FORM 2
THE PATENTS ACT 1970
(39 OF 1970)
PROVISOINAL SPECIFICATION
(SECTION 10)
"IMPROVED PROCESS FOR TRANSFORMING HUMAN INSULIN PRECURSOR TO HUMAN
INSULIN"
UNICHEM LABORATORIES LIMITED, A COMPANY
REGISTERED UNDER THE INDIAN COMPANIES ACT, 1956,
HAVING ITS REGISTERED OFFICE LOCATED AT UNICHEM
BHAVAN, PRABHAT ESTATE, OFF S. V. ROAD, JOGESHWARI
(WEST), MUMBAI - 400 102 MAHARASTRA, INDIA
The following specification describes the invention


IMPROVED PROCESS FOR TRANSFORMING HUMAN INSULIN PRECURSOR TO HUMAN INSULIN
Technical Field of the Invention:
The present invention relates to proteins and peptides from recombinant source using expression system particularly E. coli. The patent also relates to method of producing the recombinant proteins.
Background of the Invention:
The Human Insulin is a polypeptide protein which plays important role in glucose metabolism and is secreted by beta cells of pancreas. The Human insulin 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 Human Insulin Structure, the letters A and B stand for particular amino acid chain, and the number for the position of amino acid residue which is counted from the amino to carboxyl end of the particular amino acid chain

The Recombinant DNA technology is used for preparing Human Insulin via Proinsulin route. The most common expression systems used are E. coli & yeast. The three basic methods to produce Human Insulin are known from prior art;
First method involves E. coli for expression of large fusion protein in the cytoplasm. In this method Pro-insulin is produced by expressing the same in the form of fusion protein in E. coli, cutting the fusion protein with cyanogens bromide to obtain Pro-insulin, sulfonating the Pro-insulin and purifying it to obtain pure sulfonated Pro-insulin, refolding the sulfonated Pro-insulin to get correct disulphide bond, treating the refolded Pro-insulin with Trypsin and Caroboxypeptidase B and then finally purifying to obtain Insulin. However the yield of refolded Pro-insulin reduces with increase in Pro-insulin concentration probably due to mis-folding of the protein and some degree of polymerization.
Second method involves E. coli as expression vehicle and use single peptide to enable secretion of Pro-insulin into the periplasmic.
Third method utilizes yeast, particularly, Saccharomyces cerevisiae, to secrete the Pro-insulin into the fermentation medium.
Both these methods namely second & third however results in lower yield of Insulin. The Pro-insulin contains Chain A and Chain B linked together with the C-peptide. Significance of C-pcptide is that it helps in converting the Pro-insulin molecule into a properly folded Pro-insulin. However, the role of C-Peptide in the folding of Pro-insulin is not precisely known, but the presence of dibasic amino terminal at both the ends of C-peptide is important for proper processing and folding of the Pro-insulin. A known process for obtaining Human Insulin from Pro-insulin is by removal of C-peptide using Trypsin and Caroboxypeptidase B followed by reverse phase chromatography to elute biologically active Human Insulin. Trypsin is a serine protease and hydrolyses a protein or peptide at the carboxy terminal of Arginine or Lysine residue. Caroboxypeptidase B is highly specific for excising carboxy terminal basic amino acid residues from peptides and proteins with a preference for Arginine. One of the difficulties associated with this process employing a terminal di-basic amino acid sequence in the C-peptide region is the presence of substantially large amounts of difficultly removable impurities like Arg- Insulin and Des-Thr(B30) Insulin in the

reaction mixture once enzymatic cleavage with Trypsin and Caroboxypeptidase B to remove C-peptide is performed. This occurs as a result of non-specific action of Trypsin on the Pro-insulin molecule so as to remove C-peptide sequence away from the A- chain. This non specific action of Trypsin results in the unwanted by products out of which Arg-Insulin and Des Thr (B30) Insulin constitute about 2-5 % and 8-15 %, respectively. This necessitates additional purification steps to remove these impurities or seek a conversion process, condition of which minimizes formation of these impurities. Conversion to Insulin by enzymatic digestion of Pro-insulin using Trypsin and Caroboxypeptidase B can be achieved either by treating the Pro-insulin first with Trypsin, purifying digested product using Chromatography techniques to remove the unwanted impurities and then finally converting the Trypsin digested Proinsulin into Insulin by Caroboxypeptidase B or by simultaneous action of both the enzymes using reaction conditions such as there is minimum formation of impurities. In the later case the impurity Des Thr (B30) Insulin generated during the Trypsin digestion differs from the Human Insulin by the absence of single terminal amino acid only and is very difficult to separate from the Human Insulin afterwards.
A process for purification of the Insulin from Pro-insulin has been described in US Patent Application US20080146492 (Zimmerman, Ronald E; et al\ 2007). The application describes that the Pro-insulin is initially treated with Trypsin using mass ratio of protein to Trypsin as 2000: 1. Then this digested Pro-insulin is purified by CI8 reversed phase chromatography, followed by buffer exchange using a 3KDa cutoff membrane. Insulin is finally obtained by digesting it with Caroboxypeptidase B using mass ratio of protein to Caroboxypeptidase B as 1000: 1. However the process seems to be commercial unviable as it involves more number of steps, capital intensive high pressure reverse phase chromatography and use of solvents like acetonitrile in purification of Insulin. Further US5457066 (Frank, Bruce H.; et al; 1992) patent describes the process for converting Pro-insulin into Human Insulin by digesting the Pro-insulin with Trypsin and Caroboxypeptidase B simultaneously in the presence of divalent metal ions like Nickel. Under the reaction conditions used in this process very low level of Des Thr (B30) Insulin is formed. Fnzymes have been added in the reaction mixture to provide the weight ratio of Human Pro-insulin to Caroboxypeptidase B to Trypsin as 13,500:5:1.

However, the process is silent about the other impurities formed by non-specific action of Trypsin like Arg-lnsulin and it involves large quantities of enzymes particularly the expensive Caroboxypeptidase B to achieve the conversion.
Further EPO Patent EP0195691 (Thim, Lars et al; 1986) describes the process for producing human insulin using DNA sequence encoding B-X-Y-A; where B & A of the sequence encoding are the B & A chain of human insulin & X-Y is a dibasic amino acid sequence between two chains A & B & amino acid is selected from either lysine or arginine using yeast as expression vector. The Patent EP '691 also teaches us; the process of conversion of Pro-insulin to human insulin via two step conversion involving Trypsin digestion carried out at 4°C & pH range of 11 - 12 as first step; followed by digestion with carboxypeptidase B; giving about 80% of human insulin. The Patent EP '691 also describes the purification of trypsin digested pro-insulin by preparative chromatography and then digestion of Trypsin digested pro-insulin with Caroboxypeptidase B followed by final purification using anion exchange and reverse phase chromatography to get pure Insulin. However, the process is silent about the impurities formed by non-specific action of Trypsin like Des Thr (B30) Insulin & Arg-lnsulin.
As evidenced from the above literature review, a need still exists for a cost effective and efficient process for conversion of Pro-insulin to Human Insulin.
Object of the Invention:
An object of the present invention is to provide simple and cost effective method for
conversion of Pro-insulin to Human Insulin.
Another object of the present invention is to provide Human Insulin involving simple ion
exchange chromatography technique for separation of Des Thr (B30) and Arg-lnsulin
impurities from the Trypsin digestion mixture and human insulin, respectively and use of
low quantities of enzyme Caroboxypeptidase B for removal of C- peptide after trypsin
digestion.
Summary of the Invention:

The present invention is relates to a process for converting a human Insulin Precursor to Human Insulin. As used herein the term Human Insulin Precursor refers to the molecule of formula shown in Fig-2 which (1) contains human Insulin A chain and the human Insulin B chain. (2) has at least three disulphide bonds represented by joining of the Cysteine residue moieties located at A and B chain at (a) A-6 and A-l 1, (b) A-7 and B-7 and (c) A-20 and B-19, respectively, (3) has a removable connecting moiety X which is joined to the Insulin A-chain at the amino group of A-l and to the Insulin B-chain at carboxyl group of B-30, 4) the group R which is cleavable from the Insulin precursor product without loss of the integrity of the residual Insulin structure and 5) The group R] is hydroxyl group, arginine, lysine, or a peptide having arginine or lysine at its amino terminus.

Where X is a moiety having at least two amino acid residue, the moiety A-l to A-21 is the Human Insulin A-Chain, the moiety B-l to B-30 is the Human Insulin B-Chain, the moiety X is joined to the Insulin A-Chain at the amino group of A-l and the B-Chain at the carboxyl group of B-30 and is referred hereafter as C-peptide
The group R is hydrogen, an amino residue or a peptide moiety having two to fifteen amino acid residues and is cleavable from the Insulin precursor product without loss of the integrity of the residual Insulin structure. Any of a wide variety of amino acid residues or peptide moiety qualifies within the definition of group R. Examples of cleavable amino acid residues are basic amino acids such as arginine (Arg) or lysine (Lys) as well as a peptide moieties terminating at carboxyl by such amino acid residues which are susceptible to cleavage upon treatment with the proteolytic enzymes like Trypsin. Another example of a cleavable amino acid residue is methionine (Met) as well

as a peptide moiety having Methionine at its carboxyl terminal. These can be removed from the treatment of cyanogen bromide.
The group R1 is hydroxyl group, arginine, lysine, or a peptide having arginine or lysine at its amino terminus. When R.| is arginine, lysine, or a peptide having either of these residues at its amino terminus, the amino acid or peptide will be cleaved with formation of a product in which R1 is hydroxyl.
The term used herein "human pro-insulin" refers to a molecule which contains (1) a human insulin A and B chain; (2) has at least three disulphide bonds represented by joining sulphurs of each of the Cysteine residue moieties located at A and B chain at (a) A-6 and A-l 1, (b) A-7 and B-7 and (c) A-20 and B-19, respectively, and (3) has a removable connecting moiety X which is joined to the Insulin A-chain at the amino group of A-l and to the Insulin B-chain at carboxyl group of B-30, and 4) The group R1 which is hydroxyl group .
The connecting moiety, X referred as C-peptide of the Insulin precursor is a polypeptide generally having at least 35 and preferably from about 9 to about 35 and most preferably from about 2 to about 35 amino acid residues. The C-peptide is joined to the A chain at the amino group of A-l and to the B-chain at the carboxyl group of B-30. Most preferably, the C-peptide, when it is a peptide, is the naturally connecting peptide of human Pro-insulin having the formula,
-Arg-Arg-Glu-Ala-Glu-Aso-Leu-Gln-Val-Gly-Gln-Val-Glu-Leu-Gly-Gly-Gly-Pro-Gly-Ala-Gly-Ser-Lxu-Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln-Lys-Arg-
Although it is preferred to use the natural connecting sequence, as indicated above, much shorter peptide sequences can be used for connecting C-peptide. The only requirement are (1) That they be of sufficient length to permit proper disulfide bond formation between the A- and B- chains and (2) that they can be cleavable from Insulin precursor without disrupting the structure of Human Insulin.
In the Human Insulin Precursor of the present invention the Moiety X is -Arg-Lys-, where Arg is joined to the A chain at the amino group of A-l and Lys is attached to the

B-chain at the carboxyl group of B-30. R here is a polypeptide containing 21 amino acids having methionine linked to the amino terminal of B-l amino acid of B-chain. Rl is a hydroxy] group.
The precursor is prepared by cultivating the E coli containing the sequence for Insulin Precursor. E coli cells which express the protein in form of inclusion bodies are harvested and suspended in lysis buffer and are lysed at 20000 to 25000 psi using high pressure homogenizer. The inclusion bodies are centrifuged and washed by washing buffer containing Triton X 100 and EDTA and centrifuged to obtain purified inclusion bodies. The inclusion bodied are dissolved in denaturing agent like 6M GuanidineHCl or 8M Urea in the ratio of 1:4 to 1:15 (w/v) with the pH adjusted to 8 to 12 by 10 N 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 inclusion bodies extract at 35- 40"C for 1 to 5 hr, which results in substitution of -SH group of Insulin Precursor with -SS03 groups. The sulfonated Human Insulin Precursor after removal of nucleic acids and initial capture step is subjected to cyanogen bromide (CNBr) treatment by adding cyanogen bromide in the ratio of 1: 0.8 to 1: 1.5 (weight of protein to weight of CNBr) under acidic condition (pH 0.1 to 2.5) and incubating it for 15 to 48 hr at temperature of 4 to 25°C under stirring condition in dark. The solution after CNBr treatment is precipitated by adding 5-7 volumes of water and centrifuged at 8000-10000 rpm for 10-15 min. The precipitate is dissolved in 8 M Urea, pH 8.0-9.0 and loaded on DEAE Sephadex for removal of cleaved polypeptide and other byproducts generated during the CNBr treatment step. Pure sulfonated Proinsulin is eluted by passing 8 M Urea with 0.1 M Tris buffer of pH 8.0-9.0 containing 0.1 to 0.3 M sodium chloride. Refolding of the eluted sulfonated Proinsulin is carried out in folding buffer containing 0.01-0.05 M Glycine with pH in the range of 10 to 12 with final Urea concentration of 1 to 4 M. The refolded Proinsulin is purified from refolding reaction mixture by hydrophobic interaction chromatography in 0.1 M Glycine buffer of pH 9-11. The refolded Proinsulin can also be purified by reversed phase C8 preparative HPLC using Acetonitrile in the mobile phase and in this case after reverse phase chromatography buffer is exchanged with 0.1 M Glycine buffer of pH 11 or alternatively it can be lyophilized to obtain dry powder.

In the process of present invention the C-peptide in the Proinsulin is first cleaved with TPCK treated solubilised or immobilized Trypsin in 0.1 M Glycine buffer of pH 9-12 using ratio of Trypsin to Proinsulin of 1:5000 to 1:250 (weight of Trypsin to weight of protein) at 2-8°C for 5 to 10 hrs. The reaction is stopped by separating the beads of immobilized Trypsin or adding 1.0 to 2.0 M of Benzamidine in the reaction mixture if solubilised Trypsin is used. The mixture is then subjected to Anion Exchange Chromatography to remove impurities particularly Des Thr(B30) Human Insulin which constitute about 8-12 % of the total protein in reaction mixture. Trypsin treated Proinsulin is loaded onto the column packed with anion exchanger equilibrated with 0.1M glycine, at the pH of 7 to 11, more preferably at pH of 8 to 9. Separation of Des-Thr (B30) Insulin is achieved by carrying out elution using a linear gradient of 0.050 M to 0.35 M sodium chloride in 0.1M glycine buffer for 20 to 40 column volume. The Trypsin digested Proinsulin along with Arg-insulin and other impurities elutes at 0.05 to 0.175 M NaCl while Des Thr(B30) Insulin elutes at 0.175 to 0.25 M
The process of trypsin 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 human Pro-insulin is present in the aqueous medium containing 1mM calcium chloride and 0.1M Glycine 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.
The reaction is carried out at any of a wide range of temperatures, generally from about 0° C. to about 15°C. preferably; the reaction is conducted at a temperature of from about 2° C. to about 8° 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 9 to 12

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- 1-propanol], CAPS [3-(cyclohexylamino)-l-propanesulfonic acid], CHES [2-(N-cyclohexylamino) ethanesulphonic acid], TAPS [N-Tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid].
On a weight: weight basis, trypsin generally will be present in an amount relative to the human insulin precursor from about 1:20 to about 1:250, 000; preferably, from about 1:100 to about 1:20.000; and, most preferably, from about 1:250 to about 1:5000. The conversion of Trypsin treated Proinsulin free from Des-Thr(B30) Insulin to Human Insulin is then carried out with Caroboxypeptidase B. The Caroboxypeptidase B enzyme is then added in the ratio of 1:1000 to 1:40000 (weight of Caroboxypeptidase B to weight of protein), more preferably in the ratio of 1:5000 to 1:15000 into the protein solution free from Des Thr (B30) Insulin for obtaining Human Insulin and reaction is carried at 30 to 40°C, more preferably at 35 to 39°C. The pH of the reaction mixture is adjusted at 7 to 10 and more preferably at 8 to 9. The reaction is complete in a period of 5 to 10 hrs and the progress of the reaction mixture is monitored by HPLC and is terminated on complete conversion of Trypsin treated Proinsulin to Human Insulin. The reaction mixture is loaded onto the column packed with anion exchanger equilibrated with 0.1M Glycine, at the pH of 7 to 11, more preferably at ph of 8 to 9. Separation of Arg-Insulin is achieved by carrying out elution using a linear gradient of 0.050 M to 0.35 M sodium chloride in 0.1M Glycine buffer for 20 to 40 column volumes. The Human Insulin clutcs at 0.16 to 0.21 M NaCl while one unknown insulin related impurity elutes at 0.21 to 0.3 M NaCl. Arg-Insulin impurity elutes at 0.05 to 0.16 M NaCl
The conversion is carried out at any of a wide range of temperatures, generally from 30° C to 40° C preferably, the reaction is conducted at a temperature of from 35° C to 39° C

The pH of the reaction mixture can range anywhere from 7 to 10 (or 11). However, best results are obtained by careful pfl control such that the reaction is conducted at a pH in the range of 8 to 9 .
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, tricthanolamine, 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].
On a weight: weight basis, carboxypeptidase B generally will be present in an amount relative to the human insulin precursor from about 1:1000 to about 1:40,000 ; more preferably, from about 1:5000 to about 1: 15,000 .
Final polishing of the Human Insulin is carried out by reverse phase chromatography using C4, C8 and C18 columns and eluting the Human Insulin in 2 to 4 Column volumes with a linear gradient of 0 to 40% of mobile phase containing 90% acetonitrile with 0.1% Trifluoroacetic acid (TFA). In addition to acetonitrile alcohols like Isopropyl alcohol can be used in the mobile phase for clution of pure Human Insulin.
The ion exchangers used for separation of impurities like Des Thr (B30) after Trypsin digestion step and Arg-Insulin after Caroboxypeptidase B digestion step can be any from the Source Q 30, Source Q 15. QXL, Q FF and DEAE Sepharose FF, preferably Source Q 30, Source Q 1 5. QXL and Q FF and more preferably Source Q 30 and Source Q 15.

The amount of trypsin and carboxypeptidase B generally used depend upon the concentration of each enzyme and the amount of human pro-insulin. The enzymes; trypsin & carboxypeptidase B can be incorporated in the reaction mixture separately either in solution form or using recognized techniques they can be immobilized on a suitable support and thereby made available in the reaction medium.
The key discovery which forms the basis of this invention resides in the finding that the use of ion exchange chromatography especially anion exchange chromatography removes the Des-Thr (B30) & Arg-Insulin impurities formed during the reaction of pro-insulin with Trypsin due to the non-specific action of Trypsin. This is achieved by first treating the pro-insulin with Trypsin and removing the Des Thr (B30) impurity by anion exchange chromatography and then treating the Des Thr (B 30) free Trypsin digested pro-insulin mixture with Carboxypeptidase B followed by removal of Arg-Insulin impurity by anion exchange chromatography and final polishing by reverse phase chromatography to obtain >98% pure human Insulin.
Typically process of this invention is carried out by following ways; the precursor is prepared by cultivating the E coli containing the sequence for Insulin Precursor. E coli cells which express the protein in form of inclusion bodies are harvested and suspended in lysis buffer and arc lysed at 20000 to 25000 psi using high pressure homogenizer. The inclusion bodies are further centrifuged and washed by washing buffer containing Triton X 100 and EDTA and centrifuged to obtain purified inclusion bodies. The purified inclusion bodies arc dissolved in denaturing agent like 6M Guanidine HC1 or 8M Urea in the ratio of 1:4 to 1:15 (w/v) with the pH adjusted to 8 to 12 by 10 N 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 same at 35- 40°C for 1 to 5 hr. which results in substitution of-SH group of Insulin Precursor with -SS03 groups. The sulfonated Human Insulin Precursor after removal of nucleic acids and initial capture step is subjected to cyanogen bromide (CNBr) treatment by adding cyanogen bromide in the ratio of 1: 0.8 to 1: 1.5 (weight of protein to weight of CNBr) under acidic condition (pH 0.1 to 2.5) and incubating it for 15 to 48 hr 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 and centrifuged at 8000-10000 rpm for 10-15 min. The precipitate is dissolved in 8 M Urea. pH 8.0-9.0 and loaded on anionic exchange chromatographic column (DEAE Sephadex) for removal of cleaved polypeptide and other byproducts generated during the CNBr treatment step. Alternatively dia-filtration may be carried out of the solution after CNBr treatment to bring it in 8M Urea buffer of pH 8-9.0 and remove CNBr digestion products before loading onto the anion exchange column. Pure sulfonated Pro-insulin is eluted by passing 8 M Urea with 0.1 M Tris buffer of pH 8.0-9.0 containing 0.1 to 0.3 M sodium chloride. Refolding of the eluted sulfonated Pro-insulin is carried out in folding buffer containing 0.01-0.05 M Glycine with pH in the range of 10 to 12 with final Urea concentration of 1 to 4 M. The refolded Pro-insulin is purified from refolding reaction mixture by hydrophobic interaction chromatography in 0.1 M Glycine buffer of pH 8-11. The refolded Pro-insulin can also be purified by reversed phase C8 preparative HPLC using Acetonitrile in the mobile phase and in this case after reverse phase chromatography buffer is exchanged with 0.1 M Glycine buffer of pH 11 or alternatively it can be lyophilized to obtain dry powder.
The sulfonated pro-insulin is then converted to human insulin in two-step conversion process. In the process of present invention the C-peptide in the Pro-insulin is first cleaved with TPCK treated solubilised or immobilized Trypsin in 0.1 M Glycine buffer of pH 9-12 using ratio of Trypsin to Pro-insulin of 1:5000 to 1:250 (weight of Trypsin to weight of protein) at 2-8°C for 5 to 10 hrs. The reaction is stopped by separating the beads of immobilized Trypsin or adding 1.0 to 2.0 mM of Benzamidine in the reaction mixture if solubilised Trypsin is used. The mixture is then subjected to anion exchange chromatography to remove impurities particularly Des Thr (B30) Human Insulin which constitute about 8-12 % of the total protein in reaction mixture. Trypsin treated Pro-insulin is loaded onto the column packed with above mention anion exchanger equilibrated with 0.1M Glycine, at the pH of 7 to 11, more preferably at pH of 8 to 9. Separation of Des-Thr (B30) Insulin is achieved by carrying out elution using a linear gradient of 0.050 M to 0.35 M sodium chloride in 0.1 M Glycine buffer for 20 to 40 column volume. The Trypsin digested Pro-insulin along with Arg-insulin and other impurities elutes at 0.05 to 0.175 M NaCl while Des Thr (B30) Insulin elutes at 0.175 to 0.25 M NaCl.

The conversion of Trypsin treated Pro-insulin free from Des-Thr (B30) Insulin to Human Insulin is then carried out with Caroboxypeptidase B. The Caroboxypeptidase B enzyme is then added in the ratio of l: 1000 to 1:40000 (weight of Caroboxypeptidase B to weight of protein), more preferably in the ratio of 1:5000 to 1:15000 into the protein solution free from Trypsin and Des Thr (B30) Insulin for obtaining Human Insulin and reaction is carried at 30 to 40"C. more preferably at 35 to 39°C. The pH of the reaction mixture is adjusted at 7 to 10 and more preferably at 8 to 9. The reaction is complete in a period of 5 to 10 hrs and the progress of the reaction mixture is monitored by HPLC and is terminated on complete conversion of Trypsin treated Pro-insulin to Human Insulin. The Human Insulin is then purified from the reaction mixture by anion exchange chromatography. The reaction mixture is loaded onto the column packed with above mention anion exchanger equilibrated with 0.1M Glycine, at the pH of 7 to 11, more preferably at ph of 8 to 9. Separation of Arg-Insulin is achieved by carrying out elution using a linear gradient of 0.050 M to 0.35 M sodium chloride in 0.1 M Glycine buffer for 20 to 40 column volume. The Human Insulin elutes at 0.16 to 0.21 M NaCl while one unknown insulin related impurity elutes at 0.21 to 0.3 M NaCl. Arg-Insulin impurity elutes at 0.05 to 0.16 MNaCl.
Final polishing of the Human Insulin is carried out by reverse phase chromatography using C4. C8 and C18 columns and eluting the Human Insulin in 2 to 10 Column volumes with a linear gradient of 0 to 40% of mobile phase containing 90% acetonitrile with 0.1% Trifluoroacetic acid (TFA).
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: Preparation of Human Pro-insulin from Human Insulin Precursor
Recombinant E. coli BL 21 DE3 cells were grown in 10 L fermentor using fed batch fermentation. Human Insulin Precursor is expressed as inclusion bodies by adding 1 mM IPTG at the mid log phase. After fermentation cells were harvested by centrifugation. Harvested 1860g (wet weight) cells were suspended in 18.6 L lysis buffer containing 1.5

M NaCl and arc lyscd at 20000 to 25000 psi using high pressure homogenizer. The inclusion bodies were separated by centrifugation and washed twice with the 20 I, washing buffer containing Triton X 100 and EDTA. The washed and sufficiently pure inclusion bodies (790 g) were dissolved in 7.2 L extraction buffer containing 6 M Guanidinc HC1. The pH was adjusted to 8.5 with 10 N NaOH. Sulfitolysis was carried out by adding 58.1 g Sodium Tetrathionate and 119.5 g and incubating the inclusion bodies extract at 37 °C for 1 hr under stirring. After Sulfitolysis 4.32 L Isopropyl Alcohol was added and the reaction mixture was stirred for 15 minutes to precipitate nucleic acids and host cell proteins. The reaction mixture was then centrifuged to separate the precipitates and 12.4 L supernatant containing the protein of interest was obtained. An aliquot of this supernatant (61.) was diluted with 18 L purified water and pH was lowered to 4.5 with glacial acetic acid to precipitate out the protein of interest. The solution was centrifuged and 287 g protein precipitates was obtained after centrifugation. Dissolved 110 g of this protein precipitate was dissolved in 8.5 pH buffer containing 8 M urea to make the final volume to 1 L. This solution was loaded onto 200 ml Bed Volume DEAE Cellulose column at a flow rate of 15 ml/min to remove the nucleic acid impurities, protein of interests comes in the flow though. Flow through and buffer wash of the DEAE column was adjusted to pH 4.5 and loaded onto 160 ml Bed Volume CM Sephadex column at a flow rate of 10 ml/min. Protein of interest was eluted with 1020 ml 0.3 M NaCl in 8 M urea buffer of pH 9 to obtain 3.4 g of almost 90 % pure Human Insulin Precursor. An aliquot of the elutc (340 ml) containing 1.17 g of protein was precipitated with 1.7 L purified water. Protein pellet was separated by centrifugation and dissolved in 120 ml 80% Formic acid. Cyanogen bromide was added into the solution on weight to weight basis to make the final cyanogen bromide to protein ratio of 0.8:1. CNBr cleavage reaction was carried out at 8°C for 16 hrs in dark. The reaction mixture was then diluted with 600 ml 0.1 M Glycine buffer of pH 11 to precipitate out the protein of interest. Protein pellet was separated by centrifugation and washed with 60 ml Acetonitrile. Washed and dried pellet was dissolved in 8 M Urea buffer of pH 8.5 to make the final protein concentration of 10 mg/ml. This protein solution was loaded onto 50 ml bed volume DEAE Sephadex column equilibrated with 8.5 Urea buffer. Column was washed with 0.125 M Urea buffer of pH 8.5 for 10 bed volumes and finally protein of interest

was eluted with 8.5 M Urea buffer containing 0.29 M NaCl. To the 184 ml column elute was added 1.07 ml of 1 M 2-mercaptoethanol to reduce the -SS03 groups to -SH groups. The refolding of the Human Insulin Precursor was carried out by diluting the reduced protein with dilution buffer to a final protein concentration of 0.25 mg/ml and stirring at 25 °C for 4 hrs. The refolding reaction was monitored by HPLC. After completion of refolding reaction mixture was loaded onto C 8 preparative column at a flow rate of 10 ml/min and Human Pro-insulin was eluted at 33% of mobile phase containing 90 % Acetonitrile with 0.1 % Trifluoroacetic acid. The fractions containing 186 mg pure Human Pro-insulin were then pooled and lyophilized to obtain 234 mg dry powder.
Example 2: Preparation of Human Pro-insulin from Human Insulin Precursor
Another aliquot of the CM Sephadex elute (340 ml) containing 1.17 g of protein as obtained in Example 1 above was adjusted to pH 1 by concentrated HCI. Cyanogen bromide was added into the solution on weight to weight basis to make the final cyanogen bromide to protein ratio of 1.2:1. CNBr cleavage reaction was carried out at 25°C for 16 hrs in dark. The reaction mixture was then diluted with 1.85 L of 0.1 M Glycine buffer, pH 11 to precipitate out the protein of interest. Protein pellet was separated by centrifugation and washed with 55 ml Acetonitrile. Washed and dried pellet was dissolved in 8 M Urea buffer of pH 8.5 to make the final protein concentration of 10 mg/ml. This protein solution was loaded onto 44 ml bed volume DEAE Sephadex column equilibrated with 8.5 Urea buffer. Column was washed with 0.125 M Urea buffer of pH 8.5 for 10 bed volumes and finally protein of interest was eluted with 8.5 M Urea buffer containing 0.29 M NaCl. To the 132 ml column elute was added 0.840 ml of 1 M 2-mercaptoethanol to reduce the --SS03 groups to -SH groups. The refolding of the Human Insulin Precursor was carried out by diluting the reduced protein with 0.02 M Glycine buffer of pH 1 l.Oto a final protein concentration of 0.25 mg/ml and stirring at 25°C for 4 hrs. The refolding reaction was monitored by HPLC. After completion of refolding reaction, mixture was loaded onto C 8 preparative column at a flow rate of 10 ml/min and Human Proinsulin was eluted at 33% of mobile phase containing 90 % Acetonitrile with 0.1 % Triiluoroacetic acid. The fractions containing 178 mg pure Human Pro-insulin were then pooled and lyophilized to obtain 220 mg dry powder.

Example 3: Conversion of Human Pro-insulin to Purified Human Insulin
Purified Human Pro-insulin solution in 0.1M Glycine buffer of pH 11 containing 165 mg protein at a final concentration of 1.4mg/ml is kept at 4°C for 30 minutes. CaCl2 is added to a final concentration of 1 mM in the solution. TPCK treated immobilized Trypsin was added into the solution at a ratio of 25 BAE units per mg of Pro-insulin, The reaction was complete in 8 hrs. After completion of the reaction Immobilized Trypsin beads are separated by filtration. The pH of the reaction mixture was adjusted to 9.00 with 1M HC1 and was diluted with 0.1M Glycine of pH 9.00 to reach the conductivity of 5 mS/Cm. The solution was then loaded on 10 ml anion exchange column packed with Source 30Q anion exchange media of GE healthcare equilibrated with 0.1 M Glycine buffer of pH 9.0. Column bed height is kept at 20cm. Elution was carried out by a linear gradient of 0.05 to 0.35 M NaCl solution in 0.1 M Glycine buffer of pH 9.00 for 30 column volumes at flow rate of 6 ml/min. The Trypsin digested Pro-insulin along with Arg-insulin and other impurities elutes at 0.05 to 0.175 M NaCl while Des Thr(B30) Insulin elutes at 0.175 to 0.25 M NaCl. The step yield is 80 %.
The pH of the Des Thr (B30) Insulin free fraction was adjusted to 8.5 with 1M HC1. Caroboxypeptidase B enzyme was added in the ratio of 1:10000 (w/w) and the reaction mixture was incubated at 37°C for 5 hrs. The pH of the solution was adjusted to 9.00 with 10 N NaOH and diluted with 0.1M Glycine buffer of pH 9.00 to obtain the conductivity value of < 5 mS/cm. This solution was then loaded on to the anion exchange column of 7 ml Bed Volume packed with Source Q 15 anion exchange media of GE Healthcare. The bed height was kept at 18 cm and elution was done by a linear gradient of 0.1 to 0.3 M NaCl in 0.1 M Glycine buffer of pH 9.00. The Human Insulin elutes at 0.16 to 0.21 M NaCl while one unknown insulin related impurity elutes at 0.21 to 0.3 M NaCl. Arg-Insulin impurity elutes at 0.05 to 0.16 M NaCl. Step yield was 70 %. Purified Human Insulin was polished by C-8 reverse phase preparative HPLC column of YMC make. The column was first equilibrated with 10% Acetonitrile containing 0.1% TFA. Elution was done by linear gradient of 0 to 40% mobile phase containing 90% Acetonitrile with 0.1% TFA in 3 column volume at a flow rate of 10 ml/min. Fraction were analyzed on 15% Native Polyacrylamide Gel Electrophoresis and purest fractions

were pooled and lyophilized for 20 hrs to obtain Human Insulin as dry powder. Step yield was 90 %. The Final product thus obtained was analyzed as per US Pharmacopoeia. It was showing 98.6 % purity with 1.4 % related impurities thus passing US pharmacopoeia requirements of related substances. The Purified Human Insulin was also showing single band on Native Polyacrylamide Gel Electrophoresis and Iso Electric Focussing Gel.
Example 4: Conversion of Human Pro-insulin to Purified Human Insulin
Purified Human Pro-insulin solution in 0.1 M Glycine buffer of pH 11 containing 150 mg protein at a final concentration of 1.2 mg/ml is kept at 4 °C for 30 minutes. CaCl2 is added to a final concentration of 1 mM in the solution. TPCK treated Trypsin was dissolved in 0.001 M HO at a concentration of 1 mg/ml. Dissolved TPCK Treated Trypsin was added into the protein solution at a ratio of 1:500 weight by weight basis (weight of Trypsin: weight of Protein). This corresponds to 25 BAE Trypsin units per mg of Protein. The reaction was complete in 8 hrs. After eight hours reaction was stopped bye adding Benzamidine Hydrochloride into the reaction mixture at a final concentration of 1.5 mM. The pH of the reaction mixture was adjusted to 9.00 with 1M HC1 and was diluted with 0.1M Glycine of pH 9.00 to reach the conductivity of 5 mS/Cm. The solution was then loaded on 10 ml anion exchange column packed with Source 30Q anion exchange media of GE healthcare equilibrated with 0.1 M Glycine buffer of pH 9.0. Column bed height is kept at 20cm. Elution was carried out by a linear gradient of 0.05 to 0.35 M NaCl solution in 0.1 M Glycine buffer of pH 9.00 for 30 column volumes at flow rate of 6 ml/min. The Trypsin digested Pro-insulin along with Arg-insulin and other impurities elutes at 0.05 to 0.175 M NaCl while Des Thr (B30) Insulin elutes at 0.175 to 0.25 M NaCl. The step yield is 78 %
The pH of the Des Thr (B30) Insulin free fraction was adjusted to 8.5 with 1M HC1, Caroboxypeptidase B enzyme was added in the ratio of 1:10000 (w/w) and the reaction mixture was incubated at 37°C for 5 hrs. The pH of the solution was adjusted to 9.00 with 10 N NaOH and diluted with 0.1M Glycine buffer of pH 9.00 to obtain the conductivity value of < 5 mS/cm. This solution was then loaded on to the anion exchange column of 7 ml Bed Volume packed with Source Q 15 anion exchange media of GE Healthcare. The

bed height was kept at 18 cm and elution was done by a linear gradient of 0.1 to 0.3 M NaCl in 0.1 M Glycine buffer of pH 9.00. The Human Insulin elutes at 0.16 to 0.21 M NaCl while one unknown insulin related impurity elutes at 0.21 to 0.3 M NaCl. Arg-Insulin impurity elutes at 0.05 to 0.16 M NaCl. Step yield was 72 %, Purified Human Insulin was polished by C-8 reverse phase preparative HPLC column of YMC make. The column was first equilibrated with 10% Acetonitrile containing 0.1% TFA. Elution was done by linear gradient of 0 to 40% mobile phase containing 90% Acetonitrile with 0.1% TFA in 3 column volume at a flow rate of 10 ml/min. Fraction were analyzed on 15% Native Polyacrylamide Gel Electrophoresis and purest fractions were pooled and lyophilized for 20 hrs to obtain Human Insulin as dry powder. Step yield was 90 %. The Purified Human Insulin was showing single band on Native Polyacrylamide Gel Electrophoresis and Iso Electric Focussing Gel.
Dated this 10th day of July, 2009