FORM 2
THE PATENTS ACT 1970
(39 OF 1970)
COMPLETE SPECIFICATION
(SECTION 10)
IMPROVED PROCESS FOR PREPARATION OF INSULIN
LISPRO
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 PROCESS FOR PREPARATION OF INSULIN LISPRO
Technical Field of the Invention:
The present invention relates to simple and scalable process to prepare Insulin Lispro of pharm/acopoeial grade.
Background of the Invention:
The Insulin Lispro (IL) is a polypeptide protein which plays important role in glucose metabolism. Engineered through recombinant DNA technology, the penultimate lysine and proline residues on the C-terminal end of Human Insulin chain B are reversed in case of Insulin Lispro. This modification does not alter receptor binding, but blocks the formation of insulin dimers and hexamers. The Insulin Lispro is made up of two amino acid chains; "A" chain consisting of 21 amino acids & "B" chain consisting of 30 amino acids, linked to each other with two disulphide bonds at A7-B7 and A20-B19 position, and an intra disulfide bond in A Chain at A6-A11 position.
Figure 1 IL Structure, showing the A and B chains with amino acids sequence.
The process of preparation of Insulin Lispro involves the steps of preparation of the precursor of Insulin Lispro, converting it to Insulin Lispro, purification and crystallization. The processes mentioned in the prior art usually deal with improving any one step.

US5700662 discloses process to prepare Insulin analogues, including Insulin Lispro, which involves two fermentation processes and preparing separately sulfonated A chain and sulfonated B chain and then reacting them together. The process is lengthy, time consuming and results in lower yields of insulin analogues.
WO2010016069 claims a process to prepare Insulin compounds by combinatorially and concurrently treating the precursor with Trypsin and Carboxypeptidase, wherein relative concentration ratio of Trypsin to Carboxypeptidase is from about 5:1 to about 50:1 and Trypsin to protein 1:10 to 1:500 and Carboxypeptidase B to Protein 1:500. Insulin Lispro precursor is prepared using conventional methods. The process involves use of costly enzymes in high amount, thus making the process expensive. It also requires additional unit operation to clear the residual enzymes from the product.
WO2011018745 discloses the process of purification of polypeptides such as Insulin analogous by Reverse phase-High Performance Liquid Chromatography while employing ion pairing agent having concentration range from 0.01% to about 2% in combination with organic modifier having concentration ranging from 5% to about 85%. This process becomes expensive due to use of costly ion pairing agents like Tri fluro acetic acid. Also such ion pairing agents are difficult to handle at larger scale.
WO2012115641 and US20130210716 Al claim a process to prepare Insulin Lispro analogs comprising the steps of a) recombinantly producing a modified Lispro proinsulin; b) folding the modified Lispro proinsulin; c) purifying it using metal affinity chromatography; d) enzymatically cleaving the Lispro proinsulin derivative, wherein trypsin and Carboxypeptidase B ratio with protein is 1500:1 and 1000:1 respectively; and e) purifying by chromatographic column to yield Lispro proinsulin analog. The invention details the use of metal affinity chromatography where there is a chance of metal leaching into product. Further there are multiple steps of Tangential Flow Filtration which makes the process lengthy and less economical. The amount of enzymes required is high, which makes the process costly and less economical.

Object of the Invention:
An object of the present invention is to provide simple and cost effective process for production of pharmacopoeial grade Insulin Lispro (IL).
Another object of the present invention is to prepare Insulin Lispro (IL) wherein precursor protein expressed as inclusion bodies (IBs) during fermentation of recombinant bacterial cells is isolated, chemically treated, partially purified, refolded and enzymatically converted to Insulin Lispro. Chromatographic purification and crystallization of product yields pharmacopoeial grade Insulin Lispro.
Summary of the Invention:
The present invention relates to the simple and cost effective process for production of pharmacopoeial grade Insulin Lispro (IL) comprising the steps of:
a. Expressing Preproinsulin Lispro (PPL) as inclusion bodies (IBs) by Fed-batch
fermentation of recombinant E. coli cells containing plasmid bearing the gene
sequence for PPL;
b. isolation and purification of the IBs obtained in step a to get purified IBs;
c. dissolution of purified IBs obtained in step b in a denaturing agent selected from
Guanidine hydrochloride buffer, urea buffer preferably Guanidine hydrochloride
buffer and carrying out sulfitolysis to obtain dissolved sulfitolysed protein;
d. clarification of the dissolved sulfitolysed protein obtained in step c by methods
selected from dead end filtration, normal flow filtration, centrifugation or
microfiltration to get clarified PPL precursor;
e. digestion of clarified PPL precursor obtained in step d with Cyanogen bromide
(CNBr) under acidic condition with pH from 0.1 to 2.5 and in presence of buffer
selected from 8M Urea buffer and 6M Guanidine Hydrochloride;
f. precipitation of Proinsulin Lispro (PL) precursor obtained in step e with 3-6
volumes, of water or 0.02 - 0.2 M buffer with pH 2.0-3.0 to obtain precipitated
PL precursor;

g. purification of precipitated PL precursor obtained in step f using an anion exchanger selected from Cellufine max Q r, Capto DEAE, Source 30Q, Source 15Q, Q Sepharose XL, Q Sepharose FF, DEAE Sepharose FF , DEAE Sephadex, Capto DEAE, most preferably Cellufine max Q r to obtain purified PL precursor;
h. refolding of purified PL precursor obtained in step g by dilution method in the refolding buffer containing 0.01 to 0.05 M Glycine with pH 10-12 to obtain PL;
i. capturing and purification of PL obtained in step h by hydrophobic interaction chromatography (HIC) to obtain purified PL;
j. enzymatic digestion of purified PL obtained in step i to Insulin Lispro, wherein digestion is carried out directly in the elute from HIC in step i;
k. optional purification of Insulin Lispro obtained in step j using cation exchange chromatography selected from SP Sepharose FF, CM Sepharose FF, CM Sephadex, SP Sepharose BB, preferably CM Sepharose FF and elution with linear gradient from 55% to 90% of buffer containing 0.1M Glycine or 50 mM - 100 mM sodium acetate or acetate buffer with 0.5 M NaCl, 30% acetonitrile and having pH4.0- 5.0;
1. purification of Insulin Lispro obtained in step j or step k, by Reverse phase chromatography to obtain purified Insulin Lispro;
m. crystallization of Purified Insulin Lispro obtained in step 1 is to obtain pharmacopoeial grade Insulin Lispro with >98% purity.
This invention also relates to the process of enzymatic conversion of purified Proinsulin Lispro (PL) using trypsin and Carboxypeptidase B directly in the chromatographic column elutes.
The invention further also relates to the process of enzymatic cleavage of purified PL using trypsin and Carboxypeptidase B, wherein ratio of trypsin and Carboxypeptidase B to purified PL is 1:5000 to 1:20000 and 1:500 to 1:10000, respectively The present invention also relates to the process of enzymatic conversion of purified PL to obtain Insulin Lispro by trypsin and Carboxypeptidase B digestion wherein ratio

of trypsin and Carboxypeptidase B to purified PL is 1:5000 to 1:20000 and 1:500 to 1:10000, respectively and wherein the process is carried out in chromatographic column elute.
Detailed description of the Invention:
The term protein is used for Insulin Lispro or its precursor at any stage of preparation. It means IBs or Preproinsulin Lispro (PPL) Precursor or Preproinsulin Lispro or Proinsulin Lispro (PL) precursor or Proinsulin Lispro or Insulin Lispro as the context may be referring.
The phrases enzymatic cleavage and enzymatic conversion are used interchangeably. The present invention relates to the simple and cost effective process for production of pharmacopoeial grade Insulin Lispro (IL) comprising the steps of:
a. Expressing Preproinsulin Lispro (PPL) as IBs by Fed-batch fermentation of
recombinant E. coli cells containing plasmid bearing the gene sequence for PPL;
b. isolation and purification of the IBs obtained in step a to get purified IBs;
c. dissolution of purified IBs obtained in step b in a denaturing agent selected from
Guanidine hydrochloride buffer, urea buffer preferably Guanidine hydrochloride
buffer and sulfitolyzing to obtain dissolved sulfitolysed protein;
d. clarification of the dissolved sulfitolysed protein obtained in step c by methods
selected from dead end filtration, normal flow filtration, centrifugation or
microfiltration to get clarified PPL precursor;
e. digestion of clarified PPL precursor obtained in step d with CNBr under acidic
condition with pH from 0.1 to 2.5 and in presence of buffer selected from 8M
Urea buffer and 6M Guanidine Hydrochloride;
f. precipitation of PL precursor obtained in step e with 3-6 volumes, of water or
0.02 - 0.2 M buffer with pH 2.0-3.0 to obtain precipitated PL precursor;
g. purification of precipitated PL precursor obtained in step f using an anion
exchanger selected from Cellufine max Q r, Capto DEAE, Source 30Q, Source

15Q, Q Sepharose XL. Q Sepharose FF, DEAE Sepharose FF , DEAE Sephadex, Capto DEAE, most preferably Cellufine max Q r to obtain purified PL precursor;
h. refolding of purified PL precursor obtained in step g by dilution method in the refolding buffer containing 0.01 to 0.05 M Glycine with pH 10-12 to obtain PL;
i. capturing and purification of PL obtained in step h by hydrophobic interaction chromatography (HIC) to obtain purified PL;
j. enzymatic digestion of purified PL obtained in step i to Insulin Lispro, wherein digestion is carried out directly in the elute from HIC in step i;
k. optional purification of Insulin Lispro obtained in step j using cation exchange chromatography selected from SP Sepharose FF, CM Sepharose FF, CM Sephadex, SP Sepharose BB, preferably CM Sepharose FF and eluted with 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 (NaCl), 30% acetonitrile and having pH4.0 - 5.0;
1. purification of Insulin Lispro obtained in step j or step k, by Reverse phase chromatography (RPC) to obtain purified Insulin Lispro;
m. crystallization of Purified Insulin Lispro obtained in step 1 is to obtain pharmacopoeial grade Insulin Lispro with >98% purity.
In the fed-batch fermentation process 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, Na2HP04.2H20, 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, Thiamine hydrochloride, trace metal solution KH2P04, Na2HP04.2H20, (NH4)2S04, NaCl, Dextrose and MgS04.7H20 is inoculated with 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 NaOH 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 respectively.
Post fermentation, the cells are harvested using a centrifuge. The cell pellet is then suspended in lysis buffer containing 50 mM Tris Buffer, 5 mM EDTA and 150 mM Sodium Chloride (NaCl) in the presence of 100 mM 2-Mercaptoethanol and lysed at 15000-20000 psi using high pressure homogenizer. Two passes of the cell suspension are done to achieve lysis efficiency > 90%. Post lysis, the cell lysate is centrifuged and IBs pellet is resuspended in wash buffer containing 50 mM Tris Buffer, 5 mM EDTA, 1 M NaCl, 0.5% Triton X-100 in the presence of 100 mM 2-Mercaptoethanol. IBs suspension is centrifuged and the pellet obtained is resuspended in wash buffer containing 50 mM Tris Buffer, 5 mM EDTA, 4mM of 2-Mercaptoethanol and 1M NaCl. IBs suspension is then centrifuged to obtain pellet containing washed IBs. The use of centrifuge however does not obviate the use of Tangential flow or other filtration techniques to get slurry or a pellet of the desired product.
The IBs are dissolved in denaturing agent like 6M Guanidine hydrochloride Buffer (containing 0.05M - 0.1 M Tris Buffer, 2mM EDTA) or 8M Urea Buffer (0.05M-0.1M Tris Buffer, ImM EDTA, and 18 mM Ethylenediamine) in the ratio of 1:4 to 1:30 (w/v) with the pH adjusted to 8 to 12 by 5-10 N Sodium Hydroxide (NaOH). Sulfitolysis is carried out by adding Sodium Tetrathionate and Sodium Sulfite at final concentration of 40-80 mM and 0.1-0.5 M, respectively and incubating the IBs at 4°C-40°C for 2 -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 with -SSO3 groups. The IBs are then clarified either by centrifugation or by O.lu, microfiltration using microfiltration cassettes or hollow fibers in tangential flow filtration system. Clarification of the sulfitolysed protein is carried out at a TMP of 2.5 to 12 psi. The retentate is then subjected to 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 subjected to ultrafiltration using 3KDa hollow fiber at a TMP of 5-15 psi. However batch processing with or without using ultrafiltration step has no effect on product quality and quantity and hence is avoided. Alternatively, clarification is done by using solvents like ethanol, Isopropyl alcohol, and methanol. Solvent is added to the IBs in a ratio of 1:0.2 to 1:1 (v/v) (IBs to solvent ratio) and kept for mixing for 20 min. It is then centrifuged to remove the precipitated nucleic acids and other impurities and the clear solution containing the protein is precipitated with 3-5 volumes of water. The precipitate containing the protein then centrifuged and the pellet obtained is dissolved in either a chaotropic agent or formic acid for further processing. The precipitate is alternatively recovered by microfiltration by tangential flow filtration system.
The clarified reaction mixture with or without carrying out ultrafiltration containing sulfonated Insulin Lispro Precursor is subjected to CNBr treatment by adding CNBr in the ratio of 1:0.7 to 1:1.5, more preferably in the ratio of 1:1 (w/w, protein to CNBr) under acidic condition (pH 0.1 to 2.5) in 4-8M Urea buffer or in 3-6 M Guanidine Hydrochloride buffer and incubating it for 10 to 48 hrs at temperature of 4 to 25°C under stirring in dark, preferably at 15-20°C for 10-20 hrs. The CNBr treated solution is precipitated by adding 5-7 volumes of water or 0.02-0.1M Glycine, of pH 2-3, preferably pH 3 to make final pH of 2.0-3.0, preferably 2.5 and precipitate is recovered by centrifugation at 8000-10000 rpm for 10-15 min or by 0.1^ cross flow filtration using cassettes or hollow fiber, at a TMP of 2-6 psi.
The precipitate or slurry is dissolved in 8M - 9M Urea Buffer, pH adjusted to 8-9 and loaded on 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 for removal of cleaved polypeptide and other byproducts generated during the CNBr treatment. Pure sulfonated PL is eluted by passing 8M Urea with 0.05 M Tris buffer of pH 8-9 containing 0.1 to 0.35 M NaCl. Refolding of the eluted sulfonated PL is carried out in buffer containing 0.01-0.05 M

Glycine preferably 0.02 M Glycine with pH in the range of 10 to 12; preferably in pH 11 with final Urea concentration of 0.2 to 4M. Final protein concentration is maintained between 0.01 to 2.5 mg/ml, preferably between 0.1-1.0 mg/ml and protein is added slowly to the refolding buffer.
The refolded PL is purified from refolding reaction by HIC in 0.02-0.1 M Glycine buffer of pH 9-11. Preferably this is carried out at 0.1 M Glycine buffer of pH 9 in the presence or absence of an organic solvent such as 10-20% acetonitrile. The resin for purification of the refolded protein by HIC can be any one from Butyl High performance, Butyl 4 Fast flow, Butyl 6 Fast Flow, Capto butyl, Cellufine butyl, preferably Butyl High performance. The Purified PL in the elute is directly taken for enzymatic cleavage by trypsin and carboxypeptidase without separation.
Surprisingly enzymatic conversion was carried out efficiently with the use of very less amount of enzymes trypsin and Carboxypeptidase B. Unlike prior art, for 20000 and 10000 gms of protein only 1 gm of Trypsin and Carboxypeptidase B, respectively is required. As the enzymatic conversion is carried out with extremely low Trypsin or Carboxypeptidase B 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 HIC column, wherein the protein is in highly diluted form. Such process of enzymatic conversion directly in the elute is not taught, suggested or motivated by prior art. In fact in the prior art, the ratio of trypsin or Carboxypeptidase B to protein is very high, which requires the purified protein to be concentrated for enzymatic cleavage. The prior art discourages enzymatic cleavage in the diluted solution and does not make any reference to the enzymatic cleavage reaction in the elute. Therefore the process can be said to be novel and non-obvious to person skilled in the art over the prior art. Alternatively, the refolded PL can also be purified by reverse phase C8 preparative HPLC using Acetonitrile in the mobile phase. Since in the present invention enzyme cleavage is directly performed in the column elute obtained from chromatography

step, conversion of Lispro precursor to Lispro obtained in the present invention is in the range of 75-90%, which is high as compared to the processes employed currently.
In the process of the present invention the C-peptide is cleaved by double digestion using one enzyme at a time or a combination of enzymes comprising of solubilized TPCK treated animal Trypsin or recombinant Trypsin in 1:5000 to 1:20000 (w/w of Trypsin to protein), preferably in the ratio of 1:10000, and Carboxypeptidase B in the ratio of 1:500 to 1:10000, preferably in the ratio of 1:5000 (w/w Carboxypeptidase B to protein). The digestion is accelerated in presence of either 0.05-0.2 mM, preferably 0.075-0.15 mM Zinc chloride or 0.5 to 2 mM, preferably 1 mM CaCl2.
The process of Trypsin and Carboxypeptidase B digestion is conducted in an aqueous medium. 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, and the like. The PL is present in the aqueous medium containing 0.1 M Glycine, with or without 10-20% acetonitrile at protein concentration of upto 0.1 to 5 mg/ml, preferably at 0.5 to 2 mg/ml. The reaction is carried out at wide range of temperatures, generally from 8°C to 40°C, preferably; the reaction is conducted at a temperature of 15-25°C. The pH of the reaction mixture is in the range from 7 to 12. Best results are obtained at pH in the range of 8 to 10. pH control is assisted by the use of a buffering agent. Wide range of buffers employed are TRIS, ethylene diamine, triethanolamine, glycine, Ammonium Carbonate, Phosphate, Ethylenediamine, Triethanolamine, Tricine, Bicine, EPPS [(N-(2-Hydroxyethyl) piperazine-N'-(2-hydroxypropanesulfonic acid)], HEPES [N-(2-hydroxyethyl) piperazine-N'-(2-hydroxythanesulfonic acid)], MOPS [3-(N-morpholi-no) propanesulfonic acid], AMP [2-amino-2-methyl-l-propanol], CAPS [3-(cyclo-hexylamino)-l-propanesulfonic acid], CHES [2-(N-cyclohexylamino) ethanesul-phonic acid], TAPS [N-Tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid]. After complete conversion of the PL to Insulin Lispro, the reaction is inhibited by

dropping the pH of the reaction mixture to 3-4, preferably 3.5 with an organic acid like acetic acid.
The reaction mixture containing Insulin Lispro is then loaded onto a cation exchanger resin like CM Sepharose, SP Sepharose, CM Cellulose preferably CM Sepharose preequilibrated with 50-100 mM acetate buffer, pH 3-4 (Buffer A) for the removal of partially digested protein fragments, un-cleaved protein precursors and partially digested protein fragments. Post-loading, 15% of buffer B wash containing 50-100 mM acetate buffer, 1M NaCl and 20% acetonitrile is given. Elution is then carried out using a linear gradient of buffer B from 20% to 50%) buffer B containing 20% acetonitrile or N-Propanol preferably acetonitrile. The eluted protein is adjusted to pH 3.0-3.8 with acetic acid and diluted to bring down the acetonitrile concentration to <10 percent
Final polishing or purification of the Insulin Lispro is carried out by RPC using C4, C8 or CI8, preferably C8 columns with or without using the cation exchange chromatography as described above. Insulin Lispro is eluted in 2 to 4 Column volumes with a linear gradient of 15 to 20% of mobile phase containing 90% acetonitrile with 0.4% acetic acid for 15 column volumes. Alternatively other solvents, like ethanol, methanol, Isopropyl alcohol can be used in place of acetonitrile in the mobile phase for elution of pure Insulin Lispro.
Alternatively the refolded PL is first digested with soluble/immobilized TPCK (L-l-tosyl amido-2-phenyl ethyl chloromethyl ketone) treated animal Trypsin or recombinant Trypsin at the temperature of 2-8°C and pH of 10.5-12, optionally in the presence of 0.5 to 2 mM, more preferably ImM CaCl2. Trypsin digested protein is either purified by ion exchange chromatography and digested with Carboxypeptidase B enzyme or is directly digested with Carboxypeptidase B enzyme without purification. Carboxypeptidase B enzyme digestion is carried out at 35-37°C and at pH of 8 - 9 in the presence of 0.05-0.2 mM, more preferably 0.075-0.15 mM Zinc Chloride. Carboxypeptidase B enzyme digested protein is then purified as explained

above. Also the protein to enzyme ratio for both Trypsin and Carboxypeptidase B, in this alternative digestion method is same as the previous method.
The amount of enzymes; Trypsin and Carboxypeptidase B generally used depend upon the concentration of each enzyme and the amount of PL. The enzymes can be incorporated in the reaction mixture separately either in solution forms or using known techniques they can be immobilized on a suitable support and thereby made available in the reaction medium. The pure Insulin Lispro obtained is crystallized in presence of zinc and phenol, crystals obtained are filtered or centrifuged and vacuum dried or lyophilized to obtain pharmacopoeial grade Insulin Lispro.
According to this object of the invention the bacterial fed batch fermentation of E. coli BL21 DE3 Gold cells harboring plasmid pET 28a coding for PPL culture is carried out by the processes known in the prior art.
Further invention relates to the process of enzymatic conversion of purified PL using trypsin and Carboxypeptidase B directly in the chromatographic column elutes. The present invention also relates to the process of enzymatic cleavage of purified PL using trypsin and Carboxypeptidase B, wherein ratio of trypsin and Carboxypeptidase B to purified PL is 1:5000 to 1:20000 and 1:500 to 1:10000, respectively.
The present invention also relates to the process of enzymatic conversion of purified PL to obtain Insulin Lispro by trypsin and Carboxypeptidase B digestion wherein ratio of trypsin and Carboxypeptidase B to purified PL is 1:5000 to 1:20000 and 1:500 to 1:10000, respectively and wherein the process is carried out directly in chromatographic column elute.
According to the aspect of this invention Purified PL obtained by the process wherein precursor protein expressed as IBs during fermentation of recombinant bacterial cells is isolated, chemically treated, partially purified, refolded and purified using HIC, is enzymatically cleaved using trypsin and Carboxypeptidase B in the elute obtained from HIC.

In the process of the present invention the C-peptide is cleaved by double digestion using one enzyme at a time or a combination of enzymes comprising of solubilized TPCK treated animal Trypsin or recombinant Trypsin in 1:5000 to 1:20000 (weight of Trypsin to weight of protein), more preferably in the ratio of 1:10000, and Carboxypeptidase B in the ratio of 1:500 to 1:10000, more preferably in the ratio of 1:5000 (weight of Carboxypeptidase B enzyme to weight of protein). The digestion reaction can be accelerated in presence of either 0.05-0.2 mM, more preferably 0.075-0.15 mM Zinc chloride or 0.5 to 2 mM, more preferably ImM CaCl2.
The process of Trypsin and Carboxypeptidase B digestion is conducted in an aqueous medium. 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, and the like. The PL is present in the aqueous medium containing 0.1 M Glycine, with or without 10-20% acetonitrile at protein concentration of up to about 0.1 to 5 mg/ml, more preferably at concentration of 0.5 to 2 mg/ml
The reaction is carried out at any of a wide range of temperatures, generally from about 8°C to about 40°C, preferably; the reaction is conducted at a temperature of 15-25°C. The pH of the reaction mixture can range anywhere from 7 to 12. However, best results are obtained by careful pH control such that the reaction is conducted at a pH in the range of 8 to 10. The pH control generally is assisted by the use of a buffering agent. Any of a wide range of typical buffers can be employed. Examples of suitable buffers are TRIS, ethylene diamine, triethanolamine, glycine, Ammonium Carbonate, Phosphate, Ethylenediamine, Triethanolamine, Tricine, Bicine, 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 PL to Insulin Lispro, 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.
Surprisingly enzymatic conversion was carried out efficiently with the use of very less amount of enzymes trypsin and Carboxypeptidase B. Unlike prior art, for 20000 grams of protein only 1 gram of Trypsin was required and for 10000 grams of protein only 1 gm of Carboxypeptidase B is required. As the enzymatic conversion is carried out with extremely low Trypsin or Carboxypeptidase B 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 HIC 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 or Carboxypeptidase B to protein is very high, which requires the purified protein to be concentrated for enzymatic cleavage. The prior art discourages the enzymatic cleavage in the diluted solution and does not make any reference to the enzymatic cleavage 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.
Typically process of this invention is carried out by following ways:
PPL is expressed in the form of IBs by bacterial fermentation. Post fermentation the cells containing expressed protein as IBs are harvested and suspended in lysis buffer and are lysed at 15000 to 20000 psi using high pressure homogenizer by two passes of the cell suspension with lysis yield of more than 90%. The IBs are centrifuged and further washed by washing 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 8M Urea Buffer in the ratio of 1:4 to 1:30 (w/v). Sulfitolysis is carried out by adding Sodium Tetrathionate and Sodium Sulfite at final

concentration of 40-80-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 Lispro Precursor with -SSO3 groups. The reaction mixture containing sulfonated Insulin Lispro Precursor is then clarified and concentrated by 0.1 |i micro filtration using cross flow filtration and then subjecting to CNBr treatment by adding CNBr in the ratio of 1: 0.7 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°C 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 0.1 u cross flow filtration using cassettes or hollow fibers, at a TMP of 2 - 6 psi. The concentrated slurry is further washed with 20 mM Glycine; at pH 2.5 for removal of the traces of CNBr. The trapped protein precipitate in soluble form is recovered from hollow fiber by circulating 8 M - 9 M Urea with 0.05 M Tris buffer. The precipitate or concentrated slurry is dissolved in the circulated 8 M or 9 M Urea Buffer at 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 8.0 - 9.0 and remove CNBr digestion products before loading onto the anion exchange column. Pure sulfonated PL is eluted by passing 8 M Urea buffer of pH 8.0-9.0 containing 0.1 to 0.35 M NaCl. Refolding of the eluted sulfonated PL 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 11.0 with final Urea concentration of 0.2 M to 4 M and protein concentration between 0.01-2.5 mg/ml. The refolded PL is purified from refolding reaction mixture by HIC in 0.1 M Glycine buffer of pH 9-11. The refolded PL can also be purified by

reverse phase C8 preparative HPLC using Acetonitrile in the mobile phase and in this case after RPC buffer is exchanged with 0.1 M Glycine buffer of pH 11 or alternatively it can be lyophilized to obtain dry powder.
The sulfonated PL is then converted to Insulin Lispro in one-step process. The C-peptide in the PL is cleaved with TPCK treated animal Trypsin, Recombinant Trypsin or soluble TPCK treated animal Trypsin, wherein Trypsin to Protein ratio used was 1:5000 to 1:20000 (w/w), preferably 1:10000 and Carboxypeptidase B to protein ratio used was 1:500 to 1:10000 preferably 1:5000 in 0.02 - 0.1 M Glycine buffer of pH 8 -10 at 15-25°Cfor5to 15 hrs.
The reaction is complete in 5 to 15 hrs. The reaction is monitored by HPLC. On complete conversion of PL to Insulin Lispro reaction is terminated by reducing the pH to 3.5 to 4.0 with Glacial acetic acid.
The reaction mixture containing Insulin Lispro is purified either by cation exchange chromatography followed by RPC or is directly purified by reversed phase chromatography. In case of purification with cation exchanger the reaction mixture is loaded onto the column packed with above mentioned exchanger equilibrated with 100 mM acetate buffer of pH 3.0-4.0 (buffer A) for the removal of partially digested protein fragments, uncleaved protein precursors and partially digested protein fragments. Post loading, 15% of buffer B wash containing 100 mM acetate buffer, 1M NaCl and 20% acetonitrile is given.
Elution is then carried out using a linear gradient of buffer B from 20%) to 50% buffer B containing 20% acetonitrile or N-Propanol preferably acetonitrile. Final purification or polishing of the Human Insulin Lispro is carried out by RPC using C4, C8 and CI8 columns and eluting the Insulin Lispro in 2 to 4 column volumes with a 0 to 40% linear gradient of mobile phases A & B. Mobile phase A contains 10 % Acetonitrile with 0.1 % Trifluroacetic acid (TFA) and mobile phase contain 90% Acetonitrile with 0.1% Trifluroacetic acid (TFA).

Insulin Lispro can also be eluted using step gradient of 0 to 15% for 2 column volumes followed by linear gradient from 15 % to 40% for 5-15 column volumes of mobile phases A & B where Mobile phase A contains 10 % Acetonitrile with 0.4 % Acetic Acid and mobile phase B contains 90% Acetonitrile with 0.4% Acetic acid. Undigested PL and Arg-Insulin impurity with some unknown impurities elute in the step gradient and Insulin Lispro elutes at around 22-25 (16-19) % of Mobile phase B. Other unknown impurities elutes after 25 % of mobile phase B. Pure Insulin Lispro thus obtained is crystallized in presence of zinc and phenol, The crystals are separated and dried to obtain pharmacopoeial grade Insulin Lispro.
Although the invention has been described with reference to specific embodiments, this description is not meant to be construed in a limiting sense. Various modifications of the disclosed embodiments, as well as alternate embodiments of the invention, will become apparent to persons skilled in the art upon reference to the description of the invention. It is therefore contemplated that such modifications can be made without departing from the spirit or scope of the present invention as defined.
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 2490 gm was obtained. Harvest pellet was suspended in buffer containing 50 mM Tris, 5 mM EDTA, 150 mM NaCl and 100 mM B-Mercaptoethanol (PME) and kept for stirring for 2 hours. Lysis was carried out at 18000 ± 500 psi by using High Pressure Homogenizer. After two passes, centrifugation was carried out and the pellet was suspended in buffer containing 50 mM Tris, 5 mM EDTA, 1M NaCl, 0.5% Triton X-100 and 100 mM 0-ME, 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 700 gm.
Example 2: Preparation of Proinsulin Lispro from Preproinsulin Lispro
IBs 635 g isolated by using the procedure as described in example lwere dissolved in 2540 ml of 6 M Guanidine Hydrochloride containing 50 mM Tris, 2 mM EDTA. The pH of the solution was adjusted to 9.2 with 5 N NaOH and the solution was then reduced by adding 726 ju.1 of 14 M stock 2-mercaptoethanol. Sulfitolysis was carried out by adding 27.36 g Sodium Tetrathionate and 56.7 g sodium sulphite and incubating the IBs extract at 25°C for 10 hr under continuous stirring. The reaction mixture (3000 ml) was then clarified using O.lu hollow fiber filter. 7 washes of 6M Guanidine buffer were then given in step mode to extract maximum protein from the extract. This total volume was then concentrated by cross flow filtration using 3KDa hollow fiber. The pH of the concentrated extract (4200 ml) having protein concentration (measured on the basis of A280nm) of 28.95 mg/ml was adjusted to 1.00 by addition of Hydrochloric acid and was cooled to 15°C. CNBr (121 g) was then added and the reaction was carried out at 15°C for 8 hr with continuous stirring in dark. The reaction mixture was then diluted with 4 volumes of 0.02 M Glycine buffer, pH 11 to precipitate out the protein of interest. The pH of the final solution was then adjusted to 2.6 with NaOH and the precipitated protein was recovered by Cross flow filtration using O.lu. Hollow fiber cartridge, maintaining a TMP of 5 psi. Concentrated precipitates were subsequently washed with 20 L of 20mM Glycine, pH 2.5 in step mode of 2 L each to remove traces of CNBr. The cartridge was washed with 6 L of 9 M Urea Buffer containing 50mM Tris, ImM EDTA and 18mM Ethylene diamine to remove the trapped protein from the cartridge and the system. Protein slurry was slowly added in 9 M Urea wash (6000 ml) with continuous stirring. The pH of the protein solution having 60.68 g of total protein (measured at A280nm) was adjusted to 8.5 with 3N hydrochloric acid and loaded onto 1.2L

Cellufine Max 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 0.2M NaCl. 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 Elute 12.46 gm (1290 ml) was divided in two equal parts of 645ml each and pH was increased to 11.0 by 5M NaOH solution before adding to refolding buffer. Half of Elute was added to 23.6 L of refolding buffer (0.02M Glycine, pH 11.0), previously reduced with 1.785 ml of 2-mercapto ethanol (14.4 M Stock) under continuous stirring making the final protein concentration of 0.25 mg/ml at ambient temperature . Refolding reaction was monitored by HPLC and after 2 hour the second fraction under similar condition was added to the same refolding buffer solution making the final protein concentration of 0.5 mg/ml. The reaction was monitored for another 3 hr and was then terminated by addition of 460 ml of glacial acetic acid.
To the 25.22 L of refolding solution 2.02 L of 5M NaCl solution was added and stirred for 1 hour. The solution was then clarified by passing it through depth filter. The filtrate was loaded onto 1.0 L column packed with Butyl HP in BPG 100 column, pre-equilibrated with 20 mM Glycine, pH 3.8 with 0.4 M NaCl at linear velocity of 61 cm/hr. The column was then washed with equilibration buffer followed by 0.1 M Glycine buffer, pH 9.50 containing 0.5 M NaCl for 2 column volume. PL is then eluted using 0.05M Glycine buffer pH 9.50 containing 15% acetonitrile. Column is further regenerated by 2 column volume of water followed by 1.5-2 CV 6 M Urea buffer followed by water. Cleaning of column was carried out by passing 2-3column volume of 1M NaOH to remove the tightly bound protein aggregates followed by 6-8 column volume of water to remove the traces of NaOH.
Example 3: Conversion of Proinsulin Lispro to Purified Insulin Lispro
The pH of the Butyl HP column elute obtained from example 2 containing 3.72 g of purified PL with protein concentration of 1.24 mg/ml was kept for enzymatic digestion (Trypsin and Carboxypeptidase in combination) at 20°C to remove the C-

peptide and terminal arginine to form Insulin Lispro. Enzymes are used in the ratio of 1:15000 (For 15000 mg protein: lmg of Trypsin) and 1:2000 (2000mg of protein to Img Carboxypeptidase) for Trypsin and Carboxypeptidase respectively. Reaction was quenched by addition of acetic acid to reduce the pH of reaction mixture to pH 4.0 and loaded onto 250 ml column, packed with cation exchanger CM FF in XK50/20 column pre-equilibrated with 0.1M acetic acid pH 4.0. Column was then washed with equilibration buffer, followed by wash with 3CV of 20% Buffer B (0.05M Acetic acid, 1M NaCl containing 20% acetonitrile-Elution buffer) in step mode for 3 CV and elution was carried out by giving a 20 CV linear gradient from 20 to 50 %B in linear gradient. Eluted peak was fractionated and pooled CM FF protein 2.5gm 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). Elution was performed after 2column volume of equilibration by giving a 15 % step gradient of mobile phase B (10 % water, 90% Acetonitrile, 0.4% acetic acid) for 2 column volumes followed by linear gradient of 15% mobile phase B to 20% mobile phase B in 10 column volumes. Target protein peak was fractionated and fractions were analyzed on 15% Native PAGE gel and HPLC. Fractions containing the pure Human Insulin Lispro were pooled and final yield of 1.4g of Human Insulin Lispro was obtained. Purified Human Insulin Lispro was crystallized in presence of zinc chloride and phenol. Crystals were separated and dried to obtain crystalline pharmacopoeial grade Human Insulin Lispro (1.47 gm), which showed 99% of Insulin Lispro, 0.51%) Des impurity, 0.22% of high molecular impurity and assay as 96.36 %.
Example 4: Preparation of Proinsulin Lispro from Preproinsulin Lispro
IBs 680 g prepared with procedure as described in example 1 were dissolved in 2720 ml of 6 M Guanidine Hydrochloride Buffer. The pH of the solution was adjusted to 9.2 by 5 N NaOH and was then reduced by adding 780ul of 14 M stock 2-mercaptoethanol. Sulfitolysis was carried out by adding 31.0 g Sodium Tetrathionate

and 64.26 g sodium sulphite and incubating the IBs extract at 25°C for 8-10 hr under continuous stirring. The reaction mixture (3400 ml) was then clarified and concentrated by cross flow filtration. The pH of the concentrated extract (4500 ml) having protein concentration (measured on the basis of A280 nm) of 28.95 mg/ml was adjusted to 1.00 by addition of concentrated Hydrochloric acid and was cooled to 15°C. CNBr (150 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 4 volumes of 0.02 M Glycine buffer, pH 11 to precipitate out the protein of interest and the protein precipitates were recovered by Cross flow filtration using 0.1 p Hollow fiber cartridge, maintaining a TMP of 5 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 9 M Urea Buffer to remove the trapped protein from the cartridge. Protein precipitates slurry was slowly dissolved in 9 M Cartridge wash (6000 ml) with continuous stirring. The pH of the protein solution having 77 g of total protein (measured at A280nm) was adjusted to 8.5 with 3 N hydrochloric acid and loaded onto 1.2 L Cellufine Q r column pre-equilibrated with 6 M Urea Buffer of pH 8.5. Column was washed with 3 column volume of 6M Urea Buffer of pH 8.5 containing 0.2 MNaCl. 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 Elute 16.82 gm (1300 ml) was divided in two equal parts of 650ml each and pH was increased to 11.0 by 5 M NaOH solution before adding to refolding buffer. Half of Elute was added to 32.3 L of refolding buffer (0.02 M Glycine, pH 11.0), previously reduced with 2.41 ml of 2-mercapto ethanol (14.4 M Stock) under continuous stirring making the final protein concentration of 0.25 mg/ml at ambient temperature . Refolding reaction was monitored by HPLC and after 2 hour the second fraction under similar condition was added to the same refolding buffer solution making the final protein concentration of 0.5 mg/ml. The reaction was monitored for another 3 hr and was then terminated by addition of 672 ml of glacial acetic acid.

To the 32.9 L of refolding solution was added 2.63 L of 5 M NaCl and solution was stirred for 1 hour. The solution was then clarified by passing it through depth filter and the filtrate was loaded onto 1.0 L column packed with Butyl HP in BPG 100 column, pre-equilibrated with 20 mM Glycine, pH 3.8 with 0.4 M NaCl at linear velocity of 61 cm/hr. The column was then washed with equilibration buffer followed by 0.1 M Glycine buffer, pH 9.50 containing 0.5 M NaCl for 2 column volume. Pro-insulin is then eluted using 0.05M Glycine buffer pH 9.50 containing 15% acetonitrile. Column is further regenerated by 2 column volume of water followed by 1.5-2 CV 6 M Urea buffer followed by water. Cleaning of column was carried out by passing 2-3column volume of 1M NaOH to remove the tightly bound protein aggregates followed by 6-8 column volume of water to remove the traces of NaOH.
Example 5: Conversion of Proinsulin Lispro to Purified Insulin Lispro
The pH of the Butyl HP column elute obtained from example 4 containing 3.72 g of purified PL with protein concentration of 1.2 mg/ml was kept for enzymatic digestion (Trypsin and Carboxypeptidase together) at 20°C to remove the C-peptide and terminal arginine to form Insulin Lispro. Enzymes were used in the ratio of 1:15000 (For 15000 mg protein: lmg of Trypsin) and 1:2000 (2000 mg of protein to lmg Carboxypeptidase) for Trypsin and Carboxypeptidase respectively. Reaction was quenched by addition of acetic acid to reduce the pH of reaction mixture to pH 4.0 and loaded onto 250 ml column, packed with cation exchanger CM FF in XK50/20 column pre-equilibrated with 0.1M acetic acid pH 4.0. Column was then washed with equilibration buffer, followed by wash with 3CV of 20% Buffer B (0.05M Acetic acid, 1M Sodium Chloride containing 20% acetonitrile- Elution buffer) in step mode for 3 CV and elution was carried out by giving a 20 CV linear gradient from 20 to 50 %B in linear gradient. Eluted peak was fractionated and the fractions containing the protein of interest were pooled. CM FF protein 2.64 gm 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 protein loading 2 column volumes of equilibration buffer wash was performed followed by giving 2 CVs of 15 % mobile phase B (10 % water, 90% Acetonitrile, 0.4% acetic acid) in step gradient. Elution was carried by giving a linear gradient of 15% mobile phase B to 20% mobile phase B in 10 column volumes. Target protein peak was fractionated and fractions were analyzed on 15% Native PAGE gel and by HPLC. Fractions containing the pure Insulin Lispro were pooled to obtain 1.85 g of final yield of the product. Purified Insulin Lispro was crystallized in the presence of Zinc Chloride and phenol. The crystals obtained were separated and dried to obtain crystalline, pharmacopoeial grade Insulin Lispro, which showed 99.27% of Insulin Lispro, 0.23% Des impurity, 0.5%) other then des, 0.14% of high molecular impurity and assay as 95.04 %. Example 6: Preparation of Proinsulin Lispro from Preproinsulin Lispro IBs 696 g prepared with procedure as described in example 1 were dissolved in 2784 ml of 6 M Guanidine Hydrochloride Buffer. The pH of the solution was adjusted to 9.2 by 5 N NaOH and was then reduced by adding 796 u.1 of 14M stock 2-mercaptoethanol. Sulfitolysis was carried out by adding 31.92 g Sodium Tetrathionate and 66.15 g sodium sulphite and incubating the IBs extract at 25°C for 8-10 hr under continuous stirring. The reaction mixture (3500 ml) was then clarified, washed with 7 washes of guanidine buffer in step mode (1L each *7 times) and concentrated by cross flow filtration. The pH of the concentrated extract (4500 ml) having protein concentration (measured on the basis of A280nm) of 21.6 mg/ml was adjusted to 1.00 by addition of Hydrochloric acid and was cooled to 15°C. CNBr (100 g) was then added and the reaction was carried out at 15°C for 8 hr with continuous stirring in dark. The reaction mixture was then diluted with 4 volumes of 0.02 M Glycine buffer, pH 11 to precipitate out the protein of interest and the protein precipitate was recovered by Cross flow filtration using 0.1 u Hollow fiber cartridge, maintaining a TMP of 5 psi. Concentrated precipitate was subsequently washed with of 20L 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 9 M Urea Buffer to remove the trapped protein

from the cartridge and the system. Protein slurry was slowly dissolved in 9 M Urea wash (6000 ml) with continuous stirring. The pH of the protein solution having 81.6g of total protein (measured at A280nm) was adjusted to 8.5 with 3N hydrochloric acid and loaded onto 1.2L Cellufine Max Q r column pre-equilibrated with 6M Urea Buffer of pH 8.5. Column was washed with 3 column volumes of 6M Urea Buffer of pH 8.5 containing 0.2MNaCl. 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 Elute 19.1 gm (1980 ml) was divided in two equal parts of 990ml each and pH was increased to 11.0 by 5 M NaOH solution before adding to refolding buffer. Half of Elute was added to 35.85 L of refolding buffer (0.02M Glycine, pH 11.0), previously reduced with 2.728 ml of 2-mercapto ethanol (14.4 M Stock) under continuous stirring making the final protein concentration of 0.25 mg/ml at ambient temperature . Refolding reaction was monitored by HPLC and after 2 hour, the second fraction under similar condition was added to the same refolding buffer solution making the final protein concentration of 0.5 mg/ml. The reaction was monitored for another 3 hr and was then terminated by addition of 320 ml of glacial acetic acid.
To the 38.5 L of refolding solution, was added 3.08 L of 5 M NaCl and solution was stirred for 1 hour. The solution was then clarified by passing it through depth filter and the filtrate was loaded onto 1.0 L column packed with Butyl HP in BPG 100 column, pre-equilibrated with 20 mM Glycine, pH 3.8 with 0.4 M NaCl at linear velocity of 61 cm/hr. The column was then washed with equilibration buffer followed by 0.1 M Glycine buffer, pH 9.50 containing 0.5 M NaCl for 2 column volume. PL is then eluted using 0.05 M Glycine buffer pH 9.50. Column is further regenerated by 2 column volume of water followed by 1.5-2 CV 6 M Urea buffer followed by water. Cleaning of column was carried out by passing 2-3column volume of 1M NaOH to remove the tightly bound protein aggregates followed by 6-8 column volume of water to remove the traces of NaOH.
Example 7: Conversion of Proinsulin Lispro to Purified Insulin Lispro

The pH of the Butyl HP column elute obtained from example 6 containing 2.85 g of purified PL with protein concentration of 1.14 mg/ml was kept for enzymatic digestion (Trypsin and Carboxypeptidase together) at 20°C to remove the C-peptide and terminal arginine to form Insulin Lispro. Enzymes are used in the ratio of 1:15000 (For 15000 mg protein: lmg of Trypsin) and 1:2000 (2000 mg of protein to lmg Carboxypeptidase) for Trypsin and Carboxypeptidase respectively. Reaction was quenched by addition of acetic acid to reduce the pH of reaction mixture to pH 4.0 and loaded onto 250 ml column, packed with cation exchanger CM FF in XK50/20 column pre-equilibrated with 0.1M acetic acid pH 4.0. Column was then washed with equilibration buffer, followed by wash with 3CV of 20% Buffer B (0.05M Acetic acid, 1M NaCl containing 20% acetonitrile- Elution buffer) in step mode for 3 CV and elution was carried out by giving a 20 CV linear gradient from 20 to 50 %B in linear gradient. Eluted peak was fractionated and pooled CM FF protein 2.04 gm 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). Elution was performed after 2 column volume of equilibration by giving a 15 % step gradient of mobile phase B (10 % water, 90% Acetonitrile, 0.4% acetic acid) for 2 column volumes followed by linear gradient of 15% mobile phase B to 20% mobile phase B in 10 column volumes. Target protein peak was fractionated and fractions were analyzed on 15% Native PAGE gel and HPLC. Fractions containing the pure Insulin Lispro were pooled to obtain 1.3 lg of product as final yield. Pure Insulin Lispro was crystallized using zinc chloride solution in presence of phenol and crystals were separated and dried to obtain pharmacopoeial grade Insulin Lispro which showed 99.10% of Insulin Lispro with 0.32%> Des impurity, 0.57%o other related other then des were 0.15%) of high molecular impurity. The assay of the product was 98.40 %>.

We Claim:
1. A process for production of pharmacopoeial grade Insulin Lispro comprising the steps of:
a. Expressing Preproinsulin Lispro as inclusion bodies by Fed-batch
fermentation of recombinant E. coli cells containing plasmid bearing the gene
sequence for Preproinsulin Lispro;
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 selected from Guanidine hydrochloride buffer, urea buffer preferably
Guanidine hydrochloride buffer and carrying out sulfitolysis reaction to obtain
dissolved sulfitolysed protein;
d. clarification of dissolved sulfitolysed protein obtained in step c by methods
selected from dead end filtration, normal flow filtration, centrifugation or
microfiltration to get clarified Preproinsulin Lispro precursor;
e. digestion of clarified Preproinsulin Lispro precursor obtained in step d with
CNBr under acidic condition with pH from 0.1 to 2.5 and in presence of
buffer selected from 8M Urea buffer and 6M Guanidine Hydrochloride;
f. precipitation of Proinsulin Lispro precursor obtained in step e with 3-6
volumes, of water or 0.02 - 0.2 M buffer with pH 2.0-3.0 to obtain
precipitated Proinsulin Lispro precursor;
g. purification of precipitated Proinsulin Lispro precursor obtained in step f
using an anion exchanger selected from Cellufine max Q r, Capto DEAE,
Source 30Q, Source 15Q, Q Sepharose XL, Q Sepharose FF, DEAE
Sepharose FF , DEAE Sephadex, Capto DEAE, most preferably Cellufine
max Q r to obtain purified Proinsulin Lispro precursor;

h. refolding of purified Proinsulin Lispro precursor obtained in step g by dilution method in the refolding buffer containing 0.01 to 0.05 M Glycine with pH 10- ■ 12 to obtain Proinsulin Lispro;
i. capturing and purification of Proinsulin Lispro obtained in step h by hydrophobic interaction chromatography (HIC) to obtain purified Proinsulin Lispro;
j. enzymatic digestion of purified Proinsulin Lispro obtained in step i to Insulin Lispro, wherein digestion is carried out directly in the elute from HIC;
k. optional purification of Insulin Lispro obtained in step j using cation exchange chromatography selected from SP Sepharose FF, CM Sepharose FF, CM Sephadex, SP Sepharose BB, preferably CM Sepharose FF and eluted with 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 NaCl, 30% acetonitrile and having pH4.0 - 5.0;
1. purification of Insulin Lispro obtained in step j or step k, by Reverse phase chromatography to obtain purified Insulin Lispro;
m. crystallization of Purified Insulin Lispro obtained in step 1 is to obtain pharmacopoeial grade Insulin Lispro with >98% purity.
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 comprising 50 mM
Tris Buffer, 5 mM EDTA and 150 mM NaCl in presence of 100 mM 2-
Mercaptoethanol at 15000 to 20000 psi. to get cell lysate, optionally more
than two times; and
c. centrifuging the cell lysate obtained in step b to obtain isolated inclusion
bodies.
3. 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 NaCl, 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 to obtain 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 NaCl and 4 mM of 2-
Mercaptoethanol to obtain suspended Inclusion bodies;
d. Centrifuging the suspended Inclusion bodies to obtain purified Inclusion
bodies.
4. The process as claimed in claim le, wherein the protein to cyanogens bromide ratio during digestion is 1:0.7 to 1:1.5 w/w, preferably the ratio is 1:1.
5. The process as claimed in claim If, wherein precipitated Proinsulin Lispro precursor is recovered by cross flow filtration using 0.1 \i Hollow fiber cartridge or by centrifugation, preferably by hollow fiber filtration.
6. The process as claimed in claim lj, wherein enzymatic digestion is carried out using Trypsin and Carboxypeptidase B enzyme.
7. The process as claimed in claim 6, wherein trypsin digestion is carried out using recombinant Trypsin, immobilized recombinant Trypsin or soluble TPCK treated animal Trypsin.
8. The process as claimed in claim 6, wherein trypsin to protein ration is in the range of 1: 5000 to 1:20000 (w/w), preferably in the ratio of 1: 10000 (w/w).
9. The process as claimed 6, wherein Carboxypeptidase B to protein ration is in the range of 1:500 to 1:10000 (w/w), more preferably in the ratio of 1:1000 (w/w).
10. The process as claimed in claim 11 wherein reverse phase matrix used in reverse phase chromatography is C4, C8 and CI8, preferably C8 eluted with a linear gradient of 12% to 20% preferably 15% to 16% of buffer containing 70% acetonitrile with 0.4% acetic acid..

11. The process as claimed in claim lm, wherein crystallization of purified insulin Lispro is carried out in presence of zinc and phenol.
12. A process for enzymatic conversion of purified Proinsulin Lispro using trypsin and Carboxypeptidase B directly in the chromatographic column elutes.
13. The process as claimed in claim 12, wherein the source of trypsin is Recombinant Trypsin or soluble TPCK treated animal Trypsin.
14. The process as claimed in claim 12, wherein the weight ratio of trypsin to purified Proinsulin Glargine is in the range of 1:5000 to 1:20000 preferably 1:10000.
15. The process as claimed in claim 12, wherein the chromatographic column is the column comprising hydrophobic interaction resin and wherein the elute comprises of 0.1 M Glycine optionally in the presence of an 10-20% acetonitrile.
16. A process of enzymatic conversion of purified Proinsulin Lispro to obtain insulin Lispro by trypsin and Carboxypeptidase B digestion wherein ratio of trypsin and Carboxypeptidase B to purified PL is 1:5000 to 1:20000 (w/w) and 1:500 to 1:10000 (w/w), respectively.
17. The process as claimed in claim 17 wherein, preferred ratio of Trypsin to purified Proinsulin Lispro is 1:10000 (w/w) and of Carboxypeptidase B to purified Proinsulin Lispro is 1:5000 (w/w).
18. The process as claimed in claim 17 wherein, the source of trypsin is Recombinant Trypsin or soluble TPCK treated animal Trypsin
19. The process as claimed in claim 17 wherein, the enzymatic conversion is carried out directly in the chromatographic column elute.
20. The process as claimed in claim 20 wherein, the chromatographic column is the column comprising hydrophobic interaction resin and wherein the elute comprises 0.1 M Glycine optionally in the presence of an 10-20% acetonitrile.

21. A process of enzymatic conversion of purified Proinsulin Lispro to obtain insulin Lispro by trypsin digestion wherein ratio of trypsin and Carboxypeptidase B to purified Proinsulin Lispro is 1:5000 to 1:20000 (w/w) and 1:500 to 1:10000 (w/w), respectively and wherein the process is carried out directly in chromatographic column elute.
22. The process as claimed in claim 23 wherein, preferred ratio of Trypsin to purified Proinsulin Lispro is 1:10000 (w/w) and of Carboxypeptidase B to purified Proinsulin Lispro is 1:5000 (w/w).
23. The process as claimed in claim 23 wherein, the source of trypsin is Recombinant Trypsin or soluble TPCK treated animal Trypsin.
24. The process as claimed in claim 23 wherein, the chromatographic column is the column comprising hydrophobic interaction resin and wherein the elute comprises of 0.1 M Glycine optionally in the presence of an 10-20% acetonitrile.