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Recombinant Hepatitis B Dry Vaccine Formulation and In Vitro and In Vivo Potency Testing

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Hepatitis B vaccines are available in the market in the form of liquid suspensions that is heat sensitive. The study was conducted to produce the hepatitis B vaccine formula which can be managed out of the cold chain. Utilization of the drying instrument indicates that liquid vaccine formula can be dried and monitored to produce quality vaccines powder. Drying techniques used include: spray drying, freeze drying and vacuum drying. Vaccine formulas were prepared as much as 6 samples with codes F-A through F-F, which reflects the composition of fillers and drying techniques. The powder vaccine was characterized by physical, chemical and antigenic potential as well as an accelerated stability. Vaccine immunogenicity carried out by an ELISA test using a Kit Anti-HBs-Elisa. The drying technique affecting the decrease of pH and the potential of antigenic vaccine. The combination of trehalose and mannitol did not provide a significant difference to the pH and the relative potency of dried vaccine. The vaccine which was dried by freeze-drying with the composition of trehalose-mannitol (7:3) showed the relative potency in vitro at 97,78% and in vivo at 35,6%. A dry Hepatitis B Vaccine F-B has an opportunity to be managed out of the cold chain system.
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Recombinant Hepatitis B Dry Vaccine Formulation and In
Vitro and In Vivo Potency Testing
Heru Mukti*, Iskandarsyah, Maksum Radji
1 Faculty of Pharmacy. University of Indonesia. Depok 16424, West Java, Indonesia.
Abstract : Hepatitis B vaccines are available in the market in the form of liquid suspensions
that is heat sensitive. The study was conducted to produce the hepatitis B vaccine formula
which can be managed out of the cold chain.
Utilization of the drying instrument indicates that liquid vaccine formula can be dried and
monitored to produce quality vaccines powder. Drying techniques used include: spray drying,
freeze drying and vacuum drying. Vaccine formulas were prepared as much as 6 samples with
codes F-A through F-F, which reflects the composition of fillers and drying techniques. The
powder vaccine was characterized by physical, chemical and antigenic potential as well as an
accelerated stability. Vaccine immunogenicity carried out by an ELISA test using a Kit Anti-
HBs-Elisa.
The drying technique affecting the decrease of pH and the potential of antigenic vaccine. The
combination of trehalose and mannitol did not provide a significant difference to the pH and
the relative potency of dried vaccine. The vaccine which was dried by freeze-drying with the
composition of trehalose-mannitol (7:3) showed the relative potency in vitro at 97,78% and in
vivo at 35,6%.
A dry Hepatitis B Vaccine F-B has an opportunity to be managed out of the cold chain system.
Keywords : Hepatitis B Surface Antigen (HBsAg); in vitro; in vivo; relative potency; vaccine.
Introduction
Hepatitis B vaccine is a pharmaceutical preparation which is sensitive to heating and freezing. Exposure
to excessive heat can lead to decreased stability and shelf life of the vaccine, otherwise freezing the vaccine can
cause permanent loss of potency (Diminsky, Moav, Gorecki, & Barenholz, 1999). To ensure the stability and
potency during storage and distribution, the vaccine is kept at a temperature 2C - 8C known as the cold chain
system.
Indonesia, which has a tropical climate, is a challenge in the management of the stocks of vaccines for the
liquid preparation is not resistant to heating. Dried vaccine formulation may improve the stability of the
preparation and keep the potential reduction of antigen caused by chemical and physical degradation such as
hydrolysis, precipitation, clotting or fluctuations in pH (Saluja et al., 2010). Some drying techniques have been
applied in the preparation of vaccines, among others: freeze drying, spray drying and vacuum drying.
In the dried vaccine, antigen molecule movement is much smaller when compared with the liquid vaccine
as a result, protein damage will be slower. However, during the drying process, protein will be degraded and
may lose its potency. Class saccharide compounds such as mannitol, sucrose, trehalose and lactose, has been
widely used as a stabilizer to prevent the degradation of proteins during the process of drying and storage
(Tonnis et al., 2014).
International Journal of ChemTech Research
CODEN (USA): IJCRGG, ISSN: 0974-4290, ISSN(Online):2455-9555
Vol.10 No.6, pp 132-139, 2017
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et al
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Recombinant vaccine antigens are generally weak and often fail to provide the expected immune
response that should be added adjuvant. Adjuvants commonly used in vaccines are alum salts such as Al
(OH)x(PO4)y and KAl(SO4)2.12H2O and Al(OH)3. The use of aluminum hydroxide is preferred because it is
proven safe, stable and commonly used in vaccine (Vecchi, Bufali, Skibinski, O’hagan, & Singh, 2012).
Stability during storage and distribution of vaccines will affect the potential immunogenicity (Tonnis et
al., 2014). To determine the stability of the vaccine at its storage temperature it needs to be accelerated stability
test that the time required for a shorter observation.
Experimental
Materials
The active ingredient in the form of bulk antigen derived from Biofarma, Co. Ltd. with a concentration
of 0.9445 mg/ml HBsAg in Phosphate buffered saline (PBS). Comparator vaccine using a liquid hepatitis B
vaccine recombinant (HBV), Biofarma, Co. Ltd. packaging 0.5 mL Prefilled Injection Device Batch No.
3653815.
All excipient used in this study were given from Hexpharm, Co. Ltd and Dwipar L.A. Co. Ltd. with the
pharmaceutical grade or higher. The excipient included Trehalose, mannitol, polysorbate 80 and dried gel
Aluminum hydroxide (Al(OH)3) as an adjuvant.
Antigen-adjuvant adsorption
Antigen-adjuvant adsorption performed by incubating adjuvant with antigen in optimal conditions. The
process is continuous at pH 6.8 -7.2 and a temperature of 2-8 ° C and the stirring speed of 40-60 rpm (Awate,
Babiuk, & Mutwiri, 2013). The mixture is stirred at least for two hours to obtain suspension of antigen-
adjuvant.
Formulation
The vaccine formulation made by mixing the excipients in phosphate buffer with the suspension of
antigen - adjuvant. Samples are prepared in six formulas and each formula containing 20 mcg / mL HBsAg
adsorbed on 0.5 mg of adjuvant Al3+ with a ratio of carrier material trehalose: mannitol (1:1 and 7:3) and
polysorbate 80 (0,05%). The vaccine formulation is done at a temperature of about 4°C with the stirring speed
at 40-60 rpm (Awate et al., 2013).
Spray drying (SD)
The liquid vaccine formulation was sprayed using Spray Dryer SD 1000, Eyela Corp., Japan. The
Spray-drying conditions were as follows at feed rate 10 mL/min, inlet temperature at 130 ° C and its resulting
outlet temperature between 80°C - 84°C. The resulting powder is maintained so as not exposed to temperatures
above 45 ° C and placed in a vacuum desiccator with silica gel dryer at room temperature (Chen et al., 2010).
Freeze drying (FD)
The liquid vaccine formulation stored at freeze drying tube and dried using FDU Freeze Dryer 1200,
Eyela Corp., Japan. The condition run at temperature adsorption at -40 ° C. vacuum pressure of 9.5 Pa and at
least 20 hours of drying time (Li, Thakkar, Ruwona, Williams, & Cui, 2015).
Vacuum drying (VD)
The sample is introduced into a glass beaker and placed into Vacuum Dryer VOS 301 SD, Eyela Corp.,
Japan. The optimization of this method can be done with the temperature of the tool 25 ° C and slowly
increased to 30 °C, vacuum pressure at 3.15 kPa and 24 hours of drying time (Jangle & Pisal, n.d.).
Heru Mukti
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Sterilization
All prepare formulation was sterilized by 27,5 kGy Gamma Irradiation at Natural Rubber Irradiator of
National Atomic Energy Agency.
Moisture content
Briefly, testing for moisture content using a Karl Fisher Coulometer (Model 737) equipped with a
drying oven (Model 707).
pH measurement
pH measurements made after the preparation of the vaccine is reconstituted with aqua pro injection
using a pH meter (Eutech Instrument Type-510, Singapore). Observation pH should be done before In vitro
evaluation of each month.
Antigenicity of HBsAg
The potency of vaccines was determined by a quantitative enzyme immune assay using an enzyme
immunoassay(EIA) kit (Murex ® monoclonal diagnostic kit Version 3.0, Abbot lab.IL.). Each sample was
examined at four different dilutions with three repetitions against a linear fitting to the responses of control
standard dilutions. The potency was expressed as the quantity of HBsAg (μg)
Immunogenicity test
Testing is carried out on the vaccine formula which indicates a highest and lowest In vitro potency.
Four to five-weeks-old deutch democratic Yokohama (ddY) mice were used to assess the immunogenicity of
two formulas. Vaccines were reconstituted in saline and injected into animals as much as 1 ml by Intra
Peritoneal (IP) injection using a 1 ml disposable syringe with a needle size 26 G x ½ ". Injection process
starting from the smallest dose dilution (1: 256). Blood was collected via heart bleeding 28 days after the final
immunization and the antibody responses were analyzed using Anti-HBs-Elisa kit (ETI-AB-AUK 3, Diasorin®,
SPA).
Stability test
Samples are stored in the oven (Memmert®, Germany), with a temperature of 40C ± 2 C/ 75% RH
during testing range. At the end of each month evaluation, vaccine tested in vitro antigenicity and pH. Samples
that have been tested are not returned to the oven. Testing carried out during the first three months (“ICH Q5C -
Stability testing of Biotechnological / Biological products 1,” n.d.).
Results and Discussion
Table1: Summary of all formulas
Formula
Drying
methods
Excipient
ratio
( T:M:P)
Moisture
content
(%)
pH
Vaccine
sterility
F-A
Freeze drying
1:1: 0,05
3,91
6.82
Sterile
F-B
Freeze drying
7:3: 0,05
3,77
6,82
Sterile
F-C
Vacuum drying
1:1: 0,05
7,12
6,84
Sterile
F-D
Vacuum drying
7:3: 0,05
6,24
6,84
Sterile
F-E
Spray drying
1:1: 0,05
1,94
6,78
Sterile
F-F
Spray drying
7:3: 0,05
2,18
6,78
Sterile
Bulk
6.70
HBV
6,81
Notes: T: Trehalose; M: Mannitol; P: Polysorbate 80, HBV: Hepatitis B Vaccine from Biofarma Co. Ltd.
Heru Mukti
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/International Journal of ChemTech Research, 2017,10(6): 132-139. 135
Table 2: Stability test result
Formulation
Month
pH
Potency recovery
(%)
F-A
1st
6,80
89,38
2nd
6,78
82,42
3rd
6,77
72,83
F-B
1st
6,80
91,99
2nd
6,79
84,02
3rd
6,78
74,12
F-C
1st
6,80
83,53
2nd
6,76
78,10
3rd
6,73
67,74
F-D
1st
6,81
85,20
2nd
6,77
80,89
3rd
6,73
69,30
F-E
1st
6,79
2nd
6,78
3rd
6,78
F-F
1st
6,79
2nd
6,78
3rd
6,78
HBV
1st
6,77
90,45
2nd
6,72
58,72
3rd
6,65
17,03
Antigen-adjuvant adsorption
The antigen-adjuvant suspension produced during pre-formulation shows relatively clear liquid and
colorless. This is due to the dry gel Al (OH)3 is dissolved in a solution of 0.9% Na Cl and HBsAg bulk
suspension is relatively clear and colorless. Bulk antigen very easily dispersed in a solution of 0.9% Na Cl
physiological because it does not contain a grain of water-insoluble particles.
Formulation
The resulting vaccine suspension of all formulas is relatively clear and colorless. This is caused by all
the excipients used is a material that is readily soluble in water. Excipients used is a material commonly used in
parenteral preparations as well known to have a physical and biochemical stability suitable for powder
formulations.
The whole vaccines contain trehalose in two different compositions. The use of trehalose, other than as
a bulk-forming material is also known to have properties as a protein stabilizer. It is important to prevent the
degradation of antigens during the drying process (Chang & Pikal, 2009). The use of trehalose together with
mannitol as a bulk-forming material provides better powder texture. In addition, trehalose and mannitol
reducing properties were lower, thereby reducing the possibility of reaction with protein (Maa et al., 2007).
Moisture content
Measurements of water content in the dry vaccine aim to measure the humidity will affect the
flowability of powders and chemical stability during the storage process. The test results showed that F-E and
F-F have a moisture content that is relatively low compared to other formulas. This is because the primary
method, droplets of the vaccine was warming up to 130 ° C so that the dehydration process runs evenly and
quickly in the whole particle (Li et al., 2015). While the F-C and F-D contain relatively high levels of water
due to evaporation of water is not going well.
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In vitro study result
Formula F-C and F-D were dried VD showed a decrease in potency when compared with the potential
of bulk antigen but still meet the 95% confidence limit. While the vaccine F-E and F-F were dried in SD
showed significant differences in potency when compared with the HBsAg bulk. This condition occurs because
the vaccine was under pressure during the drying mainly on the process of atomization and drying droplet at
high temperature (Li et al., 2015). This condition causes the loss of potential antigens significantly during the
drying process.
Formula F-A and F-B were dried FD showed a higher in potency than others and meet the 95%
confidence limit. A freeze-drying technique produces a porous dry cake in a tube and an ideal moisture content
in the final powder. Water evaporate during the primary drying massively and after secondary drying crystals
water also eliminate (Adams, Cook, & Ward, 2015). However, the antigenicity of HBsAg decreases during
freezing.
In vivo assay
Blood of the test animals was collected by the technique bleeding heart. This technique is selected
because it can produce more blood volume and then do centrifugation at 10,000 rpm with 4C temperature for
10 minutes to obtain serum containing antibodies. Serum produced from each of the animal tests ranged from
0.3 to 0.5 ml and stored in a refrigerator at a temperature of 2-8 ° C prior to further testing.
The results showed that the relative potency of dried vaccine F-B by 35,6.0% while the F-E was 11%.
In the F-E, spray drying technique extremely reduce antigenicity of vaccine as a result, the vaccine did not show
enough potential during both in vitro and in vivo assays.
According to the British Pharmacopoeia appendix xiv k vol.5, 2013, stated that the 95% confidence
limit for In vivo test is obtained if the test results of samples are in the range of 30-300%. From the test results
obtained by the relative potency, F-B was 35,6% and meet the test requirements.
Accelerated stability test
Accelerated stability testing was conducted to predict the stability of the preparation after a certain
period and it is important to ensure the shelf life of the vaccines. During the testing range, dried vaccine
experienced a slight discoloration on the powder to become paler. In the F-C and F-D, the consistency of the
powder becomes denser but are destroyed when given vibration in the vial. Compaction of the powder occurs
because the water content decreases due to heating (Abdul-Fattah et al., 2007).
The liquid suspension HBV vaccine tends to decrease of pH. The high water content in the liquid
vaccine causes the heat retained in the medium in which the water is able to absorb and hold heat well (Abdul-
Fattah et al., 2007). Increased temperatures lead to hydrolysis of proteins and vaccine component changes and
the emergence of clotting preparations (Katdare, Chaubal, & London, n.d.). It can be observed visually by a
color change from relatively clear dosage be inclined cloudy.
Heat stability of the liquid vaccine is strongly influenced by the pH, degradation of the stocks, both
chemical and physical, are influenced by changes in pH. Chemical degradation generally involves the catalyzed
reaction such as acid-base hydrolysis of peptide bonds of proteins. Degradation in physics emerged as a result
of changes in protein content and adjuvant (Katdare et al., n.d.). So as to prevent the depletion of the liquid
vaccine that must be managed in a cold chain system.
The pH profile showed that the dried vaccine formulas were maintaining stability over a range of pH
testing. The small water content in the dried vaccine prevents a reaction catalyzed acid-base. The use of
phosphate buffer effectively kept the pH changes due to heating (Katdare et al., n.d.) . This can be observed
from the entire formula showed a decrease in pH were not significant when compared with the liquid vaccine.
The whole dried vaccine formulas show pH profiles are likely to be stable during the test but were
unable to sustain the potential antigenic HBsAg. The Stable pH profile and the potential antigenic HBsAg was
Heru Mukti
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/International Journal of ChemTech Research, 2017,10(6): 132-139. 137
not decreased sharply indicates that the dry Hepatitis B vaccine has the prospect to be managed at room
temperature.
0
1
2
3
4
5
6
-1,11 -0,51 0,09 0,69
respon
Log dos i s
HBV F-B F-E
Fig. 1: Immune respond after 28th day vaccine injected in mice
0
20
40
60
80
100
120
0 1 2 3
Potency recovery ( %
)
Month
F-A F-B F-C F-D HBV
Fig. 2: Formulation screening for the biochemical stability of four dry vaccines. HBsAg potency at each
time-point was analyzed by enzyme immunoassay(EIA) kit(Murex ® monoclonal diagnostic kit Version
3.0, Abbot lab.IL.
6,55
6,6
6,65
6,7
6,75
6,8
6,85
0 1 2 3
pH
Month
F-A F-B F-C F-D
Fig. 3: pH changes during the three months of observation
Heru Mukti
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Table 3. In vivo potential test in mice.
Dosis
HBV
F-B
F-E
Control
n
r
n
r
n
r
n
r
5
1,25
0,3125
0,07815
Lower
limit
Upper
limit
Potency
6
6
6
6
4
3
2
1
6
6
6
6
3
2
2
1
6
6
6
6
2
1
1
0
6
0
0,02
3,481
0,356
0,002
0,86
0,11
Notes : n = Samples r = Reactive samples
Conclusion
Differences in the composition of bulking agents did not show any significant differences to the pH and
potential antigenic vaccine. Drying technique effect on the decline in potential vaccine antigen in which the
freeze-dried vaccine with the potential to produce relatively high at 97,8% with a combination of trehalose:
mannitol (7:3). In vivo testing indicate potential immunogenic F-B at 35.8% and has an opportunity to be
managed out of cold chain system.
Refferences
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J. (2007). Drying-induced variations in physico-chemical properties of amorphous pharmaceuticals and
their impact on stability (I): Stability of a monoclonal antibody. Journal of Pharmaceutical Sciences.
https://doi.org/10.1002/jps.20859
2. Adams, G. D. J., Cook, I., & Ward, K. R. (2015). The principles of freeze-drying. Methods in
Molecular Biology. https://doi.org/10.1007/978-1-4939-2193-5_4
3. Awate, S., Babiuk, L. A., & Mutwiri, G. (2013). Mechanisms of action of adjuvants. Frontiers in
Immunology. https://doi.org/10.3389/fimmu.2013.00114
4. Chang, L. L., & Pikal, M. J. (2009). Mechanisms of protein stabilization in the solid state. Journal of
Pharmaceutical Sciences. https://doi.org/10.1002/jps.21825
5. Chen, D., Kapre, S., Goel, A., Suresh, K., Beri, S., Hickling, J., … Kristensen, D. (2010). Thermostable
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Thermostable vaccines promise to simplify the logistics of vaccine distribution and expand the immunization coverage. In this study, a pilot-scale spray drying process was developed and used to produce glassy state formulations of a recombinant hepatitis B (HepB) vaccine containing aluminum adjuvant and Neisseria meningitidis A (MenA) protein-polysaccharide conjugate vaccine, representing two common types of subunit vaccines in use today: the spray-dried HepB vaccine formulations were stable for at least 24 months at 37 degrees C while several MenA vaccine formulations exhibited complete stability at temperatures up to 60 degrees C. This study demonstrates the feasibility of producing thermostable vaccines with advanced processing and formulation technologies.
Article
The solid state is preferred for many proteins due to their marginal stability in solution. However, even in the solid state, chemical and physical degradation can occur on the time scale of the drying process, distribution and use. This review summarizes the major degradation pathways of proteins in the solid state and the corresponding stabilization strategies. Specially, this review discusses the mechanisms of protein stabilization in the solid state. Understanding the mechanisms of protein stabilization is critical to efficient protein formulation development in the pharmaceutical industry.
Article
Recombinant hepatitis B surface antigen (HBsAg) particles derived from Chinese hamster ovary (CHO) cells were stored at various conditions for 12-18 months in their naked form or adsorbed to alum (HBV vaccine). The physical, chemical and immunological parameters during storage at -20 degrees C, 4 degrees C, room temperature and 37 degrees C were investigated. HBsAg particles fully retained the original peptide composition when stored for 6 months, as a dispersion, at -20 degrees C and 4 degrees C; and as lyophilized powder, at all four temperatures. Ten percent sucrose preserved the size, shape and protein content of the naked particles stored at 4 degrees C and -20 degrees C for 18 months as a dispersion or lyophilized. Lyophilization in the presence of glucose, sucrose and trehalose, but not mannitol, further improved the 4 degrees C stability of size, shape and the protein content during 2 years of storage. Stability of the particle's lipid components was inferior to that of the protein components. Dispersions of naked HBsAg and of particles lyophilized in the presence of sucrose and stored at -20 degrees C were the only forms in which the lipid content and composition, including the lipid polyunsaturated acyl chains, were preserved for at least 18 months storage. The level of phospholipid and free cholesterol were most stable in the HBV vaccine which was stored at 4 degrees C; they did not change after 1 year of storage. Preservation of immunogenicity was evaluated according to dose-dependent changes in S-specific antibody titers in sera obtained from immunized BALB/c mice. The ED50 for achieving seroconversion was 0.07 microg/ml/mouse, indicating that the vaccine is very immunogenic. Freezing or freeze-drying of the HBV vaccine results in the total loss of vaccine immunogenicity (in spite of the good chemical stability), while full immunological potency was retained for at least 2.5 years at 4 degrees C. Storing formulated vaccine at 25 and 37 degrees C for 4 and 2 weeks, respectively, did not alter the vaccine potency. This study suggests that the vaccine's physical, chemical and immunological characteristics are sufficiently stable at high temperatures to reduce the need for 'cold chain' transportation.
Article
The present study was conducted to investigate the impact of drying method and formulation on the storage stability of IgG1. Formulations of IgG1 with varying levels of sucrose with and without surfactant were dried by different methods, namely freeze drying, spray drying, and foam drying. Dried powders were characterized by thermal analysis, scanning electron microscopy, specific surface area (SSA) analysis, electron spectroscopy for chemical analysis (ESCA), solid state FTIR, and molecular mobility measurements by both isothermal calorimetry and incoherent elastic neutron scattering. Dried formulations were subjected to storage stability studies at 40 degrees C and 50 degrees C (aggregate levels were measured by size exclusion chromatography initially and at different time points). Both drying method and formulation had a significant impact on the properties of IgG1 powders, including storage stability. Among the drying methods, SSA was highest and perturbations in secondary structure were lowest with the spray-dried preparations. Sucrose-rich foams had the lowest SSA and the lowest protein surface accumulation. Also, sucrose-rich foams had the lowest molecular mobility (both fast dynamics and global motions). Stability studies showed a log-linear dependence of physical stability on composition. Preparations manufactured by "Foam Drying" were the most stable, regardless of the stabilizer level. In protein-rich formulations, freeze-dried powders showed the poorest storage stability and the stability differences were correlated to differences in secondary structure. In stabilizer-rich formulations, stability differences were best correlated to differences in molecular mobility (fast dynamics) and total protein surface accumulation.