DESC:FIELD OF THE INVENTION

The present invention generally relates to a process for purification of carfilzomib using preparative high performance liquid chromatography (prep-HPLC).

BACKGROUND OF THE INVENTION

Carfilzomib is a tetrapeptide epoxyketone, also known as (2S)-N-((S)-1-((S)-4-methyl-1-((R)-2-methyloxiran2-yl)-1-oxopentan-2-yl-carbamoyl)-2-phenyl-ethyl)-2-((S)-2-(2- morpholine acetamido)-4-phenylbutanamido)-4-methyl-pentanamide, is represented by the following structure:

Carfilzomib

Carfilzomib is marketed by Onyx Pharma under the trade name Kyprolis®and isapproved as a single agent for the treatment of patients with relapsed or refractory multiple myeloma; and also approved in combination with dexamethasone or with lenalidomide plus dexamethasone for the treatment of patients with relapsed or refractory multiple myeloma who have received one to three lines of therapy.

U.S. Patent No. 7,232,818 (“the ‘818 patent”) discloses a variety of peptide based compounds and their derivatives such as carfilzomib and its process for preparation thereof.The process disclosed in the ‘818 patent is schematically represented as follows:

The processdisclosed under the ‘818 patent involves purification of finally obtained carfilzomib by dissolving in methanol and the solution was added to rapidly stirred chilled water to precipitate out carfilzomib.

Michael Screen et al. inJournal of biological chemistry vol. 285, no. 51, pp. 40125–40134, 2010 discloses preparation of carfilzomib by a different approach. The process disclosed in this article is schematically represented as follows:

The processdisclosed under this article involves purification of finally obtained carfilzomib bysilica gel columnchromatography using methylene dichloride and methanol as an eluent.

Chinese Patent application No: 104356205(“the ‘205 publication”) discloses purification of carfilzomib by silica gel column using ethyl acetate and n-hexaneas the mobile phase and followed by crystallization of the obtained compound from acetone and n-hexane.

Further, various literatures involve preparation/purification of carfilzomib by forming salts/co-crystals of carfilzomib as an intermediate. The salts reported are as follows: PCT Publication Number 2009/045497 (“the ‘497 publication”) discloses citrate, tartrate, trifluoroacetate, methanesulfonate, toluenesulfonate, chloride, and bromide salt; PCT Publication Number (s) 2016108204(“the ‘204 publication”)&2016185450(“the ‘450 publication”)and Chinese Patent application No:105985408(“the ‘408 publication”)are discloses maleate salt; and PCT Publication Number: 2016088031(“the ‘031 publication”)disclose carfilzomiboxalate.

Generally peptide coupling reactions involves formation of numerous impurities such as isomer impurities, degradant impurities and side reaction impurities.Presence of impurities in a pharmaceutical compound is undesirable, in extreme cases, might even be harmful to a patient, and health authorities in many jurisdictions (e.g. the Food and Drug Administration in the United States) have established guidelines relating to acceptable levels of impurities in pharmaceuticals. The need for and commercial utility of methods of reducing the level of impurities in any pharmaceutical are self-evident.

In order to eliminate the impurities from the carfilzomib product, most of the processes described till date in the art involved purification of carfilzomib either using a silica-gel column chromatography or by a crystallization using multiple solvents or by salt formation methods. The purification methods described in the literatures are do not eliminate all impurities completely and moreover the reported purification procedures are laborious as it involves combination of cumbersome silica column purifications and subsequent solvent crystallizations as like in the ‘205 publication or it involves saltification and subsequent desaltification steps as like in the ‘497, ‘204, ‘450, ‘408 and ‘031 literatures. Thus the processes described in the art are lengthy and results in the product with low yields.

As the selection of purification methodis important for getting high pure compound with less cumbersome techniques, the present invention aims to propose a simple purification system that avoids the aforementioned difficulties and which is more robust, user convenient and up-scalable process for the purification of carfilzomib.

The present invention provides a process for purification of carfilzomib by preparative high performance liquid chromatography; the process utilizes selective eluent system by providing easy separation of impurities and reduces the purification time with higher yield and also avoiding the solvent crystallizations and cumbersome saltification steps.

SUMMARY OF THE INVENTION

The present invention generally relates to a process for purification of carfilzomib using preparative high performance liquid chromatography (Prep-HPLC).

In accordance with one embodiment, the present invention provides a process for purification of carfilzomib, comprising subjecting the carfilzomib to a preparative high performance liquid chromatographyusing suitable eluent to obtain pure carfilzomib.

Carfilzomib

In accordance with another embodiment, the present invention provides a process for purification of carfilzomib, comprising subjecting the crude carfilzomib to a preparative high performance liquid chromatographyusing suitable eluent to obtain pure carfilzomib; wherein the suitable eluent is selected from the group consisting of:alcohols, nitriles, ethers, halogenated hydrocarbons, esters, aliphatic hydrocarbons, waterand mixtures thereof.

In accordance with another embodiment, the present invention provides a process for purification of carfilzomib, comprising subjecting the crude carfilzomib containing at least one related impurity to a preparative high performance liquid chromatographyusing suitable eluent to obtain pure carfilzomib; wherein the crude carfilzomib containing at least one related impurityidentified with following relative retention time (RRT) impurities:
RRT 0.14 0.18 0.26 0.30 0.36 0.41 0.45
0.52 0.55 0.62 0.84 0.88 0.92 1.08

In accordance with another embodiment, the present invention provides a process for purification of carfilzomib,comprising:
a) dissolving crude carfilzomib in a suitable solvent or a mixture thereof,
b) subjecting the solution to a preparative high performance liquid chromatography (Prep-HPLC)using suitable eluent,
c) collecting pure fractions of carfilzomib, and
d) isolating the pure carfilzomib.

In accordance with another embodiment, the present invention provides a process for purification of carfilzomib,comprising:
a) dissolving crude carfilzomib in a suitable solvent or a mixture thereof,
b) subjecting the solution to a preparative high performance liquid chromatography(Prep-HPLC)using suitable eluent,
c) collecting pure fractions of carfilzomib, and
d) isolating the pure carfilzomib; wherein the suitable eluent is selected from the group consisting of: alcohols, nitriles, ethers, halogenated hydrocarbons, esters, aliphatic hydrocarbons, water and mixtures thereof.

In accordance with another embodiment, the present invention provides a process for purification of carfilzomib,comprising:
a) dissolving crude carfilzomib in a suitable solvent or a mixture thereof,
b) subjecting the solution to a preparative high performance liquid chromatography(Prep-HPLC)using suitable eluent,
c) collecting pure fractions of carfilzomib, and
d) isolating the pure carfilzomib; wherein the suitable eluent is selected from the group consisting of: alcohols, nitriles, ethers, halogenated hydrocarbons, esters, aliphatic hydrocarbons, water and mixtures thereof; and wherein the crude carfilzomib containing at least one related impurityidentified with following relative retention time (RRT) impurities:
RRT 0.14 0.18 0.26 0.30 0.36 0.41 0.45
0.52 0.55 0.62 0.84 0.88 0.92 1.08

In accordance with another embodiment, the present invention provides a process for purification of carfilzomib, comprising: subjecting the carfilzomib having diol impurityof Formula A (0.36 RRT) to a preparative high performance liquid chromatography using suitable eluent to obtain a pure carfilzomib having diol impurity less than 0.15% by HPLC.
In accordance with another embodiment, the present invention provides a process for purification of carfilzomib, comprising: subjecting the carfilzomib having diol impurity of Formula A (0.36 RRT)to a preparative high performance liquid chromatography using suitable eluent to obtain a pure carfilzomib having diol impurity less than 0.15% by HPLC; wherein the suitable eluent is selected from the group consisting of:alcohols, ketones, nitriles, ethers, halogenated hydrocarbons, esters, aliphatic hydrocarbons, waterand mixtures thereof.

In accordance with another embodiment, the present invention provides carfilzomibhaving less than 0.15% by HPLC of one or more of following relative retention time (RRT) impurities:

RRT 0.14 0.18 0.26 0.30 0.36 0.41 0.45
RRT 0.52 0.55 0.62 0.84 0.88 0.92 1.08

In accordance with another embodiment, the present invention provides carfilzomibhaving less than 0.1% by HPLC of one or more of following relative retention time (RRT) impurities:

RRT 0.14 0.18 0.26 0.30 0.36 0.41 0.45
RRT 0.52 0.55 0.62 0.84 0.88 0.92 1.08

In accordance with another embodiment, the present invention provides carfilzomibhaving less than 0.05% by HPLC of one or more of following relative retention time (RRT) impurities:

RRT 0.14 0.18 0.26 0.30 0.36 0.41 0.45
RRT 0.52 0.55 0.62 0.84 0.88 0.92 1.08

In accordance with another embodiment, the present invention provides carfilzomibhaving less than 0.15% by HPLC of diol impurity of Formula A (0.36 RRT).

In accordance with another embodiment, the present invention provides carfilzomibhaving less than 0.1% by HPLC of diol impurity of Formula A (0.36 RRT).

In accordance with another embodiment, the present invention provides a pharmaceutical composition comprising carfilzomibobtained by the process of the invention and/or at least one pharmaceutically acceptable excipient.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the invention and together with the description, serve to explain the principles of the invention.

Figure-1 represents purification results of carfilzomib obtained from the known purification methods and preparative HPLC method.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a process for purification of carfilzomib using preparative high performance liquid chromatography, which process avoids the cumbersome solvent crystallizations and saltification steps thereby getting the product with high yields.

To date purification methods disclosed under the reported literature involves either silica-gel column chromatography or by solvent crystallization or by salt formation methods. By using the known purification methods it is very difficult to eliminate the process impurities completelyeven after repeated purifications either by solvent crystallization methods and/or saltification and desaltification steps.

The present inventors have observed that, preparation of carfilzomib by coupling of free amine intermediate with morpholine acetic acid as per the process disclosed by Michael Screen et al.involves formation of numerous process impurities. These impurities are very difficult to separate from carfilzomib even after repeated purifications using either silica gel column chromatography or salt formation method or crystallization method.The impurities identified bythe present inventors are listed as below:

Diol Impurity
(0.36 RRT; LC-Mass:737.9)
R,R-impurity
(0.84 RRT; LC-Mass:719.9)

N-Acetyl impurity
(0.88 RRT; LC-Mass:634.8)
Other Impurities withRRT:

RRT 0.14 0.18 0.26 0.30 0.41 0.45 0.52 0.55 0.62 0.92 1.08

Further the present inventors have also observed that one of the key impurity (diol impurity), which is formed by cleavage of epoxide ring, is very difficult to minimize or remove using the reported purifications, particularly by salt formation methods as this impurity further getting converted to corresponding ester impurity (as below) with the salt forming agents for example maleate ester or oxalate ester with the maleic acid and oxalic acid respectively. These ester impurities are not easily removed by the subsequent desaltification step/purification steps and the same retain with the carfilzomib. Therefore additional column purification is required to remove these specific impurities and this additional step adds burden to the commercial process.

Maleate ester impurity
(0.10 RRT; LC-Mass: 836)
Oxalate ester impurity

Moreover the known purification methods are not enough to get required purity and the known methods are laborious and having lessyields, as reported methods involves multiple purifications or at least two purifications comprises either solvent crystallization or saltification and subsequent desaltification steps.To overcome the difficulties associated with the art, the inventors of the present invention have surprisingly found that purification of carfilzomib using preparative high performance liquid chromatography, which involves mainly eliminating process impurities, using suitable eluents thereby getting product with more yield when compared to known multiple purification methods. Therefore, cumbersome salt formation and silica-gel column purifications of multiple purifications are avoided.

In accordance with one embodiment, the present invention provides a process for purification of carfilzomib, comprising subjecting the carfilzomib to a preparative high performance liquid chromatography using suitable eluent to obtain pure carfilzomib.

Carfilzomib

In accordance with another embodiment, the present invention provides a process for purification of carfilzomib, comprising subjecting the crude carfilzomib containing at least one related impurity to a preparative high performance liquid chromatography using suitable eluent to obtain pure carfilzomib; wherein the crude carfilzomib containing at least one related impurityidentified with following relative retention time (RRT) impurities:
RRT 0.14 0.18 0.26 0.30 0.36 0.41 0.45
0.52 0.55 0.62 0.84 0.88 0.92 1.08

In a specific embodiment, the present invention provides a process for purification of carfilzomib,comprising:
a) dissolving crude carfilzomib in a suitable solvent or a mixture thereof,
b) subjecting the solution to a preparative high performance liquid chromatography (Prep-HPLC)using suitable eluent,
c) collecting pure fractions of carfilzomib, and
d) isolating the pure carfilzomib.

All of the steps of the above process are individually described herein below.

The carfilzomib may be prepared by any known processes; including process disclosed by Michael Screen et al. inJournal of biological chemistry vol. 285, no. 51, pp. 40125–40134, 2010, the content of which is incorporated herein by reference.

In another embodiment, the "carfilzomib" in step a) refers to crude carfilzomibwhich may be having content of total impurities about 20%, and/or having at least one or more of the following impurities as identified by their RRT:
RRT 0.14 0.18 0.26 0.30 0.36 0.41 0.45
0.52 0.55 0.62 0.84 0.88 0.92 1.08

In a preferred embodiment, the "carfilzomib" in step a) refers to crude carfilzomib which may be having content of total impurities about 20%, preferably having about 15% and more preferably having about 10% and/or having at least one or more of the following impurities as identified by their RRT with the content of greater than 0.05%:
RRT 0.14 0.18 0.26 0.30 0.36 0.41 0.45
0.52 0.55 0.62 0.84 0.88 0.92 1.08

A purification process of the present invention is carried out by eluting carfilzomib through a preparative high performance liquid chromatography column, wherein the column is packed under a dynamic axial compression mode with operating pressures up to about 100 bar with a silica gel which is suitable to separate pure carfilzomib from its impurities, for exampleLuna prep C18(3) 200 x 30 mm to 500 x 60 mmwith about 5-50 µm particles, preferably 300 x 50 mmwith about 10 µm particles.Flow rate of the mobile phase may be selected from about 10 ml to 80 ml per minute, preferably about 30 ml to 60 ml per minute, more preferably about 50 ml per minute. Conditions for the preparative column chromatography are known to the person skilled in the art.

The step a) of forgoing process involves,dissolving the crude carfilzomib in a suitable solvent or a mixture thereof at a temperature of about 25-35°C to obtain a solution.

The suitable solvent used herein in step a) is selected from a solution in which carfilzomib can dissolve completely. Example for the suitable solvent include, but is not limited to alcohols, ketones, nitriles, ethers, halogenated hydrocarbons, estersand mixtures thereof. The alcohols include, but are not limited to methanol, ethanol, isopropanol, butanol and the like; ketones include, but are not limited to acetone, methyl isobutyl ketone, methyl ethyl ketone and the like; nitriles include, but are not limited to acetonitrile, propionitrile and the like; ethers include, but are not limited to tetrahydrofuran, methyl tertiary butyl ether and the like; halogenated hydrocarbons include, but are not limited to methylene chloride, ethylene chloride, chloroform and the like; esters include, but are not limited to ethyl acetate, methyl acetate, isopropyl acetate and the like and mixtures thereof; preferably a mixture of acetonitrile and tetrahydrofuran.

Optionally the step a) solution may be filtered to remove any undissolved particles present in the solution.

The step b) of forgoing process involves,subjecting the step a) solution to a preparative high performance liquid chromatography by eluting using suitable eluent.

The suitable eluent used herein in step b) is selected from the group consisting of, but is not limited to alcohols, nitriles, ethers, halogenated hydrocarbons, esters, aliphatic hydrocarbons, waterand mixtures thereof. The alcohols include, but are not limited to methanol, ethanol, isopropanol, butanol and the like; nitriles include, but are not limited to acetonitrile, propionitrile and the like; ethers include, but are not limited to tetrahydrofuran, methyl tertiary butyl ether and the like; halogenated hydrocarbons include, but are not limited to methylene chloride, ethylene chloride, chloroform and the like; esters include, but are not limited to ethyl acetate, methyl acetate, isopropyl acetate and the like; aliphatic hydrocarbons include, but are not to hexane, heptane, propane, cyclopropane, cyclobutane, cyclopentane, cyclohexane, methyl cyclohexane, cycloheptane, cyclooctane and the like; water and mixtures thereof. Preferably the suitable eluent includes, water, methanol, ethanol, acetonitrile, methylene chloride, ethyl acetate, hexane, heptane and mixture thereof.

In an embodiment, the step b) of preparative high performance liquid chromatography involvesreverse phase chromatography with a gradient elution.

Preferably, the gradient elution of step b) comprises eluent A and eluent B with predefined ratio. The eluent A is selected from water, hexane or heptane and eluent B is selected from acetonitrile, methanol, ethanol, methylene chloride or ethyl acetate. The eluent A may be ranging from 20 to 90 % (v/v) and eluent Bmay be from 10 to 80 % (v/v). More preferably, the gradient of step b) is ranging from 40 to 80 % (v/v) of eluent A, and from 20 to 60 % (v/v) of eluent B. More preferably the gradient system for the present invention is water as eluent A and acetonitrile as eluent B.

The step c) of forgoing process involves,collecting the pure fractions of carfilzomib from high performance liquid chromatography and pooling.

During elution, desired fractions are collected at regular intervals and analyzed for purity. The suitable collected fractions containing the product of similar purities may be pooled together and the pure product is extracted using a suitable organic solvent such as ethyl acetate, methylene chloride and the like and mixture thereof. The product containing layer is separated and subjected to removal of solventby the methods known in the art, for example removal of solvent by distillation under either atmospheric conditions or under vacuum at suitable temperature to obtain pure carfilzomib.

In another embodiment, the present invention provides carfilzomib, obtained by the purification process described herein, substantially free of one or more of following relative retention time (RRT) impurities:
RRT 0.14 0.18 0.26 0.30 0.36 0.41 0.45
0.52 0.55 0.62 0.84 0.88 0.92 1.08

In another embodiment, the present invention provides carfilzomib, obtained by the purification process described herein, having a purity of at least about 97%, as measured by HPLC, preferably at least about 99%, and more preferably at least about 99.5%; having total impurities less than 0.5%, as measured by HPLC,preferably less than 0.2%, more preferably less than 0.1%, still more preferably less than 0.05%; substantially free of one or more of impurities as defined by their RRT values; and substantially free of diol impurity; where in the word “substantially free” refers to said impuritiesare less than 0.10%as measured by HPLC,preferably less than 0.05%as measured by HPLC,more preferably less than the detectable value by HPLC.

The known purification methods described for carfilzomib in the art are involved either by silica-gel column chromatography or by solvent crystallization or by saltification and followed by desaltification methods, which methods are not effective in either decreasing the process impurities orobtaining the desired pure API. The present invention provides a process for purification of carfilzomib using preparative high performance liquid chromatography, which involves mainly eliminating process impurities, specifically diol impurity as this impurity can be removable only by the process of the invention and not by the saltification process as described under the reported literature. The current invention utilizes single purification for effective removal of impurities including diol impurity and getting the product with highest yield when compared to known purification methods containing solvent crystallization or salt formation and/orsilica-gel column chromatography. The results obtained from the known purification methods and preparative HPLC methodsare summarized in Figure-1.

From the Figure 1, purification using Preparative HPLC of the current invention results carfilzomib with purity about 99.9% by HPLC and having yield of 0.71w/w. whereas from other methods from salt formation and silica column chromatography method, Carfilzomib obtained with less pure and lower yield. The following table summarizes the results obtained from these two variations:

Crude Salt formation and followed by
Silica Chromatography Preparative HPLC
Yield
(wt/wt) -- 0.41 0.71
Purity
(%) 93.77 99.7 99.9

The present invention provides purification of carfilzomib by preparative HPLC method, obtained by the above process, as analyzed using the high performance liquid chromatography with the conditions described below:

Column Halo C18 (150 x 4.6) mm, 2.7 mm
Mobile phase Buffer: Potassium dihydrogen phosphate, dipotassium hydrogen phosphate andorthophosphoric acid.
Mobile phase-A: A mixture of buffer and acetonitrile
Mobile phase-B: A mixture of buffer, acetonitrile and methanol
Mobile phase-C: A mixture of acetonitrile and water
Flow rate 0.7 mL/min
Detection By UV at 210 nm
Run time 90 min
Elution mode Gradient
Time (min) Mobile phase-A
(% v/v) Mobile phase-B
(% v/v) Mobile phase-C
(% v/v)
0 60 40 0
50 40 60 0
80 20 0 80
85 60 40 0
90 60 40 0

In another embodiment, the present invention provides a pharmaceutical composition comprising carfilzomibpurified by the process of the invention and/or at least one pharmaceutically acceptable excipient.

EXAMPLES

The following non limiting examples illustrate specific embodiments of the present invention. They are not intended to be limiting the scope of the present invention in any way.

REFERENCE EXAMPLE:

Preparation of carfilzomib

A mixture of dimethyl formamide (200 ml) and 2-morpholino acetic acid hydrochloride (39.2g) were added in to a round bottom flask and allowed to cool to 5°C. To the reaction mass diisopropyl ethyl amine (37.2 g), HOBt (1.89 g) and PyBOP (112.4 g) were slowly added sequentially at 5°C. To the reaction mass was added free amine (88 g residue was dissolved in 200 mL dimethyl formamide) at 5°C and allowed to stir for 2 hrs at same temperature. After completion of the reaction, reaction mass was quenched in to water (5 lit) at 25-30°C and stirred for 15-20min at same temperature. Reaction mass was extracted with methylene chloride. Organic layer was separated and distilled completely under vacuum at below 40°C to obtaine residue. The residue was purified by silica gel columnchromatography using methylene dichloride and methanol. Thenthe product containing solvent fractions are concentrated under vacuum at below 50°C to obtain title compound. Wt: 95 g; Purity by HPLC: 93.77% (Retention time: 35.04).

Impurity profile of carfilzomib according to Reference Example (Table- 1):

TABLE-1
Impurity at RRT % by HPLC
0.14 0.7
0.18 0.2
0.26 0.16
0.30 0.15
0.36 2.88
0.41 0.2
0.45 0.4
0.52 0.09
0.55 0.6
0.62 0.09
0.84 0.3
0.88 0.11
0.92 0.2
1.08 0.15

EXAMPLE-1:

Purification of crude carfilzomib using preparative high performance liquid chromatography:

Preparative HPLC conditions:

Column Luna prep C18(3) (300×50)mm, 10µ
Mobile phase Mobile Phase A: Purified water
Mobile Phase B: Acetonitrile
Flow rate 50 mL/min
Detection UV 210nm
Injection volume 18(±3) mL
Run time 120 min
Elution Gradient
Time in min Mobile phase A
(% v/v) Mobile phase B
(% v/v)
0 80 20
10 80 20
20 45 55

Injection sample preparation: Crude carfilzomib was dissolved in a mixture of acetonitrile and tetrahydrofuran(1:2) at 25-35°C.

HPLC column packing: Prepared Luna prep C18(3) (0.35 kg) slurry by adding methanol under stirring at 25-30°C. Column was packed with the prepared silica slurry up to the bed length of ? 30 cm. Column was compressed to a pressure of 70 bars with pressure 60-80 bar.

Procedure:Loaded the sample solution onto the column with a flow rate of 50 mL/min and eluted with mobile phase of Mobile Phase A: purified water and Mobile Phase B: acetonitrile. The fractions related to pure carfilzomib was collected from 30 to 40 minutes of retention time and the purity of collected fractions was analyzed by usinga HPLC column halo C18 (150 x 4.6) mm, 2.7 mm. Pure fractions of carfilzomib were combined and the product was extracted using methylene chloride and the final product containing methylene chloride layer was concentrated under vacuum at below 40°C to obtain a pure carfilzomib.
Input wt: 4.5 g;Purity by HPLC: 93.77%;
Output wt: 3.2 g; Purity by HPLC: 99.9%; Yield: 0.71 (w/w)

Impurity profile of carfilzomib according to Example-1 (Table -2):

TABLE-2
Impurity at RRT Before purification
% by HPLC After purification
% by HPLC
0.14 0.7 Nil
0.18 0.2 Nil
0.26 0.16 Nil
0.30 0.15 Nil
0.36 2.88 Nil
0.41 0.2 0.05
0.45 0.4 Nil
0.52 0.09 Nil
0.55 0.6 Nil
0.62 0.09 Nil
0.84 0.3 Nil
0.88 0.11 0.05
0.92 0.2 Nil
1.08 0.15 Nil

EXAMPLE-2:

Purification of crude carfilzomib using preparative high performance liquid chromatography:

Preparative HPLC conditions and process is same as described in above Example-1 and the results are as follows:

Input wt: 80.0 g;Purity by HPLC:95.87%;
Output wt: 56.5 g;Purity by HPLC:99.87 %; Yield: (0.70 w/w)

Impurity profile of carfilzomib according to Example-2 (Table- 3):

TABLE- 3

Impurity at RRT Before purification
% by HPLC After purification
% by HPLC
0.14 0.5 Nil
0.18 0.2 Nil
0.26 0.1 Nil
0.30 0.06 0.01
0.36 1.98 Nil
0.41 0.02 0.02
0.45 0.38 Nil
0.52 0.30 0.01
0.55 0.08 Nil
0.62 0.13 Nil
0.84 0.07 Nil
0.88 0.10 0.09
0.92 0.13 Nil
1.08 0.08 Nil

COMPARATIVE EXAMPLE-1:

Purification of crude carfilzomib using maleate salt formation

Crude carfilzomib (90.5 g; HPLC Purity: 93.77%), acetonitrile (862 mL) and tetrahydrofuran (215 mL) were added in to a round bottom flask at 25-35°C. To the reaction mass was added maleic acid (14.4 g) at 25-35°C and stirred for 2 hrs at same temperature. Solids were filtered and washed with acetonitrile (431 mL),suck dried the solid for 5 min and dried the wet material under vacuum at 40-45°C for about 4 hrs to obtain the title compound.
Input wt: 90.5 g;Purity by HPLC: 93.77%;
Output wt: 70 g; Purity by HPLC: 99.1%; Yield: 0.77 (w/w)

Impurity profile of obtained carfilzomib maleate salt (Table-4):

TABLE-4:
Impurity at RRT Before purification
% by HPLC After salt purification
% by HPLC
0.10 0.00 0.3
0.14 0.7 0.1
0.18 0.2 0.05
0.26 0.16 Nil
0.30 0.15 Nil
0.36 2.88 0.25
0.41 0.2 0.1
0.45 0.4 0.05
0.52 0.09 Nil
0.55 0.6 Nil
0.62 0.09 Nil
0.84 0.3 Nil
0.88 0.11 0.05
0.92 0.2 Nil
1.08 0.15 Nil

The above carfilzomib maleate salt from comparative example-1 (70 g)and methylene chloride (2.1 lit) were added in to a round bottom flask and allowed to cool to 2-6°C. Reaction mass pH adjusted to 7.5-8.2 with aqueous sodium bicarbonate (21.1 g in 700 mL water) at 10-15°C. Separated the product containing organic layer and the aqueous layer was washed with methylene chloride. Combined product containing organic layer was dried with sodium sulphate (70 g) and concentrated under vacuum at below 35°C to obtain a residue. Then the residue was purified from silica gel column chromatography using silicycle silica gen IM60 and ethyl acetate as an eluent. The fractions related to pure carfilzomib was collected and the purity of collected fractions was analyzed by using a HPLC column halo C18 (150 x 4.6) mm, 2.7 mm. Pure fractions of carfilzomib were combined and concentrated under vacuum at below 40°C to obtain a pure carfilzomib.
Input wt: 70 g; Purity by HPLC: 99.1%;
Output wt: 38 g; Purity by HPLC: 99.7%; Yield: 0.41 w/w (from crude carfilzomib)

Impurity profile of carfilzomib after purification according to comparative example-1 (Table-5):

TABLE-5
Impurity at RRT Before purification
% by HPLC After purification
% by HPLC
0.10 0.3 Nil
0.14 0.1 0.05
0.18 0.05 0.05
0.26 Nil Nil
0.30 Nil Nil
0.36 0.25 0.15
0.41 0.1 Nil
0.45 0.05 Nil
0.52 Nil Nil
0.55 Nil Nil
0.62 Nil Nil
0.84 Nil Nil
0.88 0.05 0.05
0.92 Nil Nil
1.08 Nil Nil

Form the Table 3 and Table 4, it is evident that the diol impurity, which is referred to as RRT 0.36 is initially observed at about 2.88% by HPLC and after saltification the same impurity is decreased to 0.25% by HPLC and at the same time corresponding ester impurity which is referred to as RRT 0.10 is formed at the level of 0.3%. In order to remove the ester impurity additional purification using chromatography is required. Whereas the present invention proposes to remove the diol impurity by single prep-HPLC without forming corresponding ester impurity thereby multiple purification steps are avoided.

It will be understood that various modifications may be made to the embodiments disclosed herein. Therefore the above description should not be constructed as limiting, but merely as exemplifications of preferred embodiments. For example, the functions described above and implemented as the best mode for operating the present invention are for illustration purposes only. Other arrangements and methods may be implemented by those skilled in the art without departing from the scope and spirit of this invention. Moreover, those skilled in the art will envision other modifications within the scope and spirit of the specification appended hereto.
,CLAIMS:WE CLAIM:

1. A process for purification of carfilzomib, comprising subjecting the carfilzomib to a preparative high performance liquid chromatography (Prep-HPLC) using suitable eluent to obtain pure carfilzomib.

2. The process as claimed in claim 1, wherein the process specifically comprises:
a) dissolving carfilzomib in a suitable solvent or a mixture thereof,
b) subjecting the solution to a preparative high performance liquid chromatography (Prep-HPLC) using suitable eluent,
c) collecting pure fractions of carfilzomib, and
d) isolating the pure carfilzomib.

3. The process as claimed in claim 2, wherein the carfilzomib of step a) containing at least one related impurityidentified with following relative retention time (RRT):
RRT 0.14 0.18 0.26 0.30 0.36 0.41 0.45
0.52 0.55 0.62 0.84 0.88 0.92 1.08

4. The process as claimed in claim 2, wherein the suitable solvent of step a) is selected from the group consisting of alcohols, ketones, nitriles, ethers, halogenated hydrocarbons, esters and mixtures thereof.

5. The process as claimed in claim 4, wherein the suitable solvent is a mixture of acetonitrile and tetrahydrofuran.

6. The process as claimed in claim 2, wherein the suitable eluent is selected from the group consisting of alcohols, nitriles, ethers, halogenated hydrocarbons, esters, aliphatic hydrocarbons, water and mixtures thereof.

7. The process as claimed in claim 6, wherein the eluent is a mixture of water and acetonitrile.

8. The process as claimed in claim 2, wherein thepure carfilzomib is isolated by concentrating the pure fractions.
9. The process as claimed in claim 1 to 8, wherein the carfilzomib obtained by the process is substantially free of one or more relative retention time (RRT) impurities:
RRT 0.14 0.18 0.26 0.30 0.36 0.41 0.45
0.52 0.55 0.62 0.84 0.88 0.92 1.08

10. A process for purification of carfilzomib, comprising subjecting the crude carfilzomib to a preparative high performance liquid chromatography using water and acetonitrile as eluent to obtain pure carfilzomib; wherein the crude carfilzomib containing at least one related impurityidentified with following relative retention time (RRT):

RRT 0.14 0.18 0.26 0.30 0.36 0.41 0.45
0.52 0.55 0.62 0.84 0.88 0.92 1.08