Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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TITLE OF THE INVENTION
IMPROVED RECOMBINANT HEPATITIS B SURFACE ANTIGEN
CROSS-REFERENCE TO RELATED APPLICATIONS
Not applicable
STATEMENT REGARDING FEDERALLY-SPONSORED R&D
Not applicable
REFERENCE TO MICROFICHE APPENDIX
Not applicable
FIELD OF THE INVENTION
The invention relates to recombinant hepatitis B surface antigen
(rHBsAg), prophylactic and therapeutic vaccines containing HBsAg and methods
of
preparing HBsAg and vaccines.
BACKGROUND OF THE INVENTION
The major surface antigen of the hepatitis B virus is a 25 kDa protein,
HBsAg. The protein is known in three forms: preS 1, preS2 and S. The preS 1
and
preS2 forms include 14 and 39 amino acids that are cleaved from their N-
termini in
vivo to yield the 226 amino acid S form. (Valenzuela P., et al. (1979) Nature,
280:815; Valenzuela P., et al., Synthesis and assembly of hepatitis B virus
surface
antigen particles in yeast. Nature. (1982) 298:347-350. Miyanohara A., et al.,
Expression of hepatitis B surface antigen gene in yeast. PNAS U S A (1983)
80(1):1-
5)
Over the past two decades, recombinant hepatitis B surface antigen
expressed as the S form in yeast cells (rHBsAg) has superseded plasma-derived
antigen as a vaccine against hepatitis B infection (Valenzuela et al., 1982;
McAleer et
al., 1984). In the plasma of infected animals, the surface antigen protein
assembles
into 22 nm particles comprising lipids and HBsAg. However, the assembly of the
yeast produced rHBsAg into these virus-like particles has remained poorly
understood.
It has been established that reduction of the disulfide bonds in HBsAg
abolishes or greatly decreases both its antigenic and immunogenic properties
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(Reviewed by Tiollais et al., 1981, Guesser et al., 1988; Mishiro et al.,
1980; Chen et
al, 1996; Hauser, et al 1988). For example, eleven out of the total 14
cysteine residues
are conserved among three different type of S protein of Hepatitis B viruses,
namely,
HBsAg of human, woodchuck, and ground squirrel viruses. However, these three
5 types of S protein share only medium to low overall sequence homology among
(Stirk
et al, 1992). Interestingly, all 8 cysteine residues in the "a" determinant
loop (62 as
between predicted Helix B and Helix C) are fully conserved. This indicates
that
disulfide bonds may have an important role in maintaining the structural
integrity of
the antigenic determinants or epitopes (Stirk et al., 1992).
10 The importance of the presence of disulfide bonds to the functional and
structural integrity of proteins has been well documented, particularly for
ribonuclease
A (Ubuka T., (1996) Protein disulfide isomerase-catalyzed renaturation of
ribonuclease A modified by S-thiolation with glutathione and cysteine. Biochem
Mol
Biol Int. 38(6):1103-10; Fahey, R.C., (1977) Biologically important thiol-
disulfide
15 reactions and the role of cyst(e)ine in proteins: an evolutionary
perspective. Adv Exp
Med Biol. 86A:1-30; Lyles MM, et al., (1991) Catalysis of the oxidative
folding of
ribonuclease A by protein disulfide isomerase: dependence of the rate on the
composition of the redox buffer. Biochemistry. 22;30(3):613-9).
In the case of rHBsAg, the correct disulfide bond pairings are
20 important antigenic determining factors since they are most likely required
for the
integrity and stability of the major epitopes. (Wampler et al., 1985) On over-
expression in yeast cells, it is believed that the HBsAg molecules need to
find a lipid
environment for folding into the membrane-embedded structures as seen in the
plasma-derived 22 nm lipid/protein particles. However, because of a lack of
the
25 control of the cellular redox environments, unlike the propagation of the
virus in the
infected cells, during over-expression, and sub-optimal conditions during
purification
and formulation of rHBsAg, the recombinant proteins are expected to assume
certain
sub-optimal conformations as a result of mismatched disulfide bond pairings.
Although some cysteine-rich proteins or peptides are reported to have
30 strong propensity to form the correct disulfide bond pairings during
oxidative
refolding (Moroder et al., 1996; Mosiol et al., 1994), productive folding is
always in
competition with nonproductive folding. These latter pathways lead to either
wrong
disulfide bond pairings or aggregation of polypeptides.
The rate and yield of oxidative renaturation of small reduced
35 polypeptides have been reported to be influenced by the ratio of low
molecular weight
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disulfide/thiol compounds. The relationships are rather complex and do not
parallel
the redox potential of the system, as reported using reduced RNAse A as a
model
protein (Wetlaufer et al., 1987).
It is reported that a redox buffer with both forms of glutathione, i.e.,
GSSG/GSH mixture, might facilitate the conformational searching by promoting
the
formation of the correct pairings and unlocking the mismatched ones. However,
the
use of GSSG/GSH mixtures was only demonstrated for some small proteins and
peptides (Moroder, et al., 1996).
It is believed that in a natural system of cells infected by HBV, the
nascent HBsAg molecules need to find a lipid environment for the formation of
certain intramolecular and/or intermolecular disulfide bonds that lead to
proper
folding into the native conformation of the protein. In the natural system,
this process
leads to the formation of particles of approximately 22 nm in diameter made up
predominantly of HBsAg protein associated with a lipid membrane. Similarly,
when
expressed in yeast cells or other expression host , it is believed that the
nascent
rHBsAg needs to find a lipid environment prior to the spontaneous folding into
membrane-embedded structures.
However, in recombinant processes, the rHBsAg is over-expressed in a
non-natural system using a host cell. Insect, yeast and CHO cells are commonly
used
although other cell types may be employed. When overproduced in such a system,
the
rHBsAg is an assortment of aggregations of scrambled forms and non-native
conformations due to mismatched disulfide bond pairings. These artifacts of
over-
expression yield molecules locked into conformations of low antigenicity.
Therefore,
once produced, the over-expressed rHBsAg is typically processed outside a
cellular
environment to eliminate some of the undesired artifacts of over-expression.
In a method presently used in the art, once the over-expressed rHBsAg
is purified from the host cells, the antigen is treated with thiocyanate in an
oxidative
step to induce a conformational search and yield the form of rHBsAg known as
Form
III (Wampler, et al., 1985) (See FIG. 1). Thereafter, formalin treatment is
used to
lock the rHBsAg into whatever conformation it has assumed under the
chaotropic,
partially denaturing conditions of the thiocyanate treatment. Finally, the
rHBsAg it is
precipitated with adjuvant.
Previous studies reported that one could achieve an enhancement of
antigenicity of rHBsAg by incubation at elevated temperatures as well as the
inhibitory effect of formalin to the same process. Moreover, the role of the
different
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disulfides being generated (i.e., infra- and intermolecular) was also
described
(Wampler, et al., (1985)). In these studies, the conformational search for the
thermodynamically most stable forms of rHBsAg occurred spontaneously.
Therefore,
the optimal percentage of correct three-dimensional structures of the rHBsAg
could
not be obtained by those methods. Most importantly, the reported procedures
yield
product that varies for potency as well as consistency. This is possibly due
to the
poorly-controlled redox conditions, residual metals and surface contacts
during
process and formulation. Thus, vaccines including rHBsAg made by previously
reported methods varies considerably in amount of protein required to induce a
protective response in a vaccinated subject.
SUMMARY OF THE INVENTION
The present invention provides an improved recombinant hepatitis B
surface antigen, rHBsAg, that has a higher specific antigenicity than
previously
known rHBsAg. The invention also provides for the use of this improved rHBsAg
in
prophylactic and therapeutic vaccines and in combination vaccines.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. shows a schematic of the theory of conformational search as
applied to rHBsAg.
FIG. 2A. Glutathione-mediated conformational search/maturation of
rHBsAg at 37°C . Synergistic effects of GSH and GSSG. Measured by
surface
plasmon resonance in reference to a stable rHBsAg preparation and graphed
using
arbitrary units.
FIG. 2B. Glutathione-mediated conformational search/maturation of
rHBsAg at 37°C. A more antigenic conformation of HBsAg can be achieved
by
higher concentrations of GSH. Measured by surface plasmon resonance in
reference
to a stable rHBsAg preparation and graphed using arbitrary units .
FIG. 3. The nucleotide and protein sequences of the variable regions
of the mouse monoclonal antibody A1.2 (from Figures 1 and 2 of Lohman et al
1993).
FIG. 4. A flow chart showing the conduct of an ELISA.
FIG. 5. A graph of the correlation of the antigenicity of rHBsAg
measured by ELISA and immunogenicity measured by mouse potency ED50.
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FIG. 6. A graph comparing the ELISA antigenicity of rHBsAg made by
using a conformational search step with and without adding a redox buffer.
FIG. 7. A graph of in vitro relative potency (IVRP) versus the relative
antigenicity measured by surface plasmon resonance.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides an improved recombinant hepatitis B
surface antigen protein, "rHBsAg," that exhibits a higher antigenicity than
previously
known rHBsAg and a method of making the improved rHBsAg. The invention also
provides for the use of the improved rHBsAg in prophylactic and therapeutic
vaccines, and in combination vaccines.
Briefly, the present invention employs a discovery that an optimized
process of refolding rHBsAg can be used to achieve a product having a higher
level of
immunogenicity than previous rHBsAg products. The immunogenicity is measured
by determining the amount of rHBsAg required to induce an immune response in a
mouse model system. The optimized process preferably includes a redox buffer
step
to prepare the improved rHBsAg. It has been discovered that oxidative
refolding of
rHBsAg in a redox buffer produces a rHBsAg product of higher antigenicity than
previously known in the art.
As used herein, antigenicity of rHBsAg refers to the reactivity of the
rHBsAg in an in vitro test or system. The immunogenicity of rHBsAg refers to
the
ability of the rHBsAg to induce an immune reaction in an in vivo animal model
or in a
patient in a clinical setting.
Without wishing to be bound by any particular theory, it is believed
that the use of a redox buffer enables the rHBsAg polypeptides to undergo a
conformational search in a more controlled manner over a protracted period of
time.
The higher immunogenicity of the rHBsAg produced by this process may therefore
be
due to the preparation of polypeptides having a greater percentage of correct
disulfide
bond pairings. However, because the rHBsAg protein is a large polypeptide
having
14 cysteines and a complex, membrane embedded three dimensional structure,
these
hypothetical statements may not represent a complete explanation of the
factors that
lead to the improved immunogenicity of the rHBsAg products taught herein. The
interplay between polypeptides in a particle of rHBsAg may also play an
important
role.
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There are approximately 100 rHBsAg polypeptides in a particle. These
polypeptides can form dimers and trimers by intermolecular disulfide bonding.
The
process of making rHBsAg taught herein may produce particles with more, or
properly folded, multimers of polypeptides. For example, it is possible that
the
improved rHBsAg produced as taught herein is due to improved dimerization of
the
polypeptides in the particles to yield conformations that are more
immunogenetic.
Therefore, theoretically, it is possible that the improved immunogenicity of
the
rHBsAg taught herein is due to more proper infra-molecular bonds, inter-
molecular
bonds, or both.
Redox Buffers in the Foldin of rHBsA
A variety of thiol containing compounds can be used to make a redox
buffer for use in the method of making improved rHBsAg. The basic requirements
are that the thiol of the compound can interact with cysteine in rHBsAg and
that the
compound can exist in an oxidized form consisting of a dithiol linkage between
two
molecules of the compound. Examples of thiol compounds that can be made in
reduced and oxidized states are cysteine, 2-mercaptoenthanol, thiothreitol,
dithiothreitol ("DTT"), glutathione ("GSH") and diglutathione (oxidized
glutathione
"GSSG"). Our most preferred redox buffer is composed of a mixture of GSH and
GSSG due to their abundance in all mammalian cells for redox regulation. It is
preferred that one adds the reduced and oxidized compounds (e.g.: GSH/GSSG) at
a
molar ratio from about 30:1 to about 1:1, preferably, a ratio of about 25:1,
20:1, 10:1,
10:4, 5:1, 2:1 or 1:1, and most preferably at a ratio of about S:1. The final
concentration of the compounds should be between about 0.05 to 5.00 nM,
preferably
about 0.20 to about 3.0 mM, more preferably about 0.5 to about 1.5 mM, and
most
preferably about 1.0 mM.
When referring to ratios and concentrations in this specification, we
use the term "about" to acknowledge that the invention involves applying
reagents to
a complex biochemical system. Strict adherence to an absolute, time,
concentration or
ratio as explicitly recited herein is not frequently required. Moreover, one
of skill in
the art can use routine empirical testing of ratios and concentrations within
10%, 20%
25% or even 50% of more of the values explicitly stated herein to determine
the
quality of the product produced by the process when a particular thiol
compound is
employed in the conformational search step. Similarly, the times required for
incubations can vary from those explicitly stated herein.
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The appropriateness of any of these variations can be determined
routinely by testing the rHBsAg product, (e.g.: using a standard mouse potency
assay)
to determine whether the variation yields a product having the improved
antigenicity
produced when using the conditions and reagents explicitly recited herein.
Simply
stated, this specification teaches that one can in fact produce an improved
rHBsAg
through the use of a conformational search step in the presence of a redox
buffer.
Therefore, one who practices in this field can use that teaching to determine
the
parameters appropriate for employing various particular redox buffers under a
variety
of conditions to achieve the improved rHBsAg.
The redox buffer is used to induce what is believed to be a protracted
conformational search, especially under warm physiological conditions. The
concept
of conformational search, i.e., the evolution of the latent epitopes into
fully
functioning or mature ones through the optimal pathways, and the chemical
cross-
linking of the rHBsAg polypeptide chains are shown in FIG. I . Using redox
buffer,
the rHBsAg molecules are allowed to mature in a state of conformational flux
as they
search for the thermodynamically most stable conformation. The redox buffer is
believed to prevent premature locking of conformation, as well as the covalent
disulf de bonding coupled to undesired aggregation. The reducing power of the
redox
buffer is also believed to unlock the mismatched or scrambled disulfide bonds.
These
disulfide bonds are much more susceptible to reduction than the correct
disulfide
pairings. FIGS. 2A and 2B show the dramatic effects of both forms of
glutathione on
the enhancement of antigenicity of rHBsAg, particularly their synergistic
effects in
refolding this cysteine-rich protein.
Thus, it is believed that the most advantageous use of the redox step is
to use conditions under which the presence of the redox buffer compounds is
influential enough to disrupt the less stable mismatched disulfides but not so
influential as to constantly disrupt both mismatched and correctly paired
disulfide
bonds. The preferred and most preferred conditions described herein are
appropriate
when using GSH/GSSG. However, the conditions appropriate for the use of other
thiol compounds can be determined simply by empirical testing of conditions
coupled
with monitoring of the antigenicity and immunogenicity of the rHBsAg produced.
The redox buffer step is added to existing processes for the production
of rHBsAg at the point where the protein is purified from the cellular
expression
system in which it is produced. For example, using the method of Wampler et
al,
I 985, the rHBsAg is processed as described up to the point of producing the
sterile
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filtered product, or SFP. SPF should be held at 4°C until use in the
redox step. The
SPF is placed in an appropriate vessel such as a glass lined tank. A means of
mixing
the contents of the tank should be provided. If appropriate, multiple batches
of SFP
can be combined. If that is the case, we prefer to combine all batches to be
treated
S together in a redox step and to blend them gently and briefly before adding
the redox
buffer.
The redox step can be performed at a variety of temperatures. The step
should be conducted at between 4°C and 55°C. However, certain
considerations lead
one to conduct the step at between 18°C and 45°C. It should be
noted that the
rHBsAg polypeptides are embedded in a lipid membrane. The fluidity of the
membrane should be considered. For example, while one can choose to conduct
the
step cold temperatures, 4°C to 10°C, that is not preferred
because the thermodynamics
of the process would slow to the point where an enormous amount of time would
be
required to complete the step. At the upper end of the range, higher
temperatures lead
to more flexibility in the rHBsAg polypeptide and fluidity within the lipids.
While
that can lead to a faster search, it can also lead to a situation where the
correct
disulfide bonds are more easily disrupted by attack from the buffer compounds
or
other cysteines. Additionally, the protein can denature at high temperatures.
The
temperature should be high enough to allow movement of the polypeptides within
the
membrane. Therefore, we prefer to use temperatures high enough to produce a
result
in a reasonable amount of time but not so high as to lead to instability in
correctly
paired cysteines of protein denaturation. Our preferred temperature ranges are
20°C
to 45°C, more preferably 30°C to 40°C and most preferable
34°C to 38°C.
Temperature can be controlled by a variety of means including the use of a
jacketed
vessel.
We prefer to conduct the redox step without mixing beyond that
required to distribute the redox buffer through the batch at the beginning of
the step.
A dip-tube or other appropriate means of adding the redox buffer can be used
with
gentle and brief agitation sufficient to distribute the buffer through the
batch.
Thereafter, the batch is allowed to stand for a period of time.
While the optimal ratio of GSH/GSSG, their concentrations and the
duration for incubation might be difficult to predict, routine empirical
experiments
have shown that the following conditions are preferred: When using a redox
buffer of
GSH/GSSG with a ratio of 5:1 and a final concentration of 1.0 mM GSH and 0.2
mM
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GSSG, and our most preferred temperature range of 34°C to
38°C, we prefer to
incubate the mixture for about 20 to 140 hours, more preferably, about 80 to
120
hours and most preferably, about 100 hours. This incubation refers to the time
from
the beginning of the heating step to the end of the redox buffer incubation
step and
prior to any formalin treatment that may be desired.
Relevance of Tertiary Structure to Potency
It is known that the plasma-derived HBsAg is fully cross-linked by
disulfide bonds (E. Guesser et al. Model for the protein arrangement in HBsAg
Particles based on physical and chemical studies. 1988. In "Viral Hepatitis
and Liver
Disease" pp. 606-613 (ed. A. J. Zuckerman). Alan R. Liss, Inc., New York). It
is
reported in the literature that the recombinant HBsAg particles are disulfide
cross-
linked after the downstream purification (Wampler et al. 1985. Multiple
chemical
forms of hepatitis B surface antigen. Proc. Nat. Acad. Sci. 82:6830-6834).
There is
also evidence in the literature that mouse immunogenicity of HBsAg resides in
the
disulfide linkage of two 24 kDa monomeric subunits into a 49 kDa dimer (S.
Mishiro
et al. 1980. A 49,000-Dalton Polypeptide bearing all antigenic determinants
and full
immunogenicity of 22-nm Hepatitis B surface antigen particles. J. Immunology,
124:1589). Furthermore, a report has contended that MAb RF1, a neutralizing
monoclonal antibody that has been shown to protect chimpanzees after viral
infection,
reacts in Western blots only with oligomeric forms held by disulfides and not
with the
monomeric 24 kDa subunit (P. Hauser et al. 1988. Induction of neutralizing
antibodies in chimpanzees and in humans by a recombinant yeast-derived
hepatitis B
surface antigen particle. "Viral Hepatitis and Liver Disease" pp. 1031-1037
(ed. A. J.
Zuckerman). Alan R. Liss, Inc., New York). Finally, Chen et al (1966) have
reported
monoclonals that bind to epitopes that are believed to be conformational.
These reports support the notion that inter-molecular disulfides are
critical for antigenicity and immunogenicity. However, infra-molecular
disulfides may
also contribute to the product antigenicity. For example, the epitope
recognized by
monoclonal antibody H166 is reported to be linear (Chen et al., 1996.
Discontinuous
epitopes of hepatitis B surface antigen derived from a filamentous phage
peptide
library PNAS 93:1997-2001 ). However, it is believed that those 4 residues
bound by
H166 (Cys[121]-Cys[124]) represent a beta turn motif between two alpha-helix
regions that is held by an infra-molecular disulfide (Chen et al. 1996 ). In
fact, the
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sequence Cys-X-Y-Cys is contained in the active site of oxidoreductases and
their
redox potential reflect the propensity of this bis-cysteinyl motif to form
disulfide
loops (H-J. Musiol et al. 1994. Redox-active bis-cysteinyl peptides. I.
Synthesis of
cyclic cysteinyl peptides by conventional methods in solution and on solid
supports.
Biopolymers, 34:1553-1562; L. Moroder et al.. 1996. Oxidative folding of
cysteine-
rich peptides vs. Regioselective cysteine pairing strategies. Biopolymers,
40:207-234).
Presence of Cvs~ 121 ]-Cys[ 1241 in rHBsAg_
It is known that in plasma-derived HBsAg is cleaved by trypsin at the
carboxyl end of Lys[ 122] only after reduction of this intramolecular loop
(Cys[ 121 ]-
Cys[ 124)) with 2-mercaptoethanol. Therefore, the susceptibility of rHBsAg
product
to the trypsin was tested before and after the KSCN and formulation steps as a
measure of proper conformation of the protein. The rationale of the experiment
was
to determine whether this intramolecular disulfide loop is present in the
product
before conversion or is generated during the conversion to Form III. The SDS-
PAGE
analysis of different-step products revealed that the intramolecular disulfide
Cys[121]-
Cys[ 124] is already present in the product before most of the conversion
occurs.
Make-un of the Ant~,enici~ of rHBsA~
Regarding the ELISA response of rHBsAg product before and after
conversion, it could be pictured as a composite of a response towards the Cys[
121 ]-
Cys[ 124] region of the molecule. After the rHBsAg product is converted,
additional
epitopes are formed by the generation of intermolecular disulfides (and maybe
intramolecular as well) and these conformational epitopes resulting from
tertiary and
quaternary structure arrangement should then be readily detected by an
antibody
against a truly conformational epitope, e.g., monoclonal H35.
Because of the lack of a true linear relationship between generation of
intermolecular disulfides (extent of conversion) and increase in potency, it
is believed
that that during the last process steps leading to Sterile Filtered Product,
(i.e.,
conversion to form III by KSCN treatment (Wampler et al., 1985)} certain
arrays of
conformations are acquired with potentially different inter- and
intramolecular
disulfides links giving rise to different antigenic macrostructures. High
ELISA values
may then be reached by an optimal oligomerization of individual p24 protein
subunits.
This oligomerization is likely to comprise several molecular events including
(1 )
approximation of the subunits facilitated by lipid fluidity, (2) formation of
inter- and
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intradisulfides, (3) disulfide exchange or re-shuffling, and (4) final
stabilization of the
tertiary and quaternary structure. However, the present invention has revealed
that
rHBsAg with even higher in vitro relative potency values can be made when an
optimized folding step is conducted after conversion of the rHBsAg to form
III.
Measurement of the Anti enicity and Immuno~enicity of rHBsAg.
One can measure the immunogenicity of the rHBsAg product in a
variety of ways using in vivo methods currently in use in the art. Those
techniques
will not in general be described here. ~ However, they can be used to test
samples made
by the process taught herein. The antigenicity of the rHBsAg product can be
assessed
by a number of in vitro techniques. The results of these tests can be analyzed
to
determine the effectiveness of conditions that are varied from those
explicitly taught
herein as well as to monitor or track the progress of the process as adapted
to a
particular facility of equipment. The results also provide a foundation for
understanding the different effectors and inhibitors of the antigen maturation
process.
The methodologies as well as examples of the different applications used to
study the
development of the antigenicity/immunogenicity of the rHBsAg product are
discussed
below.
While in vivo animal models and in vitro ELISA assays are useful to
correlate results with records of how a process was conducted, it can be
preferable to
monitor the progression of the conformational search in a redox buffer step in
as close
to real time as possible. It is also preferable to analyze the conformational
state of the
rHBsAg as it relates to the immunogenicity of the product. Therefore, a method
has
been employed to monitor the conformational state in real time and the results
of that
analysis has been correlated to standard immunogenicity tests. The real time
method
relies on the use of a surface plasmon resonance detector (BIAcore 2000 unit
(Upsala,
Sweden)). The detector manufactured by BIAcore is preferred, but others can be
made or purchased as appropriate. We have correlated the surface plasmon
resonance
analysis of the conformational state of the rHBsAg by comparison to the widely
accepted and commercially available in vitro ELISA assay. The methods of
surface
plasmon resonance and ELISA assays are described below and the mouse potency
assay is described in the Examples.
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Standard ELISA Assay for Analysis of rHBsAQ Product
Immunoassays are used to illustrate how the rHBsAg product's
structural properties impact the immunogenic response. Examples of various
essay
values from different Hepatitis B vaccines will be used to illustrate the use
of the
assay. A standard ELISA assay has about a two day turnaround and multiple
sample
manipulations needed. This analytical method is useful for process for
assessing
antigen maturation. The process is advantageously conducted on rHBsAg that has
been formulated on an aluminum adjuvant.
Recent scientific publications provide insights into the epitopes
recognized by some monoclonal antibodies against rHBsAg (Qui et al., 1996. J
Immunol. 156:33350; Chen et al., 1996). The monoclonal H166 would be useful
for
capture because of its IgM nature (providing multiple Fab binding sites per
molecule)
and because it recognizes a linear or continuous epitope (Cys[ 121 ]-Lys-Thr-
Cys[ 124])
of the rHBsAg (Chen et al.).
The monoclonal H35 is believed to recognize a truly conformational or
epitope since it binds to 13 residues located on two discontinuous regions of
the
rHBsAg molecule (Cys[121]---Leu[175]) (Chen et al. 1996). Therefore, this
antibody
could be conjugated with HRP and used as a reporting antibody to monitor the
formation of a conformational epitope.
The amino acid sequences of these epitopes were derived from affinity-
enrichment experiments (known as "biopanning'~ using a filamentous phage
display
peptide library. (Chen et al. 1996) In addition to biopanning, the site for
H166
binding was also determined by cyclic peptide competition (L. Mimms et al.
1989.
Second generation assays for the detection of antibody to HBsAg using
recombinant
DNA-derived HBsAg. J. Viral. Methods 25:211-232.) The presence of a linear
"H 166 epitope" is also supported by the observation that H 166 binds to
monomeric
p24 in Western blots (L. Mimms et al 1989).
Enzyme immunoassays known in the art can be used to assess the
improved rHBsAg. The AUZYME monoclonal enzyme immunoassay, commercially
available from Abbott Laboratories, is preferred. To employ the AUZYME assay,
one
can follow the manufacturer's instructions for a two step procedure. This
assay has
been found to be quantitative in our hands. Briefly, beads coated with mouse
monoclonal antibodies to hepatitis B surface antigen are incubated with the
rHBsAg
product sample and then with a mouse monoclonal Anti-HBsAg peroxidase
conjugate
(Anti-HBsAg:HRPO). Unbound material is washed off, and color is developed by
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adding a 0-phenylenediamine (OPD) solution containing hydrogen peroxide.
Development of a yellow-orange color is proportional to the antigen
concentration.
The procedure is outlined in greater detail in FIG. 4. It is important to
use good testing laboratory technique at each step. After the addition of 1N
Sulfuric
acid, the absorbencies of the tubes are read in a spectrophotometer at OD492~
It is preferable to run serial dilutions of at least 5 levels for each
sample so that the resulting data can be analyzed by graphing and samples can
be
compared at the midpoints of the curves. For example, when the rHBsAg sample
is at
Smcg/ml, the sample should be diluted 1:10, 1:100, 1:500, 1:1000,
1:2000,1:4000
and 1:8000. Samples from 1:500 to 1:8000 are prepared and tested at least as
duplicates or triplicates. For samples at 10 mcg/ml the sample is diluted
further to
1:16000 and tested at 1:100-1:16000. For samples at 20 mcg/ml, 30 mcg/ml and
40
mcg/ml dilute to 1:32000, 1:48000 and 1:64000 respectively and duplicates or
triplicates are prepared at the appropriate dilutions for testing. These
dilutions are
exemplary and those of skill in the art can use other appropriate dilutions
Diluent controls should be run along with the positive and negative
controls provided in the AUZYME kit. The average of three OD492 absorbance
values for the negative control should be in the range of -0.006 to 0.001. One
should
then average two at OD492 absorbance values for the positive controls and
subtract
the negative controls from that value. If the resulting value is greater than
or equal to
0.400, then the controls are considered valid. The diluent controls should be
in the
range of at OD492 0+~-0.02. An OPD substrate control can be run and should
read
less than at OD492 0.006 and -0.100.
One can test the absolute parallelism of the lines derived from the data
for each dilution series. It is preferred that the difference in absolute
parallelism for a
sample is less than 0.17 for the test on that sample to be considered valid.
For
samples tested on different days, one can test for extravariability. It is
preferred that
the same samples tested on different days do not vary by more that 1.33 for
the test
result for a given day to be considered valid.
Herein, the units of measurement for the immunogenicity of a
preparation of rHBsAg are grounded in ED50 measured per microgram of protein
in
the mouse potency assay. However, that assay takes almost two months to
generate a
result. Therefore, it is preferred to use the ELISA assay taught herein and to
correlate
the results of that assay to the mouse potency assay. The correlation is
monitored by
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assaying a reference preparation of rHBsAg as a control with each run of the
ELISA
where the ED50 of the control is known. The correlation was established as
follows.
The initial rHBsAg reference material was tested in an in vivo mouse
potency model and generated an ED50 value of 0.26 mcg protein. It was decided
that
the OD492 of this material in the in vitro ELISA would be assigned an
arbitrary in
vitro potency value of 1Ø Thereafter, numerous tests were performed on
various
batches of rHBsAg in both the ELISA and mouse potency model. Each of the
ELISAs were run in parallel with a control rHBsAg sample whose ELISA output
was
correlated to the ELISA output of the original reference rHBsAg. Because the
expected OD492 output of the control rHBsAg was known from experience, all
ELISA test results could be correlated to the scale established by the
original
reference rHBsAg. Those values are referred to as in vitro relative potency
("IVRP")
values because they are relative to the potency of the original reference
rHBsAg in an
in vitro ELISA.
1 S A graph of in vitro relative potency versus in vivo mouse potency
(ED50) is shown in FIG. 5. Each data point represents a sample of rHBsAg that
was
tested in both the mouse model and by ELISA. These data points were used to
generate an equation that related the mouse potency value to the relative
potency
value obtained by ELISA. Once one has established the correlation of the ED50
and
ELISA output scales in this manner, one can now perform ELISA tests and expect
to
see an ED50 value in line with the output of the regression equation:
Log(ED50) _ -1.3 - (0.92xLog(RP)}
where Log is natural logarithm and RP is the relative potency obtained by
ELISA as
outlined above.
ELISA Response of other Hepatitis B Vaccines
Plasma-derived Dane particles were tested in the ELISA assay. By this
method, the Dane particles showed a higher response, of about 5Ø Therefore,
the
high values for rHBsAg product produced by the present process, i.e., values
greater
than 2.5, indicates that the rHBsAg particles are approaching an
immunogenicity
level close to that of the natural product of Hepatitis B virus infection, the
Dane
particle. However, unlike the Dane particle, particles of HbsAg produced by
recombinant methods do not contain Hepatitis DNA.
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Surface Plasmon Resonance Assa~~ a BIAcore Detector
Like the ELISA assay described above, the BIAcore assay provides
quantitative estimate of the rHBsAg's relative immunogenicity under controlled
conditions where many factors, such as protein concentrations, temperature,
and
"contact" time (related to the injection volume and flow rate) among others,
are
maintained identical for samples and reference. This technique has been used
successfully for the epitope mapping of HBsAg (J-S Tung et al. 1998.
Characterization of Recombinant Hepatitis B Surface Antigen Using Surface
Plasmon
Resonance. J. Pharmaceutical Sciences, 87:76-80).
The assay is performed in the following format. Biomolecular
interaction assay (BIA) is a technology developed by BIAcore, Inc., for
monitoring
biomolecular binding in real time. It uses a nan-invasive optical detection
principle
based on surface plasmon resonance (SPR). The SPR response reflects a change
of
refractive index as a result of mass deposition at a derivatized metallic
(gold) sensor
surface as molecules bind or dissociate. Because molecules are brought to the
surface
and the unbound molecules are washed-off by a microfluidic pumping system, the
interactions measured are actually binding kinetics. No labeling of the
interacting
components is required, and the technology is applicable to a wide range of
experimental situations.
As in any immunoassay, the proper reagents are important to the
method's response and performance. When using the BIAcore technique to monitor
the formation of the proper conformation of the improved HBsAg it is most
useful to
use an antibody that recognizes a conformational epitope. It is also
preferable that the
antibody binds to an epitope related to the neutralization of the invading
hepatitis B
virus. An example of such an antibody is a monoclonal antibody called MAb
A1.2. It
is also believed that the H35 monoclonal antibody of Chen et al 1996 would be
appropriate.
The amino acid and DNA sequences of the variable regions of the
MAb A1.2 monoclonal antibody were published in Lohman et al., 1993. Molecular
Characterization and Structural Modeling of Immunoglobin Variable Regions from
Murine Monoclonal Antibodies Specific for Hepatitis B Virus Surface Antigen.
Molecular Immunol. 30:1295-1306, and are shown in FIG. C. Using these
sequences
one can construct an appropriate antibody for use in the surface plasmon
resonance
assay. Alternatively, from the understanding of the conduct of the assay
gained from
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the descriptions provided herein, one can screen monoclonal antibodies to find
one
with an appropriate epitope specificity that performs appropriately in the
assay.
For analyzing changes in HBsAg antigenicity, the procedure involved
the following steps as outlined as follows: ('1) chemical immobilization of
rat anti-
s mouse FCgamma on the sensor chip, (2) addition of MAb A1.2 anti-HBsAg which
is
captured by the rat anti-mouse antibody; and (3) injection of antigen product
which is
finally captured by MAb A1.2. (4) the antigenicity for the rHBsAg particles is
expressed as the ratio between the amount of bound rHBsAg and the amount of
bound
MAb A1.2. For a normalized protein concentration in compared samples, the
10 difference in the measured binding represents the difference in the HBsAg
antigenicity due to the conformation of the rHBsAg. Therefore, by following
the
increase in the amount of rHBsAg bound in the surface plasmon resonance assay
one
can monitor the accumulation of rHBsAg having a conformation associated with
antigenicity. Thus, one can monitor the incubation step to determine when a
given
15 batch of rHBsAg has reached an improved antigenicity. The units used to
describe
the antigenicity or the rHBsAg are arbitrary and relate only to the control
run for the
particular assay.
One can perform a series of assays in parallel to measure the response
of improved rHBsAg product in a mouse potency model, ELISA and surface plasmon
20 resonance assay. By collecting a series of data points in each assay for
different
concentrations of a number of preparations of rHBsAg one can generate a date
set
useful to correlate the responses obtained from an ELISA, the surface plasmon
resonance assay and the mouse potency assay. Once one has gained sufficient
experience in these assays one can rely predominantly on the ELISA to for
general
25 monitoring of the antigenicity of the rHBsAg product. One can also rely on
the
surface plasmon resonance assay to monitor the progress of a folding step
while
making the product and to gauge when the product is achieving a high level of
conformationally dependent antigenicity.
30 Improved rHBsA~ in Vaccines
Vaccines including HBsAg from plasma and rHBsAg expressed in a
variety of host cells (e.g., insect, yeast, mammalian cells including CHO
cells) are in
common use in the art. The improved rHBsAg of this invention can be made by
adding the redox step to the present processes used to make rHBsAg from the
various
35 host cells. Thereafter, the improved rHBsAg can be used to make any of the
vaccines
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in which the presently made rHBsAg is used. As these vaccines and their
preparation
are known in the art they will not be described here other than by name:
RECOMBIVAX HB, ENERGIX, COMVAX, PRIMIAVAX, PENTAVAX, &
T~.
It will also be recognized by practitioners in the art that
pharmaceutically useful compositions comprising the improved rHBsAg can be
formulated according to known methods such as by the admixture of a
pharmaceutically acceptable carrier. The improved rHBsAg can be adjuvanted and
formulated using the usual practices of the pharmaceutical arts as exemplified
by
practice manuals and texts including Remington's Pharmaceutical Sciences. To
form
a pharmaceutically acceptable composition suitable for effective
administration, such
compositions will contain an effective amount of the improved rHBsAg.
Particular compositions can be made by adjuvanting the improved
rHBsAg. Many adjuvants are known in the art. Aluminum adjuvants are frequently
used because they are presently approved for use in human patients. Aluminum
adjuvants can be provided as amorphous or crystalline forms. Common aluminum
adjuvants include aluminum phosphate, aluminum hydroxide, and aluminum
hydroxyphosphate. All of these can be used with the improved rHBsAg.
Common methods of adjuvanting used in the art can be applied to the
improved rHBsAg and include co-precipitation of the adjuvant and rHBsAg and
adsorption of the rHBsAg onto the adjuvant. Other adjuvants can be used and
include
saponins, lipids including cationic lipids, oligonucleotides having a CpG
motif,
muramyl dipeptides and DNA vaccines (Donnelly, J. J., Friedman, A., Deck, R.
R.,
DeWitt, C. M., Caulfield, M. J., Liu, M. A., and Ulmer, J. B. 1997. Adjuvant
effects
of DNA vaccines. In Vaccines 97. F. Brown, F. Burton, P. Doherty, J.
Mekalanos, and
E. Norrby, eds. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.,
p.
105-111).
Therapeutic or prophylactic vaccines according to this invention
include the improved rHBsAg and are administered to an individual in amounts
sufficient to treat or prevent disorders associated with infection by
hepatitis virus.
Advantageously, vaccines of the present invention can be administered in a
single
dose, or the total dosage can be administered in divided doses of two, three
or more
doses over a period of time as determined to by effective by a skilled
physician. The
number of doses will be influenced by whether the rHBsAg containing vaccine is
being administered for prophylactic or therapeutic treatment of a patient.
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The dosage amount and regimen utilizing vaccines that include the
improved rHBsAg of the present invention are selected in accordance with a
variety of
factors including type, species, age, weight, sex and medical condition of the
patient;
the severity of the condition to be treated; the route of administration; the
renal,
hepatic and cardiovascular function of the patient; and the particular vaccine
employed. A physician or veterinarian of ordinary skill can readily determine
and
prescribe the effective amount of the drug required to prevent, counter or
arrest the
progress of the condition and determine an appropriate dosage regimen.
For combination vaccines with more than one active immunogen,
where the active immunogens can be in separate dosage formulations, they can
be
administered concurrently, or they each can be administered at separately
staggered
times. For cases of concurrent administration, improved rHBsAg can be
formulated
in a combination vaccine with other active immunogens. For example, the
improved
rHBsAg can be formulated with immunogens such as, and without limitation,
Hemophilus influenza polyribitol phosphate that has been conjugated to a
toxiod or a
protein such as the outer membrane protein complex of Neiserria meningitis;
Measles, Mumps and Rubella immunogens; Diptheria toxoid; Tetanus toxoid;
Pertussis, either whole cell or a-cellular [a-cellular meaning at least one of
the
following Pertussis immunogens: Pertussis Toxoid (also called Lymphocytosis
Promoting Factor), Filimentous Hemagglutinin, 69kd Protein (also called
Pertactin),
Agglutinogen 2 and Agglutinogen 3]; inactivated or attenuated live Yaricella
zoster;
Streptococcus pneumonia capsular polysaccharide conjugated to a toxiod or a
protein
such as Neiserria menningitis outer membrane protein complex; inactivated or
attenuated Polio virus; inactivated or attenuated Rabies virus; Lyme disease
immunogens; and Hepatitis A virus that has been inactivated, attenuated or
attenuated
and inactivated. Finally, the rHBsAg can be formulated with a DNA vaccines)
encoding an immunogen(s) alone or in addition to other immunogens listed
above.
An advantage of a vaccine formulated with the improved rHBsAg is
that one can use a lower amount of the immunogen and achieve the same efficacy
as
the vaccines presently available in the art. An additional advantage is that
one can
fonmulate a vaccine that includes an amount of improved rH$sAg equal to an
amounts used in present formulations and produce a stronger response in a
patient.
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The following examples are presented by the way of illustration and,
because various other embodiments will be apparent to those in the art, the
following
is not to be construed as a limitation on the scope of the invention.
EXAMPLE 1
Method of making_improved recombinant hepatitis B surface antigen
This embodiment of the method will be described on a lab scale.
However, it will be understood by those skilled in the art that the process
can be
scaled as appropriate.
Sterile filtered product (SFP) in phosphate buffered saline (6 mM
phosphate, 0.15 M NaCI) was manufactured as known in the art (Wampler 1985)
and
stored at 4°C. Thereafter, to a solution of 200 mL SFP in a Pyrex glass
bottle,
glutathione (GSH) and oxidized glutathione (GSSG) were introduced to the final
concentrations of 1.0 mM and 0.2 mM, respectively. Once the mixing was
completed, the batch temperature was ramped up to 37°C in an incubator.
Once temperature was reached, the batch was incubated for 44 hrs. An
amount of 300 mM formaldehyde solution, equivalent to 0.01 times the batch
weight,
was introduced, resulting a final concentration of 3 mM formaldehyde. After
the
formalin was added the batch was mixed gently for about 5 minutes to disperse
the
formalin.
After the agitation was concluded the batch was incubated further for
another 60 hrs. It is preferable that the total time from the beginning of the
heating
step through the end of this incubation should be between 100 to 105 hours. At
the
end of that time period the batch is cooled to 2 - 8°C without mixing.
When batch temperature reaches 2 - 8°C, agitation was begun and
(5.44%) potassium aluminum sulfate solution was added to the aqueous rHBsAg
solution. The adjuvanted product was precipitate by adding 1 N NaOH. The
product
was washed by settle/decant washes with 0.15 M NaCI, 6 times. 1.4% Sodium
Borate
was added to resuspend the rHBsAg to the same concentration as when it
started. We
refer to this product as Bulk Alum Product (BAP). BAPs were tested in the in
vitro
relative potency assay.
In vitro relative potency values of more than 3.0 can be obtained. In
vitro relative potency values can be expected to range from about 2.5 to about
4.0,
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more usually, from about 2.75 to about 3.5 or 3.75, and commonly from about
2.75 or
3.0 to about 3.25 or 3.5. Higher values can also be achieved if the formalin
step is
omitted. In that case, values can range as above and up to about 4.25, 4.5,
4.75, 5.0,
5.25, 5.5, 5.75 or 6Ø
It should be noted that the incubation in the presence of GSH/GSSG in
this Example was conducted for 44 hours but the incubation can be conducted
longer
as described above. An incubation of about 100 hours can be preferable to one
of 40
hours. However, while one can incubate for less than 40 hours, at least 40
hours is
preferred.
EXAMPLE 2
Measurement of antiQenicit~of recombinant hepatitis B surface antigen in vivo
An accepted measurement of the immunogenicity of rHBsAg which
will lead to an appropriate immune response in an animal is the efficacy of
rHBsAg in
1 S a live mouse model. The assay is conducted to measure the dose of a
preparation of
rHBsAg that is effective in producing an immune response in the animal. This
test is
sometimes referred to in the art as a mouse potency test.
The mouse potency (MP) test is performed by preparing a series of 2-
fold dilutions of the rHBsAg product using the adjuvant solutions (lx Alum
with
antigen, other components are the same with rHBsAg preparations) are injected
into
adult Balb/C mice. An appropriate format is to use five groups of eight mice.
Each
group is tested at one of five concentrations of rHBsAg, e.g. 1.0 mcg/mL to
0.0625
mcg/mL. As one gains experience with the assay it is preferable to provide
dilutions
such that the projected ED50 is about in the in the middle of the test range.
After
injection, the mice are held for 6 weeks, bled and the individual sera are
tested for
anti-HBsAg using "AUSAB-EIA" diagnostic kit (Abbott Lab) following the
manufacturer's instructions to determine the immuno-response by measuring the
total
anti-HBsAg B antibodies. The potency of the rHBsAg is reported as the dose
which
elicits antibody response in 50% of the mice (EDSO).
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EXAMPLE 3
~ontaneous versus redox buffer assisted refolding
To control for process parameters unrelated to the use of a redox buffer
to make an improved rHBsAg, three experiments were conducted in which sterile
filtered product was processed in parallel with and without the addition of
the redox
buffer. The process used was as described in Example 1. The incubation in the
presence and absence of redox buffer was conducted at 36°C for 40
hours.
Thereafter, the rHBsAg products were tested in the in vitro relative potency
ELISA
assay. The data is presented in the table below and graphed in FIG 6.
NRP
A 2.09
+ 3.56
g 1.69
+ 2.70
C 1.44
(+) ~ 2.47
All the experiments were done under identical conditions except that in the
(+) arms
1.0 mM GSH and 0.2 mM GSSG were included during incubation at 37°C. All
the
samples in the same group (A, B or C) were tested side by side under the same
conditions.
EXAMPLE 4
Correlation of MAb A1.2 affinity in aqueous solutions and NRP of final liquid
(FAP)
and formulated products (BAPsI
The in vitro relative potency,in vivo mouse potency and clinical
performance of bulk alum formulated rHBsAg can be monitored by appropriate
tests
as taught herein or as are in practice in the art. However, when conducting
the
process to make the rHBsAg, it is preferable to have a tool to measure the
progress of
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the production of conformationally active rHBsAg in a liquid, non-adjuvanted
sample
without having to wait the days or weeks necessary for ELISA or in vivo
methods to
report a meaningful measurement.
The surface plasmon resonance (SPR) technology embodied in the
BIAcore equipment is useful for monitoring the real time maturation of
conformationally appropriate rHBsAg. This Example describes how to use the
technology by correlating the SPR measurement of a liquid sample taken during
a
process step and to a reference standard of rHBsAg.
The binding surface of the SPR detector is made of a derivatized metal,
preferably gold. In this Example, a rat anti-mouse FCgamma is covalently
coupled to
the derivatized gold surface through an amine coupling. Then a mouse anti-
rHBsAg
monoclonal antibody that binds to a conformational epitope is contacted with
the
detector and bound to the surface by the rat anti-mouse antibody. The detector
surface
is then washed with a buffer appropriate to the binding of the rHBsAg by the
mouse
monoclonal antibody.
It is important to use an antibody that binds a conformational epitope.
Techniques for testing an antibody for binding to a conformational epitope are
known.
Several antibodies that bind conformational epitopes of rHBsAg are also known
and
include H35 (Chen et al., 1996) and MAb A1.2 (Lohman et al., 1993). The MAb
A 1.2 antibody is the preferred antibody.
The detector is calibrated by binding a reference standard of rHBsAg
for which the in vitro relative potency of the product formulated on alum is
known.
Because the SPR technology is used to measure non-adjuvanted material in a
liquid
system, reference rHBsAg material needs to be preserved in a non-adjuvanted
form,
preferably as sterile filtered product.
The detector is calibrated by binding a reference standard of rHBsAg.
Because the SPR technology is used to measure non-adjuvanted material in a
liquid
system, reference rHBsAg material needs to be preserved in a non-adjuvanted
form,
preferably as final aqueous product. It has been found that a reference
standard of
FAP (final aqueous product) can be stored at 4°C for over two years.
The reference
should be aged for at least six months and preferably aged over 2 years.
The technique is conducted according to the manufacturer's directions or as
described
in the literature (Tung et al (1998) J. Pharm. Sci. 87, 76-80 (1998); (Biacore
AB,
Sweden, BIACORE~ 2000 Instrument Handbook and BIA applications Handbook,
1996). Ethanolamine-HCI, NHS (N-Hydroxysuccinimide) and EDC (N-Ethyl-N'-(3-
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dimethylaminopropyl)-carbodiimide hydrochloride) reagents were obtained from
the
manufacturer (BIAcore) and used according to directions. Sample and
calibration
standards are diluted to 11 mcg/ml in prepared in 0.15 mM NaCI, 6 mM
phosphate,
and TWEEN 20 is added to 0.05%.
5 1) Coupling antibody: rat-anti-mouse Fcg (BIAcore, Inc.) 50 p.g/mL in 10 mM
sodium acetate buffer pH 4.8 is immobilized on the surface of sensor chip
through
amine coupling using the Ethanolamine-HCI reagent.
2) Binding monoclonal antibody: (e.g. Mab A1.2 purified at 1.0 mg/ml, and
diluted 20-fold to 50 pg/ml using HBS buffer (IOmM HEPES, O.lSmM NaCI, 3mM
EDTA, 0.005% TWEEN 20, pH 7.4 filtered and degassed) (Biacore) prior to
assay.) is
then injected and immobilized;
3) Antigen binding: rHBsAg in solutions is injected and certain amount of
antigen will be immobilized by specific antigen-antibody interactions;
4) Regeneration: The surface of the detector is exposed briefly to acid (e.g.
20
mM HCl/10 mM aspartic acid/1% TWEEN 20) to remove the non-covalently-
immobilized monoclonal antibody and antigen to regenerate the surface for the
next
cycle of assay;
$) The ratio between the amount of antigen vs. monoclonal antibody is used to
represent the antigenicity of the tested aqueous preparations of rHBsAg.
20 The 68 data points were accumulated during a period of several
months. The relative antigenicity of final aqueous products at 11 mcg/ml was
measured against a reference standard at the same concentration. The Mab A1.2
binding to rHBsAg was carried out at 20°C in 6 mM potassium phosphate
and 0.15 M
NaCI. However, during the assay the sampling block is cooled to about
10°C.
25 Between assay cycles, the detector surface is briefly exposed to acid to
remove the
non-covalently-immobilized monoclonal antibody and antigen to regenerate the
surface for the next cycle of assay. The antigenicity is expressed as the
ratio between
the amount of rHBsAg and the amount of Mab A1.2 bound to the sensor chip
surface.
Then, this ratio was normalized to the ratio for the calibration standard.
After the
30 formulation with adjuvant, ELISA was earned out on the corresponding bulk
Alum
products and IVRP values were derived. Good correlation between Mab A1.2
binding of rHBsAg by SPR and the IVRP by conventional ELISA as shown in Fig.7.
In this Example, a total of sixty eight data points were collected. Each
data point represents a sample for which the in vitro relative potency of the
alum
35 formulated rHBsAg product was determined and compared to a surface plasmon
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PCT/US99/307?0
resonance measurement of the liquid non-adjuvanted rHBsAg product. From these
data points, a linear fit yielded the theoretical line and parameters shown in
FIG.7.
The figure shows the linear relationship between IVRP of product formulated on
alum
versus the relative antigenicity of the rHBsAg in final aqueous form as a (%)
of a
reference standard as measured by SPR.
The SPR measurements of the liquid form of the rHBsAg product
correlate well with the in vitro relative potency values obtained once the
rHBsAg
product is adjuvanted with alum. Therefore, the SPR technology can be used to
monitor the real-time formation of conformational epitopes whose presence in
the
rHBsAg product result in antigenicity in vitro and immunogenicity in vivo.
EXAMPLE 5
Conformational maturation usin a redox buffer including thiol comyounds
Various concentrations and ratios of reduced and oxidized thiol
compounds can be used in the redox buffer folding step. We prefer the use of
glutathione (GSH) and oxidized glutathione (GSSG). In this Example, various
levels
of GSH and GSSH were tested in the procedure of Example 1.
All steps began by providing a liquid sterile filtered rHBsAg product
made, e.g., by the method of Wampler et al. (1985). The thiol compounds were
20 added, the liquid was mixed briefly and was warmed to between about
34°C to about
38°C and usually about 36°C. The liquid was held without
agitation. Samples were
taken at various times and compared to a reference rHBsAg standard by surface
plasmon resonance measurements.
As would be recognized in the art, a diafiltration step can be added
after the incubation, if desired, to remove the residual redox buffer agents
from the
rHBsAg.
FIG. 2A shows that there is a synergistic effect when GSH and GSSG
are combined in a redox buffer. FIG. 2B shows that there is an increase in the
amount
of rHBsAg containing important conformational epitopes as the amount of GSH
alone
is increased.
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EXAMPLE 6
Ex eriments with and without formalin
PCTlUS99/30770
Three laboratory scale batches of rHBsAg were prepared according to
the method of Example 1. In Batch A, the formalin step was included while in
5 Batches B and C formalin was omitted. The redox incubation was performed in
a
glass tank for Batches A and B and in a stainless steel tank for Batch C. The
control
batch was made without the redox buffer, with formalin and conducted in a
glass tank.
The length of the incubation step was not optimized for any of the batches.
~gp ED50
Batch
Control 2.51 0.15, 0.13
Batch A 4.32 0.074, 0.125
Batch B 4.62 0.088, 0.0725
Batch C 4.30 0.087, 0.13
The ELISA based IVRP and mouse EDso results demonstrate that the
rHBsAg produced by the method of this invention has very high antigenicity and
immunogenicity. If the above data is compared to the historical data for
rHBsAg
20 made by prior art methods shown in FIG. 5., the present data plots lower
and to the
right of the historical data.
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