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 INSULIN REFOLDING
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 cysteine bridges, the process comprises adding the solubilized insulin precursor in a diluent wherein the diluent comprises alcoholic and aprotic solvent.
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 cysteine bridges, the process comprises adding the solubilized insulin precursor in a diluent wherein the diluent comprises 5 to 40% alcoholic or aprotic solvent.
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: A 6-A 11 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 intramolecular 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. Aggregation is an assembly of non-native protein conformations into multimeric state often leading to phase separation and precipitation. Inclusion bodies are usually solubilized in a strong denaturant. When denaturant is removed during refolding the hydrophobic effect drives the unfolded protein molecule to sequester their hydrophobic groups, leading to aggregation. For industrial application it is desirable to eliminate or minimize the formation of protein aggregates.
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 No. 5,808,006 discloses a process for increasing the yield of correct refolding of an incorrectly folded polypeptide selected from the group consisting of IGF-I, growth hormone, and a neurotropin contained in host cells, said process comprising the step of contacting said incorrectly folded polypeptide with a buffer having a pH of 7-12 and comprises about 5-40% (v/v) of an alcoholic or polar aprotic solvent, about 0.2 to 3M of an alkaline earth, alkali metal, or ammonium salt, about 0.1 to 9M of a chaotropic agent, and about 0.01 to 15 .mu.M of a copper or manganese salt.
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.
Cleland, JL and Wang DIC discloses the use of polyethylene glycol (PEG) in the molecular weight range of 1000 to 8000 Daltons to effectively increase the rate of refolding and prevent aggregation of the model protein, bovine carbonic anhydrase B (CAB). (Cosolvent Assisted Protein Refolding. Biotechnology, 8, 1274 -1278 (1990))
It was observed while working on the refolding of insulin, analogues and derivatives that when proinsulin precursors are mixed with cysteine or cysteine hydrochloride and chaotropic auxiliary present simultaneously in a solution, followed by addition of the these solubilized insulin precursors in a


diluent which comprises of one or more alcoholic or aprotic solvents, 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 use of alcoholic or aprotic solvent prevents self-association of protein molecules by decreasing the hydrophobic interaction thereby decreasing the aggregation or precipitation of protein molecules during refolding or storage. This reduced aggregation results in increasing the yield of correctly folded insulins, insulin analogues or derivatives thereof.
One of the aspects of the present invention provides a process for obtaining the precursors of insulin, insulin analogs or derivatives thereof having correctly bonded cysteine bridges, the process comprises
a. mixing precursor of insulin, insulin analogs or derivatives thereof
having incorrectly bonded cystine bridges with an aqueous solution or
buffer comprising cysteine or cysteine hydrochloride and one or more
chaotropic auxiliaries, at a pH of about 7 to about 11.5 and at a
temperature of about 15-55°C;
b. diluting the reaction mixture of step (a) with a diluent which comprises
of about 5-40% (v/v) of an alcoholic or polar aprotic solvent, at a pH
of about 8-11.5 and a temperature of 2 to 40°C.
In one of the embodiments of the invention, alcoholic or an aprotic solvent can be selected from the group consisting of methanol, ethanol, iso-propanol, n-propanol, t-butanol, dimethylsulfoxide, dimethylformamide, N-methylpyrrolidone, tetrahydrofuran, dioxane, and acetonitrile.
In another embodiment of the invention, the alcoholic or polar aprotic solvent is isopropyl alcohol.
In another 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 guanidine hydrochloride.
In another embodiment of the invention, the concentration of guanidine hydrochloride varies from about 5M to 8M.
In another embodiment of the invention, an aqueous solution or buffer in step (a) and diluent in step (b) may further comprise one or more of additives Suitable additives include one or more of Ethylenediamine tetraacetic acid, Ethyleneglycol tetraacetic acid (EGTA), arginine, glycine, alanine, sugars, 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 step (a) is carried out at pH of about 8 to about 9.5 at or below room temperature.
In another embodiment of the invention, step (b) is carried out at pH of about 8 to about 9.5 at or below room temperature.
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 R2is


(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 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 He 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 R2is
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 or aspartic acid;


R 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 : 1ml/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 derivative whose cystine bridges are correctly folded
The expressed fusion protein as insoluble inclusion bodies having the proinsulin sequence 3 (SEQ ID NO: 3) was collected from E. coli cells. Proinsulin sequence 3 (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 He 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 without IPA
573g of guanidine hydrochloride, 3.5g of L-cysteine hydrochloride and 0.75g of ethylenediamine tetra acetic acid disodium salt was added to one litre of 20mM Tris buffer and 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 8g of isolated freeze dried inclusion bodies containing 3.2g of insulin precursor of sequence 3 (SEQ ID NO: 3) (the portion of insulin contain fusion protein is determined with the aid of HPLC, it is 40%) was weighed and dissolved in the above solution having both L-cysteine and guanidine hydrochloride. The solution was stirred for four hours at room temperature and transferred over a period of 4hrs to 9 Litres of precooled (10+2°C) 20mM Tris buffer at pH 9.0 containing 6.7g EDTA, 1.08 g L-cysteine and 773.79 g of guanidine hydrochloride. The diluted refolding mixture was further stirred for 24hrs at (10+2°C). After 24hrs the content of insulin precursor of sequence 3 having correctly bonded cystine bridges in the reaction mixture was determined with the aid of HPLC 1.12 g of correctly folded insulin precursor of sequence 3 (corresponding to a recovery of 35.02%) was recovered.
Example 2B: Refolding with 10 % IPA
573g of guanidine hydrochloride, 3.5g 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 8g of isolated freeze dried inclusion bodies containing 3.2g of insulin precursor of proinsulin sequence 3 (SEQ ID NO: 3) (the portion of insulin contain fusion protein is determined with the aid of HPLC, it is 40%) was weighed and dissolved in the above solution having both L-cysteine and guanidine hydrochloride. The solution was stirred for four hours at room temperature and transferred over a period of 4hrs to 9 liters of precooled (10+2°C) Tris buffer (20mM) containing 6.7g EDTA, 1.08 g L-cystine, 773.79 g of guanidine hydrochloride and 1 Litre of Iso- propyl alcohol at pH 9. The diluted refolding mixture was stirred for 24hrs at (10+2°C) . After 24hrs, the content of insulin precursor proinsulin sequence 3 having correctly bonded cystine bridges in the reaction mixture was determined with the aid of HPLC


1.39g of correctly folded insulin precursor of proinsulin sequence 3 (corresponding to a recovery of 43.55%) was recovered.
Example 2C: Refolding with 20 % IPA
573g of guanidine hydrochloride, 3.5g of L-cysteine hydrochloride and 0.75g of ethylenediamine tetra acetic acid disodium salt was added to one liter 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 8g of isolated freeze dried inclusion bodies containing 3.2g of insulin precursor of proinsulin sequence 3 (SEQ ID NO: 3) (the portion of insulin contain fusion protein is determined with the aid of HPLC, it is 40%) was weighed and dissolved in the above solution having both L-cysteine and guanidine hydrochloride. The solution was stirred for four hours at room temperature and transferred over a period of 4hrs to 9 liters of precooled (10+2°C) Tris buffer (20mM) containing 6.7g EDTA, 1.36 g, L-cystine, 773.79 g of guanidine hydrochloride and 2 Litre of Iso-propyl alcohol at pH 9. The diluted refolding mixture was stirred for 24hrs at (10+2°C). After 24hrs, the content of insulin precursor proinsulin sequence 3 having correctly bonded cystine bridges in the reaction mixture was determined with the aid of HPLC. 1.52g of correctly folded insulin precursor of proinsulin sequence 3 (corresponding to a recovery of 47.64%) was recovered.
Example 3
Process 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 5 (SEQ ID NO: 5) was collected from E. coli cells. Proinsulin sequence 5 (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 lie 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 without IPA
573g of guanidine hydrochloride, 3.5g of L-cysteine hydrochloride and 0.75g of ethylenediamine tetra acetic acid disodium salt was added to one litre of 20mM Tris buffer and 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 8g of isolated freeze dried inclusion bodies containing 3.2g of insulin precursor of sequence 5 (SEQ ID NO: 5) (the portion of insulin contain fusion protein is determined with the aid of HPLC, it is 40%) was weighed and dissolved in the above solution having both L-cysteine and guanidine hydrochloride. The solution was stirred for four hours at room temperature and transferred over a period of 4hrs to 9 Litres of precooled (10+2°C) 20mM Tris buffer at pH 9.0 containing 6.7g EDTA, 1.08 g L-cysteine and 773.79 g of guanidine hydrochloride. The diluted refolding mixture was further stirred for 24hrs at (10+2°C). After 24hrs the content of insulin precursor of sequence 5 having correctly bonded cystine bridges in the reaction mixture was determined with the aid of HPLC 1.1 g of correctly folded insulin precursor of sequence 5 (corresponding to a recovery of 34.5%) was recovered.
Example 3B: Refolding with 10 % IPA
573g of guanidine hydrochloride, 3.5g 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 8g of isolated freeze dried inclusion bodies containing 3.2g of insulin precursor of proinsulin sequence 5 (SEQ ID NO: 5) (the portion of insulin contain fusion protein is determined with the aid of HPLC, it is 40%) was weighed and dissolved in the above solution having both L-cysteine and guanidine hydrochloride. The solution was stirred for four hours at room temperature and transferred over a period of 4hrs to 9 liters of precooled


(10+2°C) Tris buffer (20mM) containing 6.7g EDTA, 1.08 g, L-cystine, 773.79 g of guanidine hydrochloride and 1 Litre of Iso- propyl alcohol at pH 9. The diluted refolding mixture was stirred for 24hrs at (10+2°C). After 24h, the content of insulin precursor proinsulin sequence 5 having correctly bonded cystine bridges in the reaction mixture was determined with the aid of HPLC 1.3g of correctly folded insulin precursor of proinsulin sequence 5 (corresponding to a recovery of 40.71 %) was recovered.
Example 3C: Refolding with 20 % IPA
573g of guanidine hydrochloride, 3.5g of L-cysteine hydrochloride and 0.75g of ethylenediamine tetra acetic acid disodium salt was added to one liter 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 8g of isolated freeze dried inclusion bodies containing 3.2g of insulin precursor of proinsulin sequence 5 (SEQ ID NO: 5) (the portion of insulin contain fusion protein is determined with the aid of HPLC, it is 40%) was weighed and dissolved in the above solution having both L-cysteine and guanidine hydrochloride. The solution was stirred for four hours at room temperature and transferred over a period of 4hrs to 9 liters of precooled (10+2°C) Tris buffer (20mM) containing 6.7g EDTA, 1.36 g L-cystine, 773.79 g of guanidine hydrochloride and 2 Litre of Iso propyl alcohol at pH 9. The diluted refolding mixture was stirred for 24hrs at (10+2°C). After 24h, the content of insulin precursor proinsulin sequence 5 having correctly bonded cystine bridges in the reaction mixture was determined with the aid of HPLC. 1.47g of correctly folded insulin precursor of proinsulin sequence 5 (corresponding to a recovery of 46.22%) 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 6 (SEQ ID NO: 6) was collected from E. coli cells. Proinsulin sequence 6 (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 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 4A: Refolding without IPA
573g of guanidine hydrochloride, 3.5g of L-cysteine hydrochloride and 0.75g
of ethylenediamine tetra acetic acid disodium salt was added to one litre of 20mM Tris buffer and 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 8g of isolated freeze dried inclusion bodies containing 2.8g of insulin precursor of sequence 6 (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 guanidine hydrochloride. The solution was stirred for four hours at room temperature and transferred over a period of 4hrs to 9 Litres of precooled (10+2°C) 20mM Tris buffer at pH 9.0 containing 6.7g EDTA, 1.08 g L-cysteine and 773.79 g of guanidine hydrochloride. The diluted refolding mixture was further stirred for 24hrs at (10+2°C). After 24h the content of insulin precursor of sequence 6 having correctly bonded cystine bridges in the reaction mixture was determined with the aid of HPLC 0.793 g of correctly folded insulin precursor of sequence 6 (corresponding to a recovery of 28.34%) was recovered.
Example 4B: Refolding with 10 % IPA
573g of guanidine hydrochloride, 3.5g 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 8g of isolated freeze dried inclusion bodies containing 2.8g of insulin precursor of proinsulin sequence 6 (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 guanidine hydrochloride. The solution was stirred for four hours at room temperature and transferred over a period of 4hrs to 9 liters of precooled (10+2°C) Tris buffer (20mM) containing 6.7g EDTA, 1.08 g L-cystine, 773.79 g of guanidine hydrochloride and 1 Litre of Iso-propyl alcohol at pH 9. The diluted refolding mixture was stirred for 24hrs at (10+2°C). After 24h, the content of insulin precursor proinsulin sequence 6 having correctly bonded cystine bridges in the reaction mixture was determined with the aid of HPLC 1.02g of correctly folded insulin precursor of proinsulin sequence 6 (corresponding to a recovery of 36.58%) was recovered.
Example 4C: Refolding with 20 % IPA
573g of guanidine hydrochloride, 3.5g of L-cysteine hydrochloride and 0.75g of ethylenediamine tetra acetic acid disodium salt was added to one liter 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 8g of isolated freeze dried inclusion bodies containing 2.8g of insulin precursor of proinsulin sequence 6 (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 guanidine hydrochloride. The solution was stirred for four hours at room temperature and transferred over a period of 4 hrs to 9 liters of precooled (10+2°C) Tris buffer (20mM) containing 6.7g EDTA, 1.36 g L-cystine, 773.79 g of guanidine hydrochloride and 2 Litre of Iso -propyl alcohol at pH 9. The diluted refolding mixture was stirred for 24hrs at (10+2°C) . After 24h, the content of insulin precursor proinsulin sequence 6 having correctly bonded cystine bridges in the reaction mixture was determined with the aid of HPLC. 1.6g of correctly folded insulin precursor of proinsulin sequence 6 (corresponding to a recovery of 48.65%) 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 analogs or
derivatives thereof having correctly bonded cysteine bridges, the process
comprises
(a) mixing precursor of insulin, insulin analogs or derivatives thereof having incorrectly bonded cystine bridges with an aqueous solution or buffer comprising cysteine or cysteine hydrochloride and one or more chaotropic auxiliaries, at a pH of about 7 to about 11.5 and at a temperature of about 15 to about 55°C;
(b) diluting the reaction mixture of step (a) with a diluent which comprises of about 5-40% (v/v) of an alcoholic or polar aprotic solvent, at a pH of about 8 to about 11.5 and a temperature of about 2°C to about 40°C.

2. The process as claimed in claim 1, wherein the alcoholic or aprotic solvent can be selected from the group consisting of methanol, ethanol, iso-propanol, n-propanol, t-butanol, dimethylsulfoxide, dimethylformamide, N-methylpyrrolidone, tetrahydrofuran, dioxane, and acetonitrile.
3. The process as claimed in claim 2, wherein the alcoholic or polar aprotic solvent is isopropyl alcohol.
4. 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.
5. 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.
6. The process as claimed in claim 5, wherein the chaotropic auxiliary is chaotropic auxiliary is guanidine hydrochloride.


7. The process as claimed in claim 6, wherein the concentration of guanidine hydrochloride varies from about 5M to about 8M.
8. The process as claimed in claim 1, wherein an aqueous solution or buffer in step (a) and diluent in step (b) 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, salts such as ammonium sulphate and magnesium chloride, and cyclodextrins or salts thereof.
9. The process as claimed in claim 1, wherein the step (a) or step (b) is carried out in pH range of about 8.0 to about 9.5 at or below room temperature.
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 R2is
a hydrogen atom,
an amino acid residue from the group consisting of lysine (Lys) and arginine (Arg), or
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 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 cysteine bridges. The process involves solubilizing inclusion bodies in a solution containing both cysteine or cysteine hydrochloride and urea followed by reverse dilution.