Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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HEPATITIS B ANTIGEN
BACKGROUND OF THE INVENTION
...
This invention relates to hepatitis B and, more
particularly, to a vaccine for hepatitis B and to a
method for purifying hepatitis B antigen for use as a
vaccine.
Hepatitis B is one of the types of viral hepa-
titis which results in a systemic infection with the
principal pathologic changes occurring in the liver.
This disease affects mainly adults and is maintained
chiefly by transfer of infection from long term carriers
of the virus. Usual methods of spread are by blood
transfusion, contaminated needles and syringes, through
skin breached by cuts or scratches, by unsterilized
dental instruments as well as by saliva, venereal contact
or exposure to aerosolized infected blood.
The incubation period of type B hepatitis is
relatively long: from 6 weeks to 6 months may elapse
between infection and the onset of clinical symptoms.
The illness usually begins with fatigue and anorexia,
sometimes accompanied by myalgia and abdominal dis-
comfort. Later jaundice, dark urine, light stools and
tender hepatomegaly may appear. In some cases, the onset
may be rapid, with appearance of jaundice early in
association with fever, chills and leukocytosis. In
other cases jaundice may never be recognized and the
patient may be aware only of a "flu-like" illness. It is
estimated that the majority of hepatitis infections
result in a mild, anicteric illness.
DETAILED DESCRIPTION
The starting material for the purified hepa-
titis B surface antigen (HBsAg) of the present invention
is plasma obtained from hepatitis B donors, e.g., by
plasmaphoresis. The level of antigen may be measured in
known manner by radioimmune assay, passive hemaggluti-
nation or complement fixation. The plasma is cooled and
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the cryoprecipitate which forms is removed by light cen-
trifugation. The HB Ag in the resulting clarified plasma
is isolated by an isopycnic banding step followed by a
rate zonal banding step.
In isopycnic banding the partially purified
concentrate is contacted with a liquid medium having a
density gradient therein which includes the density of
the specific antigen being isolated. The liquid medium
is then subjected to ultracentrifugation to attain an
equilibrium distribution of the serum components through
the density gradient according to their individual densi-
ties. Successive fractions of the medium are displaced
and those containing the desired antigen, i.e. the
fractions having a density of from about 1.21 to about
1.24 g/cc, are separated. The application of this tech-
nique to the purification of HBsAg is described in German
Specification 2,049,515 and United States Patent
3,636,191. The concentrations of the solutions forming
the gradient are selected so as to encompass the density
range of from about 1.0 to about 1.41 g/cc. The liquid
medium may be employed in the form of a linear gradient
or a step gradient. Preferably it is employed in the
form of a step gradient due to its inherent higher
capacity for fractionation.
In rate zonal banding the partially purified
concentrate is subjected to ultracentrifugation in
contact with a liquid medium having a density gradient
therein, but this time using the rate zonal technique,
i.e., at a rate and for a period such that equilibrium is
3G not attained, the HBsAg and other residual serum com-
ponents being distributed through the medium according to
their sedimentation coefficients in the medium. The
concentrations of the solutions forming the step gradient
are selected so as to encompass the density range of from
about 1.0 to about 1.28 g/cc. The rate zonal step is
carried out until the HBsAg resides in the 1.13 to 1.16
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density region. At this point the HBsAg is separated
from the bulk of the crude plasma proteins and, most
significantly, is also separated from the macroglobulin
complement of the plasma. If the rate zonal step is
carried out such that the desired HBsAg antigen reaches
its equilibrium position, i.e., about 1.18 to about 1.20
g/cc, it has been found that a plasma macroglobulin
fraction will appear as a contaminant in the desired
HBsAg antigen fraction.
The liquid media used in the isopycnic banding
and rate zonal steps may be any density gradient in the
appropriate ranges. Prior art solutes for such solutions
include, e.g. sucrose, potassium bromide, cesium
chloride, potassium tartrate and the like.
The isopycnic banding step is conveniently
carried out in a centrifuge, for example, Electro-
nucleonics-K, by filling the stationary rotor with saline
solution, then successively displacing the saline so-
lution upwards with aliquots of a liquid medium solution
of increasing density until a step gradient is formed.
The plasma is introduced at the top of the rotor dis-
placing some of the highest density solution from the
bottom. Typically, the volume of plasma is from about
15% to about 40% that of the step gradient. The centri-
fuge is brought up to speed through a programmed speed
control system which prevents mixing during the initial
reorientation phase. When equilibrium is attained and
the product is in its proper density position, the rotor
is slowed down through the same system to prevent mixing
upon reorientation to the original configuration. Then
the gradient is drained from below and the proper density
cut collected. A similar technique is used in the rate
zonal banding. The proper density cut from the rate
zonal banding is the desired concentrate of hepatitis B
antigen.
Due to the small size, approximately 20 nm, of
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HBsAg the isopycnic banding step is quite time consuming,
requiring about 18 hours of centrifuging. As a result,
even operating 24 hours a day, 7 days a week, it is
possible to prepare only about 4 batches of clarified
plasma per centrifuge. Productivity can be increased, of
course, by utilizing additional centrifuges. This in-
volves a tremendous capital investment, however, as each
centrifuge costs about $100,000.
It has now been found that substantial in-
creases in productivity and substantially reduced oper-
ating costs are obtained by multiple loading of the
isopycnic banding gradient. Multiple loading means
subjecting a sample of clarified plasma containing HBsAg
to isopycnic banding conditions for a time sufficient to
permit substantially all of the HBsAg in the clarified
plasma to pass into the gradient but insufficient to
achieve equilibrium, and repeating this step at least
once with an additional sample of clarified plasma con-
taining HBsAg, before continuing the isopycnic banding
conditions for a time sufficient to achieve equilibrium.
If desired, a gradient may be loaded with up to about 6
samples of clarified plasma. As the time required for
the HBsAg in the clarified plasma to enter the gradient
is only a fraction of that required to reach equilibrium,
and as the subsequent time required to reach equilibrium
is the same whether the gradient is single or multiply
loaded, substantial savings in time and reductions in
unit processing costs are obtained.
While the increased productivity and reduced
costs of the multiple banding technique of the present
invention may be achieved with any suitable gradient,
preferably the gradient is sodium bromide.
The isopycnic banding is carried out to equi-
librium by centrifuging at from about 40,000 x g to about
80,000 x g for about 10 hours or beyond. It has been
found, however, that by centrifuging the plasma for about
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4 hours substantially all of the HBsAg is caused to move
into the isopycnic banding gradient. Then the sample of
spent plasma is removed and a fresh sample of plasma
equal in volume to the first sample is layered onto the
gradient. Centrifuging may then be continued as previ-
ously for about 10 hours or beyond to cause the HBsAg in
both samples to move into the equilibrium density region
of the gradient (1.21 to about 1.24 g/cc) to complete the
banding. Alternatively the centrifuging may be continued
for 4 hours, the spent plasma removed and a third sample
of fresh plasma layered onto the gradient. This multiple
loading procedure may be repeated six or even more times
before completing the banding by centrifuging for about
18 hours.
The ratio of the charge (plasma) volume to the
gradient volume is from about 1:3 to about 1:6. When a
single plasma charge is applied to the gradient and cen-
trifuged under isopycnic banding conditions (e.g. for
from about 16 to about 20 hours at 30,000 rpms in the
K-II centrifuge) the resulting product generally will
have a protein content of approximately 4-10 mg/ml in a
volume of 1.0 liter, depending on the amount of protein
in the original plasma.
When a double charge of plasma is applied to
the gradient and centrifuged under isopycnic banding con-
ditions, (for from about 16 to about 20 hours at 30,000
rpms) the resulting product will have a protein content
which is additive for the charges employed, typically
from about 8-20 mg/ml in a volume of 1.0 liter, depending
on the amount of protein in the original plasma. The
level of protein increases in this manner for each subse-
quent charge of plasma applied to the gradient.
The product is then subjected to a rate zonal
banding. The rate zonal banding is carried out until the
HBsAg is in the density range of from about 1.13 to about
1.16 g/cc. Typically this takes for from about 16 hours
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to about 20 hours, preferably for from about 17 to about
18 hours, at from about 30,000 x g to about 60,000 x g.
According to one aspect of the present in-
vention the gradient is formed of sodium bromide whether
or not the multiple loading technique is used. In
contrast to heretofore used materials sodium bromide has
definite advantages. The solubility of sodium bromide
allows the use of high density solutions in the formation
of gradients at refrigerator temperatures (2-6C). There
are definite economic advantages to using sodium bromide
over a salt such as cesium chloride as well as not having
to contend with the problem of human toxicity from
residual and HBsAg bound cesium ions. In sodium bromide
gradient any ions bound to the HBsAg, due to biophysical
characteristics, will be a sodium salt which is very com-
patible with the human biological system and does not
present a toxicity problem.
The biophysical characteristics of the HBsAg
are well documented ~. Clinical Investigation 52, 1176
(1973), J. of Virology 10, 469 (1972 ~ as a negatively
charged particle. In the presence of a very high concen-
tration of positively charged sodium ions there is formed
a sodium-HBsAg salt molecule. This type of molecule is
compatible with the human biological system. In contrast
to prior art products, the HBsAg of the preferred mode of
carrying out the present invention is substantially free
of other cations, particularly added cesium and potassium
ions.
The superior solubility of NaBr at lowered
temperatures with respect to KBr permits the use of
lowered temperatures more conducive to stability of
biological materials. The use of a step gradient rather
than a linear gradient is preferred as it accumulates
impurities at the step boundaries and permits processing
a larger volume of plasma in a single gradient.
The antigen of the present invention is useful
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per se as an antigen for hepatitis B and can be used as
described in U.S. patent 3,636,191. The HBsAg antigen of
the present invention is a highly purified product. The
isopycnic banding step results in about a 100 fold puri-
fication HB Ag relative to normal plasma protein. The
rate zonal step results in about a further 20 fold puri-
fication of HBsAg relative to normal plasma protein. The
combination of the two steps result in about a 2000 fold
purification of HBsAg relative to normal plasma protein.
The resulting product has been shown to be substantially
free of blood group substances A and B as measured by
serological and electrophoresis techniques. In addition,
the antigen of the present invention can be used as the
starting material for the hepatitis B antigen of co-
pending application Serial No. 251,740, filed 4 May 1976.
The following examples illustrate the present
invention without, however, limiting the same thereto.
EXAMPLE 1
The rotor of a centrifuge, Electronucleonics K,
is filled with 8,400 ml of phosphate buffer. After
running the rotor up to 10,000 rpm to degas the system,
the following step gradient is pumped into the bottom of
the stationary rotor:
1. 2,400 ml of 10% NaBr, p=1.08
2. 1,000 ml of 20% NaBr, p=1.17
3. 1,500 ml of 30% NaBr, p=1.28
4. 3,500 ml of 40% NaBr, p=1.41
Plasma containing Australia antigen (HBsAg),
1,750 ml, is pumped into the top of the stationary rotor
displacing 1,750 ml of 40% NaBr from the bottom of the
rotor. The rotor is accelerated to 30,000 rpm and run at
this speed for 18 hours. After stopping the rotor 500 ml
of HBsAg rich material in the 1.21 - 1.24 density region,
is collected and dialyzed against phosphate buffer.
The rotor is then filled with phosphate buffer,
degassed as above, and the following step gradient pumped
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into the bottom of the stationary rotor:
1. 2,400 ml of 5% sucrose, p=1.02
2. 1,750 ml of 15% sucrose, p=1.06
3. 1,750 ml of 25% sucrose, p=l.10
4. 2,500 ml of 50% sucrose, p=1.23
The HBsAg rich material from the NaBr isopycnic
banding step, 500 ml, is pumped into the rotor top dis-
placing 500 ml. of 50~ sucrose out the rotor bottom. The
rotor is then run at 28,000 rpm for 18 hours. After
stopping the rotor, 500 ml of HBsAg rich material in the
1.135 - 1.165 density region is collected.
EXAMPLE 2
The rotor of a centrifuge, Electronucleonics K,
is filled with 8,400 ml of phosphate buffer. After
running the rotor up to 10,000 rpm to degas the system,
the following step gradient is pumped into the bottom of
the stationary rotor:
1. 2,400 ml of 10% NaBr, p=1.08
2. 1,000 ml of 20~ NaBr, p=1.17
3. 1,500 ml of 30~ NaBr, p=1.28
4. 3,500 ml of 40~ NaBr, p=1.41
Plasma containing HBsAg, 1,750 ml, is pumped
into the top of the stationary rotor displacing 1,750 ml
of 40% NaBr from the bottom of the rotor. The rotor is
accelerated to 30,000 rpm and run at this speed for 4
hours. The rotor is then stopped and 1,750 ml of 40~
NaBr are pumped into the bottom of the rotor forcing the
plasma out the top. An additional 1,750 ml of fresh
plasma containing HBsAg are pumped into the top of the
rotor displacing an equal volume of 40% NaBr out the
bottom of the rotor. The rotor is then run at 30,000 rpm
for lg hours. After stopping the rotor 1,000 ml of HBsAg
rich material in the 1.21 - 1.24 density region is col-
lected and dialyzed against phosphate buffer.
The rotor is then filled with phosphate buffer,
degassed as above, and the following step gradient pumped
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into the bottom of the stationary rotor:
1. 2,400 ml of 5~ sucrose, p=1.02
2. 1,750 ml of 15% sucrose, p=1.06
3. 1,750 ml of 25~ sucrose, P=l.10
4. 2,500 ml of 50% sucrose, P=1.23
The HBsAg rich material from the NaBr isopycnic
banding step, 1,000 ml, is pumped into the rotor top dis-
placing 1,000 ml. of 50% sucrose out the rotor bottom.
The rotor is then run at 28,000 rpm for 18 hours. After
stopping the rotor, 1,000 ml of HBsAg rich material in
the 1.135 - 1.165 density region is collected.
EXAMPLE 3
The rotor of a centrifuge, Electronucleonics K,
is filled with 8,400 ml of phosphate buffer. After
running the rotor up to 10,000 rpm to degas the system,
the following step gradient is pumped into the bottom of
the stationary rotor:
1. 2,400 ml of 10% NaBr, p=1.08
2. 1,000 ml of 20% NaBr, p=1.17
3. 1,500 ml of 30% NaBr, p=1.28
4. 3,500 ml of 40% NaBr, p=1.41
Plasma containing HBsAg, 1,750 ml, is pumped
into the top of the stationary rotor displacing 1,750 ml
of 40% NaBr from the bottom of the rotor. The rotor is
accelerated to 30,000 rpm and run at this speed for 4
hours. The rotor is then stopped and 1,750 ml of 40%
NaBr are pumped into the bottom of the rotor forcing the
plasma out the top. An additional 1,750 ml of fresh
plasma containing HBsAg are pumped into the top of the
rotor displacing an equal volume of 40% NaBr out the
bottom of the rotor. The rotor is accelerated to 30,000
rpm and run at this speed for 4 hours. The rotor is then
stopped and a third charge of 1,750 ml of fresh plasma
containing HBsAg are pumped into the top of the rotor
displacing an equal volume of 40% NaBr out the bottom of
the rotor. The rotor is then run at 30,000 rpm for 18
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hours. After stopping the rotor, 1,500 ml of HBsAg rich
material in the 1.21 - 1.24 density region is collected
and dialyzed against phosphate buffer.
The rotor is then filled with phosphate buffer,
degassed as above, and the following step gradient pumped
into the bottom of the stationary rotor:
1. 2,400 ml of 5% sucrose, p=1.02
2. 1,750 ml of 15% sucrose, p=1.06
3. 1,750 ml of 25% sucrose, p=l.10
4. 2,500 ml of 50% sucrose, p=1.23
The HBsAg rich material from the NaBr isopycnic
banding step, 1,500 ml, is pumped into the rotor top dis-
placing 1,500 ml of 50% sucrose out the rotor bottom.
The rotor is then run at 28,000 rpm for 18 hours. After
stopping the rotor 1,500 ml of HBsAg rich material in the
1.135 - 1.165 density region is collected.
EXAMPLE 4
The following table shows the marked increase
in yield per unit of time when using the multiple loading
technique of the present invention (Examples 2 and 3) as
compared with single loading (Example 1).
Total isopycnic % Increase % Increase in
Yield (m1) and rate zonal in time (with yield (with
Ex- f A centrifuging respect to respect to
ample HBs g time (hours) Example 1) Example 1)
-
1500 36
21,000 40 11.1% 100%
31,500 44 22.2% 200%
This application is a division of Application
Serial No. 272,890, filed March 1, 1977.
