|Title of Invention||
PREPARATION OF INSULIN CONJUGATES
|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.|
TITLE OF THE INVENTION
PREPARATION OF INSUUN CONJUG/totes FIELD OF THE INVENTION
The present invention relates to a process for making an insulln-oligonner conjugate as a one-pot reaction by conjugation of insulin-ester with an activated oligomer tiiereon simultaneous deblocking and conjugation is carried out. BACKGROUND OF THE INVENTION
The p-cells of the pancreatic islets secrete a single chain precursor of insulin, known as pro-insulin which upon proteolysis results in the biologically active polypeptide insulin. The insulin molecule is a highly conserved across species and generally consists of two chains of amino acids linked by disulfide bonds. The natural human insulin molecule (nnw 5,807 Daltons), has A-chain of 21 amino acid residues with glycine at the amino terminus; and a EJ-chain of 30 amino acid residues with phenylalanine at the amino terminus. Insulin may exist as a monomer or may aggregate into a dimer or a hexamer formed from three of the dimmers. The monomer has the ability to bind to receptors and is the biologically active form.
Insulin polypeptide is the primary hormone responsible for controlling the transport, utilization and storage of glucose in the body. A defect in the carbohydrate metabolism as a result of insufficient production of insulin or reduced sensitivity of the receptor to insulin leads to the biological disorder diabetes. Diabetes impairs the normal ability to use glucose as a result increases blood sugar levels (hyperglycemia). As glucose accumulates in the blood, excess levels of sugar are excreted in the urine (glycosuria). Other symptoms of diabetes include increased urinary volume and frequency, 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 build up, the patient goes into a diabetic coma, which leads to the patient's death. There are two types of diabetes. Type I is insulin-dependent diabetes mellitus, or IDDM. IDDM was formerly referred to as '^juvenile onset diabetes." In IDDM, insulin is not secreted by the pancreas and must be provided from an external source. Type II or adult-onset diabetes can ordinarily be controlled by diet, although in some advanced cases insulin is required.
Banting the use of insulin for treatment for diabetes using active extract from the pancreas in diabetic dogs "Pancreatic Extracts in the Treatment of Diabetes Mellitus" (Can. Med. Assoc. 3., 12:1-41-146 (1922);. In that same year, treatment of a diabetic patient with pancreatic extracts resulted in a dramatic, life-saving clinical improvement.
Traditionally bovine and porcine insulin were used almost exclusively to treat diabetes in humans. With the development of recombinant technology commercial scale manufacture of human i insulin was made possible by fermentation. Furthermore, genetically engineered insulin analogs having biological activity comparable to that of natural human insulin were developed to combat the disease.
However, treatment of diabetes typically requires regular injections of insulin. Due to the inconvenience of insulin injections^ various approaches have been attempted to formulate insulin for administration by non-injectable routes. A list of such disclosures include: US 4,338,306 (Kitao etaL) discloses a pharmaceutical compositions of insulin and fatty acids having 8 to 14 carbon atoms and nontoxic salts thereof for rectal administration of insulin; US 4,579,730 (Kidron etal) discloses an enter coated insulin compositions with a bile acid or alkali metal salt thereof for the oral administration of insulin n; US
5,283,236 (Chiou etaL) discloses an insulin composition with a permeation-enhancing agent to aid systemic absorption of higher molecular weight polypeptides, as well as peptidase inhibitors for systemic delivery of insulin through the eyes wherein the drug passes Into the nasolacrimal duct and becomes absorbed into circulation; US 5,658,878 (Backstrom et 31.) discloses an insulin and sodium salt of a saturated fatty acid of carbon chain length 10 (i -e., sodium caprate>, 12 (sodium laurate), or 14 (sodium myristate) which enhances the absorption of insulin in the lower respiratory tract; US 5,853,748 (M ew et aJ.) discloses an enteric-coated composition of insulin, a bile salt or bile acid, and carbonate or bicarbonate ions, used to adjust the pH of the gut to a pH of from 7.5 to 9 for the oral administration of insulin. US 6,200,602 (Watts etaf) discloses a drug delivery composition of insulin for colony delivery of insulin with an absorption promoter which includes a mixture of fatty acids having 6 to 16 carbon atoms and its salts or a mixture of mono/diglycerides of medium chain fatty acids along with a dispersing agent, in a coating to prevent the release of the insulin and absorption promoter until the tablet, capsule or pellet reaches the proximal colon-Attempts have been made to deliver insulin by oral administration. The problems associated with oral administration of insulin to achieve euglycemia In diabetic patients are well documented in pharmaceutical and medical literature. Digestive enzymes in the GI tract rapidly degrades Insulin, resulting in biologically inactive breakdown products. In the stomach, for example, orally administered insulin undergoes enzynnatic proteolysis and acidic degradation. Survival in the intestine is hindered by excessive proteolysis. In the lumen, insulin is barraged by a variety of enzymes including gastric and pancreatic enzymes, ego- and end peptidases, and brush border peptidases. Even if insulin survives
this enzymatic attack, the biological barriers that must be traversed before insulin can reach its receptors in vivo may limit oral administration of insulin. For example, Insulin may possess low membrane permeability, limiting its ability to pass from the lumen into the bloodstream.
Pharmaceutlcally active polypeptides such as insulin have been conjugated with polydispersed mixtures of polyethylene glycol or polydispersed mixtures of polyethylene glycol containing polymers to provide polydispersed mixtures of drug-oligomer conjugates; US 4,179,337 (Davis etal) discloses conjugating polypeptides such as insulin with various polyetiiylene glycols such as MPEG-1900 and MPEG-50O0 supplied by Union Carbide. US 5,567,422 (Greenwald) discloses 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.
Conjugation of polypeptides such as insulin with polyethylene glycol modified glycolipld polymers and polyethylene glycol modified fatty acid polymers are disclosed in US 5,359,030 (Ekwuribe etal
US 6,011,008 (Domb etal.) discloses 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 the dialdehyde from interfering anions and by-products; and (c) coupling the substance to the purified dialdehyde by Shift base formation to form the conjugate. Optionally, the conjugate of step (c) is reduced to an aim new conjugate by a reducing substance. Insulin was conjugated to oxidized AG (arabinogalactan) via an amine or imine bond by reacting a solution of pure oxidized AG (arabinogalactan ) in borate buffer solution at phi 8.9 with insulin at 4^C overnight. The dear solution was dialyzed through a
cellulose dialysis and the solution was lyophiilized to yield 115 mg of a white solid.
US 6,022,524 Casino et al.) Gd-DTPA was conjugated with porcine insulin in a solution of DTPA and dimethylsulfoxide (DM5O) is prepared by heating and stirring, then it is cooled at room temperature and added with a solution of 11.73 g NHS (O.102 mol) in 300 ml DMSO, then, drop by drop, with a solution of 19.6 g of N,N'-dicyclohexylcarbodiimide (0.097 mol) in 40O ml DMSO. The mixture is stirred for 16 hours, then filtered and the filtrate is concentrated by evaporation at SO.degree. C. and 5 Pa to a thick oil of an about 160 ml volume.
US 6,309,633 (Ekwuribe et al.) disclose use of solid insulin for conjugation of insulin with laurate PEG5 in presence of Triethylamine and DMSO at room temperature. The reaction was monitored via HPLC every 30 mins. The conjugate was purified using a preparative HPLC.
US 6,828,297 CEkwuribe etal.) discloses methods for making REG7-Hexyl-Insulin by using zinc or zinc free human insulin for conjugation with activated oligomer and purification of B29 modified PEG7-Hexyl-Insulin. insulin in dimethylsuifoixide and triethyl amine was reacted with activated oligomer at 22 +/- 4*^0. The crude reaction mixture is dialyzed or difiltered to remove organic solvents and small molecular weight impurities, exchanged against ammonium acetate buffer and lyophilized; which is further subjected to RR-HPLC equilibrated with 0.5 triethylamine / 0.5°/fe phosphoric acid buffer (TEAR A). The column was eluted with a gradient flow using TEAR A and TEAR B (80% acetonitrile and 20% TEAR to) solvent system. Fractions containing the conjugate were pooled and the elution buffer and solvent were removed by dialysis or infiltrations against ammonium acetate
buffer and lyophilized to produce white powder of PEG7-hexyl-insulin, B29 monoconjugate (purity>97%).
Currently, existing prior art teaches use of pure insulin powder or crystals as the starting material for making conjugated insulin wherein the insulin used is a biologically active form.
The instant invention facilitates the conjugation of insulin in its inactive ester form with an oligomer wherein the insulin ester is deblocked and conjugated to the oligomer simultaneously as a one pot reaction.
The instant invention is a more simplified and economical in the making of an insulin conjugate wherein several steps of purification to obtain pure insulin in biologically active form are circumvented- The starting material is the fermented broth containing insulin precursor. The broth containing the insulin precursor is subjected to a combines ation step of Cation exchange purification, crystallization with phenol and ZnCb, lyophilization, and transpeptidation to obtain insulin ester. The insulin-ester is subjected to conjugation with an oligomer having the general formula -OC-(CH2 -(OCH2CH2)n-OCH3 and more preferably an activated oligomer of molecular formula C14H23NO8 (CAS.no.622405-78-1), to obtain conjugated insulin. The most preferred insulin-oligomer conjugate is insulin-0C-CH2 CH2-(0CH2CH2)3-0CH3 herein after also referred to as IN 105. The overall cost of production of conjugated insulin as a result of this process is minimized. SUMMARY OF THE INVENTION
The instant inventioiq relates to a process for making an insulin-oligomer conjugate in a one-pot reaction by conjugation of insulin-ester with an activated oligomer wherein simultaneous deblocking and conjugation is done in borate buffer. The activated oligomer solubilizer
in acetonitrile is added to a solution containing insulln-ester and the pH ^ of the mixture is raised to about 11.
The instant invention discloses a one-pot reaction process for the preparation of Insulin-ollgomer conjugates comprising simultaneous deblocking and conjugation of an insulin-ester.
The insulin-ollgomer conjugate is further purified and lyophilized to a dry powder.
The process for making a insulin-ollgomer conjugate in one pot comprising:
(i) transpeptidation of insulin precursor,
(ii) deblocking of insulin ester and conjugation with an oligomer simultaneously In one pot,
(iii) affording insulin-ollgomer conjugate. The process further comprising:
(i) purification of insulin precursor by chromatography and precipitation,
(ii) transpeptidation to afford an insulin ester,
(iii) purification of the insulin ester using RP-HPLC,
(iv) treatment of the insulin ester with an oligomer in borate buffer, to effect deblocking and conjugation simultaneously,
(v) optional purification of the conjugate,
(vi) affording insulin-oligomer conjugate.
The process of making an insulin-oligomer conjugate comprising of simultaneous addition an oligomer solubilized in acetonitrile to a
solution containing insulin-ester in borate buffer and increasing the pH of the mixture.
The process wherein the pH is increased to about 11,
The process wherein the insulin precursor is proinsulin or mini-prolnsulin.
The process wherein the oligomer is an alkyl-PEG or derivative thereof.
The process wherein the oligomer is activated before conjugation.
The process wherein the activated oligomer used for conjugation isCi4H23N08.
The process wherein the alkyl-PEG has the general formula - OC-
The process wherein the insulin-oligonner conjugate is insuli n B29 INe-oligomer conjugate.
The process wherein the insulin-alkyl PEG conjugate is insulin B29 IMe -alky! PEG conjugate.
The process of wherein the conjugate is insulin -OC-CHi-CH 2-
Fermentation of recombinant yeast comtaining the insulin gene.
Inoculum of recombinant yeast containing the insulin gene is prepared by adding lOO micro litre glycerol stock culture into 50 ml of minimal glycerol (MGY) medium in 250 mi slake flasks. MGY medium contains yeast nitrogen base (YNB), glycerol, phosphate buffer and D-biotin. Seed flasks are incubated at 30 deg C, 240-hMO until 15 V-5 OD (optical density at 600 nm) is reached.
Fermentation media contains ortho-phosphoric acid, calcium sulfate di-hydrated; potassium sulfate, magmesium sulfate hepta hydrated, potassium hydroxide, glycerol, trace salts and D-biotin. Fermented is ore oared by addling all the above components except trace
salts and D-biotin and autoclaved at 121 - 124° C for one hour. Trace salts solution is prepared by filter sterilizing solution of Cupric sulfate penta hydrated. Sodium iodide, Manganese sulfate mono hydrated. Sodium molybdate di hydrated, Boric acid. Cobalt chloride hexane hydrated. Zinc chloride. Ferrous sulfate hepta hydrated. Biotin solution is also filter sterilized. Ferment or is inoculated and run at temperature 30°C, pH 5.5, air flow 0.5 Ipm and DO 30. After batch phase, glycerol feed (50 % w/w with water) is started to build the biomass. 50% glycerol w/w is prepared and autoclaved for 30 min at 121-124 deg C and then Trace salts &. Biotin solutions are added at the rate of 12 ml/i. Glycerol feed rate is gradually increased up to 20+/- 5 g/hr. Once the 300 - 400 g/l biomass is achieved, temperature is reduced to 20 - 25°C, pH is changed to 3.5 - 6.5 and methanol feed is started. Methanol is filter sterilized and trace salt and biotin solutions are added at the rate of 12 g/l. Methanol feed is increased based on consumption up to 25 +/-5 g/h. During Methanol feeding yeast extract and peptone feed is added at the rate of 0.2 - 0.5 g/h. Fermentation is continued up to 12 days. Purification of proinsulin from broth
900 mg of broth containing Insulin Precursor was adjusted to pH 4.0 by acetic acid and passed through the Cation exchange resins, pre equilibrated with the 50mM acetic acid. The column was washed with 50mM acetic acid and eluted with 50mM acetic acid with 1 M NaCI. 855 mg of product was obtained which was diluted 1:3 with water and concentration was made to 6mg/ml. Phenol was added (1.25mg/lit) and 5% (v/v) ZnCI2 of 5% (w/v) stock was added to the solution. pH of the solution was adjusted to 5.2 with IN NaOH. The solution was kept overnight at 4°C. The solid suspension was centrifuged and the pellet formed was lyophilized to dryness. Recovery in the step was 90%.
Transpeptidation and esterification of the proinsulin
400 mg of dry precursor powder was solubilized in Snort- of DMF containing 30-70% N-N Dimethyl fonnamide. 724mg of Threonine butyl ester was added to the solution. pH of the solution was adjusted to 6.5 with 3 N Acetic acid. The reaction was started with addition of 55mg of Tripping. The reaction was monitored in each hour and was stopped with 5ml of 3 N acetic acid after 4 hr when the conversion of Insulin precursor to Insulin ester was 74%. Yield of this step was 68% in terms of product conversion.
The product obtained was precipitated as above, at pH 6.0 and 228 mg of Insulin ester was recovered. The crystal pellet of Insulin ester was solubilized in 250 mM acetic acid. The filtered material was passed through C8 Kormas matrix and the 95% pure Insulin ester was recovered from the acetonitrile gradient. At the end of RPHPLC 149 mg of product was recovered.
The insulin ester so obtained in used for the preparation of the insulin conjugate as disclosed in the following examples; not to be considered as limiting. EXAMPLES Example 1
5 ml of the RP elution pool is taken as starting material and 1.2 ml of 1 M Borate buffer added to the reaction mixture; pH of the reaction mixtures were raised to 11 and the reaction mixture was stirred for 3 hr at 240C. Deblocking was monitored and when it was completed, 0.5 mg of the activated oligomer (C14H23NO8) solubilized in 300 pi of Acetonitrile was added to the reaction mixture in the same pot The reaction was stopped by bringing down the pH of the reaction to 7.5. The yield is 44%, with a chromatogram purity of 28%. Most of the product remains unconverted.
5 ml of RP elution pool is as the starting material and 1.2ml of 1 M Borate buffer added to the reaction mixture; pH of the reaction mixtures were raised to 11 and the reaction mixture was stirred for 3hr at 240C. Deblocllng was monitored and when it was completed, 2.5 mg of the activated oligomer (C14H23NO8) solubilized in 300 pi of Acetonitrile and added to the reaction mixture in title same pot The reaction was stopped by bringing down the pH of the reaction to 7.5. The yield 63% with a chromatographic product purity of 56%.
5ml of RP elution pool is taken as the starting material and 1.2 ml of 1 M Borate buffer added to the reaction mixture; pH of the reaction mixtures were raised to 11 and the reaction mixture was stirred for 3hr at 240C. Deblocking was monitored and when it was completed, 10 mg of the activated oligomer (CHHZSNOS) solubilized in 300 pi of Acetonitrile and added to tiie reaction mixture in tiie same pot The sample was analyzed from all the sets at 10 mins and Hire. The yield is 18% with a chromatographic product purity of 11%. Mostly the diconjugated product was observed.
5ml of RP elution pool is taken as the starting material! and 1.2 ml of 1 M Borate buffer added to the reaction mixture; pH adjusted to 10.5 and kept for 5hrs. 2.5 mg activated oligomer (C14H23NO8)
dissolved in 300 pi of acetonitrile and added once the deblocking was competed in the same pot of reaction mixture. Aliquot was taken and analyzed. The yield was 58% with a chromatographic purity of 51%.
5ml of RP elution pool is taken as the starting material and 1.2 ml of 1 M Borate buffer added to the reaction mixture; pH adjusted to 10.75 and kept for 4hrs. 2.5mg activated oligomer (C14H23NO8) dissolved in 300 pi of acetonitrile and added once the deblocking was completed in the same pot of reaction mixture. Aliquot was taken and analyzed. The yield was 61% with a chromatographic purity of 53%.
Example 6 (Simultaneous deblocking and conjugation)-5 ml of RP elution pool Is taken as the starting material and 1.2 ml of 1 M Borate buffer added to the reaction mixture; pH adjusted to 11 and 4 mg activated oligomer (CnHzaNOs) dissolved in 300 pi of acetonitrile was added. The sample was analyzed at 10 mins, Ihram, 2hrs, 3 hr after simultaneous deblocking with conjugation takes place in the same pot The yield 64% with a chromatographic purity of 58%
Example 7 (Simultaneous deblocking and conjugation) 5 ml of RP elution pool is taken as the starting material and 1.2 ml of 1 M Borate buffer added to the reaction mixture; pH adjusted to 11 and 2.5 mg activated oligomer (ChHzsNOs) dissolved in 300
pi of acetonitriie was added. The sample was analyzed at 10 mins,
1 hr, 2 hr, 3 hr after simultaneous deblocking with eonjugation
takes place in the same pot of reaction mixture. The yield after 3 hr
was 75% with a product purity of 73.4%.
Deblocking continued for 1 hr, 2 hr and 3 hr and Conjugation
started In each time point and allowed to continue till both
deblocking and conjugation was over for each case.
5 ml each of RP elution pool was taken In each of 4 tubes. 1.2 ml
of IM Borate buffer added to the reaction mixture. pH was
adjusted to 11. In the 1^^ Tube 2.5 mg of activated oligomer
(C14H23NO8) was added at 0 hr. deblocking was allowed to continue
for 1 hr in the 2"^^ tube and same amount of activated oligomer
(C14H23NO8) was added to the reaction mixture. Deblocking was
allowed to continue for 2 hr In the 3 tube and 2.5 mg of activated
oligomer (C14H23NO8) was added after 2 hr. In the 4* tube
deblocking was continued for 3 hr before same amount of activated
oligomer (CnHaaNOs) was added. The conjugation was allowed to
continue for each tube until it seems to be completed as confirmed
by the analytical chromatogram. Yield of the step as well as
percentage purity of the insulin conjugate was monitored by
Experiment # Yield (%) Purity of the 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
150 ml of RPHPLC elution pool in 36 ml of Borate buffer was taken
at pH 8.7. The pH was raised to 11 by adding 10 ml of 10 N NaOH
and the reaction mixture was kept at 25 ©c for 3 hrs. 135 mg of
activated oligomer (C14H23NO8) solubilized In 9ml of Acetonitrlle was
added to the reaction mixture to start the Conjugation reaction in
the same pot After Ihr, the conjugation reaction was stopped by
bringing down the pH of the reaction mixture to 7.5 by adding
glacial acetic acid. Yield of deblocking and conjugation was found
to be 61% In this reaction and the purity of the product was 62%
975 ml of the RP elution pool having concentration of 8.4 mg/ml is
taken and 234 ml of 1 M Borate buffer added at pH 8.2. The pH is
adjusted to 11 with 10 N NaOH. 975 mg of activated oligomer
(C14H23NO8) dissolved in 58.5 ml of acetonitrlle was added to the
reaction mixture and the deblocking as well as conjugation
processes were initiated together in tiie same pot
Aliquot was taken at 2 and 3 hr, analyzed in the HPLC to monitor
the reaction profile. The conjugation was stopped after 3 hr by
bringing down the pH of the reaction mixture to pH 7.5 by addition
of glacial acetic acid. Yield was found to be of 68% with a product
purity of 69%.
Example 10 (Product Recovery)
The end conjugated product Is diluted with 250mM acetic acid to make the concentration of 2.5 mg/ml. The material is loaded on Kromasil 08 RP HPLC column and eluted with acetonitrlle gradient. The
eluted pool has the IN 105 with a purity of 96.7% and with the recovery in the step of 72%.
The purified IN 105, eluted from the RPHPLC column is crystallized with Phenol and ZnCl2 at pH 5.2 at cold condition. The final crystallized pellet was collected by centrifugation. The collected crystal pellet was lyophilized and collected as dry IN 105 purified crystals.
1. A process for making a Insulin-oligomer conjugate ift one pot
comprising of -
(i) transpeptldatlon of insulin precursor,
(ii) daglocking of Insulin ester and conjugation with an
oligomer simultaneously in one pot, (iil) affording insulin-oligomer conjugate.
2. A process of claim 1, futile comprising,
(i) purification of insulin precursor by chromatography and
precipitation, (ii) transpeptldatlon to afford an insulin ester, (ill) purification of the insulin ester using RP-HPLC, (Iv) treatment of the insulin ester with an oligomer in
borate buffer, to effect deblocking and conjugation
simultaneously, (v) optional purification of the conjugate, (vi) affording insulin-oligomer conjugate.
3 A process of claim 1 for making an insulin-oligomer conjugate comprising of simultaneous addition an oligomer solubilizer in acetonitrile to a solution containing insulin-ester In borate buffer and increasing the pH of the mixture.
4. A process of claim 3, wherein the pH is increased to about
5. A process In claim 1, wherein the insulin precucsor Is
prolnsulln or minl-prolnsulln.
6. A process In claim 1, wherein the oligomers Is an alkyl-PEG or
7. A process in claim 1/ wherein the oligomer is activated before
8. A process in claim 1, wherein the activated oligomer used for
conjugation is C14H23NO8.
9. A process in claim 6, wherein the alkyl-PEG has the genera!
formula - OC-(CH2)n-(OCH2CH2)n-OCH3
10. A process in claim 1, wherein the insuiin-ollgomer conjugate
is insulin B29 Ne-oligomer conjugate.
11. A process of claim 1, wherein the insulin-alkyl PEG conjugate
is insulin B29 Ns -alkyl PEG conjugate.
12. A process of claim 1, wherein the conjugate is insulin B29 -OC-CH2-CH2-(OCH2CH2)3-OCH3.
|Indian Patent Application Number||109/CHENP/2008|
|PG Journal Number||33/2012|
|Date of Filing||08-Jan-2008|
|Name of Patentee||BIOCON LIMITED|
|Applicant Address||20TH KM, HOSUR ROAD, ELECTRONICS CITY P.O., BANGALORE, KARNATAKA 560100 INDIA|
|PCT International Classification Number||C07K 14/62|
|PCT International Application Number||PCT/IN2005/000234|
|PCT International Filing date||2005-07-08|