IOSR Journal Of Pharmacy www.iosrphr.

org
(e)-ISSN: 2250-3013, (p)-ISSN: 2319-4219
Volume 6, Issue 9 Version. 1 (Sep 2016), PP. 48-63

Development and Evaluation of Lyophilized Product of Apo-

Acetozolamide
Sandhyarani G1,Ramesh alli
University college of pharmaceutical sciences,Kakatiya university,Warangal.India

Abstract: Lyophilization or freeze drying involves the removal of water or other solvent from a frozen product
by a process called sublimation followed by desorption.Lyophilization is a multistage operation in which, quite
obviously, each step is critical. The main actors of this scenario are all well known and should be under strict to
achieve a successful operation.Freeze-dried products for parenteral use are known as Powders for injection or
infusion. As the stability of Acetazolamide in aqueous solution form was unstable it was formulated as
Lyophilized product.The objective of the present study was to develop a stable lyophilized formulation of drug
Apo-Acetazolamide for injection (500mg/vial) which is therapeutically equivalent to the reference listed
product, DIAMOX.

OBJECTIVE AND PLAN OF THE WORK:

OBJECTIVE
 As the stability of Acetazolamide in aqueous solution form was unstable it was formulated as Lyophilized
product.
 The objective of the present study was to develop a stable lyophilized formulation of drug Apo-
Acetazolamide for injection (500mg/vial) which is therapeutically equivalent to the reference listed product,
DIAMOX .

PLAN OF THE WORK

To achieve the ultimate goal of formulating lyophilized product of Apo-Acetazolamide, the present work was
designed to address the following objectives: -
 Literature survey
 Procurement of API, Excipients and Packaging materials
 Innovator sample procurement and its characterization
 Preformulation studies of the drug
 Formulation of the injectable dosage form
 Development of Lyophilization cycle
 Lyophilization of the injectable dosage form
 Evaluation of lyophilized product
 Stability studies as per ICH guidelines
METHODOLOGY
• The experimental work starts with the literature survey of innovator
product followed by the procurement and characterization of innovator
product, sourcing of raw materials (API and excipients) and packaging
materials.
• Based on the literature review, the proposed generic composition shall
contain the following ingredients:
LIST OF RAW MATERIALS AND THEIR SOURCE:

S.No INGREDIENT VENDOR

Acetozolamide sodium equivalent to Aurobindo
1
Acetozolamide Pharma Ltd, India
2 Sodium hydroxide Merck, Germany

Aurobindo
3 Water for injection Pharma Ltd, India

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 Development And Evaluation Of Lyophilized Product Of Apo-Acetozolamide

PROCUREMENT & CHARACTERIZATION OF

INNOVATOR PRODUCT
Innovator drug product is procured from the respective market of
source country and then they are characterized so as to check
whether they conform to the product specifications.
Characterization of the innovator product includes:
a) Label Information
Strength, batch number, shelf life, storage instructions,
reconstitution(if necessary) etc.
b) Primary Package Details
Vial type, dimensions, volume, fill volume, label size, head space
analysis, etc.
c) Product Analysis
Appearance, assay, related substances, impurities concentration,
sterility (endotoxin concentration.), pH etc
d) Stability study

PREFORMULATION STUDIES:
API Characterization:
• Preformulation involves the characterization of physical, chemical and microbiological
attributes of the drug substance thus providing useful information so as to develop a safe,
effective and stable dosage form.
• In the present study, the primary objective of the preformulation study is to ascertain that the
drug substance complies with the specifications or pharmacopoeial standards.
The various tests conducted on the API during the preformulation study and the specifications
set for the drug substance are tabulated below:

LIST OF PREFORMULATION TESTS AND THEIR SPECIFICATIONS:

TESTS SPECIFICATIONS
DESCRIPTION White to faintly yellowish-white, crystalline, odorless
powder
SOLUBILITY Sparingly soluble in practically boiling water, slightly
soluble in alcohol, very slightly sluble in water.
IDENTIFICATION
a) By IR The IR spectrum of sample recorded, as KBr should
exhibit transmission minima (absorption maxima)mat the
same wavelenths to these in the spectrum obtained with
the Acetazolamide working standard.

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 Development And Evaluation Of Lyophilized Product Of Apo-Acetozolamide

b) By chemical test A clear, bright yellow solution is produced. No

heavy precipitate or dark brown color results after
the mixing or heating.
WATER CONTENT Not more than 3.0
(%w/w, by KF, determined on 1g)
pH Between 9.1 and 9.6
(0.5g of sample in 25mL of
carbon dioxide free water)

HEAVY METALS (ppm) Not more than 0.002

ASSAY Not less than 98% and not more than 102%
(By HPLC, %w/w, as
C4H6N4O3S2 on anhydrous basis)

RELATED SUBSTANCES
(By HPLC, %w/w)
5-amino-1,3,4-thiadiazole-2-thiol Not more than 0.10
5-amino-1,3,4-thiadiazole-2-sulphnamide Not more than 0.10
N-(5-sulphanyl-1,3,4-thiadiazol-2-yl)acetamide Not more than 0.10
N-(5-chloro-1,3,4-thiadiazol-2-yl)acetamide Not more than 0.10
N-(1,3,4-thiadiazol-2-yl) acetaide Not more than 0.10
5-Acetamido-1,3,4-thiadiazole-2-sulphonic acid Not more than 0.10
N-[5-[5-acetamido-1,3,4-thiadiazole-2yl]sulphonyl- Not more than 0.10
1,3,4-thiadiazol-2-yl]acetamide
Any unknown Not more than 0.10
Total Not more than 0.5

RESIDUAL SOLVENTS:
(By GC-HS, µg/g)
Methanol Not more than 1000
Acetone Not more than 1000
N,N-dimethyl formamide Not more than 880
ADDITIONAL SOLVENT:
Benzene Not more than 2

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 Development And Evaluation Of Lyophilized Product Of Apo-Acetozolamide

LIST OF APPARATUS AND EQUIPMENTS:

S.No NAME OF THE MANUFACTURER USE OF EQUIPMENT
EQUIPMENT
1 Lyophilizer Tofflon Science and Technology Co To perform the process of Freeze
Ltd., Shangai, China drying
2 Freeze Drying Lyostat3, Biopharma technology To determine the collapse
Microscopy limited. temperature(Tc).

3 Weighing balance Sartorius, Japan To weigh the raw materials and

finished product
4 PH meter Thermoscientific (Model: Orion 5 star) To find out the PH of the product
before and after Lyophilization
5 Filtration unit Millipore To clarify the drug solution
6 Modulated DSC TA instruments (Model : Q 2000 series) To determine the eutectic
temperature of the drug
7 HPLC Shimadzu HPLC LC 2010 To know the assay and related
substances of the drug
8 KF titrator Metrohm To determine the water content of
lyophilized drug
9 Particle size analyzer Malvern Instruments, UK To determine the size of particles

FORMULATION DEVELOPMENT:
As the Qualitative & Quantitative composition of Acetazolamide
sodium for injection is given in the prescribing information leaflet of
DIAMOX, the drug and excipients are selected based on the innovator
formula.
PROTOTYPE FORMULA:

Sr. Present in
Ingredients Qty/Vial Rationale
No. innovator
Acetazalamide
1 Sodium equivalent 500 mg Active Yes
to Acetazolamide
For pH
3 Sodium Hydroxide qs to pH Yes
Adjustment
qs to
4 Water for Injection Solvent Yes
5mL

MANUFACTURING PROCEDURE:
• Collect required batch size of Water for Injection in SS vessel; and cool the temperature to 4-8°C.
• Transfer 80% of Water for Injection from the above SS vessel to another SS vessel.
• Add weighed quantity of Acetazolamide with continuous stirring to get a clear solution.
• Check the pH and adjust the pH to 9.6 by using 1N NaOH.
• Make up the solution to required batch size with Water for Injection and check the final pH (Adjust if
necessary).
• Filter the bulk solution through 0.2μ membrane filter.

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 Development And Evaluation Of Lyophilized Product Of Apo-Acetozolamide

• Fill the solution into to 10mL USP Type I clear glass vials and half stopper it with double slotted chlorobutyl
rubber stoppers and load for lyophilization as per optimized lyophilization cycle.
• After completion of the lyophilization, stopper the vials under vacuum and seal with aluminium flip-off seals.

EVALUATION OF LYOPHILIZED PRODUCT:

The lyophilized product was evaluated for the following formulation characteristics:
• Description
• Clarity of reconstituted solution
• Reconstitution time
• pH after reconstitution
• Water content
• Assay
• Related substances
• Particulate matter

STABILITY STUDIES:
STABILITY STUDIES AS PER ICH GUIDELINES:

Recommended Stability Condition/Station:

STORAGE CONDITION
STATION 40 C/75 % RH 30 C/65% RH 25 C/60% RH

1M #↓ - -
2M #↓ - -
3M #*↓@ #↓ #↓
6M #*↓@ #↓ #↓
9M - - -
12M - #*↓ #*↓
24 M - - @↓

# - For analysis
* - BET (Bacterial Endotoxin Test)
↓ - Invert
@ - Reconstitution stability study

COMPATIBILITY STUDIES WITH PROCESS COMPONENTS:

Definition: Compatibility is a measurement of how stable a substance when it is in contact with another
material. If there is no change in their physical and chemical properties of substance when it contacts with other
materials is considered as compatible.
If changes happen in their physical and chemical properties of substances on contact with other material are
considered as incompatible.
Types of Compatibility Studies:
• Filter compatibility
• Silicone Tubing compatibility
• SS Vessel compatibility
• ‘O’ ring compatibility
Study Design: This study can be in two ways.

Static Soak Method: In this study, the test filter shall be soaked in the drug product solution for longer period.
Dynamic Filtration: In this study, the liquid is continuously circulated through the filter membrane repeatedly.
Of these both methods, static soak method was used for the present study.
Experimental Procedure:
• The test shall be subjected to autoclaving and then use it for the compatibility studies.

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 Development And Evaluation Of Lyophilized Product Of Apo-Acetozolamide

• Place material in drug product solution and kept aside.

• At regular time intervals (0, 4, 8, 12, 24 hours) withdraw the required amount of sample and perform analysis
of it.

RESULTS AND DISCUSSION

EVALUATION OF INNOVATOR PRODUCT:
Product name : DIAMOX I.V
Drug substance : ACETAZOLAMIDE
Manufactured by : Bedford laboratories
Dosage form : Powder for injection
Storage condition : Store at room temperature
Batch number : AT2008
Expiry date : 15/1/2014

S.No TEST PARAMETER OBSERAVATION

1. DESCRIPTION White freeze dried powder for

injection
2. ASSAY 101%
3. WATER CONTENT (%w/w) 4.7

4. pH AFTER RECONSTITUTION 9.6

5. RECONSTITUTION TIME 54s

6. CONSTITUTED SOLUTION clear and free from visible particles

7. RELATED SUBSTANCES
Total impurities (%w/w) 0.5%

8. Bacterial endotoxins Not detected

PREFORMULATION STUDY:
API CHARACTERIZATION:
TESTS SPECIFICATIONS OBSERVATIONS
DESCRIPTION White to faintly yellowish-white, A white powder, crysatalline,
crystalline, odorless powder odorless powder
SOLUBILITY Sparingly soluble in practically boiling Sparingly soluble in
water, slightly soluble in alcohol, very practically boiling water,
slightly soluble in water slightly soluble in alcohol,
very slightly soluble in water
IDENTIFICATION
a) By IR The IR spectrum of sample recorded, as Concordant with that of
KBr should exhibit transmission minima Acetazolamide reference
(absorption maxima)mat the same sample
wavelenths to these in the spectrum
obtained with the Acetazolamide
workingstandard.

B) By chemical test A clear, bright yellow solution is

produced. No heavy precipitate or dark Positive
brown color results after the mixing or
heating.

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 Development And Evaluation Of Lyophilized Product Of Apo-Acetozolamide

WATER CONTENT Not more than 0.5 0.5

pH Between 9.1 and 10 9.5

HEAVY METALS (ppm) Not more than 0.002 Not more than 0.002

ASSAY Not less than 98% and not more 99.8

than 102%
RELATED SUBSTANCES
Total Not more than 0.5 Not detected

RESIDUAL SOLVENTS:
Methanol Not more than 1000 Not detected
Acetone Not more than 1000 1002
N,N-dimethyl formamide Not more than 880 400
ADDITIONAL SOLVENT:
Benzene Not more than 2 Not detected

BACTERIAL ENDOTOXINS Not more than 0.5 Not detected

ABSORBANCE OF SOLUTION Not more than 0.2 0.174

From the above results, the API was found to be within its specifications given by the
vendor and thus the drug substance Acetazolamide sodium can be used for the present study.

LYOPHILIZATION CYCLE DEVELOPMENT

DETERMINATION OF EUTECTIC TEMPERATURE
 The Eutectic temperature is determined by using Modulated Differential Scanning
Calorimeter and was found to be -23.95 C.
So in the process of lyophilization, the product should be freezed below this
temperature in order to get good cake without collapse.

LYOPHILIZATION CYCLES
TRIAL1
PRECOOLING
SL.NO FINAL T(℃) RAMP DURATION(MIN) SOAK
DURATION(MIN)
1 -25.0 90 60
2 -45.0 90 180
PRIMARY DRYING

RAMP SOAK
FINAL T(℃) PRESSURE
SL.NO DURATION(MIN) DURATION(MIN)
(µbar)

1 -32 60 900 150

2 -10 60 480 150
3 0 60 120 150

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 Development And Evaluation Of Lyophilized Product Of Apo-Acetozolamide

CYCLE 2
PRECOOLING
SL.NO FINAL T(℃) RAMP DURATION(MIN) SOAK
DURATION(MIN)
1 -25.0 90 60
2 -45.0 90 180
3 -30 30 45
4 -45 30 120
PRIMARY DRYING
SL.NO FINAL T(℃) RAMP SOAK PRESSURE
DURATION(MIN) DURATION( (µbar)
MIN)

1 -32 120 900 250

2 -10 30 300 100
3 0 120 300 150

SECONDARY DRYING
SL.NO FINAL T(℃) RAMP SOAK PRESSURE
DURATION(MIN) DURATION (µbar)
1 10 60 180 50
2 30 30 180 50
3 40 20 600 50

OBSERVATION: The moisture content observed was 10.1%.

To reduce the water content a new modified cycle was taken in
the next trial.

CYCLE 3
PRECOOLING
S.NO FINAL T(℃) RAMP DURATION(MIN) SOAK
DURATION(MIN)
1 -25.0 90 60
2 -45.0 90 180

PRIMARY DRYING
S.NO FINAL T(℃) RAMP SOAK PRESSURE
DURATION DURATION (µbar)
(MIN) (MIN)

1 -25 120 300 250

2 -10 30 900 100
3 0 120 600 150

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 Development And Evaluation Of Lyophilized Product Of Apo-Acetozolamide

SECONDARY DRYING
S.NO FINAL T(℃) RAMP SOAK PRESSURE
DURATION(MIN) DURATION (µbar)

1 10 60 240 50
2 30 30 240 50
3 40 20 240 50

OBSERVATION
• The moisture content was found to be 8.4%

CYCLE 4
PRE-COOLING
S.NO FINAL T(℃) RAMP DURATION(MIN) SOAK DURATION(MIN)

1 -25.0 90 60
2 -45.0 90 180
3 -30 30 45
4 -45 30 120

PRIMARY DRYING
S.NO FINAL T(℃) RAMP SOAK PRESSURE
DURATION(MIN) DURATION(MIN) (µbar)

1 -25 120 300 250

2 -10 30 300 100
3 0 120 300 150

SECONDARY DRYING
S.NO FINAL T(℃) RAMP SOAK PRESSURE
DURATION(MIN) DURATION (µbar)

1 10 60 240 50
2 30 30 240 50
3 40 20 240 50

OBSERVATION
• The moisture content was found to be 6.36%.

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 Development And Evaluation Of Lyophilized Product Of Apo-Acetozolamide

CYCLE 5
PRE-COOLING
S.NO FINAL T(℃) RAMP DURATION(MIN) SOAK DURATION(MIN)

1 -25.0 90 60
2 -45.0 90 180

PRIMARY DRYING
Ss.NO FINAL T(℃) RAMP SOAK PRESSURE
DURATION(MIN) DURATION(MIN) (µbar)

1 -30 120 300 250

2 -25 100 590 150
3 -15 10 600 100
4 0 120 600 150

SECONDARY DRYING
S.NO FINAL T(℃) RAMP SOAK PRESSURE
DURATION(MIN) DURATION (µbar)

1 10 60 240 50
2 30 30 240 50
3 40 20 240 50
OBSERVATION
• The moisture content was found to be 5.7%And
time taken by the cycle is 67.80hrs=2.8days.
To reduce the cycle duration a new modified
cycle was taken in the next trial.

CYCLE 6
PRE-COOLING
S.NO FINAL T(℃) RAMP DURATION(MIN) SOAK DURATION(MIN)

1 -25.0 90 60
2 -45.0 90 180

PRIMARY DRYING
S.NO FINAL T(℃) RAMP SOAK PRESSURE
DURATION(MIN) DURATION(MIN) (µbar)

1 -30 120 300 250

2 -25 120 590 150
3 -15 10 600 100
4 0 120 300 100

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 Development And Evaluation Of Lyophilized Product Of Apo-Acetozolamide

SECONDARY DRYING
S.NO FINAL T(℃) RAMP SOAK PRESSURE
DURATION(MIN) DURATION

1 10 60 240 50
2 30 30 240 50
3 40 20 240 50

OBSERVATION
• The moisture content was found to be 4.3% and
the cycle time was reduced to 62.3 hrs from
67.80hrs.

EVALUATION OF LYOPHILIZED PRODUCT:

S.No TEST PARAMETER SPECIFICATION OBSERVATION

1. DESCRIPTION White freeze dried powder for White freeze dried powder for
injection injection

2. ASSAY 98% -102% 101%

3. WATER CONTENT (%w/w) 4.7 4.3

4. pH AFTER 9.6 9.6

RECONSTITUTION
5. RECONSTITUTION TIME 54s 50sec
6. CONSTITUTED clear and free from visible clear and free from visible
SOLUTION particles particles

7. RELATED SUBSTANCES
Total impurities (%w/w) 0.5% 0.08%
8. Bacterial endotoxins NMT 0.5 USP Not detected
(EU/mg)
Based on the results obtained from the characterization product, it can be concluded
that the drug product obtained is within its specified limits of the inovator product.

OBSERVATION:
• The manufacturing process adopted for the batch provided a clear
colorless solution at the end of the bulk solution compounding.
the lyophilized product complied with the tentative specifications
for the product with respect to the tested critical parameters like
assay, related substances ph and the water content was found to be
4.3%. And time taken by the cycle is 62.30hrs = 2.5days.
• The water content of this batch was reduced and the cycle time
was reduced to 62.3 hrs from 67.80hrs.

CONCLUSION: The bulk solution manufacturing process used in

this particular trial would be used as standard manufacturing
process for all further studies unless started.
• As the water content of this batch was low, the same recipe has
been proposed as final cycle for exhibit batch.

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 Development And Evaluation Of Lyophilized Product Of Apo-Acetozolamide

RECONSTITUTION STABILITY STUDIES:

RECONSTITUTION WITH 0.9% SODIUM CHLORIDE INJECTION:

Reconstitution with 0.9%NaCl
S.No Test parameter Specification 0 hrs 6 hrs 12 hrs 24 hrs
Clear, colour Clear, Clear Clear Clear
1. Description less solution colour colour less colourless colour
less less

2. Assay 98-102% 99.7 100.4 100.1 98.9

3. pH 9.6 9.62 9.61 9.62 9.63

4. Related substances
Total impurities NMT 0.5% 0.03 0.2 0.23 0.3
5. Particulate matter (per ≥25µ - 6 14 213 42
container)
600/unit
≥10µ - 131 180 190 430
6000/unit

From the above results, it can be concluded that 0.9% SODIUM CHLORIDE
INJECTION for reconstitution.

COMPATIBILITY STUDIES WITH PROCESS COMPONENTS:

FILTER COMPATIBILITY STUDY:

PVDF Filter compatibility

S.No Test parameter Specification Initial 4 hrs 8hrs 12hrs 24hrs
Clear, colour less Clear, Clear, Clear, Clear, Clear,
1. Description solution colour less colour colour colour colou
less less less r less

2. Assay 90-102% 99.5 99.2 99.4 99.1 98.9

3. pH 9-10 9.62 9.63 9.63 9.62 9.63

4. Related substances
Total impurities NMT 1.0% 0.03 0.04 0.08 0.08 0.08
6. Particulate matter ≥10µ - 6000 224 310 320 325 957
(per container)
≥25µ - 600 6 24 26 32 62

From the above results, it can be concluded that PVDF filter can be used for the purpose of the
filtration during the experimental work.

SILICON TUBING COMPATIBILITY STUDY:

1. PHARMA PURE TUBE:
Pharma Pure compatibility
S.No Test Specification Initial (storage at 2-8 ºC)
parameter 4 hrs 8hrs 12hrs 24hrs
Clear, colour Clear, Clear, Clear, Small Small
1. Description less solution colour colour colour particles particles
less less less observed observed
2. Assay 98-102% 101 99.7 98.2 98.1 97.9

3. pH 9.6 9.62 9.63 9.64 9.63 9.64

5. Related
substances NMT 0.5% 0.03 0.01 0.1 0.1 0.1
Total
impurities
6. Particulate ≥10µ - 6000 224 367 585 1064 2020
matter (per
≥25µ - 600 6 108 120 128 310
container)

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 Development And Evaluation Of Lyophilized Product Of Apo-Acetozolamide

2. PHARMA 50 TUBE:
Pharma 50 compatibility
S.No Test parameter Specification Initial (storage at 2-8 ºC)
4 hrs 8hrs 12hrs 24hrs
Clear, colour Clear, colour Clear, Clear, Small Small
1. Description less solution less colour less colour particles particles
less observed observed

2. Assay 98-102% 101 99.3 99.2 99.2 98.9

3. pH 9-10 9.62 9.62 9.63 9.63 9.64

4. Related
substances NMT 1.0% 0.03 0.07 0.08 0.08 0.09
Total impurities
5. Particulate ≥10µ - 6000 224 260 280 630 920
matter (per ≥25µ - 600 6 17 22 33 43
container)
From the above results, it can be inferred that the silicon tubings should not be in contact
with the drug solution for more than 8hrs because beyond that period particle growth was
being observed in the drug solution.
Of both the tubings, Pharma 50 has shown better results than Pharma Pure relating to
particulate matter. So Pharma 50 can be used during the experimental work.

3. SS VESSEL COMPATIBILITY STUDY:

SS Vessel compatibility
S.No Test parameter Specification Initial (storage at 2-8 ºC)
4 hrs 8hrs 12hrs 24hrs
Clear, colour Clear, colour Clear, colour Clear, Clear, Clear,
1. Description less solution less less colour colour colour
less less less

2. Assay 98-102% 101 99.8 99.5 99.1 98.9

3. pH 9.6 9.62 9.64 9.64 9.64 9.65

4. Related substances
Total impurities NMT 0.5% 0.03 0.04 0.04 0.07 0.08
5. Particulate matter (per ≥10µ - 6000 224 180 220 280 1024
container)
≥25µ - 600 6 4 17 32 81

The above results indicate that SS Vessel was found to be compatible with the drug
solution throughout the study period and therefore it can be used for the holding the drug
solution before loading in to the lyophilizer.

‘O’ RING COMPATIBILITY STUDY:

1. SILICON ‘O’ RING:
Silicon O ring compatibility
S.No Test parameter Specification Initial (storage at 2-8 ºC)

4 hrs 8hrs 12hrs 24hrs

Clear, colour less Clear, colour Clear, Clear, Clear, Clear,
1. Description solution less colour less colour colour colour
less less less

98-102% 99.8 99.8 99.4 99.3 99.2

2. Assay

3. pH 9.6 9.62 9.63 9.63 9.64 9.63

4. Related substances NMT 0.5% 0.03 0.04 0.03 0.06 0.1

Total impurities

5. Particulate matter (per ≥10µ - 6000 224 223 240 441 820
container)
≥25µ - 600 6 7 11 22 72

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 Development And Evaluation Of Lyophilized Product Of Apo-Acetozolamide

2. PTFE ‘O’ RING COMPATIBILITY:

PTFE O ring compatibility

S.No Test parameter Specification Initial (storage at 2-8 ºC)
4 hrs 8hrs 12hrs 24hrs
Clear, colour Clear, Clear, Clear, Clear, Clear,
1. Description less solution colour colour colour colour colour
less less less less less

2. Assay 98-102% 101 99.8 99.5 99.4 98.7

3. pH 9.6 9.63 9.62 9.63 9.61 9.62

4. Related
substances NMT 0.5% 0.03 0.07 0.08 0.08 0.03
Total impurities
5. Particulate matter
≥10µ - 6000 232 290 312 351 421
(per container)
≥25µ - 600 5 7 7 10 13
Even though both the ‘O’ rings had shown satisfactory results with respect to the assay,
pH and related substances, Silicon ‘O’ ring had favored more particulate growth than
PTFE ‘O’ ring during the analysis period. So, PTFE ‘O’ ring can be selected for the
present study.

Table : Stability data of 1st and 2nd months under accelerated storage condition
S.No Test parameter Specifications Initial 40°C/75 % RH
1st Month 2nd Month
1. Description White freeze dried White freeze dried White freeze dried White freeze dried
powder powder powder powder
2. Assay 98-102% 99.7 98.9 98.7

3. pH after reconstitution 9.6 9.61 9.63 9.63

4. Reconstitution time NMT 60s 10s 11s 13s

5. Constituted solution Clear, colour less Clear, colour less Clear, colour less Clear, colour less
solution, free from solution, free from solution, free from solution, free from
visible particles visible particles visible particles visible particles

6. Related substances NMT 0.5% 0.03 0.14 0.15

Total impurities
7. Particulate matter (per ≥10µ - 6000 131 254 326
container) ≥25µ - 600 6 19 34
≥50µ 0 2 5

Table : Stability data of 3rd and 6th months under accelerated storage condition
40°C/75 % RH
Test parameter Specifications 3rd Month 6th Month
S.No
At the time of 12hrs after At the time of 12hrs after
reconstitution reconstitution reconstitution reconstitution

1. Description White freeze dried White freeze dried White freeze dried White freeze dried
White freeze dried
powder powder powder powder powder

2. Assay 98-102% 99.7 99.3 99.1 98.9

3. pH after reconstitution 9.6 9.7 9.6 9.7 9.6

4. Reconstitution time NMT 60s 50s 50s 55s 55s

5. Constituted solution Clear, color less Clear, colour less Clear, color less Clear, color less Clear, color less
solution, free from solution, free from solution, free from solution, free from solution, free from
visible particles visible particles visible particles visible particles visible particles
6. Related substance, total NMT 0 5% 0.01 0.02 0.21 0.3
impurities
7. BET NMT 0.5 EU/mg <0.5 EU/mg <0.5 EU/mg <0.5 EU/mg <0.5 EU/mg

8. Particulate matter ≥10µ - 6000 315 369 422 511

≥25µ - 600 56 69 79 96
(per container)
≥50µ 11 18 15 32

The accelerated stability study results confirm that all the test parameters were within the specifications.

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 Development And Evaluation Of Lyophilized Product Of Apo-Acetozolamide

LONG TERM AND INTERMEDIATE STORAGE CONDITION STABILITY STUDY

S.No Test parameter Specifications 25°C/60% RH 30°C/67% RH
3rd Month 6th Month 3rd Month 6th Month
1. Description White freeze White freeze White freeze White freeze White freeze
dried powder dried powder dried powder dried powder dried powder
2. Assay 98-102% 99.6 99.1 99.5 98.9
3. pH after 9.6 9.62 9.63 9.63 9.64
reconstitution
4. Reconstitution time NMT 60s 51s 52s 50s 53s
5. Constituted Clear, colour Clear, colour Clear, colour Clear, colour Clear, colour
solution less solution, less solution, less solution, less solution, less solution,
free from free from free from free from free from
visible visible visible visible visible
particles particles particles particles particles
6. Related substances, NMT 0.5% 0.01 0.02 0.2 O.3
total impurities
7. Particulate matter ≥10µ-6000 355 495 411 525
test by Light
≥25µ - 600 59 86 66 81
Obscuration test
method (per ≥50µ 15 31 20 34
container)

The above results confirm that the developed lyophilized drug was stable at long term
storage condition (25 C/60% RH) and intermediate storage condition (30 C/65% RH)
for a period of 6 months.

THERMAL CYCLING STUDY

The product was evaluated for the effects of temperature variation on the product when subjected to
temperature conditions.
S.No Test Parameter Specifications Initial Cycle 3
1. Description White freeze dried White freeze dried White freeze dried
powder powder powder
2. Assay 98-102% 99.6 98.9
3. pH after reconstitution 9.6 9.7 9.6

4. Reconstitution time NMT 60s 50 51s

5. Constituted solution Clear, colour less Clear, colour less Clear, colour less
solution, free from solution, free from solution, free from
visible particles visible particles visible particles
6. Related substances NMT 0.5% 0.01 0.2
Total impurities

7. BET NMT 0.075 EU/mg <0.075 EU/mg <0.075 EU/mg

8. Particulate matter (per ≥10µ - 6000 158 221

container)
≥25µ - 600 2 5
≥50µ 0 1

TEMPERATURE EXCURSION STUDY:

S.No Test parameter Specifications Initial 2 days at -20ºC 2 days at 60ºC

White freeze dried White freeze dried White freeze dried White freeze
1. Description powder powder powder dried powder
2. Assay 98-102% 99.7 98.4 98.2

3. Water content by KF NMT 7.0% 1.59 4.77 2.10

4. pH after reconstitution 9.2-10 9.7 99.1 98.9

5. Reconstitution time NMT 60s 50s 52s 50s

6. Constituted solution Clear, colour less Clear, colour less Clear, colour less Clear, colour
solution, free from solution, free from solution, free from less solution,
visible particles visible particles visible particles free from visible
particles
7. Related substances
Total impurities NMT 0.5% 0.01 0.04 0.2

9. Particulate matter (per 131 115 320

≥10µ - 6000
container)

≥25µ - 600 6 5 8

≥50µ 0 0 2

OBSERVATION:
From the results of the Thermal Cycling and Temperature Excursion study we can confirm that the developed
lyophilized drug product will be stable when it was subjected to or cycled through temperature conditions that
62
 Development And Evaluation Of Lyophilized Product Of Apo-Acetozolamide

simulate the short term excursions outside the proposed label storage conditions which are likely to be
encountered by the drug product during distribution.
So, it can be concluded that the drug product can be shipped between different climatic conditions without
any change in its quality.

SUMMARY AND CONCLUSION

Before the development of the formulation, preformulation studies were conducted. In that solubility studies of
the drug product, was determined the suitable conditions for the development of an optimized product.
Eutectic temperature was determined by using DSC and collapse temperature was determined which is the key
parameter in the development of lyophilisation cycle recipe. Depending upon the value of eutectic temperature
the recipes of the lyophilisation cycles were developed and optimized cycle was decided by trial and error
method. Among all the above formulations, cycle 6 produces excellent product which is having desired qualities
of the lyophilized product. This product was then compared with the innovator. And product was then subjected
to various tests like reconstitution stability, stability studies as per ICH guidelines.

REFERENCES
[1] M. J. Pikal , Takayuki doen,.Determination of end point of primary drying in freeze drying process
control,AAPS PharmSciTech,vol II( march 2010).
[2] The importance of freezing on lyophilization cycle development, biopharm march(2002),pp :16-21.
[3] Eva Meister SLObodan sasic and Henning GieselerFeeeze_dry microscopy: impact of nucleation
temperature and excipient concentration on collapse temperature data. control,AAPS PharmSciTech
vol.10 (sep 2008), PP:582-588.
[4] M. J. Pikal. Lyophilization. In J. Swarbrick and J. Boylan (eds.), Encyclopedia of Pharmaceutical
Technology, Marcel Dekker, New York, 2002, pp. 1299–1326.
[5] B. Lueckel, D. Bodmer, B. Helk, and H. Leuenberger. Formulations of sugars with amino acids or
mannitol-influence of concentration ratio on the properties of the freeze-concentrate and the lyophilizate.
Pharm. Dev. Technol. 3:325–336 (1998).
[6] M. J. Pikal, S. Shah, D. Senior, and J. E. Lang. Physical chemistry of freeze-drying: measurement of
sublimation rates for frozen aqueous solutions by a microbalance technique. J. Pharm. Sci. 72:635–650
(1983).
[7] M. J. Pikal, S. Shah, M. L. Roy, and R. Putman. The secondary drying stage of freeze drying: drying
kinetics as a function of temperature and chamber pressure. Int. J. Pharm. 60:203–217 (1990).
[8] Roy ML, Pikal MJ. Process control in freeze drying: determination of the end point of sublimation drying
by an electronic moisture sensor. J Parenter Sci Technol. 1989;43(2):60–6.
[9] Gieseler H, Kessler WJ, Finson M, Davis SJ, Mulhall PA, Bons V, et al. Evaluation of tunable diode laser
absorption spectroscopy for in-process water vapor mass flux measurements during freeze drying. J
Pharm Sci. 2007;96(7):1776–93.
[10] Mayeresse YVR, Sibille PH, Nomine C. Freeze-drying process monitoring using a cold plasma ionization
device. PDA J Pharm Sci Technol. 2007;61(3).
[11] Bardat A, Biguet J, Chatenet E, Courteille F. Moisture measurement: a new method for monitoring
freeze-drying cycles. Parenter Sci Technol. 1993;47(6):293–9.
[12] Pikal MJ, Roy ML, Shah S. Mass and heat transfer in vial freezedrying of pharmaceuticals: role of the
vial. J Pharm Sci. 1984;73 (9):1224–37.
[13] M. J. Pikal and J. E. Lang. Rubber closures as a source of haze in freeze dried parenterals: test
methodology for closure evaluation. J. Parenter. Drug Assoc. 32:162–173 (1978).
[14] M. J. Pikal, M. L. Roy, and S. Shah. Mass and heat transfer in vial freeze-drying of pharmaceuticals: role
of the vial. J. Pharm. Sci. 73:1224–1237 (1984).
[15] N. Milton, M. J. Pikal, M. L. Roy, and S. L. Nail. Evaluation of manometric temperature measurement as
a method of monitoring product temperature during lyophilization. PDA J. Pharm. Sci. Technol. 51:7–16
(1997).
[16] X. C. Tang, S. L. Nail, and M. J. Pikal. Mass transfer in freeze drying: measurement of dry layer
resistance by a non-steady state method (the MTM procedure). 1999 AAPS Anual Meeting, New
Orleans, Louisiana, 1999.
[17] J. A. Searles, J. F. Carpenter, and T. W. Randolph. Annealing to optimize the primary drying rate, reduce
freezing-induced drying rate heterogeneity, and determine Tg in pharmaceutical lyophilization.J. Pharm.
Sci. 90: 872–887.

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