FORM 2
THE PATENT ACT 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rule13)
1. TITLE OF THE INVENTION:
PROCESS FOR REFOLDING OF INSULIN, ANALOGUES AND DERIVATIVES
2. APPLICANT (S)
(a) NAME: WOCKHARDT LTD.
(b) NATIONALITY: INDIAN
(c) ADDRESS: Wockhardt Limited, D4-MIDC Area, Chikalthana,
Aurangabad - 431 210 (M.S.) INDIA.
3. PREAMBLE TO THE DESCRIPTION
The invention provides a process for obtaining a precursor for insulin, analogs or derivatives thereof having correctly bonded cystine bridges, wherein process comprises mixing of insulin precursor having incorrectly bonded cystine bridges with a solution containing both cysteine or cysteine hydrochloride and one or more chaotropic auxiliaries followed by reverse dilution resulting in increased yield of refolded protein having correctly bonded cystine bridges.
The following specification particularly describes the invention and the manner in which it is to be performed.


4. Description
The invention provides a process for obtaining a precursor for insulin, analogs or derivatives thereof having correctly bonded cystine bridges, wherein process comprises mixing of insulin precursor having incorrectly bonded cystine bridges with a solution containing both cysteine or cysteine hydrochloride and one or more chaotropic auxiliaries followed by reverse dilution resulting in increased yield of refolded protein having correctly bonded cystine bridges.
Insulin is a protein hormone consisting of an acid A-chain of 21 residues and a basic B-chain of 30 amino acids. A chain and B chain are bonded together by six cysteine residues. The A chain comprises 21 amino acid whereas B chain comprises 30 amino acid residues. Six cysteine residues are found in the two amino acid chains, each two cysteine residues being bonded to one another via a disulfide bridge. In biologically active human insulin, the A and B chains are bonded to one another via two cystine bridges, and a further cystine bridge is found in the A chain. The following cysteine residues are linked to one another in human insulin: A6-A11 A7-B7 A20-B19
The letters A and B represent the respective insulin amino acid chain and the numbers represent the position of the amino acid residue, which is counted from the amino to the carboxyl end of the respective amino acid chain. The three disulfide bonds are important in maintaining the native conformation and biological activities of the insulin molecule. Insulin folds into a unique three-dimensional structure mainly composed of three a-helical segments (A2- A8, A13-A19, and B9-B19) stabilized by its three disulfides bonds.
Insulin analogues and derivatives differ from human insulin at one or more than one amino acid positions and/or amino acid chain length. By "analogue of human insulin" (and similar expressions) as used herein is meant human insulin in which one or more amino acids have been deleted


and/or replaced by other amino acids, including non-codeable amino acids, or human insulin comprising additional amino acids, i.e. more than 51 amino acids.
By "derivative of human insulin" (and similar expressions) as used herein is meant human insulin or an analogue thereof in which at least one organic substituent is bound to one or more of the amino acids.
Human proinsulin is a protein having a linear amino acid chain, the A and B chains of the human insulin being bonded to one another via a C peptide having 11-35 amino acid residues.
Recombinant DNA processes allow precursors of insulin or insulin derivatives, in particular human proinsulin or proinsulin which has an amino acid sequence and/or amino acid chain length differing from human insulin, to be prepared in microorganisms. Either Escherichia coli or yeast are used as host to prepare insulin, its analogue or derivatives thereof. Expression of human proteins such as insulin in transformed microorganisms showed that recombinant protein did not form its native soluble and biologically active conformation. Instead of native protein, inactive inclusion bodies accumulated in host cell. These inclusion bodies contain recombinant protein in a highly enriched form with incorrect folding.
The production of human proinsulin in genetically modified Escherichia coli usually leads to the formation of inclusion bodies (proinsulins). The proinsulins prepared from genetically modified Escherichia coli cells may not have any correctly bonded cystine bridges. As a consequence, the recombinant protein must be isolated, refolded under suitable conditions, and enzymatically converted to the biologically active insulin.
There are two important issues in recovering active proteins from inclusion bodies. These include
(a) Solubilization
(b) Refolding


Chaotropic agents and detergents are commonly used as solubilizing agent. They act as protein denaturant. Chaotropic agents break hydrogen bridges in solution, thus disrupting the inter-molecular and intra-molecular interactions with partial or complete unfolding of the protein structure. These agents include one or more of ammonium sulfate, guanidine hydrochloride, ethylene carbonate, thiocyanate, dimethyl sulfoxide or urea. A key to solubilization process is the addition of reducing agent to maintain cysteine residues in the reduced state and thus prevent non-native intra- and inter- disulphide formation in highly concentrated protein solutions at alkaline pH. Typically used reducing agents include one or more of cysteine, cysteine hydrochloride, dithiothreitol, dithioerythritol or mercaptoethanol. As per the processes known in prior art (US 5,986,048 and US 6,380,355) yield of the product to be obtained depends on the order in which chaotropic auxiliaries or reducing agents are added to the suspension containing insoluble inclusion bodies.
Refolding is accomplished by removal of excessive denaturants by dilution, buffer exchange, diafiltrations, gel filtration chromatography or immobilization onto the solid support. Because of its simplicity, dilution is usually preferred for industrial scale refolding of proteins. Protein refolding is not a single reaction and competes with other reaction such as misfolding and aggregation leading to inactive protein. Ideally, protein molecules are transferred from high denaturant concentration to aqueous buffer. This addition of solubilized inclusion bodies in a buffer or an aqueous solution results in sudden change in denaturant concentration, which forces protein molecules to collapse into compact structure resulting in precipitation or aggregation. A key to refolding is in intermediate concentration of the chaotropic agents, wherein the concentration is low enough to force protein molecules to collapse, yet can allow them to stay in solution^ and be flexible to reorganize their structure in native form.
US Patent No. 5,986,048 and US Patent No. 6,380,355 discloses a process for obtaining a precursor of insulin or an insulin derivative thereof having correctly bonded, which comprises mixing an aqueous suspension of the


precursor of an insulin or an insulin derivative with an amount of cysteine or cysteine, adding the cysteine or cysteine hydrochloride containing suspension of the precursor into an solution of the chaotropic auxiliary, and diluting it in an aqueous solution.
US Patent No. 5,663,291 and US patent No. 5,473,049 discloses a reacting insulin precursor with an amount of a mercaptan. The reaction mixture is then added to an aqueous solution which contains in the presence of at least one chaotropic auxiliary at a pH of 10 to 11 wherein the concentration of the insulin precursor is 0.05 to 0.3 g per liter of aqueous medium. The resulting proinsulin having correctly linked cystine bridges is treated with trypsin or a trypsin-like enzyme.
US Patent Application 20070106063 discloses an improved process for obtaining insulins or insulin derivatives with correctly linked cystine bridges in the presence of cysteine or cysteine hydrochloride and of a chaotropic auxiliary compound, with folding being carried out in a reaction mixture in which the volume-to-surface ratio is greater than 1 and/or the oxygen concentration is 1-15 mg/l.
It was observed while working on the refolding of insulin, analogues and derivatives found when proinsulin precursors are mixed with cysteine or cysteine hydrochloride and chaotropic auxiliary present simultaneously in a solution, followed by addition of the buffer into the reaction mixture reverse dilution, the process results in an increased yield of the correctly folded proinsulins. Order of addition of cysteine or cysteine hydrochloride and chaotropic auxiliary has no impact on the final yield. Further reverse dilution of the reaction mixture containing precursor of insulin, cysteine or cysteine hydrochloride and chaotropic auxiliary, reduces the aggregation or precipitation of protein molecules, thus resulting in increasing the yield of correctly folded insulins, insulin analogues or derivatives thereof.


The whole process can be carried out in one pot, which results in decreasing the time, loss of components thus resulting in a cost during effective industrial scalable process.
One of the aspects of the present invention provides a process for obtaining the precursors of insulin, insulin analogues or derivatives thereof having correctly bonded cystine bridges, the process comprises
a. mixing precursors of insulin, insulin analogues or derivatives thereof
having incorrectly bonded cystine bridges with a solution containing
both cysteine or cysteine hydrochloride and one or more chaotropic
auxiliaries, at a pH of 8-11.5 and at a temperature of about 2°C to
about 55 °C, wherein the concentration of protein is more than
0.65g/litre,
b. adding the diluent to the reaction mixture of step (a), and
c. Isolating the precursors of insulin, insulin analogues and derivatives
thereof having correctly bonded cystine bridges.
In one of the embodiment of the invention, the concentration of cysteine or cysteine hydrochloride in step (a) varies from about 20 mM to about 60 mM.
In another embodiment of the invention, the concentration of cysteine or cysteine hydrochloride in step (a) is 50 mM.
Non-limiting examples of chaotropic auxiliaries encompassed by the invention include ammonium sulfate, guanidine hydrochloride, ethylene carbonate, thiocyanate, dimethyl sulfoxide and urea.
In another embodiment of the invention, the chaotropic auxiliary is chaotropic auxiliary is urea or guanidine hydrochloride.
In another embodiment of the invention, the concentration of chaotropic auxiliary varies from about 5M to 10M.


Guanidine hydrochloride when used as chaotropic auxiliary works well within the concentration range of about 5M to about 7M, whereas suitable concentration of urea varies between about 8M to about 10M.
Suitable solution for carrying out the reaction in step (a) and dilution in step (b) include one or more water, glycine buffer, phosphate buffer, Tris buffer, Ethanolamine buffer, C1-C4 alcohol and cystine or cystine hydrochloride solution.
In one of the embodiment of the invention, solution of step (a) and diluent in step (b) is Tris buffer.
In another embodiment of the invention, solvent or diluent is Tris buffer.
In another embodiment of the invention, solvent of step (a) may further comprise one or more of additives.
Suitable additives include Ethylenediamine tetraacetic acid, Ethyleneglycol tetraacetic acid (EGTA), arginine, methionine, proline, glycine, alanine, sugars, polyols, salts such as ammonium sulphate and magnesium chloride, and cyclodextrins or salts thereof.
In one of the embodiment of the invention, the additive added to the solvent of step (a) is ethylenediaminetetraacetic acid.
In another embodiment of the invention, the temperature of step (a) is in range of about 2°C to about 25°C.
In another embodiment of the invention, the step (b) is carried out in a temperature range of 2°C to about 25°C.
The process of present invention is performed in one pot.


In another embodiments of the invention the precursor of the insulin or an insulin derivative thereof, has the sequence according to the formula I
R2 -R1 -B2- R4-B4-B27- R5-R6 -R7-X-Gly-A2-A20-R3 wherein R2 is
(a) a hydrogen atom,
(b) an amino acid residue from the group consisting of lysine (Lys) and arginine (Arg), or
(c) a peptide having 2 to 45 amino acid residues, comprising the amino acid residue lysine (Lys) or arginine (Arg) at the carboxyl end of the peptide;
R1 is a phenylalanine residue (Phe) or a covalent bond;
R4 corresponds to position B-3 of human insulin and is an amino acid selected
form the group consisting of asparagine, lysine and proline
R5-R6 -R7 corresponds to position B-28, B-29 and B-30 of human insulin chain
respectively.
R5 can be selected from the group consisting of Asparagine, Lysine, Leucine,
proline, valine, glutamic acid, aspartic acid and alanine optionally substituted
with an acyl group having at least 10 carbon atoms.
R6 can be selected from the group consisting of lysine, glutamic acid and
proline optionally substituted with an acyl group having at least 10 carbon
atoms.
R7 can be selected from the group consisting of threonine, des threonine,
alanine, and serine.
(B2 and B4-B27) are the amino acid residues in the positions B2, B4 to B27 of
the B chain of human insulin, animal insulin or an insulin derivative thereof;
Xis
(a) an amino acid residue from the group consisting of lysine (Lys) and arginine (Arg), or
(b) a peptide having 2 to 35 amino acid residues, comprising the amino acid residue lysine (Lys) or arginine (Arg) at the N-terminal and at the carboxyl end of the peptide, or
(c) a peptide having 2 to 35 genetically encodable amino acids, comprising 1 to 5 histidine residues;


(A2-A20) are the amino acid residues in the positions A2 to A20 of the A chain of human insulin, animal insulin or an insulin derivative thereof; and R3 is a genetically encodable amino acid residue.
The amino acid sequence of peptides and proteins is indicated from N-terminal end of the amino acid chain onward. The details in formula I in brackets, e.g. A6, A20, B2, B4, B7 or B19, correspond to the position of amino acid residues in the A or B chains of the insulin.
The term "genetically encodable amino acid residue" represents the amino acids Gly, Ala, Ser, Thr, Val, Leu, lie, Asp, Asn, Glu, Gin, Cys, Met, Arg, Lys, His, Tyr, Phe, Trp, Pro and selenocysteine.
The terms "residues A2-A20" and "residues B2-B29" of "animal insulin" are understood as meaning, for example, the amino acid sequences of insulin from cattle, pigs or chickens. The terms "residues A2-A20" and "B2-B29" of insulin derivatives represent the corresponding amino acid sequences of human insulin which are formed by the replacement of amino acids by other genetically encodable amino acids.
The A chain of human insulin has the following sequence (SEQ ID NO: 1): Gly He Val Glu Gin Cys Cys Thr Ser lie Cys Ser Leu Tyr Gin Leu Glu Asn Tyr Cys Asn.
The B chain of human insulin has the following sequence (SEQ ID NO: 2): Phe Val Asn Gin His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr.
The process according to the present invention is particularly suitable for obtaining a precursor of insulin or an insulin derivative having the formula I, whose cystine bridges (not shown in formula I) are correctly folded, in which R2 is
a) a hydrogen atom, or


b) a peptide having 2 to 15 amino acid residues, at whose carboxyl end is found an arginine residue (Arg); R1 is a phenylalanine residue (Phe); R4 is asparagine or lysine; R5 is lysine, proline, glutamic acid or aspartic acid;
R6 is lysine, proline, glutamic acid optionally substituted with an acyl group having at least 10 carbon atoms; R7 is threonine or des threonine;
(B2 and B4-B27) are the amino acid residues in the positions B2, B4 to B27 of the B chain of human insulin;
X is the amino acid residue arginine (Arg) or a peptide having 2 to 35 amino acid residues, where at the beginning and at the end of the peptide there are two basic amino acid residues, in particular arginine (Arg) and/or lysine (Lys); The residue Z which codes for extra amino acid in B-chain of the insulin or insulin analogue or derivative thereof, as a rule, is part of X in the amino acid sequence of the precursor of formula I.
(A2-A20) are the amino acid residues in the positions A2 to A20 of the A chain of human insulin; and R3 is the amino acid residue asparagine (Asn), serine (Ser) or glycine (Gly).
In insulin glargine, R3 in formula I is glycine (Gly), R1 is phenylalanine (Phe), R4 is asparagine, R5 is proline, R6 is lysine, R7 is threonine and Z is an arginine residue (Arg), or a peptide residue Arg-Arg-OH.
In Insulin Lispro, R3 in formula I is Asparagine (Asn), R1 is phenylalanine (Phe), R4 is asparagine, R5 is lysine, R6 is proline, R7 is threonine.
The precursor of formula I can be produces in microorganism with the aid of a genetic construct, which are expressed in Escherichia coli or Streptomycetes during fermentation using the process known in the art.
Example 1 Quantification of insulin precursor in inclusion bodies


After completion of fermentation, the cells were separated off by centrifugation and disrupted by customary high-pressure homogenization. The fusion protein inclusion bodies released were isolated by centrifugation. The isolated inclusion bodies having proinsulin sequence were freeze dried. Quantity of insulin precursor in inclusion bodies was determined by HPLC.
100 mg of inclusion bodies were dissolved in 100 ml of a solution of 8 M urea containing 100 mm Dithiothreitol. The solution was mixed properly and then heated at 95°C for 5min. The solution was centrifuged for 10 min at 10000 rpm and 0.002 ml was applied on to a HPLC column for quantification.
Analytical HPLC conditions:
Flow rate : 1 ml/min
UV detection : 214nm
Column : Waters Spherisorb C 18 , 4.6 X 250 mm 5 micron 120 A
Buffer A : 90% water, 10 % acetonitrile and 0.1 % TFA
Buffer B : 20% Water, 80 % acetonitrile and 0.15 TFA
Column temperature: 40° C
Column equilibrated with 10% buffer B prior to injection of the sample. Gradient elution starts after 2min of injection and increase to 100% B in 25minuites. Total analysis time was 30 min.
Example 2 Process for obtaining a precursor of insulin whose cystine bridges are correctly folded
The expressed fusion protein as insoluble inclusion bodies having the proinsulin sequence 1 (SEQ ID NO: 3) was collected from E. coli cells. Proinsulin sequence 1 (SEQ ID NO: 3)
Ala Thr Thr Ser Thr Gly Asn Ser Ala Arg Phe Val Asn Gin His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu Asp Leu Gin Val Gly Gin Val Glu Leu Gly Gly Gly Pro Gly Ala Gly Ser Leu Gin Pro Leu Ala Leu Glu Gly Ser Leu Gin Lys


Arg Gly lie Val Glu Gin Cys Cys Thr Ser lie Cys Ser Leu Tyr Gin Leu Glu Asn Tyr Cys Asn
X is C-peptide from human insulin (SEQ ID NO: 4);
Arg Arg Glu Ala Glu Asp Leu Gin Val Gly Gin Val Glu Leu Gly Gly Gly Pro Gly
Ala Gly Ser Leu Gin Pro Leu Ala Leu Glu Gly Ser Leu Gin Lys Arg.
Example 2A: Refolding by dilution
480g of Urea, 9.08g of L-cysteine hydrochloride and 0.75g of ethylenediaminetetraacetic acid disodium salt was added to one litre of 20mM Tris buffer and the pH of the solution was adjusted to 8.5 with 5N sodium hydroxide solution. This solution was poured in a pot. An amount equal to 40 g of isolated freeze dried inclusion bodies containing 16g of insulin precursor of proinsulin sequence 1 (SEQ ID NO: 3) (the portion of insulin contain fusion protein was determined with the aid of HPLC, it was 40%) was weighed and dissolved in the above solution containing both L-cysteine and urea. The solution was stirred for one hour at room temperature. The pH of the solution was adjusted to 10.6 with 5N sodium hydroxide and the stirring was continued further for 1h at room temperature. The solubilized mixture was slowly added to 29 liters of precooled (10+2°C) Tris buffer (20mM) containing 2mM EDTA at pH 10.6. The pH of the reaction mixture was adjusted to 10.6 with 5N sodium hydroxide solution. The diluted refolding mixture was stirred for 24h. After 24h, the content of insulin precursor of proinsulin sequence 1 having correctly bonded cystine bridges in the reaction mixture was determined with the aid of HPLC. 8.0g of correctly folded insulin precursor of proinsulin sequence 1 (corresponding to a recovery of 50%) was recovered.
Example 2B: Refolding following reverse dilution
480g of Urea, 9.08 g of L-cysteine hydrochloride and 0.75g of ethylenediaminetetra acetic acid disodium salt was added to one litre of 20mM Tris buffer and the pH of the solution was adjusted to 8.5 with 5N sodium hydroxide solution. This solution was poured in a pot. An amount equal to 40 g of isolated freeze dried inclusion bodies containing 16g of insulin precursor of proinsulin sequence 1 (the portion of insulin contain fusion


protein was determined with the aid of HPLC, it was 40%) was weighed and dissolved in the above solution having both cysteine and urea. The solution was stirred for 1 h at room temperature. The pH of the solution was adjusted to 10.6 with 5N sodium hydroxide and the stirring was continued further for 1h at room temperature. To the above solubilized mixture 29 litres of precooled (10+2°C) Tris buffer (20mM) containing 2mM EDTA at pH 10.6 was added slowly. The pH of the reaction mixture was adjusted to 10.6 with 5N sodium hydroxide solution. The refolding mixture was stirred for 24h at (10+2°C). After 24h, the content of insulin precursor of proinsulin sequence 1 having correctly bonded cystine bridges in the reaction mixture was determined with the aid of HPLC. 9.83 g of correctly folded insulin precursor of proinsulin sequence 1 (corresponding to a recovery of 61.6%) was recovered.
Example 3
for obtaining a precursor of insulin derivative, whose cystine bridges are correctly folded
The expressed fusion protein as insoluble inclusion bodies having the proinsulin sequence 2 (SEQ ID NO: 5) was collected from E. coli cells. Proinsulin sequence 1 (SEQ ID NO: 5)
Ala Thr Thr Ser Thr Gly Asn Ser Ala Arg Phe Val Asn Gin His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu Asp Leu Gin Val Gly Gin Val Glu Leu Gly Gly Gly Pro Gly Ala Gly Ser Leu Gin Pro Leu Ala Leu Glu Gly Ser Leu Gin Lys Arg Gly He Val Glu Gin Cys Cys Thr Ser lie Cys Ser Leu Tyr Gin Leu Glu Asn Tyr Cys Gly
X is C-peptide from human insulin (SEQ ID NO: 4);
Arg Arg Glu Ala Glu Asp Leu Gin Val Gly Gin Val Glu Leu Gly Gly Gly Pro Gly
Ala Gly Ser Leu Gin Pro Leu Ala Leu Glu Gly Ser Leu Gin Lys Arg.
Example 3A: Refolding by dilution
480g of Urea, 9.08g of L-cysteine hydrochloride and 0.75g of ethylenediamine tetra acetic acid disodium salt was added to one litre of 20mM Tris buffer and the pH of the solution was adjusted to 8.5 with 5N sodium hydroxide solution.

This solution was poured in a pot. An amount equal to 40 g of isolated freeze dried inclusion bodies containing 14g of insulin precursor of proinsulin sequence 2 (SEQ ID NO: 5) (the portion of insulin contain fusion protein is determined with the aid of HPLC, it is 35%) was weighed and dissolved in the above solution having both L-cysteine and urea. The solution was stirred for one hour at room temperature. The pH of the solution was adjusted to 10.6 with 5N sodium hydroxide and the stirring was continued further for 1h at room temperature. The solubilized mixture was slowly added to 29 litres of precooled (10+2°C) Tris buffer (20mM) containing 2mM EDTA at pH 10.6. The pH of the reaction mixture was adjusted to 10.6 with 5N sodium hydroxide solution. The diluted refolding mixture was stirred for 24h. After 24h, the content of insulin precursor proinsulin sequence 2 having correctly bonded cystine bridges in the reaction mixture was determined with the aid of HPLC. 6.74 g of correctly folded insulin precursor of proinsulin sequence 2 (corresponding to a recovery of 48.28%) was recovered.
Example 3B: Refolding following reverse dilution
480g of Urea, 9.08g of L-cysteine hydrochloride and 0.75g of ethylenediaminetetra acetic acid disodium salt was added to one litre of 20mM Tris buffer and the pH of the solution was adjusted to 8.5 with 5N sodium hydroxide solution. This solution was poured in a pot. An amount equal to 40 g of isolated freeze dried inclusion bodies containing 14g of insulin precursor of proinsulin sequence 2 (SEQ ID NO: 5) (the portion of insulin contain fusion protein is determined with the aid of HPLC, it is 35%) was weighed and dissolved in the above solution containing both L-cysteine and urea. The solution was stirred for 1 h at room temperature. The pH of the solution was raised to 10.6 with 5N sodium hydroxide and the stirring was continued further for 1 h at room temperature. To the above solubilized mixture 29 litres of precooled (10+2°C) Tris buffer (20mM) containing 2mM EDTA at pH 10.6 was added slowly. The pH of the reaction mixture was adjusted to 10.6 with 5N sodium hydroxide solution. The refolding mixture was kept under stirring for 24h at (10+2°C). After 24h, the content of insulin precursor of proinsulin sequence 2 (SEQ ID NO: 5) having correctly bonded cystine bridges in the reaction mixture was determined with the aid of HPLC. 7.7 g of


correctly folded insulin precursor of proinsulin sequence 2 (corresponding to a recovery of 55.7%) was recovered.
Example 4
Process for obtaining a precursor of insulin analogue, whose cystine bridges are correctly folded
The expressed fusion protein as insoluble inclusion bodies having the proinsulin sequence 3 (SEQ ID NO: 6) was collected from E. coli cells. Proinsulin sequence 1 (SEQ ID NO: 6)
Ala Thr Thr Ser Thr Gly Asn Ser Ala Arg Phe Val Asn Gin His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Lys Pro Thr Arg Arg Glu Ala Glu Asp Leu Gin Val Gly Gin Val Glu Leu Gly Gly Gly Pro Gly Ala Gly Ser Leu Gin Pro Leu Ala Leu Glu Gly Ser Leu Gin Lys Arg Gly lie Val Glu Gin Cys Cys Thr Ser He Cys Ser Leu Tyr Gin Leu Glu Asn Tyr Cys Asn.
X is C-peptide from human insulin (SEQ ID NO: 4);
Arg Arg Glu Ala Glu Asp Leu Gin Val Gly Gin Val Glu Leu Gly Gly Gly Pro Gly
Ala Gly Ser Leu Gin Pro Leu Ala Leu Glu Gly Ser Leu Gin Lys Arg.
Example 4A: Refolding by dilution
480g of Urea, 9.08g of L-cysteine hydrochloride and 0.75g of ethylenediaminetetra acetic acid disodium salt was added to one litre of 20mM Tris buffer and the pH of the solution was adjusted to 8.5 with 5N sodium hydroxide solution. This solution was poured in a pot. An amount equal to 40 g of isolated freeze dried inclusion bodies containing 14g of insulin precursor of proinsulin sequence 3 (SEQ ID NO: 6) ( the portion of insulin contain fusion protein is determined with the aid of HPLC, it is 35%) was weighed and dissolved in the above solution having both L-cysteine and urea. The solution was stirred for one hour at room temperature. The pH of the solution was adjusted to 10.6 with 5N sodium hydroxide and the stirring was continued further for 1h at room temperature. The solubilized mixture was slowly added to 29 liters of precooled (10+2°C) Tris buffer (20mM) containing 2mM EDTA at pH 10.6. The pH of the reaction mixture was adjusted to 10.6

with 5N sodium hydroxide solution. The diluted refolding mixture was stirred for 24h. After 24h, the content of insulin precursor of proinsulin sequence 3 having correctly bonded cystine bridges in the reaction mixture was determined with the aid of HPLC. 7.14g of correctly folded insulin precursor of proinsulin sequence 3 (corresponding to a recovery of 51%) was recovered.
Example 4B: Refolding following reverse dilution
480g of Urea, 9.08g of L-cysteine hydrochloride and 0.75g of ethylenediaminetetra acetic acid disodium salt was added to one litre of 20mM Tris buffer and the pH of the solution was adjusted to 8.5 with 5N sodium hydroxide solution. This solution was poured in a pot. An amount equal to 40 g of isolated freeze dried inclusion bodies containing 14g of insulin precursor of proinsulin sequence 3 (SEQ ID NO: 6) ( the portion of insulin contain fusion protein is determined with the aid of HPLC, it is 35%) was weighed and dissolved in the above solution having both L-cysteine and urea. The solution was stirred for 1 h at room temperature. The pH of the solution was adjusted to 10.6 with 5N sodium hydroxide and the stirring was continued further for 1h at room temperature. To the above solubilized mixture 29 liters of precooled (10+2°C) Tris buffer (20mM) containing 2mM EDTA at pH 10.6 was added slowly. The pH of the reaction mixture was adjusted to 10.6 with 5N sodium hydroxide solution. The refolding mixture was stirred for 24h at (10+2°C). After 24h, the content of insulin precursor of proinsulin sequence 3 having correctly bonded cystine bridges in the reaction mixture was determined with the aid of HPLC. 7.5 g of correctly folded insulin precursor of proinsulin sequence 3 (corresponding to a recovery of 54%) was recovered.
While the invention has been described in terms of its specific embodiments, certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the invention.


WE CLAIM:
1. A process for obtaining the precursors of insulin, insulin analogues or
derivatives thereof having correctly bonded cystine bridges, the process
comprising,
(a) mixing precursors of insulin, insulin analogues or derivatives thereof having incorrectly bonded cystine bridges with a solution containing both cysteine or cysteine hydrochloride and one or more chaotropic auxiliaries, at a pH of about 8 to about 11.5 and at a temperature of about 2°C to about 55 °C, wherein the concentration of protein is more than 0.65g/litre,
(b) adding the diluent to the reaction mixture of step (a), and
(c) Isolating the precursors of insulin, insulin analogues and derivatives thereof having correctly bonded cysteine bridges.

2. The process as claimed in claim 1, wherein the concentration of cysteine or cysteine hydrochloride in step (a) varies from about 20 mM to about 60 mM.
3. The process as claimed in claim 1, wherein one or more chaotropic auxiliaries are selected from the group consisting of ammonium sulfate, guanidine hydrochloride, ethylene carbonate, thiocyanate, dimethyl sulfoxide and urea.
4. The process as claimed in claim 3, wherein the chaotropic auxiliary is chaotropic auxiliary is urea or guanidine hydrochloride.
5. The process as claimed in claim 1, wherein the concentration of chaotropic auxiliary varies from about 5M to 10M.
6. The process as claimed in claim 1, wherein the solution in step (a) or diluent in step (b) can be selected from the group consisting of water, glycine buffer, phosphate buffer, carbonate buffer, Tris buffer, Ethanolamine buffer, C1-C4 alcohol and cystine or cystine hydrochloride solution.


7. The process as claimed in claim 6, wherein the solution in step (a) or diluent in step (b) is Tris buffer.
8. The process as claimed in claim 1, wherein the solvent or diluent may further comprise one or more of additives selected from the group consisting of, Ethylenediamine tetraacetic acid, Ethyleneglycol tetraacetic acid (EGTA), arginine, glycine, alanine, sugars, polyols, salts such as ammonium sulphate and magnesium chloride, and cyclodextrins or salts thereof.
9. The process as claimed in claim 1, wherein step (a) or step (b) is carried out in a temperature range of about 2°C to about 25°C.
10. The process as claimed in claim 1, wherein the precursor of the insulin
or an insulin derivative thereof, has the sequence according to the formula
I
R2 -R1 -B2- R4-B4-B27- R5-R6 -R7-X-Gly-A2-A20-R3 wherein R2 is
(a) a hydrogen atom,
(b) an amino acid residue from the group consisting of lysine (Lys) and arginine (Arg), or
(c) a peptide having 2 to 45 amino acid residues, comprising the amino acid residue lysine (Lys) or arginine (Arg) at the carboxyl end of the peptide;
R1 is a phenylalanine residue (Phe) or a covalent bond;
R4 corresponds to position B-3 of human insulin and is an amino acid
selected form the group consisting of asparagine, lysine and proline
R5-R6 -R7corresponds to position B-28, B-29 and B-30 of human insulin
chain respectively.
R5 can be selected from the group consisting of Asparagine, Lysine,
Leucine, proline, valine, aspartic acid, glutamic acid and alanine


optionally substituted with an acyl group having at least 10 carbon
atoms.
R6 can be selected from the group consisting of lysine, glutamic acid
and proline optionally substituted with an acyl group having at least 10
carbon atoms.
R7 can be selected from the group consisting of threonine, des
threonine, alanine, and serine.
(B2 and B4-B27) are the amino acid residues in the positions B2, B4 to
B27 of the B chain of human insulin, animal insulin or an insulin
derivative thereof;
Xis
(a) an amino acid residue from the group consisting of lysine (Lys) and arginine (Arg), or
(b) a peptide having 2 to 35 amino acid residues, comprising the amino acid residue lysine (Lys) or arginine (Arg) at the N-terminal and at the carboxyl end of the peptide, or
(c) a peptide having 2 to 35 genetically encodable amino acids, comprising 1 to 5 histidine residues;
(A2-A20) are the amino acid residues in the positions A2 to A20 of the
A chain of human insulin, animal insulin or an insulin derivative thereof;
and
R3 is a genetically encodable amino acid residue.




Abstract
The invention provides a process for obtaining a precursor for insulin, analogs or derivatives thereof having correctly bonded cystine bridges. The process involves solubilizing inclusion bodies in a solution containing both cysteine or cysteine hydrochloride and urea followed by reverse dilution.