FORM 2 THE PATENTS ACT 1970
(39 OF 1970)
COMPLETE SPECIFICATION
(SECTION 10)
"IMPROVED HIGH CELL DENSITY
FERMENTATION PROCESS FOR HIGH YIELD AND
INSOLUBLE EXPRESSION OF RECOMBINANT
INSULIN AND INSULIN ANALOGUES"
UNICHEM LABORATORIES LIMITED, A COMPANY
REGISTERED UNDER THE INDIAN COMPANY ACT, 1956,
HAVING ITS REGISTERED OFFICE LOCATED AT
UNICHEM BHAVAN, PRABHAT ESTATE OFF. S. V.
ROAD, JOGESHWARI (W) MUMBAI 400102
MAHARASHTRA, INDIA

IMPROVED HIGH CELL DENSITY FERMENTATION PROCESS FOR
HIGH YIELD AND INSOLUBLE EXPRESSION OF RECOMBINANT
INSULIN AND INSULIN ANALOGUES
FIELD OF INVENTION
The present invention relates to the preparation of Insulin and Insulin Analogues in insoluble form using fed batch fermentation process.
BACKGROUND OF THE INVENTION
Insulin is a polypeptide hormone essential for the control of glucose metabolism and it is administered daily to patients suffering from diabetes mellitus, a metabolic disorder characterized by an inadequate supply of insulin.
Bovine insulin and porcine insulin may be considered as the first clinically used insulin analogs due to lack of manufacturing methods of human insulin. Recombinant human insulin (HUMULIN-R) was later developed by Eli Lilly and was approved by FDA on October 28, 1982.
At present Recombinant Human Insulin and its analogues are produced by genetic engineering using expression systems such as bacterial (E Coli) or yeast DNA. In general process the Preproinsulin produced by fermentation of E coli or Yeast is isolated, purified and cleaved by CNBr in order to release the proinsulin polypeptide. The latter is further modified by oxidative sulfitolysis to proinsulin S-sulfonate. The proinsulin S-sulfonate is then purified and folded, under reducing conditions, to proinsulin. Conversion of the proinsulin to insulin is achieved by the combined action of trypsin and carboxypeptidase B. The insulin obtained is purified by chromatography method and crystallized to obtain Pharmacopoeial grade Insulin. For the high yields of Insulin all the above listed steps are modified to certain extent. However high cell density, fed-batch fermentation of E. coli gives Preproinsulin in

high yields; thus increasing the yields of the final product. There are several methods reported for High cell density, fed-batch fermentation for the preparation of Insulin. Michael Schmidt et al. (Journal of Biotechnology 68 (1999) 71-83) published a process of production of the insulin, wherein the recombinant cells are grown in high-cell density fed-batch cultures using a synthetic medium with glucose as sole carbon and energy source and the induction of recombinant protein production is carried out by a temperature-shift from 30 to 42°C.
Young-Jin Son et al. (Biotechnology and Bioprocess Engineering Oct 2007) studied the effect of temperature shift strategies on human Preproinsulin production in the fed-batch fermentation of recombinant E Coli. The studies showed that the three-step shift of culture temperature during fermentation from 30°C to 37°C for 2h, gave the best results. It is silent on the effect of feeding rates^
US6001604 (A) disclosed process for the production of recombinant human insulin by folding of a proinsulin hybrid polypeptide, wherein Fermentation of is. coli in the presence of any one of glucose, glycerol, galactose or a combination thereof as carbon source facilitated the expression of the SOD-proinsulin hybrid polypeptides. It is silent on fermentation temperatures. The feeding rates in the patent are strikingly different from those in the present invention.
WO2008139496 (Al) discloses the process of production of recombinant human insulin in Pichia pastoris and increasing its yield by optimization of codons in pichia expression system and the process parameters. The publication describes the use of vectors other than E. coli.
High cell density and high expression level of Preproinsulin is important for improving the productivity of recombinant human insulin and its analogues. To obtain high concentrations of recombinant E. coli cells, it is necessary to supply key nutrients in a controlled manner, and at the same time to reduce the accumulation of acetate, a well known inhibitory metabolite for cell growth. In case of fermentation of E coli control of substrate supply may produce a challenge to the cells as the ability of the substrate uptake usually declines along with a decrease in the specific growth

rate during fed-batch culture and even cell lysis may occur in the final stage of fed-batch culture. Moreover, the overproduction of target protein is known to be toxic to the host cell. It is important to maintain the culture conditions and the feeding rate for high production of Insulin and its analogues.
The processes in the prior art limit themselves by resolving few issues in the fed-batch fermentation for production of Insulin and its analogues. The processes involve alteration of the expression system or change in the temperature or pH or the feeding rates. There are no methods reported that would account to all the variables in the fermentation process and lead to higher productivity of the target protein along with the efficiency and the cost-effectiveness of the process.
OBJECT OF THE INVENTION
The main object of the present invention is the optimization of culture conditions and the feeding rates during fermentation so as to obtain the greatest possible productivity of insulin and its analogues.
Another object of the invention is to develop an efficient, cost-effective and easily scalable process for the preparation of Insulin and insulin analogues.
SUMMARY OF THE INVENTION:
The present invention relates to a process of fed-batch fermentation comprising E. coli BL21 Gold cells in a suitable medium at an initial temperature of 35-40°C, having a predetermined feeding rate during fermentation as:
a. for Carbon source; increasing feed flow rate from 3.38 to 27.6 gmL/'h"1 till 6th hour, and decreasing feed flow rate from 20.9 to 6.75 gmL/'h"1 from 7th to 12th hour of fermentation; and

b. for Nitrogen source increasing feed flow rate of 3.30 to 27.0 gmL'V till 6th hour, and decreasing feed flow rate from 20.45 to 6.60 gmL/'h"1 from 7th to 12th hour of fermentation.
The present invention also relates to the process of preparation of recombinant insulin and insulin analogues by aerobic fed-batch fermentation of recombinant E.coli BL21 Gold cells, wherein growth of the culture is carried out with 1- 2.5 vvm aeration.
The present invention describes the efficient, cost-effective and easily scalable process for the preparation of Insulin and insulin analogues
DETAILED DESCRIPTION OF THE INVENTION
Recombinant Insulin refers to the Human insulin prepared by recombinant DNA technology and Insulin analogues include Recombinant Insulin Lispro and Recombinant Insulin Glargine.
The unit of time is considered as hour and is represented by h or hr, the plural being hrs.
Efficient process and cost effectiveness is to be interpreted in terms of yield and costs of input to output. Efficiency is also to be interpreted as absence of impurity.
Suitable mediums are to be interpreted as mediums as described herein the present invention and the examples. Suitable medium comprises of seed medium and culture medium.
The recombinant E. coli BL21 DE3 Gold cells containing plasmid pET 28a harboring a gene coding for Insulin and Insulin analogues cloned in between the sites Nde I and Bam HI, were grown in the seed medium comprising 2% Luria HiVeg broth (w/v), 0.75% Na2HP04 (w/v), 0.5% Dextrose Anhydrous (w/v), 0.1% MgS04.7H20 (w/v), Kanamycin sulphate to a final concentration of 20 ng/ml and 0.1% (v/v) trace metal solution comprising FeSO4.7H20, ZnSO4.7H20, CoCl2.6H20 NaMoO42H20,

CaCl2.2H20, MnCl24H20, CuSO4.5H20 and H3B03. Incubation of seed culture is carried out for 09-15 hrs at 35°C - 40°C, preferably 37°C at shaker speed 110 ± 10 rpm. When inoculum ODgoonm reached around 10.0 - 14.0, 10% (v/v) of culture medium was transferred to Fermenter containing sterile medium.
The fed-batch fermentation of recombinant E. coli BL21 DE3 Gold cells was carried out in culture medium comprising 1% yeast extract (w/v), 1% Dextrose Anhydrous (w/v), 0.3% KH2P04 (w/v), 0.7% Na2HP04 (w/v), 0.2% (NH4)2S04 (w/v), 0.033% NaCl (w/v), 0.1% MgS04.7H20 (w/v) and 0.1% (v/v) of trace metal solution. Kanamycin sulphate is added to a final concentration of 20ug/ml and 10^g/ml Thiamine Hydrochloride solutions.
The present invention describes a process for the fed-batch fermentation of recombinant E. coli cells, preferably E coli BL21 DE3 Gold cells for the expression of Insulin and Insulin analogues in insoluble forms.
The main aspect of the present invention is a process of fed-batch fermentation comprising E. coli BL21 Gold cells in a suitable medium at an initial temperature of 35-40°C, having a predetermined feeding rate during fermentation as:
a. for Carbon source; increasing feed flow rate from 3.38 to 27.6 gmL'h"1 till 6th
hour, and decreasing feed flow rate from 20.9 to 6.75 gml/'rf'from 7th to 12th hour
of fermentation; and
b. for Nitrogen source increasing feed flow rate of 3.30 to 27.0 gmL/'h"' till 6th hour,
and decreasing feed flow rate from 20.45 to 6.60 gmL"'h"' from 7th to 12th hour of
fermentation.
The process for the fed-batch fermentation of recombinant E coli BL21 DE3 Gold cells was carried out at a temperature of 35°C - 40°C, preferably at 37°C. Fermentation runtime was at least 12 ±1 hrs, preferably, 12 hrs.

To increase the cell mass during fed-batch fermentation, feeding was initiated with a suitable carbon source like glucose or glycerol, preferably glycerol, at a concentration of 75% (w/v) and a nitrogen source like tryptone, peptone or yeast extract, preferably yeast extract at a concentration of 40% (w/v). Feeding may be initiated after 1.5 log hrs, at predetermined feeding rates maintaining the C: N ratio of 3:1 to 6:1. Those skilled in the art may vary the rates and the amounts as per their suitability, since they are specific for the parameters demonstrated for the particular batch sizes. Inventive step resides in the initiation of the feeding after 1.5 log hours.
The feeding rate for optimal growth of the culture during fermentation was surprisingly observed when Carbon source was supplied from 1.5 fermentation log hours by feeding glycerol with increasing feed flow rate from 3.38 to 27.6 gmL"'h"' till 6th hour and decreasing feed flow rate of 20.9 to 6.75 gmL"V from 7th to 12th hour of fermentation. Nitrogen source was supplied from 1.5 fermentation log hours by feeding Yeast Extract with increasing feed flow rate of 3.30 to 27.0 gmLV'h"1 till 6th hour, and decreasing feed flow rate of 20.45 to 6.60 gmL/'h"1 from 7th to 12th hour of fermentation.
Another aspect of the present invention is the process of preparation of recombinant insulin and insulin analogues by aerobic fed-batch fermentation of recombinant E.coli BL21 Gold cells, wherein initial growth of the culture is carried out with 1- 2.5 vvm aeration.
During the initial growth of the culture, dissolved oxygen was maintained at 50% -70%) before induction and 30% - 60% after induction with air and oxygen mixing. The pH of the culture during growth was maintained between 6.6 - 7.0 with alkali such as sodium hydroxide solution. Foam produced was subsided with antifoaming agent. The expression of Insulin and Insulin analogues was carried out by appropriately inducing the cells with an inducer, like lactose, isopropyl thio-galacto-pyranoside (IPTG), preferably IPTG at a concentration of 500 uM to 1500 uM, preferably 1000 uM. Induction is optimally performed at mid log phase, when the

cells are active, with the cell density measuring at least 120 - 160 OD, preferably 125 - 150 OD at 600 nm. Final culture OD at 600nm was >180 and expression > 5 g/L and expression percentage > 20% as demonstrated with densitometry by SDS-PAGE.
Fed-batch fermentation of recombinant E. coli BL21 Gold cells when carried out as per the conditions of the present invention surprisingly provided high level expression. Recombinant proteins were produced as unfolded, partially folded insoluble protein aggregates called inclusion bodies.
The novel and inventive aspect of the present invention resides in the process for the insoluble expression of Insulin and Insulin analogues using fed-batch fermentation process, wherein the fermentation process is carried out at 35°C - 40°C, preferably at 37°C with novel feeding rates of Carbon and Nitrogen source that surprisingly resulted in high yields of insoluble Insulin and Insulin analogues. None of the prior art gives the specific feeding rate or the aeration conditions that would result in the high expression of the protein to yield Insulin and Insulin analogues. Surprisingly the specific feeding rate and the maintenance of dissolved Oxygen at the specific level, as disclosed in the present invention, during fermentation lead to high density expression of protein corresponding to Insulin and insulin analogues.
Thus the inventors have accomplished first object of the present invention by optimizing of culture conditions and the feeding rates during fermentation that would lead to high productivity of insulin and its analogues.
In the present invention the products obtained in high yield makes the process efficient and cost effective. Also the cheap and easy to handle raw materials used in the process leads to cost-effective and easily scalable process. Thus the inventors of the present invention have also accomplished second object of the invention by developing an efficient, cost-effective and easily scalable process for the preparation of Insulin and insulin analogues.

The present invention further describes a process for the isolation of proteins (e.g. Preproinsulin) and their conversion to Insulin. The following process demonstrates the process specifically for insulin and person skilled in the art can easily extrapolate the process to Insulin analogues.
Isolation of Inclusion bodies (IB) from the fermentation broth was carried out by harvesting cells by centrifugation to obtain the cell pellet, which was re-suspended in a suitable buffer, pre-chilled to a temperature of NMT 10°C before lysis to avoid denaturation of protein during cell disruption. Lysis was carried out using homogenizer under high pressure of 16000-20000 psi.
The cell lysate containing inclusion bodies was treated with Tris buffer, EDTA, Sodium chloride and Beta Mercaptoethanol, Triton x 100. Two buffer washes were given to the inclusion bodies (IB) followed by centrifugation to isolate the inclusion bodies in pellet form. SDS PAGE (15%) analysis confirms the results depicting minimum loss of protein of interest in the supernatant. The inclusion bodies are then processed further to isolate the protein.
The inclusion bodies were dissolved in 6M Guanidine hydrochloride Buffer in 1:4 to 1:15(w/v) ratio and pH was adjusted to 8 to 12 by 5N - ION sodium Hydroxide to get inclusion bodies extract. Sulfitolysis was carried out by adding Sodium Tetrathionate and Sodium Sulfite at final concentration of 20-80 mM and 0.1-0.5 M, respectively and incubating the inclusion bodies extract at 4°C- 40°C for 2 to 24 hrs to obtain sulfonated precursor protein. The sulfonated p recursor protein was then clarified either by centrifugation or using solvents like ethanol, isopropyl alcohol and methanol or using O.lu. hollow fiber filtration system. Clarification of the sulfitolysed IB extract using hollow fiber was carried out at a TMP of 5-10 psi. The retentate was then subjected to 5-7 washes of the 3-6 M concentration of guanidine hydrochloride buffer or 4-8M of Urea buffer in step mode to extract maximum amount of the protein in the permeate. The clear permeate was then subjected to ultrafiltration using 3KDa hollow fiber at a TMP of 5-10 psi to obtain clarified reaction mixture

containing sulfonated IB. Batch processing with or without using ultrafiltration step had no effect on product quality and quantity and hence can be avoided.
The clarified reaction mixture with or without carrying out ultrafiltration containing sulfonated precursor was subjected to Cyanogen bromide (CNBr) treatment by adding cyanogen bromide in the ratio of 1: 0.8 to 1: 1.5 (weight of protein to weight of CNBr) under acidic condition (pH 0.1 to 2.5) in 3-6 M Guanidine Hydrochloride buffer and incubating it at 15-20°C for 10-20 hours. The solution after CNBr treatment was precipitated by adding 5-7 volumes of water or 0.02-0.1M Glycine, of pH 3 to make the final pH of around 2.5 and precipitate was recovered by centrifugation at 8000-10000 rpm for 10-15 min or by 0. l\i cross flow filtration using hollow fiber, at a TMP of 2-6 psi to obtain Proinsulin precipitate.
The Proinsulin precipitate was dissolved in 8M - 9M Urea Buffer, pH was adjusted to 8.0-9.0 and loaded onto Cellufine max Q-r for removal of cleaved polypeptide and other byproducts generated during the CNBr treatment. Refolding of Proinsulin was carried out in refolding buffer containing 0.01-0.05 M Glycine with pH in the range of 10 to 12 with final Urea concentration of 0.2 to 4 M to obtain refolded Proinsulin. Final refolded proinsulin concentration was maintained between 0.01 to 2.5 mg/ml.
The refolded Proinsulin was purified from refolding reaction mixture by hydrophobic interaction chromatography in 0.02-0.1 M Glycine buffer of pH 9-11. Alternatively, the refolded Proinsulin can also be purified by reverse phase C8 preparative HPLC using Acetonitrile in the mobile phase with 0.1 M Glycine buffer of pH 11 or can be lyophilized to obtain dry powder of refolded Proinsulin.
The refolded pro Insulin is digested to the final product (Insulin) by enzymatic conversion. The digested product is then partially purified from the partially digested protein and other related impurities by ion exchange chromatography to obtain Insulin.

Examples given below serve as references concerning the preparation of insoluble Insulin and Insulin analogues using fed-batch fermentation.
Example 1: Fermentation process for the expression of Preproinsulin
30 uL of the recombinant E. coli BL21 DE3 Gold cells containing plasmid pET 28a harboring gene coding for human Insulin were inoculated in 200ml of seed (inoculum) medium. Incubation was carried out for I I hrs at 37°C at shaker speed 110 ± 10 rpm. When inoculum ODeoonm reached 11.0, 10% (v/v) of culture medium was transferred to Fermenter containing sterile medium.
Fermentation was carried out for run time of 12 hrs with 1-2.5 VVM aeration. Dissolved oxygen was maintained at 50% -70% before induction and 30% - 60 % after induction with air and oxygen mixing, pH was maintained at 6.8 ±0.2 using 10M sodium hydroxide (Alkali) solution. Temperature was maintained at 37 ±2 °C. Feeding rate for optimal growth of the culture during fermentation was predetermined. Carbon source; increasing feed flow rate from 3.38 to 27.6 gmL"'h"' till 6th hour, and decreasing feed flow rate from 20.9 to 6.75 gmL/'h"1 from 7th to 12th hour of fermentation; and Nitrogen source increasing feed flow rate of 3.30 to 27.0 gmL"'h"' till 6th hour, and decreasing feed flow rate from 20.45 to 6.60 gmL"'h~' from 7th to 12th hour of fermentation.
Induction was done with 1000 uM 1PTG at 6 log hours and cell density at OD 600nm 144. Final cell density achieved was 212 as measured by absorbance at 600nm at 12 log hours with final protein expression of 5.5 gm/L and expression 25.54 percent.
Example 2: Fermentation process for the expression of Preproinsulin Lispro
30uL of the recombinant E. coli BL21 DE3 Gold cells containing plasmid pET 28a harboring a gene coding for Insulin Lispro were inoculated in 200ml of seed (inoculum) medium. Incubation was carried out for 11 hrs at 37°C at shaker speed

110 ± 10 rpm. When inoculum OD600nm re ached around 12.0, 10% (v/v) of culture medium was transferred to Fermenter containing sterile medium.
Fermentation was carried out for the preparation of Human Insulin where in 10% inoculum was transferred to culture medium comprising 1% yeast extract (W/V),l% Dextrose (W/V) , 0.3% KH2P04 (W/V),0.7% Na2HP04 (W/V) 0.2% (NH4)2S04(W/V), 0.033% Sodium Chloride,0.1% MgS04 .7H20 (W/V), 0.1% (v/v) trace metal solution, Kanamycin sulphate final concentration of 20 ixg/ml and 10 |ig/ml of Thiamine Hydrochloride solution. Heat labile materials were sterilized by filtration.
Fermentation was carried out for run time of 11 hrs with 1-2.5 VVM aeration, dissolved Oxygen was maintained at 50% - 70% before induction and 30% - 60 % after induction with air and oxygen, pH was maintained at 6.8 ±0.2 using sodium hydroxide (Alkali) solution. Temperature was maintained at 37 ±2 °C. Feeding rate for optimal growth of the culture during fermentation was predetermined. Carbon source; increasing feed flow rate from 3.38 to 27.6 gmL^h"' till 6th hour, and decreasing feed flow rate from 20.9 to 6.75 gmL"'h"' from 7th to \2U hour of fermentation; and Nitrogen source increasing feed flow rate of 3.30 to 27.0 gml/'h"1 till 611 hour, and decreasing feed flow rate from 20.45 to 6.60 gml/'h"1 from 7th to 12th hour of fermentation.
Induction was done with lOOOuM IPTG at 6 log hours and cell density at OD600nm 142. Final cell density achieved was 206 as measured by absorbance at 600nm at 11 log hours with final protein expression of 5.4 gm/L and expression 23 percent.
Example 3: Fermentation process for expression of Preproinsulin Glargine
30(0.1 of the recombinant E. coli BL21 DE3 Gold cells containing plasmid pET 28a harboring a gene coding for Insulin Glargine were inoculated in 200ml of seed (inoculum) medium. Incubation was carried out for 11 hrs at 37°C at shaker speed

110 ± 10 rpm (BRDP0513011002). When inoculum OD600nm reached around 14.0, 10% (v/v) of culture medium was transferred to Fermenter containing sterile medium.
Fermentation was carried out for the preparation of Human Insulin where in 10% inoculum was transferred to culture medium comprising 1% yeast extract (W/V), 1% Dextrose (W/V) , 0.3% KH2P04 (W/V), 0.7% Na2HP04 (W/V) 0.2% (NH4)2S04(W/V). 0.033% Sodium Chloride, 0.1% MgS04.7H20 (W/V), 0.1% (v/v) trace metal solution, Kanamycin sulphate final concentration of 20 (ig/ml and 10 |j.g/ml of Thiamine Hydrochloride solution. Heat labile materials were sterilized by filtration.
Fermentation was carried out for run time of 11 hrs with 1-2.5 VVM aeration, dissolved Oxygen was maintained at 50% - 70% before induction and 30% - 60 % after induction with air and oxygen , pH was maintained at 6.8 ±0.2 using 10M. Sodium hydroxide (Alkali) solution. Temperature was maintained at 37 ±2 °c. Feeding rate for optimal growth of the culture during fermentation was predetermined Carbon source; increasing feed flow rate from 3.38 to 27.6 gmU'h"1 till 6th hour, and decreasing feed flow rate from 20.9 to 6.75 gmL/'h"' from 7th to 12th hour of fermentation; and Nitrogen source increasing feed flow rate of 3.30 to 27.0 gmL"'h"' till 6th hour, and decreasing feed flow rate from 20.45 to 6.60 gmL'V1 from 7lh to 12th hour of fermentation.
Induction was done with 1000 uM IPTG at 6 log hours and cell density at OD 600nm 142. Final cell density achieved was 208 as measured by absorbance at 600nm at 11 log hours with final protein expression of 5.5 gm/L and expression 25 percent.

CLAIM
We claim,
1. A process of fed-batch fermentation comprising E. coli BL21 DE3 Gold cells
containing plasmid pET 28a in a suitable medium at an initial temperature of
35°C - 40°C, having a predetermined feeding rate during fermentation as:
a. for Carbon source; increasing feed flow rate from 3.38 to 27.6 gmL/'h"' till
6th hour, and decreasing feed flow rate of 20.9 to 6.75 gmL"'h"' from 7th to
12th hour of fermentation; and
b. for Nitrogen source increasing feed flow rate of 3.30 to 27.0 gmL" h" till 6U
hour, and decreasing feed flow rate of 20.45 to 6.60 gmL"'h"' from 7th to 12lh
hour of fermentation.
2. The process for fed-batch fermentation as claimed in claim 1, wherein the suitable medium comprises of seed medium and culture medium.
3. The process for fed-batch fermentation as claimed in claim 2, wherein seed medium comprises 2% Luria Hi-Veg broth (w/v), 0.75% Na2HP04 (w/v), 0.5% Dextrose Anhydrous (w/v), 0.1% MgS04.7H20 (w/v), Kanamycin sulphate to a final concentration of 20 ug/ml and 0.1% (v/v) trace metal solution comprising FeSO4.7H20, ZnSO4.7H20, CoCl2.6H20, NaMoO42H20, CaCl22H20, MnCl24H20, CuS045H20 and H3B03.
4. The process for fed-batch fermentation as claimed in claim 2, wherein culture medium comprises: 1% yeast extract (w/v), 1% Dextrose Anhydrous (w/v), 0.3% KH2P04 (w/v), 0.7% Na2HP042H20 (w/v), 0.2% (NH4)2S04 (w/v), 0.033% NaCl (w/v), 0.1% MgS04.7H20 (w/v) and 0.1% (v/v) of trace metal solution. Kanamycin sulphate is added to a final concentration of 20fig/ml and lO^ig/ml Thiamine Hydrochloride solutions.

5. The process for fed-batch fermentation as claimed in claim 1, wherein the preferred temperature is 37°C.
6. The process for fed-batch fermentation as claimed in claim 1, wherein the Carbon source is selected from glucose and glycerol, preferably glycerol at a concentration of 75% (w/v).
7. The process for fed-batch fermentation as claimed in claim 1, wherein the "Nitrogen source is selected from tryptone, peptone or yeast extract, preferably yeast extract at a concentration of 40% (w/v).
8. A process of preparation of recombinant insulin and insulin analogues by aerobic fed-batch fermentation of recombinant E.coli BL21 DE3 Gold cells containing plasmid pET 28a, wherein growth of the culture is carried out with 1 - 2.5 vvm aeration.
9. The process as claimed in claim 8, wherein dissolved oxygen in the fermenter before induction is maintained at 50% - 70% and after induction it is maintained at 30% - 60% with air and oxygen mixing.
10. The process as claimed in claim 9, wherein inducer used during induction phase is selected from IPTG or Lactose, preferably 1PTG at a concentration of 500 uM to 1500 uM, preferably 1000 uM.
11. The process as claimed in claim 9, wherein induction is optimally performed at mid log phase, when the cells are active, with the cell density measuring at least 120 - 160 OD, preferably 125 - 150 OD at 600 nm.
12. The process as claimed in claim 9, wherein final culture OD at 600nm was >180 and expression > 5 g/L and expression percentage > 20%.