M Pharm Major Project

SPP SPTM-NMIMS University 11
EFFECT OF VARIOUS FREEZE DRYING
PARAMETERS ON THE STABILITY OF DRUG-X
R. Deepak Chandra
M.Pharm (Pharm Analysis)+MBA
Roll no:15
Under the Guidance of
Dr. Dipti Gatne
Professor & HOD of Pharm Analysis
SPP SPTM
NMIMS University
Under the Guidance of
Dr. Subash gore
General Manager, R&D Formulations
Dr. Ajeet K singh
Sr. Manager, R&D Formulations
Mylan laboratories limited, Hyderabad

Background
 Drug-X has the stable forms among its polymorphs, it available in the form of
lyophilized injection, stable form-Monohydrate & unstable form- Anhydrous
form.
 During the process of lyophilization it converts monohydrate form of Drug-X to
anhydrous form where complete water is removed there by, monohydrate form of
Drug-X to anhydrous which is unstable not suitable for the formulation.

Aim & Objective of the Work
 To differentiate stable and unstable forms of Drug-X .
 To characterize solid state of monohydrate form of Drug-X in freeze drying
formulation.
 Determine the effects of various relative humidity conditions on Drug-X & ideal
storage conditions for stable Drug-X monohydrate form i.e. Temperature and
humidity conditions.

S. no Materials Category, mechanism of action & use
1 Drug-X (API)  Alkylating agent; Nitrogen Mustards, synthetic antineoplastic drug. Very
Narrow Therapeutic Index.
 Prodrug; Activation requires cytochrome P450 enzyme.
 Reactive carbonium ion intermediates transfer alkyl groups by forming
covalent bonds results in cross linking/ abnormal base pairing/ scission of
DNA strand.
Current Therapies:
1. Regulatory T-cell modulation
2. Treatment of Multiple Sclerosis.
3. Choice for acute & chronic leukaemias, myeloid leukaemia, Breast
Cancer
2 Mannitol
(Excipient)
 In lyophilized preparations-Mannitol as a carrier to produce a stiff,
homogeneous cake that improves the appearance of the lyophilized cake
 Improved dissolution rates; Improved thermal stability
 Bulking agent
Materials

S. No Equipment Purpose of Use
1. Differential Scanning Calorimeter
Determine the freeze drying parameters, and
characterization of API and Excipient.
2. Thermo Gravimetric Analysis To determine the weight loss in API.
3. X-Ray Diffraction D-8 Advance
Characterization of API & Mannitol, solid state
characterization of API, Lyophilized cakes, polymorphs of
API
4.
Gravimetric sorption Analyzer
(Intelligent gravimetric analysis)
Study the humidity effect on API
(Sorption-Desorption studies)
5. Karl Fisher Titration Determine the water content
6. Lyophilizer Perform the process of lyophilization/freeze drying.
Methods

The project was planned as follows-
 Characterization of Drug-X :
 Determination of Melting point by Differential scanning calorimeter.
 Determination of Powder Characteristics by Powder X-ray diffraction.
 Determination of Water content by Thermogravimetric analyzer.
 Characterization of Mannitol:
 Determination of Melting point by Differential scanning calorimeter.
 Determination of Powder Characteristics by Powder x-ray diffraction.
 Lyophilization of Drug-X Formulation:
 Drug-X for Injection, USP.
 Optimization of lyophilization conditions in stable lyophilized Drug-X formulation by Differential Scanning
Calorimetry.
 Lyophilization process
 Characterization of Freeze Dried Formulations:
 Determination of Water content by Karl fischer Titration
 Characterization of Freeze Dried Formulations by Powder X-ray Diffraction
 Effect of Relative humidity on Drug-X:
 Effect of Relative humidity on Drug-X by Gravimetric sorption analyzer
 Effect of Relative humidity on Drug-X by Powder x-ray diffraction.
Research Envisaged &Plan of Work

 Melting point at 49.3°C.
characteristic for monohydrate form of Drug-X
wrt the reference in the USP monograph
specifications. (49°C-52°C)
 Peaks for at 6.973°, 14.755° and 17.785,
which are the characteristic for Drug-X.
Characterization of Drug-X - DSC, XRD & TGA
DSC Thermogram of Drug-X Diffractogram of Drug-X

Loss of weight as 6.15% which is characteristic for Drug-X
TGA Thermogram of Drug-X

 Melting point at 170.9°C which is
characteristic wrt the reference in the USP
monograph (165°C-170°C)
 Observed peaks for at, 9.402°, 13.659°,
and 17.263° which are characteristic
Mannitol
Characterization of Mannitol - DSC & XRD
DSC Thermogram of Mannitol Diffractogram of Mannitol

Dissolving
Drug and
excipients in
a suitable
solvent,
generally
WFI.
Sterilizing
bulk solution
by passing it
0.22 micron
bacteria-
retentive
filter.
Filling into
individual
sterile
containers
and partially
stoppering
the containers
under aseptic
conditions.
Transporting
containers to
lyophilizer
and loading
into the
chamber
Freezing the
solution by
placing in
shelves in a
freeze-drying
chamber or
pre-freezing
in another
chamber.
Applying
vacuum to
chamber ,
heating
shelves to
evaporate the
water from
the frozen
state.
Stoppering of
the vials
installed in
lyophilizers.
Lyophilization generally includes the following steps:
Drug-X is available as parenteral dosage formulations consisting of
sterile packaged dry powder blend admixtures of the Monohydrate from
of Drug-X, sodium chloride and mannitol.

Laboratory Freeze Dryer- AdVantage freeze dryer

 Exotherm which represents crystallization temperature at -20°C and e
 Endotherm which represents at ice melting at -1.60°C and melting at 4.37°C.
Optimization of freeze drying parameters –
Differential Scanning Calorimeter

 The freeze drying parameters are optimized by using DSC
Freezing temperature = -20°C-25°C
Primary drying = 1-5°C
Secondary drying = 20°C-25°C.
The freezing can be set to more than -25°C ; as lyophilizer runs for long time and also
to avoid any fluctuations.
 Different lyophilization batches are operated varying in vacuum, freezing time,
primary drying time, secondary drying time. all the batches yields cakes with good
appearance and greater stability.

Batch Thermal Treatment Primary Drying Secondary Drying
Batch-1 Freezing to -40°C 150 min High vacuum
(500 m Torr)
1330 min High vacuum
(500 m Torr)
100 min
Batch-2 Freezing to -40°C 240 min Moderate
vacuum
(380 m Torr)
(500 m Torr)
120 min
Batch-3 Freezing to -40°C 360 min Less vacuum
(200 m Torr)
1100 min Less vacuum
(200 m Torr)
1200 min
(200 m Torr)
(500 m Torr)
500 min
(200 m Torr)
(500 m Torr)
240 min
Batch-6 Freezing to -
40°C
360 min Less vacuum
(200 m Torr)
(500 m Torr)
40 min
Freeze dried formulations are analyzed by using XRD & KF
Freeze Drying cycle- Lyophilizer
Freeze dried formulation of 6 different Batches

Water content - Karl Fisher Titration
Water content of Freeze dried formulations
S.NO Sample/Batch Percentage of Water Content
1 Drug-X (API) 6.18%
2 Batch-1 0.002%
3 Batch-2 0.002%
4 Batch-3 0.004%
5 Batch-4 0.004%
6 Batch-5 0.002%
7 Batch-6 4.35%

5 Batches - overlayed and compared with monohydrate form of Drug-X and mannitol
peaks, Batch-3 shows significant decrease in intensities .
Solid pattern studies-XRD
Diffractogram of Freeze dried formulations

S. No RELATIVE HUMIDITY (%)
RANGE
TIME INFERENCE
1 Zero % (Desorption) 16 hrs Physical form of drug changes to powder to sticky liquid.
2 Zero % (Desorption) 4 hrs 7.1% weight loss
Sticky, semisolid form
3 0-90% 20 hrs
4.5 % weight gain
1.2 % loss at 40-50% RH
4
0-20%
Zero % (Desorption) for an hr
20 % for 2 hrs
3 hrs 4.28% weight loss
5 5% RH 15 hrs 5.2% weight loss
6 20-30% RH 10 hrs 0.8 % weight loss
Powder form
7 80 % RH 6 hrs 0.5% weight gain
Powder form
Effect of Relative humidity- Gravimetric Sorption Analyzer

Continued…
 Monohydrate from of Drug-X is subjected 0% RH to 90% RH with changes in time
intervals
 At lower RH: loss in weight ; anhydrous form ; physical nature : semisolid and sticky in
nature; unstable
Back to room RH: weight gain ; physical nature remains same.
 At higher RH: weight gain; monohydrate nature; physical nature remains same;
crystalline powder form; stable.
Back to room RH: Weight loss; physical nature remains same.
Therefore optimum RH conditions for the Monohydrate from of Drug-X to be stored are
20-30% RH, where negligible weight loss is seen that withstands monohydrate nature.

 Drug-X is subjected to various relative humidity conditions -reduced in intensities
Variations in relative humidity conditions
Diffractogram of Drug-X at various RH

Drug-X is subjected to higher relative humidity conditions where crystalline form
is unchanged. It means monohydrate form of Drug-X is stable in its crystal form at
higher relative humidities.
Higher Relative Humidity
Diffractogram of Drug-X at higher RH

Non-ambient PXRD Study
Diffractogram of Drug-X using Non-ambient PXRD study
Monohydrate form of Drug-X is subjected to Non-ambient powder x-ray
diffraction study and diffractograms are observed at different intervals of time,
characteristic peaks for monohydrate form slowly disappearing after 2 hrs.
Gradually the monohydrate form converted in to anhydrous after 14 hrs under zero
percent relative humidity.

Summary
 Characterization of Drug and excipient are done by DSC, XRD which helps to
determine the nature of drug and excipient
 Monohydrate from of Drug-X is thermally and hydrolytically susceptible and
exhibit poor stability in aqueous solution hence the technique lyophilization is
employed.
 Presence of water content in the Drug-X formulation after lyophilization process is
an characteristic feature, as the stable form of Drug-X is monohydrate, therefore
determination of water content by Karl fisher
 Studied the changes in the solid state patterns of freeze dried lyophilized cakes and
determines the changes in the nature of solid state patterns with variations in the
freeze drying parameters.
 Drug-X is sensitive to humidity variations therefore the solid state characterization
of Monohydrate from of Drug-X by XRD at various humidity conditions and effect
of various relative humidity conditions are done in order to determine the
temperature and relative humidity conditions.

Conclusion
 Powder X-ray diffraction studies able to identify between
monohydrate and anhydrous forms of Drug-X.
 By varying the secondary drying conditions in the lyophilizer
an optimum of 4% moisture is achieved in the vial.
 Relative humidity effects studies on Monohydrate from of
Drug-X reveals that optimum storage conditions.
 Temperature = less than 25°C.
 Relative Humidity = 25%-30%.

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