetically remains fully active in receptor binding and its activation. For optimal receptor binding activity, the N-terminal portion, 1 - 27 amino acids of PTH1-34 polypeptide chain are found to be essential for biological activity. The N- terminal portion of PTH1-34 causes stimulation of cAMP upon binding to its receptor, whereas the C-terminal portion of PTH1"34 helps in providing most of the binding energy without leading to cAMP activation.

PTH plays an important role in Ca2+ homeostasis. Release of PTH is triggered from parathyroid cells via a plasma membrane bound calcium sensor, when concentration of Ca2+ is low in circulating blood (hypocalcaemia). If the hypocalcaemia is sustained, then hypertrophy and hyperplasia of the parathyroid gland occur. On the other hand, an increased concentration of Ca in plasma inhibits the release of PTH by a negative feed-back mechanism.

The present invention is related to purification of recombinant PTH. There are several purification processes known in prior art. Such purification processes include use of high performance liquid chromatography (HPLC) which is expensive and requires a large amount of organic solvent during operation. The high cost of the instrument, requirement of flame-proof manufacturing plant and requirement of large amount of costly good quality organic solvents used as mobile phase are the major limitations in the case of purification of PTH by HPLC at industry scale. WO2009019715 discloses two steps orthogonal purification process for rhPTH (1-34) comprising of cation exchange chromatography optionally followed by preparative chromatography selected from HIC or RP- HPLC to yield a target protein of >98% purity. WO2003102132 relates to a method for protein purification that involves the combination of non-affinity chromatography with HPTFF.

An Indian application 2991 /MUM/2010 discloses purification process of PTH comprising cation exchange chromatography and gel filtration chromatography. The process described in the present invention for purification of PTH does not include any column chromatography wherein organic solvents are used as mobile phase or any HPLC column chromatography during purification process of the said polypeptide molecule. Thus, the present invention discloses a simple, cost- effective, highly scalable, industrially viable and environmentally favorable process of purification to obtain highly purified rhPTH1""34. The process of purification disclosed in the present invention can be used for purifying PTH from a crude mixture containing rhPTH1-34 generated by any process.