m pharm major project

SPP SPTM-NMIMS University 1 1

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

SPP SPTM-NMIMS University 8

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%.

m pharm major project

Documents