FIELD OF THE INVENTION

The present invention relates to lyophilized pharmaceutical compositions comprising nitrogen mustards, particularly the nitrogen mustard is bendamustine. The invention also relates to the use of lyophilized pharmaceutical compositions of Bendamustine for the treatment of neoplastic diseases.

BACK GROUND OF THE INVENTION

The following description includes information that may be useful in understanding the present invention. It is not an admission that any such information is prior art, or relevant, to the presently claimed inventions, or that any publication specifically or implicitly referenced is prior art.

Bendamustine also known as (4-{5-[Bis (2-chloroethyl) amino]-l-methyl-2-benzimidazolyl} butyric acid, is an active nitrogen mustard (see Formula I, which shows bendamustine hydrochloride).

Bendamustine was initially synthesized in 1963 in the German Democratic Republic (GDR) and was available from 1971 to 1992 in that location under the name Cytostasan®. Since that time, it has been marketed in Germany under the trade name Ribomustin®. It has been widely used in Germany to treat chronic lymphocytic leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, and breast cancer.

Due to its degradation in aqueous solutions (like other nitrogen mustards), bendamustine is supplied as a lyophilized product. Earlier, the lyophilized formulation of bendamustine (Ribomustin®) contains bendamustine hydrochloride and mannitol in a sterile lyophilized form as a white powder for intravenous use following reconstitution. The finished lyophilisate is unstable when exposed to light. Therefore, the product is stored in brown or amber-colored glass bottles. The current lyophilized formulation of bendamustine contains degradation products that may occur during manufacturing of the drug substance and/or during the lyophilization process to make the finished drug product.

Currently, the bendamustine product is sold in the United States by Cephalon, Inc. as TREANDA® for injection, a lyophilized powder in a single-use vial indicated for the treatment of patients with chronic lymphocytic leukemia and indolent B-cell non-Hodgkin's lymphoma. A 25 mg dose vial contains 25 mg of bendamustine hydrochloride and 42.5 mg of mannitol, and a 100 mg dose vial contains 100 mg of bendamustine hydrochloride and 170 mg of mannitol. TREANDA® is intended for intravenous infusion only after reconstitution with sterile water for injection USP, and then further dilution with either 0.9% Sodium Chloride injection, USP, or 2.5% Dextrose/0.45% Sodium Chloride injection, USP. The pH of the reconstituted solution is 2.5-3.5. TREANDA® is supplied as a sterile non-pyrogenic white to off-white lyophilized powder, in a single-use vial.

Bendamustine hydrochloride is very unstable in an aqueous solution. The bis-2-chloroethylamino bond is found labile to get hydrolyzed in weak acid, neutral, or alkaline solution. Monohydroxy-bendamustine [HP-1] is formed as a hydrolyzed product rapidly in the presence of water.

Another compound- Bendamustine ethyl ester [hereinafter referred as BM1EE] is also known to be formed when bendamustine reacts with ethyl alcohol, where ethanol was utilized in penultimate steps of either API or formulation. BM1EE can be formed during drug substance manufacturing, e.g., during recrystalization and/or purification processes. BM1EE is reported to be one of the potential cytotoxic compound than bendamustine.

Following reconstitution with 40 mL sterile water for Injection, vials of bendamustine are stable for a period of 7 hours under room temperature storage or for 6 days upon storage at 2-8°C. The 500 mL admixture solution must be administered to the patient within 7 hours of vial reconstitution (assuming room temperature storage of the admixture).

Reports from the clinic indicate that reconstitution can require at least fifteen minutes and may require as long as thirty minutes. Besides being burdensome and time-consuming for the healthcare professional responsible for reconstituting the product, the lengthy exposure of bendamustine to water during the reconstitution process increases the potential for loss of potency and impurity formation due to the hydrolysis of the product by water.

German (GDR) Patent No. 80967 discloses an injectable preparation of y-[l-methyl-5-bis-(P-chloroethyl)-amino-benzimaidazolyl-(2)-]-butric acid hydrochloride.

German (GDR) Patent No. 159289 discloses an injectable solution of bendamustine.

Ribomustin® bendamustine Product monograph (updated January 2002) http://www.ribosepharm.de/pdf/ribomustin bendamustin/productmonogra- ph.pdf provides information about Ribomustin® including product description.

Ni et al. report that the nitrosourea SarCNU was more stable in pure tertiary butanol than in pure acetic acid, dimethyl sulfoxide, methylhydroxy, water or in TBA/water mixtures (Ni et al. (2001) Intl. J. Phamaceutics 226:39-46).

The lyophilized composition of nitrogen mustard Ifosfamide is disclosed in International Publication No. WO 2003/066027; U.S. Pat. Nos. 6,613,927; 5,750,131; 5,972,912; 5,227,373; and 5,204,335.

Teagarden et al. disclose lyophilized formulations of prostaglandin E-l made by dissolving PGE-1 in a solution of lactose and tertiary butyl alcohol (U.S. Pat. No. 5,770,230).

U.S. Patent Application Publication No. 2006/0159713 discloses a lyophilized pharmaceutical composition comprising bendamustine or bendamustine HC1, mannitol, tertiary-butyl alcohol and water.

The said lyophilized pharmaceutical composition contains not more than about 0.5% bendamustine ethylester.

U.S. Patent Application Publication No. 2013/0041003 discloses a pharmaceutical composition comprising bendamustine or bendamustine hydrochloride, mannitol, water, and a list of plethora solvents selected from ethanol, n-propanol, n-butanol, isopropanol, methanol, ethyl acetate, dimethyl carbonate, acetonitrile, dichloromethane, methyl ethyl ketone, methyl isobutyl ketone, acetone, 1-pentanol, methyl acetate, carbon tetrachloride, dimethyl sulfoxide, hexafluoroacetone, chlorobutanol, dimethyl sulfone, acetic acid, cyclohexane, or a combination thereof. Surprisingly, the application even covers lyophilized compositions with solvents, wherein bendamustine or bendamustine hydrochloride as well as mannitol are even insoluble. However, in the entirety of the specification, applicant only discloses solvents particularly ethanol, propanol, isopropanol and tertiary butanol along with mannitol and water.

Chandrasekhar et al WO2012/103226 A2 discloses a lyophilized pharmaceutical formulation of bendamustine, comprising not more than about 0.4% by weight of HP-1 compound, based on the bendamustine content, of a compound having the structure:

the formulation being prepared by lyophilizing a solution that does not contain tertiary-butyl alcohol or ethanol. The lyophilized pharmaceutical formulation being prepared by lyophilizing a solution containing necessarily water and at least one organic solvent or a mixture thereof. This application summarizes that the lyophilized powders containing bendamustine formulations are difficult to make and organic solvents were found to be more suitable to avoid degradation of bendamustine HCI in pre-lyophilization bulk solutions. As mannitol cannot be dissolved in completely organic solvent systems, water should necessarily be a part of solvent system for preparing bulk solution. In view of other difficulties for such lyophilized formulation to lower the rate of degradation of bendamustine HCI, other critical factors include- solvent systems for manufacturing bulk solution, the sequence of addition of ingredients, temperatures, duration of critical steps in lyophilization, and the like.

Palepu Nagesh R et al in US 2011/0184036 discloses long term storage stable bendamustine-containing solution compositions. The compositions include bendamustine or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable fluid which can include in some embodiments PEG, PG or mixtures thereof and an antioxidant or chloride ion source. The bendamustine-containing compositions have less than about 5% total impurities, on a normalized peak area response ("PAR") basis as determined by high performance liquid chromatography ("HPLC") at a wavelength of 223 nm, after at least about 15 months of storage at a temperature of from about 5°C to about 25°C.

Drager Anthony et al US8344006 discloses a stable, non-aqueous liquid, pharmaceutical formulation comprising from about 5 mg/ml to about 120 mg/mL of bendamustine, or a pharmaceutically acceptable salt thereof, solubilized in about 66% (v/v) of dimethylacetamide and about 34% (v/v) of propylene glycol, wherein said formulation, following dilution with a pharmaceutically acceptable diluent, is suitable for injection into a patient without lyophilization.

Cooper Martin Ian et al in US 2012/0071532 discloses novel solid forms of bendamustine hydrochloride form I, form III, form IV, amorphous and mixtures thereof as well as methods of their preparation and use.

The application also disclose lyophilized compositions of the said forms or its mixtures derived from lyophilzation excipient mannitol and organic solvent selected from methanol, ethanol, n-propanol, iso-propanol, n-butanol, tert-butanol, or a mixture thereof.

Because of their high reactivity in aqueous solutions, bendamustine hydrochloride is preferred to be supplied in lyophilized form. Also it is well-known that bendamustine hydrochloride is hygroscopic in nature ; it is always preferred to select diluents as sugar alcohols like mannitol, for preparing such lyophilized bendamustine compositions.

Despite various disclosures as discussed, it is evident that such lyophilized bendamustine products may not be easily made due to one or more of the following reasons:

a) Bendamustine or bendamustine hydrochloride, and mannitol are soluble in few organic solvent-water mixtures like ethanol, n-propanol, n-butanol, isopropanol etc; and form clear solutions in specific ratios of these mixtures; however, it is difficult to achieve the stable solutions for desired time period of lyophilization cycles which in turn may increase the impurity levels.

b) Bendamustine or bendamustine hydrochloride, and mannitol are insoluble in few solvents like dimethyl carbonate, dichloromethane, methyl ethyl ketone, methyl isobutyl ketone etc. Hence, it is difficult to optimize lyophilization cycles for such pre-lyophilized bendamustine dispersions.

c) Difficulties in selecting optimizing appropriate ratio of pre-lyophilized solvent mixture containing organic solvent and water, which may be amenable to scale up.

d) Difficulties in optimizing lyophilization parameters.

e) At higher batch size, there is a difficulty to optimize lyophilization parameters for preparing safe lyophilized bendamustine products, with trace amounts of organic solvents, of a permitted daily exposure (mg/day), intended for human use.

f) To lower the rate of degradation of bendamustine HCI, other critical factors include-the sequence of addition of ingredients, temperatures, duration of critical steps in lyophilization, and the like.

The lyophilized bendamustine products also require reconstitution in water, by skilled hospital personal prior to administration, and it is always needed to ensure quicker reconstitution time of bendamustine lyophilized cakes in water.

However, due to the aforementioned reasons, it shows impact on lyophilized bendamustine product, with increase in the reconstitution time of bendamustine lyophilized cakes, and further, if reconstitution time increases, drug degradation by hydrolysis cannot be avoided.

Thus, a need exists for a simple lyophilized formulation of bendamustine that is much easier to prepare, having better reconstitution results with better impurity profile.

SUMMARY OF THE INVENTION

Surprisingly, the present invention is directed to a lyophilized bendamustine composition comprising bendamustine or bendamustine hydrochloride, and mannitol, derived from dimethyl sulfoxide.

The present invention is directed to a lyophilized bendamustine composition derived from pre-lyophilized solution, devoid of water. The present invention is directed to a lyophilized bendamustine composition derived from pre-lyophilized solution of dimethyl sulfoxide.

The present invention is directed to a pre-lyophilized bendamustine composition comprising bendamustine or bendamustine hydrochloride, mannitol, and dimethyl sulfoxide.

The present invention is directed to a pre-lyophilized bendamustine composition, wherein Bendamutsine HCI is crystalline Form-SM characterized by X-ray powder diffraction pattern comprising atleast 7 characteristic peaks selected from the XRPD 2 theta degrees peaks at 7.42, 10.60, 11.17, 16.43, 17.94, 22.89, 26.33, 28.77, 30.28, 31.92, 40.89 ± 0.1 20°.

The present invention is directed to a pre-lyophilized bendamustine composition, wherein Bendamutsine HC1 is crystalline Form II.

The present invention is directed to a process for preparing a lyophilized pharmaceutical composition of comprising:

a) dissolving bendamustine or bendamustine hydrochloride, and mannitol, in dimethyl sulfoxide; and

b) lyophilizing the pre-lyophilization solution.

The present invention is directed to a pre-lyophilized bendamustine composition comprising bendamustine or bendamustine hydrochloride, mannitol, and dimethyl sulfoxide.

The present invention is directed to a lyophilized pharmaceutical composition, derived from a pre-lyophilization solution containing bendamustine or bendamustine hydrochloride at a concentration of about 5 to 50 mg/ml, and mannitol at a concentration of about 10 to 80 mg/ml.

The present invention is directed to a lyophilized bendamustine pharmaceutical composition, containing not more than about 0.2% (area percent of bendamustine) HP1 as shown in Formula II, wherein said HP1 is the amount of HP1 present at release.

The present invention is directed to a lyophilized bendamustine pharmaceutical composition, containing no detected levels of bendamustine ethylester present at release.

The present invention is directed to a method of treating a medical condition selected from chronic lymphocytic leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, breast cancer, small cell lung cancer, and an autoimmune disease, by administering therapeutically effective amount of the dissolved preparation of lyophilized pharmaceutical composition.

BRIEF DESCRIPTION OF THE DRAWING
Not limited to Bendamustine hydrochloride Monohydrate Form-SM characterized by X-ray powder diffraction pattern comprising atleast 7 characteristic peaks selected from the XRPD 2 theta degrees peaks at 7.42, 10.60, 11.17, 16.43, 17.94, 22.89, 26.33, 28.77, 30.28, 31.92, 40.89 ± 0.1 20°, and Bendamustine hydrochloride Form II, disclosed under US20120071532. Various other crystalline forms of Bendamustine hydrochloride, mentioned under prior art, can be included here for preparing the invention lyophilized compositions.

Fig.l is illustration of X-ray powder diffraction (XRPD) pattern of Bendamustine hydrochloride monohydrate Form-SM, disclosed under WO201205993 5.

Below table provides X-ray powder diffraction (XRPD) pattern of Bendamustine hydrochloride Form II, disclosed under US20120071532.

DETAILED DESCRIPTION OF THE INVENTION

In emboidment of the present invention, provides a lyophilized bendamustine composition comprising bendamustine or bendamustine hydrochloride, and mannitol, derived from dimethyl sulfoxide.

In emboidment of the present invention, provides a lyophilized bendamustine composition derived from pre-lyophilized solution, devoid of water. The present invention is directed to a lyophilized bendamustine composition derived from pre-lyophilized solution of dimethyl sulfoxide.

In emboidment of the present invention, provides a pre-lyophilized bendamustine composition comprising bendamustine or bendamustine hydrochloride, mannitol, and dimethyl sulfoxide.

In emboidment of the present invention, provides a pre-lyophilized bendamustine composition, wherein Bendamutsine HC1 is crystalline Form-SM characterized by X-ray powder diffraction pattern comprising atleast 7 characteristic peaks selected from the XRPD 2 theta degrees peaks at 7.42, 10.60, 11.17, 16.43, 17.94, 22.89, 26.33, 28.77, 30.28, 31.92, 40.89 ± 0.1 20°.

In emboidment of the present invention, provides a pre-lyophilized bendamustine composition, wherein Bendamutsine HC1 is crystalline Form II.

In emboidment of the present invention, provides a process for preparing a lyophilized pharmaceutical composition of comprising:

a) dissolving bendamustine or bendamustine hydrochloride, and mannitol, in dimethyl sulfoxide; and
b) lyophilizing the pre-lyophilization solution.

In emboidment of the present invention, provides a pre-lyophilized bendamustine composition comprising bendamustine or bendamustine hydrochloride, mannitol, and dimethyl sulfoxide.

The present invention is directed to a lyophilized pharmaceutical composition, derived from a pre-lyophilization solution containing bendamustine or bendamustine hydrochloride at a concentration of about 5 to 50 mg/ml, and mannitol at a concentration of about 10 to 80 mg/ml.

In emboidment of the present invention, provides a lyophilized bendamustine pharmaceutical composition, containing not more than about 0.2% (area percent of bendamustine) HP1 as shown in Formula II, wherein said HP1 is the amount of HP1 present at release.

In emboidment of the present invention, provides a lyophilized bendamustine pharmaceutical composition, containing no detected levels of bendamustine ethylester present at release.

In emboidment of the present invention, provides a method of treating a medical condition selected from chronic lymphocytic leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, breast cancer, small cell lung cancer, and an autoimmune disease, by administering therapeutically effective amount of the dissolved preparation of lyophilized pharmaceutical composition.

As used herein, the term "bendamustine" includes the compound bendamustine, pharmaceutically acceptable salts of bendamustine, isomers, solvates, complexes and hydrates, anhydrous forms thereof, and any polymorphic or amorphous forms or combinations thereof.

The salt bendamustine hydrochloride will be discussed herein as a representative of any of these, although the disclosure is not limited to the use of only this salt.

The term "formulation" refers to preparing a drug, e.g., bendamustine, in a form suitable for administration to a patient, such as a human. Thus, a "formulation" can include pharmaceutically acceptable excipients, including diluents or carriers.

As used herein, the term "excipient" means the substances used to formulate active pharmaceutical ingredients (API) into pharmaceutical formulations; in a preferred embodiment, an excipient does not lower or interfere with the primary therapeutic effect of the API. Preferably, an excipient is therapeutically inert. The term "excipient" encompasses carriers, diluents, vehicles, solubilizers, stabilizers, bulking agents, and binders. Excipients can also be those substances present in a pharmaceutical formulation as an indirect or unintended result of the manufacturing process. Preferably, excipients are approved for or considered to be safe for human and animal administration, i.e., GRAS substances (generally regarded as safe). GRAS substances are listed by the Food and Drug administration in the Code of Federal Regulations (CFR) at 21 CFR section 182 and 21 CFR section 184, incorporated herein by reference. Preferred excipients include, but are not limited to, hexitols, including mannitol and the like.

The term "organic solvent" means an organic material, usually a liquid, capable of dissolving other substances.

The term "pharmaceutically acceptable" as used herein describes substances or components that do not cause unacceptable losses of pharmacological activity or unacceptable adverse side effects. Examples of pharmaceutically acceptable ingredients are those having monographs in United States Pharmacopeia (USP 29) and National Formulary (NF 24), United States Pharmacopeial Convention, Inc, Rockville, Maryland, 2005 ("USP/NF"), or a more recent edition, and the components listed in the continuously updated Inactive Ingredient Search online database of the FDA. Other useful components that are not described in the USP/NF, etc. may also be used.

The term "pharmaceutical composition" as used herein shall mean a composition that is made under conditions such that it is suitable for administration to humans, e.g., it is made under GMP conditions and contains pharmaceutically acceptable excipients, e.g., without limitation, stabilizers, bulking agents, buffers, carriers, diluents, vehicles, solubilizers, and binders. As used herein pharmaceutical composition includes but is not limited to a pre-lyophilization solution or dispersion as well as a liquid form ready for injection or infusion after reconstitution of a lyophilized preparation.

As used herein, the term "lyophilized composition" or "lyophilized bendamustine pharmaceutical composition"; can be used in contrary, refers to any solid material obtained by lyophilization i.e., freeze-drying of non-aqueous solution or freeze-drying of aqueous solution; non-aqueous solution refers to a solution composed of one or more non-aqueous solvent(s). Preferably, a lyophilized preparation is one in which the solid material is obtained by freeze-drying a solution composed of one or more non-aqueous solvents, more preferably the non¬aqueous solvent is Dimethyl sulfoxide.

By "degraded" or "degradation" is meant that the active has undergone a change in chemical structure.
The term "therapeutically effective amount" as used herein refers to that amount of the compound being administered that will relieve to some extent one or more of the symptoms of the disorder being treated. In reference to the treatment of neoplasms, a therapeutically effective amount refers to that amount which has the effect of (1) reducing the size of the tumor, (2) inhibiting (that is, slowing to some extent, preferably stopping) tumor metastasis, (3) inhibiting to some extent (that is, slowing to some extent, preferably stopping) tumor growth, and/or, (4) relieving to some extent (or, preferably, eliminating) one or more symptoms associated with the cancer. Therapeutically effective amount can also mean preventing the disease from occurring in an animal that may be predisposed to the disease but does not yet experience or exhibit symptoms of the disease (prophylactic treatment). Further, therapeutically effective amount can be that amount that increases the life expectancy of a patient afflicted with a terminal disorder. Typical therapeutically effective doses for bendamustine for the treatment of non-Hodgkin's lymphoma can be from about 60-120 mg/m2 given as a single dose on two consecutive days. The cycle can be repeated about every three to four weeks. For the treatment of chronic lymphocytic leukemia (CLL) bendamustine can be given at about 80-100 mg/m2 on days 1 and 2. The cycle can be repeated after about 4 weeks. For the treatment of Hodgkin's disease (stages II-IV), bendamustine can be given in the "DBVBe regimen" with daunorubicin 25 mg/m2 on days 1 and 15, bleomycin 10 mg/m2 on days 1 and 15, vincristine 1.4 mg/m2 on days 1 and 15, and bendamustine 50 mg/m2 on days 1-5 with repetition of the cycle about every 4 weeks. For breast cancer, bendamustine (120 mg/m2) on days 1 and 8 can be given in combination with methotrexate 40 mg/m2 on days 1 and 8, and 5-fluorouracil 600 mg/m2 on days 1 and 8 with repetition of the cycle about every 4 weeks. As a second-line of therapy for breast cancer, bendamustine can be given at about 100-150 mg/m2 on days 1 and 2 with repetition of the cycle about every 4 weeks.

As used herein, the term "vial" refers to any walled container, whether rigid or flexible.

As used herein, "trace amount of an organic solvent" means an amount of solvent that is equal to or below recommended levels for pharmaceutical products, for example, as recommended by ICH guidelines (International Conferences on Harmonization, Impurities—Guidelines for Residual Solvents. Q3C. Federal Register. 1997; 62(247):67377). The lower limit is the lowest amount that can be detected.
The term "release" or "at release" means the drug product has met the release specifications and can be used for its intended pharmaceutical purpose.

The term "ND" or "Not detected" or "No detected limits of means the drug or drug product has no detected content of a particular impurity as compared to bendamustine content.

Bendamustine impurities:
Impurity-A: Monohydroxy Bendamustine or BND- HP1.
Impurity-B: Bendamustine Dimer.

Impurity-C: Isopropyl 4-(5-(bis (2-chIoroethyl) amino)-l-methyl-l H-benzo[d]imidazol-2-yl) butanoate (Formula V).
Impurity-D or BND-Ethyl Ester:

Example 1
Bendamustine hydrochloride for injection 25mg/vial and lOOmg/vial with DMSO.

Manufacturing process:

1. 90% of total batch quantity of DMSO collected in a processing vessel of suitable capacity.

2. Mannitol was added to the above step and stirred for 5min.

3. Weighed batch quantity of Bendamustine HC1 was added to above step 2.0 and Stir for 10 minutes to get solution.

4. Volume was made up to 100% with DMSO, and stirred for 5 minutes.

5. The solution from above step was filtered through 0.2u membrane filter; the solution was filled in to 10 mL Amber vial with 2.0 mL fill volume, and in 20mL Amber vial with 8.0 mL fill volume, half stoppered the vials then loaded to lyophilizer to freeze dry the contents.

6. Lyophilization recipe is as follows: Precooling of shelf to -5° C. Freezing at -20° C for 2 to 5 hours. Freezing at -45° C for 6 to 8 hours.

Primary drying at -40° C for 10 to 12 hours at 220mtorr. Primary drying at -20° C for 10 to 14 hours at220mtorr. Primary drying at 0° C for 8 to 12 hours at 150mtorr. Primary drying at 15° C for 4 to 6 hours at 150mtorr. Primary drying at 30° C for 4 to 8 hours at 70mtorr. Secondary drying at 40° C for 10 to 12 hours at 70mtorr.

7. After completion of lyophilization, the vials were completely stoppered under Nitrogen,
unloaded and sealed with flip off aluminium seals.

Lyophilized cakes obtained in example 1 is characterized for cake appearance & solution clarity, after dissolving in WFI to get 5mg/mL concentration of bendamustine.

100 mg/vial lyophilized cake of example 1 is characterized by HPLC for assay & related substances.
Analytical method description for Assay by HPLC: Reversed phase Liquid Chromatography with Gradient elution and UV Detection.

Buffer preparation: Weigh and transfer 3.85g of ammonium acetate in lOOOmL of water, dissolve and add lmL of triethylamine, adjust pH 4.7 with dilute glacial acetic acid. Filter and degas.

Mobile phase A: Transfer 580mL of buffer solution 420mL of acetonitrile into a lOOOmL of beaker and mix and degas.

Mobile Phase B: Acetonitrile, filter and degas.
Chromatographic conditions:
Column : Symmetry shield RP18, 250mm x 4.6mm, 5 urn or Equivalent
Wavelength : 233nm
Flow rate : 1.0 ml/ minute

Injection volume : 20 uL
Diluent : Dimethyl sulphoxide
Coloumn temperature: 25°C
Sampler temperature : 25°C
Run Time : 15 minutes
Standard Solution:

Weigh and transfer 4.0mg of Bendamustine Hydrochloride working standard into lOOmL volumetric acid dissolve and dilute to volume with dimethyl sulphoxide. (Or prepare equivalent to 0.04mg/ml).
Sample preparation for lOOmg/Vial:

Take 2 vials and reconstitute each vial with 5ml of diluent and transfer into 100ml of volumetric flask, mix well and dilute to volume with diluent. Further, transfer 1ml of the above solution into 50ml volumetric flask and dilute to volume with diluent.

Procedure:
Separately inject Blank (diluent) (one injection), Standard solution (five injections) into chromatograph and check the system suitability.

System Suitability Parameters:

1. The Tailing factor for Bendamustine peak is NMT 2.0.

2. The relative standard deviation (RSD) for 5 injections of standard preparation is NMT 2.0%.
Analytical method description for Related Substances by HPLC: Reversed phase Liquid Chromatography with Gradient elution and UV Detection.

Buffer Preparation: Transferred 1ml of Trifluro acetic acid in 1000ml volumetric flask containing 500ml water, mixed well and diluted with water.

Mobile phase A: Prepared a mixture of Buffer solution & Acetonitrile in the ratio of 90:10.
Mobile Phase B: Prepared a mixture of Buffer solution & Acetonitrile in the ratio of 50:50.
Chromatographic conditions:
Column : Kinetex CI8; 100 mm X 4.6mm, 2.6um
Wavelength : 230nm
Flow rate : 1.0 ml / minute
Injection volume : 10 uL
Diluent : Dimethyl sulfoxide (DMSO)
Run Time : 20 minutes
Column Temperature : 30°C
Sampler Temperature: 25°C
Gradient Program:
Diluted Standard preparation (2 PPM):

Weigh and transfer 2.0mg of Bendamustine HC1 into 50ml volumetric flask add 25ml of diluent dissolve and dilute to volume with diluent. Further transfer 1ml of the above solution into 20ml volumetric flask, diluent to volume with diluent.(or prepare equivalent) Sample preparation for 100 mg/Vial: Take 1 vial and reconstitute with 20 ml of diluent and transfer into 50ml volumetric flask and dilute to volume with diluent.
Procedure: Separately inject Blank (diluent) (one injection), Diluted Standard Preparation (six injections) into chromatograph and check the system suitability.

System Suitability Parameters:

1. The Tailing factor for Bendamustine HC1 peak is NMT 2.0.

2. The relative standard deviation (%RSD) for 6 injections of standard preparation is NMT 5.0%.
Retention time and elution order of impurities mentioned below:
Assay & Related substances results of bendamustine lyophilized cake lOOmg/vial of example 1, shown under below table:

ND - Not detected.

Claims

1. A pre-lyophilized bendamustine composition comprising bendamustine or bendamustine hydrochloride, mannitol, and dimethyl sulfoxide.

2. A pre-lyophilized bendamustine composition according to claim 1, wherein Bendamutsine HC1 is crystalline Form-SM characterized by X-ray powder diffraction pattern comprising atleast 7 characteristic peaks selected from the XRPD 2 theta degrees peaks at 7.42, 10.60, 11.17, 16.43, 17.94,22.89,26.33,28.77, 30.28, 31.92, 40.89 ±0.1 29°.

3. A pre-lyophilized bendamustine composition according to claim 1, wherein Bendamutsine HC1 is crystalline Form II.

4. A lyophilized bendamustine composition comprising bendamustine or bendamustine
hydrochloride, and mannitol, derived from dimethyl sulfoxide.

5. A process for preparing a lyophilized pharmaceutical composition of comprising:

a) dissolving bendamustine or bendamustine hydrochloride, and mannitol, in dimethyl sulfoxide; and

b) lyophilizing the pre-lyophilization solution.

6. A lyophilized pharmaceutical composition according to claim 5, wherein the pre-
lyophilization solution contains bendamustine or bendamustine hydrochloride at a
concentration of about 5 to 50 mg/ml, and mannitol at a concentration of about 10 to 80 mg/ml.

7. A lyophilized pharmaceutical composition according to according to claims 4-6, contains not
more than about 0.2% (area percent of bendamustine) HP1 as shown in Formula II, wherein
said HP1 is the amount of HP1 present at release.

8. A lyophilized pharmaceutical composition according to according to claims 4-7, containing no detected levels of bendamustine ethylester present at release.

9. A method of treating a medical condition selected from chronic lymphocytic leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, breast cancer, small cell lung cancer, and an autoimmune disease, by administering therapeutically effective amount of the dissolved preparation of lyophilized pharmaceutical composition of claims 4-8.