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FormulationDevelopment GemcitabineHCl DryPowder For IntravenousInfusion
FormulationDevelopment GemcitabineHCl DryPowder For IntravenousInfusion
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Department of Pharmaceutical Sciences, Coimbatore Medical College, Coimbatore
Abstract: Gemcitabine is an anticancer nucleoside analogue active against various solid tumors. How ever, it
possesses important draw backs like a poor biological half life and induction of resistance. In this work, the
techniques for the lyophilization were described, lyophilized injections were readily reconstituted. Lyophilisation
resulted in the preparation with excellent storage characteristics. Dry powder of Gemcitabine HCl was prepared
by lyophilization method. In this work seven formulations were prepared by using Mannitol as a bulking agent
and as a non hygroscopic material. All formulations were prepared by changing the process parameters
temperature and the time of the process. The maximum temperature used in the optimized formulation (F7) is
470C and the minimum temperature is -350C, optimized for 41.66 hours and the moisture content in this formulation
was found as 0.4%. All the formulations were evaluated for moisture content, pH, assay, clarity of reconstituted
solution, particulate matter and microbial analysis for bacterial endotoxin test by gel clot method and sterility
test. Stability studies were performed for the optimized batch (F7) one month and evaluated for physical
appearance, moisture content, particulate matter and assay. It was with in the limits with good stability.
Keywords: Lyophilization, Sublimation, Stability, Gemcitabine HCl, Mannitol.
MATERIALS AND METHODS Test Procedure for Assay and Relative Substances
Gemcitabine HCl, Mannitol, Sodium acetate, Chromatographic Condition
Sodium hydroxide/ Hydrochloric acid, Water for Column: 4.6 mm X 25 cm column, packed with
injection are procured by Zenotech laboratories, C18, 5.
Hyderabad. Flow rate: 1.2 ml per minute
Detection: 275 nm
ANALYSIS OF GEMCITABINE HCL
Load: 20 l for assay
Physico Chemical Evaluation 20 l for relative substances
Description Column temperature: Ambient
Sample was taken in a watch glass and Mobile phase: Mobile phase contained 13.8g of
spreaded with spatula and the material was mono basic sodium phosphate and 2.5 ml of
observed physically. The results are shown in phosphoric acid 1000 ml of water. pH to 2.42.6
table 4. Run time: 20 min
The results are shown in table no.4
Residue on Ignition
Microbial analysis
Procedure
Bacterial Endotoxin test
The substance was weighed about 1.0gm, Product: Gemcitabine in inj. 200mg
examined in a crucible that previously has been
Potency: 40mg/ml
ignited, cooled and weighed. The crucible
containing sample heated till substance was Endotoxin limit: NMT 0.05 USP EU/mg of
thoroughly charred. The residue was cooled and Gemcitabine
moistened with 1ml of sulfuric acid and heated Method: LAL Test or Gel clot method
till white fumes were evolved and ignited at 800 MVD Calculation
+ 25 C, until the carbon was consumed. After
cooling it weighed and calculated the percentage 40mg / ml 0.05 EU / mg
= 66
of residue. 0.03EU / ml
Formulation Development and Evaluation of Gemcitabine Hydrochloride Dry Powder... 25
this test; they were positive control (pathogen Dissolving the Drug and Excipients in WFI
+ medium), negative control (only medium)
and test control (test sample + medium). Sterilizing the Solution by Filtration Through
Incubated at 35 C for three days, after 0.22 Filter
incubation it was confirmed that there was no
growth in negative control and test control of
all batches. The sample is free from pathogens. Filling in to Sterile Vials, Partial Stoppering the Vials with
Slotted Rubber Stoppers
(3) Salmonella species
Medium used: Brilliant green agar medium
Transporting the Vials to the Lyophilizer
Procedure: Three Petri plates were taken for
this test; they were positive control (pathogen
+ medium), negative control (only medium) Processing the Lyophilisation
and test control (test sample + medium).
Incubated at 35 C for three days, after
Complete Stoppering the Vials Inside of the Lyophilizer by
incubation it was confirmed that there was no Hydraulic Pressure
growth in negative control and test control of
all batches. The sample is free from pathogens.
(4) Escherichia coli Figure 1: Diagrammatic Representation for Lyophilisation
Medium used: MacConkey Agar medium Method
The aim of lyophilization is to reduce the moisture Estimation of moisture content in sample:
content to improve the stability of the product. Weighed quantity of sample was introduced in
The lyophilized product must contain very less to the vessel and titrated with K.F. Reagent up to
amount of moisture content as much as possible. the end point. The percentage of moisture content
For Gemcitabine HCl dry powder it must be was calculated by,
Formulation Development and Evaluation of Gemcitabine Hydrochloride Dry Powder... 27
Vol. of K.F. reagent consumed K. F. Factor 100 then allowed to stand in undisturbed position
= until it was free from air bubbles. Three aliquots
1000 Wt. of sample taken ( gm) are withdrawn, each aliquot contain 10ml in to
Results are shown in table 16 the light obscuration counter sensor for analysis.
The particulate matter was calculated by,
Test for pH
= PVP/VAn
pH is the value given by a suitable, properly
Where, P = Avg. particle count obtained from
standardized, potentiometric instrument (pH
the portions analyzed.
meter). The electrodes and cell were rinsed several
times with the test solution containing 40 mg of VP = Volume of pooled sample
gemcitabine in each ml of 0.9% sodium chloride VA = Volume of each portion analyzed
solution. The cell was filled with test solution, the N = No. of containers pooled.
pH value is recorded. Results are shown in table Results are shown in table 16
16.
Microbial Analysis
Assay and % Relative Substances was Carried
using HPLC (1) Bacterial endotoxin test
Product: Gemcitabine inj. 200mg
Chromatographic Condition
Sample taken: 1ml
Column: 4.6 mm X 25 cm column, packed with
, 5. Potency: 40mg/ml
18
C
Flow rate: 1.2 ml per minute Endotoxin limit: NMT 0.05 USP EU/mg of
Detection: 275 nm Gemcitabine
Load: 20 l for assay Method: LAL Test or Gel clot method
20 l for relative substances MVD Calculation
Column temperature: Ambient 40mg / ml 0.05 EU / mg
Mobile phase: Mobile phase contained 13.8g of = 66
0.03 EU / ml
mono basic sodium phosphate and 2.5 ml of
phosphoric acid 1000 ml of water. pH to 2.4 2.6 MVD: 1:66
Run time: 20 min Test con. /Dilution: 1:33 (MVD/2)
Results are shown in table no.16 Reagent information
Clarity Test (1) LAL reagent: 0.03 EU/ml
(2) Controlled Std. Endotoxin
The clarity test was performed by visual
observation of each externally cleaned container (3) LAL Reagent water
under a good light and viewed against a black Materials
and white back ground, with the contents set in (4) Test tubes
motion with a swirling action. Results are shown
(5) Incubator
in table 16.
Test Data
Test for Particulate Matter
Temperature: Start 37.1 C
The test was performed by light obscuration End 37.1 C
particle count test.
Procedure
Procedure: 10 containers (vials) were taken
and opened. The vials were reconstituted with Solutions are prepared in test tubes as mentioned
water for injection and replaced with closers. The below.
containers were agitated and inverted 20 times To dissolve the sample (dry powder) LAL
to suspend the particulate matter, combine all the Reagent water was used. The pH of solutions was
contents of containers in cleaned container and adjusted to 6 before performing the LAL test.
28 Margret Chandira, B. Jayakar, Debjit Bhowmik & K. P. Sampath Kumar
Table 1
Preparation of Solutions for LAL Test
Solution No. of tubes (marks on tubes) Type of tubes Contents in tubes
A 2(1,2) Negative control LAL Reagent water, LAL reagent
B 2(3,4) Positive control LAL reagent water, LAL reagent and
Controlled standard Endotoxin (CSE)
C 2(5,6) Negative product control LAL reagent water, LAL reagent and sample
D 2 (7,8) Positive product control LAL reagent water, LAL reagent,
CSE and sample
A and B tubes were prepared to test the LAL mentioned earlier. After incubation filter the
reagent water (LRW) to confirm, the LRW doesnt contents through filter paper. A small piece was
contain endotoxins. C and D tubes were prepared taken from the filter paper and the microbial test
to test the product. After preparation of solutions performed as mentioned above in microbial
all tubes are incubated for one hour at 37 C. The analysis as per in house specification. The growth
results are shown in table 17. was absent in test control and ve control.
(2) Sterility Test Test for fungi: 100mg of sample was taken
Name of the sample: Gemcitabine injection from the container and reconstituted in sterile
200 mg water for injection. Transfers it in to Soy-Bean
Casein Digested Medium present in the conical
Method: Membrane filtration method
flask which is already pre incubated, the contents
Medium for Bacteria: Fluid Thioglycollate are incubated for 14 days at 22 C. Positive control
medium and negative controls were also prepared in
Incubation: 35 2 C for 14 days conical flasks and incubated in same manner as
Media for fungus: Soy-Bean Casein Digested mentioned earlier. After incubation filter the
Media contents through filter paper. A small piece was
Incubation: At 22 C for 14 days taken from the filter paper and the microbial test
performed as mentioned above in microbial
Volume of medium: 150 50 ml analysis as per in house specification. The growth
Requirements was absent in test control and ve control.
(1) Petri plates The results are shown in table 17
(2) Measuring cylinder (250 ml)
(3) Distilled water STABILITY STUDIES
(4) Bunsen burner Stability Studies
(5) Laminar flow bench Describing the studies as per ICH requirements
(6) Isopropyl alcohol with related to climatic zones describing of
(7) Cotton, Forceps conditions and environment of stability chambers.
(8) Incubator
Introduction
Procedure Stability study of a drug has been defined as the
(1) Test for bacteria: 100mg of sample was taken stability of a particular formulation, in a specific
from the container and reconstituted in sterile container, to remain within its physical, chemical,
water for injection. Transfers it in to Fluid therapeutic and toxicological specifications
Thioglycollate medium present in the conical flask throughout its shelf life.
which is already pre incubated, the contents are
incubated for 14 days at 37. Positive control and Objective of the Study
negative controls were also prepared in conical The purpose of the stability testing is to provide
flasks and incubated in same manner as evidence on the quality of a drug substance or its
Formulation Development and Evaluation of Gemcitabine Hydrochloride Dry Powder... 29
Method
The selected formulation was stored at 40 C / 75% Figure 1: IR Spectrum of Gemcitabine HCl
RH for one month and evaluated for its moisture
content, assay and particulate matter. Also stored
at 60 C for one month and its physical appearance Table 3
was observed. The results are shown in table 18. Interpretation Results for IR spectrum
S. No Group Frequency Range
RESULTS AND DISCUSSION (cm-1)
Table 8 Table 9
Process Parameters used for Formulation-3, Process Parameters used for Formulation-4,
Batch Size: 50 vials Batch Size: 50 Vials
Description Step R/H Set temp. Product Time Pressure Description Step R/H Set temp. Product Time Pressure
(C) temp. (min) (Milli (C) temp. (min) (Milli
(C) torr) (C) torr)
observed observed
Freeze 1 R -40 -12.6 90 0 Freeze 1 R -37 -12.3 90 0
drying 2 H -40 -38.2 250 0 drying 2 H -37 -35.8 250 0
Extra freeze 1 H -35 -35.8 20 0 Extra freeze 1 H -35 -35.6 20 0
Primary 1 R -30 -34 30 600 Primary 1 R -30 -33.8 30 600
drying 2 H -30 -30.2 180 600 drying 2 H -30 -30.6 220 600
3 R -20 -27.2 30 600 3 R -20 -27.3 30 600
4 H -20 -22 600 600 4 H -20 -21.8 600 600
5 R -10 -15.4 30 600 5 R -10 -14.8 30 600
6 H -10 -5.3 180 600 6 H -10 -11.3 200 600
7 R 5 4.5 180 600 7 R 5 4.2 150 600
8 H 5 6.8 30 600 8 H 5 6.6 50 600
9 R 20 18.2 120 600 9 R 20 17.6 160 600
10 H 20 19.3 50 600 10 H 20 18.2 80 600
11 R 47 22.4 30 50 11 R 47 21.4 30 50
12 H 47 44 360 50 12 H 47 45.2 300 50
Secondary 1 R 25 28.3 30 50 Secondary 1 R 25 31.4 30 50
drying drying
Total time (in min) 2210 Total time (in min) 2270
Total time (in hrs) 36.83 Total time (in hrs) 37.83
Table 10 Table 11
Process Parameters used for Formulation-5, Process Parameters used for Formulation- 6 & 7,
Batch Size: 50 vials Batch Size: F6-50 vials, F7-60 vials
Description Step R/H Set temp. Product Time Pressure Descrip- Step R/H Set Product Product Time Pressure
(C) temp. (min) (Milli tion temp. temp. temp. (min) (Milli
(C) torr) (C) (C) F6 (C) F7 torr)
observed
Freeze 1 R -35 -10 -10.5 90 0
Freeze 1 R -35 -11.4 90 0 drying 2 H -35 -33.4 -33.8 120 0
drying 2 H -35 -33.6 120 0 Extra 1 H -35 -34.1 -34.7 20 0
Extra freeze 1 H -35 -34.3 20 0 freeze
Primary 1 R -20 -26.8 30 600 Primary 1 R -18 -24.7 -24.5 30 600
drying 2 H -20 -22.1 120 600 drying 2 H -18 -17.5 -19 120 600
3 R -10 -19.8 10 600 3 R -10 -18.9 -18.1 10 600
4 H -10 -11.9 480 600 4 H -10 -12.1 -11.8 480 600
5 R -3 -11.6 -11 10 600
5 R -5 -10.6 10 600
6 H -3 -5.3 -5.2 480 600
6 H -5 -7.2 420 600
7 R 5 -3.1 -3 10 600
7 R 5 -5 10 600
8 H 5 5 5 380 600
8 H 5 2 360 600
9 R 20 7.3 7.5 15 600
9 R 20 7.3 15 600 10 H 20 20.3 20.5 210 600
10 H 20 17.8 210 600 11 R 47 27.2 27.4 15 50
11 R 47 26.2 15 50 12 H 47 45.1 45.4 480 50
12 H 47 44.8 480 50 Secondary 1 R 25 26.1 26.3 30 50
Secondary 1 R 25 29.1 30 50 drying
drying Total time (in min) 2500
Total time (in min) 2420 Total time (in hrs) 41.66
Total time (in hrs) 40.33
Table 12
Comparative Data of Parameters for Formulations 1-3
Step Ramp/ F1(32.66 hrs) F2 (32.66 hrs) F3 (36.83 hrs) Vacuum
Hold Time Set Temp. Prod. Temp. Time Set Temp. Prod. Temp. Time Set Temp. Prod. Temp. (M.T)
(min) (C) (C) (min) (C) (C) (min) (C) (C)
F.D R 90 -40 -12.5 90 -40 -11.8 90 -40 -12.6 O
1 H 250 -40 -37.8 180 -40 -37.8 250 -40 -38.2 O
2
E.F H 30 -35 -35.9 20 -35 -37 20 -35 -35.8 0
1
P.D
1 R 30 -30 -33.8 30 -30 -33.6 30 -30 -34 600
2 H 180 -30 -30.6 180 -30 -31.3 180 -30 -30.2 600
3 R 30 -20 -27.3 30 -20 -27.3 30 -20 -27.2 600
4 H 400 -20 -21.8 600 -20 -22.1 600 -20 -22 600
5 R 30 -15 -6.2 30 -15 -10.2 30 -10 -15.4 600
6 H 180 -15 -3.3 180 -15 -9.5 180 -10 -5.3 600
7 R 180 5 7.2 10 5 -5.2 180 5 4.5 600
8 H 30 5 6.6 50 5 -2.2 30 5 6.8 600
9 R 120 20 17.2 20 20 2.3 120 20 18.2 600
10 H 50 20 18.2 160 20 10.6 50 20 19.3 600
11 R 30 47 21.4 30 47 25.5 30 47 22.4 50
12 H 300 47 45.2 320 47 41.4 360 47 44 50
S.D
1 R 30 25 30.2 30 25 28.8 30 25 28.3 50
Table 13
Comparative Data of Parameters for Formulations 4-7
F4 (37.83 hrs) F5 (40.33 hrs) F6 & F7
(41.66 hrs) F6 F7
Step Ram Time Set temp. Prod. Time Set Prod. Time Set Prod. Prod. Vacuum
P/Hold (min) (C) temp. (min) Temp. Temp. (min) Temp. Temp. Temp. (M.T.)
(C) (C) (C) (C) (C) (C)
1 R 90 -37 -12.3 90 -35 -11.4 90 -35 -10 -10.5 O
2 H 250 -37 -35.8 120 -35 -33.6 120 -35 -33.4 -33.8 O
E.F
1 H 20 -35 -35.6 20 -35 -34.3 20 -35 -34.1 -34.7 0
P.D
1 R 30 -30 -33.8 30 -20 -26.8 30 -18 -24.7 -24.5 600
2 H 220 -30 -30.6 120 -20 -22.1 120 -18 -17.5 -19 600
3 R 30 -20 -27.3 10 -10 -19.8 10 -10 -18.9 -18.1 600
4 H 600 -20 -21.8 480 -10 -11.9 480 -10 -12.1 -11.8 600
5 R 30 -10 -14.8 10 -5 -10.6 10 -3 -11.6 -11 600
6 H 200 -10 -11.3 420 -5 -7.2 480 -3 -5.3 -5.2 600
7 R 150 5 4.2 10 5 -5 10 5 -3.1 -3 600
8 H 50 5 6.6 360 5 2.6 380 5 5 5 600
9 R 160 20 17.6 15 20 7.3 15 20 7.3 7.5 600
10 H 80 20 18.2 210 20 17.8 210 20 20.3 20.5 600
11 R 30 47 21.4 15 47 26.2 15 47 27.2 27.4 50
12 H 300 47 45.2 480 47 44.8 480 47 45.1 45.4 50
S.D
1 R 30 25 31.4 30 25 29.1 30 25 26.1 26.3 50
34 Margret Chandira, B. Jayakar, Debjit Bhowmik & K. P. Sampath Kumar
Table 14
Physicochemical Evaluation of Formulations
S.No Test F1 F2 F3 F4 F5 F6 F7 Limits
1 Moisture content (%) 1.85% 1.65% 1.13% 1.00% 0.80% 0.50% 0.40% NMT 1%
2 pH 2.95 2.92 2.8 2.85 2.96 2.9 2.93 2.7-3.3
3 Assay 102.10% 102.90% 102.40% 103.30% 102.90% 103.10% 102.90% 95%-105%
4 Relative substances
a.% of Cytosine 0.09% 0.08% 0.07% 0.08% 0.06% 0.08% 0.07% NMT 0.1%
b.% of Gemcitabine -anomer 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% NMT 0.1%
c. % of individual impurities 0.07% 0.05% 0.05% 0.06% 0.05% 0.06% 0.07% NMT 0.2%
d. %Total impurities 0.17% 0.14% 0.13% 0.15% 0.12% 0.15% 0.15% NMT 0.3%
5 Clarity test Clear Clear Clear Clear Clear Clear Clear Should be
clear
6 Particulate matter
(1) > 10 m 2430 2380 2400 2120 1980 1920 1860 6000/vial
(2) > 25 m 180 160 180 170 160 150 160 600/vial
Microbial Analysis of Dry Powder Formulations bacteria for all formulations and it was confirmed
that all formulations were sterile.
(a) Bacterial Endotoxin Test
All formulations are evaluated for bacterial Stability Studies
Endotoxin test by LAL test (gel-clot method) and
The formulation-7 was stored at 40 C / 75% RH
according to the procedure as mentioned earlier.
for one month and evaluated for its moisture
It was found out that all formulations contain
content, assay and particulate matter. Also stored
Endotoxin not more than 0.05% EU/mg.
at 60 C for one month and its physical appearance
(b) Sterility test was observed.
Sterility test was performed according to the The optimized formulation was stored at 600C
procedure as mentioned earlier for fungi and and checked for physical appearance in three
Table 15
Microbial Analysis of Dry Powder of F1-F7
S. No Test Parameter F1 F2 F3 F4 F5 F6 F7 Limit
1 Bacterial <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 NMT
Endotoxin test EU/mg EU/mg EU/mg EU/mg EU/mg EU/mg EU/mg <0.05
EU/mg
2 Sterility test Sterile Sterile Sterile Sterile Sterile Sterile Sterile Must be
Sterile
Table 16
Stability Studies Parameters for Formulation-7 at 40 C / 75% RH
Parameter Time in days
0 15 days 30 days
intervals at initial, 15 days and 30 days. There was It was concluded that in lyophilization
no significant change in the physical appearance method to prepare Gemcitabine dry powder, 230
of formulation. min for freezing, 2240 min for primary drying, 30
min for secondary drying and the lowest
SUMMARY AND CONCLUSION temperature is -35 C and higher temperature is
47 C were best process parameters and it was
The present work was an attempt to formulate
also confirmed that F7 was the best formulation.
and evaluate drug powder for IV infusion of an
Further investigations are needed to confirm the
anticancer drug Gemcitabine. In the present study
long term stability studies and in vivo efficiency.
lyophilization was used to formulate dry
powder.The dry powder was formulated by
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