CA1100038A - 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

llo~o~ 15605A

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 ~ ~ ~ 15605A

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 11 0 ~ ~ 15605A

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 i l~ ~ ~ 15605A

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 1 ~O ~ ~ 15605A

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 1 ~O ~ ~ 15605A

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 llOQ~ 15605A

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.

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 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.

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 1 1O ~ ~ 15605A

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.

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 liO~ 15605A

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.

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) -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.

Claims (6)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for multiple loading a density gradient comprising subjecting clarified plasma contain-ing HBsAg to partial isopycnic banding in a density gradient under conditions effective to pass substantially all of the HBsAg from the clarified plasma into the density gradient but ineffective to complete the iso-pycnic banding of the HBsAg, removing the spent clarified plasma and repeating the first step with a fresh sample of clarified plasma at least once.

2. The process of Claim 1, wherein the density gradient is NaBr.

3. A process according to claim 1 wherein a fraction of the density gradient rich in HBsAg is recovered.

4. A process according to claim 4 wherein a fraction of the density gradient rich in HBsAg is recovered.

5. A process for isopycnic banding HBsAg from clarified plasma which comprises multiple loading a density gradient according to Claim 1 and then continuing the isopycnic banding under conditions effective to reach equilibrium.

6. The process of Claim 5 wherein the density gradient is NaBr.