MX2008000344A - Preparation of insulin conjugates - Google Patents

Abstract

The present invention discloses a process for making an insulin-oligomer conjugate as a one-pot reaction by conjugation of insulin-ester with an activated oligomer wherein simultaneous deblocking and conjugation is carried out.

Description

PREPARATION OF INSULIN CONJUGATES FIELD OF THE INVENTION The present invention relates to a process for producing an oligomeric insulin conjugate as a reaction in a container by conjugation of insulin ester with an activated oligomer wherein deblocking and conjugation are carried out simultaneously.

BACKGROUND OF THE INVENTION Pancreatic islet β-cells secrete a precursor of a single chain of insulin, known as proinsulin which upon proteolysis results in the biologically active insulin polypeptide. The insulin molecule is highly conserved throughout the species and generally consists of two chains of amino acids linked by disulfide bonds. The natural human insulin molecule (mw of 5.807 Daltons), has an A chain of 21 residual amino acids with glycine in an amino terminal; and a B chain of 30 residual amino acids with phenylalanine at the amino terminus. Insulin can exist as a monomer or can be added in a dimer or a hexamer formed of three of the dimers. The monomer has the ability to bind to receptors and is the biologically active form. The insulin polypeptide is the main hormone responsible for controlling the transport, utilization and storage of glucose in the body. A defect in the metabolism of carbohydrates as a result of insufficient insulin production or reduced sensitivity of the receptor to insulin leads to the biological disorder known as diabetes. Diabetes damages the normal ability to use glucose as a result of increased blood sugar levels (hyperglycemia). As glucose builds up in the blood, high levels of sugar in the urine (glycosuria) are excreted. Other symptoms of diabetes include increased and frequent urine volume, thirst, itching, hunger, weight loss, and weakness. Diabetes when left untreated leads to ketosis, followed by acidosis with nausea and vomiting. As the toxic products continue to accumulate, the patient goes into a diabetic coma, which leads to the death of the patient. There are two types of diabetes, type I is insulin dependent diabetes mellitus, or IDDM. IDDM was previously referred to as "juvenile onset diabetes". In IDDM, insulin is not secreted by the pancreas and can be provided from an external source. Type II or adult onset diabetes can commonly be controlled by diet, although in some advanced cases insulin is required. Banting et al describes the use of insulin for the treatment of diabetes using the active extract of the pancreas in diabetic dogs "Pancreatic Extracts in the Treatment of Diabetes Mellitus" (Can. Med. Assoc. J., 12: 141: 146 (1922 In that same year, the treatment of a diabetic patient with pancreatic extracts resulted in a dramatic clinical improvement that saved his life.Bovine and porcine insulin was traditionally used almost exclusively (to treat diabetes in humans. The development of recombinant technology is commercial, the manufacture of human insulin by fermentation became possible, and genetically engineered insulin analogues have been developed that have a biological activity comparable to that of natural human insulin to fight the disease. The typical treatment of diabetes requires regular injections of insulin, due to the inconvenience of insulin injections, Several methods have been tried to formulate insulin - to be administered by non-injectable routes. A list of such descriptions includes: US 4,338,306 (Kitao et al.) Which describes a pharmaceutical composition of insulin and fatty acids having from 8 to 14 carbon atoms and non-toxic salts thereof for rectal administration of insulin; US 4,579,730 (Kidron et al) discloses enteric-coated insulin compositions with a bile acid or alkali metal salt thereof for oral administration of insulin; US 5,283,236 (Chiou et al) describes an insulin composition with a permeation enhancing agent to aid the systemic absorption of higher molecular weight polypeptides, as well as peptidase inhibitors for the systemic delivery of insulin through the eyes, where the drug passes into the nasolacrimal duct and is absorbed into the circulation; US 5,658,878 (Backstrom et al) describes an insulin and sodium salt of a saturated fatty acid with a carbon chain length of 10 (ie, sodium caprate), 12 (sodium laurate), or 14 (sodium myristate) ) which improves the absorption of insulin in the lower respiratory tract; US 5,853,748 (New et al.) discloses an enteric coated insulin composition, a bile salt or bile acid, and carbonate or bicarbonate ions, used to adjust the pH of the intestine to a pH of 7.5 to 9 for oral administration of insulin; US 6,200,602 (Watts et al) describes an insulin drug release composition for the colonic release of insulin with an absorption promoter, which includes the mixture of fatty acids having from 6 to 16 carbon atoms and their salts or a mixture of mono- / diglycerides of medium-chain fatty acids together with a dispersing agent, in a coating to prevent the release of insulin and the absorption promoter until the tablet, capsule or granule reaches the proximal colon.

Attempts have been made to release insulin by oral administration. The problems associated with the oral administration of insulin to achieve euglycemia in diabetic patients are well documented in the pharmaceutical and medical literature. The digestive enzymes of the Gl tract rapidly degrade insulin, resulting in biologically inactive degradation products. In the stomach, for example, insulin administered orally undergoes enzymatic proteolysis and acid degradation. Survival in the intestine is impeded by excessive proteolysis. In light, insulin is attacked by a variety of enzymes including gastric and pancreatic enzymes, exo and endopeptidases, and ciliary boundary peptidases. Even if insulin survives this enzyme attack, the biological barriers that must be traversed before insulin can reach its receptors in vivo to limit the oral administration of insulin. For example, insulin may have low membrane permeability, limiting its ability to pass from light into the bloodstream. Pharmaceutically active polypeptides such as insulin have been conjugated with polydisperse polyethylene glycol mixtures or polydisperse mixtures of polyethylene glycol-containing polymers to provide polydisperse mixtures of drug-oligomer conjugates; US 4,179,337 (Davis et al) describes the conjugation of polypeptides such as insulin with various polyethylene glycols such as MPEG-1900 and MPEG-5000 distributed by Union Carbide. US 5, 567, 422. ((Greenwald) describes the conjugation of biologically active nucleophiles with polyethylene glycols such as m-PEG-OH (Union Carbide), which has a number average molecular weight of 5,000 Daltons. such as insulin with glycolipid polymers modified with polyethylene glycol and fatty acid polymers modified with polyethylene glycol is described in US 5,359,030 (Ekwuribe et al.) US 6,011,008 (Domb et al) describes a method for producing a water soluble polysaccharide conjugate of an oxidation-sensitive substance comprising activating the polysaccharide to a dialdehyde by periodate oxidation; (b) purifying dialdehyde from interfering anions and by-products; and (c) coupling the substance to the purified dialdehyde by forming a Schiff base to form the conjugate. Optionally, the conjugate of step (c) is reduced to an amine conjugate by a reducing substance. Insulin was conjugated to oxidized AG (arabinogalactan) via an amine or imine linkage by reacting a pure oxidized AG (arabino galactane) solution in borate buffer at pH 8.9 with insulin at 4 ° C overnight. The clear solution was dialyzed through dialysis with cellulose and the solution was lyophilized to yield 115 mg of a white solid. US 6,022,524 ', (Maisano et al.) Was prepared Gd-DTPA conjugated with porcine insulin in a solution of DTPA and dimethylsulfoxide (DMSO) by heating and stirring, then cooled to room temperature and a solution of 11.73 g of NHS was added. (0.102 mol) in 300 ml of DMSO, then drop by drop, with a solution of 19.6 g of N, N'-dicyclohexylcarbodiimide (0.097 mol) in 400 ml of DMSO. The mixture was stirred for 16 hours, then filtered and the filtrate was concentrated by evaporation at 50 ° C and 5 Pa to a thick oil of a volume of about 160 ml. US 6,309,633 (Ekwuribe et al.) Describes the use of solid insulin for the conjugation of insulin with PEG5 laurate in the presence of triethylamine and DMSO at room temperature. The reaction was verified via CLAP every 30 minutes. The conjugate was purified using a preparative CLAP. US 6,828,297 (Ekwuribe et al) describes the methods for producing PEG7-Hexyl-Insulin using zinc or zinc free human insulin to conjugate with an activated oligomer and the purification of PEG7-Hexyl-Insulin modified with B29. The insulin in dimethylsulfoxide and triethylamine was reacted with activated oligomer at 22 +/- 4 ° C. The crude reaction mixture was dialyzed or diafiltered to remove organic solvents and small molecular weight impurities, exchanged against ammonium acetate buffer and lyophilized; which was additionally subjected to RP-CLAP balanced with 0.5% triethylamine / 0.5% phosphoric acid buffer (TEAP A). The column was eluded with a gradient flow using the solvent system TEAP A and TEAP B (80% acetonitrile and 20% TEAP A). The fractions containing the conjugate were pooled and elution buffer and solvent were removed by dialysis or diafiltration against ammonium acetate buffer and lyophilized to produce a white powder of PEG7-hexyl insulin, monoconjugate with B29 (purity> 97 %). Currently, there are prior techniques that use pure insulin powder or crystals as the starting material to produce conjugated insulin, where the insulin used is a biologically active form. The present invention facilitates the conjugation of the insulin in its inactive ester form with an oligomer wherein the insulin ester is unblocked and conjugated to the oligomer simultaneously as a reaction in a single vessel. The present invention is more simplified and economical to produce an insulin conjugate, where several purification steps are avoided to obtain pure insulin in a biologically active form. The starting material is the fermented broth that contains an insulin precursor. The broth containing the insulin precursor is subjected to a combination step of cation exchange purification, crystallization with phenol and ZnCÍ2, lyophilization, and transpeptidation to obtain insulin ester. The insulin ester is subjected to conjugation with an oligomer having the general formula -0C- (CH2) n- (OCH2CH2) n-OCH3 and more preferably an activated oligomer of molecular formula C? 4H23N08 (CAS No. 622405-78-1), to obtain conjugated insulin. The most preferred insulin-oligomer conjugate is insulin -OC-CH2CH2- (OCH2CH2) 3-OCH3 hereinafter also referred to as IN 105. The total cost of conjugated insulin production as a result of this process is minimized.

SUMMARY OF THE INVENTION The present invention relates to a process for producing an insulin-oligomer conjugate in a single container reaction by conjugation of insulin ester with an activated oligomer, where simultaneous deblocking and conjugation are effected in buffer of borate. The activated oligomer solubilized in acetonitrile is added to a solution containing insulin ester and the pH of the mixture is raised to approximately 11.

DETAILED DESCRIPTION The present invention describes a single-vessel reaction process for the preparation of insulin-oligomer conjugates comprising unblocking and simultaneous conjugation of an insulin ester. The insulin-oligomer conjugate is further purified and lyophilized to a dry powder. The process for producing an insulin-oligomer conjugate in a single container comprises: (i) transpeptidation of the insulin precursor, (ii) unblocking of the insulin ester and conjugation with an oligomer simultaneously in a container, (iii) providing the conjugate of insulin-oligomer. The process further comprises: (i) purification of the insulin precursor by chromatography and precipitation, (ii) transpeptidation to give an insulin ester; (iii) purification of the insulin ester using the RP-CLAP, (iv) treatment of the insulin ester with an oligomer in borate buffer, to effect deblocking and configuration simultaneously, (v) optional purification of the conjugate, (vi) provide the oligomeric insulin conjugate. The process for producing the insulin-oligomer conjugate comprises the simultaneous addition of an oligomer solubilized in acetonitrile or a solution containing insulin ester in borate buffer and increasing the pH thereof. The process where the pH is increased to approximately 11. The process where the insulin precursor is proinsulin or miniproinsulin. The process where the oligomer is an alkyl-PEG or derivative thereof. The process where the oligomer is activated before conjugation. The process where the active oligomer used for conjugation is C? 4H23N08. The process where the alkyl-PEG has the general formula -OC- (CH2) n- (OCH2CH2) n-OCH3. The process where the insulin-oligomer conjugate is conjugated B29 insulin Ne-oligomer. The process where the insulin-alkyl PEG conjugate is conjugated B29 insulin Ne-alkyl PEG. The process where the conjugate is insulin -OC-CH2-CH2- (OCH2CH2) 3-OCH3.

Fermentation of recombinant yeast containing the insulin gene The recombinant yeast inoculum containing the insulin gene is prepared by adding 100 microliters of glycerpl standard culture in 50 ml of minimum glycerol medium (MGY) in shake flasks in 250 ml. The MGY medium contains yeast nitrogen base (YNB), glycerol, phosphate buffer and D-biotin. The seed flasks are incubated at 30 ° C, until they are reached (optical density at 600 nm) of 240 +/- 10 to 15 +/- 5. The fermentation media contains orthophosphoric acid, calcium sulfate dihydrate, sulphate potassium, magnesium sulfate heptahydrate, potassium hydroxide, glycerol, trace salts and D-biotin. The fermentor is prepared by adding all the above components except the trace salts and D-biotin and autoclaving at 121-124 ° C for one hour. Trace salts solution is prepared by filter sterilizing a solution of cupric sulfate pentahydrate, sodium iodide, manganese sulfate monohydrate, sodium molybdate dihydrate, boric acid, cobalt chloride hexahydrate, zinc chloride, ferrous sulfate heptahydrate. The biotin solution is also sterilized by filtration. The fermenter is inoculated and operated at a temperature of 30 ° C, pH 5.5, air flow of 0.5 lpm and OD 30. After the batch phase, the glycerol feed begins (50% w / w with water) to accumulate biomass. The glycerol 50% w / w is prepared and placed in an autoclave for 30 min, at 121-124 ° C and then the solutions in traces and biotin are added at a rate of 12 ml / 1. The glycerol feed rate is gradually increased to 20 +/- 5 g / hr. Once a biomass of 300 - 400 g / 1 is reached, the temperature is reduced to 20-25 ° C, the pH is changed to 3.5 - 6.5 and the methanol feed begins. The methanol is sterilized by filtration and the solutions of trace salts and biotin are added at a rate of 12 g / 1. The methanol feed is increased on the basis of consumption up to 25 +/- 5 g / h. During the feeding of methanol the feeding of yeast extract and peptone is added at a speed of 0.2 - 0.5 g / h. It continues fermenting up to 12 days.

Purification of broth proinsulin 900 mg of broth containing insulin precursor were adjusted to pH 4.0 with acetic acid and passed through cation exchange resins, pre-equilibrated with a 50 mM acetic acid. The column was washed with 50 mM acetic acid and eluted with 50 mM acetic acid with NaCl IM. 855 mg of product was obtained which was diluted 1: 3 with water and the concentration was brought to 6 mg / ml. Phenol (1.25 mg /, lit) and ZnC12 at 5% (v / v) a 5% (w / v) standard was added to the solution. The pH of the solution was adjusted to 5.2 with 1N NaOH. The solution was maintained overnight at 4 ° C. The solid suspension was centrifuged and the granules formed were lyophilized to dryness. The recovery in the passage was 90%.

Transpeptidation and esterification of proinsulin 400 mg of dry precursor powder was solubilized in 30 ml of DMF containing 30-70% of N-N Di-ethylformamide. 724 mg of threonine butyl ester was added to the solution. The pH of the solution was adjusted to 6.5 with 3 N acetic acid. The reaction began with the addition of 55 mg trypsin. The reaction was checked every hour and was stopped by 5 ml of 3 N acetic acid after 4 hours when the conversion of the Insulin precursor to the Insulin ester was 74%. The performance of this step was 68% in terms of the product conversion. The product obtained was precipitated as above, at pH 6.0 228 mg of Insulin ester were recovered. The insulin ester crystal granules were solubilized in acetic acid. at 250 mM. The filtrate was passed through a matrix of Kromasil C8 and 95% pure Insulin ester of acetonitrile gradient was recovered. At the end of the RPCLAP, 149 mg of product was recovered. The insulin ester thus obtained was used for the preparation of the insulin conjugate as described in the following examples; It is not considered limiting.

EXAMPLES Example 1 5 ml of RP elution was taken as starting material and 1.2 ml of 1 M Borate buffer was added to the reaction mixture; the pH of the reaction mixture was raised to 11 and the reaction mixture was stirred for 3 hr at 24 ° C. It was verified that the unblocking and when it was completed, 0.5 mg of activated oligomer (C? 4H23N08) solubilized in 300 μl of Acetonitrile in the reaction mixture was added in the same vessel. The reaction was stopped by diluting the pH of the reaction - to 7.5. The yield is 44%, with a chromatographic purity of 28%. The majority of the product remains uncovered.

Example 2 5 ml of RP elution was taken as the starting material and 1. 2 ml of 1 M Borate buffer was added to the reaction mixture; the pH of the reaction mixture was raised to 11 and the reaction mixture was stirred for 3 hr at 24 ° C. It was verified that the unblocking and when it was completed, 2.5 mg of activated oligomer (C? 4H23N08) in 300 μl of Acetonitrile was solubilized is added to the reaction mixture in the same vessel. The reaction was stopped by lowering the pH of the reaction to 7.5. The yield was 63%, with a chromatographic purity of the product of 56%.

Example 3 5 ml of RP elution was taken as the starting material and 1.2 ml of 1 M Borate buffer was added to the reaction mixture; the pH of the reaction mixture was raised to 11 and the reaction mixture was stirred for 3 hr at 24 ° C. It was verified that the unblocking and when it was completed, 10 mg of activated oligomer (C? 4H23NOa) in 300 μl of Acetonitrile were solubilized and added to the reaction mixture in the same vessel. All samples were analyzed in 10 min in 1 hour. The yield was 18%, with a chromatographic purity of the product of 11%. A majority of the biconjugated product was observed.1, Example 4 5 ml of RP elution was taken as the starting material and 1.2 ml of 1 M Borate buffer was added to the reaction mixture; the pH was adjusted to 10.5 and maintained for 5 hrs. 2.5 mg of activated oligomer (C? 4H23N? 8) in 300 μl of acetonitrile and once the unblocking in the same vessel of the reaction mixture is complete. Aliquots were taken and analyzed. The yield was 58%, with a chromatographic purity of 51%.

Example 5 5 ml of RP elution was taken as the starting material and 1.2 ml of 1 M Borate buffer was added to the reaction mixture; the pH was adjusted to 10.75 and maintained for 4 hrs. 2.5 mg of activated oligomer (C? 4H23N08) is dissolved in 300 μl of acetonitrile and once the unblocking in the same vessel of the reaction mixture is complete. Aliquots were taken and analyzed. The yield was 61%, with a chromatographic purity of 53%.

Example 6 (Simultaneous unblocking and conjugation) 5 ml of RP elution was taken as the starting material and l was added. < 2 ml of 1 M Borate buffer to the reaction mixture; the pH was adjusted to 11 and 4 mg of activated oligomer (C? 4H23N? 8) was dissolved in 300 .mu.l of acetonitrile and added. The mixture was analyzed at 10 min, 1 hr, 2 hrs, 3 hr after unblocking and simultaneous conjugation took place in the same container. The yield was 64%, with a chromatographic purity of 58%.

Example 7 (Simultaneous unblocking and conjugation) 5 ml of RP elution was taken as the starting material and 1.2 ml of 1 M Borate buffer was added to the reaction mixture; the pH was adjusted to 11 and 2.5 mg of activated oligomer (C? 4H23N08) was dissolved in 300 μl of acetonitrile and added. The mixture was analyzed at 10 min, 1 hr, 2 hrs, 3 hr after simultaneous unblocking and conjugation took place in the same vessel of the reaction mixture. The yield after 3 hr was 75%, with a product purity of 73.4%. Unlocking continued for 1 hr, 2 hr and 3 hr and the Conjugation started at each time point and was allowed to continue until the unblocking and conjugation for each case was observed. 5 ml of each elution was taken in each of the 4 tubes. 1.2 ml of Borate ImM buffer was added to the reaction mixture. The pH was adjusted to 11. In the first tube, 2.5 mg of activated oligomer (C? 4H23NOs) was added at 0 h, the release was allowed to continue for 1 hr in the 2nd. Tube and the same amount of activated oligomer (C? 4H23N08) was added into the reaction mixture. The unlocking was allowed to continue for 2 hr in the 3rd. tube and 2.5 mg of the activated oligomer (C? 4H23N? 8) was added after 2 hr. In the 4th. Tube was allowed to continue unblocking for 3 hours before an amount of activated oligomer was added (C? 4H23NOs). The conjugation was allowed to continue for each tube until it seemed to be completed as confirmed by the analytical chromatogram. The yield of the passage as well as the percentage of purity of insulin conjugate was verified by analytical chromatography.

Experiment # Performance (%) Purity of IN 105 (%) Tube 1 74.7 73.0 Tube 2 71.0 70.1 Tube 3 67.6 65.7 Tube 4 64.8 59.0 Example 8 150 ml of RPCLAP elution was taken in 36 ml of borate buffer at pH 8.7. The pH was raised to 11 by adding 10 ml of 10 N NaOH and the reaction mixture was maintained at 25 ° C for 3 hours. 135 mg of activated oligomer (C? 4H23N? 8) solubilized in 9 ml of acetonitrile was added to the reaction mixture to begin the conjugation reaction in the same vessel. After 1 hour, the conjugation reaction was stopped by lowering the pH of the reaction mixture to 7.5 by adding glacial acetic acid. It was found that the unblocking and conjugation were 61% in this reaction and the purity of the product was 62%.

Example 9 975 ml of RP elution having a concentration of 8.4 mg / ml and 234 ml of 1 M borate buffer added at pH 8.2 were taken. The pH was adjusted to 11 with 10 N NaOH. 975 mg of activated oligomer (C? 4H23NOs) dissolved in 58.5 ml of acetonitrile was added to the reaction mixture and the deblocking as well as the conjugation process were started together in the same vessel . An aliquot was taken at 2 and 3 hr, analyzed in CLAP to verify the reaction profile. The conjugation was stopped after 3 hr bringing a pH of the reaction mixture to pH 7.5 by adding glacial acetic acid. It was found that . the yield was 68% with a product purity of 69%.

Example 10 (Product Recovery) The final conjugate was diluted with 250 mM acetic acid to produce a concentration of 2.5 mg / ml.

The material was isolated on a Kromasil C8 RP CLAP column and eluted with acetonitrile gradient. The product eluted has the IN 105 with a purity of 96.7% and with the recovery in the step of 72%. The purified IN 105 was eluted from the RPCLAP column, crystallized with phenol and ZnC12 at pH 5.2 in cold condition. The final crystallized granule was collected with centrifugation. The collected crystallized granule was lyophilized and harvested in purified crystals of dried IN 105.

Claims (12)

1. CLAIMS 1. A process for producing an insulin-oligomer conjugate, in a container, characterized in that it comprises: (i) transpeptidation of the insulin precursor, (ii) unblocking of the insulin ester and conjugation with an oligomer simultaneously in a container, ( iii) provide the insulin-oligomer conjugate.
2. 2. The process in accordance with the claim 1, characterized in that it further comprises: (i) purification of the insulin precursor by chromatography and precipitation, (ii) transpeptidation to give an insulin ester; (iii) purification of the insulin ester using the RP-CLAP, (iv) treatment of the insulin ester with uri oligomer in borate buffer, to effect deblocking and conjugation simultaneously, (v) purification of the conjugate, (vi) provide the insulin-oligomer conjugate.
3. The process according to claim 1 for producing an insulin-oligomer conjugate comprising the simultaneous addition of oligomer solubilized in acetonitrile to a solution containing insulin ester in borate buffer and increasing the pH of the mixture.
4. 4. The process according to claim 3, characterized in that the pH is increased to approximately 11.
5. The process according to claim 1, characterized in that the insulin precursor is proinsulin or miniproinsulin.
6. 6. The process in accordance with the claim 1, characterized in that •, the oligomer is an alkyl-PEG or derivative thereof.
7. 7. The process according to claim 1, characterized in that the oligomer is activated before conjugation.
8. 8. The process according to claim 7, characterized in that the activated oligomer used for the conjugation is C? 4H23N08.
9. 9. The process according to claim 6, characterized in that the alkyl-PEG has the general formula -OC- (CH2) n- (OCH2CH2) n- () CH3.
10. The process according to claim 1, characterized in that the insulin-oligomer conjugate is the insulin conjugate, B29 Ne-oligomer.
11. 11. The process according to claim 1, characterized in that the insulin-alkyl PEG conjugate is conjugated B29 insulin Ne-alkyl PEG.
12. 12. The process according to claim 1, characterized in that the conjugate is insulin B29 Ne -0C-CH2-CH2- (OCH2CH2) 3-OCH3.