US20050080000A1 - Method of purifying preproinsulin - Google Patents

Method of purifying preproinsulin Download PDF

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Publication number
US20050080000A1
US20050080000A1 US10/632,414 US63241403A US2005080000A1 US 20050080000 A1 US20050080000 A1 US 20050080000A1 US 63241403 A US63241403 A US 63241403A US 2005080000 A1 US2005080000 A1 US 2005080000A1
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Prior art keywords
gly
leu
glu
ser
preproinsulin
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US10/632,414
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Horst Thurow
Hans Blumenstock
Chantalle Havenith
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Sanofi Aventis Deutschland GmbH
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Aventis Pharma Deutschland GmbH
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Priority to US10/632,414 priority Critical patent/US20050080000A1/en
Assigned to AVENTIS PHARMA DEUTSCHLAND GMBH reassignment AVENTIS PHARMA DEUTSCHLAND GMBH ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BLUMENSTOCK, HANS, HAVENITH, CHANTALLE, THUROW, HORST
Publication of US20050080000A1 publication Critical patent/US20050080000A1/en
Assigned to SANOFI-AVENTIS DEUTSCHLAND GMBH reassignment SANOFI-AVENTIS DEUTSCHLAND GMBH CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: AVENTIS PHARMA DEUTSCHLAND GMBH
Priority to US11/305,508 priority patent/US7803763B2/en
Abandoned legal-status Critical Current

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    • 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

  • Insulin preparations are pharmaceutical preparations whose active substance is the hormone insulin.
  • insulin analogs and insulin derivatives are used in addition to naturally occurring insulins.
  • Human insulin which is produced in the human pancreas is a polypeptide comprising 51 amino acid residues which divide into two peptide chains: the A chain having 21 amino acid residues and the B chain having 30 amino acid residues.
  • the sequence of the amino acid residues in both peptide chains has been genetically determined and is known. Both chains are connected to one another by two disulfide bridges. In addition, the A chain also contains an intrachain disulfide bridge.
  • Insulin analogs differ from human insulin by substitution of at least one amino acid residue and/or addition or removal of at least one amino acid residue. Insulin analogs may either occur naturally in species other than humans or may have been prepared artificially. Insulin derivatives contain chemically modified amino acid residues which contain, for example, additional ester or amido groups but otherwise show the human or an analog amino acid sequence.
  • insulin analogs or insulin derivatives exhibit an altered action kinetics compared to unmodified human insulin.
  • a genetic method for preparing human insulin comprises the following method steps:
  • the amino acid sequence (of the A and B chains) in the appropriate regions of preproinsulin has already been predetermined.
  • Enzymic cleavage of the various preproinsulins is carried out using proteases such as, for example, the enzyme trypsin and in addition, if necessary, the enzyme carboxypeptidase B.
  • the preproinsulin is a protein of the formula 1, in which
  • the preproinsulin is preferably a protein of the formula 1 in which
  • the process stage dissolving the fusion protein with correct folding of the peptide chain and with simultaneous closure of the disulfide bridges to give preproinsulin—produces, in addition to the desired monomeric preproinsulin, also polymeric forms of preproinsulin in a competing reaction.
  • Said polymeric preproinsulins can be detected, owing to their higher molecular weight, by HPLC-GPC analysis or by the method of dynamic light scattering.
  • the initial concentration of the fusion protein needs to be as low as possible (De Bernadez et al., Meth. Enzym. 309:217, 1999).
  • this process stage produces preproinsulin at a concentration of from approx. 0.5 to 1 g/l, with approx. 40% of higher molecular weight proportions being found in addition.
  • the higher molecular weight proportions include the polymeric preproinsulins.
  • the polymeric forms of preproinsulins adversely affect 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, a first reversible step produces, from the dissolved monomeric insulin molecules, linear aggregates in which physical adhesive forces hold together the repeated units. An irreversible subsequent reaction produces, from the dissolved aggregates, stable insoluble aggregate bundles (fibrils) which in turn induce the denaturation of native insulins in an autocatalytic process. These insoluble insulin fibers are not only biologically inactive but may also cause blockage of injection needles during application of the pharmaceutical insulin preparations.
  • preproinsulin is converted to human insulin with the aid of the enzymes trypsin and carboxypeptidase B (see Kemmler, W., Peterson, J. D., and Steiner, D. F., J. Biol. Chem., 246 (1971) 6786-6791).
  • the linker peptide between the A and B chains (X in the formula 1) and the pre part at the amino end of the B chain (R 1 in the formula 1) are removed.
  • the enzymic reaction with trypsin cleaves not only those peptide bonds whose cleavage produces human insulin but also, in a competing reaction, other peptide bonds whose cleavage produces a plurality of undesired byproducts.
  • the present invention therefore relates to a method for effectively removing the higher molecular weight substances from an aqueous solution of preproinsulin with simultaneous high concentration of the monomeric preproinsulin.
  • a diluted aqueous solution of a preproinsulin as is produced during the preparation process of insulin, is pumped at pH 7.0 to 9.0, preferably at pH 7.5 to 8.5, and a conductivity of from 5 to 7 mS/cm through a precolumn packed with an anion exchanger resin, for example Source 30 Q.
  • an anion exchanger resin for example Source 30 Q.
  • the monomeric preproinsulin is not bound to the resin but runs through the column together with the permeate.
  • the majority of the higher molecular weight substances, including the polymeric preproinsulins is adsorbed to the resin and thus removed from preproinsulin.
  • the permeate from this precolumn which contains the substance of interest, is adjusted in line to pH 3.0 to 5.5, preferably to pH 4.0 to 5.0, using hydrochloric acid and then pumped directly onto a second column packed with a cation exchanger resin, for example Source 30 S.
  • Preproinsulin adsorbs to this resin and impurities are washed out of the column together with the permeate.
  • Preproinsulin is desorbed with the aid of an elution buffer containing sodium chloride at a linearly increasing concentration of from 1 to 20 g/l, preferably 2.5 to 15.0 g/l.
  • the purified preproinsulin is collected in a main fraction, whereas further impurities are removed in a prefraction and a postfraction.
  • preproinsulin purified in this way can be isolated from the solution intermediately by crystallization or the solution can be fed directly to the enzymic cleavage process stage.
  • the present invention thus relates to a method for the chromatographic purification of preproinsulin of the formula 1, in which
  • the invention further relates to a method as described above for separating foreign substances from the solutions of preproinsulins which induce insulin denaturation.
  • the invention further relates to a method as described above, wherein the second chromatography is carried out at a pH of from 3.0 to 5.5.
  • the invention further relates to a method as described above, wherein the second chromatography is carried out under a pressure of from 1 to 30 bar.
  • the invention further relates to a method for preparing insulin by expressing nonfolded preproinsulin, comprising the steps:
  • the E. coli cells formed inclusion bodies which contained the fusion protein having the amino acid sequence of the preproinsulin.
  • the cells are isolated by centrifugation and disrupted by means of the usual high-pressure homogenization (process stage b).
  • the insoluble inclusion bodies released in the process were isolated by centrifugation and washed with water in the centrifuge (process stage c).
  • the fusion protein inclusion bodies were dissolved in an 8 M guanidine hydrochloride solution at pH 10.8. After diluting with water and adding cysteine hydrochloride, the fusion protein was folded with closure of the 3 disulfide bridges at pH 10.8 and 4° C.
  • preproinsulin of the formula 1 The solution was then adjusted to pH 5 using 10% strength hydrochloric acid, as a result of which foreign proteins were precipitated which were removed by centrifugation. The supernatant after centrifugation contained 0.6 to 0.8 g/l monomeric preproinsulin.
  • HPLC-GPC analysis determined a proportion of approx. 45% by area higher molecular weight impurities.
  • the peak area of preproinsulin in the analyzed sample was divided by the corresponding peak area of a standard substance.
  • the peak area of preproinsulin was divided by the sum of the peak areas of all elutable substances in the analyzed sample.
  • the peak areas of all higher molecular substances which were eluted prior to monomeric preproinsulin were divided by the sum of the peak areas of all elutable substances.
  • the retention time for monomeric preproinsulin was determined using a standard substance.
  • Said preproinsulin corresponds to the formula 1, in which X is a peptide chain having 35 amino acid residues with the sequence of simian C peptide, R1 is a peptide chain having 10 amino acid residues of the sequence: Ala-Thr-Thr-Ser-Thr-Gly-Asn-Ser-Ala-Arg (SEQ ID NO: 5) R2 is the amino acid residue Asn (identical to A21 of the A chain of human insulin) A1-A20 is the peptide chain having the sequence (only A1 to A20) of the A chain of human insulin B1-B30 is the peptide chain having the sequence of the B chain of human insulin.
  • the preproinsulin solution was purified using an apparatus which comprised primarily two chromatography columns arranged in series and a stirred vessel arranged in between.
  • the stirred vessel was used to change the pH of the solution in line between the two columns.
  • a gel bed (bed height: 14 cm, bed volume: 275 ml) was prepared using the anion exchanger resin DEAE-Sepharose fast flow (manufacturer: Pharmacia Biotech; Prod. No. 17-0709-05).
  • the column was operated from top to bottom and at atmospheric pressure of 1 bar.
  • the flow rate was 2 000 ml/h.
  • a multiway valve, a loading pump (Ismatec MV) and a bubble trap were installed upstream of the column. The following solutions were pumped onto the column successively via the multiway valve:
  • a UV probe (275 nm, with data recording) and another multiway valve were installed downstream of the column. Via the second multiway valve, approx. 10.2 l of permeate fraction were conducted into the abovementioned stirred vessel and, subsequently, approx. 1 l of washing fraction was conducted into a collecting vessel. The remaining permeates were discharged into the biological waste channel via the multiway valve.
  • the solutions used had the following composition: Starting solution for column 1: Starting solution (supernatant 8.0 l from centrifugation) Sodium chloride solution, 25% 100 ml 12.5 ml/l strength (w/w) Sodium hydroxide solution, approx. 4.5 ml 0.6 ml/l 10% (w/w) pH 8.3 Conductivity 6.1 mS/cm Temperature approx. 5° C. Purified water 1 l Tris(hydroxymethyl) 4.0 g/l aminomethane Sodium chloride 2.5 g/l Hydrochloric acid, approx. 2.5 ml/l 25% strength (w/w) pH 8.0 Conductivity approx.
  • the permeate fraction containing the valuable substance preproinsulin and the washing fraction containing the majority of the higher molecular weight impurities were collected at the column outlet: 1. approx. 10.2 l permeate fraction (at start of loading solution, from UV value 20% (ascending) to UV value 35% (descending), during product displacement) 2. approx. 1 l washing fraction (during loading of washing buffer, from UV value 30% (ascending slope) to UV value 40% (descending))
  • FIG. 1 depicts the UV diagram measured at the outlet of column 1.
  • the permeate fraction of the first column was adjusted to pH 3.5 with 90% strength lactic acid inline in the intermediate vessel (nominal volume: 4 l, with stirrer, pH probe and inlet tube) and then pumped directly onto the second chromatography column.
  • a UV probe (275 nm, with data recording) and another multiway valve were installed downstream of the column. Approx. 1 l of the main fraction was conducted via the second multiway valve into a collecting vessel. The remaining permeates were discharged via the multiway valve into the biological waste channel.
  • the cation exchanger chromatography was operated in adsorption mode, i.e. the valuable substance preproinsulin was adsorbed to the gel during product application and (after displacing the loading solution) desorbed again using the elution buffer A/B.
  • a linearly increasing sodium chloride gradient was applied in the elution buffer.
  • Elution buffer B for column 2 Purified water 1 l Lactic acid, 90% strength 8.3 ml 0.1 mol/l Sodium chloride 15.0 g 15.0 g/l Sodium hydroxide solution, approx. 7 ml 10% strength (w/w) pH 3.5 Conductivity approx. 25 mS/cm Temperature room temperature Regeneration solution for columns 1 and 2: Purified water 0.91 l Sodium chloride 40 g 40 g/l Sodium hydroxide solution, 0.09 l 1 mol/l 33% strength (w/w) Equilibrium buffer for column 2: Purified water 1 l Lactic acid, 90% strength 8.3 g 0.1 mol/l Sodium chloride 2.9 g/l Sodium hydroxide solution approx. 9 ml 10% strength (w/w) pH 3.5 Conductivity approx. 8.5 mS/cm Temperature room temperature
  • the main fraction which contained the valuable substance preproinsulin was collected at the column outlet: approx. 1.0 l main fraction (during elution, from UV value 65% (ascending) to UV value 76% (descending))
  • FIG. 2 depicts the UV diagram measured at the outlet of column 2.
  • Said preproinsulin corresponds to the formula 1, in which X is a peptide chain having 35 amino acid residues with the sequence of simian C peptide, R1 is a peptide chain having 10 amino acid residues of the sequence: Ala-Thr-Thr-Ser-Thr-Gly-Asn-Ser-Ala-Arg (SEQ ID NO: 5) R2 is the amino acid residue Gly A1-A20 is the peptide chain having the sequence (only A1 to A20) of the A chain of human insulin B1-B30 is the peptide chain having the sequence of the B chain of human insulin.
  • the preproinsulin solution was purified by again using an apparatus which comprised primarily two chromatography columns arranged in series and a stirred vessel arranged in between.
  • the stirred vessel was used to change the pH of the solution inline between the two columns.
  • the apparatuses for the second chromatography stage were designed for pressure stability.
  • a gel bed (bed height: 10 cm, bed volume: 196 ml) was prepared using the cationic exchanger Source 30 S (manufacturer: Pharmacia Biotech; prod. No.: 17-1273-04).
  • the column was operated from top to bottom and at a working pressure of 10 bar.
  • the flow rate was 3 500 ml/h.
  • a multiway valve, a loading pump (manufacturer: Besta; type: HD2-300) were installed upstream of the column. The following solutions were pumped onto the column successively via the multiway valve:
  • a UV probe (275 nm, with data recording) and another multiway valve were installed downstream of the column.
  • the main fraction containing the purified preproinsulin was conducted via the second multiway valve into a collecting vessel.
  • the remaining permeates were discharged via the multiway valve into the biological waste channel.
  • the cation exchanger chromatography was operated in adsorption mode, i.e. the valuable substance preproinsulin was adsorbed to the gel during product application and (after displacing the loading solution) desorbed again using the elution buffer A/B.
  • a linearly increasing sodium chloride gradient was applied in the elution buffer.
  • Elution buffer B for column 2 Purified water 1 l Lactic acid, 90% strength 8.3 ml 0.1 mol/l Sodium chloride 15.0 g 15.0 g/l Sodium hydroxide solution, approx. 27 ml 10% strength (w/w) pH 4.6 Conductivity approx. 25 mS/cm Temperature room temperature Regeneration solution for columns 1 and 2: Purified water 0.91 l Sodium chloride 40 g 40 g/l Sodium hydroxide solution, 0.09 l 1 mol/l 33% strength (w/w) Equilibrium buffer for column 2: Purified water 1 l Lactic acid, 90% strength 8.3 g 0.1 mol/l Sodium chloride 2.9 g/l Sodium hydroxide solution approx. 26 ml 10% strength (w/w) pH 4.6 Conductivity approx. 8.7 mS/cm Temperature room temperature
  • the main fraction which contained the valuable substance preproinsulin was collected at the column outlet: approx. 0.9 l main fraction (during elution, from UV value 65% (ascending) to UV value 76% (descending))
  • Said preproinsulin corresponds to the formula 1, where X is a peptide chain having 29 amino acid residues with the sequence: 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 (SEQ ID NO: 1) R1 is a peptide chain having 10 amino acid residues with the sequence: Ala-Thr—Thr-Ser-Thr-Gly-Asn-Ser-Ala-Arg (SEQ ID NO: 5), R2 is the amino acid residue Asn (A21 of the A chain of human insulin), A1-A20 is a peptide chain with the sequence (only A1 to A20) of the A chain of human insulin, B1-B30 [lacuna] peptide chain with a sequence similar to the B chain of human insulin, i
  • the preproinsulin solution was purified using the same apparatus used in example 1.
  • the anion exchanger resin Source 30 Q (manufacturer: Pharmacia Biotech; Prod.-No.: 17-1275-04) was used for the chromatography on column 1. Regeneration of this gel required twice the amount of regenerating solution compared to examples 1 and 2. The remaining parameters of the first chromatography, such as composition and volumes of the solutions, were the same as those described in examples 1 and 2.
  • the second chromatography was this time carried out with a working pressure of 15 bar. All other parameters of the second chromatography were identical to those described in example 2.
  • the denaturation assay shows that the higher molecular weight, polymeric forms of preproinsulins, as produced during the folding reaction, can induce denaturation of native insulin.
  • the solution was filtered through a membrane filter with a pore width of 0.1 ⁇ m.
  • this acidic standard solution was admixed with solutions of the various assay substances: the washing fraction of column 1 which contained the removed polymeric forms of preproinsulins at a concentration of 5 g/l or the main fraction of column 2 which contained purified preproinsulin at a concentration of 15 and, respectively, 17 g/l.
  • 10 ml of a 0.1% strength aqueous stock solution of Poloxamer 171 were added. Poloxamer 171 is known to be able to suppress insulin denaturation 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% strength sodium hydroxide solution with stirring, resulting in the precipitation of amorphous insulin.
  • the amorphous suspension was stirred at 26° C. for 50 hours. After this time, all mixtures contained insulin crystals.
  • each mixture was divided into two parts of approximately the same size. The first part was introduced into a 250 ml measuring cylinder in order to investigate the sedimentation behavior, and after leaving the mixtures at room temperature for 60 min, sediment volume and supernatant clarity were evaluated. The second part was adjusted to pH 3 with 1 N hydrochloric acid, and, after the insulin crystals had dissolved, the clarity of the resulting solution was evaluated.
  • Table 1 shows the result of the denaturation assay.
  • the samples 174 A, 188 A and 174 C in the case of which 1 ml and, respectively, 5 ml of polymer fraction had been added to the crystallization mixture, showed a distinct denaturation of insulin glargine. Under the microscope, an amorphous veil was visible between the crystals.

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US10/632,414 2002-08-01 2003-08-01 Method of purifying preproinsulin Abandoned US20050080000A1 (en)

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DE10235168A DE10235168A1 (de) 2002-08-01 2002-08-01 Verfahren zur Reinigung von Preproinsulin
DE10235168.6-43 2002-08-01
US43372602P 2002-12-16 2002-12-16
PCT/EP2003/007820 WO2004013176A1 (de) 2002-08-01 2003-07-18 Verfahren zur reinigung von preproinsulin
WOPCT/EP03/07820 2003-07-18
US10/632,414 US20050080000A1 (en) 2002-08-01 2003-08-01 Method of purifying preproinsulin

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