CA1088426A - Purification of hepatitis b antigen - Google Patents

Abstract

A B S T R A C T

There is disclosed an improved process for concentrating HBsAg from clarified plasma of human hepatitis B donors which comprises subjecting the clarified plasma to isopycnic banding in sodium bromide density gradient and recovering a fraction rich in HBsAg.

Description

11~84Z6 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.

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 .,,: .

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 4 ~
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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

- 5 -1~84~6 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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: ' : , ~ 15605 ,, ~Q8~ Z6 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 - 7 - ~
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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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~05 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 - .:
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 . .
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 , _ 10 , 1560~
884;26 1 stopping the rotor, 1,000 ml of H~sAg rich matexial in the 2 1.135 - 1.165 density region is collected.

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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1~8~4Z6 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. ~:

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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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Claims (4)

WHAT IS CLAIMED IS:

1. In a process for concentrating HBsAg from clarified plasma of human hepatitis B donors, the improvement comprising subjecting the clarified plasma to isopycnic banding in sodium bromide density gradient and recovering a fraction rich in HBsAg.

2. A process according to claim 1 wherein the density gradient is a step gradient.

3. In a process for purifying HBsAg from a plasma that has been subjected to an isopyenic banding in sodium bromide gradient, the improvement comprising subjecting the isopycnic banding fraction having a density region of from about 1.21 g/cc to about 1.24 g/cc to a rate zonal fractionation until the HBsAg is found in the density region of from about 1.13 g/cc to about 1.16 g/cc.

4. In a process according to claim 1 the improvement comprising subjecting the isopycnic banding product containing ABsAg to a rate zonal fractionation until the HBsAg is found in the density region of from about 1.13 g/cc to about 1.16 g/cc.