MXPA98006666A - Improved procedure for the obtaining of insulin precursors with cistina bridges correctly uni - Google Patents

Abstract

The present invention relates to an improved process for obtaining a precursor of insulins or insulin derivatives with correctly bound cystine bridges, in the presence of cysteine or cysteine hydrochloride and a caotropic adjuvant substance.

Description

Improved procedure for obtaining insulin precursors with cystine bridges correctly attached The present invention relates to an improved process for obtaining a precursor of insulin-derived insulins with correctly linked cystine bridges in the presence of cysteine or cysteine hydrochloride of a chaotropic coadjuvant substance. Human insulin is a protein with two chains of 10 amino acids with 51 amino acid residues together. 6 cysteine residues are present in the amino acid chains, the cysteine residues being linked to each other, < through a disulfide bridge. In biologically active human insulin, the A and B chains are linked together via two cystine bridges, and another cystine bridge is present in the A chain. Within a molecule of human insulin there are, from the statistical point of view, 15 possibilities for the formation of disulph bridges. E biologically active human insulin only one of the 15 possibilities is present. The following cysteine residues are linked to human insulin: A6-A11 A7-B7 - ^ A20-B19 25 The letters A and B represent the respective amino acid chain of insulin, the number represents the amino acid residue position, which is counted from the amino terminus to the carboxyl of the respective amino acid chain. S can also form disulfide bridges between two molecules of human insulin, so that many different disulfide bridges can easily be originated. A known method for the preparation of human insulin is based on the use of human proinsulin. Human proinsulin is a protein with a linear amino acid chain of 86 amino acid residues, the B and A chains of human insulin being linked together via a peptide. with 35 amino acid residues. The formation of the disulfide bridges present in human insulin is effected through an intermediate product, the cysteine residues of human insulin being provided with a sulfur protecting group, for example, an S-sulfonate group (- S-S03 ~) (EP 0 037 255). In addition, a method for obtaining proinsulin with correctly bound cystine bridges (Biochemistry, 60, (1968), pages 622 to 629) which part of proinsulin obtained from pig pancreas, in which 10 cysteine residues are present, is known. as thiol residues (-SH). By the term "correctly linked cystine bridges" is meant the disulfide bridges that are present in the insulin of biologically active mammals. Genetic engineering methods make it possible to prepare insulin precursors or insulin derivatives in microorganisms, especially human proinsulin or proinsulin having an amino acid sequence and / or p-amino acid chain length that differs from human insulin. The proinsulins prepared from genetically engineered Escherichia coli 20 cells do not have correctly linked cystine bridges. A method for obtaining human insulin with E. coli (EP 0 055 • "945) is based on the following process steps: Fermentation of the microorganisms - opening of cells - isolation of the fusion protein - dissociation of fusion protein with cyanogen halide - isolation of the dissociation product with the sequence of proinsulin - protection of the cysteine residues of proinsulin by S-sulfonate groups - chromatographic purification of 30 S-sulfonate - formation of cystine bridges correctly - desalination of proinsulin - chromatographic purification of proinsulin with correctly bound cystine bridges - concentration of proinsulin solution - chromatographic purification of concentrated proinsulin solution - enzymatic dissociation of proinsulin to obtain human insulin - purification Chromatographic measurement of human insulin In this procedure, the number of stages of the procedure and the losses in the purification stages are disadvantageous., which lead to a small yield in insulin. Due to the multistage process, considerable losses must be taken into account. From the stage of the isolated fusion protein, through dissociation with cyanogen halide, sulfitolysis and purification of proinsulin, a loss of up to 40% of proinsulin must be counted (EP 0 055 945). High similar losses can occur during the subsequent purification stages up to the final product. Therefore, in the genetic engineering of human insulin or insulin derivatives, yield increases can be achieved if the number of necessary process steps can be substantially reduced. From EP 0 600 372 A1 (US Pat. No. 5,473,049) and EP 0668 292 A2 a correspondingly improved method for obtaining insulins or insulin derivatives is known, in which the insulin precursor or precursor of the derivative of Insulin, whose cystine bridges are not correctly bound, is reacted in the presence of a mercaptan, for example cysteine, and at least one chaotropic coadjuvant substance, for example urea or guanidine hydrochloride, to give an insulin precursor or precursor of the Insulin derivative with correctly linked cystine bridges. In the known process, these proteins are dissolved first, in a very low concentration, in aqueous solutions of a chaotropic coadjuvant substance or mixtures of various chaotropic coadjuvant substances. Next, the protein mixture is mixed with an aqueous mercaptan solution. Surprisingly, it has now been found that yields in insulin precursors or insulin derivatives correctly folded increase and the reaction times for the process of shedding can be reduced if in the first stage the precursor is not brought into solution by the adjuvant substance. chaotropic but, firstly, the mercaptan, ie cysteine or cistern hydrochloride, is incorporated into the aqueous suspension of the precursor, and only at a later stage dissolution of the precursor occurs by incorporation into an aqueous solution of the substance chaotropic adjuvant and, finally, the correct folding of the precursor by diluting the mixture to a preferred concentration of cysteine or cysteine hydrochloride with the incorporation of the mixture into a corresponding amount of water. Accordingly, the present invention relates to a method for obtaining a precursor of insulins or insulin derivatives with correctly bound cystine bridges, in the presence of cysteine or cysteine hydrochloride and a chaotropic coadjuvant, - crimped because the following 20 steps are successively carried out: (a) Mixing an aqueous suspension of the insulin precursor or insulin derivatives with an amount of cysteine or cysteine hydrochloride that provides ^? from 1 to 15 SH residues of the cysteine or cysteine hydrochloride per cysteine residue of the precursor, (b) incorporation of the suspension of the precursor, which contains cysteine or cysteine hydrochloride, to a solution of 4 to 9 molar of the substance chaotropic adjuvant, at a pH value of about 8 to about 11.5 and at a temperature of about 15SC to about 559C, keeping the mixture obtained for about 10 to 60 minutes at this temperature and (c) incorporating the mixture, at a pH value from about 8 to about 11.5 and a temperature from about 5SC to about 30C, to an amount of water that provides a dilution of the concentration of the cysteine or the cysteine hydrochloride in the mixture of about , 1 to 5 M and the chaotropic coadjuvant substance from 0.2 to 1.0 M. Preferably, the process is characterized in that in step (a) the amount The amount of cysteine or cysteine hydrochloride corresponds to that which provides from 1 to 6 SH residues of cysteine or cysteine hydrochloride per residue. cysteine of the precursor, 10 in step (b) is carried out the incorporation of the suspension of the precursor containing cysteine or cysteine hydrochloride to a solution of 4 to 9 molar of the substance - chaotropic adjuvant at a pH value of 8 to 11 and at a temperature of 30 to 45 ° C, maintaining the mixture obtained during 20 to 40 minutes at this temperature, and in step (c) the incorporation of the mixture is carried out , at a pH value of 8 to 11 and at a temperature of 15 to 20 ° C, a quantity of water that provides a dilution of the concentration of cysteine or cysteine hydrochloride in the mixture of about 1 to 5 mM and a concentration of ": the chaotropic coadjuvant substance from 0.2 to 1.0 M. The chaotropic coadjuvant substances are compounds which break the bonds in aqueous solution by hydrogen bonds, for example ammonium sulphate, hydrochloride ^ 25 guanidine, ethylene carbonate, thiocyanate, dimethylsulfoxide and urea. In the process according to the present invention, guanidine hydrochloride or, with special preemption, urea is preferably used as chaotropic coadjuvant substance. The concentration of the chaotropic coadjuvant in step (b) of the process according to the invention is preferably from 7.0 to 9 M, the temperature in step (b), preferably 40 ° C, and the pH value in step 35 (b), preferably, from 10 to 11. In the process according to the invention, the value * of the pH in step i c) is preferably from 10 to 11. In step (c) of the method according to To the present invention, the amount of water to which the mixture is incorporated is preferably chosen such that it provides a dilution of the concentration of cysteine or cysteine hydrochloride in the mixture of 2.5 to 3 mM and a concentration of of the 0, 5 M chaotropic coadjuvant substance. Particularly preferably, the process according to the invention is characterized in that the concentration of the chaotropic coadjuvant substance in step (b) is approximately 8 M, the temperature of the step ( b) is approximate At 40 ° C., the pH value in step (b) is about 10.6, the pH value in step (c) is about 10.6, and in step (c), the amount of water provides a dilution of the cysteine or cysteine hydrochloride concentration in the mixture of approximately 2.5 to 3 mM and a concentration of the adjuvant substance. - & _ _ 3 & chaotropic 0.5 M. The result of the process according to the present invention is a precursor of insulins or insulin derivatives, especially a proinsulin, whose cystine bridges are correctly bound. The insulin derivatives are derivatives of insulins present in nature, namely human insulin (see SEQ ID NO 1 = human insulin A chain); see SEQ ID NO 2 = human insulin B chain, sequence protocol) or animal insulins, which differ by substitution of at least one amino acid residue present in nature and / or addition of at least one amino acid residue and / or remainder organic of 30 the corresponding insulins present in nature, otherwise equal. From the precursor of insulins or insulin derivatives with correctly bound cystine bridges, obtained with the aid of the process according to the present invention, it can finally be prepared, according to the process described in EP 0 600 372 Al ( or * US 5,473,049) or in EP 0 668 292 A2, by enzymatic dissociation with trypsin or a trypsin-like enzyme and, optionally, additionally with carboxypeptidase B and subsequent purification1 in an adsorption resin, an insulin or an insulin derivative with correctly linked cystine bridges. Insulin or insulin derivative preparable from the precursor can preferably be described by the formula I (I), And Z (B30) where Y is a genetically encodable amino acid residue, Z a) an amino acid residue of the His group, Arg or Lys, b) a peptide with 2 or 3 amino acid residues containing the amino acid residue Arg or Lys at the carboxyl terminus of the peptide , c) a peptide with 2-35 genetically encodable amino acids, containing 1 to 5 histidine moieties, od) OH, Rx a phenylalanine residue (Phe) or a covalent bond, R3 a genetically encodable amino acid residue, corresponding to residues A2-A20, not shown for the purpose of formula I, the amino acid sequence of the • A chain of human insulin, animal insulin or an insulin derivative, and residues B2-B29, not shown for simplification of formula I, to the amino acid sequence of the B chain of the numan insulin, animal insulin or an insulin derivative. t 'The amino acid sequence of peptides and proteins is designated from the N-terminus end of the amino acid chain. The indications in parentheses made in formula I, for example A6, A20, Bl, B7 or B19, correspond to the position of amino acid residues in the A or B chains of insulin. The expression "genetically encodable amino acid residue" represents the amino acids Gly, Ala, Ser, Thr, Val, Leu, He, Asp, Asn, Glu, Gln, 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, for example, as the amino acid sequences of cows, pigs or chickens. The terms "residues A2-A20" and "residues B2-B29" of insulin derivatives represent the corresponding amino acid sequences of human insulin that are formed by the exchange of amino acids by other genetically encodable amino acids. The A chain of human insulin has, for example, the following sequence (SEQ ID NO.:l): Gly He Val Glu Gln Cys Cys Thr Ser lie Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn. The B chain of human insulin has the following sequence (SEQ ID NO .: 2): Phe Val Asn Gln 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 . In this case, in the formula I R3 is asparagine (Asn), R1 is phenylalanine (Phe), Y is threonine (Thr) and Z is OH. Accordingly, the process according to the present invention is especially suitable for obtaining a precursor of insulins or insulin derivatives with the general formula II, whose cystine bridges (not shown in Formula II) are correctly folded, R2 -R1- (B2-B29) -YX-Gly- (A2-A20) -R3 (II), in which R2 a) denotes an atopy of hydrogen, 5 b) an amino acid residue of the group lysine (Lys) or arginine (Arg), or c) a peptide with 2 to 45 amino acid residues containing the amino acid residue, lysine (Lys) or arginine (Arg) at the carboxyl terminus of the peptide, R1 a phenylalanine residue (Phe) or a covalent bond, ( B2-B29) the amino acid residues at positions B2 to B29 of the B chain of human insulin, animal insulin or an insulin derivative, optionally differentiated in one or several of these positions, and a remainder genetically encodable amino acid, X a) an amino acid residue of the lysine (Lys) group or rginine (Arg), b) a peptide with 2 to 35 amino acid residues containing the amino acid residue lysine (Lys) or arginine (Arg) at the N-terminus and at the carboxyl terminus of the peptide, or .. ' c) a peptide with 2 to 35 genetically usable amino acids containing from 1 to 5 residues histidi-amino acid residues at positions A2 to A20 of the B chain of human insulin, animal insulin or an insulin derivative, optionally differentiated into one or several of these positions, and 30 R3 a genetically encodable amino acid residue. 1. In formula II they preferably mean: R2 a) a hydrogen atom, or b) a peptide with 2 to 25 amino acid residues containing the amino acid residue arginine (Arg) at the carboxyl end of the peptide, R1 a phenylalanine residue (Phe), PL-- (B2-B29) the amino acid residues in positions B2 to B29 of the B chain of human insulin, and an amino acid residue of the alanine (Ala), treoni? A group Thr) or serine (Ser), 5 X the amino acid residue arginine (Arg) or a peptide with the amino acid sequence of the C chain of human insulin, • (A2-A20) the amino acid residues at positions A2 to A20 of the B chain of human insulin, and 10 R3 an amino acid residue of the group asparagine (Asn), serine (Ser) or glycine (Gly). The C chain of human insulin has the following sequence (SEQ ID NO .: 3): Arg Arg Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly 15 Gly Gly Pro Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys Arg. 2. In the formula II they preferably mean: R2 a) a hydrogen atom, or b) a peptide with 2 to 15 amino acid residues, at the carboxyl end of which there is an arginine residue (Arg), R1 a phenylalanine residue (Phe), (B2-B29) the amino acid residues in positions B2 to B29 "of the B chain of human insulin, 25 Y a threonine residue (Thr), X the amino acid residue arginine (Arg) or a peptide with 2 to 35 amino acid residues, being located at the beginning and at the end of the peptide two amino acid residues of basic character, especially arginine 30 (Arg) and / or lysine (Lys), (A2-A20) the amino acid residues at positions A2 to A20 of the B chain of human insulin, and R3 the amino acid residue asparagine (Asn ) or glycine (Gly) The Z radical of the insulin or insulin derivative of the formula I is generally part of the amino acid sequence of X of the precursor of formula II and is formed by the activity of proteases such as trypsin, enzymes analogous to trypsin or carboxypeptidase The radical R3 is the aminoacid moiety that is at position A21 of the 5A chain of insulin.The radical Y is the amino acid residue that is at position B30 of the B chain of insulin, trypsin or enzymes analogous to tr ipsin are proteases, which dissociate the amino acid chains by the arginine or lysine residue. Carboxypeptidase B is an exoprotease which dissociates basic amino acid residues such as Arg or Lys which are found at the carboxyl terminal end of amino acid chains. (Kemmler et al., J. Biol. Chem. 246, pages 6787-6791). From the precursor mentioned in section 1, it is possible to obtain, for example, an insulin or an insulin derivative of formula I with correctly linked cystine bridges, having Y, R1, R2, R3, A2-A20 and B2- B29 the meaning mentioned in section 1 and Z means a remainder arginine (Arg), a peptide residue Arg-Arg u -OH. From the precursor mentioned in section 2, it is possible to obtain, for example, an insulin or an insulin derivative of formula I with correctly linked cystine bridges, having Y, R1, R2, R3, A2-A20 and B2-B29 the meaning mentioned in section 2 and Z means an arginine residue (Arg), a peptide residue Arg-Arg or Lys-Lys or -OH. The precursor of formula II can be formed with the aid of a plurality of genetically engineered constructs in microorganisms (EP 0 489 780, EP 0 347 781, EP 0 453 969). Genetically engineered constructs are expressed in microorganisms such as Escherichia coli or streptomycetes during fermentation. The proteins formed are deposited inside the microorganisms (EP 0 489 780) or are secreted into the solution of fermentation. For the process according to the invention, insulin precursors or insulin derivatives of formula II can be used, which immediately after the opening of cells are still contaminated with a plurality of proteins coming from the fermentation solution and from the microorganisms . However, the precursors of formula II can also be used in prepurified form, for example after precipitation or chromatographic purification.

Example 1 (Comparative example, state of the art) By fermentation of genetically modified Escherichia coli cells (EP 0 489 780) a fusion protein with the following amino acid sequence is obtained. 15 Sequence of proinsulin 1 (SEQ ID N0.:4): Wing Thr Thr Ser Thr Gly Asn Wing Arg Phe Val Asn G n His Leu & CY3 G1Y Be Hls Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg Glu Wing Glu Asp Leu Gln 20 Val G1Y Gln Val Glu Leu Gly Gly Gly Pro Gly Wing Gly Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys Arg Gly lie Val Glu Gln Cys Cys Thr Ser lie Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn The sequence of proinsulin 1 corresponds to formula 25 II, where X is the C peptide of human insulin (SEQ ID NO.:3) :, Y THr (B30), R1 Phe (Bl), R2 a peptide with 10 amino acid residues , 30 R3 Asn (A21), and A2-A20 the amino acid sequence of the A chain of human insulin (amino acid residues 2 to 20) and B2-B29 the amino acid sequence of the B chain of human insulin (amino acid residues 2 to 29). In the E. coli cells, the fusion protein expressed with the proinsulin 1 sequence accumulates and forms inclusion corpuscles. After the completion of the fermentation, the cells are separated by centrifugation and opened by usual homogenization at high pressure. Fusion protein inclusion bodies released 5 are isolated by mediating centrifugation. 20 kg of the isolated fusion protein inclusion bodies are dissolved (referred to the dry mass after lyophilization; the fraction of the fusion protein containing insulin is determined with the aid of HPLC; amounts to 50%) with the sequence of proinsulin 1 in 550 1 of an 8 M urea solution, at pH 10.6. Eventually, after centrifugation of small amounts of cloudy material, the clear solution is incorporated with stirring into 9,000 1 of an aqueous solution of cysteine (5 kg of hydrochloride of cysteine hydrate) at a pH value of 10.6 and at a temperature of 49C. After completion of the folding reaction after approx. 24 hours is determined in the reaction batch, with the help of analytical HPLC, the sequence content of proinsulin I with correctly bound cystine bridges with 3.0 kg, corresponding to a conversion of 30%. The 9,500 1 solution is adjusted with IN HCl to a pH of 5.0 and separated. Then the adjustment is made to pH 9 by Hr the addition of sodium hydroxide IN. They are incorporated into the solution 3 g of trypsin. 1.25 kg of an insulin with 2 arginine residues are originated at the carboxyl terminal ends, according to the determination by HPLC. After dissociation with carboxypeptidase B results in human insulin, which is further purified with the help of chromatographic methods. Human insulin corresponds to Formula I, where Y is Thr (B30), Z OH, R3- Phe (Bl), 35 R3 Asn (A21) and A2-A20 the amino acid sequence of the A chain of human insulin (amino acid residues 2 to 20) and B2-B29 the amino acid sequence of the B chain of human insulin (rest amino acids 2 to 29). The insulin fhumana 2 is composed of SEQ ID N0.:1 and 2, 5 which are united1; each other through correctly joined cistin bridges. The solution is concentrated and purified by means of adsorption resin as described in EP 0 66 292. The eluted material, which contains insulin 2, after dilution with water and pH adjustment, can be further purifi ed immediately. on a chromatographic column.

HPLC analysis 0.5 g of protein are dissolved in 40 ml of a solution of 6 M guanidine hydrochloride, 50 mM Tris, pH 8.5, 5 mM ethylene diamine tetraacetate (EDTA), 1% 2-mercaptoethanol, dithiothreitol 10 ItiM, at 95 SC for 2 min and after & .ÍÍJ --- SS. centrifuge at 14,000 g for 20 min. From the transparent supernatant 0.02 ml is applied on a chromatographic column of high pressure liquids. Column: RNucleogel RP 300-5 / 46 (Macherey &Nagel, Aachen, Germany) Gradient: Buffer A: 0.1% trifluoroacetic acid (TFA) "t Buffer B: 0.09% TFA in acetonitrile 25 Temperature: 55aC Total elution time: 40 min The gradient is characterized by the following quantities of buffer B after the corresponding elution times: 30 10 min 25% , 12 min 60%, 13 min 90%, 15 min 100% Flow rate: 1 ml / min Detection: 215 nm Insulin retention time: approximately 19 min.

Example 2 (Method according to the present invention) - By fermentation of genetically modified Escherichia coli cells (EP 0 489 780) a fusion protein with the amino acid sequence represented in Ejeijiplo 1 (sequence of proinsulin 1, SEQ ID 5 NO .: 4). In the E. coli cells, the fusion protein expressed with the sequence of proinsulin 1 is accumulated and forms inclusion corpuscles. After the completion of the fermentation, the cells are separated by centrifugation and opened by means of usual homogenization under high pressure. Fusion protein inclusion bodies released are isolated by centrifugation. To the aqueous suspension of fusion protein containing 40 kg of fusion protein (determined by lyophilization of an aliquot), 5 kg of cysteine hydrochloride hydrate are added. The suspension with the sequence of proinsulin 1 (the fraction of the fusion protein containing insulin is determined with the aid of HPLC, it is 50%) is dissolved in 20 550 1 of an 8M urea solution at pH 10.2, a 409C. The clear solution is incorporated with stirring in 9,000 1 of water at a pH value of 10, 6 and at a temperature of 15 C. After completion of the folding reaction after approx. After 5 hours, the sequence content of proinsulin 1 with correctly bound cystine bridges with 10.0 kg, corresponding to a conversion of 50%, is determined in the reaction batch, with the aid of analytical HPLC. The 9.500 1 solution is adjusted with 1N HCl to a pH of 30.0, and separated. The adjustment is then made to pH 9 by the addition of 1N sodium hydroxide solution. 10 g of trypsin are incorporated into the solution. 4 kg of an insulin with 2 arginine residues are originated at the terminal carboxy ends. After dissociation with carboxypeptidase B, human insulin is generated (SEQ ID NO .: 1 and 2 with correctly bound cystine bridges).

The solution is concentrated and purified by means of adsorption resins. The eluted material, which contains human insulin, after dilution with water and pH adjustment, can be further purified immediately on a chromatographic column.

Example 3 (Comparative example, state of the art) By fermentation of genetically modified Escherichia coli cells (EP 0 489 780) a fusion protein with the following amino acid sequence is prepared.

Sequence of proinsulin 2 (SEQ ID NO .: 5): Wing Thr Thr Ser Thr Gly Asn Wing Arg Phe Val Asn Gln 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 Wing Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro Gly Wing Gly Ser Leu Gln Pro Leu Wing Leu Glu Gly Being Leu Gln Lys Arg Gly lie Val Glu Gln Cys Cys Thr Ser lie Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Gly The sequence of proinsulin 2 corresponds to formula II, where X is the C peptide of human insulin (SEQ ID No. 3), and Thr (B30), R1 Phe (Bl), R2 a peptide with 10 amino acid residues, R3 Gly (A21) and A2-A20 the amino acid sequence of the A chain of human insulin (amino acid residues 2 to 20) and B2-B29 the amino acid sequence of the B chain of human insulin (amino acid residues 2 to 29). In the E. coli cells, the fusion protein expressed with the proinsulin 2 sequence accumulates and forms inclusion corpuscles. After completion of the fermentation, the cells are separated by centrifugation * and opened by usual homogenization under high pressure. Fusion protein inclusion bodies released are isolated by centrifugation. 20 kg of the inclusion bodies of the isolated fusion protein are dissolved (referred to the dry mass after lyophilization, the fraction of the fusion protein containing insulin is determined with the aid of HPLC, amounts to 50%) with the proinsulin 2 sequence in 550 1 of an 8M urea solution at pH 10.6, at 20SC. The clear solution is incorporated with stirring in 9,000 1 of an aqueous solution of cysteine (5 kg of cysteine hydrochloride hydrate) at a pH value of 10.6 and at a temperature of 4aC. ~ * After completion of the folding reaction after approx. 24 hours, the sequence content of proinsulin 2 with cystine bridges correctly linked with 3.0 kg, corresponding to a conversion of 30%, is determined in the reaction batch with analytical HPLC aid. The 9,500 1 solution is adjusted with HCl to IN at a pH of 5.0 and separated. Then the adjustment to pH 9 is made by the addition of sodium hydroxide solution. 3 g of trypsin are incorporated into the solution. 0.98 kg of an insulin derivative with 2 arginine residues are originated at the carboxyl termini. ':' •: - terminals, as determined by HPLC ppr. -β This insulin derivative corresponds to formula I, where Y is Thr (B30), Z Arg-Arg, R1 Phe (Bl) R3 Gly (A21) and 30 A2-A20 the amino acid sequence of the A chain of insulin human (amino acid residues 2 to 20) and B2-B29 the amino acid sequence of the B chain of human insulin (amino acid residues 2 to 29), and consists of SEQ ID NO: 6 and 7, which are linked together through cystine bridges 35 correctly attached. The solution is concentrated and purified by means of adsorption resins. The eluted material, which contains the insulin derivative, after dilution with water and pH adjustment, can be followed by immediately impinging on a chromatographic column.

Example 4 (Method according to the present invention) By fermentation of genetically modified Escherichia coli 10 cells (EP 0 489 780), the fusion protein is prepared with the sequence of proinsulin 2 (SEQ ID NO: 5), according to Example 3. In the cells of E. coli accumulates the fusion protein expressed with the proinsulin 2 sequence and forms 15 inclusion bodies. After the completion of the fermentation, the cells are separated by centrifugation and opened by usual homogenization under high pressure. The f- | f-ÍÍf fusion protein inclusion corpuscles released They are isolated by centrifugation. To the aqueous suspension of fusion protein that '' '. • "contains 40 kg of fusion protein (determined by lyophilization of an aliquot part), 5 kg of cysteine hydrate hydrochloride is added, and the suspension with the sequence of proinsulin 2 (the fraction of the insulin-containing fusion protein) is added. it is determined with the help of HPLC (50% increase) is dissolved in 550 1 of an 8M urea solution at pH 10.2 at 40 ° C. The clear solution is incorporated with stirring in 9,000 1 of water at a pH value of 10.6 and at a temperature of 30 ° C. After the conclusion of the folding reaction after about 5 hours, the sequence content of proinsulin 1 with cystine bridges is determined in the reaction run, using analytical HPLC. correctly added with 10.0 kg, corresponding to a conversion of 50% 35 The solution of 9,500 1 is adjusted with IN HCl to a pH of • 5.0 and separated, followed by adjustment to pH 9 by addition of soda lye. add 10 g of trypsin to the solution. 2.8 kg of the insulin derivative (determination by HPLC) consisting of the sequences SEQ ID NO .: 6 and 7, which are / are linked to each other through correctly joined cystine bridges 5, originate. The solution is concentrated and purified by means of adsorption resins.; The eluted material, which contains the insulin derivative, after dilution with water and adjustment of the pH, can be followed by purification immediately on a chromatographic column. *

Claims (13)

1. Procedure for obtaining a precursor of insulins or insulin derivatives with correctly bound cystine bridges, in the presence of cysteine or of cysteine hydrochloride and of a chaotropic coadjuvant substance, characterized in that the following steps are carried out successively: (a) ) Mixing an aqueous suspension of the insulin or insulin derivative precursor with an amount of cysteine or cysteine hydrochloride which provides from 1 to 15 SH residues of the cysteine or cysteine hydrochloride by the cysteine residue of the precursor, (b) incorporation of the suspension of the precursor, containing cysteine or cysteine hydrochloride, to a solution of 4 to 9 molar of the chaotropic coadjuvant substance, at a pH value of from about 8 to about 11.5 and at a temperature of about 15. BC to approximately 55 SC, maintaining the mixture obtained for approximately 10 to 60 minutes at this temperature ura and (c) incorporation of the mixture, at a pH value from about 8 to about 11.5 and a temperature from about 53C to about 30 SC, to an amount of water that provides a dilution of the cysteine concentration. or of the cysteine hydrochloride in the mixture of about 1 to 5 mM and of the chaotropic coadjuvant substance of 0.2 to 1.0 M.

2. Method according to claim 1, characterized in that in step (a) the amount of cysteine or cysteine hydrochloride corresponds to that which provides from 1 to 6 SH residues of cysteine or cysteine hydrochloride per cysteine residue of the precursor, in step (b) the incorporation of the suspension of the precursor containing cysteine is carried out or cysteine hydrochloride to a solution of 4 to 9 molar of the chaotropic coadjuvant substance at a pH value of 8 to 11 and at a temperature of 0.05 to 45 ° C, maintaining the mixture obtained over 2 hours. 0 to 40 minutes at this temperature, and in step (c) the mixture is incorporated, at a pH value of 8 to 11 and a temperature of 15 to 20BC, to an amount of water that provides a dilution of the . concentration of cysteine or cysteine hydrochloride in the mixture of about 1 to 5 mM and a concentration of the chaotropic coadjuvant substance of 0.2 to 1.0 M. Method according to claim 1 or 2, characterized in that the substance Caotropic adjuvant is guanidine or guanidine hydrochloride. 4. Process according to claim 1 or 2, characterized in that the chaotropic coadjuvant substance is urea. 5. Procedure according to one or several of claims -5§j. 1 to 4, characterized in that the concentration of the chaotropic coadjuvant in step (b) is from 7.0 to 20 9 M. Method according to one or more of claims 1 to 5, characterized in that the temperature in the stage (b) is 409C. The process according to one or more of claims 1 to 6, characterized in that the pH value in step (b) is from 10 to 11. The method according to one or more of claims 1 to 7. , characterized in that the pH value in step (c) is from 10 to 11. Process according to one or more of claims 1 to 8, characterized in that in step (c), the amount of water provides a dilution. of the concentration of cysteine or cysteine hydrochloride in the mixture of 2.5 to 3 mM and a chaotropic substance concentration of 0.5 M. 10. Method according to one or more of claims 2 to 9. , characterized in that the concentration of the chaotropic coadjuvant substance in step (b) is about 8 M, the temperature in step (b) is about 40 ° C, the va! < The pH of the step (b) is about 10.6, the pH value in step (c) is about 10.6 and, in step (c), the amount of water provided a dilution of the concentration of cysteine or cysteine hydrochloride in the mixture of approximately 2.5 to 3 mM and a concentration of the chaotropic substance 10 of 0.5 M. 11. Process according to one or more of the claimed claims. -Nations 1 to 10, characterized in that the precursor of insulins or insulin derivatives exhibits the sequence according to general formula (II) R-R1- (B2-B29) -YX-Gly- (A2- A20) -R3 (II), in which R2 a) means a hydrogen atom, b) an amino acid residue of the group lysine (Lys) or arginine (Arg), or 20 c) a peptide with 2 to 45 amino acid residues that contains the amino acid residue lysine (Lys) or argi-, nina (Arg) at the carboxyl end of the peptide, R1 a phenylalanine residue (Phe) or a covalent bond, (B2-B29) the amino acid residues at positions B2 to B29 of the B chain of human insulin, animal insulin or an insulin derivative, optionally differentiated at one or several of these positions, and a genetically encodable amino acid residue, X a) an amino acid residue of the group lysine (Lys) or arginine ( Arg), or b) a peptide with 2 to 35 amino acid residues that contains the amino acid residue lysine (Lys) or arginine (Arg) in the ext N-terminal and at the carboxyl end of the peptide, or c) a peptide with 2 to 35 genetically encodable amino acids containing from 1 to 5 histidi- residues (A2-A20) the amino acid residues at positions A2 to A20 of the B chain of human insulin, animal insulin or an insulin derivative, optionally differentiated at one or several of these positions, and R3 a genetically encodable amino acid residue. 12. Process according to claim 11, characterized in that in formula (II) they mean R2) a) a hydrogen atom, or b) a peptide with 2 to 25 amino acid residues containing the amino acid residue arginine (Arg) at the carboxyl terminus of the peptide, R1 a phenylalanine residue (Phe), 15 (B2-B29) the amino acid residues at positions B2 to B29 of the B chain of human insulin, and an amino acid residue of the alanine (Ala), threonine (Thr) group or serine (Ser), í £ ^ f S! X the amino acid residue arginine (Arg) or a peptide with the amino acid sequence of the C chain of human insulin, (A2-A20) the amino acid residues at positions A2 to A20 of the B chain of human insulin, and R3 an amino acid residue of the group asparagine (Asn), serine (Ser) or glycine (Gly). 1

3. Process according to claim 11, characterized in that in formula (II) R2 denotes a) a hydrogen atom, or b) a peptide with 2 to 15 amino acid residues, at the carboxyl end of which there is an arginine residue (Arg), R1 a phenylalanine residue (Phe), (B2-B29) the amino acid residues at positions B2 to B29 of the B chain of human insulin, and a threonine residue (Thr), X the amino acid residue arginine (Arg) or a peptide with 2 to 35 amino acid residues, being located at the beginning and end of the peptide two amino acid residues of basic character, especially arginine (Ar? f) and / or Usin (Lys), 5 (A2-A20) amino acid residues in positions A2 to A20 of the B chain of human insulin, and R3 the amino acid residue asparagine (Asn) or glycine (Gly). Item * r L SUMMARY OF THE INVENTION The present invention; refers to an improved process for obtaining a precursor of insulins or insulin derivatives with cystine bridges correctly 10 bound in the presence of cysteine or cysteine hydrochloride and a chaotropic coadjuvant substance.