FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
Complete Specification [See Sections 10 and rule 13]
Title: Stable lyophilizcd formulation of Bendamustine
Applicant: (a) Astron Research Limited
(b) "Nationality: Indian
(c) 10th Floor. Premier House Bodakdev, Opp. Gurudwara Sarkhej - Gandbinagar Highway Ahmedabad 380054
Gujarat. India.
The following specification particularly describes the invention and the manner in which it is to be performed.

FIELD OF THE INVENTION
This invention relates to a stable lyophilized Bendamustine composition having better impurity profile and process for its preparation.
BACKGROUND OF THE INVENTION
Bendamustine chemically known as 4-[5-[Bis(2-chloroethyl)amino]-l-methylbenzimidazoI-2-yl]butanoic acid is a nitrogen mustard used in the treatment of chronic lymphocytic leukemia and lymphomas. It belongs to the family of drugs called alkylating agents.
Bendamustine is marketed under the trade names Ribomustin® and Treanda® Bendamustine degrade in aqueous solutions (like other nitrogen mustards) and hence it is supplied as a lyophilized product. The current 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 lyophilizate 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. Various impurities are found in the finished formulation like dimmer form (Impurity B) and other unknown impurities such as impurity A. impurity C etc.
German Patent DE 80967 discloses an injectable preparation of gamma.-[1-methyl-5-bis-(.beta.-chloroethyl)-amino-benzimaidazolyl-(2)- ]-butric acid hydrochloride.

German Patent DE 159289 discloses an injectable solution of Bendamustine in anhydrous monovalent or polyvalent alcohol (polyol).
US20060159713 discloses methods of producing lyophilized Bendamustine having lower impurity profile.
So there is a need for stable lyophilized Bendamustine composition having better impurity profile and process for preparation of such formulation.
OBJECTS OF THE INVENTION
The main objective of the present invention is to obtain stable lyophilized Bendamustine composition having better impurity profile.
Another object of the invention is to develop a process for the preparation of stable lyophilized Bendamustine composition having better impurity profile.
Another object of the present invention is to develop a process for the preparation of stable lyophilized Bendamustine composition by controlled lyophilization.
Another object of the present invention is to develop a process for the preparation of stable lyophilized Bendamustine composition having better impurity profile by controlling moisture content of the end product at optimum level in order to reduce impurity.
Another object of the present invention is to develop a process for the preparation of stable lyophilized Bendamustine comprising annealing step followed by controlled

iyophilization to obtain porous uniform cake which is easy to reconstitute at the time of administration.
SUMMARY OF THE INVENTION
Invention relates to stable lyophilized Bendamusline composition having better impurity profile.
Further invention relates to stable lyophilized Bendamustine composition having optimum moisture content and better impurity profile.
Further invention relates to stable lyophilized Bendamustine composition having better impurity profile and easy to reconstitute.
Further invention relates to process for preparing stable lyophilized Bendamustine composition having optimum moisture content and better impurity profile.
Further invention relates to process for preparing stable lyophilized Bendamustine composition which is easy to reconstitute by performing annealing step prior to Iyophilization.
Further invention relates to process for preparing stable lyophilized Bendamustine composition having optimum moisture content and better impurity profile and easy to reconstitute at the time of administration.

DETAILED DESCRIPTION Definitions:
"Stable pharmaceutical composition" is meant any pharmaceutical composition having sufficient stability to have utility as a pharmaceutical product. Preferably, a stable pharmaceutical composition has sufficient stability to allow storage at a convenient temperature, preferably between -20 C and 40 C. more preferably about 2 C to about 8 C, for a reasonable period of time, e.g.. the shelf-life of the product which can be as short as one month but is typically six months or longer, more preferably one year or two year. For purposes of the present invention stable pharmaceutical composition includes reference to pharmaceutical compositions with specific ranges of impurities as described herein. Preferably, a stable pharmaceutical composition is one which has minimal degradation of the active ingredient, e.g.. it retains at least about 80% of un-degraded active, preferably at least about 90%, and more preferably at least about 95%, after storage at 2-30 °C for a 1 to 3 year period of time.
"Controlled lyophilization" means controlling different parameters of iyophilization like lyophilization cycle time/duration, temperature, vacuum, etc.
"Lyophilization" is the technique used for the drug product may be susceptible to physical and chemical degrade in solution phase. Lyophilization also known as freeze-drying technique is a technique used to remove water from a solution to leave a dry 'cake' as an end product. In lyophilization or freeze-drying process water is removed from a product after it is frozen and placed under a vacuum, allowing the ice to change directly from a solid to a vapor, without passing through a liquid phase. The process consists of three separate, unique, and interdependent processes; a freezing phase, a primary drying phase (sublimation), and a secondary drying phase (desorption).

"Annealing" is generally a thermal treatment process wherein product temperature is cycled, for example freezing the drug solution to -40 °C and holding at this temperature for specific time period followed by raising temperature up to -10 °C and holding at this temperature for specific time period and repeating the same steps in cyclic manner to get desired results.
The term "Bendamustine" mentioned herewith refers to Bendamustine or its pharmaceutically acceptable salts thereof, preferably Bendamustine hydrochloride salt.
Bendamustine degrade in aqueous solutions (like other nitrogen mustards) and hence it is supplied as a lyophilized product to avoid exposure to water. However, the inventors of the present invention have surprisingly found that reducing moisture below certain level the stability of the lyophilized product found to be less as amount of impurity B increases upon storage for 2-3 months. So. lyophilized Bendamustine product having optimum moisture level shows better stability upon storage for 2-3 months. Such optimum moisture level in lyophilized product found to be 1-10 % preferably 1 -5% and more preferably 1 -4%.
In the present invention inventors have surprisingly found that keeping moisture content in the lyophilized powder of Bendamustine to optimum level up to 1 -5% and more preferably 1-4% increases the stability of the end product as level of impurity B over a period of time does not increases significantly as compared to the lyophilized powder of Bendamustine containing moisture content below 1%.
The product produced with less aggressive secondary drying which probably could not remove bound water, stabilizes the product with respect to degradation into

Impurity B (Bendamustine Dimer). The reason for the same could be the role of water molecule to make the chlorine end group less reactive. It is known that chlorine end group is very reactive and would always tend to react. Presence of water molecule makes the reactive chlorine less reactive thereby would reduce or avoid further reaction. In Bendamustine also there are two free chlorine free groups which in absence of water become more reactive and leads high amount of Impurity B which is dimmer.
The moisture content of the final formulation can be controlled in many ways. Such as controlling different parameters of the lyophilization cycle like process time; vacuum, etc. Further person skilled in the art can also achieve the desired moisture content into final formulation by humidifying the lyophilizate by exposing it to water vapor.
Further, inventors of the present invention have also found that performing annealing during lyophilization gives more uniform porous cake as end product which takes less time to reconstitute as compare the product prepared without performing annealing step during lyophilization.
The iyophilized formulations of the present invention may be reconstituted with water, preferably Sterile Water for Injection, or other sterile fluid such as co-solvents. with shaking for appropriate time duration to obtain appropriate solution of Bendamustine for administration, as through parenteral injection following further dilution into an appropriate intravenous admixture container, for example, normal saline.

Examples: In the following section embodiments are described by a way of examples to illustrate the process of invention. However, these are not intended in any way to limit the scope of present invention.
Example-!:

Sr. No. Ingredients Quantity (mg/mL)
1. Bendamustine hydrochloride 15.0
2. Mannitol 25.5
j, Tert-Butanol (TBA) 314.0
4. Water for Injection 595.5
5. Nitrogen q.s. to sparge
Manufacturing Process:
1. A batch quantity of Water for Injections of 2°C-8°C was taken into the Stainless Still manufacturing vessel and temperature was maintain to 2°C-8°C by Brian / chilled water circulation followed by nitrogen sparging for 30 minutes.
2. A Mannitol was added to the above Water for Injection and stirred for 10 min.
3. A batch quantity of Tertiary Butyl Alcohol was added into the solution of step-2 and stirred for 15 minutes with nitrogen sparging.
4. Bendamustine hydrochloride was added to the bulk solution of step-3 and the bulk solution was stirred with nitrogen sparging for 60 minutes with maintaining the temperature at 2°C-8°C.
5. Tertiary Butyl Alcohol if required was added to the final solution of the step-4. to make up the batch weight and stirred for 5 minutes.
6. The solution of the step-5 was then lyophilized in suitable container under
controlled conditions.
Example-2:
The lyophilized compositions of Bendamustine were prepared using procedure explained in Example-1 with controlling different parameters of the process to obtain final product having different initial moisture contents eg. 0.2% and around 3.0%

and kept them for stability testing. The impurity profile of these lyophilized compositions obtained at different time interval and temperature conditions are disclosed below in respective Tables,
Table - 1: Stability data of more aggressive secondary drying [Initial Water Content - 0.2%]

Parameters Initial 1 M/40OC 1M/30°C IM/25°C 2 M/40°C 2M/30°C 2M/25°C
Description White Lyz. Pwd. White Lyz. Pwd. White Lyz. Pwd. White Lyz. Pwd. White Lyz. Pwd. White Lyz. Pwd. White Lyz. Pwd.
Assay of BND HCI 100.9 100.8 NA NA NA NA NA
Related substances
Impurity A 0.233 0.315 0.297 0.195 0.25 0.244 0.316
Impurity B 0.067 0.727 0.329 0.235 1.191 0.633 0.379
Impurity C 0.079 0.072 0.071 0.071 0.076 0.079 0.083
Single unknown Impurity 0.118 0.204 0.232 0.171 0.21 1 0.142 0.132
Total Impurity 0.572 1.670 1.130 0.828 2.248 1.331 1.066
White Lyz, Pwd. - White lyophilized powder BND HCI - Bendamustine hydrochloride
Table - 2A: Stability data of less aggressive secondary drying [lnitial Water Content - Around 3.0%|

Parameters Initial 1 M/40°C 2M/40°C 3M/40°C 3M/25PC 6M/40°C 6M/25°C
Description White Lyz. Pwd. White Lyz. Pwd. White Lyz. Pwd. White Lyz. Pwd. White Lyz. Pwd. White Lyz. Pwd. White Lyz. Pwd.
Assay of BND HCI 97.3 NA NA NA NA NA NA
Related substances
Impurity A 0.186 0.105 0.22 0.18 0.12 0.17 0.15
Impurity B 0.016 0.037 0.05 0.02 0.07 0.012 ND
impurity C 0.063 0.069 0.08 0.08 0.08 0.046 0.054
Single unknown Impurity 0.079 0.046 0.072 0.137 0.091 0.071 0.032
Total Impurity 0.367 0.317 0.51 0.48 0.46 0.40 0.29
White Lyz. Pwd. - White lyophilized powder BND HCI - Bendamustine hydrochloride

Table - 2B: Stability data of less aggressive secondary drying (Initial Water Content - Around 3.0%]

Parameters Initial IM/40°C 2M/40°C 3M/40°C 3M/25°C 6M/40°C 6M/25°C
Description White Lyz. Pwd. White Lyz. Pwd. White Lyz. Pwd. While Lyz. Pwd. White Lyz. Pwd. White Lyz. Pwd. White Lyz. Pwd.
Assay of BND HCl' 99.1% 99.2 98.7 101.2 100.7 100.2 99.0
Related substance s
Impurity A 0.101% 0.246% 0.143% 0.133% 0.095% 0.131% 0.087%
Impurity B 0.010% 0.042% 0.062% 0.036% 0.029% 0.026% 0.017%
Impurity C 0.069% 0.081% 0.050% 0.073% 0.076% 0.071% 0.072%
Single unknown Impurity 0.041% 0.060% 0.052% 0.035% 0.039% 0.081% 0.031%
Total Impurity 0.234% 0.513% 0.482% 0.339% 0.274% 0.540% 0.285%
White Lyz. Pwd. - White lyophilized powder BND HCl - Beiidamustinc hydrochloride
Table — 2C: Stability data of less aggressive secondary drying [Initial Water Content- Around 3.0%]

Parameters Initial 3M/40°C 3M/25°C 6M/40°C 6M/25°C
Description White Lyz. Pwd. White Lyz. Pwd. White Lyz. Pwd. White Lyz. Pwd. White Lyz. Pwd.
Assay of BND HCl 102.2 102 101.8 10!. 6 102.6
Related substances
Impurity A 0,147% 0.330% 0.205% 0.264% 0.181%
Impurity B 0.015% 0.008% ND 0.008% 0.008%
Impurity C 0.087% 0.064% 0.065% 0.074% 0.076%
Single unknown Impurity 0.037% 0.059% 0.045% 0.067% 0,032%
Total Impurity 0.33% 0.617% 0.375% 0.592% 0.40%
White Lyz. Pwd. - White lyophiSized powder BND HCl -- Bcndamustine hydrochloride
Above mentioned results show that the products produced with less aggressive secondary drying which probably could not remove bound water stabilizes the product with respect to degradation into Impurity B (Bendamustine Dimer).

Example-3:
The lyophilized compositions of Bendamustine prepared using procedure explained in example-1. The physical characteristics and reconstitution time of the final product was determined.
Lyophilization cycle without Annealing:

THERMAL TREATMENT
Step No. Temperature (°C) Time (min.) Ramp/Ho ld


1 -45 180 R


2 -45 180 H




Freeze -45°C

Extra Freeze 0


Condenser -55


Vacuum 150


PRIMARY DRYING
Step No. Temperature (°C) Time (min.) Vacuum (mTorr) Ramp /Hold
] -10 120 100 R
2. -10 1200 100 H
3 -10 1200 100 H
4. 0 120 100 R
5. 0 480 100 H
6. 20 120 100 R
7. 20 240 100 H
8. 45 120 50 R

SECONDARY DRYING
Step No. Temperature (°C) Time (min.) Vacuum (mTorr) Ramp/Hold
1 | 45 1200 50 H
Observation

Sr. no. Tests Observation/results
01 | Description A white to off white lyophilized cake
02 1 Reconstitution time 3-5 minutes*
+ Even after 5 minutes, there was slight haziness.
In trials without annealing process, hard cake was observed and even after reconstitution the reconstituted solution was slightly hazy/bluish tinge in solution.

Example-4:

Sr. No. Ingredients Quantity (mg/mL)
1. Bendamustine hydrochloride 15.0
2. Marmitol 25.5
3. Tert-Butanol (TBA) 314.0
4. Water for Injection 595.5
5. Nitrogen q.s. to sparge
Manufacturing Process:
1. A batch quantity of Water for Injections of 2°C-8°C was taken into the Stainless Still manufacturing vessel and temperature was maintain to 2°C-8°C by Brian / chilled water circulation followed by nitrogen sparging for 30 minutes.
2. Marmitol was added to the above Water for Injection and stirred for 10 min.
3. A batch quantity of Tertiary Butyl Alcohol was added into the solution of step-2 and stirred for 15 minutes with nitrogen sparging.
4. Bendamustine hydrochloride was added to the bulk solution of step-3 and the bulk solution was stirred with nitrogen sparging for 60 minutes with maintaining the temperature at 2°C-8°C.
5. Tertiary Butyl Alcohol if required was added to the final solution of the step-4, to make up the batch weight and stirred for 5 minutes.
6. Annealing of the solution of step-5 was performed by providing thermal treatment as described in Table-6.

7. The solution of the step-6 was then lyophilized in suitable container under controlled conditions.
8. The moisture content, physical characteristics and re-constitution time of the final product was determined.

Lyophilization cycle with Annealing:

THERMAL TREATMENT
Step No. Temperature (°C) Time (min.) Ramp/Hold
1 -40 120 R
2 -15 90 R
3 -15 30 H
4 -45 120 R
5 -45 180 H

Freeze -45°C
Extra Freeze 0

Condenser -55

Vacuum 300

PRIMARY DRYING
Step No. Temperature (°C) Time (min.) Vacuum (mTorr) Ramp/Hold
1 -10 120 250 R
-10 1200 250 H
3. 0 60 250 R

SECONDARY DRYING
Step No. Temperature (°C) Time (min.) Vacuum (mTorr) Ramp/Hold
1 0 120 250 H
Observation

Sr. no. Tests Observation/results
01 Description A white to off white lyophilized porous cake/powder
02 Reconstitution time Less than 1 minutes
The above examples example-3 and example-4 clearly indicates that performing annealing during lyophilization reduces the reconstitution time as compare to the product prepared with out performing annealing during lyophilization.
All of the compositions and methods disclosed in this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the spirit and scope of the invention.

We Claim:
1. A stable lyophilized bendamustine composition comprising bendamustine
lyophilized powder with an optimum level of moisture content less than 10%.
2. The composition according to claim 1. wherein the moisture content is 1-5 % in the lyophilized powder of bendamustine,
3. The composition according to claim 2. wherein the moisture content is 1-4 % in the lyophilized powder of bendamustine.
4. A process of preparing a stable lyophilized bendamustine composition comprising the steps of:

a) dissolving bendamustine in a suitable solvent to form a solution; and
b) controlled lyophilization of the drug solution;
wherein the obtained bendamustine lyophilized powder comprises an optimum level of moisture content of 1 - 10%.
5. The process according to claim 4. wherein the obtained bendamustine lyophilized
powder with optimum level of moisture comprises reduced impurity as compared to
bendamustine lyophilized powder with moisture content less than 1%.

6. A process of preparing a stable lyophilized bendamustine composition comprising
a lyophilization cycle with annealing, comprising the steps of:
a) freezing the drug solution at or below -40 °C and holding at this temperature for a specific time period;
b) raising temperature at or below 0 °C and holding at this temperature for a specific time period; and
c) repeating the same above steps in a cyclic manner to obtain lyophilized
bendamustine composition.
7. The process according to claim 6. wherein the lyophilization cycle with annealing
comprises the steps of:
i) freezing the drug solution to a temperature at or below -40.degree. C, to form a
frozen solution;
ii) holding the frozen solution at or below -40.degree. C. for at least 30 minutes;
iii) ramping the frozen solution to a primary drying temperature between about -40.
degree. C. and about -10.degree. C. to form a dried solution;
iv) holding for at least 2 hours;
v) ramping the dried solution to a secondary'' drying temperature between about -10.
degree. C, and about 0.degree. C; and
vi) holding for at least 2 hours; to obtain lyophilized bendamustine composition.
8. The process according to claim 7. wherein the said lyophilized bendamustine
powder is obtained as uniform porous cake.

9. The process according to claim 8. wherein the obtained lyophilized bendamustine
powder reconstitutes within 1 minute to form an aqueous solution.
10. A stable lyophilized bendamustine composition with reduced impurity as
described according to the description.