IMPROVED PROCESS FOR PREPARATION OF PHARMACEUTICAL GRADE INSULIN GLARGINE
Technical Field of the Invention:
The present invention relates to the simple and cost effective process to prepare pharmacopoeial grade Insulin Glargine.
Background of the Invention:
The Insulin Glargine is a long acting basal Insulin analog with a following substitution of the amino acids in the human insulin: i) Glycine in the place of Asparagine at positional in A chain; and ii) Two additional Arginines added to the carboxy terminal of B chain. These substitutions in human insulin amino acid sequence leads to the formation of Insulin Glargine with a shift of isoelectric point from 5.4 to 6.7. Due to the near neutral isoelectric point, reduced solubility is seen at the physiological pH. Insulin Glargine forms amorphous microprecipitates at the neutral pH of the subcutaneous tissue and is slowly released to provide basal Insulin supplementation. Glargine thus does not exhibit a peak in its activity at any point of time and is found to give a plateau within 6-8 hours with its action seen up to 24
Primary Structure of Insulin Glargine.
The general method of preparation of insulin Glargine involves preparing a precursor by genetic engineering method using either bacteria or yeast, isolating the precursor,

sulfonating and refolding the precursor followed by enzymatic digestion and purification. The prior art processes reported to prepare insulin Glargine mainly involves improvement of any one of the steps listed above.
US7396903 B2 discloses a process to prepare insulin compounds from their precursors in two steps; first cleavage step using enzymes and a second coupling step by adding nucleophile compound to the cleavage product. In this patent there are cleavage, coupling and hydrolysis reactions to form the insulin like compounds. Such reactions yield undesirable impurities and multiple steps are required to clear these impurities. This makes the process tedious, less economical and lengthy. J. D. Wade et al. in an article in Bioconjugate Chemistry (Use of a Temporary "Solubilizing" Peptide Tag for the Fmoc Solid-Phase Synthesis of Insulin Glargine via Use of Regioselective Disulfide Bond Formation Year: 2009, 20 (7), pp 1390— 1396; Publication Date (Web): June 24, 2009) reported a method of increasing the solubility of peptide using penta-lysine "tags". Thus insulin Glargine was prepared by synthesizing A and B chains of insulin glargine via solid-phase peptide synthesis, wherein the poorly soluble chain A is tagged. The two chains were combined in solution via regioselective disulfide bond formation. At the conclusion of the chain combination, the solubilizing peptide tag was removed from the A-chain to provide synthetic human glargine in nearly 10% overall yield. The article details the synthesis of A and B chains, along with tag which is difficult to control in solid phase synthesis in large scale operation and less economical as compared to the fermentation process that is used in the present invention.
US8802816 B2 discloses a method of obtaining purified heterologous Insulins in Yeast. Although many advantages like ease of growing, speed and scalability of the process involving yeast cells are seen, major disadvantage seen in case of yeast cells is the post translational modification which is not seen in the case of bacterial cells. WO/2009/104199A1 mentions a crystallization process of column chromatography elute before carrying out trypsinization. The process of crystallization, recovery and further solubilization of the crystals of protein becomes a tedious activity before

carrying out trypsinization. This becomes an extra and unnecessary step in the process to prepare insulin Glargine, thus increasing the cost of the process. Further it is also mentioned that conversion of Glargine precursor to Insulin Glargine is carried out by using trypsin in the presence of different solvents like DMSO, DMF, acetonitrile, ethyl acetate and ethanol. Different percentage of different solvents is shown to give different yields. These experiments in different solvent give maximum yield of 40% to 45%, which is low thus making the process inefficient. US20120214965A1 describes a process for the conversion of Glargine Precursor to Glargine by trypsin in the presence of Citraconic anhydride. The process is both tedious and time consuming. Firstly, blocking by citraconic anhydride is carried out followed by buffer exchange. Trypsinization is then carried out followed by the last step of deblocking. The steps in this process, are lengthy, time consuming and uneconomical.
Object of the Invention:
An object of the present invention is to provide simple, economic and cost effective
method for production of pharmacopoeial grade Insulin Glargine.
Another object of the present invention is to prepare pharmacopoeial grade Insulin
Glargine from a bacterial fermentation process followed by isolation of inclusion
bodies (IBs), refolding, enzymatic conversion and chromatographic purification.
Finally Pure Insulin Glargine is lyophilized or crystallized to obtain pharmacopoeial
grade Insulin Glargine.
Yet another object of the present invention is to perform initial enzymatic cleavage of
purified Proinsulin Glargine (PG) using trypsin directly in the chromatographic
column elute.
Summary of the Invention:
The present invention relates to a process for producing pharmacopoeial grade Insulin Glargine from Preproinsulin Glargine comprising the steps of:

a. bacterial fed batch fermentation of E. coli, harboring plasmid coding for
Preproinsulin Glargine to express the Preproinsulin Glargine in the form of
Inclusion bodies;
b. isolation and purification of the Inclusion bodies obtained in step a to get purified
Inclusion bodies;
c. dissolution of purified Inclusion bodies, obtained in step b, in a denaturing agent
and sulfitolization using Sodium Tetrathionate and Sodium Sulfite to obtain
dissolved sulfitolysed protein;
d. clarification of the dissolved sulfitolysed protein obtained in step c at a TMP of
3-12 psi using a solvent selected from ethanol, Isopropyl alcohol and methanol,
preferably Isopropyl alcohol to get clarified Preproinsulin Glargine precursor;
e. digestion of clarified Preproinsulin Glargine precursor obtained in step d with
Cyanogen bromide under acidic condition with pH 0.1 to 2.5 in presence of
buffer selected from 4-8 M Urea buffer and 3-6 M Guanidine Hydrochloride
buffer to obtain Proinsulin Glargine precursor;
f. precipitation of Proinsulin Glargine precursor obtained in step e with 3-6
volumes of water or with 0.02 - 0.2 M Glycine buffer with pH of 2.0-3.0,
preferably pH 3.0 and recover precipitated Proinsulin Glargine precursor by cross
flow filtration to obtain filtered Proinsulin Glargine precursor;
g. purification of filtered Proinsulin Glargine precursor obtained in step f using an
anion exchanger selected from Capto DEAE, Source 30Q, Source 15Q, Q
Sepharose XL, Q Sepharose FF, DEAE Sepharose FF or Cellufine max Q-r,
preferably Cellufine max Q-r to get purified Proinsulin Glargine precursor;
h. refolding purified Proinsulin Glargine precursor obtained in step g and convert it to Proinsulin Glargine by dilution method in the refolding buffer containing 0.01 to 0.05 M Glycine with pH 10-12;

i. capturing and purifying Proinsulin Glargine obtained in step h, by hydrophobic interaction chromatography to obtain purified Proinsulin Glargine;
j. digestion of purified Proinsulin Glargine obtained in step i, to convert it to Insulin Glargine using Recombinant Trypsin or soluble TPCK treated animal Trypsin, wherein the process is carried out directly in the elute from hydrophobic interaction chromatography in step h;
k. optionally purifying Insulin Glargine, obtained in step j, using cation exchange chromatography with resin selected from CM Sepharose, SP Sepharose, CM Cellulose preferably CM Sepharose FF;
1. purification of Insulin Glargine obtained in step k or j by silica based C4, C8 and CI8 Reverse phase chromatography to obtain pure Insulin Glargine with purity >98%;
m. lyophilization or crystallization of pure Insulin Glargine obtained in step 1 to obtain pharmacopoeial grade.
The present invention relates to the process of enzymatic conversion of purified Proinsulin Glargine (PG) using trypsin directly in the chromatographic column elutes
The present invention also relates to the process of enzymatic conversion of purified PG using trypsin, wherein ratio of trypsin to purified PG is 1:500 to 1:2000
The present invention also relates to the process of enzymatic conversion of purified PG to obtain insulin Glargine by trypsin digestion wherein ratio of trypsin to purified PG is 1:500 to 1:2000 and wherein the process is carried out in chromatographic column elute.
Detailed description of the Invention:
The term protein is used for Glargine or its precursor at any stage of preparation. It means inclusion bodies or Preproinsulin Glargine (PPG) Precursor or Proinsulin

Glargine (PG) precursor or Proinsulin Glargine or Insulin Glargine as the context may be referring.
The phrases enzymatic cleavage and enzymatic conversion are used interchangeably.
The present invention relates to a process for producing pharmacopoeial grade Insulin Glargine from Preproinsulin Glargine comprising the steps of:
a. bacterial fed batch fermentation of E. coli, harboring plasmid coding for
Preproinsulin Glargine to express the Preproinsulin Glargine in the form of
Inclusion bodies;
b. isolation and purification of the Inclusion bodies obtained in step a to get purified
Inclusion bodies;
c. dissolution of purified Inclusion bodies, obtained in step b, in a denaturing agent
and sulfitolization using Sodium Tetrathionate and Sodium Sulfite to obtain
dissolved sulfitolysed protein;
d. clarification of the dissolved sulfitolysed protein obtained in step c at a TMP of
3-12 psi using a solvent selected from ethanol, Isopropyl alcohol and methanol,
preferably Isopropyl alcohol to get clarified Preproinsulin Glargine precursor;
e. digestion of clarified Preproinsulin Glargine precursor obtained in step d with
Cyanogen bromide under acidic condition with pH 0.1 to 2.5 in presence of
buffer selected from 4-8 M Urea buffer and 3-6 M Guanidine Hydrochloride
buffer to obtain Proinsulin Glargine precursor;
f. precipitation of Proinsulin Glargine precursor obtained in step e with 3-6
volumes of water or with 0.02 - 0.2 M Glycine buffer with pH of 2.0-3.0,
preferably pH 3.0 and recover precipitated Proinsulin Glargine precursor by cross
flow filtration to obtain filtered Proinsulin Glargine precursor;
g. purification of filtered Proinsulin Glargine precursor obtained in step f using an
anion exchanger selected from Capto DEAE, Source 30Q, Source 15Q, Q

Sepharose XL, Q Sepharose FF, DEAE Sepharose FF or Cellufine max Q-r, preferably Cellufine max Q-r to get purified Proinsulin Glargine precursor;
h. refolding purified Proinsulin Glargine precursor obtained in step g and convert it to Proinsulin Glargine by dilution method in the refolding buffer containing 0.01 to 0.05 M Glycine with pH 10-12;
i. capturing and purifying Proinsulin Glargine obtained in step h, by hydrophobic interaction chromatography to obtain purified Proinsulin Glargine;
j. digestion of purified Proinsulin Glargine obtained in step i, to convert it to Insulin Glargine using Recombinant Trypsin or soluble TPCK treated animal Trypsin, wherein the process is carried out directly in the elute from hydrophobic interaction chromatography in step h;
k. optionally purifying Insulin Glargine, obtained in step j, using cation exchange chromatography with resin selected from CM Sepharose, SP Sepharose, CM Cellulose preferably CM Sepharose FF;
1. purification of Insulin Glargine obtained in step k or j by silica based C4, C8 and CI8 Reverse phase chromatography to obtain pure Insulin Glargine with purity >98%;
m. lyophilization or crystallization of pure Insulin Glargine obtained in step I to obtain pharmacopoeial grade.
Glycerol stock of the E. coli BL21 DE3 Gold cells harboring plasmid pET 28a coding for PPG culture is inoculated in the inoculum medium comprising of Luria HiVeg broth, Na2FIP04 2H20, Dextrose, MgS04.7H20, Kanamycin and trace metal solution under continuous shaking at 35-40°C for 9-15 hrs.
10% of the production medium (inoculum) comprising Yeast Extract, KH2PO4, Na2HP04.2H20, (NH4)2S04; NaCl, Dextrose, MgS04.7H20, Thiamine hydrochloride and trace metal solution is inoculated with this inoculum. Fermentation is carried out

at 37°C with air flow 1.0 VVM throughout the process. pH is maintained at 6.8 ± 0.2 with Sodium Hydroxide solution. Dissolved oxygen and agitation is maintained throughout the batch run. Induction of the cells is carried out at mid log phase with IPTG. Foaming during the process is controlled by antifoaming agent. Feeding is carried out with carbon and nitrogen source like glycerol and yeast extract. According to the object of the invention post fermentation, IBs are isolated by harvesting the cells using a centrifuge and suspending the cell pellet obtained after centrifugation in lysis buffer containing 50-100 mM Tris Buffer, 2-5 mM EDTA and 100-200 mM Sodium Chloride in the presence of 100 mM 2-Mercaptoethanol and lysing at 15000 to 20000 psi using high pressure homogenizer. Two passes of the lysis broth are done to achieve a lysis efficiency of > 90%. Post lysis, centrifuging of the cell lysate is done to isolate the IBs. The IBs are purified by first resuspending the pellet in wash buffer containing 50-100 mM Tris Buffer, 2-5 mM EDTA, 1-1.5 M Sodium Chloride, 0.5-1.0% Triton X-100 in the presence of 100 mM 2-Mercaptoethanol. Centrifugation of the IB suspension is then carried out and the pellet is again suspended in wash buffer containing 50-100 mM Tris Buffer, 2-5 mM EDTA, 1-1.5 M Sodium Chloride and 4 mM of 2-Mercaptoethanol. Centrifugation of the suspension is carried out to obtain purified IBs.
The purified IBs are dissolved in denaturing agent like 6 M Guanidine hydrochloride Buffer (containing 50-100 mM Tris Buffer, 2-5 mM EDTA) or 8 M Urea Buffer (containing 50-100 mM Tris Buffer, 1-2 mM EDTA, and 15-20 mM Ethylenediamine) in the ratio of 1:4 to 1:15 (weight of IBs to volume of denaturant) with the pH adjusted to 8 to 12 by 5-10 N Sodium Hydroxide (NaOH). Further sulfitolysis is 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 protein at 4°C - 40°C for 2 to 24 hrs, preferably 20°C-40°C for 2-15 hrs, more preferably at 25°C - 30°C for 4-12 hrs which results in substitution of -SH group of Insulin Glargine Precursor with -SSO3 groups, thus giving dissolved sulfitolysed protein.

The dissolved sulfitolysed protein is then clarified either by centrifugation or using 0.1 |i hollow fiber filtration system. Clarification of the dissolved sulfitolysed protein is carried out at a Trans Membrane Pressure of 3-12 psi. The retentate is then subjected to 5-7 washes of the 3-6 M concentration of guanidine hydrochloride buffer or 4-8 M of Urea buffer in step mode to extract maximum amount of the protein in the permeate. To reduce the handling volume for ease of operation and to increase the protein concentration, the clear permeate is then subjected to ultrafiltration using 3 KDa hollow fiber at a TMP of 5-10 psi. However batch processing with or without using ultrafiltration step has no effect on product quality and quantity and hence can be avoided. Alternatively, clarification is done by using solvents like ethanol, Isopropyl alcohol and methanol. Solvent is added to the protein in a ratio of 1:0.2 to 1:1 (v/v) (protein to solvent ratio) and kept for mixing for 20 minutes. It is then centrifuged to precipitate out the nucleic acids and the clear solution containing the protein of interest is precipitated with 3-5 volumes of water. The precipitate containing the protein of interest is then centrifuged. The pellet obtained containing clarified PPG precursor is dissolved in chaotropic agent like Guanidine hydrochloride buffer, Urea buffer, or formic acid for further processing steps using CNBr cleavage. The clarified PPG precursor with or without carrying out ultrafiltration, is subjected to cyanogen bromide (CNBr) treatment. In this clarified PPG precursor is treated by adding CNBr in the ratio of 1: 0.8 to 1: 1.5, more preferably in the ratio of 1:1 (weight of protein to weight of CNBr) under acidic condition (pH 0.1 to 2.5) in 4-8 M Urea buffer or in 3-6 M Guanidine Hydrochloride buffer and incubating it for 10 to 48 hr at temperature of 4 to 25°C under stirring condition in dark, preferably at 15-20°C for 10-20 hours to give solution containing PG precursor. After CNBr treatment, the solution, containing PG precursor, is precipitated by adding 5-7 volumes of water or 0.02-0.1M Glycine of pH 2-3, more preferably pH 3 to make the final pH of 2.0-3.0, more preferably 2.5 and precipitate is recovered by centrifugation at 8000-10000 rpm for 10-15 min or by 0.1 (j. cross flow filtration using hollow fiber, at a TMP of 2-6 psi to get filtered PG precursor. Cross flow filtration

method used in this to process to prepare insulin Glargine is a novel method which makes the process more simple and cost effective as compared to the other conventional methods mentioned in the prior art.
The filtered PG precursor is purified by dissolving in 8-9M Urea Buffer with pH 8.0-9.0 and loading onto anion exchangers like Capto DEAE, Source 30Q, Source 15Q, Q Sepharose XL, Q Sepharose FF, DEAE Sepharose FF or Cellufine max Q-r, preferably Cellufine max Q-r to remove cleaved polypeptide and other byproducts generated during the CNBr treatment. Eluting purified PG precursor by passing 8M Urea with 0.05 M Tris buffer of pH 8.0-9.0 containing 0.1 to 0.35 M sodium chloride. Refolding of the eluted purified PG precursor is carried out in refolding buffer containing 0.01-0.05 M Glycine preferably 0.02 M Glycine with pH in the range of 10 to 12; more preferably with pH 11.2 with final Urea concentration of 0.2 to 4 M. Final protein concentration is maintained between 0.01 to 2.5 mg/ml, more preferably between 0.1-1.0 mg/ml to obtain PG.
PG is purified from refolding reaction mixture by hydrophobic interaction chromatography (HIC) in 0.02-0.1 M Glycine buffer of pH 9-11. Preferably this is carried out at 30 mM Glycine buffer of pH 9.5 to obtain purified PG. The resin for purification of the refolded protein by HIC is selected from Butyl High performance, Butyl 4 Fast flow, Butyl 6 Fast Flow, Capto butyl, Cellufine butyl; preferably Butyl High performance. PG in the column elute is reacted with Trypsin enzyme using either solubilized or immobilized TPCK treated animal Trypsin or recombinant Trypsin in the ratio of 1:500 to 1:2000 (weight of Trypsin to weight of protein) in presence of organic solvent such as acetonitrile or Dimethyl Sulphoxide (DMSO), more preferably DMSO at a concentration of 20% to 60%, preferably 40% to 50%. As the process of separation of the purified PG from the chromatographic column elute is not performed the steps in the process of preparing insulin Glargine are reduced. The loss during separation by lyophilization or filtration is also avoided. The time required for filtration or lyophilization is circumvented, therefore reduced. Thus

the direct enzymatic reaction without separation of the purified PG makes the process economical, high yielding and efficient.
Alternatively, the refolded PG is purified by reverse phase C8 preparative HPLC using Acetonitrile in the mobile phase. Trypsinization is then carried out in the buffer exchanged protein or in the lyophilized protein after dissolving it in 0.1M Glycine buffer of pH 11.0 with trypsin using either solubilized or immobilized TPCK treated animal Trypsin or recombinant Trypsin in the ratio of 1:500 to 1:2000 (weight of Trypsin to weight of protein) in presence of organic solvent such as acetonitrile or Dimethyl Sulphoxide (DMSO), more preferably DMSO at a concentration of 20% to 60%, preferably 40% to 50%.
The process of Trypsin digestion is conducted in an aqueous medium containing 0.1 M Glycine and PG at concentration of up to about 0.1 to 5 mg/ml, more preferably at concentration of 0.5 to 2 mg/ml and most preferably from about 1 to 1.5 mg/ml, and organic solvent such as acetonitrile or Dimethyl Sulphoxide (DMSO), more preferably DMSO at a concentration of 20% to 60%, preferably 40% to 50%. The term "aqueous medium" requires the presence of water; it does not, however, preclude the presence of water-miscible organic solvents such as methanol, ethanol, acetone, N, N-dimethylformamide, DMSO and the like.
Surprisingly enzymatic conversion was carried out efficiently with the use of very less amount of Trypsin. Unlike prior art, for 2000 grams of protein only 1 gram of Trypsin was required for the conversion. As the enzymatic conversion is carried out with extremely low Trypsin to protein ratio, the process of enzymatic conversion could be performed even in diluted solution of protein. Surprisingly this inventive process can be applied for enzymatic conversion of protein in the elute obtained from the column, wherein the protein is in highly diluted form. To perform the process of enzymatic conversion directly in the chromatographic elute is not taught, suggested or motivated by any of the prior art. In fact in the prior art, the ratio of trypsin to protein is very high, which requires the purified protein to be concentrated for trypsinization. The prior art discourages the trypsinization in the diluted solution and

does not make any reference to the trypsinization reaction in the elute. Thus the process is novel and non-obvious to the person skilled in the art over the prior art. Since in the present invention trypsinization process is directly performed in the column elute obtained from chromatography step conversion of Glargine precursor to Glargine obtained in the present invention is in the range of 60-75%, which is high as compared to the processes employed currently.
The reaction is carried out at any of a wide range of temperatures, generally from about 2°C to about 40°C, preferably at a temperature of 2°C-10°C. The pH of the reaction mixture is in the range of 7 to 12. However, best results are obtained when the reaction is conducted at a pH in the range of 11.0 to 12.0. The pH control is assisted by the use of a buffering agent. Typical buffers employed are TRIS, ethylene diamine, Bicine, triethanolamine, glycine, Ammonium Carbonate, Phosphate, Ethylenediamine, Triethanolamine, Tricine, EPPS [(N-(2-Hydroxyethyl) piperazine N'-(2-hydroxypropanesulfonic acid)], HEPES [N-(2-hydroxyethyl) piperazine-N'-(2 hydroxythanesulfonic acid)], MOPS [3-(N-morpholino) propanesulfonic acid], AMP [2-amino-2-methyl-l-propanol], CAPS [3-(cyclohexylamino)-l-propanesulfonic acid], CHES [2-(N-cyclohexylamino) ethanesulphonic acid], TAPS [N-Tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid]. After complete conversion of the PG to Insulin Glargine, diluted DMSO concentration to 10-20% with water and the reaction is inhibited by dropping the pH of the reaction mixture to 3.0-4.0, preferably 3.5 with an organic acid like acetic acid.
Further Insulin Glargine is purified by loading Insulin Glargine onto a cation exchanger resin like CM Sepharose, SP Sepharose, CM Cellulose preferably CM Sepharose FF pre-equilibrated with 50-100 mM acetate buffer having pH 3.5 - 4.0 (Buffer A) for the removal of partially digested protein fragments, un-cleaved PG and other related impurities from Insulin Glargine. Post loading, 2-3 column volumes (CVs) of equilibration buffer wash is given followed by 55% of buffer B wash containing 0.1M Glycine or 50-100 mM sodium acetate or acetate buffer, 0.5 M sodium chloride, 30% acetonitrile, and having pH 4.0-5.0. Elution is then carried out

using a linear gradient of buffer B from 55% to 90% buffer for 15 CVs. The eluted protein is adjusted to pH 3.0-4.0 with acetic acid and further purified on a silica based C4, C8, CI8 Reverse Phase Column (RPC) to obtain a final purity of >98%. Alternatively protein can also be purified directly using C4, C8, CI8 RPC column after the enzymatic digestion step without affecting the purity levels. To the protein onto the RPC, equilibration buffer (10% acetonitrile with 0.4% acetic acid) wash was given, followed by 15% buffer B (70% acetonitrile with 0.4% acetic acid) wash in step mode to remove the bound impurities from the column. Elution of Insulin Glargine is carried out with a linear gradient of 12% to 20%, preferably 15% to 16% of buffer. In addition to acetonitrile, alcohols like Isopropyl alcohol can be used in the mobile phase for elution of pure pharmacopoeial grade Insulin Glargine. The present invention also relates to the process of enzymatic conversion of purified Proinsulin Glargine (PG) using trypsin directly in the chromatographic column elutes The present invention also relates to the process of enzymatic conversion of purified PG using trypsin, wherein ratio of trypsin to purified PG is 1:500 to 1:2000 The present invention further relates to the process of enzymatic conversion of purified PG to obtain insulin Glargine by trypsin digestion wherein ratio of trypsin to purified PG is 1:500 to 1:2000 and wherein the process is carried out in chromatographic column elute.
PG is prepared from PPG precursor comprising the steps of fermentation process to express the PPG precursor in the form of IBs. The IBs are then isolated and purified from the fermentation broth and protein is further digested, precipitated and refolded to obtain PG by the process known in the art. According to the aspect of the invention the PG prepared is purified from refolding reaction mixture by hydrophobic interaction chromatography in 0.02-0.1 M Glycine buffer of pH 9-11. Preferably this is carried out at 30 (0.03) mM Glycine buffer of pH 9.5 to obtain purified PG. The resin for purification of the refolded protein by Hydrophobic Interaction Chromatography is selected from Butyl High performance, Butyl 4 Fast flow, Butyl 6 Fast Flow, Capto butyl, Cellufine butyl; preferably Butyl High performance. The

purified PG in the elute is then reacted with the trypsin enzyme without separation from the elute. In the process the PG is reacted with Trypsin using either solubilized or immobilized TPCK treated animal Trypsin or recombinant Trypsin in presence of organic solvent such as acetonitrile or Dimethyl Sulphoxide (DMSO), more preferably DMSO at a concentration of 20% to 60%, preferably 40% to 50%. According to another aspect of the invention Trypsin to Purified PF ratio is from 1:500 to 1:2000. The process of Trypsin digestion is conducted in an aqueous medium containing 0.1 M Glycine and PG at concentration of up to about 0.1 to 5 mg/ml, more preferably at concentration of 0.5 to 2 mg/ml and most preferably from about 1 to 1.5 mg/ml, and organic solvent such as acetonitrile or DMSO, more preferably DMSO at a concentration of 20% to 60%, preferably 40% to 50%. The term "aqueous medium" requires the presence of water; it does not, however, preclude the presence of water-miscible organic solvents such as methanol, ethanol, acetone, N, N-dimethylformamide, DMSO and the like.
Surprisingly enzymatic conversion was carried out efficiently with the use of very less amount of enzyme i.e. Trypsin. Unlike prior art, for 2000 grams of protein only 1 gram of Trypsin was required for the conversion. As the enzymatic conversion is carried out with extremely low Trypsin to protein ratio, the process of enzymatic conversion could be performed even in diluted solution of protein. Surprisingly this inventive process can be applied for enzymatic conversion of protein in the elute obtained from the column, wherein the protein is in highly diluted form. To perform the process of enzymatic conversion directly in the chromatographic elute is not taught, suggested or motivated by any of the prior art. In fact in the prior art, the ratio of trypsin to protein is very high, which requires the purified protein to be concentrated for trypsinization. The prior art discourages the trypsinization in the diluted solution and does not make any reference to the trypsinization reaction in the elute. Therefore the process can be said to be novel and non-obvious to the person skilled in the art over the prior art.

Since in the present invention trypsinization process is directly performed in the column elute obtained from chromatography step conversion of Glargine precursor to Glargine obtained in the present invention is in the range of 60-75%, which is high as compared to the processes employer currently. Typically process of this invention is carried out by following ways:
The PG precursor is prepared by cultivating the E coli harboring a plasmid containing the sequence for Insulin Glargine Precursor by Fed Batch fermentation to obtain the precursor as IBs. The IBs are centrifuged and further washed by buffer containing Triton X 100 and EDTA and centrifuged to obtain purified IBs. The purified IBs are dissolved in denaturing agent like 6 M Guanidine HC1 Buffer or 8 M Urea Buffer in the ratio of 1:4 to 1:15 (w/v). Sulfitolysis is 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 same at 4°C- 40°C for 2 to 24 hrs, preferably 20°C-40°C for 2-15 hrs, most preferably at 22°C-30°C for 4-12 hrs which results in substitution of -SH group of Insulin Glargine Precursor with -SSO3 groups. The reaction mixture containing sulfonated Insulin Glargine Precursor is then clarified and concentrated by microfiltration using cross flow filtration and then subjecting to cyanogen bromide (CNBr) treatment by adding cyanogen bromide in the ratio of 1: 0.8 to 1: 1.5 more preferably in the ratio of 1:1 (weight of protein to weight of CNBr) under acidic condition (pH 0.1 to 2.5) in 8 M Urea buffer or in 6 M Guanidine Hydrochloride buffer and incubating it for 15 to 48 hrs at temperature 4 to 25°C under stirring condition in dark. The solution after CNBr treatment is precipitated by adding 5-7 volumes of water or 0.02-0.1M Glycine of pH 2.0-3.5 more preferably pH 3.0 to make the final pH of 2.0-2.8, most preferably 2.5 and precipitate is recovered by centrifugation at 8000-10000 rpm for 10-15 min or by cross flow filtration using O.lu hollow fibers. The concentrated slurry is further washed with 20 mM Glycine; pH 2.5 for removal of the traces of CNBr. Protein is recovered from hollow fiber by circulating 8-9 M Urea with 0.05 M Tris buffer to recover the trapped protein

precipitate in soluble form. The precipitate or concentrated slurry is dissolved in the circulated 8 M or 9 M Urea Buffer, pH 8.0-9.0 and loaded on anion exchange chromatographic column for removal of cleaved polypeptide and other byproducts generated during the CNBr treatment step. Alternatively diafiltration may be carried out for the solution after CNBr treatment to bring it in 8 M Urea buffer of pH 8 - 9.0 and remove CNBr digestion products before loading onto the anion exchange column. Pure sulfonated precursor for PG is eluted by passing 8 M Urea buffer of pH 8.0-9.0 containing 0.1 to 0.35 M sodium chloride. Refolding of the eluted sulfonated PG is carried out in folding buffer containing 0.01-0.05 M Glycine, preferably 0.02 M Glycine with pH in the range of 10 to 12 more preferably in pH 11.2 with final Urea concentration of 0.2 M to 4 M and protein concentration between 0.01-2.5 mg/ml. The refolded PG is purified from refolding reaction mixture by hydrophobic interaction chromatography in 0.1 M Glycine buffer of pH 8-11. The refolded PG can also be purified by reverse phase C8 preparative HPLC using Acetonitrile as mobile phase and in this case after reverse phase chromatography buffer is exchanged with 0.1 M Glycine buffer of pH 11 or it can be lyophilized to obtain dry powder. The sulfonated PG is then converted to Insulin Glargine in one-step conversion process. In the process of present invention the PG is converted to Insulin Glargine with solubilized or immobilized TPCK treated animal Trypsin, Recombinant Trypsin in the Trypsin to Protein ratio of 1:500 to 1:1000 (w/w), more preferably 1:500 at 6 10°C for 5 to 15 hrs in the presence of 50% DMSO.
The reaction is complete in a period of 5 to 15 hrs and the progress of the reaction is monitored by HPLC. Diluted DMSO concentration to 10-20% with water and the reaction is terminated by and reducing the pH to 3.0 to 4.0 with Glacial acetic acid on complete conversion of PG to Insulin Glargine.
Insulin Glargine is then purified either by cation exchange chromatography followed by reverse phase chromatography or can be directly purified by reversed phase chromatography. In case of purification with cation exchanger, Insulin Glargine is loaded onto the column packed with the cation exchanger equilibrated with 50-100

mM acetate buffer, pH 3.0 - 4.0 (Buffer A) for the removal of partially digested protein fragments, uncleaved and related impurities from Insulin Glargine. Post loading, equilibration buffer wash was given followed by 55% of buffer B wash containing 0.1 M Glycine or 50-100 mM sodium acetate buffer or acetate buffer, 0.5 M sodium chloride, 20% acetonitrile with pH 4.0 to 5.0. Elution is then carried out using a linear gradient of buffer B from 55% to 90%, buffer B containing 20% acetonitrile or N-Propanol preferably acetonitrile for 15 column volumes. Final purification or polishing of the Insulin Glargine is carried out by reverse phase chromatography using C8 columns and Insulin Glargine is eluted first by using step gradient of 15% for 2-3 column volumes to remove the bound impurities followed by linear gradient from 15% to 16% for 10-20 column volumes of mobile phases A & B where Mobile phase A contains 10% Acetonitrile with 0.4% Acetic Acid and mobile phase B contains 70% Acetonitrile with 0.4% Acetic acid. The elute is then directly lyophilized to obtain Insulin Glargine powder. Examples:
The following examples are presented for illustration only, and are not intended to limit the scope of the invention or appended claims.
Example 1: IB Preparation:
From the cells obtained from fermentation, harvesting was carried out in batch
centrifuge at 9000 RPM for 11 ± 1 minute. Harvest pellet of 720g was obtained. Harvest pellet was suspended in buffer containing 50 mM Tris, 5mM EDTA, 150 mM Sodium Chloride and 100 mM B-Mercaptoethanol (PME) and kept for stirring for 2 hours. Lysis was carried out at 18000 ±1000 psi by using High Pressure Homogenizer. After two passes, centrifugation was carried out and the pellet was suspended in buffer containing 50mM Tris, 5mM EDTA, 1M Sodium Chloride, 0.5%) Triton X-100 and lOOmM BME, pH8.5 and kept for stirring for lhr. Centrifugation was carried out and pellet was suspended in same buffer except Triton X-100. It was

then reduced with 4 mM (3-ME and kept for mixing for 2 hours. Centrifugation was carried out and pellet weight obtained was 180 gm.
Example 2: Preparation of PG from PPG
IBs 180g isolated by using the procedure as described in example 1 were dissolved in 720 ml of 6 M Guanidine Hydrochloride containing 50mM Tris, 2 mM EDTA. The pH of the solution was adjusted to 9.2 with 5 N Sodium hydroxide and the solution was then reduced by adding 242 jxl of 14 M stock 2-mercaptoethanol. Sulfitolysis was carried out by adding 7.75 g Sodium Tetrathionate and 16.08 g sodium sulphite and incubating the protein at 37°C for 3hr under continuous stirring. The reaction mixture (850 ml) was then clarified by centrifugation after addition of 510 ml of Isopropyl alcohol and stirring for 30 minutes. The pellet contains undissolved or partially solubilized IBs, nucleic acids and few other proteins. The clear supernatant was then added to 3.4L of water to precipitate the protein. The precipitate was then recovered by centrifugation and the pellet obtained was dissolved in 600 ml of 6 M Guanidine hydrochloride buffer. The pH of the clear protein solution (700 ml) having protein concentration (measured on the basis of A280 nm) of 21.8 mg/ml containing a total of 15.26 g was adjusted to 1.0 by addition of Hydrochloric acid and was cooled to 8°C. Cyanogen bromide (CNBr) (15.5 g) was then added and the reaction was carried out at 8°C for 12 hr with continuous stirring in dark. The reaction mixture was then diluted with 5 volumes (3.5L) of water to precipitate out the protein. It was then centrifuged to recover the protein pellet and the pellet was suspended in 20mM Glycine buffer pH 2.5 to wash off CNBr from the protein pellet. It was then re-centrifuged to obtain a tight pellet and the pellet obtained was dissolved in 8M urea buffer containing 50mM Tris, ImM EDTA, 18mM EDA. pH of the clear solution containing 8.8g was then adjusted to 8.6 with 3N hydrochloric acid and loaded onto 110ml Cellufine Max Q r column pre-equilibrated with 6M Urea Buffer containing 20mM tris, ImM EDTA, 18mM EDA pH 8.5. After complete loading, column was washed with 3 column volumes of 6M Urea Buffer pH 8.5 containing 0.1M sodium

chloride. The protein of interest was eluted by passing 5 column volumes of 8M Urea Buffer of pH 8.5 containing 0.3 M NaCl. The pH of the elute 1.65g (155 ml) was adjusted to 10.8 with 5M Sodium hydroxide solution and added to 3.58L refolding buffer containing 20mM Glycine pH 11.5 and 228.0|il of 2-Mercaptoethanol maintaining final urea concentration of 0.8M and final protein concentration of 0.4mg/ml. Refolding reaction was carried out for a period of 3 Hrs and was monitored hourly by HPLC. It was then terminated by addition of 40 ml of glacial acetic acid to drop the pH of the reaction to 3.8.
To the 4.0 L of refolding solution 320 ml of 5 M sodium chloride solution was added and stirred for 1 hour at room temperature. The solution was then clarified by centrifugation to give clear solution thus separating the pellet containing protein aggregates and other impurities. Clear protein solution was loaded onto 90 ml column bed of Butyl HP, pre-equilibrated with 20 mM Glycine, pH 3.8 with 0.4 M sodium chloride, pH 4.0. The column was then washed to remove the unbound impurities with equilibration buffer followed by 0.1 M Glycine buffer, pH 9.50 containing 0.5 M sodium chloride for 2 column volume. PG was eluted using 30mM Glycine buffer pH 9.50. Total of 520 mg protein was obtained in elute.
Example 3: Conversion of PG to Purified Insulin Glargine
To the elute from Butyl HP column (390 ml) from example 2 containing 520 mg of purified pro-Glargine with protein concentration of 1.21 mg/ml, DMSO in 1:1 ratio was added and the pH was adjusted to 11.5 with sodium hydroxide. The protein solution was then cooled to 6°C and treated with Trypsin in the ratio of 1:500 (w/w). Reaction was quenched by addition of purified water to reduce DMSO concentration to 20% and acetic acid to reduce the pH of reaction mixture to pH 4.0. 60% of digested protein as analyzed by HPLC was obtained after 13 Hrs and loaded onto 20 ml column, packed with cation exchanger CM FF column pre-equilibrated with 50 mM acetic acid pH 4.0. Column was then washed with equilibration buffer, followed by wash with 5CV of 55% Buffer B (0.1M Glycine, 0.5M Sodium chloride

containing 30% acetonitrile) and elution was carried out by giving a 15 CV linear gradient from 55 to 90 % of Buffer B. Eluted peak was fractionated and pooled. CM FF protein 304mg was further polished from related impurities by RPC C8 by loading onto YMC make C8 reversed phase preparative column pre-equilibrated with mobile phase A (90% water, 10% Acetonitrile, 0.4% Glacial acetic acid). Post loading, 15% Buffer B (30% water, 70% ACN, 0.4% Glacial Acetic Acid) wash of 3 CVs given followed by elution with a linear gradient from 15% - 16% Buffer B in 15 CVs. Target protein peak was fractionated and fractions were analyzed on 15% Native PAGE gel and HPLC. Fractions containing pure Insulin Glargine were pooled to obtain 75mg final yield. Insulin Glargine powder was obtained after lyophilization.
Example 4: Conversion of PG to Purified Insulin Glargine
935 mg of PG was loaded onto HIC column Butyl Sepharose HP column. 568 mg of purified PG was obtained in the elute at a concentration of 0.8g/L. To the elute 710 ml of DMSO was added to get a final DMSO concentration of 50%). The solution was then cooled to 6°C and pH adjusted to 11.5. 1.13 mg recombinant trypsin was added to the protein solution and kept for stirring at 6°C for 12 hours. Reaction progress was monitored by HPLC. 60% conversion of PG to Insulin Glargine was achieved. Example 5: IB Preparation:
From the cells obtained from fermentation, harvesting was carried out in batch centrifuge at 9000 RPM for 11 ± 1 minute. Harvest pellet of 2250 g was obtained. Harvest pellet was suspended in buffer containing 50 mM Tris, 5mM EDTA, 150mM Sodium Chloride and 100 mM B-Mercaptoethanol (pME) and kept for stirring for 2 hours. Lysis was carried out at 18000 ±1000 psi by using High Pressure Homogenizer. After two passes, centrifugation was carried out and the pellet was suspended in buffer containing 50mM Tris, 5mM EDTA, 1M Sodium Chloride, 0.5% Triton X-100 and lOOmM, pH 8.5 and kept for stirring for 1 hour. Centrifugation was carried out and pellet was suspended in the same buffer except Triton X-100. It was

then reduced with 4mM p-ME and kept for mixing for 2 hours. Centrifugation was carried out and pellet weight obtained was 540 gm.
Example 6: Preparation of PG from PPG
IBs 540 g prepared with procedure as described in example 4 were dissolved in 2160 ml of 6M Guanidine Hydrochloride Buffer and kept for solubilization for 8 hours. The pH of the solution was adjusted to 9.0 by 5 N Sodium hydroxide and was then reduced by adding 800 JJ.1 of 14 M stock 2-mercaptoethanol. Sulfitolysis was carried out by adding 25.5 g Sodium Tetrathionate and 52.9 g sodium sulphite and incubating the protein at 25°C for 9 hr under continuous stirring. The reaction mixture (2800 ml) was then clarified by cross flow filtration on 0.1 \i hollow fiber filter. 8 washes of 6M Guanidine buffer were given to the retentate in step mode and the washes were pooled with the permeate to get a volume of 7L. The clarified protein was then concentrated on 3KDa hollow fiber membrane at a TMP of 7 psi to get a final volume of 3.2L. The pH of the concentrated protein (3200 ml) having protein concentration (measured on the basis of A280 nm) of 22.2 mg/ml was adjusted to 1.1 by addition of concentrated Hydrochloric acid and was cooled to 15°C. Cyanogen bromide (CNBr) (71 g) was then added and the reaction was carried out for 8 hr with continuous stirring in dark. The reaction mixture was then diluted with 5 volumes of water to precipitate out the protein followed by adjustment of pH to 2.5 with pH 11.0 Glycine. Protein precipitates were recovered by Cross flow filtration using 0.1 \i Hollow fiber cartridge, maintaining a TMP of 4 psi. Concentrated precipitates were subsequently washed with of 20 L ml of 0.02M Glycine, pH 2.5 in step mode of 2 L each to remove traces of CNBr. The cartridge was washed with 6 L of 8 M Urea Buffer to remove the trapped protein from the cartridge. Protein precipitates slurry was slowly dissolved in 8 M Cartridge wash (6000 ml) with continuous stirring. The pH of the protein solution having 53 g of total protein (measured at A280nm) was adjusted to 8.6 with 3N hydrochloric acid and loaded onto 1.2L Cellufine Q r column pre-equilibrated with 6M Urea Buffer of pH 8.5. Column was washed with 3 column

volume of 6M Urea Buffer of pH 8.5 containing 0.11M sodium chloride. The protein of interest was eluted by passing 5 column volumes of 8 M Urea Buffer of pH 8.5 containing 0.3 M NaCl. 10.0 g of elute was obtained from the column. Refolding was then carried out for the elute after adjustment of its pH to 10.8. It was then added to 25000 ml buffer containing 1.43 ml of 2 mercaptoethanol, pH 11.0 maintaining final urea concentration of 0.8M and final protein concentration of 0.4mg/ml. Refolding reaction was carried out for a period of 3 Hrs and was monitored hourly by HPLC. It was then terminated by dropping pH to 3,8 using 270 ml of glacial acetic acid. To the 27.0 L of refolding solution was added 2.16 L of 2 M sodium chloride and solution was stirred for 1 hour. The solution was then clarified by passing it through depth filter and the filtrate was loaded onto 500ml column packed with Butyl HP, pre-equilibrated with 20 mM Glycine, pH 3.8 with 0.4 M sodium chloride. The column was then washed with equilibration buffer followed by 0.1 M Glycine buffer, pH 9.50 containing 0.5 M sodium chloride for 2 column volume. 1.56g of PG was eluted using 30mM Glycine buffer pH 9.50.
Example 7: Conversion of PG to Purified Insulin Glargine
To the elute from Butyl HP column (2800 ml) from example 5 containing 2.04 g of purified pro-Glargine, DMSO (2800 mL)was added and the pH was adjusted to 11.5 with sodium hydroxide. The protein solution was then cooled to 6°C and treated with Trypsin in a ratio of 1:500 (w/w). Reaction was quenched by addition of water to reduce DMSO concentration to 10% and acetic acid to reduce the pH of reaction mixture to pH 4.0. 60% of digested protein as analyzed by HPLC was obtained after 13 Hrs and loaded onto 100 ml column, packed with cation exchanger CM FF column pre-equilibrated with 50mM acetic acid pH 4.0. Column was then washed with equilibration buffer, followed by wash with 5CV of 55% Buffer B (0.1M Glycine, 0.5M Sodium chloride containing 30% acetonitrile) and elution was carried out by giving a 15 CV linear gradient from 55 to 90 % of Buffer B. Eluted peak was fractionated and fractions containing protein of interest were pooled.

CM FF protein 1205mg was further polished from related impurities by reverse phase C8 column by loading onto YMC make C-8 reversed phase preparative column pre-equilibrated with mobile phase A (90% water, 10% Acetonitrile, 0.4% Glacial acetic acid). Post loading, 15% Buffer B (30% water, 70% ACN, 0.4% Glacial Acetic Acid) wash of 3 CVs given followed by elution with a linear gradient from 15% - 16% Buffer B in 15 CVs. Target protein peak was fractionated and fractions were analyzed on 15% Native PAGE gel and HPLC. Fractions containing the pure Insulin Glargine were pooled to obtain final yield of 912 mg. The Insulin Glargine solution was then lyophilized to get a powder weighing 940 mg.

We Claim:
1. A process for producing pharmacopoeial grade Insulin Glargine from Preproinsulin Glargine comprising the steps of:
a. bacterial fed batch fermentation of E. coli, harboring plasmid coding for
Preproinsulin Glargine to express the Preproinsulin Glargine in the form of
Inclusion bodies;
b. isolation and purification of the Inclusion bodies obtained in step a to get
purified Inclusion bodies;
c. dissolution of purified Inclusion bodies, obtained in step b, in a denaturing
agent and sulfitolization using Sodium Tetrathionate and Sodium Sulfite to
obtain dissolved sulfitolysed protein;
d. clarification of the dissolved sulfitolysed protein obtained in step c at a TMP
of 3-12 psi using solvent selected from ethanol, Isopropyl alcohol and
methanol to get clarified Preproinsulin Glargine precursor;
e. digestion of clarified Preproinsulin Glargine precursor obtained in step d with
Cyanogen bromide under acidic condition with pH 0.1 to 2.5 in presence of
buffer selected from 4-8 M Urea buffer and 3-6 M Guanidine Hydrochloride
buffer to obtain Proinsulin Glargine precursor;
f. precipitation of Proinsulin Glargine precursor obtained in step e with 3-6
volumes of water or with 0.02 - 0.2 M Glycine buffer with pH of 2.0-3.0,
preferably pH 3.0 and recover precipitated Proinsulin Glargine precursor by
cross flow filtration to obtain filtered Proinsulin Glargine precursor;
g. purification of filtered Proinsulin Glargine precursor obtained in step f using
an anion exchanger selected from Capto DEAE, Source 30Q, Source 15Q, Q
Sepharose XL, Q Sepharose FF, DEAE Sepharose FF or Cellufine max Q-r,
preferably Cellufine max Q-r to get purified Proinsulin Glargine precursor;

h. refolding purified Proinsulin Glargine precursor obtained in step g and
convert it to Proinsulin Glargine by dilution method in the refolding buffer
containing 0.01 to 0.05 M Glycine with pH 10-12; i. capturing and purifying Proinsulin Glargine obtained in step h, by
hydrophobic interaction chromatography to obtain purified Proinsulin
Glargine; j. digestion of purified Proinsulin Glargine obtained in step i, to convert it to
Insulin Glargine using Recombinant Trypsin or soluble TPCK treated animal
Trypsin, wherein the process is carried out directly in the elute from
hydrophobic interaction chromatography in step h; k. optionally purifying Insulin Glargine, obtained in step j, using cation
exchange chromatography with resin selected from CM Sepharose, SP
Sepharose, CM Cellulose preferably CM Sepharose FF; 1. purification of Insulin Glargine obtained in step k or j by silica based C4, C8
and CI8 Reverse phase chromatography to obtain pure Insulin Glargine with
purity >98%; m. lyophilization or crystallization of pure Insulin Glargine obtained in step 1 to
obtain pharmacopoeial grade.
2. The Process as claimed in claim lb, wherein the process of isolation of Inclusion
bodies comprises the steps of:
a. Harvesting the fermented cell using centrifuge to obtain cell pellet;
b. Lysing the cell pellet obtained in step a in a lysis buffer to get cell lysate,
optionally more than two times; and
c. Centrifuging the cell lysate obtained in step b to get isolated Inclusion bodies.
3. The process as claimed in claim 2b, wherein the Lysing of the cell pellet is carried
out in lysis buffer comprising 50-100 mM Tris Buffer, 2-5 mM EDTA and 100-
200 mM Sodium Chloride in the presence of 100 mM 2-Mercaptoethanol at
15000 to 20000 psi.

4. The process as claimed in claim lb, wherein the Inclusion bodies are purified by:
a. resuspending the pellet in wash buffer containing 50-100 mM Tris Buffer, 2-5
mM EDTA, 1-1.5 M Sodium Chloride, 0.5-1.0% Triton X-100 in the presence
of 100 mM 2-Mercaptoethanol to obtain resuspended Inclusion bodies;
b. centrifuging the resuspended Inclusion bodies obtained in step a to get
prepurified Inclusion bodies;
c. suspending the prepurified Inclusion bodies in wash buffer containing 50-100
mM Tris Buffer, 2-5 mM EDTA, 1-1.5 M Sodium Chloride and 4 mM of 2-
Mercaptoethanol to obtain suspended Inclusion bodies;
d. Centrifuging the suspended Inclusion bodies to obtain purified Inclusion
bodies.
5. The process as claimed in claim lc, wherein the denaturing agent is selected from 6 M Guanidine hydrochloride Buffer containing 50-100 mM Tris Buffer, 2-5 mM EDTA and 8 M Urea Buffer containing 50-100 mM Tris Buffer, 1-2 mM EDTA, and 15-20 mM Ethylenediamine in the ratio of 1:4 to 1:15 (w/v) with the pH adjusted to 8 to 12
6. The process as claimed in claim Id, wherein protein to solvent ratio of 1:0.2 to 1:1 (v/v).
7. The process as claimed in claim le, wherein in the process of digestion the ratio of weight of protein to weight of Cyanogen Bromide is in the range of 1:0.8 to 1:1.5, more preferable ratio of 1:1.
8. The process as claimed in claim lg, wherein anion exchanger is eluted with 8M Urea with 0.05 M Tris buffer of pH 8.0-9.0 containing 0.1 to 0.35 M sodium chloride.
9. The process as claimed in claim lh, wherein preferred refolding buffer is 0.02 M Glycine with pH in the range of 10 to 12; more preferably in pH 11.2 with Urea concentration of 0.2 to 4 M.

10. The process as claimed in claim li, wherein Hydrophobic Interaction resin is selected from Butyl High performance, Butyl 4 Fast flow, Butyl 6 Fast Flow, Capto butyl and Cellufine butyl, preferably Butyl High performance resin.
11. The process as claimed in claim li, wherein the process is performed in 0.02-0.1 M Glycine buffer of pH 9-11.
12. The process as claimed in claim lj, wherein the weight ratio of trypsin to purified Proinsulin Glargine is in the range of 1:500 to 1:2000 and the organic solvent is selected from acetonitrile and Dimethyl Sulphoxide (DMSO), more preferably DMSO at a concentration of 20% to 60%, preferably 40% to 50%..
13. The process as claimed in claim lj, wherein the range of temperature during the process is from 2° C to 40° C, preferably from 2° C to 10° C and the range of pH of the reaction is from 7 to 12, preferably from 11.0 to 12.0.
14. The process as claimed in claim Ik, wherein Insulin Glargine is eluted using linear gradient from 55% to 90% of buffer containing 0.1M Glycine or 50 mM -100 mM sodium acetate or acetate buffer, 0.5 M sodium chloride, 30% acetonitrile, and having pH 4.0-5.0.
15. The process as claimed in claim 11, wherein Insulin Glargine is eluted using a linear gradient of 12% to 20%, preferably 15%) to 16% of buffer containing 70% acetonitrile with 0.4% acetic acid.
16. A process for enzymatic conversion of purified Proinsulin Glargine using trypsin directly in the chromatographic column elutes.
17. The process as claimed in claim 16, wherein the source of trypsin is Recombinant Trypsin or soluble TPCK treated animal Trypsin.
18. The process as claimed in claim 16, wherein the weight ratio of trypsin to purified Proinsulin Glargine is in the range of 1:500 to 1:2000.

19. The process as claimed in claim 16, wherein the chromatographic column is the column comprising hydrophobic interaction resin and wherein the elute comprises 0.1M Glycine and DMSO to a final concentration of 50% (v/v).
20. A process of enzymatic conversion of purified Proinsulin Glargine to obtain insulin Glargine by trypsin digestion wherein the ratio of trypsin to purified Proinsulin Glargine is 1:500 to 1:2000.
21. The process as claimed in claim 20 wherein, the source of trypsin is Recombinant Trypsin or soluble TPCK treated animal Trypsin
22. The process as claimed in claim 20 wherein, the trypsin digestion is carried out in the chromatographic column elute.
23. The process as claimed in claim 22 wherein, the chromatographic column is the column comprising hydrophobic interaction resin and wherein, the elute comprises of 0.1M Glycine and DMSO to a final concentration of 50% (v/v).
24. A process of enzymatic conversion of purified Proinsulin Glargine to obtain insulin Glargin by trypsin digestion wherein trypsin to purified Proinsulin Glargine is 1:500 to 1:2000 and wherein the process is carried out in chromatographic column elute.
25. The process as claimed in claim 24 wherein, the source of trypsin is Recombinant Trypsin or soluble TPCK treated animal Trypsin.
26. The process as claimed in claim 24 wherein, the chromatographic column is the column comprising hydrophobic interaction resin and wherein, the elute comprises of 0.1 M Glycine and DMSO to a final concentration of 50% (v/v).