Post on 21-Jan-2017
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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
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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.
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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.
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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
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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
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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
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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
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Loss of weight as 6.15% which is characteristic for Drug-X
TGA Thermogram of Drug-X
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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
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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.
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Laboratory Freeze Dryer- AdVantage freeze dryer
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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
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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.
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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)
1180 min High vacuum
(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
Batch-4 Freezing to -40°C 1270 min Less vacuum
(200 m Torr)
2810 min High vacuum
(500 m Torr)
500 min
Batch-5 Freezing to -40°C 260 min Less vacuum
(200 m Torr)
2540 min High vacuum
(500 m Torr)
240 min
Batch-6 Freezing to -
40°C
360 min Less vacuum
(200 m Torr)
1100 min High vacuum
(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
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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%
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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
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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
Sticky, semisolid form
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
Sticky, semisolid form
5 5% RH 15 hrs 5.2% weight loss
Sticky, semisolid form
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
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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.
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Drug-X is subjected to various relative humidity conditions -reduced in intensities
Variations in relative humidity conditions Diffractogram of Drug-X at various RH
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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
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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.
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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.
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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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