Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/62—Insulins

Definitions

• Type 1 diabetes mellitus which is characterized by inadequate production of the hormone insulin by the body.
• the substitution of the missing endocrine insulin secretion by the application of insulin preparations is the only possible form of therapy.
• Insulin preparations are pharmaceutical preparations that contain the hormone insulin as an active ingredient. Not only naturally occurring insulins are used, but also insulin analogues and insulin derivatives.
• the human insulin produced in the human pancreas is a polypeptide consisting of 51 amino acid residues, which are distributed over 2 peptide chains: the A chain with 21 amino acid residues and the B chain with 30 amino acid residues.
• the sequence of the amino acid residues in both peptide chains is genetically determined and known. Both chains are linked by two disulfide bridges. In addition, there is an intrachainar disulfide bridge in the A chain.
• Insulin analogs differ from human insulin by substituting at least one amino acid residue and / or adding or removing at least one amino acid residue. Insulin analogs can either occur naturally in species other than human, or they can be made artificially. Insulin derivatives contain chemically modified amino acid residues, e.g. contain additional ester or amido groups, but otherwise show the human or an analogous amino acid sequence.
• insulin analogues or insulin derivatives show a modified kinetic effect compared to the unmodified human insulin.
• human insulin and the insulin analogues or insulin derivatives have been produced by recombinant DNA technology.
• a corresponding precursor of formula 1 preproinsulin (PPI) is produced, are produced human insulin or the insulin analogues from the by enzymatic cleavage '.
• PPI preproinsulin
• a genetic engineering process for the production of human insulin consists of the following process steps:
• the amino acid sequence (the A and B chain) is already specified in the corresponding areas of the preproinsulin.
• Proteases such as the enzyme trypsin and, if necessary, the enzyme carboxypeptidase B are used for the enzymatic cleavage of the various preproinsulins.
• Preproinsulin is a protein of formula 1, X
• R 1 a) a hydrogen atom, b) a genetically encodable amino acid residue or c) a peptide with 2 to 15 amino acid residues, R 2 is a genetically encodable amino acid residue and
• residues A1 - A20 correspond to the amino acid sequence of the A chain of human insulin or an insulin analogue and residues B1 - B30 correspond to the amino acid sequence of the B chain of human insulin or an insulin analogue.
• the preproinsulin is preferably a protein of formula 1 in which
• X is a peptide with 35 amino acid residues with the sequence of the C chain of human insulin or monkey insulin, or a peptide with 29 amino acids with the sequence: Arg-Asp-Val-Pro-Gln-Val-Glu-Leu-Gly-Gly-GIy- Pro-Gly-Ala-Gly-Ser-Leu-Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln-Lys-Arg (SEQ ⁇ Ü NO: 1)
• R 1 is a peptide with 2 to 15 amino acid residues, at the carboxyl end of which
• R 2 represents the amino acid residue Asn or Gly, ⁇ . and residues A1 - A20 correspond to the amino acid sequence of the A chain of human insulin and residues B1 - B30 correspond to the amino acid sequence of the B chain of human insulin or an insulin analogue in which Lys instead of Asn in the B3 position and in the B29 position Glu instead of Lys.
• preproinsulin is produced in this process stage in a concentration of approx. 0.5 to 1 g / L and in addition approx.
• the polymeric preproinsulins are among the higher molecular weight fractions. It has surprisingly been found in the context of this invention that the polymeric forms of the preproinsulins negatively influence the stability of the insulins in the subsequent process stages by inducing the denaturation of the native insulins. It is known that during the denaturation reaction chain, in a first reversible step, the dissolved monomeric insulin molecules form linear aggregates in which the repeating units are held together by physical adhesive forces. In an irreversible subsequent reaction, stable, insoluble aggregate bundles (fibrils) arise from the dissolved aggregates, which in turn induce the denaturation of native insulins in an autocatalytic process.
• insoluble insulin fibrils are not only biologically inactive, but can also lead to the clogging of injection needles when the pharmaceutical insulin preparations are applied. In addition, they are also held responsible for immunological intolerance reactions that can occasionally occur during therapy with insulin preparations (J.Brange et al, J. Pharm. Sc. 1997, 86, 517-525; RE Ratner et al., Diabetes, 39, 728-733, 1990).
• the preproinsulin is converted into human insulin using the enzymes trypsin and carboxypeptidase B (see Kemmler, W., Peterson, JD, and Steiner, DF, J. Biol. Chem., 246 (1971) 6786 -6791).
• the connection peptide between the A and B chain (X in Formula 1) and the pret part at the amino end of the B chain (R 1 in Formula 1) are split off.
• trypsin not only are those peptide bonds cleaved whose cleavage leads to human insulin, but in a competing reaction also other peptide bonds whose cleavage leads to several undesirable by-products.
• a cation exchange resin With a cation exchange resin, the higher molecular fractions were separated down to approx. 1%, but the binding capacity of the resin for the preproinsulin proved to be unsatisfactory.
• the present invention therefore relates to a method for effectively separating the High molecular weight substances from an aqueous solution of the preproinsulin with simultaneous high concentration of the monomeric preproinsulin.
• a dilute aqueous solution of a preproinsulin as it arises during the production process of insulin, is at pH 7.0 to 9.0, preferably at pH 7.5 to 8.5 and a conductivity of 5 to 7 mS / cm over a Guard column pumped with an anion exchange resin, e.g. Source 30 Q, is filled.
• the monomeric preproinsulin is not bound to the resin, but runs through the column with the permeate.
• the majority of the higher molecular substances, including the polymeric preproinsulin is adsorbed on the resin and thus separated from the preproinsulin.
• the permeate of this precolumn which contains valuable substances, is adjusted to pH 3.0 to 5.5, preferably to pH 4.0 to 5.0, in hydrochloric acid and then pumped directly onto a second column which is coated with a cation exchange resin, e.g. Source 30 S, is filled.
• the preproinsulin is adsorbed onto this resin and impurities are flushed out of the column with the permeate.
• the preproinsulin is desorbed with the aid of an elution buffer which contains sodium chloride with a linearly increasing concentration of 1 to 20 g / L, preferably 2.5 to 15.0 g / L.
• the purified preproinsulin is collected in a main fraction, whereas other impurities are separated in a pre and post fraction.
• concentration factor F 20-25.
• the higher molecular weight substances were separated apart from ⁇ 0.1%.
• the preproinsulin purified in this way can be isolated from the solution by crystallization or the solution • can be fed directly to the process step of the enzymatic cleavage.
• the invention thus relates to a process for the chromatographic purification of preproinsulin of the formula 1, (B1) (B7) (B19) (B30)
• R 1 a a hydrogen atom, b) a genetically encodable amino acid residue or c) a peptide with 2 to 15 amino acid residues,
• R is a genetically encodable amino acid residue and and residues A1 - A20 correspond to the amino acid sequence of the A chain of human insulin or an insulin analogue and residues B1 - B30 correspond to the amino acid sequence of the B chain of human insulin or an insulin analogue;
• preproinsulin can have the following amino acid sequence:
• Another object of the invention is a method as described above for the separation of foreign substances from the solutions of preproinsulins which induce the denaturation of insulin.
• Another object of the invention is a method as described above, characterized in that the second chromatography is carried out at a pH of 3.0 to 5.5.
• Another object of the invention is a method as described above, characterized in that the second chromatography is carried out at a pressure of 1 to 30 bar.
• Another object of the invention is a method for producing insulin by expression of unfolded preproinsulin, comprising the steps: a) fermentation of genetically modified microorganisms which express unfolded preproinsulin b) harvesting of the microorganisms and cell disruption c) isolation of the inclusion bodies with undissolved, unfolded preproinsulin d) Dissolving the preproinsulin with correct folding of the peptide chain and simultaneously closing the disulfide bridges to preproinsulin and then going through a process for the chromatographic purification of preproinsulin of the formula 1 as described above. e) Enzymatic cleavage of preproinsulin to human insulin f) Cleaning human insulin g) Crystallization of human insulin and drying
• inclusion bodies were formed in the E. coli cells which contained the fusion protein with the amino acid sequence of the preproinsulin.
• the cells were isolated by centrifugation and disrupted by the usual high-pressure homogenization (process stage b).
• the insoluble inclusion bodies released in the process were isolated by centrifugation and washed on the centrifuge with water (process stage c).
• the fusion protein inclusion bodies were in an 8 M
• Guanidine hydrochloride solution dissolved at pH 10.8. After dilution with water and addition of cysteine hydrochloride, the fusion protein was folded at pH 10.8 and 4.degree. C. to close the 3 disulfide bridges to the preproinsulin of the formula 1. The solution was then adjusted to pH 5 with 10% hydrochloric acid and foreign proteins were precipitated, which were separated off by centrifugation.
• Centrifuge supernatant contained 0.6 to 0.8 g / L of monomeric preproinsulin.
• the purity of the preproinsulin was determined by the HPLC-RP analysis to be about 65 area%.
• the HPLC-GPC analysis determined a proportion of approx. 45 area% higher molecular impurities.
• the quotient was formed from the peak area of the preproinsulin in the analysis sample and the corresponding peak area of a standard substance. For the determination of the degree of purity, the quotient was formed from the peak area of the preproinsulin and the sum of the peak areas of all elutable substances in the analysis sample.
• the quotient was formed from the peak areas of all higher molecular weight substances that eluted before the monomeric preproinsulin and the sum of the peak areas of all elutable substances.
• the retention time for the monomeric preproinsulin was determined using a standard substance.
• This preproinsulin corresponds to Formula 1, where is
• R1 a peptide chain with 10 amino acid residues with the sequence:
• Human insulin B1-B30 peptide chain with the sequence of the B chain from human insulin An apparatus was used to purify the preproinsulin solution, which mainly consisted of two chromatography columns arranged in series and a stirring vessel arranged between them. The pH of the solution between the two columns was changed inline in the stirred vessel.
• the solutions used had the following composition: Feed solution for column 1: starting solution 8.0 L (centrifuge supernatant) sodium chloride solution 25% 100 ml ml / L (w / w) 12.5 sodium hydroxide solution 10% (w / w) approx. 4.5 ml ml / L
• the permeate fraction of the first column was in the intermediate vessel (nominal volume: 4 L, equipped with a stirrer, pH probe, and inlet tube) is set in line with 90% lactic acid to pH 3.5 and then pumped directly to the second chromatography column 15th
• the chromatography on the cation exchanger was operated in the adsorption mode, i.e.
• the valuable substance preproinsulin was adsorbed onto the gel during the product application and (after the feed solution had been displaced) was desorbed again with the elution buffer A / B.
• a linearly increasing sodium chloride gradient was used in the elution buffer.
• Displacement buffer for column 2 Purified water 1 L lactic acid 90% 8.3 ml moI / L
• Elution buffer A is identical to the displacement buffer for column 2.
• This preproinsulin corresponds to Formula 1, where is
• X is a peptide chain with 35 amino acid residues with the sequence of
• An apparatus was again used to purify the preproinsulin solution, which mainly consisted of two chromatography columns arranged in series and a stirring vessel arranged between them. The pH of the solution between the two columns was changed inline in the stirred vessel.
• the apparatus for the second chromatography stage was designed to be pressure-stable.
• the chromatography on the cation exchanger was operated in the adsorption mode, i.e.
• the valuable substance preproinsulin was adsorbed onto the gel during the product application and (after the feed solution had been displaced) was desorbed again with the elution buffer A / B.
• a linear sodium chloride gradient in the elution buffer was used to achieve an optimal cleaning effect.
• Feed solution for column 2 permeate fraction of column 1 approx. 10.2 L lactic acid 90% 12.2 ml 1, 2 ml / L pH 4.6
• Elution buffer A is identical to the displacement buffer for column 2.
• Elution buffer B for column 2 Purified water 1 L lactic acid 90% 8.3 ml mol / L
• a solution of preproinsulin with the following amino acid sequence is obtained from the correspondingly genetically modified E. coli cells: Ala-Thr-Thr-Ser-Thr-Gly-Asn-Ser-Ala-Arg-Phe-Val-Lys-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-Glu-Thr- Arg- Asp-Val-Pro-Gln-Val-Glu-Leu-Gly-Gly- Gly-Pro-Gly-Ala-Gly-Ser-Leu-Gln-Pro-Leu- Ala-Leu-Glu-Gly-Ser-Leu-Gln-Lys-Arg-Gly-Ile-
• This preproinsulin corresponds to Formula 1, where is
• X is a peptide chain with 29 amino acid residues with the sequence: Arg-Asp-Val-Pro-Gln-Val-Glu-Leu-Gly-Gly-Gly-Pro-Gly-Ala-Gly-Ser-Leu-
• Human insulin B1-B30 peptide chain with an analogous sequence to the B chain of human insulin i.e. with an exchange of Lys for Val in position B3 and an exchange of Glu for Lys in position B29.
• the same apparatus used in Example 1 was used to clean the preproinsulin solution.
• the anion exchange resin Source 30 Q (manufacturer: Pharmacia Biotech; product no .: 17-1275-04) was used for the chromatography on column 1.
• the anion exchange resin Source 30 Q manufactured by Pharmacia Biotech; product no .: 17-1275-04
• twice the amount of regeneration solution compared to Examples 1 and 2 was used.
• Example 2 The change in pH in the intermediate vessel was also carried out as described in Example 1.
• the second chromatography was carried out this time at a working pressure of 15 bar. All other parameters of the second chromatography were the same as described in Example 2.
• the denaturation test (Table 1) shows that the higher molecular weight polymeric forms of preproinsulins, such as those formed during the folding reaction, can induce the denaturation of native insulin.
• the solution was filtered through a membrane filter with a pore size of 0.1 ⁇ m.
• the various test substances were added to this acidic standard solution in the denaturation test: the washing fraction of column 1, which contained the separated polymeric forms of preproinsulins in a concentration of 5 g / L, or the main fraction of column 2, which contained the purified preproinsulin in a concentration of 15 or 17 g / L contained.
• the addition of 10 ml of 0.1% aqueous solution of Poloxamer 171 served as further proof that the observed phenomena were caused by the denaturation of insulin. It is known that Poloxamer 171 can suppress the denaturation of insulin at hydrophobic interfaces (H. Thurow and K. Geisen, Diabetologia (1984) 27, 212-218 and EP 0 018 609).
• the solutions were then heated to 26 ° C. and adjusted to pH 6.1 with 10% sodium hydroxide solution while stirring, the insulin precipitating amorphously.
• the amorphous suspension was stirred at 26 ° C for 50 hours. After this time, insulin crystals were formed in all batches.
• each batch was divided into two roughly equal parts. The first part was placed in a 250 ml measuring cylinder to investigate the sedimentation behavior, the volume of the sediment and the clarity of the supernatant being assessed after standing for 60 minutes at room temperature. The second part was adjusted to pH 3 with 1N hydrochloric acid, the clarity of the resulting solution being assessed after the insulin crystals had dissolved.
• Example 2 from Example 2 contained 17 g / L preproinsulin

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