JPS6313969B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for purifying hepatitis B antigen. This purified hepatitis B antigen can be used as a vaccine. Hepatitis B is a type of viral hepatitis.
The infection is systemic, but the main lesions appear in the liver. The disease primarily affects adults and is maintained primarily by transmission of infection from long-term carriers of the virus. Common routes of transmission include blood transfusions, contaminated needles or syringes, cuts or scratches on the skin, unsterilized dental instruments, saliva, sexual intercourse, and contact with aerosolized infected blood. Hepatitis B has a relatively long incubation period, which can take from 6 weeks to 6 months from infection to clinical onset.
The illness usually begins with fatigue and loss of appetite, and may be accompanied by muscle pain and abdominal discomfort. Later, jaundice, dark urine, light stools, and mild liver swelling may occur. In some cases, the onset is rapid, with fever, nausea,
Jaundice may appear with an increase in white blood cells.
In some cases, jaundice never appears and the patient only feels flu-like symptoms. Most hepatitis infections are thought to result in mild, non-jaundice symptoms. The present invention is effective in obtaining clarified plasma containing HBsAg, in which substantially all of the HBsAg enters the density gradient from the clarified plasma, but ineffective in completing isopycnic banding of HBsAg. subject the clarified plasma depleted of HBs antigen to partial isopycnic banding in a density gradient under conditions and repeat the first step at least one more time with a new sample of clarified plasma. A method for purifying HBs antigen from clarified plasma, characterized in that multiple loadings are carried out, followed by isopycnic banding under conditions effective to reach equilibrium, and then subjected to velocity zone fractionation. It provides: The starting material for the hepatitis B surface antigen (HBs antigen) purified according to the present invention is plasma obtained from a donor with hepatitis B by plasmaphoresis or the like. The antigen titer can be measured by known methods such as radioimmunoassay, passive coagulation reaction, or complement fixation reaction. The plasma is cooled and the resulting cryoprecipitate is removed by mild centrifugation. HBs antigen in cleared plasma is isolated by an isopycnic banding step followed by a rate zonal banding step. In isopycnic banding, a partially purified concentrate is contacted with a liquid medium that creates a density gradient containing the same density as the antigen to be isolated. The liquid medium is then subjected to ultracentrifugation to achieve an equilibrium distribution of the serum components in the density gradient according to their respective densities. Separate successive fractions of the liquid medium and select the fraction containing the desired antigen, i.e., with a density of approximately
A fraction of 1.21 to about 1.24 g/cc is collected. Application of this method to the purification of HBs antigens has been published in German Patent Specification No. 2049515 and US Pat.
It is stated in No. 3636191. The concentration of the solution forming the density gradient is selected so that the density ranges from about 1.0 to about 1.41 g/cc. The liquid medium may be a linear gradient or a stepped gradient. More preferably, it is used in the form of a stepwise gradient due to its inherent higher capacity for fractionation. In the velocity zone banding method, a partially purified concentrate is brought into contact with a liquid medium with a density gradient and subjected to ultracentrifugation, but this time we will use the velocity zone method. That is, it is carried out at a rate and time such that equilibrium is not reached, so that the HBs antigen and other serum components are distributed in the medium according to the sedimentation constant in the medium.
The concentration of the solution forming the gradient has a density range of approximately 1.0 to approximately
Select to cover 1.28g/cc. The speed zone method is
Repeat until HBsAg reaches a density range of 1.13 to 1.16. At this point, the HBsAg is separated from most of the crude plasma proteins and, most importantly, from the macroglobulin complement in the plasma. When the velocity zone method step is carried out until the desired HBsAg reaches a position of equilibrium, i.e., a density of about 1.18 to 1.20 g/cc, the macroglobulin fraction in the plasma becomes the desired HBsAg fraction. It was found that it was mixed in as an impurity. The liquid medium used in the isopycnic banding and velocity zoning steps may be any suitable range of density gradients. In the prior art, solutes in such solutions included sucrose, potassium bromide, cesium chloride, potassium tartrate, and the like. To easily carry out the isopycnic banding process, use a centrifugal separator such as Electronucleonics K.
A salt solution (saline) is placed in the rotor when it is not moving.
solution) and then aliquots
A stepwise gradient is formed by pushing up the salt solution with liquid medium solutions of increasing density. Plasma is introduced from the top of the rotor by removing a portion of the densest solution from the bottom. Typically, the volume of plasma is 15% to 40% of the volume of the step gradient. The centrifuge is sped up by a speed control system programmed to prevent mixing between the first reorientation faces. When equilibrium is reached and the product is at the proper density, the rotor is slowed down by the same speed control system so that it does not mix up during reorientation back to the original position. Next, cut down the gradient from the bottom and collect the appropriate cut density range. A similar method is used in the velocity zone method. Fractions of the appropriate density range by velocity zone banding are concentrated solutions of the desired hepatitis B antigen. Since the size of HBs antigen is small at 20 nm, the isopycnic banding process is time-consuming, requiring 18 hours of centrifugation. As a result, even if the centrifuge is operated 24 hours a day, seven days a week, only four batches of clarified plasma can be processed per centrifuge. Increasing the number of centrifuges would increase productivity, but each centrifuge costs about $100,000, so it would require a huge capital investment. The gradient for isopycnic banding is loaded multiple times.
It was found that productivity was greatly improved and operating costs were significantly reduced by loading the system. Multiple loading is a method of loading clarified plasma samples containing HBsAg under conditions of isopycnic banding, which is effective in getting substantially all of the HBsAg into the gradient, but is difficult to reach equilibrium. was treated for an ineffective period of time, and the process was repeated at least one more time with another sample of clarified plasma containing HBsAg, followed by a period of time sufficient to allow the isopycnic banding process to reach equilibrium. Just do it. If desired, the same gradient can be loaded up to six times with cleared plasma. Since the time required for HBsAg in clarified plasma to enter the gradient is a small fraction of the time required to reach equilibrium, and also the time required to reach equilibrium thereafter, the gradient is run only once. Since the load is the same whether it is loaded or loaded multiple times, time can be significantly reduced and unit processing costs can be reduced. The increased productivity and reduced cost of the multiple banding method of the present invention can be achieved using any suitable gradient, but preferably the gradient is based on sodium bromide. The isopycnic banding method is performed by centrifuging at about 40,000 g to about 80,000 g for about 10 hours or more until equilibrium is reached. However, it has been found that when plasma is centrifuged for about 4 hours, almost all of the HBs antigen enters the isopycnic banding gradient. The sample of plasma used is then removed and the same volume of plasma preparation used initially is layered on top of the gradient. After that, centrifugation is performed for about 10 hours or more under the same conditions as before, so that the HBs antigen in both preparations can enter the equilibrium density region (about 1.21 to about 1.24 g/cc) in the gradient and complete band formation. . Alternatively, centrifugation can be performed for 4 hours, the used plasma removed and a third fresh plasma layered on top of the gradient. After performing this multiple loading method 6 or more times, approximately 18
Banding may be completed after centrifugation for an hour. The ratio of the volume of charged plasma to the volume of the gradient is approximately
1.3 to about 1.6. Plasma is charged once onto the gradient and centrifuged under isopycnic banding conditions (e.g., 30,000 revolutions per minute in a K-centrifuge;
Depending on the amount of protein in the initial plasma, the product typically has a protein content of about 4-10 mg/ml in a 1.0 volume. Plasma was charged twice onto the gradient and centrifuged under isopycnic banding conditions (approximately 30,000 revolutions per minute).
Depending on the amount of protein in the initial plasma, the amount of protein in the product is additive with the number of charges performed, typically about 8-20 mg/ml in a volume of 1.0 becomes. The amount of protein thus increases with each charge of plasma into the gradient. The product is then subjected to a velocity zone banding process.
Velocity zone banding is approximately 1.13 to 1.16 for HBsAg.
Continue until you reach the g/cc density region. Typically this will take about 16 hours to about 20 hours, more preferably about 17 hours at about 30,000 g to about 60,000 g, and about
It is 18 hours. In one embodiment of the invention, the gradient can be created using sodium bromide. Sodium bromide has distinct advantages over materials used to date. The solubility of sodium bromide is suitable for the use of dense solutions in creating gradients at refrigeration temperatures (2-6°C). Compared to the case of using salts such as cesium chloride, not only is there no need to solve the problem of toxicity to the human body due to residual or bound cesium ions to the HBs antigen, but it is clearly economically advantageous. . In the sodium bromide gradient, due to product physics, all ions that bind to the HBs antigen are sodium ions, which are well tolerated by the human biological system and do not pose toxicity problems. do not have. There are already sufficient reports on the biophysical properties of HBs antigen [Clinical Research Journal (J. Clinical
Investigation), Vol. 52, p. 1176 (1973), J. of Virology, Vol. 10, p. 469 (1972)], and are negatively charged particles. When there are high concentrations of positively charged sodium ions,
A sodium-HBs antigen salt molecule is formed.
HBs obtained by the preferred method of carrying out the invention compared to products obtained by previous techniques
The antigen is substantially free of other cations, especially exogenously added cesium and potassium ions. The greater solubility of sodium bromide at low temperatures compared to potassium bromide allows the use of conducive low temperatures for the stability of biological materials. The use of stepwise gradients is preferable to linear gradients because impurities collect at the step interfaces and a larger amount of plasma can be processed with one gradient. The antigens related to the present invention can be used as such as hepatitis B antigens and are disclosed in US Pat.
Can be used as described in No. 3636191. The HBs antigen according to the present invention is highly purified. By isodensity banding, HBs antigen is purified approximately 100 times compared to normal plasma protein. In the velocity zone process, HBsAg is further detected with respect to normal plasma proteins.
Purified 20 times. By combining the two steps, HBs antigen is purified 2000 times over normal plasma proteins. The resulting product was shown to be substantially free of blood group substances A and B by serological and electrophoretic methods. Furthermore, the antigen of the present invention can be used as a starting material for the hepatitis B antigen of JP-A-51-139619. The present invention will be explained more specifically by the following examples, but these are not intended to limit the scope of the present invention. Example 1 (Reference Example) The rotor of a centrifuge, Electronucleonics K, is filled with 8400 ml of phosphoric acid buffer. After rotating the rotor to 10,000 revolutions per minute for degassing, the following stepwise gradient is injected into the bottom of the stopping rotor. 1 10% Sodium Bromide ρ = 1.08 2400ml 2 20% Sodium Bromide ρ = 1.17 1000ml 3 30% Sodium Bromide ρ = 1.28 1500ml 4 40% Sodium Bromide ρ = 1.41 3500ml Plasma containing Australian antigen (HBs antigen)
Add 1750 ml to the top of the stationary rotor while removing 1750 ml of 40% sodium bromide from the bottom of the rotor. Accelerate the rotor to 30,000 revolutions per minute,
Drive at this speed for 18 hours. After stopping the rotor, 500 ml of the HBs antigen-rich portion in the density region of 1.21-1.24 is collected and dialyzed against phosphate buffer. The rotor is then filled with phosphate buffer, degassed as described above, and the stepwise gradient described below is injected into the bottom of the stationary rotor. 1 5% sucrose ρ = 1.02 2400ml 2 15% sucrose ρ = 1.06 1750ml 3 25% sucrose ρ = 1.10 1750ml 4 50% sucrose ρ = 1.23 2500ml Obtained by banding process in sodium bromide
Add 500 ml of the HBs antigen enriched fraction to the top of the rotor while removing 500 ml of 50% sucrose from the bottom of the rotor. The rotor then rotates at 28,000 revolutions per minute to 18
drive for hours. After stopping the rotor, 1.135 ~
Collect 500 ml of the HBs antigen-enriched fraction in the 1.165 density region. Example 2 The rotor of a centrifuge, Electronucleonics K, is filled with 8400 ml of phosphate buffer. To degas, rotate the rotor at up to 10,000 revolutions per minute and then inject the stepwise gradient described below into the bottom of the stationary rotor. 1 10% Sodium Bromide ρ = 1.08 2400ml 2 20% Sodium Bromide ρ = 1.17 1000ml 3 30% Sodium Bromide ρ = 1.28 1500ml 4 40% Sodium Bromide ρ = 1.41 3500ml 1750ml of plasma containing HBs antigen was added to 40% Add 1750 ml of sodium bromide to the top of the stationary rotor while removing it from the bottom of the rotor. rotor per minute
Accelerate to 30,000 rpm and operate at this speed for 4 hours.
Then stop the rotor and add 40% sodium bromide 1750
Push the plasma out from the top by injecting ml into the bottom of the rotor. Add another 1750ml of fresh plasma containing HBsAg to 40% of the same volume from the bottom of the rotor.
Remove the sodium bromide and add to the top of the rotor. The rotor then rotates at 30,000 revolutions per minute for 18 hours. After stopping the rotor, 1000 ml of the HBs antigen-enriched fraction in the density region of 1.21-1.24 is collected and dialyzed against phosphate buffer. The rotor is filled with phosphate buffer, degassed as described above, and the stepwise gradient described below is injected into the bottom of the stationary rotor. 1 5% sucrose ρ = 1.02 2400ml 2 15% sucrose ρ = 1.06 1750ml 3 25% sucrose ρ = 1.10 1750ml 4 50% sucrose ρ = 1.23 2500ml HBs antigen obtained by isopycnic banding process in sodium bromide 1000ml of enriched fraction, 50% sucrose
Pour 1000ml into the top of the rotor, removing it from the bottom. Then the rotor at 28000 rpm
Runs for 18 hours on rotation. After stopping the rotor,
HBs antigen-enriched fraction in the 1.135-1.165 density region
Collect 1000ml. Example 3 The rotor of a centrifuge, Electronucleonics K, is filled with 8400 ml of phosphate buffer. For degassing, the rotor is rotated at a maximum of 10,000 revolutions per minute, and then the following graded gradient is injected into the bottom of the stationary rotor. 1 10% Sodium Bromide ρ = 1.08 2400ml 2 20% Sodium Bromide ρ = 1.17 1000ml 3 30% Sodium Bromide ρ = 1.28 1500ml 4 40% Sodium Bromide ρ = 1.41 3500ml 1750ml of plasma containing HBs antigen was added to 40% Inject 1750 ml of sodium bromide into the top of the stationary rotor while removing it from the bottom of the rotor. The rotor is accelerated to 30,000 revolutions per minute and operated at this speed for 4 hours. Stop the rotor and 1750ml of 40% sodium bromide.
The plasma is forced out of the top of the rotor by injecting it into the bottom of the rotor. Furthermore, 1750ml
Fresh plasma containing HBsAg is added to the top of the rotor while removing the same amount of 40% sodium bromide from the bottom of the rotor. The rotor is accelerated to 30,000 revolutions per minute and operated at this speed for 4 hours. Stop the rotor and use new plasma containing HBs antigen for the third time.
Inject 1750 ml into the top of the rotor while removing the same amount of 40% sodium bromide from the bottom of the rotor. The rotor rotates at 30,000 revolutions per minute for 18 hours. After stopping the rotor, 1500 ml of the HBs antigen-enriched fraction in the density region of 1.21-1.24 is collected and dialyzed against phosphate buffer. The rotor is then filled with phosphate buffer, degassed as described above, and the stepwise gradient described below is injected into the bottom of the stationary rotor. 1 5% sucrose ρ = 1.02 2400ml 2 15% sucrose ρ = 1.06 1750ml 3 25% sucrose ρ = 1.10 1750ml 4 50% sucrose ρ = 1.23 2500ml 1500ml of the HBs antigen-rich fraction obtained in the isopycnic banding step, Remove 1500ml of 50% sucrose from the bottom of the rotor and inject it into the top of the rotor. The rotor is then operated at 28,000 revolutions per minute for 18 hours. After stopping the rotor, collect 1500 ml of the HBs antigen-enriched fraction in the density region of 1.135-1.165. Example 4 The table below shows that when using the multiple loading method according to the present invention (Examples 2 and 3), the yield per unit time was increased compared to when only one loading was used (Example 1). It shows that it gets better. 【table】

Claims (1)

[Claims] 1. Cleared plasma containing HBs antigen is effective for substantially all of the HBs antigen to enter the density gradient from the cleared plasma, but it is difficult to complete isopycnic banding of HBs antigen. is subjected to partial isopycnic banding in a density gradient under ineffective conditions to remove cleared plasma depleted of HBsAg and repeat the first step with a fresh sample of cleared plasma to at least HBs antigen from clarified plasma characterized by multiple loading consisting of one repetition, followed by isopycnic banding under conditions effective to reach equilibrium, and then subjected to velocity zone fractionation. How to refine. 2. The method of claim 1, wherein the density gradient comprises sodium bromide.