MATERIALS AND METHODS - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/13225/12... · API and...

Methods and Materials 2012

Lyophilized Formulation Development with Novel Excipients Derived from Cassia Roxburghiifor a PEGylated Therapeutic Protein

MATERIALS AND METHODS



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



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



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



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



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



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


6 Water for injection Q.S Water for injection Q.S Vehicle



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


5 Monobasic sodium phosphate

10 mM Monobasic sodium phosphate


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



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



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:



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:



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



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.



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



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.



• 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



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



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.



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



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,



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.



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,



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


rom Cassia

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


rom Cassia

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