Title of Invention	A PROCEDURE FOR THE END-POLISHING OF AN INSULIN IN AN INSULIN PURIFICATION PROCESS
Abstract	The present invention relates to an improved procedure for the chromatographic purification of insulins wherein a pressure-stable organic polymeric chromatography material is used as a stationary phase, and the mobile phase contains at least one water-miscible organic solvent and at least one buffer substance and the pH is from about 7 to about 11.

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Procedure for the chromatographic purification of insulins The present invention relates to an improved procedure for the chromatographic purification of insulins. In addition to enzymatic and/or genetic engineering procedures, the preparation procedures for insulins essentially comprise chromatographic procedures in order to fulfill the extremely high purity demands. The term insulins is understood here as meaning insulins originating from natural sources or recombinant insulins (i.e. expressed by genetically modified microorganisms) of animal or human origin (e.g. porcine insulin, bovine insulin or human insulin), proinsulins (e.g. insulin precursors, proinsulin), or insulin derivatives. Insulin derivatives are designated below as derivatives of naturally occurring insulins, namely human insulin or animal insulins, which differ by substitution of at least one naturally occurring amino acid radical and/or addition of at least one amino acid radical and/or organic radical from the corresponding, otherwise identical naturally occurring insulin. Human insulin is a polypeptide which is constructed of 51 amino acids. The so-called A (acidic) chain consists of 21, and the B (basic) chain of 30 amino acid radicals. In both amino acid chains 6 cysteine radicals occur, each two cysteine radicals being bonded to one another via a disulfide bridge (the two chains are linked to one another by two cysteine bridges). In biologically active human insulin, the A and B chains are bonded to one another via two cysteine bridges, and a further bridge occurs in the A chain. The following cysteine radicals are linked to one another in the (biologically active) human insulin: A6 - A11 A7-B7 A20-B19. The letters A and B stand for the particular insulin amino acid chain and the number for the position of the amino acid radical which is counted from the amino to the carboxyl end of the respective amino acid chain. The preparation of recombinant insulin is customarily carried out in the steps of fermentation and cell disruption, followed by protein chemistry and process technology processes, customarily chromatographic processes, for the purification of the product. Genetic engineering procedures allow human proinsulin or proinsulin (proinsulin of insulin derivatives) which has an amino acid sequence and/or amino acid chain length differing from human insulin to be prepared in microorganisms. The proinsulins prepared from genetically modified Escherichia coli cells do not have correctly bonded cysteine bridges. A procedure for the obtainment of human insulin having correctly bonded cysteine bridges using E. coli is disclosed, for example, in EP 0 055 945. Improved procedures for the preparation of human insulin and insulin derivatives having correctly bonded cysteine bridges are described in EP 0 600 372 A1 (US 5,473,049) and in EP 0 668 292 A2 (US 5,663,291). The proinsulin, a precursor of insulin, prepared from genetically modified microorganisms is first isolated from the cells, correctly folded and then converted enzymatically to human insulin. In addition to undesired by-products, the cleavage mixture obtained in the enzymatic perpetuation processes contains both the valuable substance and undesired insulin-like impurities, which do not markedly differ from the valuable product either in molecular weight or in other physical properties, which makes separation and purification very difficult, in particular on a large industrial scale. The process technology processes for purification are a series of various chromatography procedures (e.g. adsorption chromatography, ion-exchange chromatography, reversed phase or reverse-phase high-pressure chromatography or combinations thereof) in some cases in a number of stages using different support materials, in some cases with subsequent crystallization, the actual purification being achieved by chromatography. The removal of the insulin-like impurities in this case takes place on ion exchangers or on reversed phase silica supports. The end-polishing (removal of very minor impurities, as the last purification stage) is customarily carried out in the high-pressure range using chromatography on reversed phase silica gel (RP-HPLC = reversed phase high-pressure liquid chromatography). Reversed phase (or reverse-phase, i.e. lipophilically modified, that is hydrophobic) silica gel is understood as meaning a silica material to which a hydrophobic matrix has been applied. Examples of a hydrophobic matrix are alkanes having a chain length of 3 to 20 carbon atoms, in particular 4 to 18 carbon atoms. The particle sizes are in the range from 10 to 50 //m, the pore widths 50 to 300 A. Examples of chromatography procedures which, according to the prior art, utilize RP-silica gels (lipophilically modified silica gels) are EP 0 547 544 A2 (US 5,621,073) or EP 0 474 213 A1 (US 5,245,008). According to the prior art, the high demands on the purity of the insulins to be prepared can only be fulfilled by the use of reversed phase silica gels. The use of reversed phase silica gel, however, has crucial disadvantages: Reversed phase silica gels are only stable in the range from pH 2 to pH 10. In the chromatography of fermentation products, high molecular weight by-products are always contained which are persistently adsorbed and cannot be desorbed using the customary elution. These by-products concentrate on the RP silica gel with time (referred to as aging of the adsorbent). Regeneration or cleaning in place (CIP) is usually carried out only by rinsing with dilute sodium hydroxide solution. Thus, in each CIP process a part of the RP silica gel is destroyed, whose continuous replacement is very cost-intensive. The danger of denaturation furthermore exists for insulins on silica gels. Many attempts are known to replace RP silica gels based on silica. Attempts using RP material based on alumina or titanium dioxide (both materials are not completely pH-stable, but at least more stable than silica gel) have shown that the separation is only inadequate and that the required specifications with respect to purity cannot be achieved. A further necessary property of chromatography materials is their pressure stability. Pressure-stable polymeric chromatography materials are understood as meaning particles (which can occur in all possible forms such as in rod form, in the form of fragments or preferably in spherical form and preferably have diameters between 10 and 35 //m) of organic polymers, whose deformation under the action of pressure (up to 70 bar) is only slight. The material located in the chromatography column must be so well packed that no cavities are present (the quality of the packing decides the separation result). For the packing of columns, in principle two different techniques are known which can also be used in combination. There is the method of compressing the packing by means of a (usually hydraulically operated) ram (DAC = direct axial compression), or of packing the column hydro dynamically by means of a high-pressure pump, i.e. of pressing a suspension of liquid and particles into the column. In both cases, practice shows pressures of up to 70 bar must act on the cross section of the column in order to be able to pack the particles as tightly as possible and without resulting in cavities. Many organic polymer particles are not pressure-stable and deform under the action of pressure to the extent that spheres become flat disks which overlap and suppress the flow through the packing. In contrast, reversed phase silica gels are considerably harder by nature, and hardly deform under the pressures mentioned. The object of the present invention is to provide a process for the chromatographic purification of insulins on suitable chromatography materials which are pressure-stable and can be employed in the entire pH range from 1 to 14, which produces such a high separation efficiency that, instead of the customary one or more chromatography stages in series according to the prior art for the obtainment of the necessary purity of the insulin with simultaneous increase in the yield of this purification step, only one stage is needed. The object is achieved by a procedure for the chromatographic purification of insulins, which is distinguished in that a pressure-stable organic polymeric chromatography material is used as a stationary phase, the mobile phase contains at least one water-miscible organic solvent and at least one buffer substance and the pH is 7 to 11. Surprisingly, it was found that with chromatography in the pH range from 7 to 11, i.e. in the basic range, a very good separation is achieved on pressure-stable organic polymeric chromatography materials. The pH is preferably 9 to 10. A particular advantage of the procedure according to the present invention is that in this basic pH range the formation of diamide insulin, an impurity which is customarily formed in the acidic medium and, according to the specifications of insulin preparations, is to be removed to very small residual amounts, is suppressed. The mobile phases which are employed for the elution contain water-miscible organic solvents, for example alcohols having 1 to 4 carbon atoms, ketones, methyl acetate or acetonitrile. Preferred alcohols are those such as 1- or 2-propanol (n- or iso-propanol), methanol or ethanol. The concentration of the water-miscible organic solvents is between 1 and 90% by volume, preferably between 10 and 50% by volume. The mobile phases furthermore contain a buffer substance in order to keep the pH of the eluent constant. Suitable buffer substances are, for example, phosphates, alkali metal or alkaline earth metal salts, such as sodium citrate or potassium acetate, ammonium citrate, acetate, sulfate or chloride. The pH is adjusted by the addition of hydrochloric acid or sodium hydroxide solution. The elution can be carried out isocratic ally, i.e. with constant concentration of the buffer substances and with a constant proportion of the organic solvent, or preferably with a linear gradient, i.e. with an increase in the proportion of solvent. The average particle size of the pressure-stable organic polymeric chromatography material should advantageously be 5 to 300 ^m, preferably 10 to 50 ^im. The smaller the particle size, the sharper and better the separation. However, the pressure stability of smaller particles is lower. Insulin is a relatively small polypeptide (molecular weight about 6000) and can diffuse without problems into pores having a diameter of 10 nm (no steric hindrance). Materials having small pore diameters are more suitable, since the specific surface area and thus the adsorption capacity are larger. The average pore size of the pressure-stable organic polymeric chromatography material is advantageously 5 to 500 nm, preferably 10 to 50 nm. For the procedure according to the present invention, pressure-stable organic polymeric chronnatography materials which preferably consist of polystyrene/divinylbenzene or of polymethacrylate are particularly suitable. Examples of commercially available pressure-stable organic polymeric chromatography materials which can be advantageously employed in the procedure according to the present invention are compiled in Table 1. PMA = polymethacrylate Stove = styrene/divinylbenzene The procedure according to the invention is suitable for analytical, for semi preparative and in particular for preparative chromatography. The term "preparative chromatography" is understood as meaning the preparation of pure products on the technical scale. In order to achieve the purity necessary for insulin preparations, it is necessary to insert at least one further reversed phase chromatography or a cation exchange chromatography and, if appropriate, a crystallization step before the reversed phase chromatography, for example according to EP 0 547 544 A2 (US 5.621,073) or EP 0 474 213 A1 (US 5,245,008). In the procedure according to the invention, the same result is achieved with a single stage chromatography on the polymer support. Using the procedure according to the invention, the total yield in the insulin preparation is therefore significantly improved, as losses in yield are eliminated by combining several process stages into one stage. The procedure according to the present invention is suitable for the chromatographic purification of all insulins according to the definition introduced at the outset, namely insulins originating from natural sources or recombinant insulins (i.e. expressed by genetically modified microorganisms) of animal or human origin (e.g. porcine insulin, bovine insulin or human insulin), proinsulins (e.g. insulin precursors, preinsulins), or insulin derivatives, insulin derivatives being understood as meaning derivatives of naturally occurring insulins, namely human insulin or animal insulins, which differ from the corresponding, tokenize identical naturally occurring insulin by substitution of at least one naturally occurring amino acid radical and/or addition of at least one amino acid radical and/or organic radical. Examples of such insulins are human insulin, bovine insulin, porcine insulin, insulins according to EP 0 368 187 (US 5,656,722), for example Gly(A21), Arg(B31), Arg(B32) human insulin, insulins according to EP 0 821 006 (ZA 97/6645) or the insulins described in EP 0 547 544 A1 (US 5,621,073), EP 0 474 213 A1 (US 5,245,008), EP 0 600 372 A1 (US 5,473,049) or in EP 0 668 292 (US 5,663,291). (The letters A and B stand for the respective insulin amino acid chain, the number for the position of the naturally occurring amino acid radical, which is replaced by the amino acid radical given before the brackets.) Examples Example 1: Variation of the pH In Example 1, tests were made on a semi preparative column of the dimensions 10 mm in diameter and 120 mm length, which is packed with PLRP-S 10-15/ym 100A (Polymer Laboratories). The object was to purify reunified insulin which has a purity of 95 area % in such a way that the purity was greater than 98.5 area %. The amount applied was adjusted such that a loading of the polymer chromatography material of 6 g/liter [bed volume] resulted. The application buffer and the mobile phase are water/propanol mixtures, containing 0.05 M ammonium acetate and 0.1 M glycine, which were adjusted to the respective pH using hydrochloric acid or NaOH. The empty tube rate was 150 cm/h. The three pH values pH 3.5 - pH 6.8 - pH 9 were adjusted. The eluate was collected in fractions. Table 2 shows the results. Purities above 98 area % are achieved only at pH 9, and the specifications are thus fulfilled. It can be clearly seen that insulin purification on polymeric chromatography materials only has the required efficiency in the basic medium. Example 2: Pressure stability and packing of a column A chromatography column (Prochrom® LC50, of 50 mm diameter) was packed using the DAC (Direct Axial Compression) technique. The chromatography material (stationary phase) was introduced into 100% methanol and packed into the column. The pressure loss of an 11% n-propanol mixture was measured at various flow rates. The tamping pressure of the chromatography column was varied between 5 and 80 bar. The following chromatography materials were investigated: PLRP-S 10-15 100® (Polymer Laboratories) Source® 15 RPC (Marsha Pharmacia Biotech) Kromasil® 13-120 (Akzo Nobel) The materials have approximately identical particle diameters (spherical particles). PLRP® and Source® are polymers; Kromasil® is a high-grade RP-silica gel. A specific pressure loss of 1 bar/cm means a pressure fall of 30 bar in a 30 cm high packing, which is customary in technical chromatography. Example 3: Purification of human insulin on the preparative scale A total of 3 examples are described below, in which human insulin is purified in a technical column which is packed according to the DAC principle using a movable ram. A Prochrom© column, type LC50, was used. For all experiments, the packing in each case has identical dimensions of diameter 50 mm, bed length 110 to 120 mm. Human insulin having a purity of 95 area % should be brought to a purity of greater than 98.5 area %. Three chromatography supports were used: PLRP-S 10-15 100® (Polymer Laboratories) Source® 15 RPC (Marsha Pharmacia Biotech) Kromasil® C4 13-120 (Akzo Nobel) As already described in Example 2, PLRP and Source® are polymer materials, while Kromasil® is a high-grade RP silica gel support. The application buffer and the mobile phase correspond to the details in Example 1. The loading is indicated in grams of human insulin per liter of bed volume. Yield is understood as meaning the proportion of the eluate which has a purity of greater than 98.5 area %. The yields achieved are compared in Table 4. The values of 60 to 70% are surprisingly good for a bed length of about 12 cm. The crucial difference between the prior art (purification using a silica gel support, in this case Kromasil®) and chromatography using a polymer support is the pH difference: yields of this level can only be achieved at a pH of 9. Example 4: Purification of bolus insulin on the preparative scale In this example, a bolus insulin (fast-acting insulin) is to be purified. The example is moreover intended to demonstrate that even relatively poor qualities are permissible as starting conditions for this chromatography stage. The prior art is that the final purification of insulin is customarily carried out in two chromatography stages. If the end-polishing stage is directly loaded with a poor, i.e. severely contaminated, material, the required purities and, at the same time, high yields can no longer be achieved. Surprisingly, it was now found that the polymer material investigated achieves this purity in a single chromatography step, which can be attributed to the small particle diameter of 10 to 15 /ym and the excellent adsorption properties. Four tests were made, with the following starting conditions: 75 area % purity 85 area % purity 89 area % purity 93 area % purity In Table 5, the yields achieved (i.e. that proportion of the insulin employed which was eluted with a purity of greater than 98.5 area %) are compiled. If the starting condition has only 75 area %, a purity of above 98.5 area % is not achieved. The purity is in the range of only 98.0 area %. However, if the starting conditions are above 85 area %, the required purities of greater than 98.5% are reliably achieved, with yields between 60 and 80%. The preparative conditions were as described in Example 1, All tests were carried out in a Prochrom® column type LC50 of 50 mm diameter and 12 cm packing height. The loading was 6 g/l of BV in each case, the pH was adjusted to 9 and the tamping pressure was measured at 35 bar. With the aid of Table. 5, it can be readily seen how the yield in the chromatography increases depending on the quality of the starting condition. The tests clearly show that the purification is certainly possible in a single chromatography stage. 1. A procedure for the chromatographic purification of insulins, wherein a pressure-stable organic polymeric chromatography material is used as a stationary phase, the mobile phase contains at least one water-miscible organic solvent and at least one buffer substance and the pulls 7 to 11. 2. The procedure as claimed in claim 1, wherein the pH is 9 to 10. 3. The procedure as claimed in claim 1 or 2, wherein the water-miscible organic solvent is an alcohol having 1 to 4 carbon atoms, 4. The procedure as claimed in claim 3, wherein the alcohol is 1-propanol or 2-propanol. 5. The procedure as claimed in claim 3, wherein the alcohol is ethanol. 6. The procedure as claimed in claim 3, wherein the alcohol is methanol. 7. The procedure as claimed in claim 2, wherein the water-miscible organic solvent is a ketone. 8. The procedure as claimed in claim 2, wherein the water-miscible organic solvent is methyl acetate. 9. The procedure as claimed in claim 2, wherein the water-miscible organic solvent is acetonitrile. 10. The procedure as claimed in one or more of claims 1 to 9, wherein the concentration of the water-miscible organic solvent in the mobile phase is 1 to 90% by volume. 11. The procedure as claimed in claim 10, wherein the concentration of the water-miscible organic solvent in the mobile phase is 10 to 50% by volume. 12. The procedure as claimed in one or more of claims 1 to 11, wherein the elution is carried out isocratically. 13. The procedure as claimed in one or more of claims 1 to 11, wherein the elution is carried out using a linearly increased gradient of the proportion of the water-miscible organic solvent. 14. The procedure as claimed in one or more of claims 1 to 13, wherein the pressure-stable organic polymeric chromatography material has an average particle size of 5 to 300 /ym. 15. The procedure as claimed in claim 14, wherein the average particle size is from 10 to 50/ym. 16. The procedure as claimed in one or more of claims 1 to 15, wherein the average pore size of the pressure-stable organic polymeric chromatography material is 5 to 500 nm. 17. The procedure as claimed in claim 16 , wherein the average pore size is 10 to 50 nm. 18. The procedure as claimed in one or more of claims 1 to 17, wherein the pressure-stable organic polymeric chromatography material consists of a polymethacrylate. 19. The procedure as claimed in one or more of claims 1 to 17, wherein the pressure-stable organic polymeric chromatography material consists of a polystyrene/divinylbenzene. 20. Procure futile dismount purified foundlings substantially as herd above desorbed and amphfied

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