COMPARITIVE EVALUATION OF HEAT & FREEZE THAW STABILITY OF BIODEGRADABLE MICROSPHERE BASED & CONVENTIONAL VACCINE

1. Presented by
PRIYA DIWEDI
M Pharm ( Pharmaceutics)
Bhopal Institute of
technology and science ,
pharmacy Bhopal(M.P)

2. VACCINES
 T-cell memory is very important for long-lasting immunity,
because T-cells control both humoral and cell mediated
immunity.
 When the immune system recognizes a foreign antigen for the
first time, an immune response is produced. When T cells are
involved, immunological T-cell memory is produced.
 When the body encounters same antigen subsequently, a
stronger immune response is produced. This is because of
existing immunological memory against that antigen.
 Further antigenic stimulus increases the immune response.
First antigenic stimulus is “priming” whereas subsequent
stimuli are “booster”. This is the principle of active
immunization.

3. The term “vaccine” was coined by Louis Pasteur to
commemorate first successful immunization against
small pox by Edward Jenner.
The term vaccine was derived from “vacca”, meaning
cow, since Edward Jenner used cowpox virus
(Vaccinia) to prevent smallpox infection.
Vaccination involves deliberate exposure to antigen
under conditions where disease should not result.

4. Vaccination is aimed at inducing active immunity in an individual, so
that subsequent contact with the microorganism following natural
infection induces strong protective immune response.
The protective immunity may involve secretion of neutralizing
antibodies or production of memory CTL or Th1 cells.
 The use of vaccines is now being extended to immunize against
tumors or to block fertilization (contraceptive vaccines).
 A vaccine is a suspension of whole (live or inactivated) or
fractionated bacteria or viruses that have been rendered nonpathogenic,
and is given to induce an immune response and prevent disease

5. DISEASES COMMONLY TARGETED BY ROUTINE
IMMUNIZATION IN INDUSTRIALIZED COUNTRIES
EXCLUDING DISEASES TARGETED BY TRAVEL VACCINE
Bacterial diseases Viral diseases
Pertussis Measles
Pneumococcal diseases (pneumonia,
meningitis, otitis media, and others)
Mumps
Meningococcal diseases
(meningitis and others)
Rubella
Tuberculosis Polio
Diphtheria Influenza A and B
Tetanus Hepatitis B

6. Bacterial diseases Viral diseases
Heamophilus influenzae type b
diseases (pneumonia, meningitis
and others
Chickenpox
Herpes zoster
Hepatitis A
Human Papilloma Virus diseases
(genital/cervical/oral warts and
cancers)
Japanese encephalitis (regional
importance)
Rabies (in at-risk groups)
Rotavirus

7. VACCINE COMPONENT
 In addition to the bulk antigen that goes into a vaccine, vaccines
are formulated (mixed) with other fluids (such as water or saline),
additives or preservatives, and sometimes adjuvants.
Collectively, these ingredients are known as the excipients.
 These ensure the quality and potency of the vaccine over its
shelf-life.
 Vaccines are usually formulated as liquids, but may be freeze-
dried (lyophilized) for reconstitution immediately prior to the
time of injection.

8.  Active components
 Adjuvants
 Diluents
 Stabilisers
 Preservatives
 Trace components

9. TYPE OF VACCINE
 Live, Attenuated Vaccines
 Killed Or Inactivated Vaccines
 Toxoids
 Conjugate Vaccine
 Recombinant Vector Vaccines
 Anti-idiotypic Vaccine

10. Vaccine stability
All vaccines are sensitive biological substances that progressively
lose their potency (i.e., their ability to give protection against
disease).
This loss of potency is much faster when the vaccine is exposed to
temperatures outside the recommended storage range.
 Once vaccine potency has been lost, returning the vaccine to
correct storage condition cannot restore it.
Any loss of potency is permanent and irreversible.
Thus, storage of vaccines at the correct recommended temperature
conditions is vitally important in order that full vaccine potency is
retained up to the moment of administration.
Although all vaccines are heat-sensitive, some are far more
sensitive than others are.

11. Those listed in section given below can be arranged in
order of decreasing sensitivity to heat as follows:
Least sensitive
•Adsorbed Diphtheria-Tetanus vaccine (DT, Td)
•BCG (Lyophilized) *
•Tetanus Toxoid (TT)
•Adsorbed Diphtheria-Pertussis-Tetanus vaccine (DPT)
Most sensitive•Live oral polio vaccine (OPV)
•Measles (Lyophilized)
• Hepatitis B Pertussis and Mumps (Lyophilized)

12. Vaccines damaged by freezing Vaccines unaffected by
freezing
DPT BCG *
DT OPV
TT Measles *
Hepatitis B Mumps
Td
Note: Vaccines freeze at temperatures just below zero.
•BCG and measles vaccines must not be frozen after reconstitution
•diluent for any vaccine must never be frozen.
Sensitivity of vaccines to freezing

13. Vaccine Vial Monitors (VVM)
VVMs, which measure exposure to heat, are time and
temperature sensitive labels attached to vials of vaccine at the
time of manufacture. Through a gradual colour change they warn
about the falling potency.
Importance of the cold chain
To ensure the optimal potency of vaccines, storage and
handling need careful attention adequate electrical power
and refrigeration are often lacking in developing countries,
where storage, handling and the heat stability of vaccines
are consequently matters of great concern.
New products have been developed for safe transport
and storage, while the reliability of vaccine supply has
been increased by the introduction of improved
management techniques.

14. Substantial drops in vaccine potency caused by
unsatisfactory conditions of delivery and storage have
been reported. The most commgon deficiencies in cold
chain performance reported from developed countries
are:
 high temperatures during storage or transport;
exposure of adsorbed vaccine to freezing
temperatures; refrigerators without thermometers;
failure to take and record temperature readings
regularly; storage of drugs,
drinks, food and pathology specimens with vaccines
and failure to discard unused vaccine after sessions at
ambient temperature.

15. Freeze - Thaw Stability Testing
Freeze-thaw cycle testing is a part of stability testing that allows you to determine
if your formula will remain stable under various conditions. This type of test puts
your sample through a series of extreme, rapid temperature changes that it may
encounter during normal shipping and handling processes.
Freeze-thaw testing is conducted by exposing the product to freezing
temperatures (approximately -10°C) for 24 hours, then allowing to thaw at
room temperature for 24 hours.
The sample is then placed in a higher temperature (approximately 45°C) for 24
hours, and then placed at room temperature again for 24 hours. The sample is
analyzed for significant changes. This completes one cycle. If, after three cycles
of freeze-thaw testing, no significant changes are observed, you can be
confident that the stability of your product is sufficient for transport.

16. Biodegradable Polymers
Biodegradable materials are natural or synthetic in origin and are
degraded in vivo, either enzymatically or non-enzymatically or
both, to produce biocompatible, toxicologically safe by-products
which are further eliminated by the normal metabolic pathways.
The basic category of biomaterials used in drug delivery can be
broadly classified as:
 synthetic biodegradable polymers, which includes relatively
hydrophobic materials such as the hydroxy acids (a family that
includes poly lactic-co-glycolic acid, PLGA), polyanhydrides,
and others, and
(naturally occurring polymers, such as complex sugars
(hyaluronan, chitosan) and inorganics (hydroxyapatite)

17. Microparticle Preparation Techniques
Solvent Evaporation Method
Double (Multiple) emulsion process
Phase Separation (Coacervation)
Phase separation of the coating polymer
solution,
Adsorption of the coacervate around the
drug particles, and
Quenching of the microspheres.
Spray Drying
Single emulsion process

18. Antigen profile (Hepatitis B Vaccine)
Hepatitis - B vaccine (r DNA) is a non-infectious
recombinant DNA Hepatitis B vaccine.
 It contains purified surface antigen of the virus obtained
by culturing genetically-engineered Hansenula polymorpha
yeast cells having the surface antigen gene of the Hepatitis
B virus.
 The Hepatitis-B surface antigen (HBs Ag) expressed in
the cells of Hansenula polymorpha is purified through
several chemical steps and formulated as a suspension of the
antigen adsorbed on aluminium hydroxide and thiomersal is
added as preservative.
The vaccine does not contain any material of human or
animal origin.

19. Stability of Hepatitis B Vaccine
Hepatitis B vaccine is one of the most heat-stable children’s
vaccines, maintaining stability for up to four years at temperatures
between 2°C and 8°C. T, for months at 20°C to 25°C, for weeks at
37°C and for days at 45°C.
As with other vaccines adsorbed on aluminum salts, freezing of
Hep B vaccine may cause a significant reduction of potency. The
freezing point of Hep B vaccine is about -0.5°C.
A 50 percent loss of potency of the vaccine has been reported to
be observed after 9 months at 20°C to 26°C, after one month at
36°C to 40°C and after three days at 45°C.
The vaccine should always be protected from being frozen,
especially at the end of the cold chain when it is transported in cold
boxes and may come into close contact with cold packs.
 Freeze damage is the greatest threat to its integrity, and strategies
to mitigate the risk of freezing should be employed.

20.  Maintaining optimal potency of vaccine is the prime
concern during storage and handling. Drop in vaccine
potency due to unsatisfactory conditions of storage
have been reported. In developing countries, lack of
adequate infra-structural logistics, uninterrupted power
supply and refrigeration are often lacking. Thus
stability of vaccine outside cold chain is important.
Apart from this, accidental freezing of vaccine during
maintenance of cold chain have also been reported.
 This motivated us to microencapsulate model antigen
and assess heat shock and freeze thaw stability of this
microencapsulated antigen vis-à-vis marketed vaccine
formulation.

21.  Literature survey
 Characterization of antigen
• UV Scanning
• FTIR Spectroscopy
• Total protein content determination
 Characterization of polymer
• FTIR
 Preparation of antigen containing microsphere
 Characterization of prepared microsphere.
• Particle size
• Antigen loading
• In-vitro release profile
• Surface morphology
 Heat shock stability assessment
• By keeping microsphere based formulation and marketed formulation at 37 degree c
for1 month.
 Freeze thaw stability
• Both formulations will be subjected to three cycle of freeze thaw.
 Compilation of work

22.  All vaccines are sensitive to heat, necessitating
use of a vaccine cold chain a global distribution
network of equipment and procedures for
maintaining vaccine quality during transport and
storage.
 Many vaccines, including hepatitis B vaccine, are
also sensitive to freezing. Maintaining vaccines
within a specified temperature range, usually 2–8
◦C, to protect them from excessive heat and
freezing is a difficult task in both developing and
developed countries. Temperature excursions,
either too cold or too hot, during transport and
storage are frequently observed when
temperatures are monitored closely.

23. Moreover, there is evidence indicating that a single
vaccine shipment can be exposed to several episodes
of freezing temperatures within different segments of
the cold chain. Temperature monitoring studies also
suggest that the deviations from ideal storage
temperatures are more likely to happen in the
periphery of the cold chain (i.e., at the local storage
center or the immunization clinics) as opposed to the
central storage facility or at the vaccine
manufacturer’s site Among the factors contributing to
the problems are inappropriate cold chain equipment,
insufficient training, human error, and power
shortages.

24. Consequences of cold chain failure may include increases in
the cost of immunization due to vaccine wastage and staff time,
as well as immunization recall and potentially inadequate
protection of patients if affected products are not identified.
Therefore, a thermostable vaccine that could tolerate repeated
temperature excursions or be stored at ambient temperature for
all or a portion of its shelf life is highly desirable.
A microsphere preparation method and characterization of
prepared PLGA microparticles was described.

25.  MATERIALS:
 PLGA was purchased from Boennge Hmen.
 Hepatitis B Antigen was purchased from Bharat
Biotech International Limited, Hyderabad.
 Dichloromethane was purchased from HiMedia
Laboratories pvt.Ltd.Mumbai.
 Potassium Dihydrogen phosphate was phosphate was
purchase from Ranbaxy fine chemicals Ltd.
 Other reagent grade chemicals.

26. EQUIPMENTS:
UV/Visible-1800 spectrophotometer(SHIMADZU,Japan)
FT-IR
Remi Mechanical stirrer(Remi India Pvt.Ltd,New Delhi)
Remi cooling centrifuge(Remi India Pvt.Ltd,New Delhi)
Differential Scanning Calorimeter(DSC)
Zeta sizer(Malvern Instrument,UK)
CHARACTERIZATION OF ANTIGEN
UV Scanning:
HBs Ag was scanned in 200-400 nm and peaks were
recorded.
FTIR Spectroscopy:
 FTIR Spectroscopy of hepatitis B vaccine was performed
at Sagar Institute of Research And Technology, Bhopal.

27. TOTAL PROTEIN CONTENT DETERMINATION:
Preparation of Standard Curve
Bradford reagent was prepared by dissolving 100 mg Coomassie
Brilliant Blue G-250 in 50 ml, 95% ethanol; thereafter 100 ml 85%
(w/v) phosphoric acid was added. Volume was made up to 1 liter
when the dye has completely dissolved, and was filtered through
Watman paper just before use.
Standards were prepared containing a range of 10µg/ml to
100µg/ml of protein (Bovine serum albumin). 5 ml of Bradford
reagent was added to each and incubated for 5 minutes. The
absorbance was measured at 595 nm.
Unknowns were diluted till we got absorbance in standard. 5 ml of
Bradford reagent was added and incubated for 5 minutes. The
absorbance was measured at 595 nm.
A standard curve of absorbance versus micrograms protein was
prepared and the amount of protein was determined from the curve.

28. CHARACTERIZATION OF POLYMER
FTIR Spectroscopy of PLGA microsphere
FTIR Spectroscopy PLGA microsphere was performed at
Sophesticated Analytical Instrument Laboratory, School of
Pharmaceutical Science, RGPV, Bhopal.
Preparation of PLGA microspheres containing Hepatitis B
Vaccine
Microspheres were prepared by W/O/W double emulsion solvent
evaporation techniques.
100 mg of PLGA was dissolved in 10 ml of dichloromethane.
The initial W/O emulsion was formed by adding 1ml of HBs Ag by
sonication.
W/O primary emulsion thus prepare was added drop wise through
syringe into 50 ml. of 5% PVA solution with constant stirring for 2 hrs
in mechanical stirrer.
 The resultant microspheres were collected and separated by
filtration, washed with distilled water and finally air dried over a period
of 12 hrs.

29. CHARACTERIZATION OF PREPARED
MICROSPHERE
I. Particle size, Distribution and Zeta potential
Particle size, Distribution and Zeta potential was measured by
Malvern Instruments “Zetasizer” (DTSVer.5.03, Serial Number
MAL1023461).
II. Loading efficiency of the formulation:
Accuratly weight sample of microsphere sample (5mg) was taken
into 1ml, 5% SDS-0.1 M NaoH solution in an effenorf tube and
shaken in an incubator shaker at 37ºC till, it got clear solution.
After centrifugation at 3000 rpm for 4 min the supernatant was
collected and analysed.

30. III. In-vitro Antigen release profile of microsphere
In vitro release studies were carried out by suspending 100 mg
of microspheres in 60 ml of phosphate buffered saline (PBS, pH
7.4) containing 0.02% sodium azide as bacteriostatic agent and
0.01% Tween 80 to prevent the microspheres from aggregation
in the dissolution medium in stoppered flasks.
Amount of the protein was calculated from the calibration
curve. Protein-loading percentage and protein- loading
efficiency were calculated using the following formula.
Loading efficiency = weight of protein in
microspheres*100/ weight of
protein taken initially for
preparation of formulation.

31. The flasks were placed in a reciprocal shaking water bath
maintained at 37±0.5° at a speed of 60 cycles/min. At
predetermined time intervals of 2, 12, 24, 72, 120 and 168 h,
samples were collected and centrifuged at 1726 RPM for 15
min.
The supernatant was assayed for the protein release using
UV-Vis spectrophotometer at a detection wavelength of 280
nm.
 The collected amount of supernatant was replaced with fresh
PBS to maintain sink conditions.
The percentage of protein release at different intervals was
calculated by using a freshly prepared calibration curve using
the standard samples which were run along with test samples.
Release experiments were done independently in triplicate
for each batch of microspheres.

32. IV. Size and Surface morphology
The prepared microspheres were subjected to light microscope
analysis to study the surface morphology. A drop of freshly prepared
microsphes was placed in a clean and dried glass slides which was
then covered with a coverslip to focus under the100X magnification.
This was carried out to study the morphology, size uniformity and
aggregation or coalescence of microsphere being prepared.
The surface morphology was visualized by scanning electron
microscopy. The samples for SEM were prepared by sprinkling the
microsphere powder on double adhesive tapes that was fixed on
aluminium stab. The stab was then coated with gold to a thickness of
about 300º by using sputter coater. The samples were then randomly
scanned and photograph were taken.
Surface morphology of prepared microsphere was performed at
MANIT, Bhopal.

33. V. Percentage yield of microsphere
The yield of microspheres was determined by comparing the whole
weight of microspheres formed against the combined weight of the co-
polymer and drug.
HEAT SHOCK STABILITY ASSESSMENT
By keeping microsphere based formulation and marketed formulation at 37⁰C
for 1 month and stability evaluated by FTIR and UV Spectroscopy.
FREEZE THAW STABILITY STUDIES
Both formulations were subjected to three cycle of freeze thaw and stability
evaluated by FTIR and UV Spectroscopy.

34. RESULTS AND DISCUSSION
Characterization of antigen
I. UV Scanning
 Hepatitis B vaccine in Potassium Di hydrogen phosphate
buffer (1µg/ml) was scanned in range 200-400nm and
wavelength maxima was found at 280nm as shown in (Fg.no.
10) which was same as given in IP 1996.
II.FTIR Spectroscopy
 HBs Ag show chracterstic peak at 1636.63 cm-1 which
indicates the presence of amide group. As shown in fig.11

35. III. Total Antigen content determination:
Standard curve of BSA in PBS (7.4)
Calibration graph was constructed by plotting absorbance on
ordinate and different concentrations of BSA on abscissa (Table &
Figure) For BSA the graph was found to be linear in the
concentration range of 10 µg/ml to 100 µg/ml with the following
regression equation
Y= 0.0138 x + 0.0523, R2 =0.9922
Visual inspection of the plotted calibration curves and the
correlation coefficient >0.99 confirmed the linearity over the
concentration range of 10 µg/ml to 100 µg/ml. As shown in
fig.no.12

36. Characteristic of polymer
I.FTIR Spectroscopy of PLGA polymer
PLGA show characteristic peaks such as OH stretching (3200-
3500 cm-1), –CH (2850-3000 cm-1), carbonyl –C=O stretching
(1700-1850 cm-1) and C–O stretching (1050-1250 cm-1)
Characterisation of prepared microsphere
I.FTIR of prepared microspheres
The FT IR spectrum illustrates the characteristics bands of
poly (DL-lactide-co-glycolide) microspheres and hepatitis B
vaccine at 1750 cm (-1) and 1650 cm (-1), respectively. Clearly
indicates the presence carbonyl group, saturated aliphatic esters
and primary amines.

37. II. In-vitro release profile of microspheres
The in vitro release profile showed that the entrapped
antigen was released gradually from d 1 and the peak antigen
was released on d 42. However, the percentage of total protein
and antigenic active protein and antigenic active protein
released from microsphere were entirely different.
It clearly indicates that there might be some degradation of
hepatitis B vaccine during micro encapsulation process, which
increases the loading capacity but unfortunately it results in
particle aggregation.
With respect to the release of antigen from PLGA
microsphere in vitro, the in-vitro results also confirmed the
release of antigen.

38. III. Average particle size and entrapment efficiency of
HBs Ag loaded PLGA microsphere
The size of the prepared microsphere was measured by light
microscopy-stage micrometer method. The values were
tabulated and average size of each formulation was calculated
with standard deviation.
The average size range of micrometer varied from 2.56µm to
41.27µm.On increasing the PLGA concentration by keeping
the PVA concentration as constant, the size of microsphere
increasing from 2.95µm to 37.18µm.
But on increasing the PVA concentration by keeping PLGA
concentration as constant, the size of microsphere decreasing
from 41.27µm to 2.56µm.

39. AVERAGE PARTICLE SIZE, % ENTRAPMENT
EFFICIENCY OF HBS AG LOADED PLGA MICROSPHERE
S. No. Formulatio
n code
PLGA
Concentrati
on(% w/v)
PVA
Concentrati
on (%w/v)
Average
size(µm)*
%
Entrapmen
t efficiency*
1. PLGA1 3 6 2.95±0.06 28.36±3.4
2. PLGA2 4 6 5.86±0.22 30.56±2.0
3. PLGA3 5 6 14.93±0.08 30.88±2.4
4. PLGA4 6 6 19.22±0.31 31.28±3.0
5. PLGA5. 7 6 25.15±0.07 29.24±1.8
6. PLGA6 8 6 27.82±0.31 28.84±2.1
7. PLGA7 9 6 37.18±0.30 29.48±3.1
8. PVA1 4 2 41.27±0.24 29.34±2.8
9. PVA2 4 3 31.12±0.52 28.56±4.2

40. S. No. Formulatio
n code
PLGA
Concentrati
on(% w/v)
PVA
Concentrati
on (%w/v)
Average
size(µm)*
%
Entrapmen
t efficiency*
10. PVA3 4 4 23.98±0.62 30.35±5.2
11. PVA4 4 5 21.83±0.17 32.42±2.8
12. PVA5 4 6 19.01±0.13 34.54±2.4
13. PVA6 4 7 15.01±0.40 33.46±4.2
14. PVA 7 4 8 13.18±0.43 33.56±2.6
15. PVA8 4 9 11.31±0.12 34.46±1.8
16. PVA9 4 10 4.72±0.16 32.13±2.8
17. PVA10 4 11 2.56±0.24 31.87±2.3

41. Heat shock stability assessment
I.FTIR Spectroscopy of Hepatitis B Vaccine by keeping at 37ºc
for 1 month
Intensity of antigen was decreased as compare to native antigen that is from
1636.63cm-1 to 1636.25cm-1
II. UV Spectroscopy of Hepatitis B Vaccine by keeping at 37ºc
for 1 month
Hepatitis B vaccine show absorbance at 0.1 nm and absorbance is measured at 280
nm wavelength
Freeze thaw stability
I. FTIR Spectroscopy of Hepatitis B Vaccine subjected to three
cycle of freeze thaw
Intensity of HBs Ag after freeze thaw was reduced that is from 1636.63cm-1 to
1636.24 cm-1
II. UV Spectroscopy of Hepatitis B Vaccine subjected to three
cycle of freeze thaw
Hepatitis B vaccine show absorbance at 0.064 nm and absorbance is measured at
280 nm.

42. UV absorption spectrum of hepatitis B Vaccine

43. Spectroscopy of antigen (Hepatitis B Antigen)

44. y = 0.0138x + 0.0523
R² = 0.9922
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
0 20 40 60 80 100 120
Absorbance
Concentratrtion (mg/ml)
lmax=595nm
y = 0.0138x + 0.0523
R² = 0.9922
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
0 20 40 60 80 100 120
Absorbance
Concentratrtion (mg/ml)
lmax=595nm
Standard curve of BSA in PBS (7.4)

45. FTIR Spectroscopy of PLGA polymer

46. Microscopic view of microsphere in phase contrast microscope

47. Scanning electronic photomicrograph of plga microspheres obtained by
the multiple emulsion w/o/w method.bar = 5 μm.

48. Scanning electronic photomicrograph of plga
microspheres obtained by the multiple emulsion w/o/w
method.bar = 5 μm.

49. Size distribution of microspheres

50. Zeta potential of microspheres

51. FTIR Spectra of prepared PLGA microsphere

52. In-vitro release profile of HBs Ag loaded
microsphere

53. FTIR Spectroscopy of Hepatitis B Vaccine by keeping at 37ºc
for 1 month

54. : FTIR Spectroscopy of Hepatitis B Vaccine subjected to three cycle of freeze thaw

55.  From the present study, it was concluded that hepatitis B
Microsphere can be prepared by W/O/W double emulsion
solvent evaporation technique by using PLGA polymer. By
encapsulating vaccine by a biodegradable polymer stability of
vaccine improved as well as also provides ease of
transportation.
 Stability studies such as heat shock assessment and freeze
thaw stability studies reveals that intensity decreased as
compared to native antigen.
 The above study demonstrated the possibility of making a
novel drug delivery system for hepatitis B vaccine, which will
be more efficacious and acceptable than the conventional drug
delivery of hepatitis B Vaccine it could be a drug delivery of
choice for treatment of hepatitis B infection.

56. Characterization parameters like size, zeta
potential, shape, drug release were
mechanically stable in all formulation.
In-vitro release rate study shows that the
drug release were more in selected
formulation show minimum drug release.
Formulation was found to be stable over
the storage period and condition tested.