Note: Descriptions are shown in the official language in which they were submitted.
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1 HE~ATITIS B A~ITIGEN
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2 BACKGROUND O~ THE INVENTION
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3 This invention relates to hepatitis B and, more
4 particularly, to a vaccine for hepatitis B and to a m~thod
~or ~uriying hepatitis B antigen for use as a vaccine.
6 Hepatitis B is one of the types of viral hepatitis
7 which results in a systemic infection with the principal path-
8 ologic changes occurring in the liver. This disease affects
9 mainly adults and is maintained chiefly by transfer o~ in-
10 fection from long term carriers o' the virus. Usual methods ;
11 of spread are by blood transfusion, contaminated needles and
12 syringes, through skin breached by cuts or scratches, by
13 unsterilized dental instruments as well as by saliva~
14 venereal contact or exposure to aerosolized infected blood.
The lnaubation poriod o~ type B hepatitis is re-
16 lativel~ long: ~rom 6 weeks to 6 months may elapse between
li infoction and the onset of clinical symptoms. The illness
18 usually begins with ~atigue and anorexia, sometimes accom-
l9 panied by myalgia and abdominal discomrort. Later jaundice,
dark urine, light stools and tender hepatomega.l.y ~ay appear.
21 In some cases, the onset may be rapid, with appearance of
22 jaundice early in association with faver, chills and lau~octr-
23 ~osis. In other aases jaundice may never be recognized and
24 the patient may be aware only of a "flu-like" illness. It is
estima~ed that the majority of hepati~is inections result in
26 a mild, anicteric illness.
27 DETAILED DESCRIPTION
28 ~he starting material or the purified hepatitis
29 B surface antigen ~HBsAg) OI the present inven~ion is plasma
obtained from hepatitis B donors, e.g., by plasmaphoresis.
31 The level of antigen may be measured in known manner by
32 radioimmune assay, passive hemagglutination or complement
33 ~ixation. The plasma is cooled and the cryoprecipitate
34 which forms is removed by light centriguation. The
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1 HBcAg in the resulting clarified plasma is isolated by an
2 isopycnic banding step followed by a rate æonal banding step.
3 In isopycnic banding the partial:Ly purified
4 concentrate is contacted with a liquid medium having a
density gradient therein which includes the density of
6 the ~pecific antigen being isolated. The li~uid medium is
7 then subjected to ultracentrifugation to attain an
8 equilibrium distribution of the serum components through
9 the density gradient according to their individual den~ities.
Sllccessive fractions o~ the medium are displaced and those con-
11 taining the desired antigen, i.e. the fractions ha~ing a density
12 of ~rom about 1.21 to about 1.24 g/cc, are separated. The
13 appllcaeion of this technique to the purification o~ HBsAg
lq i~ de5aribed in German Speci~lcation 2,049,515 and United
States Patent 3,636,191. The concentrations of the solutions
16 forming the gradient axe selected so as to encompass the density
17 range of from about 1.0 to about 1.4I g/cc. The liquid medium
18 may be employed in the form of a linear gradient or a step
19 gradient. Preferably it is employed in the form of a step
gradient due to its inherent higher capacity or fractionation.
21 In rate zonal banding the partially puri~ied
22 concentrate i5 subjected to ultracentriugation in contact
23 with a liquid medium having a density gradient therein, but
24 this time using the rate zonal technique, i.e., at a rate
and for a period such that equilibrium is not attained, the
26 HBsAg and other residual serum components being distributed -
27 through the medium according to their sedimentation coefficients ~
-i8 in the medium. The concentrations of the solutions ; ~-
29 ~orming the step gradient are selected so as to ~
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1 encompas3 the density range of from about 1.0 to about 1.28
2 g/cc. The rate 20nal step is carried out until the ~BsAg re-
3 sides in the 1.13 ~o 1.16 density region~ At this point
4 the H~sAg is separated from the bulk of the crude plasma
proteins and, most significantly, is also separated from the
6 macroglobulin complement of the plasma. If the rate zonal step ~;
7 is carried out ~uch that the desired HBsAg ar.tigen reaches its
8 equilibrium position, i.e., about 1.18 to about 1.20 g/cc,
g it has been found that a plasma macroglobulin fraction will
appear as a contaminant in the desired H~sAg antigen fraction.
11 The li~uid media used in the isopycnic banding and
12 rate zonal step9 may be any density gradient in the approprlate
13 ranges. Prior art solutes for such solutions include, e.g.
14 sucro~e, pota8sium bromide, ce9ium chloride, potassium tartrate
15 and the like. -
16 The isopycnic banding step is conveniently carried out
17 in a centrifuge, for example, Electronucleonics-K, by filling
18 the 9tationary rotor with saline solution, then successively
19 displacing the saline solution upwards with ~liquots Gf a
liquid medium solution of increasing density until a step
21 gradient is formed. The plasma is introduced at the top of the
22 rotor displacing some of the h:Lghest density solutlon fxom the
23 bottom. Typiaally, the volume o plasma is from about 15~ to
24 about 40~ that of the step gradient. The centrifuge is
brought up to speed through a programmed speed control
26 system which prevents mixing during the initial reorienta-
27 tion phase. When e~uilibrium is attained and the product
28 is in its proper density position, the rotor is slowed down
29 through the same system to prevent mixlng upon reorientation
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to the original configuration. Then the gradient i.s drained
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1 from below and the propQr density cut collected. A s~milar
2 technique is used i~ the rate zonal banding. The proper density
3 cut from the rate zonal banding is the desired concentrate of
4 hepatitis B antigen.
Due to the small size, approximately 20 nm,of ~B~Ag
6 the isopycnic banding step is quite time consuming, requiring
7 about 18 hours of centrifuging. As a resu:lt, even operating
8 24 hours a day, 7 days a weeX, it is possible to prepare only
9 about 4 batches of clariied plasma per centrifuge. Produc-
tivity can be increased, of course, by utilizing additional
11 centrifu~es. This in~volves a tremendous capital investment,
12 however, as each centrifuge costs about $100,000. ~-
13 It ha9 now been found that substantial increa~es in
14 producti~ity and substantially reduaed operating costs are ob-
tained by multiplo loadin~ o~ the isopycnic bandln~ ~radient.
16 Multiple loading means subjecting a sample of clarified plasma
17 containing HBsAg to isopycnic banding conditions for a time -~
18 sufficient to permit substantially all of the HBSA~ in the
19 clariied plasma to pass into the gradient but insufficient to
achieve e~uilibrium, and repeating this step at least once
21 with an additional sample of clarified plasma containin~ HBsAg,
22 b~ore aontinuing the isopycnia bandin~ conditions for a time
Z3 sufficient to achieve equillbrium. If desired, a gradient may
24 be loaded with up to about 6 samples of clarified plasma. As
the time required for the HBsAg in the clarified plasma to
26 enter the gradient is only a frac`tion of that required to
27 reach eguilibrium, and as the subsequent time required to
28 reach equilibrium is the same whether the gradient is slngle or
29 multiply loaded, substantial savings in time and reductions in
unit processing costs are obtained.
31 While the increased productivity and reduced costs
32 of the multiple banding techni~ue of the present invention
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1 may be achieved with any suit~ble gradient, preferably the
2 gradient is sodium bromide.
3 The isopycnic banding is carried out ~o equili-
4 brium 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,
6 however, that by centrifuging he plasma for about 4 hours sub-
7 stantially all of the HBsAg is caused to move into the isopycnic
8 banding gradient. Then the sample of spent plasma is removed
9 ~ and a fresh sample of plasma equal in volume to the first
sample is layered onto the gradient. Centrifuging may then be
11 continued as previously for about 10 hours or beyond to cause
12 the HBsAg in both samples to move into the equillbrium density
13 region of the gradient ~1.21 to about 1.24 g/cc) to complete
14 the bandin~. Alternatively the centrifuging may be continued
~or 4 hours, the qpent plasma removed and a third sample o
16 fresh plasma layered onto the gradient. This multiple loading
17 procedure may be repeated six or even more times before com~
18 pleting the banding by centrifuging for about 18 hours.
19 The ratio of the charge ~plasma) volume to the grad-
ient volume is from about 1:3 to about 1:6. When a single
21 plasma charge is appli~d to the gradient and centrifu~ed under
22 isopycnic bandihg conditions (2-g- for rom about 16 to about
23 20 hours at 30,000 rpms in the K-II centrifuge) the resulting
24 product generally will have a protein content of approxmately -~
4-10 mg/ml in a volume of 1.0 liter, depending on the amount
26 of protein in the original plasma.
27 When a double charge of plasma is applied to the
28 gradient and centrifuged under isopycnic banding conditions,
29 (for from about 16 to about 20 hours at 30,000 rpms) the re-
sulting product will have a protein content which is additive
31 for the charges employed, typically rom about 8-20 mg/ml in a
32 volume of 1.0 liter, depending on th~ amount of protein in the
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1 original plasma. The level of protein increases in this ,'
2 manner for each subsequent charge of plasm~ applied to the
3 gradient.
4 The product is then subjected to a rate zonal banding.
S The rate zonal banding is carried out unti:L the HBsAg is in the ~'
6 density range of from about 1.13 to about :L.16 g/c-c. Typically
7 this takes for from about 16 hours to about 20 hours, preferably
8 for from about 17 to,about 18 hours, at from about 30,000 x g ~'
3 to about 60,000 x g. , ' ,,
According ~o one aspect of the present invent:ion the
11 gradient is formed o sodium bromide whether or not the
12 multiple loading technique is used. In contrast to heretofore ,
13 used materials sodium bxomide has definite advantages. The
14 i301ubillty of 30dium bromide allows the use of high density
15 solutions in the formation of gradients at refrigerator ~'
16 temperatures t2-6oC). There are definite economic advantages
17 to using ~odium bromide over a salt such as cesium chloride as
18 well as not having to contend with the problem of human ~oxicity ''~,
19 from residual and ~BsAg bound cesium ions. In i30dium bromide
gradient'any ions bound to the HBsAg, due to biophysical
21 characteristics, will be a sodium salt which is very compatible
22 with the human biological system and does not present a toxicity
23 problem. ,,~ ~'
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1 The biophysical characteristics of the HBsAg are
2 well documented (J. Clinical Investigation 52, 1176 (1973),
3 J. of Virology 10, 469 (1972)] as a negatively charged
4 particle. In the presence of a very high concentration of
positively charged sodium ions there is fo~med a sodium-HBsAg
6 salt molecule. This type of molecule is compatible with the
7 human biological system. In contrast to pxior art products,
8 the ~BsAg of the preferred mode of carrying out the present
9 invention is substantially ree of other cations, particularly
added cesium and potassium ions.
11 The superior so}ubility of ~aBr at lowered temper-
12 atures with respect to KBr permits the use of lowered temper-
13 atures more conducive to stability of biological materials.
1~ The use o~ a step gradient rathe~ than a linear gradient is
preferred as it accumulates impurities at the step boundaries
16 and permits processing a larger volume o~ plasma in a single
17 gradient~
18 The antigen of the present invention is useful
19 per se as an antigen for hepatitis B and can be used as des-
cribed in U.S. patent 3,636,191. The HBsAg antigen of the
21 present ~nvention is a highly purified product. ~he isopycnic
22 banding step results in about a 100 fold purification H~sAg
23 relative to normal plasma protein. The rate zonaL step results
24 in about a further 20 fold purification of HBsAg relative to
normal plasma protein. The combination of the two steps
26 result in about a 2000 fold purification of HBsAg relative to
~7 normal plasma protein. The resulting product has been shown
28 to be substantially free of blood group substances A and B as
29 measured by serological and electrophoresis techniques. In
addition, the antigen of the present invention can be used as
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the starting material for the hepatitis B antigen of copending
application Serial No. 251,740, filed 4 May 1976.
The following examples illustrate the present
invention without, however, limiting the same thereto.
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1 EXAMPLE 1
2 The rotor of a centrifuge, Electronucleonics X,
3 is filled with 8,400 ml of phosphate buffer. After run-
ning the rotor up to 10,000 rpm to degas the system, the
foilowing step gradient is pumped into the bottom of the
6 stationary rotor:
7 1. 2,400 ml of 10% ~aBr,~ =1.08
8 2. 1,000 ml of 20~ NaBr,~ =1.17
9 3. 1,500 ml of 30% NaBr,~ =1.28
4. 3,500 ml of 40% NaBr,fO=1.41
11 Plasma containing Australia antigen ~HBsAg),
12 1,750 ml, is pumped into the top of the stationary rotor
13 displacing 1,750 ml of 404 NaBr rom the bottom o the ;
14 rot~r. The rotor is accelerated to 30,000 rpm and run
at this speed or 18 hours. A~ter stopping the rotor
16 500 ml of HBsAg rich material in the 1.21 - 1.24 density
17 region, is collected and dialyzed against phosphate bufer.
18 The rotor i5 then filled with phosphate bufer,
19 degassed as above, and the following step gradient pumped
into the bottom of the stationary rotor:
21 1. 2,400 ml of 5~ sucrose, ~ =1.02
22 2. 1,750 ml of 15% sucrose,~ ~1.06
23 3~ 1,750 ml of 25% sucrose,~ =1.10
~4 4. 2,500 ml of 50% sucrose,~ -1.23
The HBsAg rich material from the NaBr isopycnic
26 banding step, 500 ml, is pumped into the rotor top dis-
27 placing -SOo ml. of 50% sucrose out the rotor bottom. The
28 rotor is then run at 28,000 rpm for 18 hours. After
29 stopping the rotor, 500 ml of HBsAg rich material in the
1.135 - 1.165 density region is collected.
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1 E~AMPLE 2
3 The rotor of a centrifuge, Electronucleonics K,
4 is filled with 8,400 ml of phosphate buffer. After xun~
ning the rotor up to 10,000 rpm to degas the system, the
6 following step gradient is pumped into the bottom of the
-.
7 stationary rotor:
8 1. 2,400 ml of 10~ NaBr, ~=1.08
9 2. 1,000 ml o 20% NaBr,~ -1.17
3. 1,500 ml of 30% NaBr,~ =1.28
11 4. 3,500 ml of 40% NaBr,~ =1.41
12 Plasma containing HBsAg, 1,750 ml, is pumped into
13 the top of the stationary rotor displacing 1,750 ml of
14 40~ NaBr ~rom the bottom of the rotor. The rotor is
accelerated to 30,000 rpm and run at this speed for 4 hours.
1~ The roto~ ig then stopped and 1,750 ml o 40% NaBr are
17 pumped into the bottom of the rotor forcing the plasma
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18 out the top. An additional 1,750 ml of fresh plasma con-
19 taining HBsAg are pumped into the top of the rotor displacing
an equal volume of 40% NaBr out the bottom of the -otor. The
21 xotor is then run at 30,000 rpm for 18 hours. After stopping
22 the rotor 1,000 ml of H~sA~ rich material in the 1.21 -
23 1.24 density region i9 collected and dialyzed against phos-
24 phate bufer.
The rotor i8 then filled with phosphate buferr
26 degassed as abo~e, and the following step gradient pumped
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27 into the bottom of ~he stationary rotor~
28~ 1. 2,400 ml of 5~ sucrose,~ =1.02
29 2. 1,750 ml of 15% sucrose,~ =1.06
3. 1,750 ml of 25% sucrose,~ =1.10
31 4. 2,500 ml of 50% sucrose,~ =1.23
,
32 The ~BsAg rich material from the NaBr isopycnic
.
33 banding step, 1,000 ml, is pumped into the rotor top dis-
34 placing 1,000 ml. o 50% sucrose out the rotor bottom. The -
rotor is then run at 28,000 rpm for 18 hours. After
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1 stopping the rotor, 1,000 ml of H~sAg rich matexial in the
2 1.135 - 1.165 density region is collected.
4 EXAMPLE 3
The rotor of a centrifuge, Elect:ronucleonics K, ~
6 is filled with 8,400 ml of phosphate buffe~r. Afer running ~ -
7 the rotor up to 10,000 rpm to degas the system, the follow-
8 ing step gradient is pumped into the bottom of the station~
9 ary rotor:
1. 2,400 ml of 10% NaBr, ~ =1.08
11 2. 1,000 ml of 20% NaBr, ~ =1.17
12 3. 1,500 ml of 30~ NaBr, ~ =1.28
13 4. 3,500 ml of 40% NaBr, ~ -1.41
14 Plasma containing HBSA~, 1,750 ml, i9 pumped into
the top o~ the stationary rotor di~placin~ 1,750 ml of 40%
16 NaBr ~rom ~he bottom o~ the rotor. The rotor i9
17 accelerated to 30, oao rpm and run at this speed for 4 hours.
18 The rotor is then stopped and 1,750 ml of 40% NaBr are
19 pumped into the bottom of the rotor forcing the plasma
out the top. An additional 1,750 ml of fresh plasma con-
21 taining HBsAg are pumped into the top of the rotor dis-
22 placing an equaL volume of 40% NaBr out the bottom o
23 the rotor. The rotor is accelerated to 30,000 rpm ~`
24 and run at this speed for 4 hours. The rotor is then
stopped and a third charge of 1,750 ml o~ fresh plasma
26 contalning HBsAg are pumped into the top of the rotor dis-
27 placing an equal volume of 40% NaBr out the bottom of the
28 rotor. The rotor is then run at 30,000 rpm for 18 hours.
29 After stopping the rotor, 1,500 ml of H~sAg rich material
in the 1.21 ~ 4 density region is collected and dialyzed
31 against phosphate buffer.
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1 The rotor is then filled with phosphate buffer,
2 degassed as above, and the following step ~radient pumped
3 into the bottom of the stationary rotor~
4 1. 2,40Q ml of 5% sucrose, ~ =1.02
2. 1,750 ml of 15% sucrose,~ =1.06
6 3. 1,750 ml of 25% sucrose,~O =1.10 :~
7 4. 2,500 ml of 50% sucrose, ~ =1.23
8 The HBsAg rich material from the NaBr isopycnic
9 banding step, 1,500 ml, is pumped into the rotor top dis-
placing 1,500 ml of 50% sucrose out the rotor bottom. The
11 rotor is then run at 28,000 rpm for 18 hours. After stop- : .
12 ping the rotor 1,500 ml of HBsAg rich material in the 1.135
13 1.165 density region is collected. ~:
14
EXAMPLE 4
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16 The following table shows the marked increase in
17 yield p~r unit o~ time when using the multiple load:Lng
18 technique o the present invention ~Examples 2 and 3) as
19 compared with single loading (Example 1). : .
. Total isopycnic % InGrease % Increa3e
21 and rate zonal in time~with in yield(with : .
22 Yield (ml) centrifuging respect to respect to : .
23 Example of HBsAg time (hours) Example 1) Example ~
24 1 500 36 - .. ...
.25 21,000 40 11.1 % 100%
26 3 1,500 44 22.2~ 200~ .
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