Title of Invention	PROCESS FOR PREPARING INSULIN COMPOUNDS
Abstract	ABSTRACT 1089/CHENP/2004 "Process for preparing insulin compounds" The present invention relates to a process for preparing an insulin compound wherein a) in a reaction mixture containing at least about 55 %, preferably at least about 60 %, more preferred at least 70 %, water (weight/weight), an insulin precursor is subjected to an enzymatic cleavage and, thereafter, b) the intermediate product is coupled with a nucleophile compound in the reaction mixture used for the enzymatic cleavage reaction with the proviso that the composition of the reaction mixture has been modified so that the content of water in the reaction mixture is in the range from about 10 % to about 50 % water (weight/weight), preferably in the range from about 20 % to about 40 % water (weight/weight), and c), if desired, removing the protecting group(s), and wherein no isolation of the intermediate product is performed between the cleavage step (a) and the coupling step (b).

Title of Invention

PROCESS FOR PREPARING INSULIN COMPOUNDS

Abstract

ABSTRACT 1089/CHENP/2004 "Process for preparing insulin compounds" The present invention relates to a process for preparing an insulin compound wherein a) in a reaction mixture containing at least about 55 %, preferably at least about 60 %, more preferred at least 70 %, water (weight/weight), an insulin precursor is subjected to an enzymatic cleavage and, thereafter, b) the intermediate product is coupled with a nucleophile compound in the reaction mixture used for the enzymatic cleavage reaction with the proviso that the composition of the reaction mixture has been modified so that the content of water in the reaction mixture is in the range from about 10 % to about 50 % water (weight/weight), preferably in the range from about 20 % to about 40 % water (weight/weight), and c), if desired, removing the protecting group(s), and wherein no isolation of the intermediate product is performed between the cleavage step (a) and the coupling step (b).

Full Text	The present invention relates to an improved process for converting an insulin precursor into an insulin compound, optionally via an insulin ester. BACKGROUND OF THIS INVENTION Insulin is a pancreatic hormone involved in the regulation of blood-glucose concentrations. For example, human, porcine, and bovine insulin, insulin analogues and mixed insulins are given to patients with insulin-dependent diabetes mellitus to control their blood-glucose concentrations. Porcine and bovine insulin are, usually, prepared from pancreas glands. Human insulin can, semisynthetically, be prepared from porcine insulin. Alternatively, human insulin, as well as many insulin analogues, can be prepared by genetic engineering. By genetic engineering, which may, for example, be performed in bacteria or in yeast, an insulin precursor is prepared which, thereafter, is to be converted into the desired product. This conversion can be performed in different ways. One possibility is the so-called transpeptidation where a peptide cleavage and a peptide coupling takes place consecutively in the same reaction mixture, under the same reaction conditions, vide, for example, US patent No. 4,343,898 (Novo Industri). Another possibility is, in the first step, to cleave the insulin precursor, wofe, for example, Hoppe-Seyler's Z. Physiol. Cliem. 359 (1978), 799, thereafter, to isolate the intermediate product and, then, to perform the desired coupling in another reaction mixture than that used in the first step, vide, for example. Nature 280 (1979), 412. According to EP 87,238, a transpeptidation reaction is performed in a solvent system comprising between about 75% and 97% (vol/vol) of at least one non-aqueous reaction miscible solvent including at least about 50% (vol/vol) butane-1,4-diol. According to US 4,579,820, the transpeptidation process is performed using an L- specific serine carboxypeptidase enzyme, for example carboxypeptidase Y. According to US 4,601,979 (Nordisk Insulinlaboratorium), the transpeptidation or only the peptide coupling is performed in an aqueous reaction medium substantially free of organic solvent. According to WO 83/00504 (Nordisk insulinlaboratorium), a porcine product was treated with carboxypeptidase A, the resulting des-alanine-B30 insulin product was suspended in a lower alcohol, and this suspension was mixed with a solution of an L- threonine ester and trypsin. In all the specific examples, the des-alanine-B30 insulin product was isolated, either by freeze-drying or by precipitation. 2 The object of this invention is to overcome or ameliorate at least some of the disadvantages of the prior art. Hence, not all the more detailed objects mentioned below may be fully overcome or ameliorated. DEFINITIONS The term "amino acid" as used herein, refers to amino acids which can be coded for by nucleotide sequences. Analogously, this applies to the term amino acid residue which is an amino acid from which hydroxy has been removed from a carboxy group and/or hydrogen has been removed from an amino group. Similarly, the terms peptide and peptide residue consists of amino acid residues. Preferably, the peptide contains not more than 10 amino acid residues. The term amino acid amide, as used herein, refers to an amino acid having an optionally substituted C terminal carboxamide group. The term peptide amide, as used herein, refers to a peptide having an optionally substituted C terminal carboxamide group. The term "insulin precursor", as used herein, refers to a polypeptide consisting of two peptide chains (corresponding to the A and B chains of insulin and, hereinafter, designated the A and B chains) which, similarly with insulin, are connected with each other via two disulphide bridges (from one cysteine (Cys) residue to another cysteine residue) between the two peptide chains and wherein, lilce in insulin, there is an disulphide bridge from one cysteine residue in the A chain to another cysteine residue in the A chain. In this insulin precursor there is, at least, one lysine or arginine residue in the B chain. Optionally, in this insulin precursor, the A and B chains are connected with each other via a third peptide chain (corresponding to the connecting peptide in insulin) between the C terminal end of the B chain and the N terminal end of the A chain. In case the A and B chains are connected with each other via this third peptide chain, lysine is present at the C terminal end of this third peptide. Optionally, in this insulin precursor, a fourth peptide chain may be connected to the N terminal end of the B chain. In case this fourth peptide chain is connected to the N terminal end of the B chain, lysine is present at the C terminal end of this fourth peptide chain. Furthermore, in this insulin precursor, there is an identity of the amino acid residues of at least 80 %, preferably at least 85 %, more preferred at lest 90 %, and even more preferred at least 95%, compared with human insulin, with the proviso that the third and fourth peptide chains are to be disregarded for this calculation. In human insulin, there are 3 disulphide bridges between Cys'^® and Cys^\ between Cys^^ and Cys^^ and between Cys^° and Cys^^^ and there is lysine in the B29 position. The term "amino acid ester", as used herein, refers to an amino acid carrying a C terminal carboxy protecting group and, optionally, a hydroxy protecting group. The term "peptide ester", as used herein, refers to a peptide wherein at least the C terminal carboxy group carries a carboxy protecting group. Optionally, any hydroxy group is protected and, optionally, the e-amino group of any lysine residues is derivatised, preferably with a hydrophobic group, for example an acyl group having at least 10 carbon atoms. Preferably, the peptide ester contains not more than 10 amino acid residues. The term nucleophile compound, as used herein, refers an amino acid ester, an amino acid amide, a peptide, a peptide ester, and a peptide amide. In any of these amino acid esters, amino acid amides, peptides, peptide esters, and peptide amides, the amino group in any lysine group is, optionally, derivatised, preferably with a hydrophobic group, for example, an acyl group having at least 10 carbon atoms. The term "insulin compound", as used herein, refers to insulin from any species such as porcine insulin, bovine insulin, and human insulin and salts thereof such as zinc salts, and protamin salts. Furthermore, the term "insulin compound", as used herein, refers to what could briefly be designated "insulin analogues". Insulin analogues, as used herein, refers to insulin compounds wherein one or more of the amino acid residues have been exchanged with another amino acid residue and/or from which one or more amino acid residue has been deleted and/or from which one or more amino acid residue has been added, provided that said insulin analogue has a sufficient insulin activity. Examples of insulin analogues are described in the following patents and equivalents thereto: US 5,618,913; EP 254,516; EP 280,534; US 5,750,497; and US 6,011,007. Examples of specific insulin analogues are insulin aspart (i.e., [Asp^^^] human insulin), insulin lispro (i.e., [Lys^^^Pro^^®] human insulin), and insulin glargin (i.e., [Gly^\Arg^^\Arg^^^] human insulin). The term "insulin analogue", as used herein also covers what could be designated insulin derivatives, i.e., compounds which a skilled art worker would generally considers derivatives of insulin, vide general textbooks, for example, insulin having a substituent not present in the parent insulin molecule. Examples of insulin derivatives are insulins or insulin analogues having an optionally substituted carboxamide group. Also compounds which can be considered being both an insulin derivative and an insulin analogue are herein covered by the term insulin analogue. Examples of such compounds are described in the following patents and equivalents thereto: US 5,750,497 and US 6,011,007. Hence, a further example of a specific insulin analogue is insulin detemir (i.e., des-Thr^^° human insulin y Lys^^^ tetradecanoyi). The insulin compounds prepared by this invention have an anti-diabetic activity sufficiently high to be 4 used to treat diabetic patients. The anti-diabetic activity can be determined using the so- called free fat cell assay. The term pH value, as used herein, refers to the value measured with a pH meter by immersing a calomel combination glass electrode connected to the pH meter directly in the solution, the pH value of which is to be measured. The pH meter is calibrated with an aqueous standard buffer. BRIEF DESCRIPTION OF THE FIGURES SEQ ID NO.: 1 is the peptide moiety Glu-(Glu-Ala)3-Pro-Lys-; SEQ ID NO.: 2 is the peptide moiety Glu-Glu-Gly-Glu-Pro-Lys-; and SEQ ID NO.: 3 is the peptide moiety Gly-Phe-Phe- Tyr-Thr-Lys-Pro-Thr. BRIEF DESCRIPTION OF THIS INVENTION The present invention relates to a process for preparing insulin compounds. These insulin compounds can be used as medicaments. In a preferred embodiment of this invention, insulin compounds having threonine (Thr) in the C terminal end of the 8 chain are prepared. Any sl of the insulin compounds to administer to a diabetic patient, and when. The starting material for the process of this invention is an insulin precursor which is subjected to both a peptide cleavage and a peptide coupling at conditions favoring both reactions but where no isolation of the intermediate product takes place. In other words, the insulin precursor is subjected to a peptide cleavage and the resulting product, i.e., the intermediate, is subjected to a peptide coupling. The conditions favoring a peptide cleavage are not identical with the conditions favoring a peptide coupling. Hence, in the first step of this invention, i.e., the cleavage step or the cleavage reaction, the reaction conditions in the reaction mixture are chosen so as to favor the peptide cleavage and, in the second step of this invention, i.e., the coupling step or the coupling reaction, the reaction conditions in the reaction mixture are altered so as to favor the peptide coupling. In one embodiment of this invention, the insulin precursor is, in the first step, dissolved in a predominantly aqueous medium and the enzyme used for cleavage is added. This reaction mixture may be free or substantially free of organic solvent. Alternatively, the reaction mixture may contain a certain amount of organic solvent which may ensure a proper 5 solubility of the insulin precursor. However, it is desired not to use so much organic solvent that it has an undesired influence on the enzymatic cleavage. In the first step of the process of this invention, the reaction parameters such a pH value, temperature, and time, are chosen so that they are favorable to cleavage at the lysine residue(s) or arginine residue(s). When the cleavage reaction has taken place to a certain, desired degree, a nucleophile compound and an organic solvent is mixed with the reaction mixture (without previous isolation of the intermediate product), so that the coupling of the nucleophile compound to the lysine or arginine residue of the desired intermediate product takes place. In this step, the reaction parameters are set so as to be favorable to the coupling reaction. In a preferred embodiment of this invention, the nucleophile compound is an amino acid ester, for example a threonine ester, or a peptide ester. Thereafter, the protecting group(s) may, if desired, be removed from the resulting compound. Compared with the known transpeptidation reaction, the advantages obtained by the process of this invention is a shorter, over all reaction time with the same amount of enzyme and a similar or higher yield. Compared with a two pot reaction with cleavage in an aqueous medium, isolation of the intermediate product, and coupling in a mixture of organic solvent and water, the advantages obtained by the process of this invention is a shorter, over all reaction time, the use of a lower amount of enzyme, and an easier process flow. More precisely, this invention relates to the following embodiments: DETAILED DESCRIPTION OF THIS INVENTION As appears from claim 1, first a peptide cleavage takes place and, thereafter, a coupling reaction takes place. Briefly, the cleavage reaction (i.e., the enzymatic cleavage) is performed as follows: The enzymatic cleavage of the insulin precursor (i.e., the peptide cleavage) takes place in a reaction mixture containing at least about 55%, preferably at least about 60%, more preferred at least 70%, water (weight/weight). In a preferred embodiment of this invention, the concentration of the insulin precursor in the reaction mixture wherein the enzymatic cleavage takes place is at least 2 %, preferably in the range from about 5 to about 10% (weight/vol). 6 The cleavage reaction is performed in a neutral or alkaline medium, preferably having a pH value in the range from about 6 to about 11, more preferred in the range from about 8 to about 10. In a preferred embodiment of this invention the amounts of enzyme compared with the amount of insulin precursor is in the range from approximately 0.05 to approximately 5% (weight/weight), preferably from approximately 0.1 to approximately 2%. The tryptic enzyme is not material to practice of this invention. Trypsin is a well- characterized enzyme available in high purity, notably from bovine or porcine origin. From microbial origin, Acromobacter lyticus protease I (hereinafter designated ALP) can be obtained. Moreover, the enzyme form, whether it is a native enzyme or an active immobilized enzyme or an enzyme derivative, is not material to practice of this invention, if it is desired to split at the C terminal end of arginine, trypsin can be used and if it is desired to split at the C terminal end of lysine, either trypsin or ALP can be used. For the splitting at the C terminal end of lysine, ALP is preferred. As examples of active enzyme derivatives can be mentioned acetylated trypsin, succinylated trypsin, glutaraldehyde treated trypsin, and immobilized trypsin or ALP derivatives. If an immobilized trypsin or ALP is used, it is suspended in the reaction mixture or may be packed into a column. To a great extent, the action of the enzyme is controlled by an interrelation of water and solvent content, the pH value, and the reaction temperature. Increasing the concentration of organic solvent in the reaction mixture and lowering of the pH value to around neutral shifts the usual enzymatic reaction from cleavage towards coupling. Reducing the temperature reduces the reaction rate, but might also reduce biproduct formation and enzyme denaturation. In a preferred embodiment of this invention, the insulin precursor is dissolved in an aqueous medium having a concentration of acetate ions in the range from about 5 mM to about 500 mM, preferably in the range from about 20 mM to about 200 mM. For example, sodium, potassium, ammonium acetate ortriethyl ammonium acetate can be used. According to one embodiment of this invention, the insulin precursor (being a peptide) can be illustrated by the following general formula I: R'-Cys-Zn-Cys-R^ I I (I) R'-Cys-Z,„-Cys-R' wherein Zn and Z^, independent of each other, represents two peptide moieties each containing n and m amino acid residues, respectively, R^ represents a peptide residue which peptide residue optionally contains a lysine or arginine residue, R^ represents an amino acid residue or a peptide residue, R^ represents a peptide residue which peptide residue optionally contains a lysine or arginine residue, R" represents a lysine or arginine residue or a peptide residue which peptide residue contains a lysine or arginine residue, or R^ and R" are together a peptide residue containing a lysine or arginine residue, the two vertical lines indicate the disulphide bonds between the two cysteine residues and, furthermore, there is an disulphide bond between two cysteine residues present in R^ and in Zn. Preferably, the amino acid residues present in the insulin precursor of formula I are those which can be coded for by the nucleotide sequences. According to a preferred embodiment of this invention, an insulin precursor, wherein the number of amino acid residues in R^ and R" together is in the range from about 8 to about 50, is used. In another preferred embodiment of this invention, Zn contains 12 amino acid residues. In another preferred embodiment of this invention, Zm contains 11 amino acid residues. In another preferred embodiment of this invention, R^ contains 1 amino acid residue, for example, Asn or Gly. In another preferred embodiment of this invention, R^ contains 6 amino acid residues. In a preferred embodiment of this invention, the insulin precursor is a single chain precursor, i.e. a compound of formula I wherein R^ and R"* together are a peptide residue containing a lysine or arginine residue. Hence, preferably, the insulin precursor is not mammalian insulin such as porcine insulin, rabbit insulin, dog insulin or whale insulin. According to another embodiment of this invention, the insulin precursor of formula I contains the same amino acid residues in positions A1 through A21 and in positions B1 through 829 as are present in human insulin in the same positions. According to another embodiment of this invention, the insulin precursor of formula I contains the same amino acid residues in positions A1 through A21 and in positions 81 through 829 with the proviso that the 828 amino acid residue is Asp. According to another embodiment of this invention, the insulin precursor of formula 1 contains the same amino acid residues in positions A1 through A21 and in positions 81 through 829 as are present in human insulin in the same positions with the proviso that the 828 amino acid residue is Lys and the 829 amino acid residue is Pro. According to another embodiment of this invention, the insulin precursor of formula I contains the same amino acid residues in positions A1 through A21 and in positions 81 through 829 as are present in human insulin in the same positions with the proviso that the A21 amino acid residue is Gly and the 831 and 832 amino cid residues both are Arg. 8 Examples of specific insulin precursors which can be use in the process of this invention are human proinsulin; monkey proinsulin; tAla^\Lys^^]-des(33-63) porcine proinsulin; porcine insulin; [Asp^^]-des(30-65) human proinsulin being N-terminally extended with Glu-(Glu-Ala)3-Pro-Lys- (SEQ ID NO.: 1); and [Asp'^Met'°,Trp'\Lys']-des(33-65) human proinsulin being N-terminally extended with Glu-Glu-Gly-Glu-Pro-Lys- (SEQ ID NO.: 2). The insulin precursors of formula I can be prepared as described in or analogously as described in the International applications having publication numbers WO 01/49742, WO 01/49870, WO 01/079250, and WO 02/079254, the content of which is hereby incorporated by reference. The desired intermediate product (i.e., the desired cleaving product) corresponds to the insulin precursor wherein at least one lysine or arginine residue has been cleaved to form a lysyl or arginyl moiety, respectively. Furthermore, in the desired intermediate product, the A and B chains which are connected with each other via two disulphide bridges are not connected with each other via a peptide chain between the C terminal end of the B chain and the N terminal end of the A chain. In a preferred embodiment of this invention, the number of amino acid residues present in the desired intermediate product is in the range from about 48 to about 52, preferably in the range from about 49 to about 51, even more preferred 50. In another preferred embodiment of this invention, there are not more than 4, preferably not more than 3, more preferred not more than 2, and even more preferred not more than 1, of the amino acid residues present in the desired intermediate product which are not present at the corresponding position in human insulin. According to one embodiment of this invention, the desired intermediate product (the desired cleaving product) can be illustrated by the general formula II R'^-Cys-Zn-Cys-R' 1. I (II) R'^-Cys-Zm-Cys-R'" wherein Zn and Zm, independent of each other, represents two peptide moieties each containing n and m amino acid residues, respectively, R'^ represents a peptide residue, R'^ represents an amino acid residue or a peptide residue, R'^ represents a peptide residue, R"* represents lysine or arginine or a peptide residue containing a lysine or arginine residue in the C terminal end, the two vertical lines indicate the disulphide bond between the two cysteine residues and, furthermore, there is an disulphide bond between two cysteine residues present in R'^ and in Zn. In a preferred embodiment of this invention, R'^ is the amino acid residues A1 through A6 in human insulin in this order in which, optionally, one or two of the amino acid residues have been exchanged with another amino acid residue or wherein one or two of the amino acid residues are not present. In another preferred embodiment of this invention, R'^ is -Asn or -Gly. In another preferred embodiment of this invention, R'^ is the amino acid residues B1 through B6 in human insulin in this order in which, optionally, one or two of the amino acid residues have been exchanged with another amino acid residue or wherein one or two of the amino acid residues are not present. In another preferred embodiment of this invention, R'" is the amino acid residues B20 through B29 in human insulin in this order, the amino acid residues B20 through B29 in human insulin in this order with the proviso that it has Asp in B28 and Lys in B29, and the amino acid residues B20 through B28 in human insulin in this order with the proviso that it has Lys in B28, in each of which, optionally, one or two of the amino acid residues have been exchanged with another amino acid residue or wherein one or two of the amino acid residues are not present or a part of any of these peptide residues leaving out one or more consecutive amino acid residues from the C terminal end thereof. In another preferred embodiment of this invention, Zn is the amino acid residues AS through A19 in human insulin in this order in which, optionally, one or two of the amino acid residues have been exchanged with another amino acid residue or wherein one or two of the amino acid residues are not present. In another preferred embodiment of this invention, Zm is the amino acid residues B8 through B18 in human insulin in this order in which, optionally, one or two of the amino acid residues have been exchanged with another amino acid residue or wherein one or two of the amino acid residues are not present. During both the cleavage reaction and the coupling reaction, the reaction temperature is in the range from the freezing point of the reaction mixture to about 50°C. The preferred temperature is in the range from about 0°C to about 25°C. Briefly, the coupling reaction is performed as follows: When at least about 25 %, preferably at least 50 %, more preferred at least 75 %, preferably at least 85 %, more preferred at least 95 %, of the insulin precursor has been cleaved to the desired intermediate product, on one hand, the nucleophile compound and, on the other hand, organic solvent is mixed with the reaction mixture in which the cleavage took place so as to obtain reaction conditions which are convenient or favorable to the coupling step. The percentage of cleavage (conversion) is based upon the equilibrium possible in the reaction mixture used for cleavage. Usually, from the beginning of the enzymatic cleavage reaction and until a certain period of time has lapsed, the yield of the desired intermediate product, 10 i.e., the desired cleavage product, increases and reaches a maximum concentration. Thereafter, the concentration of the desired cleavage product may decrease. In a preferred embodiment of this invention, no components are removed from the reaction mixture resulting from the cleavage reaction before the coupling reaction takes place. A simple way of doing this is, after the cleavage reaction, to add the nucelophile compound and a sufficient amount of organic solvent. In this way, for example, the enzyme used in the cleavage step is also used in the coupling step. The process of this invention also covers coupling reactions in a reaction mixture which besides the desired intermediate product contains a small amount of partially cleaved insulin precursor and/or unreacted insulin precursor. In another preferred embodiment of this invention, the nucleophile compound is an amino acid amide or a peptide amide wherein the carboxamide group isn't substituted or is mono or disubstituted with an alkyl group with not more than 16 carbon atoms which alkyl group(s), together with the adjacent nitrogen atom, may form a ring or the carboxamide group is mono or disubstituted with an aryl group. The aliphatic substituents are preferred. Examples of substituted carboxamide groups are N,N-dimethylcarboxamide, N,N-diethyl- carboxamide, and N-hexylcarboxamide. In a preferred embodiment of this invention, the nucleotide compound is an amino acid ester wherein the carboxyl group is protected and any hydroxy group optionally is protected. In a further preferred embodiment of this invention, the nucleotide compound is a threonine ester wherein the carboxyl group is protected and, optionally, the hydroxy group is protected. Hence, an L-threonine ester can be illustrated by the following general formula Ilia: Thr(R')-OR' (Ilia) wherein R® represents a carboxyl protecting group, and R^ represents hydrogen or a hydroxyl protecting group. To make it more clear, a threonine ester can be illustrated by the general formula CH3-CH(OR^)-CH(NH2)COOR^ wherein R^ and R^ are as mentioned above. Some nucleophile compounds are known compounds and the remaining nucleophile compounds can be prepared in analogy with the preparation of known compounds or in analogy with known methods. The nucleophile compounds may be employed in the form of the free base or soluble salts thereof such as hydrochlorides, acetates, propionates, and butyrates. When the coupling reaction starts, it is desirable that a substantial excess of nucleophile compound is present in the coupling reaction mixture solution, with the molar 11 ratio I , , ^ ^ ^ exceeding about 5;1. When the coupling reaction starts, the concentration of the nucleophile compound in the reaction mixture should preferably exceed 0.1 molar, the upper concentration limit being the solubility thereof. To obtain a 60% yield considered herein as an important aspect to practice of this invention, the reaction temperature, water content and pH value are interrelated within the described ranges. The organic solvents suited to practice of this invention are polar solvents which are miscible with water and preferably such that are capable of containing therein high concentrations of the desired intermediate product (for example of formula II) and the nucleophile compound. Examples of suitable organic solvents are aprotic solvents, such as N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone-2, and dimethyl sulfoxide, and protic solvents, such as acetic acid, ethanol, methanol, 2-propanol, 1- propanol, butanol and 1,4-butanediol. Dioxane, acetone, tetrahydrofuran, formamide, and acetonitrile may also be used and even an amino acid ester used as the nucleophile compound can, fully or partially, be used as the organic solvent. The nature of the solvent does affect the system as a whole, and interrelationships suited to one solvent productive of high yields may not apply with a different solvent. Best yield results have been obtained with aprotic solvents, and aprotic solvents are most preferred for practice of this invention. Obviously, when calculating or determining the content of water in the reaction mixture, the nucleophile compound is considered an organic solvent. The addition of an acid, such as hydrochloric acid, formic acid, acetic acid, propionic acid, or butyric acid, or of a base, such as pyridine, TRIS, N-methylmorpholine, triethylamine, or N-ethylmorpholine, is optional. They are included in the reaction mixture to bring about a suitable pH value. Although mineral acids or bases may be used in practice of this invention, organic acids and bases are preferred, particularly those identified above. Organic acids are most preferred. When the coupling reaction starts, the weight ratio between trypsin or ALP (calculated as crystalline trypsin or ALP or an amount of trypsin or ALP derivative corresponding thereto) and the desired intermediate product in the reaction mixture is preferably in the range from about 1:1000 to about 1:10, more preferred in the range from about 1:200 to about 1:50. In some cases, the enzyme added in the cleavage step is sufficient for performing the coupling reaction and, in such case, there is no need for adding a further amount of enzyme during the coupling step. In other cases, it may be desirable to add an additional amount of enzyme during the coupling step. 12 Inasmuch as high concentrations of the desired intermediate product and of nucleophile compound in solution promote high conversion rates, solvent selection is biased towards those solvents in which the reactants are very soluble. The solubility of the nucleophile compound in particular is important, because that reactant should be present in high concentration. When the coupling reaction starts, the molar ratio of the nucleophile compound to the desired intermediate product should preferably exceed 5:1, preferably exceed 50:1. When the coupling reaction starts, the concentration of the nucleophile compound in the reaction mixture should preferably be at least 0.1 molar. In a preferred embodiment of this invention, a nucleophile compound having carboxy protecting group(s) which can be removed from the resulting insulin compound under conditions, which do not cause substantial irreversible alterations in the insulin molecule, is used. As examples of such carboxyi protecting groups can be mentioned lower alkyl, for example, methyl, ethyl, and tert-butyl, substituted benzyl groups such as p-methoxybenzyl, diphenylmethyl, and 2,4,6-trimethylbenzyl, and groups of the general formula -CH2-CH2-S02R^ wherein R^ represents lower alkyl, such as methyl, ethyl, propyl, and n- butyl. Suitable hydroxyl protecting groups are those which can be removed under conditions which do not cause substantial irreversible alteration in the insulin molecule. As an example of such a group can be mentioned ferf-butyl. Further protection groups usually used are described by Wunch; Metoden der Organischen Chemie (Houben-Weyl), Vol. XV/1, editor: Eugen Muller, Georg Thieme Verlag, Stuttgart 1974. According to one embodiment of this invention, the process of this Invention will result in a compound of the general formula IV: R'^-Cys-Zn-Cys-R'^ I I (IV) R'-Cys-Zm-Cys-R'^-R' wherein Zn and Zm, independent of each other, represents two peptide moieties each containing n and m amino acid residues, respectively, R'^ represents a peptide residue, R'^ represents an amino acid residue or a peptide residue, R'^ represents a peptide residue, R'" is as mentioned above, and R'® is an amino acid carrying a carboxy protecting group or a peptide residue, optionally carrying a carboxy protecting group. Any carboxy protecting group (for example, R®) and any hydroxy protecting group (for example, R^) present in an insulin compounds can be removed by known methods or methods known per se. In case the carboxy protecting group is methyl, ethyl, or a group of 13 the general formula -CH2-CH2-S02R^ wherein R^ is as defined above, said protecting group can be removed at gentle basic conditions in an aqueous medium, preferably at a pH value in the range from about 8 to about 12, for example, at about 9.5. As the base can be used strong bases, for example, a tertiary amine, for example triethylamine, hydroxides of alkali metals such as sodium hydroxide or hydroxides of alkaline earth metals such as calcium, or magnesium hydroxide, in case the carboxy protecting group is fert-butyl, substituted benzyl such as p-methoxybenzyl or 2,4,6-trimethylbenzyl, or diphenylmethyl, said group can be removed by acidolysis, preferably with trifluoroacetic acid. The trifluoroacetic acid may be nonaqueous or may contain some water, or it may be diluted with an organic solvent, such as dichloromethane. In case the hydroxy protecting group (for example, R^) is tert-butyl, said group can be removed by acidolysis, vide above. Preferably, the insulin compounds prepared have no hydroxy protecting group. In a preferred embodiment of this invention, the process of this invention converts the insulin precursor (for example, of formula I) into an insulin compound (for example, formula IV), having a carboxy protecting group in the C terminal amino acid residue in the B chain which, then, can be deblocked to form an insulin compound having no carboxy protecting group. When selecting the reaction conditions according to the above explanation and considering the results obtained in the following examples it is possible to obtain a yield of insulin compound which is higher than 60%, and even higher than 80%, and under certain preferred conditions higher than 90%. By the process of this invention, insulin compounds of an acceptable purity can be obtained and be further purified, if desired, for therapeutic purpose. More specifically, insulin aspart may, for example, be prepared by enzymatic cleavage with ALP of an insulin precursor such as [Asp^^]-des(30-65) human proinsulin being N-terminally extended with Glu-(Glu-Ala)3-Pro-Lys- (SEQ ID NO.: 1) and coupling with a nucleophile compound such as L-threonine methyl ester, followed by hydrolysis. insulin lispro may, for example, be prepared by enzymatic cleavage with trypsin of a precursor such as porcine insulin and coupling with a nucleophile compound such as Gly- Phe-Phe-Tyr-Thr-Lys-Pro-Thr (SEQ ID NO.: 3). Insulin glargin may, for example, be prepared by enzymatic cleavage with ALP of an insulin precursor such as [Gly^®]-des(30-65) human proinsulin and coupling with a nucleophile compound such as Thr-Arg-Arg-OMe, followed by hydrolysis. Abbreviations used herein are in accordance with the rules approved (1974) by the lUPAC- lUB Commission on Biochemical Nomenclature, w'cfe Collected Tentative Rules & 14 Recommendations of the Commission on Biochemical Nomenclature lUPAC-lUB, 2"" edition, Maryland 1975. The mentioning herein of a reference is no admission that it constitutes prior art. Herein, the word "comprise" Is to be interpreted broadly meaning "include", "contain" or "comprehend" (vide, the EPO guidelines C 4.13). The following examples are offered by way of illustration, not by limitation. Example 1. 200 mg [Ala^\Lys^^]-des(33-63) porcine proinsulin was suspended in 1.35 ml water and the pH value was adjusted to 9 with 10 ^1 triethylamine. A mixture of 375 \|j\| N,N-dimethyl- acetamide and 460 \|jl water was added with slightly agitation and to the resulting solution was added 315 \|j\| of a 5.4 mg/ml aqueous solution of Achromobacter lyticus lysyl specific protease (EC 3.4.21.50) (herein designated ALP). The pH value was adjusted to 9.8 with 20 \|j\| triethylamine and the reaction solution was left for 1 hour at 23°C. The reaction solution was acidified by addition of 70 (JI 4 N hydrochloric acid and cooled in an ice bath. A solution of 300 mg L-threonine methyl ester in 4.85 ml N,N-dimethylacetamide was added and the pH value was adjusted to 6.5 by addition of 450 \|jl 4 N hydrochloric acid. The reaction solution was left for 4 hours at 23°C after which the reaction was stopped by addition of hydrochloric acid to a pH value 5 pm C18 silica column with an ethanol-water eluent containing 0.125 M ammonium sulphate adjusted to a pH value 4, a conversion yield of 86% to human insulin methyl ester was found after a total reaction time of 5 hours. For comparison, a one-step conversion was performed: 100 mg [Ala^\Lys^^]-des(33-63) porcine proinsulin was suspended in a mixture of 887 \|jl water and 175 \|JI N,N-dimethylacetamide. 150 mg L-threonlne methyl ester was dissolved in 2.265 ml N,N-dimethylacetamide and was slowly added to the ice-cooled mixture. The pH value was adjusted to 6.5 with 340 \|jl acetic acid and 158 \^\ of a 5.4 mg/ml aqueous solution of ALP was added. The conversion reaction was followed by RP-HPLC analysis of acidified samples. After 5 hours, a 53% conversion to human insulin methyl ester was found and after 24 hours the conversion reached a maximum of 87%. 15 The isolated human insulin methyl ester was converted into human insulin by dissolution in water at a pH value of 10 at a concentration of 10 mg/ml. The reaction was terminated after 24 hours by adjusting the pH value to 5.2 with 1 N hydrochloric acid and the precipitated human insulin was isolated by centrifugation and purified by reverse phase high performance liquid chromatography. At the same reaction time, i.e., 5 hours, the yield by the process of this invention, compared with the per se known process, was improved with 62 %. The two processes obtained almost the same yield, if the reaction time of the per se known one-step conversion was extended almost 5 times, compared with the reaction time for the process of this invention. Example 2. 200 mg porcine insulin was suspended in 1.37 ml water and a mixture of 294 [i\ N-methyl-2- pyrrolidon and 326 \|jl water was added with slightly agitation. The pH value was adjusted to 9.0 with 10 \|jl 2 N sodium hydroxide and to the resulting solution was added 315 M' of a 5.4 mg/ml aqueous solution of ALP. The pH value was adjusted to 9.8 with 12 \|jl 2 N sodium hydroxide and the reaction solution was left for 4 hours at 23°C. The reaction solution was acidified by addition of 70 \|jl 4 N hydrochloric acid and cooled in an ice bath. A solution of 300 mg L-threonine methyl ester in 4.4 ml N-methyl-2-pyrrolidon was acidified with 500 \|jl 4 N hydrochloric acid. The insulin solution was slowly added and the pH value was adjusted to 6.5 with 50 \|jl 2 N hydrochloric acid. The reaction solution was left for 4 hours at 23°C after which the reaction was stopped by addition of hydrochloric acid to a pH value reversed phase HPLC analysis on a 4mm x 250 mm 5 \|jm C18 silica column with an ethanol-water eluent containing 0.125 M ammonium sulphate adjusted to a pH value 4, a conversion yield of 86% to human insulin methyl ester was found after a total reaction time of 8 hours. For comparison, a one-step conversion was performed: 100 mg porcine insulin was suspended in a mixture of 848 pi water and 147 pi N- methyl-2-pyrrolidon. 150 mg L-threonine methyl ester was dissolved in 2.2 ml N-methyl-2- pyrrolidon and was slowly added to the ice-cooled mixture. The pH value was adjusted to 6.5 with 300 pi acetic acid and 158 pi of a 5.4 mg/ml aqueous solution of ALP was added. The reaction solution was left at 23°C and the conversion reaction was followed by RP-HPLC analysis of acidified samples. After 8 hours, the conversion was found to 54% and after 48 hours a conversion maximum of 86% to human insulin methyl ester was reached. 16 The isolated human insulin methyl ester can be converted to human insulin by alkaline hydrolysis. At the same reaction time, i.e., 8 hours, the yield by the process of this invention was improved with 59 %, compared with the perse known process. The two processes obtained the same yield, if the reaction time of the per se known one-step conversion was extended 6 times, compared with the reaction time for the process of this invention. Example 3. 200 mg [Asp^^]-des(30-65) human proinsulin, N-terminally extended with the peptide Glu- (Glu-Ala)3-Pro-Lys- (SEQ ID NO.: 1), was suspended in 1.35 ml water. A mixture of 350 ^1 N,N-dimethylformamide and 425 \|jl water was added with slightly agitation and the pH value was adjusted to 9 with 45 \|jl triethylamine. To the resulting solution was added 200 pi of a 8.5 mg/ml aqueous solution of ALP and the pH value was adjusted to 9.8 with 20 pi triethyl- amine. The reaction solution was left for 1 hour at 23°C. The reaction solution was acidified by addition of 70 pi 4 N hydrochloric acid and cooled in an ice bath. A solution of 300 mg L- threonine methyl ester in 4.95 ml N,N-dimethylformamide was added and the pH value was adjusted to 6.5 by addition of 470 pi 4 N hydrochloric acid. The reaction solution was left for 4 hours at 23°C after which the reaction was stopped by addition of hydrochloric acid to a pH value with an ethanol-water eluent containing 0.125 M ammonium sulphate adjusted to a pH value 4, a conversion yield of 87% to [Asp^^^j-human insulin methyl ester was found after a total reaction time of 5 hours. For comparison, a one-step conversion was performed: 90 mg [Asp^^]-des(30-65) human proinsulin, N-terminally extended with the peptide Glu-(Glu-Ala)3-Pro-Lys- (SEQ ID NO.: 1), was suspended in a mixture of 887 pi water and 175 pi N,N-dimethylformamide. 150 mg L-threonine methyl ester was dissolved in 2.13 ml N,N-dimethyiformamide and was slowly added to the ice-cooled mixture. The pH value was adjusted to 6.5 with 250 pi acetic acid and 118 pi of a 8.5 mg/ml aqueous solution of ALP was added. The conversion reaction was followed by RP-HPLC analysis of acidified samples. After 5 hours, the conversion was found to 47% and after 24 hours the conversion to [Asp^^^j-human insulin methyl ester reached a maximum of 81%. The isolated insulin methyl ester can be converted to [Asp^^^j-human insulin by alkaline hydrolysis. 17 At the same reaction time, i.e., 5 hours, the yield by the process of this invention was almost doubled, compared with the perse known process. Comparable yields ware obtained by the two processes, if the reaction time of the per se known one-step conversion was extended nearly 5 times, compared with the reaction time for the process of this invention. Example 4 1.5 g insulin aspart precursor [Asp^^Met^^Trp^^Lys^^]-des(33-65) human proinsulin, N- terminally extended with the peptide Glu-Glu-Gly-Glu-Pro-Lys- (SEQ ID NO.: 2) was suspended in 3.5 g water. With slight agitation and at ambient temperature, the precursor was dissolved by gradually adding 4M sodium hydroxide to a pH value of 10.67. 3.7 g of a 45 % (weight/weight) solution of ethanol in water was added.1.5 ml of a 5.8 mg/ml aqueous solution of ALP was added and the mixture was left to react for 2 hours. The pH value was adjusted to 4.7 by addition of 4 N hydrochloric acid. 2.025 g L-threonine ethyl ester was dissolved in 16.2 ml ethanol and the solution was added at a maximum temperature of 15°C. The pH value was adjusted to 6.5 with 4 N hydrochloric acid. The temperature was adjusted to ambient temperature and the reaction mixture was left for 20 hours at this temperature. By reversed phase HPLC analysis on a 4mm x 250 mm 5 pm CI8 silica column with an acetonitrile-water eluent containing 200mM sodium sulphate adjusted to a pH value of 3.6, a conversion yield of 89.1 % insulin aspart ethyl ester was found after 1 hours reaction time and after 20 hours reaction time, a conversion yield of 90.5% was found. The isolated insulin aspart ethyl ester can be converted to insulin aspart by alkaline hydrolysis. Example 5 10.9 g insulin aspart precursor [Asp^^Met^°,Trp^^Lys^^]-des(33-65) human proinsulin, N- terminally extended with the peptide Glu-Glu-Gly-Glu-Pro-Lys- (SEQ ID NO.: 2) was suspended in 49.3 g water. With slight agitation and at ambient temperature, the precursor was dissolved by gradually adding 37.6 g from a mixture containing 0.36 M sodium hydroxide, 0.27 M sodium acetate and 36 % N-methyl-2-pyrrolidon. The pH value was adjusted to 9.7 with 9.2 ml 0.5 M sodium hydroxide. 7.1 mL of a 7.1 mg/mL aqueous solution of ALP was added, and the mixture was left to react for 5 hours. The pH value was kept constant at 9.7 by adding more 0.5 M sodium hydroxide throughout the reaction. The 18 reaction mixture was cooled to 5 °C and the pH value was adjusted to 5.7 by addition of 2.73 g 4 N hydrochloric acid. 14.02 g L-threonine ethyl ester was added and the pH value was adjusted to 6.0 with 4 N hydrochloric acid. 344 g cold (4 °C) N-methyl-2-pyrrolidon was added. The temperature was adjusted to 22 °C and the pH value adjusted to 6.5 with 4 N hydrochloric acid. The reaction mixture was left for 9 hours at this temperature. By reversed phase HPLC analysis on a 4mm x 250 mm 5 pm C18 silica column with an acetonitrile-water eluent containing 200mM sodium sulphate adjusted to a pH value of 3.6, a conversion yield of 87.5 % to insulin aspart ethyl ester was found after a total reaction time of 14 hours. The isolated insulin aspart ethyl ester can be converted to insulin aspart by alkaline hydrolysis. WE CLAIM: 1. A process for preparing an insulin compound wherein a) in a reaction mixture containing at least about 55 %, preferably at least about 60 %, more preferred at least 70 %, water (weight/weight), an insulin precursor is subjected to an enzymatic cleavage and, thereafter, b) the intermediate product is coupled with a nucleophile compound in the reaction mixture used for the enzymatic cleavage reaction with the proviso that the composition of the reaction mixture has been modified so that the content of water in the reaction mixture is in the range from about 10 % to about 50 % water (weight/weight), preferably in the range from about 20 % to about 40 % water (weight/weight), and c), if desired, removing the protecting group(s), and wherein no isolation of the intermediate product is performed between the cleavage step (a) and the coupling step (b). 2. The process, according to claim 1, wherein the enzyme used for the cleavage step is also present in the coupling step. 3. The process, according to any one of the preceding claims, wherein, before the coupling reaction is initiated, at least about 25 %, preferably at least 50 %, more preferred at least 75 %, preferably at least 85 %, more preferred at least 95 %, of the insulin precursor is cleaved to the intermediate product. 4. The process, according to any one of the preceding claims, wherein the enzyme used for the enzymatic cleavage is trypsin or a lysyl specific protease, preferably Achromobacter lyticus protease I. 5. The process, according to any one of the preceding claims, wherein the nucleophile compound is an amino acid ester. 20 6. The process, according to the preceding claim, wherein the nucleophile compound is a threonine ester. 7. The process, according to any one of the claims 1 to 4, wherein the nucleophile compound is an amino acid amide, 8. The process, according to any one of the claims 1 to 4, wherein the nucleophile compound is a peptide. 9. The process, according to any one of the claims 1 to 4, wherein the nucleophile compound is a peptide ester. 10. The process, according to any one of the claims 1 to 4, wherein the nucleophile compound is a peptide amide. 11. The process, according to any one of the preceding claims, wherein the protection group(s) from the insulin compound is removed. 12. The process according to any one of the preceding claims, wherein the resulting insulin compound has threonine in the B30 position. 13. The process according to any one of the preceding claims, wherein the resulting compound is human insulin, insulin aspart, insulin lispro, insulin glargin, or insulin detemir.

Full Text

The present invention relates to an improved process for converting an insulin precursor into
an insulin compound, optionally via an insulin ester.
BACKGROUND OF THIS INVENTION
Insulin is a pancreatic hormone involved in the regulation of blood-glucose concentrations.
For example, human, porcine, and bovine insulin, insulin analogues and mixed insulins are
given to patients with insulin-dependent diabetes mellitus to control their blood-glucose
concentrations.
Porcine and bovine insulin are, usually, prepared from pancreas glands. Human
insulin can, semisynthetically, be prepared from porcine insulin. Alternatively, human insulin,
as well as many insulin analogues, can be prepared by genetic engineering. By genetic
engineering, which may, for example, be performed in bacteria or in yeast, an insulin
precursor is prepared which, thereafter, is to be converted into the desired product. This
conversion can be performed in different ways.
One possibility is the so-called transpeptidation where a peptide cleavage and a
peptide coupling takes place consecutively in the same reaction mixture, under the same
reaction conditions, vide, for example, US patent No. 4,343,898 (Novo Industri).
Another possibility is, in the first step, to cleave the insulin precursor, wofe, for
example, Hoppe-Seyler's Z. Physiol. Cliem. 359 (1978), 799, thereafter, to isolate the
intermediate product and, then, to perform the desired coupling in another reaction mixture
than that used in the first step, vide, for example. Nature 280 (1979), 412.
According to EP 87,238, a transpeptidation reaction is performed in a solvent system
comprising between about 75% and 97% (vol/vol) of at least one non-aqueous reaction
miscible solvent including at least about 50% (vol/vol) butane-1,4-diol.
According to US 4,579,820, the transpeptidation process is performed using an L-
specific serine carboxypeptidase enzyme, for example carboxypeptidase Y.
According to US 4,601,979 (Nordisk Insulinlaboratorium), the transpeptidation or only
the peptide coupling is performed in an aqueous reaction medium substantially free of
organic solvent.
According to WO 83/00504 (Nordisk insulinlaboratorium), a porcine product was
treated with carboxypeptidase A, the resulting des-alanine-B30 insulin product was
suspended in a lower alcohol, and this suspension was mixed with a solution of an L-
threonine ester and trypsin. In all the specific examples, the des-alanine-B30 insulin product
was isolated, either by freeze-drying or by precipitation.
2

The object of this invention is to overcome or ameliorate at least some of the disadvantages
of the prior art. Hence, not all the more detailed objects mentioned below may be fully
overcome or ameliorated.
DEFINITIONS
The term "amino acid" as used herein, refers to amino acids which can be coded for by
nucleotide sequences. Analogously, this applies to the term amino acid residue which is an
amino acid from which hydroxy has been removed from a carboxy group and/or hydrogen
has been removed from an amino group.
Similarly, the terms peptide and peptide residue consists of amino acid residues.
Preferably, the peptide contains not more than 10 amino acid residues.
The term amino acid amide, as used herein, refers to an amino acid having an
optionally substituted C terminal carboxamide group.
The term peptide amide, as used herein, refers to a peptide having an optionally
substituted C terminal carboxamide group.
The term "insulin precursor", as used herein, refers to a polypeptide consisting of two
peptide chains (corresponding to the A and B chains of insulin and, hereinafter, designated
the A and B chains) which, similarly with insulin, are connected with each other via two
disulphide bridges (from one cysteine (Cys) residue to another cysteine residue) between
the two peptide chains and wherein, lilce in insulin, there is an disulphide bridge from one
cysteine residue in the A chain to another cysteine residue in the A chain. In this insulin
precursor there is, at least, one lysine or arginine residue in the B chain. Optionally, in this
insulin precursor, the A and B chains are connected with each other via a third peptide chain
(corresponding to the connecting peptide in insulin) between the C terminal end of the B
chain and the N terminal end of the A chain. In case the A and B chains are connected with
each other via this third peptide chain, lysine is present at the C terminal end of this third
peptide. Optionally, in this insulin precursor, a fourth peptide chain may be connected to the
N terminal end of the B chain. In case this fourth peptide chain is connected to the N
terminal end of the B chain, lysine is present at the C terminal end of this fourth peptide
chain. Furthermore, in this insulin precursor, there is an identity of the amino acid residues of
at least 80 %, preferably at least 85 %, more preferred at lest 90 %, and even more
preferred at least 95%, compared with human insulin, with the proviso that the third and
fourth peptide chains are to be disregarded for this calculation. In human insulin, there are
3

disulphide bridges between Cys'^® and Cys*^\ between Cys^^ and Cys^^ and between
Cys*^° and Cys^^^ and there is lysine in the B29 position.
The term "amino acid ester", as used herein, refers to an amino acid carrying a C
terminal carboxy protecting group and, optionally, a hydroxy protecting group.
The term "peptide ester", as used herein, refers to a peptide wherein at least the C
terminal carboxy group carries a carboxy protecting group. Optionally, any hydroxy group is
protected and, optionally, the e-amino group of any lysine residues is derivatised, preferably
with a hydrophobic group, for example an acyl group having at least 10 carbon atoms.
Preferably, the peptide ester contains not more than 10 amino acid residues.
The term nucleophile compound, as used herein, refers an amino acid ester, an
amino acid amide, a peptide, a peptide ester, and a peptide amide. In any of these amino
acid esters, amino acid amides, peptides, peptide esters, and peptide amides, the amino
group in any lysine group is, optionally, derivatised, preferably with a hydrophobic group, for
example, an acyl group having at least 10 carbon atoms.
The term "insulin compound", as used herein, refers to insulin from any species such
as porcine insulin, bovine insulin, and human insulin and salts thereof such as zinc salts, and
protamin salts. Furthermore, the term "insulin compound", as used herein, refers to what
could briefly be designated "insulin analogues". Insulin analogues, as used herein, refers to
insulin compounds wherein one or more of the amino acid residues have been exchanged
with another amino acid residue and/or from which one or more amino acid residue has
been deleted and/or from which one or more amino acid residue has been added, provided
that said insulin analogue has a sufficient insulin activity. Examples of insulin analogues are
described in the following patents and equivalents thereto: US 5,618,913; EP 254,516; EP
280,534; US 5,750,497; and US 6,011,007. Examples of specific insulin analogues are
insulin aspart (i.e., [Asp^^^] human insulin), insulin lispro (i.e., [Lys^^^Pro^^®] human insulin),
and insulin glargin (i.e., [Gly*^\Arg^^\Arg^^^] human insulin). The term "insulin analogue", as
used herein also covers what could be designated insulin derivatives, i.e., compounds which
a skilled art worker would generally considers derivatives of insulin, vide general textbooks,
for example, insulin having a substituent not present in the parent insulin molecule.
Examples of insulin derivatives are insulins or insulin analogues having an optionally
substituted carboxamide group. Also compounds which can be considered being both an
insulin derivative and an insulin analogue are herein covered by the term insulin analogue.
Examples of such compounds are described in the following patents and equivalents
thereto: US 5,750,497 and US 6,011,007. Hence, a further example of a specific insulin
analogue is insulin detemir (i.e., des-Thr^^° human insulin y Lys^^^ tetradecanoyi). The insulin
compounds prepared by this invention have an anti-diabetic activity sufficiently high to be
4

used to treat diabetic patients. The anti-diabetic activity can be determined using the so-
called free fat cell assay.
The term pH value, as used herein, refers to the value measured with a pH meter by
immersing a calomel combination glass electrode connected to the pH meter directly in the
solution, the pH value of which is to be measured. The pH meter is calibrated with an
aqueous standard buffer.
BRIEF DESCRIPTION OF THE FIGURES
SEQ ID NO.: 1 is the peptide moiety Glu-(Glu-Ala)3-Pro-Lys-; SEQ ID NO.: 2 is the peptide
moiety Glu-Glu-Gly-Glu-Pro-Lys-; and SEQ ID NO.: 3 is the peptide moiety Gly-Phe-Phe-
Tyr-Thr-Lys-Pro-Thr.
BRIEF DESCRIPTION OF THIS INVENTION
The present invention relates to a process for preparing insulin compounds. These insulin
compounds can be used as medicaments. In a preferred embodiment of this invention,
insulin compounds having threonine (Thr) in the C terminal end of the 8 chain are prepared.
Any sl of the insulin compounds to administer to a diabetic patient, and when.
The starting material for the process of this invention is an insulin precursor which is
subjected to both a peptide cleavage and a peptide coupling at conditions favoring both
reactions but where no isolation of the intermediate product takes place. In other words, the
insulin precursor is subjected to a peptide cleavage and the resulting product, i.e., the
intermediate, is subjected to a peptide coupling. The conditions favoring a peptide cleavage
are not identical with the conditions favoring a peptide coupling. Hence, in the first step of
this invention, i.e., the cleavage step or the cleavage reaction, the reaction conditions in the
reaction mixture are chosen so as to favor the peptide cleavage and, in the second step of
this invention, i.e., the coupling step or the coupling reaction, the reaction conditions in the
reaction mixture are altered so as to favor the peptide coupling.
In one embodiment of this invention, the insulin precursor is, in the first step,
dissolved in a predominantly aqueous medium and the enzyme used for cleavage is added.
This reaction mixture may be free or substantially free of organic solvent. Alternatively, the
reaction mixture may contain a certain amount of organic solvent which may ensure a proper
5

solubility of the insulin precursor. However, it is desired not to use so much organic solvent
that it has an undesired influence on the enzymatic cleavage. In the first step of the process
of this invention, the reaction parameters such a pH value, temperature, and time, are
chosen so that they are favorable to cleavage at the lysine residue(s) or arginine residue(s).
When the cleavage reaction has taken place to a certain, desired degree, a
nucleophile compound and an organic solvent is mixed with the reaction mixture (without
previous isolation of the intermediate product), so that the coupling of the nucleophile
compound to the lysine or arginine residue of the desired intermediate product takes place.
In this step, the reaction parameters are set so as to be favorable to the coupling reaction. In
a preferred embodiment of this invention, the nucleophile compound is an amino acid ester,
for example a threonine ester, or a peptide ester.
Thereafter, the protecting group(s) may, if desired, be removed from the resulting
compound.
Compared with the known transpeptidation reaction, the advantages obtained by the
process of this invention is a shorter, over all reaction time with the same amount of enzyme
and a similar or higher yield. Compared with a two pot reaction with cleavage in an aqueous
medium, isolation of the intermediate product, and coupling in a mixture of organic solvent
and water, the advantages obtained by the process of this invention is a shorter, over all
reaction time, the use of a lower amount of enzyme, and an easier process flow.
More precisely, this invention relates to the following embodiments:
DETAILED DESCRIPTION OF THIS INVENTION
As appears from claim 1, first a peptide cleavage takes place and, thereafter, a coupling
reaction takes place.
Briefly, the cleavage reaction (i.e., the enzymatic cleavage) is performed as follows:
The enzymatic cleavage of the insulin precursor (i.e., the peptide cleavage) takes place in a
reaction mixture containing at least about 55%, preferably at least about 60%, more
preferred at least 70%, water (weight/weight).
In a preferred embodiment of this invention, the concentration of the insulin precursor
in the reaction mixture wherein the enzymatic cleavage takes place is at least 2 %,
preferably in the range from about 5 to about 10% (weight/vol).
6

The cleavage reaction is performed in a neutral or alkaline medium, preferably
having a pH value in the range from about 6 to about 11, more preferred in the range from
about 8 to about 10.
In a preferred embodiment of this invention the amounts of enzyme compared with
the amount of insulin precursor is in the range from approximately 0.05 to approximately 5%
(weight/weight), preferably from approximately 0.1 to approximately 2%.
The tryptic enzyme is not material to practice of this invention. Trypsin is a well-
characterized enzyme available in high purity, notably from bovine or porcine origin. From
microbial origin, Acromobacter lyticus protease I (hereinafter designated ALP) can be
obtained. Moreover, the enzyme form, whether it is a native enzyme or an active
immobilized enzyme or an enzyme derivative, is not material to practice of this invention, if it
is desired to split at the C terminal end of arginine, trypsin can be used and if it is desired to
split at the C terminal end of lysine, either trypsin or ALP can be used. For the splitting at the
C terminal end of lysine, ALP is preferred.
As examples of active enzyme derivatives can be mentioned acetylated trypsin,
succinylated trypsin, glutaraldehyde treated trypsin, and immobilized trypsin or ALP
derivatives.
If an immobilized trypsin or ALP is used, it is suspended in the reaction mixture or
may be packed into a column.
To a great extent, the action of the enzyme is controlled by an interrelation of water
and solvent content, the pH value, and the reaction temperature. Increasing the
concentration of organic solvent in the reaction mixture and lowering of the pH value to
around neutral shifts the usual enzymatic reaction from cleavage towards coupling.
Reducing the temperature reduces the reaction rate, but might also reduce biproduct
formation and enzyme denaturation.
In a preferred embodiment of this invention, the insulin precursor is dissolved in an
aqueous medium having a concentration of acetate ions in the range from about 5 mM to
about 500 mM, preferably in the range from about 20 mM to about 200 mM. For example,
sodium, potassium, ammonium acetate ortriethyl ammonium acetate can be used.
According to one embodiment of this invention, the insulin precursor (being a
peptide) can be illustrated by the following general formula I:
R'-Cys-Zn-Cys-R^
I I (I)
R'-Cys-Z,„-Cys-R'

wherein Zn and Z^, independent of each other, represents two peptide moieties each
containing n and m amino acid residues, respectively, R^ represents a peptide residue which
peptide residue optionally contains a lysine or arginine residue, R^ represents an amino acid
residue or a peptide residue, R^ represents a peptide residue which peptide residue
optionally contains a lysine or arginine residue, R" represents a lysine or arginine residue or
a peptide residue which peptide residue contains a lysine or arginine residue, or R^ and R"
are together a peptide residue containing a lysine or arginine residue, the two vertical lines
indicate the disulphide bonds between the two cysteine residues and, furthermore, there is
an disulphide bond between two cysteine residues present in R^ and in Zn.
Preferably, the amino acid residues present in the insulin precursor of formula I are
those which can be coded for by the nucleotide sequences.
According to a preferred embodiment of this invention, an insulin precursor, wherein
the number of amino acid residues in R^ and R"* together is in the range from about 8 to
about 50, is used. In another preferred embodiment of this invention, Zn contains 12 amino
acid residues. In another preferred embodiment of this invention, Zm contains 11 amino acid
residues. In another preferred embodiment of this invention, R^ contains 1 amino acid
residue, for example, Asn or Gly. In another preferred embodiment of this invention, R^
contains 6 amino acid residues.
In a preferred embodiment of this invention, the insulin precursor is a single chain
precursor, i.e. a compound of formula I wherein R^ and R"* together are a peptide residue
containing a lysine or arginine residue. Hence, preferably, the insulin precursor is not
mammalian insulin such as porcine insulin, rabbit insulin, dog insulin or whale insulin.
According to another embodiment of this invention, the insulin precursor of formula I
contains the same amino acid residues in positions A1 through A21 and in positions B1
through 829 as are present in human insulin in the same positions.
According to another embodiment of this invention, the insulin precursor of formula I
contains the same amino acid residues in positions A1 through A21 and in positions 81
through 829 with the proviso that the 828 amino acid residue is Asp.
According to another embodiment of this invention, the insulin precursor of formula 1
contains the same amino acid residues in positions A1 through A21 and in positions 81
through 829 as are present in human insulin in the same positions with the proviso that the
828 amino acid residue is Lys and the 829 amino acid residue is Pro.
According to another embodiment of this invention, the insulin precursor of formula I
contains the same amino acid residues in positions A1 through A21 and in positions 81
through 829 as are present in human insulin in the same positions with the proviso that the
A21 amino acid residue is Gly and the 831 and 832 amino cid residues both are Arg.
8

Examples of specific insulin precursors which can be use in the process of this
invention are human proinsulin; monkey proinsulin; tAla^\Lys^^]-des(33-63) porcine
proinsulin; porcine insulin; [Asp^^]-des(30-65) human proinsulin being N-terminally extended
with Glu-(Glu-Ala)3-Pro-Lys- (SEQ ID NO.: 1); and [Asp'^Met'°,Trp'\Lys']-des(33-65)
human proinsulin being N-terminally extended with Glu-Glu-Gly-Glu-Pro-Lys- (SEQ ID NO.:
2).
The insulin precursors of formula I can be prepared as described in or analogously
as described in the International applications having publication numbers WO 01/49742, WO
01/49870, WO 01/079250, and WO 02/079254, the content of which is hereby incorporated
by reference.
The desired intermediate product (i.e., the desired cleaving product) corresponds to
the insulin precursor wherein at least one lysine or arginine residue has been cleaved to
form a lysyl or arginyl moiety, respectively. Furthermore, in the desired intermediate product,
the A and B chains which are connected with each other via two disulphide bridges are not
connected with each other via a peptide chain between the C terminal end of the B chain
and the N terminal end of the A chain. In a preferred embodiment of this invention, the
number of amino acid residues present in the desired intermediate product is in the range
from about 48 to about 52, preferably in the range from about 49 to about 51, even more
preferred 50. In another preferred embodiment of this invention, there are not more than 4,
preferably not more than 3, more preferred not more than 2, and even more preferred not
more than 1, of the amino acid residues present in the desired intermediate product which
are not present at the corresponding position in human insulin.
According to one embodiment of this invention, the desired intermediate product (the
desired cleaving product) can be illustrated by the general formula II
R'^-Cys-Zn-Cys-R'
1. I (II)
R'^-Cys-Zm-Cys-R'"
wherein Zn and Zm, independent of each other, represents two peptide moieties each
containing n and m amino acid residues, respectively, R'^ represents a peptide residue, R'^
represents an amino acid residue or a peptide residue, R'^ represents a peptide residue, R"*
represents lysine or arginine or a peptide residue containing a lysine or arginine residue in
the C terminal end, the two vertical lines indicate the disulphide bond between the two
cysteine residues and, furthermore, there is an disulphide bond between two cysteine
residues present in R'^ and in Zn.

In a preferred embodiment of this invention, R'^ is the amino acid residues A1
through A6 in human insulin in this order in which, optionally, one or two of the amino acid
residues have been exchanged with another amino acid residue or wherein one or two of the
amino acid residues are not present. In another preferred embodiment of this invention, R'^
is -Asn or -Gly. In another preferred embodiment of this invention, R'^ is the amino acid
residues B1 through B6 in human insulin in this order in which, optionally, one or two of the
amino acid residues have been exchanged with another amino acid residue or wherein one
or two of the amino acid residues are not present. In another preferred embodiment of this
invention, R'" is the amino acid residues B20 through B29 in human insulin in this order, the
amino acid residues B20 through B29 in human insulin in this order with the proviso that it
has Asp in B28 and Lys in B29, and the amino acid residues B20 through B28 in human
insulin in this order with the proviso that it has Lys in B28, in each of which, optionally, one or
two of the amino acid residues have been exchanged with another amino acid residue or
wherein one or two of the amino acid residues are not present or a part of any of these
peptide residues leaving out one or more consecutive amino acid residues from the C
terminal end thereof. In another preferred embodiment of this invention, Zn is the amino acid
residues AS through A19 in human insulin in this order in which, optionally, one or two of the
amino acid residues have been exchanged with another amino acid residue or wherein one
or two of the amino acid residues are not present. In another preferred embodiment of this
invention, Zm is the amino acid residues B8 through B18 in human insulin in this order in
which, optionally, one or two of the amino acid residues have been exchanged with another
amino acid residue or wherein one or two of the amino acid residues are not present.
During both the cleavage reaction and the coupling reaction, the reaction
temperature is in the range from the freezing point of the reaction mixture to about 50°C. The
preferred temperature is in the range from about 0°C to about 25°C.
Briefly, the coupling reaction is performed as follows:
When at least about 25 %, preferably at least 50 %, more preferred at least 75 %, preferably
at least 85 %, more preferred at least 95 %, of the insulin precursor has been cleaved to the
desired intermediate product, on one hand, the nucleophile compound and, on the other
hand, organic solvent is mixed with the reaction mixture in which the cleavage took place so
as to obtain reaction conditions which are convenient or favorable to the coupling step. The
percentage of cleavage (conversion) is based upon the equilibrium possible in the reaction
mixture used for cleavage. Usually, from the beginning of the enzymatic cleavage reaction
and until a certain period of time has lapsed, the yield of the desired intermediate product,
10

i.e., the desired cleavage product, increases and reaches a maximum concentration.
Thereafter, the concentration of the desired cleavage product may decrease.
In a preferred embodiment of this invention, no components are removed from the
reaction mixture resulting from the cleavage reaction before the coupling reaction takes
place. A simple way of doing this is, after the cleavage reaction, to add the nucelophile
compound and a sufficient amount of organic solvent. In this way, for example, the enzyme
used in the cleavage step is also used in the coupling step.
The process of this invention also covers coupling reactions in a reaction mixture
which besides the desired intermediate product contains a small amount of partially cleaved
insulin precursor and/or unreacted insulin precursor.
In another preferred embodiment of this invention, the nucleophile compound is an
amino acid amide or a peptide amide wherein the carboxamide group isn't substituted or is
mono or disubstituted with an alkyl group with not more than 16 carbon atoms which alkyl
group(s), together with the adjacent nitrogen atom, may form a ring or the carboxamide
group is mono or disubstituted with an aryl group. The aliphatic substituents are preferred.
Examples of substituted carboxamide groups are N,N-dimethylcarboxamide, N,N-diethyl-
carboxamide, and N-hexylcarboxamide.
In a preferred embodiment of this invention, the nucleotide compound is an amino
acid ester wherein the carboxyl group is protected and any hydroxy group optionally is
protected. In a further preferred embodiment of this invention, the nucleotide compound is a
threonine ester wherein the carboxyl group is protected and, optionally, the hydroxy group is
protected. Hence, an L-threonine ester can be illustrated by the following general formula
Ilia:
Thr(R')-OR' (Ilia)
wherein R® represents a carboxyl protecting group, and R^ represents hydrogen or a
hydroxyl protecting group. To make it more clear, a threonine ester can be illustrated by the
general formula CH3-CH(OR^)-CH(NH2)COOR^ wherein R^ and R^ are as mentioned above.
Some nucleophile compounds are known compounds and the remaining nucleophile
compounds can be prepared in analogy with the preparation of known compounds or in
analogy with known methods.
The nucleophile compounds may be employed in the form of the free base or soluble
salts thereof such as hydrochlorides, acetates, propionates, and butyrates.
When the coupling reaction starts, it is desirable that a substantial excess of
nucleophile compound is present in the coupling reaction mixture solution, with the molar
11

ratio I , , ^ ^ ^
exceeding about 5;1. When the coupling reaction starts, the concentration of the nucleophile
compound in the reaction mixture should preferably exceed 0.1 molar, the upper
concentration limit being the solubility thereof.
To obtain a 60% yield considered herein as an important aspect to practice of this
invention, the reaction temperature, water content and pH value are interrelated within the
described ranges.
The organic solvents suited to practice of this invention are polar solvents which are
miscible with water and preferably such that are capable of containing therein high
concentrations of the desired intermediate product (for example of formula II) and the
nucleophile compound. Examples of suitable organic solvents are aprotic solvents, such as
N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone-2, and dimethyl
sulfoxide, and protic solvents, such as acetic acid, ethanol, methanol, 2-propanol, 1-
propanol, butanol and 1,4-butanediol. Dioxane, acetone, tetrahydrofuran, formamide, and
acetonitrile may also be used and even an amino acid ester used as the nucleophile
compound can, fully or partially, be used as the organic solvent. The nature of the solvent
does affect the system as a whole, and interrelationships suited to one solvent productive of
high yields may not apply with a different solvent. Best yield results have been obtained with
aprotic solvents, and aprotic solvents are most preferred for practice of this invention.
Obviously, when calculating or determining the content of water in the reaction
mixture, the nucleophile compound is considered an organic solvent.
The addition of an acid, such as hydrochloric acid, formic acid, acetic acid, propionic
acid, or butyric acid, or of a base, such as pyridine, TRIS, N-methylmorpholine,
triethylamine, or N-ethylmorpholine, is optional. They are included in the reaction mixture to
bring about a suitable pH value. Although mineral acids or bases may be used in practice of
this invention, organic acids and bases are preferred, particularly those identified above.
Organic acids are most preferred.
When the coupling reaction starts, the weight ratio between trypsin or ALP
(calculated as crystalline trypsin or ALP or an amount of trypsin or ALP derivative
corresponding thereto) and the desired intermediate product in the reaction mixture is
preferably in the range from about 1:1000 to about 1:10, more preferred in the range from
about 1:200 to about 1:50.
In some cases, the enzyme added in the cleavage step is sufficient for performing
the coupling reaction and, in such case, there is no need for adding a further amount of
enzyme during the coupling step. In other cases, it may be desirable to add an additional
amount of enzyme during the coupling step.
12

Inasmuch as high concentrations of the desired intermediate product and of
nucleophile compound in solution promote high conversion rates, solvent selection is biased
towards those solvents in which the reactants are very soluble. The solubility of the
nucleophile compound in particular is important, because that reactant should be present in
high concentration. When the coupling reaction starts, the molar ratio of the nucleophile
compound to the desired intermediate product should preferably exceed 5:1, preferably
exceed 50:1. When the coupling reaction starts, the concentration of the nucleophile
compound in the reaction mixture should preferably be at least 0.1 molar.
In a preferred embodiment of this invention, a nucleophile compound having carboxy
protecting group(s) which can be removed from the resulting insulin compound under
conditions, which do not cause substantial irreversible alterations in the insulin molecule, is
used. As examples of such carboxyi protecting groups can be mentioned lower alkyl, for
example, methyl, ethyl, and tert-butyl, substituted benzyl groups such as p-methoxybenzyl,
diphenylmethyl, and 2,4,6-trimethylbenzyl, and groups of the general formula
-CH2-CH2-S02R^ wherein R^ represents lower alkyl, such as methyl, ethyl, propyl, and n-
butyl.
Suitable hydroxyl protecting groups are those which can be removed under
conditions which do not cause substantial irreversible alteration in the insulin molecule. As
an example of such a group can be mentioned ferf-butyl.
Further protection groups usually used are described by Wunch; Metoden der
Organischen Chemie (Houben-Weyl), Vol. XV/1, editor: Eugen Muller, Georg Thieme Verlag,
Stuttgart 1974.
According to one embodiment of this invention, the process of this Invention will
result in a compound of the general formula IV:
R'^-Cys-Zn-Cys-R'^
I I (IV)
R'-Cys-Zm-Cys-R'^-R'
wherein Zn and Zm, independent of each other, represents two peptide moieties each
containing n and m amino acid residues, respectively, R'^ represents a peptide residue, R'^
represents an amino acid residue or a peptide residue, R'^ represents a peptide residue, R'"
is as mentioned above, and R'® is an amino acid carrying a carboxy protecting group or a
peptide residue, optionally carrying a carboxy protecting group.
Any carboxy protecting group (for example, R®) and any hydroxy protecting group (for
example, R^) present in an insulin compounds can be removed by known methods or
methods known per se. In case the carboxy protecting group is methyl, ethyl, or a group of
13

the general formula -CH2-CH2-S02R^ wherein R^ is as defined above, said protecting group
can be removed at gentle basic conditions in an aqueous medium, preferably at a pH value
in the range from about 8 to about 12, for example, at about 9.5. As the base can be used
strong bases, for example, a tertiary amine, for example triethylamine, hydroxides of alkali
metals such as sodium hydroxide or hydroxides of alkaline earth metals such as calcium, or
magnesium hydroxide, in case the carboxy protecting group is fert-butyl, substituted benzyl
such as p-methoxybenzyl or 2,4,6-trimethylbenzyl, or diphenylmethyl, said group can be
removed by acidolysis, preferably with trifluoroacetic acid. The trifluoroacetic acid may be
nonaqueous or may contain some water, or it may be diluted with an organic solvent, such
as dichloromethane. In case the hydroxy protecting group (for example, R^) is tert-butyl,
said group can be removed by acidolysis, vide above.
Preferably, the insulin compounds prepared have no hydroxy protecting group.
In a preferred embodiment of this invention, the process of this invention converts the
insulin precursor (for example, of formula I) into an insulin compound (for example, formula
IV), having a carboxy protecting group in the C terminal amino acid residue in the B chain
which, then, can be deblocked to form an insulin compound having no carboxy protecting
group.
When selecting the reaction conditions according to the above explanation and
considering the results obtained in the following examples it is possible to obtain a yield of
insulin compound which is higher than 60%, and even higher than 80%, and under certain
preferred conditions higher than 90%.
By the process of this invention, insulin compounds of an acceptable purity can be
obtained and be further purified, if desired, for therapeutic purpose.
More specifically, insulin aspart may, for example, be prepared by enzymatic
cleavage with ALP of an insulin precursor such as [Asp^^]-des(30-65) human proinsulin
being N-terminally extended with Glu-(Glu-Ala)3-Pro-Lys- (SEQ ID NO.: 1) and coupling with
a nucleophile compound such as L-threonine methyl ester, followed by hydrolysis.
insulin lispro may, for example, be prepared by enzymatic cleavage with trypsin of a
precursor such as porcine insulin and coupling with a nucleophile compound such as Gly-
Phe-Phe-Tyr-Thr-Lys-Pro-Thr (SEQ ID NO.: 3).
Insulin glargin may, for example, be prepared by enzymatic cleavage with ALP of an
insulin precursor such as [Gly^®]-des(30-65) human proinsulin and coupling with a
nucleophile compound such as Thr-Arg-Arg-OMe, followed by hydrolysis.
Abbreviations used herein are in accordance with the rules approved (1974) by the lUPAC-
lUB Commission on Biochemical Nomenclature, w'cfe Collected Tentative Rules &
14

Recommendations of the Commission on Biochemical Nomenclature lUPAC-lUB, 2""
edition, Maryland 1975.
The mentioning herein of a reference is no admission that it constitutes prior art.
Herein, the word "comprise" Is to be interpreted broadly meaning "include", "contain" or
"comprehend" (vide, the EPO guidelines C 4.13).
The following examples are offered by way of illustration, not by limitation.
Example 1.
200 mg [Ala^\Lys^^]-des(33-63) porcine proinsulin was suspended in 1.35 ml water and the
pH value was adjusted to 9 with 10 ^1 triethylamine. A mixture of 375 |j| N,N-dimethyl-
acetamide and 460 |jl water was added with slightly agitation and to the resulting solution
was added 315 |j| of a 5.4 mg/ml aqueous solution of Achromobacter lyticus lysyl specific
protease (EC 3.4.21.50) (herein designated ALP). The pH value was adjusted to 9.8 with 20
|j| triethylamine and the reaction solution was left for 1 hour at 23°C. The reaction solution
was acidified by addition of 70 (JI 4 N hydrochloric acid and cooled in an ice bath. A solution
of 300 mg L-threonine methyl ester in 4.85 ml N,N-dimethylacetamide was added and the
pH value was adjusted to 6.5 by addition of 450 |jl 4 N hydrochloric acid. The reaction
solution was left for 4 hours at 23°C after which the reaction was stopped by addition of
hydrochloric acid to a pH value 5 pm C18 silica column with an ethanol-water eluent containing 0.125 M ammonium
sulphate adjusted to a pH value 4, a conversion yield of 86% to human insulin methyl ester
was found after a total reaction time of 5 hours.
For comparison, a one-step conversion was performed:
100 mg [Ala^\Lys^^]-des(33-63) porcine proinsulin was suspended in a mixture of
887 |jl water and 175 |JI N,N-dimethylacetamide. 150 mg L-threonlne methyl ester was
dissolved in 2.265 ml N,N-dimethylacetamide and was slowly added to the ice-cooled
mixture. The pH value was adjusted to 6.5 with 340 |jl acetic acid and 158 \^\ of a 5.4 mg/ml
aqueous solution of ALP was added. The conversion reaction was followed by RP-HPLC
analysis of acidified samples. After 5 hours, a 53% conversion to human insulin methyl ester
was found and after 24 hours the conversion reached a maximum of 87%.
15

The isolated human insulin methyl ester was converted into human insulin by
dissolution in water at a pH value of 10 at a concentration of 10 mg/ml. The reaction was
terminated after 24 hours by adjusting the pH value to 5.2 with 1 N hydrochloric acid and the
precipitated human insulin was isolated by centrifugation and purified by reverse phase high
performance liquid chromatography.
At the same reaction time, i.e., 5 hours, the yield by the process of this invention,
compared with the per se known process, was improved with 62 %. The two processes
obtained almost the same yield, if the reaction time of the per se known one-step conversion
was extended almost 5 times, compared with the reaction time for the process of this
invention.
Example 2.
200 mg porcine insulin was suspended in 1.37 ml water and a mixture of 294 [i\ N-methyl-2-
pyrrolidon and 326 |jl water was added with slightly agitation. The pH value was adjusted to
9.0 with 10 |jl 2 N sodium hydroxide and to the resulting solution was added 315 M' of a 5.4
mg/ml aqueous solution of ALP. The pH value was adjusted to 9.8 with 12 |jl 2 N sodium
hydroxide and the reaction solution was left for 4 hours at 23°C. The reaction solution was
acidified by addition of 70 |jl 4 N hydrochloric acid and cooled in an ice bath. A solution of
300 mg L-threonine methyl ester in 4.4 ml N-methyl-2-pyrrolidon was acidified with 500 |jl 4
N hydrochloric acid. The insulin solution was slowly added and the pH value was adjusted to
6.5 with 50 |jl 2 N hydrochloric acid. The reaction solution was left for 4 hours at 23°C after
which the reaction was stopped by addition of hydrochloric acid to a pH value reversed phase HPLC analysis on a 4mm x 250 mm 5 |jm C18 silica column with an
ethanol-water eluent containing 0.125 M ammonium sulphate adjusted to a pH value 4, a
conversion yield of 86% to human insulin methyl ester was found after a total reaction time
of 8 hours.
For comparison, a one-step conversion was performed:
100 mg porcine insulin was suspended in a mixture of 848 pi water and 147 pi N-
methyl-2-pyrrolidon. 150 mg L-threonine methyl ester was dissolved in 2.2 ml N-methyl-2-
pyrrolidon and was slowly added to the ice-cooled mixture. The pH value was adjusted to 6.5
with 300 pi acetic acid and 158 pi of a 5.4 mg/ml aqueous solution of ALP was added. The
reaction solution was left at 23°C and the conversion reaction was followed by RP-HPLC
analysis of acidified samples. After 8 hours, the conversion was found to 54% and after 48
hours a conversion maximum of 86% to human insulin methyl ester was reached.
16

The isolated human insulin methyl ester can be converted to human insulin by
alkaline hydrolysis.
At the same reaction time, i.e., 8 hours, the yield by the process of this invention was
improved with 59 %, compared with the perse known process. The two processes obtained
the same yield, if the reaction time of the per se known one-step conversion was extended 6
times, compared with the reaction time for the process of this invention.
Example 3.
200 mg [Asp^^]-des(30-65) human proinsulin, N-terminally extended with the peptide Glu-
(Glu-Ala)3-Pro-Lys- (SEQ ID NO.: 1), was suspended in 1.35 ml water. A mixture of 350 ^1
N,N-dimethylformamide and 425 |jl water was added with slightly agitation and the pH value
was adjusted to 9 with 45 |jl triethylamine. To the resulting solution was added 200 pi of a
8.5 mg/ml aqueous solution of ALP and the pH value was adjusted to 9.8 with 20 pi triethyl-
amine. The reaction solution was left for 1 hour at 23°C. The reaction solution was acidified
by addition of 70 pi 4 N hydrochloric acid and cooled in an ice bath. A solution of 300 mg L-
threonine methyl ester in 4.95 ml N,N-dimethylformamide was added and the pH value was
adjusted to 6.5 by addition of 470 pi 4 N hydrochloric acid. The reaction solution was left for
4 hours at 23°C after which the reaction was stopped by addition of hydrochloric acid to a pH
value with an ethanol-water eluent containing 0.125 M ammonium sulphate adjusted to a pH value
4, a conversion yield of 87% to [Asp^^^j-human insulin methyl ester was found after a total
reaction time of 5 hours.
For comparison, a one-step conversion was performed:
90 mg [Asp^^]-des(30-65) human proinsulin, N-terminally extended with the peptide
Glu-(Glu-Ala)3-Pro-Lys- (SEQ ID NO.: 1), was suspended in a mixture of 887 pi water and
175 pi N,N-dimethylformamide. 150 mg L-threonine methyl ester was dissolved in 2.13 ml
N,N-dimethyiformamide and was slowly added to the ice-cooled mixture. The pH value was
adjusted to 6.5 with 250 pi acetic acid and 118 pi of a 8.5 mg/ml aqueous solution of ALP
was added. The conversion reaction was followed by RP-HPLC analysis of acidified
samples. After 5 hours, the conversion was found to 47% and after 24 hours the conversion
to [Asp^^^j-human insulin methyl ester reached a maximum of 81%.
The isolated insulin methyl ester can be converted to [Asp^^^j-human insulin by
alkaline hydrolysis.
17

At the same reaction time, i.e., 5 hours, the yield by the process of this invention was
almost doubled, compared with the perse known process. Comparable yields ware obtained
by the two processes, if the reaction time of the per se known one-step conversion was
extended nearly 5 times, compared with the reaction time for the process of this invention.
Example 4
1.5 g insulin aspart precursor [Asp^^Met^^Trp^^Lys^^]-des(33-65) human proinsulin, N-
terminally extended with the peptide Glu-Glu-Gly-Glu-Pro-Lys- (SEQ ID NO.: 2) was
suspended in 3.5 g water. With slight agitation and at ambient temperature, the precursor
was dissolved by gradually adding 4M sodium hydroxide to a pH value of 10.67. 3.7 g of a
45 % (weight/weight) solution of ethanol in water was added.1.5 ml of a 5.8 mg/ml aqueous
solution of ALP was added and the mixture was left to react for 2 hours. The pH value was
adjusted to 4.7 by addition of 4 N hydrochloric acid. 2.025 g L-threonine ethyl ester was
dissolved in 16.2 ml ethanol and the solution was added at a maximum temperature of 15°C.
The pH value was adjusted to 6.5 with 4 N hydrochloric acid. The temperature was adjusted
to ambient temperature and the reaction mixture was left for 20 hours at this temperature. By
reversed phase HPLC analysis on a 4mm x 250 mm 5 pm CI8 silica column with an
acetonitrile-water eluent containing 200mM sodium sulphate adjusted to a pH value of 3.6, a
conversion yield of 89.1 % insulin aspart ethyl ester was found after 1 hours reaction time
and after 20 hours reaction time, a conversion yield of 90.5% was found.
The isolated insulin aspart ethyl ester can be converted to insulin aspart by alkaline
hydrolysis.
Example 5
10.9 g insulin aspart precursor [Asp^^Met^°,Trp^^Lys^^]-des(33-65) human proinsulin, N-
terminally extended with the peptide Glu-Glu-Gly-Glu-Pro-Lys- (SEQ ID NO.: 2) was
suspended in 49.3 g water. With slight agitation and at ambient temperature, the precursor
was dissolved by gradually adding 37.6 g from a mixture containing 0.36 M sodium
hydroxide, 0.27 M sodium acetate and 36 % N-methyl-2-pyrrolidon. The pH value was
adjusted to 9.7 with 9.2 ml 0.5 M sodium hydroxide. 7.1 mL of a 7.1 mg/mL aqueous solution
of ALP was added, and the mixture was left to react for 5 hours. The pH value was kept
constant at 9.7 by adding more 0.5 M sodium hydroxide throughout the reaction. The
18

reaction mixture was cooled to 5 °C and the pH value was adjusted to 5.7 by addition of 2.73
g 4 N hydrochloric acid. 14.02 g L-threonine ethyl ester was added and the pH value was
adjusted to 6.0 with 4 N hydrochloric acid. 344 g cold (4 °C) N-methyl-2-pyrrolidon was
added. The temperature was adjusted to 22 °C and the pH value adjusted to 6.5 with 4 N
hydrochloric acid. The reaction mixture was left for 9 hours at this temperature. By reversed
phase HPLC analysis on a 4mm x 250 mm 5 pm C18 silica column with an acetonitrile-water
eluent containing 200mM sodium sulphate adjusted to a pH value of 3.6, a conversion yield
of 87.5 % to insulin aspart ethyl ester was found after a total reaction time of 14 hours.
The isolated insulin aspart ethyl ester can be converted to insulin aspart by alkaline
hydrolysis.

WE CLAIM:
1. A process for preparing an insulin compound wherein a) in a reaction mixture
containing at least about 55 %, preferably at least about 60 %, more preferred at least
70 %, water (weight/weight), an insulin precursor is subjected to an enzymatic
cleavage and, thereafter, b) the intermediate product is coupled with a nucleophile
compound in the reaction mixture used for the enzymatic cleavage reaction with the
proviso that the composition of the reaction mixture has been modified so that the
content of water in the reaction mixture is in the range from about 10 % to about 50 %
water (weight/weight), preferably in the range from about 20 % to about 40 % water
(weight/weight), and c), if desired, removing the protecting group(s), and wherein no
isolation of the intermediate product is performed between the cleavage step (a) and
the coupling step (b).
2. The process, according to claim 1, wherein the enzyme used for the cleavage step is
also present in the coupling step.
3. The process, according to any one of the preceding claims, wherein, before the
coupling reaction is initiated, at least about 25 %, preferably at least 50 %, more
preferred at least 75 %, preferably at least 85 %, more preferred at least 95 %, of the
insulin precursor is cleaved to the intermediate product.
4. The process, according to any one of the preceding claims, wherein the enzyme
used for the enzymatic cleavage is trypsin or a lysyl specific protease, preferably
Achromobacter lyticus protease I.
5. The process, according to any one of the preceding claims, wherein the nucleophile
compound is an amino acid ester.
20

6. The process, according to the preceding claim, wherein the nucleophile compound
is a threonine ester.
7. The process, according to any one of the claims 1 to 4, wherein the nucleophile
compound is an amino acid amide,
8. The process, according to any one of the claims 1 to 4, wherein the nucleophile
compound is a peptide.
9. The process, according to any one of the claims 1 to 4, wherein the nucleophile
compound is a peptide ester.
10. The process, according to any one of the claims 1 to 4, wherein the nucleophile
compound is a peptide amide.
11. The process, according to any one of the preceding claims, wherein the protection
group(s) from the insulin compound is removed.
12. The process according to any one of the preceding claims, wherein the resulting
insulin compound has threonine in the B30 position.
13. The process according to any one of the preceding claims, wherein the resulting
compound is human insulin, insulin aspart, insulin lispro, insulin glargin, or insulin
detemir.

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Patent Number

232707

Indian Patent Application Number

1089/CHENP/2004

PG Journal Number

13/2009

Publication Date

27-Mar-2009

Grant Date

20-Mar-2009

Date of Filing

17-May-2004

Name of Patentee

NOVO NORDISK A/S

Applicant Address

NOVO ALLE, DK-2880 BAGSVAERD,

Inventors:

#	Inventor's Name	Inventor's Address
1	BOGSNES, ARE	SOLENGEN 14, DK-2990 NIVA,
2	CHRISTIANSEN, INGUN	LAERKEBAKKEN 15, DK-3460 BRIKEROD,
3	BALSCHMIDT, PER	TIBBERUP ALLE 20, DK-3060 ESPERGAERDE,

PCT International Classification Number

C12P 21/00

PCT International Application Number

PCT/DK02/00765

PCT International Filing date

2002-11-15

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	2001 01716	2001-11-19	Denmark