Post on 04-May-2020
Methods and Materials 2012
Lyophilized Formulation Development with Novel Excipients Derived from Cassia Roxburghiifor a PEGylated Therapeutic Protein Page 1
MATERIALS AND METHODS
Methods and Materials 2012
Lyophilized Formulation Development with Novel Excipients Derived from Cassia Roxburghiifor a PEGylated Therapeutic Protein Page 2
I. MATERIALS AND METHODS
1.1 EQUIPMENT’S USED
Table 1 - Equipment’s used
S. No Equipment’s Make
1 Analytical Balance Mettler Toledo
2 Precision Balance Mettler Toledo
3 Microbalance Sartorius
4 PH Meter Lab India
5 Hot Plate Schott Instruments
6 Slab Gel Dryer Hoefer
7 Osmometer Advanced Instruments
8 Conductivity Meter LabIndia
9 Stirrer Spinot
10 Orbital Shaker Barnstead/Heidolph
11 Centrifuge Sigma/Eppendorf
12 Phast System Amersham Biosciences
13 Filters Millipore
14 UV Spectrophotometer Shimadzu
15 Vortex Mixer Spinix
16 Incubator Cintex
17 HPLC Agilent/Shimadzu
18 Freezers (-20C) Vest Frost
19 Refrigerator (2-8C) Samsung
20 Vial Kaisha
21 Sonicator Transonics
22 Syringe & Needles Disopovan
23 SDS Page Atto corporation
24 Lyophilizer BOC Edwards
Methods and Materials 2012
Lyophilized Formulation Development with Novel Excipients Derived from Cassia Roxburghiifor a PEGylated Therapeutic Protein Page 3
S. No Equipment’s Make
25 Digital hardness tester Campbell electronic, Bombay
26 Disintegration test apparatus Tab machine, Bombay
27 Friability tester Campbell electronic, Bombay
28 Hot air oven Tecnico, Chennai
29 FTIR JASCO, Japan
30 ESI-LC-MS Mass spectrometer FLEX-PC [Bruker Daltonics –
MALDI TOF/TOF]
31 Ion Trap detector 1200-HPLC from Agilent having
ESI
32 CD Spectrometer Jasco J-815
33 DSC Micro calorimeter VP-DSC from Microcal
34 DLS Zetasizer Nano series
35 Fluoro spectrometry Perkin elmer LS55
36 DSC - Q200 TA Instruments
Methods and Materials 2012
Lyophilized Formulation Development with Novel Excipients Derived from Cassia Roxburghiifor a PEGylated Therapeutic Protein Page 4
1.2 LIST OF CHEMICALS USED
Table 2 – Chemicals Used
S. No Ingredient Manufacture
1 Sucrose JT Baker
2 Polaxamer 188 Sigma
3 Mono Sodium Phosphate JT Baker
4 Di Sodium Phosphate JT Baker
5 Sodium Dodecyl Sulphate Sigma
6 Tris base Sigma
7 Acrylamide Sigma
8 TEMED Sigma
9 Coomasiee Blue Sigma
10 Glacial Acetic acid Merck
11 Methanol Merck
12 Acetic acid Merck
13 Sodium thiosulphate Merck
14 Silver nitrate Merck
15 Sodium carbonate Merck
16 Formaldehyde Merck
17 Na EDTA JT Baker
18 Glycerol Merck
19 Urea Sigma
20 Fehling solution SRL
21 Benedict’s solution SRL
22 Molish reagent SRL
23 Seliwanoff’s solution Biored
24 Millons reagent SRL
25 Dragendorff’s reagent Biored
Methods and Materials 2012
Lyophilized Formulation Development with Novel Excipients Derived from Cassia Roxburghiifor a PEGylated Therapeutic Protein Page 5
S. No Ingredient Manufacture
26 Mayer’s solution Biored
27 Borntragor solution SRL
28 Libermann Bruchard solution Biored
29 FeCl3 SRL
30 Ethanol Sigma
31 Keller Killiani reagent Biored
1.3 NATURAL SOURCE FOR NOVEL EXCIPIENTS:
1.3.1 PLANT IDENTIFIED: C. ROXBURGHII SAMPLE:
The C. roxburghii, seeds were obtained from Department of Siddha, Tamil University,
Thanjavur and Authenticated Botanical Survey of India (BSI).
1.3.2 DRUG SUBSTANCE USED:
PEGylated therapeutic protein was selected and received as gift samples from Intas
Biopharmaceuticals Limited, Ahmedabad, Gujarat.
1.4 COLLECTION OF SEEDS AND ISOLATION OF SEED GUM POWDE R
(PLANT EXCIPIENT)
4.4.1 COLLECTION:
The collected seeds were dried at room temperature (250C) for 24 hours and powdered by
using grinder.
4.4.2 ISOLATION OF SEED GUM:
• Defatted Seed Gum:
The seeds of C. roxburghii were broken by mechanical impounding followed by
powdering with elite grinder. The powders followed defat process. This method utilizes
Methods and Materials 2012
Lyophilized Formulation Development with Novel Excipients Derived from Cassia Roxburghiifor a PEGylated Therapeutic Protein Page 6
soxhlet apparatus and extraction procedure. The solvent used for this extraction process
were petroleum ether at temperature conditions between 60 oC - 80 oC. the same process
were getting cycled multiple times to ensure complete extraction. Hot water used as allied
to get maximum yield. Following the final mucilage were filtered by using sterile muslin
cloth to ensure its purity. The same were getting precipitated by using acetone. To get
fine powder, the precipitates were freeze dried under the blanket of inert atmosphere.
1.5 DRUG PRODUCT FORMULATION PROCESS:
4.5.1 REQUIREMENTS FOR PREPARING FORMULATION:
• Drug Substance (DS): It is the active protein ingredient (API) present in buffer
solution along with or without stabilizer in it.
• Excipient solution (ES): It is a solution containing excipients such as stabilizer,
osmolites, etc other than active ingredients.
• Drug Product (DP): It is the formulated and filtered solution containing both
API and excipients in buffer solution.
4.5.2 PREPARATION OF DRUG SUBSTANCE (API):
The drug substance was obtained from the downstream processing laboratory of Intas
Biopharmaceutical’s Limited, Ahmedabad, Gujarat. The concentration of protein used for
the study was 0.39 mg/ml as determined by RP-HPLC.
4.5.3 FORMULATION PROCESS DEVELOPMENT:
Since the identified novel excipient contains mixed population of polymers and
monosaccharides in variable amounts as part of its components, the intended role of the
same in lyophilized formulations could be,
• Stabilizer (Cryoprotectant and/ Lyoprotectant)
• Surfactant
Methods and Materials 2012
Lyophilized Formulation Development with Novel Excipients Derived from Cassia Roxburghiifor a PEGylated Therapeutic Protein Page 7
Hence the novel excipient was formulated with following different formulation
compositions and the stability of novel formulation composition was compared with
reference formulation. The reference formulation composition selection were done as
following basis,
• All formulation components are from USFDA approved excipients in the
same intended role in lyophilized formulation category
• All the comparisons made by qualitative and quantitative analysis of both the
formulations at different conditions
1.6 IDENTIFIED FORMULATION COMPOSITIONS (Tim J. Kamerze ll, et al.,
(2011)):
4.6.1 RESEARCH COMPARISON STUDY - I
Objective:
Effect of stabilizing property of novel excipient in lyophilized formulation composition
was scrutinized by comparing with the well-established USFDA approved stabilizer
Sucrose.
Table 3 – Formulation Composition 1
S. No
FORMULATION COMPOSITION IDENTIFIE
D ROLE Novel Formulation A Reference formulation Component Qty./mL Component Qty. / mL
1 PEGylated Therapeutic protein
100 µg PEGylated Therapeutic protein
100 µg Drug substance
2 Novel excipient 20 mg Sucrose 80 mg Stabilizer 3 Polaxmer 188 0.5 mg Polysorbate 80 0.1 mg Surfactant
4 Dibasic sodium phosphate 10 mM Dibasic sodium phosphate
10 mM Buffer component
5 Monobasic sodium phosphate
10 mM Monobasic sodium phosphate
10 mM Buffer component
6 Water for injection Q.S Water for injection Q.S Vehicle
Methods and Materials 2012
Lyophilized Formulation Development with Novel Excipients Derived from Cassia Roxburghiifor a PEGylated Therapeutic Protein Page 8
4.6.2 RESEARCH COMPARISON STUDY - II
Objective:
Effect of Surfactant property of novel excipient in lyophilized formulation composition
was scrutinized by comparing with the well-established USFDA approved Surfactant
Polysorbate 80.
Table 4 – Formulation composition 2
S. No
FORMULATION COMPOSITION IDENTIFIED
ROLE Novel Formulation B Reference formulation Component Qty./mL Component Qty./ mL
1 PEGylated Therapeutic protein
100 µg PEGylated Therapeutic protein
100 µg Drug substance
2 Sucrose 80 mg Sucrose 80 mg Stabilizer 3 Novel excipient 0.2 mg Polysorbate 80 0.1 mg Surfactant
4 Dibasic sodium phosphate 10 mM Dibasic sodium phosphate
10 mM Buffer component
5 Monobasic sodium phosphate
10 mM Monobasic sodium phosphate
10 mM Buffer component
6 Water for injection Q.S Water for injection Q.S Vehicle
4.6.3 DRUG PRODUCT FORMULATION PROCESS:
The batch size of the drug product was 100 mL. The batch formulae were detailed in
table 4 and table 5 and API requirement were as shown in table 6.
4.6.4 CALCULATION FOR API REQUIREMENT FOR DP MANUFACTURING
Table 5 – API Requirement for DP manufacturing
S. No Content Concentration (mg/mL) Required Amount for 100 mL
1 DP 0.1 10 mg
2 API 0.39 25.64 mL
Methods and Materials 2012
Lyophilized Formulation Development with Novel Excipients Derived from Cassia Roxburghiifor a PEGylated Therapeutic Protein Page 9
The DP was formulated in the final concentration of DS as 0.1 mg/mL. The batch
formulae calculation was worked out in order to get the desired concentration as per the
above table. The API was taken away from freezer (stored in a temperature range of
-20 ± 2 ºC) carefully and thawing the same at room temperature of 25 ± 2 ºC. Care should
be taken to minimize the protein degradation while thawing the material. The thawing
time was extended till to get uniform clear solution; it should be free from ice crystals.
4.6.5 MANUFACTURING PROCESS FOR FORMULATION COMPOSITION I:
The manufacturing process of drug product typically involved steps as explained in detail
below:
Step-1: Preparation of formulation buffer
Initially the required quantity of formulation components includes mono and disodium
phosphate, Novel excipient (as shown in table 4) were weighed and dissolved in 100 mL
beaker containing water for injection (WFI) with continuous stirring. The solution was
transferred to a 100 mL measuring cylinder. A 1 % (10 mg/ mL) stock solution of
poloxamer 188 was prepared by weighing and dissolving 1 gm in 20 mL WFI in a 100 mL
volumetric flask, followed by making up the volume to 100 mL with WFI. Required
volume of poloxamer 188 from the above stock solution was withdrawn and added to the
above 100 mL measuring cylinder, followed by making up the volume to 100 mL with
WFI. The entire formulation buffer was stirred at 100 rpm for 10 minutes and checked for
pH and osmolality.
Step-2: Preparation of formulated bulk solution
Required volume of formulation buffer to dilute the API (drug substance) as per batch
calculation was withdrawn and added into a clean, dry glass beaker. Required volume of
API (drug substance) as per batch calculation was withdrawn and added to the above
formulation buffer contained in the beaker for dilution. The solution was mixed by
stirring at 100 rpm for 10 minutes to obtain uniform formulated bulk solution (unfiltered)
followed by checking for pH, Osmolality and protein content.
Step-3: Sterile Filtration of formulated bulk solution
Methods and Materials 2012
Lyophilized Formulation Development with Novel Excipients Derived from Cassia Roxburghiifor a PEGylated Therapeutic Protein Page 10
The formulated bulk solution was filtered using a sterile 0.22 µm NUPORE
Polyethersulphone (PES) filters into a sterile, clean and dry Scott Duran glass bottle.
Step-4: Filling and loading vials into Lyophilizer:
Than DP was aseptically transferred in to the sterile 2 ml USP type 1 Kaisha glass vial.
The filling was done by manually and aseptically with use of micropipette by sterile
pipetting aids. Care should be taken to fill accurately in each vial at the fill volume of
700µl in each vial. Followed by filled vials were getting half stoppered with bromo-butyl
lyo stopper. Then the vials were loaded aseptically into the lyophilization chamber with
metal ring protection and preferably in every corner and center of the shelf place
temperature probe to monitor the temperature fluctuations and process flow inside the lyo
vial. The flow of lyophilization cycle illustrated in figure no: 7.
4.6.6 MANUFACTURING PROCESS FOR FORMULATION COMPOSITION II:
Step-1: Preparation of formulation buffer
Initially the required quantity of formulation components includes mono and disodium
phosphate, Novel excipient and Sucrose (as shown in table 5) were weighed and dissolved
in 100 mL beaker containing water for injection (WFI) with continuous stirring. The
solution was transferred to a 100 mL measuring cylinder. Followed by make up to the final
volume (100 mL) with WFI. The entire formulation buffer was stirred at 100 rpm for
10 minutes and checked for pH and osmolality.
Step’s 2, 3 and 4 were followed as per the above mentioned section 4.6.5.
4.6.7 MANUFACTURING PROCESS FOR REFERENCE COMPOSITION:
Step-1: Preparation of formulation buffer
Initially the required quantity of formulation components includes mono and disodium
phosphate, Sucrose (as shown in table 5) were weighed and dissolved in 100 mL beaker
containing water for injection (WFI) with continuous stirring. The solution was transferred
to a100 mL measuring cylinder. A 1 % (10 mg/ mL) stock solution of polysorbate 80 was
Methods and Materials 2012
Lyophilized Formulation Development with Novel Excipients Derived from Cassia Roxburghiifor a PEGylated Therapeutic Protein Page 11
prepared by weighing and dissolving 1 gm in 20 mL WFI in a 100 mL volumetric flask,
followed by making up the volume to 100 mL with WFI. Required volume of polysorbate
80 from the above stock solution was withdrawn and added to the above 100 mL measuring
cylinder, followed by making up the volume to 100 mL with WFI. The entire formulation
buffer was stirred at 100 rpm for 10 minutes and checked for pH and osmolality.
Step’s 2, 3 and 4 were followed as per the above mentioned section 4.6.5.
1.7 LYOPHILIZATION PROCESS DEVELOPMENT( Wei Wang, (2000)):
4.7.1 CRITICAL PHYSIO-CHEMICAL VALUES:
Different lyophilization cycles were adopted based on the Physico-chemical analysis of
the novel formulation compositions 1 and 2.
4.7.2 DESIGN OF LYOPHILIZATION PROCESS:
By considering the above physio-chemical values, the freezing was done from 4 ± 2 0c to
-45 ± 2 0c for 6 hrs. Followed by evacuation of lyochamber was done in order to down
the chamber pressure from 9999 µbar to 300 µbar. Followed by primary drying was
applied 0±2 0c to 15±2 0c with the vacuum around 260 ubar to 150 ubar for 28 hours,
continued by the secondary drying at 15±2 0c to 30±2 0c with reduced pressure of
150 ubar to 90ubar for 13 hours. The detailed flow of lyocycle was explained in
lyographs figure no: 7.
At the end of lyophilization cycle the lyophilized vials were carefully stoppered under
vacuum (it’s powered by hydraulic system) followed by vacuum break. Vials were than
unloaded from the lyophilization chamber and ascetically sealed by aluminum sealing
material by hand sealing method. During sealing care should be taken to maintain the
vacuum inside lyophilized vials. The sealing should be done by appropriate technique to
have a uniform sealing in all lyovials.
Methods and Materials 2012
Lyophilized Formulation Development with Novel Excipients Derived from Cassia Roxburghiifor a PEGylated Therapeutic Protein Page 12
1.8 CHARACTERIZATION OF NOVEL EXCIPIENT:
The obtained novel excipients were characterized by various physical, phytochemical and
structural identifications techniques.
4.8.1 PHYSICAL PROPERTIES OF SEED GUM:
The separated gum powder was evaluated for solubility, swelling index, loss on drying,
bulk density, tapped density, angle of repose, carr's index. The evaluated methods were
briefed below,
• SOLUBILITY (A Glomme, et al.,(2005)):
• SWELL INDEX
• LOSS ON DRYING (LOD)
• BULK DENSITY (DB):
• TAPPED DENSITY (DT):
• ANGLE OF REPOSE:
• CARR’S INDEX (I):
4.8.2 PHYTOCHEMICAL STUDIES (Kokate, C.K., (1994)):
The extracted seed gum was subjected to some preliminary tests to confirm the nature of
the obtained mucilage. The tests performed were to determine the presence of
carbohydrates, starch, lipids, proteins and aminoacids, Gelatins, alkaloids, glycosides,
terpenoids, volatile oils, tannins and resins.
i. Test for Carbohydrates o Fehling solution test
o Benedict’s test
o Molish test
o Seliwanoff’s test
ii. Chemical Test for Starch o Jelly test
iii. Chemical tests for lipids o Grease spot test
Methods and Materials 2012
Lyophilized Formulation Development with Novel Excipients Derived from Cassia Roxburghiifor a PEGylated Therapeutic Protein Page 13
o Emulsification test
iv. Chemical test for proteins and amino acids: o Biuret test
o Millons test
o Xanthoprotic test
v. Chemical test for gelatin: o Solubility test
o Soda lime test
vi. Chemical test for alkaloids: o Dragendorff’s test
o Mayer’s Test
o Tannic Acid Test
vii. Chemical Tests for Glycosides o Chemical Tests for Anthraquinone Glycosides - Borntragor’s Test:
o Chemical Tests for Saponin Glycosides - Haemolysis test:
o Chemical tests for steroid and triterpenoid glycosides - Libermann
Bruchard test:
o Chemical tests for cardiac glycosides - Keller Killiani test:
o Chemical tests for Coumarin glycosides - FeCl3 test:
o Chemical tests for Cynophoric glycoside - Sodium picrate test:
o Chemical tests for flavonoid glycosides - Ammonia test:
viii. Chemical test for Terpenoids ix. Chemical tests for Volatile oils x. Chemical Tests of Resins
o Turbidity test
xi. Chemical tests for Tannins o Test with Iron salts
4.8.3 STRUCTURAL IDENTIFICATIONS OF NOVEL EXCIPIENTS
To understand the novel excipients structural components FT-IR, MALDI and DSC were
used.
Methods and Materials 2012
Lyophilized Formulation Development with Novel Excipients Derived from Cassia Roxburghiifor a PEGylated Therapeutic Protein Page 14
• MOLECULAR CHARACTERIZATION BY FT-IR STUDIES:
The spectra were recorded for C. roxburghi, and formulation. Samples were prepared in
KBr disc (2 mg sample in 200 mg KBr).The scanning range was 400-4000 cm-1,
resolution was 4 cm-1.
• MATRIX-ASSISTED LASER DESORPTION/IONIZATION (MALDI)
ANALYSIS:
MALDI is a soft ionization technique used in mass spectrometry, allowing the analysis of
biomolecules (biopolymers such as DNA, proteins, peptides and sugars) and large
organic molecules (such as polymers, dendrimers and other macromolecules), which tend
to be fragile and fragment when ionized by more conventional ionization methods.
o MS Method:
Table 6 – MS Method for Characterization
MSD Parameters
Source ESI
Mode Auto
Polarity Positive
Trap ICC
Target 5000
Max Accu .Time 100 ms
Scan Range 100-2200 m/z
Averages and rolling averages 5 & 0
Target Mass 1000m/z
Compound stability 80%
Trap drive level 100%
Optimise Normal
Nebuliser 50 psi
Dry gas 12 l/min
Methods and Materials 2012
Lyophilized Formulation Development with Novel Excipients Derived from Cassia Roxburghiifor a PEGylated Therapeutic Protein Page 15
Dry Temperature 350 oC
o Data analysis
After the LC run peak was selected and deconvoluted using these parameters
Charge deconvolution parameter
Table 7 – MS Data analysis
Adduct Ions H+
Low mass 1000
Higher Mass 5000
Abundance cut off 0.1%
o Related Ion Deconvolution
Table 8 – MS Related Ion Deconvolution
Maximum charge 75
Minimum Peak in Compound 2
Max No of Compound 10
Abundance cut off 10%
MW aggregate 0.01%
Peak width (m/z) 0.1%
S/N threshhold 1
Absolute intensity threshold 100%
Alogrithm Version 2
• DSC ANALYSIS (Tg):
Tg of the novel excipient with freeze-dried formulations was measured by using a DSC
(Q200) differential scanning calorimeter (TA Instruments).
• Heating rate : 1°C/min
• Scanning temperature: 20 oC to 150 oC
Methods and Materials 2012
Lyophilized Formulation Development with Novel Excipients Derived from Cassia Roxburghiifor a PEGylated Therapeutic Protein Page 16
1.9 QUALITATIVE AND QUANTITATIVE ANALYSIS OF DRUG PRODU CT:
The finished lyophilized vials were subjected into following qualitative and quantitative
analysis and the results of novel formulations were compared with the available reference
formulation composition.
Table 9 – Qualitative and Quantitative Analysis
S. No
Tests done Purpose Volume of sample/ Vials required
1 Visual appearance (Cake and reconstituted product)
To check visual clarity and uniformity in appearance /structure.
3 vials
2 Reconstitution time Check dissolution time 2 vials
3 pH For acidic or basic changes due to lyophilization
200µl
4 RP-HPLC To check protein content and its purity changes due to lyophilization
120µl
5 SEC-HPLC Check aggregation before and after lyophilization
120µl
6 IEC To detect change in isomers profile before and after lyophilization.
120µl
7 Moisture analysis To check the moisture content present in lyophilized vial
5 vials
8 Osmolality To check biological compatibility of the drug product
200µl
9 SDS-PAGE Check aggregation before and after lyophilization
100µl
4.9.1 PHYSICAL APPEARANCES:
Based on the lyophilized cake appearance, the lyophilized products are grouped into
excellent, good and bad lyo cakes.
Methods and Materials 2012
Lyophilized Formulation Development with Novel Excipients Derived from Cassia Roxburghiifor a PEGylated Therapeutic Protein Page 17
Excellent cake represents white elegant cake structure, no physical adherence to the glass
vials and uniform in texture. Good lyo cakes are white color good in appearance, uniform
cake / product distribution with slight or no adherence to the glass vials. Bad lyo cakes
are partially or totally collapsed compositions. It may be due to various reasons.
4.9.2 MOISTURE ANALYSIS:
Water contents were determined by Karl Fischer (KF) coulometric technique. Here the
end point detection (Metrohm – 860 KF Thermoprep) was kept in automatic mode. All
measurements were carried out in duplicates.
4.9.3 HPSEC - HIGH PERFORMANCE SIZE EXCLUSION CHROMATOGRAPHY:
It’s used to measure soluble aggregates. Agilent 1100 and 1200 series HPLC system
consisting of Quaternary Solvent pump, auto sampler and VWD UV detector was used
for this study.
4.9.4 HPIEC - HIGH PERFORMANCE ION EXCHANGE CHROMATOGRAPHY
It is used to determine purity of drug product in terms of positional isomers. Agilent 1100
and 1200 series HPLC system consisting of Quaternary Solvent pump, auto sampler and
VWD UV detector was used.
4.9.5 RPHPLC- REVERSE PHASED HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
It is used to determine purity of drug product in terms of oxidized or reduced protein
related impurities. Agilent 1100 and 1200 series HPLC system consisting of Quaternary
Solvent pump, auto sampler and VWD UV detector was used.
4.9.6 SDS PAGE - SODIUM DODECYL SULPHATE - POLYACRYLAMIDE GEL
ELECTROPHORESIS
Methods and Materials 2012
Lyophilized Formulation Development with Novel Excipients Derived from Cassia Roxburghiifor a PEGylated Therapeutic Protein Page 18
Used 14% homogenous SDS for resolution and 4% for stacking. All samples were
analyzed by using reducing and non-reducing conditions.
4.9.7 RECONSTITUTION TIME:
The lyophilized vials were reconstituted with sterile Water for Injection and time taken
for reconstitution was recorded. It was observed that all the formulation composition /
combinations got reconstituted within 13 seconds. The reconstituted solution was
analyzed visually for presence of any fibers or visible particulate matter against white and
black back ground. The reconstituted solution appeared clear without any visible fibers or
particulate matter. This indicates that there are no visual aggregates (insoluble) produced
during and after the lyophilization.
4.9.8 FREEZE DRYING INDUCED pH CHANGES:
The lyophilization induced pH changes were identified by measuring the pH before and
after lyophilization.
1.10 CHARACTERIZATION OF LYOPHILIZED DRUG PRODUCT:
The structural level impact of Novel excipient and PEGylated therapeutic protein was
analyzed by following techniques,
Methods and Materials 2012
Lyophilized Formulation Development with Novel Excipients Derived from Cassia Roxburghiifor a PEGylated Therapeutic Protein Page 19
Table 10 – Characterization of Lyophilized Drug Product
Technique Measurement Interpretation
DSC analysis Temperature of unfolding
(Tm)
Tm must be comparable or greater than Ref
drug product (RDP)
DLS analysis Hydrodynamic radius
Smaller the radius, more compact the drug.
Comparable radius as RDP indicative
similar conformation in solution
Fluorescence analysis λmax and Intensity of
emission
Intrinsic fluorescence of molecule due to
aromatic residues. Comparable intensity and
λmax indicative of similar tertiary fold as
RDP
pH measurement pH Could impact stability
UV analysis λmax and Intensity UV absorbance profile should be
comparable with RDP
4.10.1 THERMODYNAMIC DENATURATION OR UNFOLDING BY DSC:
DSC measurement of both formulation compositions was performed using VP-DSC
instrument at heating scan rate of 1oC/min. in scanning range of 10 oC to 100 oC. The
baseline was established by running multiple buffer-buffer scans. Both formulations were
done at 0.1 mg/mL concentration. Data evaluation was done with origin software, version
7.0 VP-DSC 2000. The buffer-buffer baseline was subtracted from sample data. The data
were corrected and fitter by using non two-state transition model. A comparison was
established for melting transition temperature (Tm) and change in enthalpy (DelH) for
both the formulations.
Methods and Materials 2012
Lyophilized Formulation Development with Novel Excipients Derived from Cassia Roxburghiifor a PEGylated Therapeutic Protein Page 20
4.10.2 DLS ANALYSIS:
Both formulations were used in the concentration of 0.1 mg/mL. the set parameters were
refractive index of material and dispersant an 1.34 and 1.33 respectively, viscosity of
dispersant at 1.0cP, dielectric constant of dispersant at 75 and absorption of coefficient
material at 0.86. The samples were then analyzed by Zetasizer Nano series.
4.10.3 FLUORESCENCE ANALYSIS:
The fluorescence analysis of both the formulations was done using fluorescence
spectrophotometer (Perkin elmer LS55). The sample concentration was used as
0.1 mg/mL for intrinsic and extrinsic fluorescence. Both the formulations were analyzed
in 0.1M Tris-Cl, pH 6.8. for extrinsic fluorescence analysis, 1-Anilino-8-napthalene
sulfonate (ANS) was added to a final concentration of 0.5 mM. The excitation
wavelength for intrinsic fluorescence was 280 nm, so both the formulations were scanned
from 300 nm to 500 nm. The excitation wavelength for extrinsic fluorescence was
380 nm, so both the products were scanned from 400 nm to 600 nm.
4.10.4 DRUG PRODUCT STABILITY ANALYSIS (ICH –Q5C):
In order to check the compatibility, stability and effect of novel excipient with API, the
final lyophilized vials were subjected for stability analysis by keeping at different
temperature conditions. The study temperatures were Stress condition (40 oC) for 15 days
and accelerated condition (25oC) for 6 months and each time point study samples were
compared with corresponding reference standard. The typical study plan was depicted in
below table,
Methods and Materials 2012
Lyophilized Formulation Development with Novel Excipients Derived from Cassia Roxburghiifor a PEGylated Therapeutic Protein Page 21
Table 11 – Drug Product Stability Plan
S. No Study condition Time points* Analytical tests
1 Stress condition (40 oC) 3 D, 7 D & 15 D SDS-PAGE, SEHPLC & Moisture content
2 Accelerated condition (25 oC) 1 M, 3M & 6 M SDS-PAGE, SEHPLC & Moisture content
3 Real Condition (5 oC) Initial (0D) & 6 M SDS-PAGE, SEHPLC & Moisture content
*D – Days: M – Months
Lyophilized Formulation Development wRoxburghiifor a PEGylated Therapeutic Protein
1.11 LYOPHILIZATION
Figure 1 -
Methods and Materials
ilized Formulation Development with Novel Excipients Derived from Cassia PEGylated Therapeutic Protein
LYOPHILIZATION PROCESS REPRESENTATION
Schematic Diagram of operation of Lyophilizer
Methods and Materials 2012
rom Cassia Page 22
Diagram of operation of Lyophilizer
Lyophilized Formulation Development wRoxburghiifor a PEGylated Therapeutic Protein
Figure
Methods and Materials
ilized Formulation Development with Novel Excipients Derived from Cassia PEGylated Therapeutic Protein
Figure 2 - Lyophilization Operation System
Methods and Materials 2012
rom Cassia Page 23