FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)
PROCESS FOR THE PRODUCTION OF HUMAN GROWTH 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 relates to a fed-batch fermentation process for high level production of recombinant human growth hormone (rhGH) and its analogs in the form of inclusion bodies. The high level production of inclusion bodies can be achieved by the specific type of fed batch strategy which includes maintaining the particular DO level during an induction phase and the growth phase and the production of rhGH is induced by raising the temperature during fermentation.
BACKGROUND OF THE INVENTION
Human growth hormone (hGH) is an anterior pituitary hormone, a protein consisting of 191 amino acids (21.5 kilo Dalton) and two intermolecular disulfide linkages. Because of a wide variety of biological activities, the hGH has been extensively used for the treatment of dwarfism, bone fractures, skin burns, bleeding ulcers, enhancing the physiological functions of middle-aged or elderly persons as a youth hormone and AIDS. Since non glycosylated hGH is a biologically active protein, prokaryotic expression systems are preferred in the production of rhGH. Although heat-induced production of hGH in fed-batch cultures at high cell concentrations of recombinant E. coli producing hGH constitutively or under chemical induction in complex and/or defined media has been studied, getting high yield of rhGH in the form inclusion body by maintaining the DO level at a particular range during the growth phase and the induction phase has not yet been investigated.
The rhGH synthesized in an E. coli expression system is called somatropin. Many brands of somatropin are commercially available namely Humatrope® from Eli Lilly, Nutropin AQ® from Genentech, Norditropin Nordiflex® from Novo Nordisk etc., and two biosimilars are also approved in U.S. and in Europe - Valtropin® and Omnitrope®.
U.S. Patent no. 6410270 discloses about an invention related to a fed-batch fermentation process which uses special E. coli host/vector systems for the purpose of efficiently forming recombinant proteins, in particular recombinant antibody molecules, preferably antibody fragments such as mini antibodies. Under the given conditions, the E. coli cells are able to grow at a maximum specific growth rate up to very high cell densities. After the recombinant product formation has been switched on, it is only the formed product which restricts growth; there is no growth restriction due to limitation of substrates or accumulation of any metabolic by-products in the said invention. High space-time yields of recombinant proteins can be achieved in this manner.

U.S. Patent no. 7189811 discloses about an invention related to a process for solubilization and recovery of bioactive proteins from host cells, the process comprising:- (a) providing a source of a host cell incorporating an insoluble synthesized or expressed protein in the form of an inclusion body, (b) isolating and treating the inclusion body with a denaturating solution consisting of a mercapto-organic solvent at a concentration of 6-8 M and urea at a concentration of 1-2 M to solubilize the inclusion body and obtain solubilized protein, and (c) refolding the solubilized protein by treatment with a refolding buffer to obtain a protein in its bioactive form.
Yee. L,, and Blanch. H. W., (Nat. Biotechnol. 1992, vol. 10, pp no. 1550 - 1556) reported that cell concentrations 5-10 grams DC W"' (Dry Cell Weight) are typical of batch cultures, fed batch techniques can be used to achieve concentrations greater that 50 grams DCW'1. Feeding strategies for fed-batch cultures include feed-back control as well as determined feeding profiles. The volumetric yield of recombinant products can be improved by controlling specific growth rate and the substrate concentration. Furthermore, inhibitory by product formation can be minimized in fed-batch cultures. This review focuses on the use of fed-batch techniques to produce recombinant products in E coli. The modes of nutrient feeding that have been employed (constant and exponential feeding), and the factors important in attaining high ceil concentrations as well as high specific yields of recombinant product are described.
Tabandeh. F., Yakhchali B., et al (Iranian J Sci & Tech Trans A. 2004, vol. 28, 11 - 17) studied about the batch fermentation production of human growth hormone in a new recombinant Escherichia coli. The effect of different levels of dissolved oxygen and glucose concentration on cell density and substrate utilization were evaluated. The results indicated that the optimum level of dissolved oxygen was 25% of air saturation. The specific growth rate reached a maximum for an optimal glucose concentration of Sgl"1, the maximum yield was 0.58 gl-1 and glucose was completely consumed after 10 hours. The growth of recombinant E. coli on semi-defined and complx media was compared. The results demonstrated that dry cell weight and maximum specific growth rate were 2.35gr' and 0.4h-1 on a complex medium, respectively, but they were 3.3 gl-1 and 0.24h"' on semi-defined medium. The plasmid stability and hGH production during batch fermentation were investigated. The results suggested that the specific growth rate is the most important factor on hGH production and should be investigated in fed batch cultivation to get high cell density.
Patra A. K., Mukhopadhyay R., et al., (Protein Expr Purif. 2000, vol. 18, 182 - 192) reported that recombinant human growth hormone (rhGH) was expressed in Escherichia coli as inclusion bodies. In 10 hours of fed-batch fermentation, 1.6 g/L (gram/litre) of rhGH was produced at a cell concentration of 25

grams dry cell weight/Litre. Inclusion bodies from the cells were isolated and purified to homogeneity. Various buffers with and without reducing agents were used to solubilize rhGH from the inclusion bodies and the extent of solubility was compared with that of 8M (Molar) urea as well as 6M Guanidine Hydrochloride. Hydrophobic interactions as well as ionic interactions were found to be the dominant forces responsible for the formation of rhGH inclusion bodies during its high-level expression in E. coli. Complete solubilization of rhGH inclusion bodies was observed in 100 mM (milli Molar) Tris buffer at pH 12.5 containing 2M urea. Solubilization of rhGH inclusion bodies in the presence of low concentrations of urea helped in retaining the existing native-like secondary structures of rhGH, thus improving the yield of bioactive protein during refolding. Solubilized rhGH in Tris buffer containing 2M urea was found to be less susceptible to aggregation during buffer exchange and thus was refolded by simple dilution. The rhGH was purified by use of DEAE-Sepharose ion-exchange chromatography and the pure monomelic rhGH was finally obtained by using size-exclusion chromatography. The overall yield of the purified monomelic rhGH was approximately 50% of the initial inclusion body proteins and was found to be biologically active in promoting growth of rat Nb2 lymphoma cell lines.
None of the above mentioned prior art describes about a fermentation process, wherein by maintaining dissolved oxygen (DO) level during an induction phase and growth phase increases the production of recombinant human growth hormone and its analogs in the form of inclusion bodies in a fed batch process as addressed by the present invention.
OBJECT OF THE INVENTION
The principal object of the present invention is to develop a process for obtaining high level expression of the human growth hormone gene in a recombinant E. coli as inclusion bodies by fed-batch fermentation process in an animal source free culture medium.
SUMMARY OF THE INVENTION
The objective of the present invention is to develop a process comprising
• Culturing of E.coli in a fermenter through fed-batch process
• Maintaining dissolved oxygen level in a particular range during induction phase and growth phase
• Inducing the production of inclusion bodies by changing the temperature during fermentation, to reduce further the deamidation and oxidation of the expressed product.

In the context of the present invention, wherein the feeding strategy used is constant feed fed-batch strategy.
In one embodiment of the present invention, wherein the dissolved oxygen level is maintained between 0 to 10% during an induction phase and 25 to 35% during the growth phase of the fermentation process to obtain high yield of inclusion bodies.
In one embodiment of the present invention, wherein the animal source free medium is free from Transmissible Spongiform Encephalopathy (TSE) virus and Bovine Spongiform Encephalopathy (BSE) virus and used for the production of recombinant human growth hormone as inclusion bodies.
In the context of the present invention, wherein the volumetric yield of inclusion bodies is more than 25g/l (gram/ liter) with more than 80% purity which is achieved through primary down stream process.
In the context of the present invention, wherein the inclusion body contains recombinant human growth hormone and the expression level of recombinant human growth hormone at the end of the production is at least 20% of the total protein.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the profile of DO concentration (%) and turbidity (AU) during two-step increased feeding of fed-batch fermentation process of rhGH.
Figure 2 shows the reverse phase chromatogram of the rhGH Inclusion Bodies.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a fermentation process(s) for high level production of Inclusion bodies that has recombinant human growth hormone and its analogs. The high level production of inclusion bodies can be achieved by the specific type of fed batch strategy which includes maintaining the particular DO level during an induction phase and the growth phase and the production of recombinant human growth hormone is induced by raising the temperature during fermentation. Also the present invention is economically viable and feasible by virtue of reduction in the number of batches to be taken.

EXPERIMENTS
The following experiments will further illustrate certain aspects and embodiments of the invention in greater detail and are not intended to limit the scope of the invention.
Inoculums development
1.6-2.0 % Select Soytone, 1.0-1.3 % yeast extract and 0.5-0.8 % glycerol containing 100 mg/ml of kanamycin is used for stock and seed culture. The culture broth of E.coli cells is mixed with 20% glycerol and stored at -800C for culture stock. Seed culture is carried at 30-32 0C and 100-150 rpm in a shaker incubator in a 500 ml (milliliter) round flask containing 200 ml of medium inoculated with 1X10-7 of these cells for 12-18 hours, and the resulting cells which are in the mid exponential phase, are transferred to a 9.0 L (liter) fermenter directly.
Media and Culture Conditions
2.5-3.0 % select Soytone, 5-6 % yeast extract, 2.5-3.0 % Glycerol, 0.84% Di-potassium hydrogen phosphate, 0.42% potassium dihydrogen phosphate, 0.01% Strucktol 673, and 0.0125% of Kanamycin are mixed in Water for injection and charged into the fermenter. Fed batch cultivations are carried out in a 15.0 L fermenter with 9.0 L working volume. The pH is maintained at 7.0 by controlled addition of acid and base.
Temperature is controlled at 30-32°C; tip speed 2.75 m/s, aeration is done through a perforated pipe sparger ring and is maintained at 1.4-1.8wm. Filter sterilized kanamycin is added at 100 mg/ml (milligram/liter) at the beginning of the fermentation.
The minimum DO is maintained between 20% - 40 % of air saturation by using pure oxygen is blended with the sparged at growth phase and 5-10% of air saturation at induction phase. 10% (w/v -weight/volume) Strucktol is used as antifoaming agent and is sensed using a conductivity probe mounted 5 cm above the culture level.
Feed media preparation: FEED:
14-18 % Select Soytone, 28-32 % yeast extract, 42-48 % glycerol, 0.8% antifoam, 0.1% Kanamycin, are dissolved in 1000 ml of WFI (Water for Injection) and sterilize 9th hour of fermentation process, feeding is started. Feeding is done as per the details given in Table. 1.

S.No Fermentation Time Hour (hr) L/hr
1 9 0.25
2 10 0.25
3 11 0.25
4 12 0.125
5 13 0.125
Table 1
At 9th hour of fermentation, FEED is given in five different lots and dissolved oxygen concentration (20-40%) is maintained till 11.5th hours of fermentation by oxygen enrichment.
At 11.5 hrs of fermentation, induced the cells by increasing fermentation temperature to 35-37°C, reduced the tip speed to 1.66 m/s and then maintained the dissolved oxygen concentration to 5-15%.
At the end of the fermentation process (i.e) at 25th hour, the batch is harvested and the observed OD6oo value is 90 to 100.
Harvesting:
Culture broth is dispensed in the centrifuge bottles and the cells are separated by centrifugation. Total broth is centrifuged (RCF 10800 X g and time 20 min) and the pellet is recovered. The weight of recovered cell pellet is (120-lSOgl-1)
Inclusion bodies Isolation:
Cell disruption:
To get the Inclusion bodies, cells has to be lysed by high pressure cell disruption (Constant systems 2.2 KW). Cell pellet is homogenized in cold (below 10°C) WFI to get uniform suspension using a hand held homogenizer. Volume of WFI required is calculated by the following formula:
Volume of WFI required = (cell pellet weight (g) X 5ml)

Before cell lysis homogenized cell suspension is filtered through 30-µ membrane.
Cells are lysed by passing through a cell disrupter twice. Cell disrupter is kept cool during operation; by connecting it with a circulating chiller (set temperature is -4°C). During lysis set pressure is 18-22KPSI.
Lysate centrifugation:
Cell lysate is then centrifuged (RCF 10800 X g and time 30 min) to recover inclusion bodies as pellet. After centrifugation the supernatant is discarded.
Resuspension Buffer washes:
Re-suspension Buffer : ImM DTT, 10 mM EDTA and 50mM Tris pH 8.0.
IB pellet contains some unwanted lipids and proteins etc. To remove these impurities, buffer wash is given. Add 10 X of resuspension buffer for each gram of above IB pellet into the centrifuge bottle containing the IB pellet. Resuspend the pellet into resuspension buffer using hand held homogenizer. Stir the resulting suspension for 30 minutes at room temperature (18-25°C) using magnetic stirrer. After 30 min, remove the magnetic bead and centrifuge (RCF 10800 X g and time 30 min) by Sigma 8 K. After centrifugation the supernatant is discarded
2-3% of Sodium deoxycholate buffer wash:
Add 10 % sodium deoxycholate buffer for each gram of above IB pellet into the centrifuge bottle containing IB pellet. Resuspend the pellet into sodium deoxycholate buffer using hand hold homogenizer. Then centrifuge (RCF 10800 X g and time 30 min) by Sigma 8 K. After centrifugation supernatant is discarded.
Water wash:
Water wash helps to remove remaining contaminants left from buffer wash from the protein of interest and to make it compatible for further purification by chromatography. The weight of pellet obtained after water wash is 25-28g/L. Water wash is repeated with the pellet for three times & centrifugation during water wash is done at RCF 11285X g.

Analysis of Inclusion bodies by UPLC:
Inclusion bodies obtained after the primary purification is dissolved in 8M Gu-HCl and is injected into UPLC system (waters Acquity, USA) equipped with a 100 X 3 mm 3-µm column (ACE). A gradient of 100% (v/v) acetonitrile (HPLC grade,) (Merck, Germany) and 100% water for injection along with 0.1%. Tri flouro Acetic acid is used as mobile phase and the flow rate is kept constant at 0.4 mL/min. The analysis is carried out at ambient temperature with a variable wavelength of UV detector at 220 nm. The retention time for GH is 7.7 min.

We Claim
1. A process for obtaining high level expression of the human growth hormone gene in a
recombinant E. coli as inclusion bodies by fermentation process in an animal source free culture
medium comprising
o Culturing of E. coli in a fermenter through fed-batch process
o Maintaining dissolved oxygen level in a particular range during induction phase and
growth phase o Inducing the production of inclusion bodies by temperature which reduces deamidation
and oxidation.
2. The process claimed in claim 1, wherein the fed batch mode is constant feed fed- batch strategy.
3. The process claimed in claim 1, wherein the dissolved oxygen level is maintained between 0 to 10% during an induction phase and 25 to 35% during the growth phase of the fermentation process.
4. The process claimed in claim 1, wherein the animal source free medium is free from Transmissible Spongiform Encephalopathy (TSE) virus and Bovine Spongiform Encephalopathy (BSE) virus and used for the production of recombinant human growth hormone as inclusion bodies.
5. ,The process of claim 1 wherein pH ranges between 6.8 - 7.2, agitation ranges between 450rpm-750rpm, temperature ranges between 37±2°C, airflow from lvvm to 1.5 vvm, and back pressure from 0.2 bar to 0.3 bar.
6. A process claimed in any preceding claims, wherein the volumetric yield of inclusion bodies is more than 25gl_1 (gram/litre) with 80% purity.
7. A process claimed in claim 7, wherein the 80% purity is achieved through primary down stream process.

8. A process claimed in any preceding claims, wherein the inclusion body contains recombinant human growth hormone and its analogs.
9. A process for claimed in any preceding claims, wherein the inclusion body contains recombinant human growth hormone and the expression level of recombinant human growth hormone at the end of the production is at least 20% of the total protein.