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Formulation Development and Evaluation of


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Intravenous Infusion

Article July 2010

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International Journal of Pharmacology and Technology
2(1), June 2010, pp. 23-36

Formulation Development and Evaluation of Gemcitabine


Hydrochloride Dry Powder for Intravenous Infusion

Margret Chandira1, B. Jayakar2, Debjit Bhowmik3 and K. P. Sampath Kumar*


1
Vinayaka Mission College of Pharmacy, Salem, Tamilnadu
Rajiv Gandhi College of Pharmacy, Nautanwa, Maharajganj, Uttar Pradesh
2

3
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.

INTRODUCTION intramuscular injection may prove very fatal if it


Parenteral preparations are those pharmaceutical is given by intravenous route.A substance must
products that are given by other than oral route. be transported from the site of entry to the part
Transfusion fluids and injections are parenteral of the body where its action is desired to take
preparations. Injections are sterile solutions or place (even if this only means penetration through
suspension of drugs in aqueous or oily vehicle the stratum corneum into the skin). Using the
meant for introduction into the body by means bodys transport mechanisms for this purpose,
of an inject able needle under or through one or however, is not trivial. The pharmacokinetic
more layers of the skin or mucous membrane. properties of a drug (that is, those related to
Injections should be sterile, isotonic and free from processes of uptake, distribution, and elimination)
foreign particles, such as dust, fibers etc. They are critically influenced by the route of
should be introduced through the same route for administration. Formulation and evaluation of
which they are intended. For example, an oily dry powder of Gemcitabine hydrochloride for
suspension meant for intramuscular injection intravenous infusion by Lyophilization method.
may be very dangerous if it is administer by The objective of the present study is to develop a
intravenous injection. Similarly those potent pharmaceutically stable and robust formulation
drugs which are required to be given through of Gemcitabine hydrochloride dry powder 200mg
comparable with innovator. To achieve this goal
various prototype trials are taken & evaluated
* Corresponding author: E-mail: margretchandira@yahoo.com with respect to various quality parameters. The
24 Margret Chandira, B. Jayakar, Debjit Bhowmik & K. P. Sampath Kumar

formulation shall be finalized by comparing the Calculation: The percentage of residue on


moisture content with that of the innovator. And ignition in the sample was calculated by,
all the results of quality parameters are with in
the limits specified in 2007-USP. Most frequently Residue onignition % =
Weight of residue after ignition
100
prescribed drug in the treatment of specific type Weight of sample taken
cancers such as Breast cancer, Non-small cell lung
The results are shown in table 4
cancer, and pancreatic cancer. It may lose its
viability in the liquid, so Lyophilization has been
Determination of pH
selected to convert the liquid form into solid
form.Bioavailability of drug is 100% by intra 250mg of substance was weighed and dissolved
venous (Parenteral) route. Lyophilized drugs can in carbon dioxide free water and diluted to 250ml
easily dissolve in water for injections while with same solvent and mixed. The pH of sample
reconstituting them. The reconstituted dosage was determined by the pH meter. The results are
form is administered along with saline (NaCl) shown in table 4.
through intra venous route to facilitate the Identification by infrared spectrophotometer:
isotonicity with blood.In the present work, Gemcitabine hydrochloride is subjected to IR
Gemcitabine HCl was chosen as a model drug, is spectral analysis. The functional groups of
an antineoplastic and an antimetabolite. It is used Gemcitabine HCl are identified as shown in
in the treatment of Non-small cell lung cancer, graph. The results are shown in Figure no.2 &
breast cancer and pancreatic cancer. table 5.

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

MVD: 1:66 The results are shown in table 6


Test con. /Dilution: 1:33 (MVD/2)
Total Molds and Yeasts Count
Reagent information:
(1) LAL reagent: 0.03 EU/ml Requirements
(2) Controlled Std. Endotoxin 1. Sabouraud Dextrose agar media
(3) LAL Reagent water 2. Petri plates
Materials 3. Conical flask (250 ml)
(1) Test tubes 4. Measuring cylinder (250 ml)
(2) Incubator 5. Distilled water
Test Data 6. Pipette (1ml)
Temperature: Start 37.1 C 7. Bunsen burner
End 37.1 C 8. Laminar flow bench
The results are shown in table 6 9. IPA (70%)
10. Cotton, Forceps
As per in house specification
11. Auto clave
Total aerobic microbial count
12. Incubator
Requirements:
13. Colony counter
1. Soybean-Casein Digest agar media
Procedure: 10g of sample was taken and
2. Petri plates
dissolved in 100ml (10mg/ml) of Sabouraud
3. Conical flask (250 ml) Dextrose agar media, after 20 min from this 1ml
4. Measuring cylinder (250 ml) was taken and transferred into Petri plates (P1,
5. Distilled water P2) then incubated for 5 days at the condition of
6. Pipette (1ml) 22 C 2 then the formed colonies are counted.
7. Bunsen burner Limit: NMT 50 CFU/gm
8. Laminar flow bench Calculation
9. IPA (70%) No. of colonies formed = 1 colony/100mg
10. Cotton, Forceps Total colonies/gm = 110 = 10 colonies/g
11. Auto clave
The results are shown in table 6
12. Incubator
13. Colony counter Test for Pathogens
Procedure (1) Staphylococcus aureus
Medium used: Mannitol-salt agar medium
10g of sample was taken and dissolved in 100ml
(10mg/ml) of soya bean casein digest media, after Procedure: Three Petri plates were taken for
20 min from this 1ml was taken and transferred this test; they were positive control (pathogen
into Petri plates (P1, P2) then incubated for 5 days + medium), negative control (only medium)
at the condition of 37 C 2 then the formed and test control (test sample + medium).
colonies are counted. Incubated at 35 C for three days, after
incubation it was confirmed that there was no
Limit: NMT 100 CFU/gm growth in negative control and test control of
all batches. The sample is free from pathogens.
Calculation
(2) Pseudomonas aeruginosa
No. of colonies formed = 3 colonies/100mg Medium used: Cetrimide agar medium
Total colonies/gm = 310 = 30 colonies/g Procedure: Three Petri plates were taken for
26 Margret Chandira, B. Jayakar, Debjit Bhowmik & K. P. Sampath Kumar

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

Procedure: Three Petri plates were taken for


this test; they were positive control (pathogen below 1%. In this work the moisture content was
+ medium), negative control (only medium) reduced up to 0.4%. Moisture content was
and test control (test sample + medium). determined by using Karl-fisher auto-titrator.
Incubated at 35 C for three days, after
incubation it was confirmed that there was no
Procedure
growth in negative control and test control of
all batches. The sample is free from pathogens. Standardization: Before the use of K. F. titremeter
Results are shown in table 6 standardization of K.F. Reagent done, in a manner
as follows, 35 ml of methanol was added in to
Formulation of Dry Powder by Lyophilisation titration vessel to dip the platinum electrode in
it, automatic titration done to continue with K.F.
The dry powder was prepared by employing
Reagent until the titration reaches the end point.
lyophilization method using various process
For the determination of trace amount of water
parameters like duration time of the process,
di-sodium tartrate (DST) di hydrate was used as
temperature and vacuum. Mannitol was used as
a convenient water reference substance. 150mg of
bulking and non hygroscopic agent to prevent the
DST weighed and added to the titration vessel
absorption of moisture from the outer
and titrated up to end point.
environment into vials. The process parameters
of all formulations are shown in table 8-15. K.F. Reagent factor (mg/ml) was calculated by,
= W 0.1566
Analysis of Lyophilized Product
Titer value
Physicochemical Evaluation Where,W is the Wt. of DST dihydrate in mg
Test for Moisture Content 0.1566 is the factor of DST

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

product, with varies with time under the influence


of a variety of environmental factors such as
temperature, humidity and light. Recommended
storage conditions, re test periods and shelf lives
are to be established.
The international conference on
harmonization (ICH) guidelines titled stability
testing of new drug substances and products
describes the stability test requirement for drug
registration applications in the European Union,
Japan and United States of America.
Stability studies were carried out at 40 C / 75%
RH and at 60 C for optimized formulation i.e.F7.

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)

Analysis of Gemcitabine HCl 1 C=O Stretching 1705-1725


2 O-H Bending 1050-1150
Physicochemical Evaluation 3 N-H Stretching 3400-3500

Table 2 4 N-H Bending 1500-1650


Physico Chemical Evaluation of Gemcitabine HCl 5 C-N Vibrations 1000-1400
S. Test Result Specificity
No Microbial Analysis of Gemcitabine HCl
1 Description White crystalline White crystalline
Table 4
powder powder
Microbial Analysis of Gemcitabine HCl
2 Residue on 0.098% NMT 0.1%
ignition S. Test Result Specificity
3 pH(10mg/ 2.65 Between 2.0 and No
ml solution) 3.0
4 Identification Complies Spectrum should 1 Bacterial
by IR match with Endotoxin test < 0.05 EU/mg NMT 0.05 EU/mg
spectroscopy absorption ranges 2 Total aerobic 30 CFU/gm NMT 100 CFU/gm
of functional microbial count
groups. 3 Total molds & 10 CFU/gm NMT 30 CFU/gm
5 Relative yeasts count
substances
4 Pathogen test
a. % of Cytosine 0.06% NMT 0.1%
b. % of 0.01% NMT 0.1% 1. Staphylococcus Absent Should be absent
Gemcitabine aureus
anomer 2. Pseudomonas Absent Should be absent
c. % Individual 0.04% NMT 0.1% aeruginosa
impurities
3. Salmonella Absent Should be absent
d. Total 0.11% NMT 0.2%
species
impurities
6 Assay 98.94% Between 97.5% 4. Esherichia Absent Should be absent
and 101.5 Coli
30 Margret Chandira, B. Jayakar, Debjit Bhowmik & K. P. Sampath Kumar

Formulation of Dry powder of Gemcitabine by


Lyophilization Method
Table 5
Formula
S. No Ingredient Quantity/
VIAL (mg)
1 Gemcitabine Hydrochloride 200 mg
2 Mannitol 200 mg
3 Sodium acetate 12.5 mg
4 Sodium hydroxide/ Quantity Figure 2: Graphical Data of Formulation-1
Hydrochloric acid sufficient
5 Water for injection 5 ml Table 7
Process Parameters used for Formulation-2,
Batch size: 50 vials
The formulation was developed by Description Step R/H Set temp. Product Time Pressure
optimizing the lyophilisation method with (C) temp. (min) (Milli
various Para meters such as time of the process, (C) torr)
temperature, and vacuum. All the parameters observed
were used to develop the formulation were shown Freeze 1 R -40 -11.8 90 0
in tables 8-15. drying 2 H -40 -37.8 180 0
Extra freeze 1 H -35 -37 20 0
Table 6 Primary 1 R -30 -33.6 30 600
Process Parameters used for Formulation-1, drying 2 H -30 -31.3 180 600
Batch Size: 50 vials 3 R -20 -27.3 30 600
Description Step R/H Set temp. Product Time Pressure 4 H -20 -22.1 600 600
(C) temp. (min) (Milli 5 R -15 -10.2 30 600
(C) torr) 6 H -15 -9.5 180 600
observed 7 R 5 -5.2 10 600
Freeze 8 H 5 -2.2 50 600
drying 1 R -40 -12.5 90 0 9 R 20 2.3 20 600
2 H -40 -37.8 250 0 10 H 20 10.6 160 600
Extra freeze 1 H -35 -35.9 30 0 11 R 47 25.5 30 50
12 H 47 41.4 320 50
Primary 1 R -30 -33.8 30 600
drying 2 H -30 -30.6 180 600 Secondary 1 R 25 28.8 30 50
drying
3 R -20 -27.3 30 600
Total time 1960
4 H -20 -21.8 400 600 (in min)
5 R -15 -6.2 30 600 Total time 32.66
(in hrs)
6 H -15 -3.3 180 600
7 R 5 7.2 180 600
8 H 5 6.6 30 600
9 R 20 17.2 120 600
10 H 20 18.2 50 600
11 R 47 21.4 30 50
12 H 47 45.2 300 50
Secondary 1 R 25 30.2 30 50
drying
Total time (in min) 1960
Total time (in hrs) 32.66
Figure 3: Graphical Data of Formulation-2
Formulation Development and Evaluation of Gemcitabine Hydrochloride Dry Powder... 31

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

Figure 4: Graphical Data of Formulation-3 Figure 5: Graphical Data of Formulation-4


32 Margret Chandira, B. Jayakar, Debjit Bhowmik & K. P. Sampath Kumar

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

Figure 7: Graphical Data of Formulation-6

Figure 6: Graphical Data of Formulation-5

Figure 8: Graphical Data of Formulation-7


Formulation Development and Evaluation of Gemcitabine Hydrochloride Dry Powder... 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

Moisture content (%) 0.4 0.5 0.5


Assay (%) 102.9 102.7 102.5
Particulate matter
(1) 10 m 1860 1864 1870
(2) 25 m 160 164 170
Stability studies result for Formulation-7 at 60 C
Formulation Development and Evaluation of Gemcitabine Hydrochloride Dry Powder... 35

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
lyophilisation method using mannitol (200 mg) REFERENCE
as bulking agent and non hygroscopic agent. [1] Afzal R. Mohammed. Lyophilisation and Sterilization
Sodium acetate was used as buffering agent to of Liposomal Vaccines to Produce Stable and Sterile
Products. International J. Pharmaceutics. 2006; 40(1): 30-
attain the stable pH in the product. Sufficient 38.
amount of sodium hydroxide / HCl were used to [2] A. D. Woolfson. Stabilisation of Hydrotropic
adjust the pH of the formulation and 5 ml sterile Temazepam Parenteral Formulations by
water for injection in each vial was used as Lyophilization. International J. Pharmaceutics, 1986, 34(1-
vehicle.Total 7 formulations were prepared and 2): 17-22.
evaluated for moisture content, pH, assay, relative [3] Antonello A. Barresi. In-line Control of the
substances, clarity of solution, particulate matter, Lyophilization Process. A Gentle PAT Approach using
Soft Ware Sensors. International Journal of Refrigeration
and microbial analysis for bacterial endotoxin test 2009, 32(5): 1003-1014.
and sterility test.The results of all formulations [4] Sam Corveleyn. Formulation and Production of Rapidly
for pH, assay, relative substance, particulate Disintegrating Tablets by Formulation and Production
matter and microbial analysis were found to be of Rapidly Disintegrating Tablets by Lyophilization
within the United States Pharmacopoeia limits. using Hydrochlorothiazide as a Model Drug;
International J. Pharmaceutics, 1997, 152, 2(26): 215-225.
The amount of % moisture content for all
formulations was found to be 1.85% - 0.4%. The [5] Zhai. S. Measurement of Lyophilisation Primary Drying
Rates by Freeze-drying Microscopy. Chemical
F7 contains lowest amount of moisture content Engineering Science, 2003, 58 (11): 2313-2323.
of 0.4% so the F7 was found as the best [6] Sui Lin. Thermal Control of Freeze-drying Processes in
formulation as compared with other formulations a Porous Medium with Predetermined Rate of Drying.
by comparing the % moisture content.The time International Journal of Refrigeration, 1995, 18 (3): 161-167.
required for each formulations of F1 and F2 is [7] Wassim Abdelwahed. Freeze-drying of Nanoparticles:
32.66 hrs and they contains the moisture content Formulation, Process and Storage Considerations;
of 1.85% and 1.65%. The time required for F3 was Advanced Drug Delivery Reviews, 2006, 58(15), 30, 1688-
1713.
36.83 hrs, for F4 37.83 hrs, for 40.33 hrs and for F6
[8] Ward, K. R. Effects of Lyophilization on the Formation
and F7, 41.66 hrs was required. There was no and Stability of Stealth Liposomes; European Journal of
change in the vacuum in all formulations. The Pharmaceutical Sciences, 1996, 4(1): 173.
time and temperature in the primary drying were [9] Chengjun Chen. An Overview of Liposome
increased from formulations 1 to 7. Because high Lyophilization and its Future Potential; Journal of
freezing caused to form the melt back vials, which Controlled Release, In Press, Corrected Proof, 2009, 28(2):
will causes to poor solubility while reconstituting 1-13.
the dry powder. All the formulations were passed [10] Sam Corveleyn. Moisture Absorption and Desorption
of Different Rubber Lyophilisation Closures;
in the microbial analysis.Stability studies for International Journal of Pharmaceutics. 1997 159, 1(15): 57-
optimized formulation F7 were carried out at 400c 65.
and 75% RH and at 60 C. There was no significant [11] Barbara Stella. Encapsulation of Gemcitabine Lipophilic
variation found in % moisture content, assay, Derivatives into Polycyanoacrylate Nanospheres and
particulate matter at 40 C & 75% RH. There was Nanocapsules; International Journal of Pharmaceutics,
no significant variation in the physical appearance 2007, 344(1-2): 71-77.
of dry powder at 60 C. [12] Roberta Cavalli. Sterilization and Freeze-drying of
Drug-free and Drug-loaded Solid Lipid Nanoparticles;
36 Margret Chandira, B. Jayakar, Debjit Bhowmik & K. P. Sampath Kumar

International Journal of Pharmaceutics, 1997, 148, 1(14): [15] Dirk L. Teagarden. Practical Aspects of Lyophilization
47-54. using Non-aqueous Co-solvent Systems; European
[13] Aurelie Hottot. Freeze Drying of Pharmaceuticals in Journal of Pharmaceutical Sciences, 2002, 15(2): 115-133.
vials: Influence of Freezing Protocol and Sample [16] Mitra Mosharraf. Formulation, lyophilization and
Configuration on Ice Morphology and Freeze-dried Solid-state Properties of a Pegylated Protein;
Cake Texture; Chemical Engineering and Processing, 2007, International J. Pharmaceutics. 2007, 336(2): 215-232.
46(7): 666-674. [17] Tsinontides. S. C. Freeze DryingPrinciples and Practice
[14] Morgan, C. Freeze-Drying of Microorganisms; for Successful Scale-up to Manufacturing; International
Encyclopedia of Microbiology, 2009, 28(1-2): 162-173. Journal of Pharmaceutics, 280(1-2), 2004: 1-16.
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