FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents Rules, 2003
PROVISIONAL SPECIFICATION (See section 10 and rule 13)
A PROCESS FOR THE PREPARATION OF PARATHYROID HORMONE (1-34)


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 describes the invention.


FIELD TO THE INVENTION
The present invention relates to the process for obtaining high yield of highly purified recombinant proteins in the form of inclusion body proteins from the host cells.
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, parathyroid hormone is synthesized as a preprohormone. After intracellular processing, the mature hormone is packaged within the Golgi into secretory vesicles, then secreted into blood by exocytosis. Parathyroid hormone is secreted as a linear protein of 84 amino acids.
Indian Patent Application No. 912/DEL/2002 describes about the invention relates to the solubilization and recovery in high yield, of inclusion body proteins from host cells using an appropriate denaturizing agent. The process avoids the use of high concentration of chaotropic agents such as guanidine hydrochloride or urea.
It was also reported by SURINDER MOHAN SINGH and AMULYA KUMAR PANDA in Journal of Bioscience & Bioengineering, vol.99 (4), 2005, 303-310 that the inclusion bodies produced in Escherichia coli are composed of densely packed denatured protein molecules in the form of particles. Refolding of inclusion body proteins into bioactive forms is cumbersome, results in poor recovery and accounts for the major cost in production of recombinant proteins from E. coli. With new information available on the structure and function of

protein aggregates in bacterial inclusion bodies, it has been possible to develop improved solubilization and refolding procedures for higher recovery of bioactive protein. Inclusion bodies are formed from partially folded protein intermediates and are composed of aggregates of mostly single types of polypeptide. This helps to isolate and purify the protein aggregates to homogeneity before solubilization and refolding. Proteins inside inclusion body aggregates have native-like secondary structures. It is assumed that restoration of this native-like secondary structure using mild solubilization conditions will help in improved recovery of bioactive protein in comparison to solubilization using a high concentration of chaotropic agent. Analysis of the dominant forces causing aggregation during inclusion body formation provides information to develop suitable mild solubilization procedures for inclusion body proteins. Refolding from such solubilized protein will be very high due to restoration of native-like secondary structure. Human growth hormone inclusion bodies were purified to homogeneity from E. coli cells before solubilization and refolding. Pure inclusion bodies were solubilized at alkaline pH in the presence of 2M urea solution. The solubilized proteins were refolded using a pulsatile renaturation process and subsequently purified using chromatographic procedures. More than 40% of the Inclusion body proteins could be refolded back to the bioactive native conformation. Mild solubilization is thus the key for high recovery of bioactive protein from inclusion bodies.
In the present invention the process to obtain recombinant human PTH(1-34) from E.coli through batch fermentation process by using animal source free media which increases the yield of recombinant human PTH(1-34) in the form of inclusion bodies and the Urea wash step increases the purity of the inclusion bodies were found.

SUMMARY OF THE INVENTION
It is the objective of the present invention to provide a process to obtain a recombinant human PTH(1-34) or analogues of PTH(1-34) by batch fermentation process using E.coli so as to get high yield of inclusion bodies and henceforth recombinant human PTH (1-34).
It is another objective of the present invention to provide a process to obtain >75% purified recombinant protein in the form of inclusion bodies.
DESCRIPTION OF THE INVENTION
The present invention relates to process to obtain recombinant human PTH (1-34) or analogues of recombinant human PTH (1-34). The present invention was also found to be economically viable and feasible by virtue of reduction in the number of batches to be taken.
In an embodiment of the present invention, batch fermentation process of E.coli with animal source free media was done to get high yield of inclusion bodies.
Parathyroid hormone was produced using batch fermentation and the expression level was around 40% of the cellular protein. E. coli cells were used for inclusion body preparation and subsequent refolding. E. coli cells were lysed by a French press at 22 Thousand Pounds per Square Inch (KPSI) and the inclusion bodies were isolated by centrifugation at approximately 7000rpm. As the inclusion bodies have higher densities, high speed centrifugation helps in separating them from contaminating cellular fragments/proteins. Using an appropriate centrifugation and washing process, more that 80% pure inclusion bodies containing recombinant human PTH(1-34) were isolated from E.coli cells. Such purified inclusion bodies were used for subsequent solubilization and refolding for the recovery of biologically active recombinant protein.

After lysis step the inclusion body is washed with a buffer (phosphate buffer) to remove some unwanted proteins, which is present as insoluble protein along with inclusion bodies. At the particular pH of the buffer several insoluble proteins gets solubilized and thus removed from the inclusion bodies.
The next step is the Urea wash. This step is for the removal of various unnecessary proteins which gets solubilized at lower concentration of urea as urea solubilizes and denatures the proteins by unfolding them through disruption of non-covalent interactions. Thus the solubilized proteins can be removed from insoluble inclusion bodies by centrifugation and helps in obtaining highly purified inclusion bodies which can be solubilized and refolded to obtain a biologically active recombinant human PTH(1-34) for therapeutic use.
Dated 18th day of March 2009 For Intas Biopharmaceuticals Limited

To
The Controller of Patents, The Patent Office, at Mumbai.