FORM 2
THE PATENT ACT 1970
(39 of 1970)
&
THE PATENT RULES, 2003 COMPLETE SPECIFICATION (See Section 10 and rule 13)
1. A PROCESS FOR SEPARATION OF IMPURITIES FROM PURE TACROLIMUS.
2. (a) Name: CONCORD BIOTECH LIMITED

(b) Nationality: An Indian Company
(c) Address: 1482-1486, Trasad Road,
Dholka, Dist. Ahmedabad-387810. INDIA
3. The following specification particularly describes the
invention and the manner in which it is to be performed.
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Field Of Invention:
The present invention relates to a process for separation of impurities from marcolides such as tacrolimus, rapamycin, ascomycin and immunomycin
The present invention also includes the preparation of a novel polymer resin and its use along with transition metal ions in column chromatography for the purification of macrolides.
More particularly, it relates to process for preparing highly pure Tacrolimus with separation of four impurities. Separation is obtained by adsorbing the crude Tacrolimus over nonionic adsorption resin pretreated with cupric ions.
Background of the Invention:
Macrolides such tacrolimus, rapamycin, ascomycin and immunomycin have difficulties associated with removal of impurities from the final compound. Tacrolimus (FK 506) is macrolide compound, known for its immunosuppressant activity. This compound is used for the treatment of autoimmune disorder, infectious diseases and prevention of resistance occurs during organ transplantation.
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Tacrolimus was first described in US 4,894366 and EP184162, and the compound was produced from Streptomyces tsukubaensis, Streptomyces hygroscopicus subsp. Yakushimaenis. The compound contained several impurities which were purified with column chromatography using reverse phase silica gel column, non-ionic silica gel column, non-ionic HP-20 resin with silver ions, cation exchange resin with silver ions and using preparative HPLC with different solvents for elution.
Removal of impurities from the active pharmaceutical compound is very important for the prevention of health hazards to the patients. Many conventional techniques were used to remove the impurities from the active compound. J. Chromatography 149(1978)417 describes the use of silver ions for separating czs-trans isomers of unsaturated aliphatic acid having same number of carbon atoms.
Higaki et al in WO 01/18007 claimed a method for separating of lactone containing high molecular weight compound Tacrolimus compound having at least one of a lower alkenyl group and lower alkoxy group as its side-chain were separated from analogues of Tacrolimus in one or two steps; first step of adsorption was through a
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nonionic adsorption resin, eluting with silver ion containing aqueous solvent, and in the second step adsorbing to a basic active alumina eluting with organic solvent or in reverse order.
However, the method involved silver ions are difficult to use at production scale due to their light sensitive nature and being highly corrosive.
Yutaka Nishihara et al., in US patent No. 6,492,513 Bl claimed separation and purification method of Tacrolimus from its analogues by using a sulfonic acid group-containing cation exchange resin pretreated with silver ions. Nevertheless, use of silver ion in purification process has its drawbacks.
WO/2005/010015 Al describes method of purifying Tacrolimus includes loading macrolide to a bed of sorption resin and eluting with water and organic solvent tetrahydrofuran and acetonitrile.
WO 05/019226 claims a process for the recovery of substantially pure form of rapamycin, tacrolimus or immunomycin. In this process, the macrolide was treated with water immiscible solvent followed by concentration; mixed with water, water immiscible solvent or mixture thereof then passed through hydrophobic interaction chromatography, fractions collected thereof were
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concentrated, further water miscible solvent was added, thereafter passed through silica gel chromatography and from the collected fractions macrolide was isolated in pure form. This method did not work well in our hands as silica gel works better with water immiscible solvents rather than aqueous solvents.
WO 05/054253 describes a process for the purification of macrolides. In this process, the: crude macrolide was treated with water immiscible solvent followed by optional concentration; treated with ammonia gas, separated the impurities and concentrate it, thereafter loaded on silica gel chromatography optionally reverse phase or pretreated with silver and pure form of macrolide were collected, further optional repetition of silica gel and reverse phase chromatography to obtain pure macrolide.
US 2006/0142565 Al describes pure Tacrolimus and method for purifying by loading charge of tacrolimus in juxtaposition on bed of resin, eluting with tetrahydrofuran (THF), acetonitrile or combination thereof, water and optionally one additional organic solvent. The heart cut of eluent is collected and tacrolimus is collected then crystallized and recrystallized until reduction in the impurity level. This method suffers serious drawbacks regarding yield.
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WO 06/031664 Al describes a process for separation of tacrolimus and its analogues, ascomycin and tsucubamycin B; wherein purification of tacrolimus is accomplished in two steps using two different chromatographic sorbents. One is accomplished on normal phase chromatography and another on silica gel column with silver nitrate and organic solvents.
As mentioned in foregoing prior art, reverse phase system and use of silver ion in chromatography is inconvenient. Moreover, in the ! purification process, use of resin HP-20 with silver nitrate to obtain pure form of Tacrolimus is also not economical. In addition to this, passing the crude Tacrolimus material multiple times through chromatography is a tedious procedure and commercially not feasible.
Further, the procedure for purification of Tacrolimus through polymer resin containing silver ions describes separation of only two analogues such as Ascomycin and Dihydro Tacrolimus, while the procedure described in the present invention using cupric nitrate over the non-ionic adsorption resin segregates other analogues such as Delta lactone, Methyl derivative, Demethomycin and Pyrrole analogue (FK-525). In the prior art purification procedures for Tacrolimus, there is no mention of such analogues.
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In addition to this, cupric nitrate is easy to handle as compared to all types of silver ion metal salts during use.
In the present invention, an economical process for purification of macrolides such as tacrolimus, rapamycin, ascomycin and immunomycin is described which involves use of a polymer resin and a transition metal salt.
In the said invention the resin used contains polyvinyl pyrrolidone, methyl acrylate, divinyl benzene based compounds and the metal salt used for purification belongs to transition metals like copper, nickel, lead etc. The same metal salts can also be used along with other neutral non ionic adsorption resins of polyethylene i.e. HP20, HP20SS, Sepabeads SP207 and the like. In the above said process Tacrolimus obtained after extraction and final crystallization contains individual impurity below 0.15% (as per ICH guidelines 0.15% is the qualification limit) and total impurity level is less than 0.50% excluding Tautomers.
Summary of Invention:
The invention presents an economical and efficient process to remove impurities from macrolides.
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The invention discloses a unique resin to be used for column chromatography in the process of purification of macrolides.
The main object of the present invention is to provide a process for separation of analogues of Tacrolimus such as Demethomycin, Pyrrole analogue, Ascomycin, Delta lactone, dihydro and Methyl derivative as shown in following structures:.

Delta Lactone
1) R1, R3 = H; n =2 Demethomycin
2) R1 =CH3; R3 = H; R2 = CH2 - CH2 - CH3 n =2 Dihydro
3) R1 =CH3; R3 = H; R2 = CH2 - CH3 n =2 Ascomycin
4) R1 =CH3; R3 = H; R2 = allyl; n =1 Pyrrole Analogue
5) R1 =CH3; R3 = CH3; R2 = allyl; n =2 Methyl Derivative
Another object of the present invention is to provide a process of purification of crude Tacrolimus using a novel polymer resin of Polyvinyl pyrrolidone (K-30) type loaded with aqueous solvent
solution of a reagent containing cupric ions. As a copper salt contain
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in the copper ion containing aqueous solvent preferred one is cupric nitrate, cupric chloride or the like.
Yet another object of the present invention is to provide a process for purification of crude Tacrolimus using a nonionic adsorbent resin of polyethylene like HP20 loaded with aqueous solvent containing cupric salts.
The polymer resin used can be polyvininyl pyrrolidone resins having partial structure of divinyl benzene and polyvinyl pyrrolidone.
The polymer resin used can be a polyethylene resin having partial structure of Divinyl benzene.
The concentration of copper ions may be generally 0.20 to 1.5 mol/L, preferably 0.30 to 0.40 mol/L.
As an aqueous medium for the copper ion containing aqueous solvent, an aqueous acetone, an aqueous alcohol, an aqueous acetonitrile or the like may be exemplified.
The quantity of adsorbent used is about 40 times more than the wt. of the compound. The flow rate of the eluting solvent varies from 0.5 bed volume to 1.2 bed volume per hour, preferably at 0.9 to 1.0 bed volume per hour.
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The fractions containing pure product analyzed through HPLC are combined and extracted with water immiscible solvent like ethyl acetate. The solvent is evaporated under reduced pressure and a dried powder obtained is further purified to yield Tacrolimus pure in solvents like Diisopropyl ether and Acetonitrile -water. Detailed Description of the Invention:
In one embodiment, the present invention encompasses a process of purification of macrolides such as Tacrolimus.
In another embodiment, the present invention discloses a novel resin to be used in column chromatography for purification of macrolides.
In a preferred embodiment of the present invention, a non ionic adsorption polymer resin is prepared and loaded over a column and the macrolide compound is adsorbed over the polymer resin and eluted with solvents such as aqueous solvents containing transition metal ions. The level of individual impurity from the present process is less than that present in crude Tacrolimus and the process gives a highly pure product.
The preferred embodiment of the invention covers a process for purification of tacrolimus by adsorbing tacrolimus over non ionic
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adsorption resin and eluting it with an aqueous solvent containing cupric ions to separate impurities.
The process for the preparation of a novel polymer resin comprises the following steps:
Charge around 159 L D.M. Water in a 500 L reactor. To this Poly Vinyl Pyrrolidone K-30 (PVPK) is added at around 30° C temperature. In the monomer aqueous phase Glycidine Methyl Acrylate is charged along with Divinyl Benzene, Acrylonitirile, Azobis Iso Butyro Nitrile and Cyclohexanol. Both the mass are combined and stirred well at 30° C for 30 minutes. After this the reaction mass is heated to 70° C for 70-90 minutes and then cooled to 30° C. The cooled mass converts to polymerized beads.
Polymerized beads from the reactor are transferred into grading vessel by opening the bottom valve. The beads are washed thoroughly with water and then with methanol in the grading vessel. The polymerized beads are now filtered through Nutsche Filter (NF) under vacuum. The filtered slurry containing polymerized beads and methanol is used as polymer resin in the column chromatography.
In the preferred embodiment of the present invention, polymer
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resin is charged in column then loaded with crude Tacrolimus which is produced by fermentation process using mutated culture strain of Streptomyces tsukubaensis; under submerged aerobic fermentation conditions culturing in aqueous nutrient medium containing vegetable oil as carbon source and nitrogen, mineral salts and other defoaming agents at temperature between 20 - 40° C, for 48 - 216 hours at pH 7. The fermentation broth obtained is extracted in toluene. The toluene extract is then concentrated, column chromatographed over silica gel using ethyl acetate: hexane eluent to obtain crude Tacrolimus.
Crude Tacroliums recovered from the broth is further purified over the polymer resin in column chromatography. Purification is done with using aqueous solvents like acetone water, methanol water, THF water and acetonitrile water, preferably with acetone water.
Methanol is charged to a column. Polymer resin is added slowly in methanol. Resin is then allowed to stand for 24 hours for activation. After 24 hrs methanol is drained and resin is washed thoroughly with R.O. water till neutral pH is attained. The resin is then equilibrated with a solution of 4BV acetone: water (40:60) at a flow rate of 0.6 -1.5 BV/hr. After equilibration, a solution of crude Tacrolimus is prepared at a concentration of 0.30 - 0.60 g/L preferably at 0.30 -
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0.50 g/L and most preferably at 0.5 g/L. The solution of compound is then charged over the resin. The column is then washed with 4 BV of acetone : water (40:60 ) at a flow rate of 0.6 - 1.5 BV/hr preferably at 0.8-1.1 BV/hr and most preferably at 1 BV/hr. The elute is collected and analysed using HPLC. The compound is eluted with a 8 BV solution of acetone : water (50:50) containing cupric salts preferably cupric nitrate, cupric chloride and the like.
A solution of copper nitrate at a concentration of 0.25-1.20mole/L in 8 BV acetone : water (50:50) is prepared, preferably at 0.3-0.70mole/L and most preferably at 0.30-0.40 mole/L. The solution is then added over the column and fractions are collected of around half bed volume and analyzed through HPLC to check impurity profile of Tacrolimus. The fractions containing pure Tacrolimus are pooled and further diluted with water. The compound is then extracted into ethyl acetate and then concentrated to yield Tacrolimus pure in oil form. To the oil, Diisopropyl ether is added and a few mL of water is added, and the mixture is stirred to give Tacrolimus monohydrate as crystalline solid. The compound is filtered and washed with Diisopropyl ether and then dried to give Tacrolimus pure having chromatographic purity more than 98%. Chromatogram purity
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analysis showed higher content of tautomer and impurity at RRT 1.12 i.e. delta lactone. The compound is then dissolved in acetonitrile (3X) and water (10X). The mixture is stirred for 2 hours at RT and then filtered, dried to yield Tacrolimus having total impurity content less than 0.5% excluding Tautomers.
Pure Tacrolimus obtained has the content of Demethomycin impurity and Pyrrole analogue is less than about 0.30%, preferably less than about 0.10%, most preferably non detectable. '
Other impurity Delta lactone in pure Tacrolimus is present at less than about 0.30%, preferably less than 0.10%, most preferably non detectable.
Methyl derivative content in pure Tacrolimus is present at less than about 0.3%, preferably non detectable.
The present invention is further described in detail with reference to the following examples, which are given for illustration only and therefore should not be construed as limitation to the scope of invention.
Example 1:
150 L of D.M. water was charge in a 500 L reactor. 2.2 kg. polyvinyl pyrrolidone was added into the reactor at RT. To the
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monomer phase, 12.01 kg Glycidine Metha Acrylate, 2.85 kg Acrylonitrile, 8.0 kg ,Divinyl benzene and 0.6 kg Azobis Iso butyronitrile were added at RT simultaneously. The mixed phase solution was heated to 70° C for about 1 hr. After heating the reaction mixture was cooled to RT. Upon cooling the polymer converted into polymer beads. The beads were then filtered and washed thoroughly with water. The beads were then graded through 100 mesh and then washed thoroughly with methanol to give resin of particle size of around 150 micron.
Example 2:
Approx. 70 L methanol was charged in a 130 L column. Polymer resin prepared in example 1 was added slowly in methanol to make bed volume (BV) around 100 L. Resin was allowed to stand for 24 hours in methanol for activation. Methanol was drained and matrix was washed thoroughly with 600 -1000 L R. O. Water. The resin was equilibrated with a solution of 400 L (4BV) acetone: water (40:60) at flow rate of 100 L/hrs. After equilibration the resin was charged with the solution of crude Tacrolimus prepared in acetone : water (40:60) at concentration of 50 g/L, without disturbing the bed. The column was washed with 4BV of methanol : water (40:60) at flow rate of lBV/hr.
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The elute was collected and checked by HPLC. The column was eluted with a solution of 8BV acetone : water (50:50) at flow rate of 1 BV/hrs. Fractions of 0.5 BV were collected and checked through HPLC. Pure fractions from 10-19 were pooled and compound was obtained by extracting it in ethyl acetate and concentrating the solvent to yield oily residue which can be crystallized in Di-isopropyl ether to give Tacrolimus in pure form. The Tacrolimus obtained contained Tautomer around 2.77% and delta lactone impurity around 0.25%. The compound was then dissolved in acetonitrile (3X) and crystallized by adding water (10X) at room temperature. Pure Tacrolimus obtained after filtration and drying contained Tautomer 0.38% and delta lactone impurity below detection level. The product purity of compound was more than 99.0%.
The results of the fractions obtained are tabulated in below mentioned Table 1.
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Table 1:

Fraction# Tautomer Impurity (Tl & T2)RRT-0.62, 0.85 Demethomy cinRRT-0.66 Pyrrole analogueRRT-0.72 AscomycinImpurityRRT-0.95 Tacrolim us DeltalactoneRRT-1.12 Dihydro ImpurityRRT-1.18 Methyl Derivativ e ImpurityRRT-1.22
Crude Sample - -0.20% -0.20% -9.00% -80.00% -2.0 % -10.05 % -0.35%
Fraction No. 1-9 ND ND ND ND ND ND ND ND
10,d - - - - - - - -
11th 4.81 ND ND ND 70.73 ND ND ND
12th 6.48 ND ND ND 82.92 ND ND ND
13th ND ND ND ND ND ND
14th 6.89 ND ND ND 84.68 0.45 ND ND
15th 6.70 ND ND ND 85.75 0.56 ND ND
16th 6.81 ND ND ND 85.49 0.73 ND ND
17th 6.24 ND ND ND 86.48 0.90 ND ND
18th 6.58 ND ND ND 84.10 1.08 ND ND
19th 5.87 ND ND ND 85.72 1.29 ND ND
20th 5.86 ND ND ND 81.81 1.42 0.11 0.30
21st 5.88 ND ND ND 81.90 1.57 0.16 0.38
22nd 4.77 ND ND ND 79.73 1.66 0.21 0.55
23rd 6.08 ND ND ND 79.63 1.54 0.25 0.65
24th 4.92 ND ND ND 80.11 1.34 0.30 0.69
25th 5.94 ND ND ND 80.81 1.11 0.33 0.70
26th 5.99 ND ND ND 81.93 0.92 0.37 0.70
ND=Not detectable
It is to be noted that if following crystallization, the concentration of the impurity in the final product is greater than
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desired, one or more additional crystallization steps may be performed to remove the impurity.
Example 3:
70 L methanol was charged in a 130 L column. HP 20 resin was added slowly in methanol to make bed volume (BV) around 100 L. Resin was allowed to stand fro 24 hours I methanol for activation. Methanol was drained and resin was washed thoroughly with 600-1000 L R.O. water. The resin was equilibrated with a solution of 400 L (BV) acetone:water (40:60) at flow rate of 100 L/hrs. After equilibration the resin was charged with the solution of crude Tacrolimus prepared in acetone : water (40:60) at concentration of 50 g/L, without disturbing the bed. The column was washed with 4BV of acetone : water (40:60) at flow rate of lBV/hr. The elute was collected and checked using HPLC. The column was eluted with a solution of 8BV acetone : water (50:50) at flow rate of 1 BV/hrs. Fractions of 0.5 BV were collected and checked through HPLC. Pure fractions were pooled and compound is obtained in pure form as per the procedure in example-2.
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We claim:
1. A process for purification of a macrolide comprising: adsorbing a crude macrolide over non ionic adsorption resin and eluting with a solvent containing transition metal ions to separate impurities.
2. A process as claimed in claim 1 wherein said macrolide is selected from a group comprising tacrolimus, rapamycin, ascomycin and immunomycin.
3. A process as claimed in claim 1 wherein said macrolide is tacrolimus.
4. A process as claimed in claim 1 wherein said transition metal ions are cupric ions.
5. " A process as claimed in claim 1 wherein said solvent is an
aqueous solvent.
6. A process as claimed in claim 1 wherein said solvent is an aqueous solvent selected from the group comprising aqueous acetone, aqueous alcohol, aqueous acetonitrile or combinations thereof.
7. A process as claimed in claim 1 wherein said impurities are selected from the group comprising Demethomycin, Pyrrole
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analogue, Ascomycin, Delta lactone, Dihydro and Methyl derivative of Tacrolimus.
8. A process as claimed in claim 1 wherein the final level of an individual impurity is less than about 0.30%.
9. A process as claimed in claim 1 wherein the final level of individual impurity is preferably less than about 0.10%.
10. A resin, for use in column chromatography, prepared by:
a) Mixing water and Poly Vinyl Pyrrolidone K-30 (PVPK);
b) Adding Glycidine Methyl Acrylate, Divinyl Benzene,
Acrylonitirile, Azobis ho Butyro Nitrile and Cyclohexanol to
the product of step [a];
c) Heating the mixture of step [c] to approx 70° C;
d) Cooling the mixture to 30° C resulting in formation of beads.

11. A process for purification of Tacrolimus involving use of cupric ions and separation of impurities as claimed as herein described with foregoing description and examples.
12. Tacrolimus having a purity of more than 99.7%
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Dated this on 21st day of March, 2007.

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Abstract:
Process for purification of macrolides such as Tacrolimus by adsorbing crude macrolide over non ionic adsorption resin and eluting with an aqueous solvent containing cupric ions to separate process impurities. The invention covers Tacrolimus having a purity of more than 99.7%. The invention also covers preparation of a resin to purify macrolides.
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