US3790552A

US3790552A - Method of removing hepatitis-associated antigen from a protein fraction using polyethylene glycol

Info

Publication number: US3790552A
Application number: US00235211A
Authority: US
Inventors: A Johnson; J Newman
Original assignee: US Department of Health and Human Services
Current assignee: US Department of Health and Human Services
Priority date: 1972-03-16
Filing date: 1972-03-16
Publication date: 1974-02-05
Anticipated expiration: 1991-02-05
Also published as: DK133292B; GB1375811A; DK133292C; CA1061251A; DE2306107A1; AT321465B; BE796770A; FR2175969B1; FR2175969A1

Abstract

A METHOD OF REMOVING HEPATITIS-ASSOCIATED ANTIGEN FROM A PROTEIN FRACTION BY REGULATING PROTEIN CONCENTRATION, IONIC STRENGTH OF THE SOLUTION, AND PH, AND FRACTIONATING THE MIXTURE BY THE USE OF POLYETHYLENE GLYCOL IN A PARTICULAR CONCENTRATION RANGE IS DISCLOSED. THE PH OF THE SOLUTION SHOULD BE REMOVED FROM THE ISOELECTRIC POINT OF THE MATERIAL BEING PRECIPITATED.

Description

United States Patent 3,790,552 METHOD OF REMOVlNG HEPATITIS-ASSOCI- ATED ANTIGEN FROM A PROTEIN FRACTION USING POLYETHYLENE GLYCOL Alan J. Johnson, New York, and Jack Newman, Pelham, N .Y., assiguors to the United States of America as represented by the Secretary, Department of Health, Education, and Welfare No Drawing. Filed Mar. 16, 1972, Ser. No. 235,211 Int. Cl. A23j 1/06; C07g 7/00 US. Cl. 260-112 B 28 Claims ABSTRACT OF THE DISCLOSURE A method of removing hepatitis-associated antigen from a protein fraction by regulating protein concentration, ionic strength of the solution, and pH, and fractionating the mixture by the use of polyethylene glycol in a particular concentration range is disclosed. The pH of the solution should be removed from the isoelectric point of the material being precipitated.

BACKGROUND OF THE INVENTION This invention relates to the separation of blood proteins, and more particularly this invention relates to the removal of hepatitis-associated antigen from other protein fractions.

Hepatitis is a relatively common disease, but the disease is sometimes diflicult to diagnose and there is, as yet, no specific treatment. Even though as many as five percent of the reported cases become chronically ill and another two percent become cirrhotic, it has been estimated that three out of every thousand persons who become infected with the serum hepatitis virus do not become ill, but nevertheless carry the disease. For this reason hepatitis poses a serious problem in detecting blood donors who may transmit the disease.

The known methods for detecting serum hepatitis rely on detection of virus products in the body fluids but are time-consuming and burdensome as well as being rela tively insensitive. In fact, it has been estimated that most of the prior art tests can detect only twenty to thirty percent of the carriers of the disease. Moreover, prior tests have not been sensitive enough to detect antibodies that develop after a single exposure to hepatitis virus or primary hepatitis infection.

A specific antigen, popularly known as Australia antigen has been found in the serum of many patients with serum hepatitis. The exact pathogenic role of the Australia antigen is a current problem of great academic interest as well as urgency in view of the importance of recognizing the source and/or cause of hepatitis. The Australia antigen is also known as hepatitis-associated antigen (HAA), which terminology will be used herein. Most of the common methods used for the detection of hepatitis rely on the presence of the HAA, or the antibody thereto, for their specificity.

At present, the neutralizing effect of immune serum, that is, the serum of an individual who has recovered from hepatitis and contains antibodies specific against the disease, is used to diagnose hepatitis by various tests. In these tests HAA antibody is added to the same, forming a precipitate or complex with the antigen present in the sam ple.

Existing techniques for detecting and diagnosing hepatitis include: irnmunodiffusion, complement fixation, electrophoretic modifications of precipitin techniques, radioimmunoassays, and hemagglutination procedures. Diffusion methods are the simplest but the least sensitive and slowest. The electrophoretic modifications of precipitin techniques are rapid, as rapid as complement fixation, but

3,790,552 Patented Feb. 5, 1974 not quite as sensitive. Of the available techniques, complement fixation and hemagglutination procedures appear to be the most rapid and are also moderately sensitive. The complement fixation technique involves adding a biologic material such as blood or plasma to the appropriate antibody for HAA. The reaction mixture is incubated with a predetermined amount of complement to fix the latter. The amount of HAA or antibody in the body fluid can be determined through titrating the amount of remaining non-fixed complement by incubation with a standard cell solution. The complement fixation technique is only moderately sensitive for detecting antigen and antibody, gives anti-complementary reactions with plasma fractions, and is not routinely available in many hospitals and blood banks. The various radioimmunoassays used are the most sensitive tests available at present but usually require at least one day to run, and the reproducibility is only fair. The hemagglutination test with antibody-coated red cells appears to combine both sensitivity and rapidity.

Epidemiological surveys following transfusion therapy reveal a very high incidence of hepatitis, in the order of 0.l2l2 percent with pooled plasma or whole blood. The incidence of hepatitis with fibrinogen transfusion has been variously estimated at nine percent to more than thirtysix percent, whereas one investigator believed the incidence was thirty-five times as high as with transfusion of blood or plasma fractions. The fibrinogen is considered to be contaminated because it is isolated from very large plasma pools (5000 or more units, some of which contain HAA) but probably contains only about one percent of the hepatitis-associated antigen present in the original plasma. The tendency to transmit hepatitis has severely limited the usefulness of fibrinogen or fibrinogen-containing fractions, e.g., when Fraction 1-0 containing sixty to eighty percent fibrinogen was used as a source of AHF in this country, it caused hepatitis in severty-five percent of hemophiliacs not previously given massive transfusions of fresh-frozen plasma and in fifty percent of those who were previously transfused. It is estimated that more than ten percent of patients who develop hepatitis die from its effects.

Prior screening of all blood donors by present assays will probably reduce the incidence by only twenty to thirty percent since the available assays for the antigen associated with serum hepatitis (HAA) are relatively insensitive and plasma with barely detectable HAA content by in vitro assay usually produces overt disease. Furthermore, injection of a 1,000,000 dilution of such plasma may cause demonstrable viremia without icterus. Since professional blood donors, who have a ten times higher incidence of hepatitis than volunteer blood donors, furnish almost half the countrys blood supply, it would be very difiicult to exclude them as donors and indeed would be unnecessary if the HAA could be removed during blood collection. Patients receiving transfusions could be given ordinary gamma globulin but it has proved to be helpful only against infections hepatitis; or they could be given high-titer immune gamma globulin from recovered hepatitis patients, but the supply is exceedingly limited and its usefulness in preventing clinical disease in patients infused with large amounts of HAA contaminated plasma fractions has not yet been proved conclusively.

The HAA or serum hepatitis (SH) antigen probably participates in the pathogenesis of serum hepatitis, and is viewed primarily as a marker in plasma for the disease. The HAA particles found in infected blood are predominantly of two morphologic types: those about 22 my. in diameter which seem to represent incomplete virions (empty capsids), and particles about 42 me in diameter which may represent the entire virus.

For many years the risk of hepatitis has prevented the widespread use of fractions such as fibrinogen and slowed the commercial production of a concentrate of Factors II, VII, IX and X as well as Factor VIII (antihemophilic factor-AI-I F), from large pools of human blood. Because the Cohn procedure has been widely used for many years and little or no serum hepatitis develops after treatment with Cohn Fraction II and albumin, the method has been considered more or less sacred or definitive."

These considerations have mitigated against the commercial use of new fractionation methods such as the zinc method XIII and DEAE adsorption in the purification of gamma globulin, and polyethylene glycol (PEG) in the purification of serum albumin although these methods would undoubtedly result in products of higher purity and yield, lower aggregation potential and greater stability. Even new anticoagulants such as citrate phosphatedextro'se (CPD) cannot be used without prior extensive clinical trials of each plasma fraction derived from blood collected with CPD and fractionated by the Cohn proce dure, to ascertain its potential for transmitting hepatitis a long, tedious, expensive and time-consuming process. Thus, in addition to demonstrating that new fractionation techniques or use of CPD blood result in plasma fractions of higher purity and yield than the Cohn fractions, it is necessary to show that they are no more likely to cause hepatitis.

Some therapeutically valuable plasma fractions are often contaminated with HAA. In the hands of most investigators, commercially prepared concentrates of Fractors II, VII, IX and X have been associated with a high incidence of hepatitis, at least seventeen percent, while one study reports forty to seventy percent. This material is therefore reserved primarily for the treatment of a small group of bleeding patients with Factor IX deficiency and a history of multiple transfusions. Many other bleeding patients defincient in one or more of the four factors but with no history of transfusions would probably benefit greatly from therapy with plasma, plasma fractions, and whole blood free of HAA. This group includes patients with hepatitis, cirrhosis, liver poisoning or other parenchyrnal liver damage, or with dicumarol overdosage or biliary obstruction.

Possible HAA contamination of AHF concentrates and fibrinogen also seems to be a pressing problem. Recent emphasis on HAA by the Division of Biologics Standards (DBS) has pointed up the need for screening donors before the plasma is fractionated as well as three months after blood collection to make sure that they are still free of the antigen. It is very difficult, however, to predict whether donors will develop HAA-positive blood six weeks to three months after giving blood. According to recent estimates, a fairly high percentage do so. It is also diflicult to rule out circulating antigen-antibody complexes that give negative results with present HAA assays and may subsequently prove to be infective.

The high morbidity and mortality attending post-transfusion hepatitis can probably be reduced to some extent by modifying present plasma fractionation methods. About 50,000 units of unselected plasma were fractionated from 1966 to 1969 to produce American National Red Cross (ANRC) intermediate-purity AHF which was used with other materials to treat 153 episodes in 96 patients with hemophilia .A or von Willebrands disease. Although some of them received very large amounts, only two developed hepatitis and they had also received large amounts of other Factor VIII concentrates and plasma. Similarly, about 50,000 units of unselected plasma were processed for the ANRC high-purity AHF which was used with other Factor VIII preparations to treat 92 episodes in 57 patients. One of these developed hepatitis and he, too, had received large amounts of other AHF-containing materials. Since other patients given intermediateor high-purity AHF from the same lots did not develop the disease, the level of any contaminating HAA present in the preparations seems to have been very low indeed.

In order to prevent transmission of hepatitis by blood donors, most transfusion centers are setting up one of the new assay methods for detecting HAA in the donors blood. If it is found, the HAA-positive plasma is usually sterilized and discarded. As mentioned above, these methods will detect the HAA in only 20 to 30 percent of the contaminated plasma units.

Polyethylene glycol in the fractionation and concentration of proteins and viruses: Albertson used polythylene glycol (PEG) to fractionate and concentrate cells, viruses, microsomes, proteins, nucleic acids, and antigen-antibody complexes in two-phase systems with dextrans or ammonium sulfate and water. This synthetic, non-reactive polymer has also been used as a vehicle for intramuscular and intravenous administration of various hormones. Polson et a1. fractionated albumin, gamma globulin and fibrinogen with PEG of 6000 molecular weight, and a procedure for the preparation of high-purity AHF with PEG of 4000 and 6000 M.W. is described and claimed by Johnson et al. in co-pending US. patent application Ser. No. 663,507, filed Aug. 28, 1967, now 'U.S. Pat. No. 3,652,530. In general, PEGs mode of action is believed to be based on removal of water from the hydrophilic region of proteins; as a result, their hydrophobic bonds presumably interact causing aggregation and a decrease in solubility.

Extensive acute and chronic toxicity studies at the Mel- Ion Institute from 1947 to 1970, principally with PEG- 4000, further attested the nontoxic nature of the polymer. In animals, intravenous doses as high as 16 gm./kg. were given to four species without evidence of toxicity. In 52 hemophiliacs and Von Willebrand patients, high-puriyt AHF precipitated with PEG was administered during 92 treatment episodes with no toxic effects. PEG is rapidly excreted by the kidneys, with a glomerular clearance rate virtually the same as that of inulin and creatinine.

PEG has also been used by virologists to concentrate and purify a number of plant viruses, bacteriophages, and some animal organisms:

(a) Hebert precipitated partially purified soil-borne wheat mosaic virus (WMV) from a growth medium with three percent PEG-6000 and centrifugation for ten minutes at 10,000 r.p.m. In other studies, he precipitated rodshaped WMV particles in 0.1 M phosphate buffer at pH 7.5 with two percent PEG. Tobacco mosaic virus (TMV) was also precipitated from extracted, infected tobacco leaves by four percent PEG in 0.1 M NaCl and by two percent PEG in 0.3 M NaCl. In contrast, eight percent PEG was required to precipitate two plant viruses with spherical particles: tobacco ringspot virus (precipitated by treating the juice with PEG in 0.3 M NaCl) and the bean pod mottled virus (precipitated in 0.2 M NaCl).

(b) When purifying African horse-sickness virus for electronmicroscopy, Polson and Becks precipitated the virus from emulsified mouse brain tissue using three to four percent PEG-6000 and 0.066 M phosphate bufier at pH 7.0.

(c) Leberman described precipitation of four highly purified viruses from solutions: turnip crinkle virus (TCV), turnip yellow mosaic virus (TYMV), a nitrous acid mutant of tobacco mosaic virus (TMV), and bacteriophage T-4 with PEG-6000 at varying pH and NaCl concentrations. Similar studies were later carried out with only partially purified virus solutions. Precipitation with PEG required a higher N aCl concentration as the pH was raised.

(d) In earlier studies, viruses with a nucleic acid core and a protein coat were precipitated with PEG. However, McSharry and Benzinger applied the method to a virus with a lipiprotein coat: vesicular stomatitis virus. The

virus was effectively precipitated with a six to eight percent concentration of PEG-6000 and 0.5 M NaCl; these researchers concluded that PEG precipitation concentrates and purifies representatives of all major virus classes except the pox-virus and herpesvirus groups (not yet tested) without loss of infectivity.

(e) In experiments by Yamomoto et al., seven bacteriophages were readily concentrated from crude lysates of infected bacteria by two to ten percent PEG-6000 and 0.5 M NaCl. The efiicacy of the method was relatively unaffected by changes in pH ionic strength. They also showed that the asymmetric particles of TMV and bacteriophage fd were especially susceptible to PEG and could easily be purified from the more symmetric phage particles at low PEG concentrations. In addition, they found that a relatively constant percentage of phage was precipitated by a fixed concentration of PEG over a phage concentration range of nearly and concluded that the exact mechanism of precipitation is unknown but a phase partition rather than a normal precipitation reaction seems to be involved.

(f) Juckes precipitated proteins with PEG-6000 in an effort to determine the mechanism (5) involved. He varied the pH, ionic strength, protein concentration, and temperature, and concluded that each variable had an eifect on precipitability of the proteins, studied: carboxyhemoglobin, ovalbumin and bovine serum albumin, as well as bromegrass mosaic virus. He concluded that precipitability was related primarily to the molecular weight of the protein and the pH, as measured by difierences in the Stokes radius of the proteins, but could not relate the other effects to changes in the Stokes radius. These principles did not obtain when viruses and other proteins in low concentration and at low ionic strength were precipitated.

(g) Pert reported that antibody-antigen reactions can be enhanced with protein-precipitating agents such as PEG. In one phase of these experiments, he added eight percent PEG to eliminate fibrinogen, alpha macroglobulin, cryoglobulins and some other large-molecular-weight proteins, then used twelve percent PEG to precipitate and concentrate most of the HAA together with many other moderate-sized proteins such as gamma globulin, alpha 1 and alpha 2 globulins, beta globulins, and their associated lipiproteins.

SUMMARY OF THE INVENTION Since the heptatitis-associated antigen (HAA) has been associated with, and may be involved in the pathogenesis of, serum heptatitis, absence of the antigen has been interpreted as indicating relative freedom from the infectious agent causing serum hepatitis. The primary object, therefore, is to provide a method to eliminate the antigen from ful plasma fractions-albumin, gamma globulins, coagulation Factors II, V, VII, IX, X, XI, XII and XHI, fibrinoful plasma fractions-albumin, gamma globulins, coagulation Factors II, V, VII, IX, X, XI, XII and X11, fibrinogen, antihemophilic factor (Factor VIII), plasminogen, ceruloplasmin, transferrin, thyroxin-biding protein, antithrombin HI, cal antitrypsin, a2 macroglybulin. Ci inactivator, inter-u trypsin inhibitor, as well as sewage-by fractionation with polyethylene glycol (PEG).

It is another primary object of the prsent invention to provide a method for removing the contaminating HAA from plasma fractions that have been partially purified.

It is still another object of the present invention to provide a superior fractionation method using an agent which does not denature or combine with plasma proteins, but selectively concentrates the HAA from albumin, gamma globulin, and fibrinogen, as well as concentrates of AHF and Factors II, VII, DC, and X. Variations of this method are used to remove the antigen from albumin, gamma globulin, fibrinogen, a concentrate of Factors H, VH, and IX, and a concentrate of Factors II, VH, IX and Xall prepared for therapeutic purposes.

In accordance with the foregoing objects, a method is provided which comprises the essential steps of: (1) maintaining the solubility of the HAA-containing protein fraction at a pH away from its isoelectric point, (2) adding polyethylene glycol with a molecular weight ranging from about 200 to 6000 to a concentration of from about 12 to about 30 percent to thereby precipitate the HAA, and (3) separating the HAA from the protein.

The major factors that are varied to remove the HAA from the various fractions are the final PEG concentration of the mixture, the pH, the ionic strength, and the protein concentration. The PEG concentration is varied from about 12-30 gm./ ml. of solution, depending on the molecular weight of the polymer. The pH is adjusted so that it is removed as far as possible from the isoelectric point of the fraction remaining in solution, but without denaturing the protein and still within the precipitability range for the HAA. The pH is generally separated by 1.0 or 2.0 pH units from the isoelectric point, which is well known for most of the protein fractions and in any event can be easily determined by one of ordinary skill in the art, using known methods. As examples, the isoelectric point of fibrinogen is about 5.5, of gamma globulin from 6.5 to 7.5, nominally 7.2, of albumin about 4.9, of Factor 1X about 4.3, and Factor 11 about 4.7.

The solutions are made with any suitable buffer or the like, provided that the small amounts which may be carried along in the fractionation procedure are physiologically tolerable 0n I.V. or LP. injection. Typical suitable materials for these solutions are a glycine-citrate buifer, a phosphate buifer, a saline solution, a tris-citrate buffer with or without other additives such as urea, alone or in various combinations. The ionic strength may vary from 0.20 to 0.001.

After the HAA is precipitated, it is removed from the supernatant by any suitable procedure such as centrifugation or filtration or both. The filter should have pores of less than 0.6 mg, preferably between about 0.45 and 0.2 mil. Suitable filters are commercially supplied by Millipore or Cox.

Temperature is not a factor in the method of the present invention, although for practical purposes it may be conveniently practiced at room temperature or from about 15 to 25 C. At lower temperatures, lower concentrations of PEG are used.

While the method of the present invention is preferably practiced by selectively precipitating the HAA to re move it from a solution of the same with another protein fraction, it should be understood that all the proteins could be precipitated by PEG and the fraction to be separated from the HAA then solubilized with a suitable buffer at a proper pH.

The foregoing objects of the present invention and other objects will in part be obvious and in part be pointed out as the description of the invention proceeds.

DESCRIPTION OF THE PREFERRED EMBODIMENT Concentration of HAA from fractions for assay One milliliter of serum or partially purified HAA (isolated and washed by centrifugation) with an HAA concentration barely detectable by counter-immunoelectrophoretic assay, was diluted up to 10,000 times with fibrinogen, albumin, or gamma globulin, prepared by Cohn methods 6 and 9, or purified concentrates of Factors 11, VII, IX and X or Factor VIII. The added HAA was precipitated from the plasma fraction by PEG, reconstituted to its original volume in buffer and assayed (Table 1). Since only a minute amount of precipitate was obtained, it was collected by batch centrifugation at moderate speedabout 10,000 relative centrifugal force TABLE 1 RECOVERY OF UNDETECTABLE AMOUNTS OF HAA FROM ALBUMIN BY PRECIPITATION AND CONCENTRATION WITH POLYETHYLENE GLY- COL (PEG) PEG fraction: Percent HAA recovered Supernatan (containing albumin) Precipitate 100 *One ml. or partially purified HAA, positive on counterimmuno-electrophoresis at a dilution of 1 35, proved negatwo by HAA assay when diluted with 2000 ml, of five percent human serum albumin.

Removal of HAA from fractions by PEG precipitation In other experiments, HAA-rich serum or a serum fraction (complement-fixation titer over 1000) or partially purified HAA (isolated and washed by ultra-centrifugation) was mixed with a partially purified concentrate of Factors II, VII, IX and X prepared by DEAE adsorption of plasma and subsequent elution, or with AHF prepared by cryoethanol precipitation, or with fibrinogen, albumin or gamma globulin prepared by Cohn methods 6 and 9. The added HAA was precipitated from the plasma fraction by PEG, and the desired fraction itself was then precipitated and concentrated by PEG. The yield of the plasma fraction was 90-100 percent for all except fibrinogen and AHF (Tables 2-4).

TABLE IL-REMOVAL OF HAA FROM A MIXTURE OF THE ANTIGEN AND A CONCENTRATE OF COAGULATION FACTORS II, VII. IX AND X BY POLYETHYLENE GLYCOL (PEG) PRECIPITATION TABLE III-REMOVAL OF HAA FROM A MIXTURE OF THE ANTIGEN AND HUMAN ALBUMIN BY POLYETHYLENE GLYCOL (PEG) PRECIPITATION HAA titer Total protein (countenmmnnoelectro- Fractionation step Mg. Percent phoresis) tate=final product) 2, 900 93 b Neg. 2d PEG supernatant (discard) 130 4 b Neg.

e Complement fixation titer-1/410. b Neg. undilute.

1 RCF: e2 2 X gravity wherein rzradius (in feet) of the rotor and n=revolutions per second=r.p.m. 60.

TABLE IV.REMOVAL OF HAA FROM A MIXTURE OF THE ANTIGEN AND HUMAN GAMMA GLOBULIN BY POLY EIHYLENE GLYOOL (PEG) PRECIPITATION HAA titer Total protein (counterimmunoelectro- Fractionation step Mg. Percent phoresis) Gammaglobulin plus HAA (starting material 546 1 1/24 1st PEG precipitate (discard) 11.1 2 1/20 2d PEG precipitate (solubilized pr cipitate =fina1 product) 507 93 b Neg. 2 PEG supernatant (discard) 26 5. 1 b Neg.

! Complement fixation titer-U614. b Undilute.

EXAMPLE 1 Concentration of HAA from a plasma fraction containing prothrornbin complex (Factors II, VII, IX and X) and preparation of the fraction free of hepatitis-associated antigen (HAA) for clinical use The starting material commonly used for this procedure may be partially purified prothrombin complex obtained by DEAE-Sephadex or DEAE column chromatography or from calcium phosphate adsorption and elution methods. The ionic strength for an Optimal yield of these fractions ranges from 0.15 to 0.001, and the protein concentration ranges from 3 to 27 mg./ml. at about neutral pH, with water and a suitable buffer, e.g., sodium citratesodium chloride. The temperature of this solution is maintained at 15-25 C. Polyethylene glycol (PEG), molecular weight 200-6000, is added to a final concentration of 12-30 grams per 100 ml. The exact percentage is increased for PEG of low molecular weight and decreased for the high molecular weight material. After sufi'icient mixing to dissolve the PEG and precipitate the HAA, usually 30 minutes, the solution is centrifuged at room temperature for at least 10 minutes at a relative centrifugal force (RCF) of approximately 10,000 and the supernatant in separated cleanly from the precipitate by decantation or vacuum aspiration and reserved.

The HAA precipitate is reconstituted with three volumes of buffer or saline solution, and the walls of the centrifuge bottle are washed, bringing the total volume to about 10 percent of the starting material, or to a volume which readily permits transfer of the reconstituted precipitate from the large centrifuge tube to a small one. The HAA is then reprecipitated from the buifer or saline solution with 12-30 percent PEG 200-6000 and centrifuged. Since the precipitate contains the HAA, special care must be taken in this separation and in disposition of the precipitate after it has been reconstituted in 0.2-1.0 ml. of water or saline solution and assay. This optional procedure of redissolving and reprecipitating the HAA provides a purified, concentrated HAA for assay.

If the II, VII, IX and X concentrate is for laboratory or clinical use, the reserved superantant containing it is adjusted to a pH of about 5.2 and the PEG concentration is raised, according to the molecular weight of the PEG used, e.g., 30 grams per 100 ml. with PEG-4000. After suflicient mixing to dissolve the PEG and precipitate the concentrate, usually 30 minutes, the solution is centrifuged at about 10,000 RCF for 10 minutes at room temperature. The precipitate is collected, washed with 30 percent ethanol at 5 C. in buffer or in water and dissolved at neutral pH in a suitable buffer, e.g., sodium citrate-sodium chloride. Alternatively, Souliers method of precipitation and concentration with ethanol may be used instead of PEG in the second precipitation step, but the yield is moderately decreased with this procedure.

EXAMPLE 2 Concentration of HAA from gamma globulin, and/or preparation of gamma globulin free of hepatitis-associated antigen (HAA) for clinical use The starting material for this procedure can be Fractron II+III (from Cohn Method VI), Fractions 11-3,

II-l, 2 and II (from Method X) or fractions obtained from similar or derivative techniques. The protein in these fractions is diluted in a suitable bufl er, e.g., glycinesodium citrate-sodium chloride at about neutral pH to a concentration of 12-22 mg./ ml. The temperature is maintained at 15-25 C. The solution is adjusted to pH 3.0- 3.7 by slowly adding acid, e.g., acetic, hydrochloric or citric acid, with mixing. Polyethylene glycol (PEG), 200-6000 molecular weight, is added to a final concentration of 12-30 grams per 100 ml. of solution. The exact percentage is increased for PEG of low molecular weight and decreased for materal of high molecular weight. After the PSG has been dissolved by mixing and the HAA has been precipitated, which usually requires 30 minutes, the solution is centrifuged at room temperature for at least 10 minutes at a relative centrifugal force of about 10,000, and the supernatant is separated cleanly from the HAA precipitate by decantation or vacuum aspiration and reserved.

The precipitate is reconstituted with three voldmes of buffer or saline solution, and the walls of the centrifuge bottle are washed, bringing the total volume to about 10 percent of the starting material, or to a volume which readily permits transfer of the reconstituted precipitate from the large centrifuge tube to a small one. The HAA is then reprecipitated from the buffer or saline solution with 12-30 percent PEG 2006000 and centrifuged. Since the precipitate contains the HAA, special care must be taken with this separation and with disposal of the precipitate after it has been reconstituted in 0.2-1.0 ml. water, saline, or buffer, and assayed. This optional procedure of redissolving and reprecipitating the HAA provides a purified, concentrated HAA for assay.

If the gamma globulin recovered is for laboratory or clinical use, the reserved supernatant containing it is adjusted to about pH 7.0 by slowly adding a base, e.g., sodium hydroxide, with mixing. The precipitate is collected, washed with 20-40 percent ethanol at -5 to l0 C. in buffer or in water and then dissolved in buifer, usually a glycine-sodium citrate-saline buffer at pH 7.0. As an alternative, this procedure can be introduced into Method 6 after supernatant I has been collected and just before the precipitation of Fraction II-III, or introduced into Method 9 after collection of Supernatant HI.

EXAMPLE 3 Concentration of HAA from albumin and/or preparation of albumin free of hepatitis-associated antigen (HAA) for clinical use The starting material for this procedure can be Cohn supernatant V or Fraction V precipitate (from Method 6), or fractions obtained from derivative techniques or other methods. The protein in these fractions is diluted in a solvent, e.g., 0.9 percent sodium chloride, to a final concentration up to 50-60 mg./ml. and adjusted to about pH 7.0 by slowly adding acid or base, e.g., hydrochloric, citric 0r acetic acid, or sodium hydroxide. The temperature of this solution is maintain at 15-20 C. Polyethylene glycol (PEG), molecular weight 2006000, is added to a final concentration of 12-30 grams per 100 ml. The exact percentage is increased for the low-molecular-weight PEG and decreased for the high-molecular-weight material. After sufiicient mixing to dissolve the PEG and precipitate the HAA, usually 30 minutes, the solution is centrifuged at room temperature for at least minutes at a relative centrifugal force of about 10,000, and the supernatant is separated cleanly from the HAA precipitate by decantation or vacuum aspiration and reserved.

The precipitate is reconstituted with three volumes of buffer or saline solution, and the walls of the centrifuge bottle are washed, bringing the total volume to about 10 percent of the starting material, or to a volume which readily permits transfer of the reconstituted precipitate from the large centrifuge tube to a small one. The HAA is then reprecipitated from the buffer or saline solution with 12-30 percent PEG 200-6000 and centrifuged'Since the precipitate contains the HAA, special care must be taken in this separation and in disposal of the precipitate after it has been reconstituted in 0.2-1.0 ml. of water or saline solution and assayed. This optional procedure of redissolving and reprecipitating the HAA provides a purified, concentrated HAA for assay.

If the albumin is for laboratory or clinical use, the pH of the reserved supernatant containing it is adjusted to approximately 4.8 by slowly adding an acid, e.g., acetic, hydrochloric or citric acid. The precipitate is collected washed with 40 percent ethanol in water or 0.9 percent saline at -5 C., and a suitable solvent is added, e.g., water or 0.9 percent sodium chloride. As an alternative, the Fraction V precipitate can be further fractionated by Method 6 and the resulting albumin can then be processed as described above.

EXAMPLE 4 Concentration of HAA for assay from fibrinogen or AHF concentrates and/ or preparation of fibrinogen or AHF concentrates free of hepatitis-associated antigen (HAA) for clinical use Freshly prepared, fresh frozen or lyophilized fibrinogen or AHF concentrate prepared for clinical usereconstituted in sterile water or a suitable buifer-is diluted to a protein concentration or 2-12 mg./ml. with physiologic saline solution, 0-.02 M tris-0.02 M citrate buffer, or other buffer. Urea is added to a final concentration of 2.0-5.0 M. The procedure may be carried out at a high pH or at a low PH.

High pH method: The pH of the solution is adjusted to 9.8-10.5 with any suitable base. After the diluted fibrinogen or AHF has been mixed for 30 minutes at pH 9.8-10.5 at room temperature, PEG 2006000 molecular weight is added to a final concentration of 12-30 grams/ ml. and mixing is continued for 30-120 minutes at room temperature to dissolve the PEG and precipitate the HAA. The pH must be adjusted at this stage to prevent any precipitation of the fibrinogen or AHF, or to dissolve any that has already precipitated. Such precipitation may occur at pH 10.0 or lower, but rarely above pH 10.3.

Low pH method: The pH of the solution is adjusted to 3.0-4.5 with any suitable acid. After the diluted fibrinogen or AHF has been mixed for 30 minutes at room temperature, PEG 200-6000 molecular weight is added to a final concentration of 12-30 grams/ml. and mixing is continued for 30-120 minutes at room temperature. The pH must be adjusted at this stage to prevent any precipitation of the fibrinogen or AHF, or to dissolve any that has already precipitated. Such precipitation may occur at a pH of 4.0 or more, but rarely below 4.2.

With either the high or low pH method, the mixture is then centrifuged for at least 10 minutes at about 10,000 RCF at 20 C. to bring down the HAA, and the supernatant is aspirated carefully to avoid disturbing any minute quantities of precipitate that may be visible at the bottom of the centrifuge cup or tube and reserved. The precipitate is reconstituted with three volumes of buffer or saline solution, and the walls of the centrifuge bottle are washed, bringing the total volume to about 10 percent of the starting material, or to a volume which readily permits transfer of the reconstituted precipitate from the large centrifuge tube to a small one. The HAA is then reprecipitated from the buffer or saline solution with 12- 30 percent PEG 2006000, and centrifuged at pH 9.8- 10.5 for the high pH method and pH 3.0-4.5 for the low pH method, as described above. The final precipitate is dissolved in 0.2-l.0 ml. of saline, water or tris-citrate or other buffer for optimal concentration and then assayed. This optional procedure of redissolving and reprecipitating the HAA provides a purified, concentrated HAA fOr assay.

The fibrinogen or AHF in the aspirated reserved supernatant may be precipitated by adding suflicient NaOH or HCl to bring the pH to 6.0. The precipitate is collected, washed with 10 percent ethanol in water at 2 C. and a suitable solvent is added, e.g., 0.02 M tris citrate plus 0.1 M NaCl.

EXAMPLE 5 Concentration of HAA from a plasma fraction containing prothrombin complex (Factors 11, VII, IX and X) and preparation of the fraction free of hepatitis-associated antigen (HAA) for clinical use Partially purified prothrombin complex (500 mg), obtained by DEAE column chromatography, was dissolved in 0.03 M glycine-0.0005 M sodium citrate-0.01 percent sodium chloride buffer, pH 7.0, and diluted with additional buffer to an ionic strength of 0.003 and a protein concentration of 5.0 mgJml. The temperature of this solution was maintained at 25 C. Polyethylene glycol (PEG) molecular weight 4000 was added to a final concentration of 20 grams per 100 ml. After 30 minutes of mixing to dissolve the PEG and precipitate the HAA, the solution was centrifuged at room temperature for at least minutes at a relative centrifugal force of approximately 10,000, and the supernatant was separated cleanly from the HAA precipitate by vacuum aspiration and reserved.

EXAMPLE 6 The precipitate from Example 5 was reconstituted with three (10 ml.) volumes of saline, and the walls of the centrifuge bottle were washed with an additional 5 ml., bringing the total volume to 35 ml.all of which was pooled in a small test tube. The HAA was then reprecipitated from the saline at pH 7.0 with percent PEG- 4000 and centrifuged. Since the precipitate contains the HAA, special care must be taken in this separation and in disposition of the precipitate after it has been reconstituted in 0.2-1.0 ml. normal saline and assayed.

EXAMPLE 7 The reserved supernatant from Example 5 containing Factors II, XII, XI and X was adjusted to a pH of 5.2 by adding 1 N hydrochloric acid, and the PEG concentration was raised to 30 grams per 100 ml. with PEG- 4000. After 30 minutes of mixing to dissolve the PEG and precipitate the HAA, the solution was centrifuged at a RCF of 10,000 for at least 10 minutes at room temperature. The precipitate was collected, washed with 30 percent ethanol at 5 C. in buffer (pH 5.2) and dissolved in 20 ml. of 0.3 M glycine-0.005 M sodium citrate-0.1 percent sodium chloride buffer, at pH 7.0.

EXAMPLE 8 Concentration of HAA from gamma globulin and/or preparation of gamma globulin free of hepatitis-associated antigen (HAA) for clinical use Fraction 11-3 (500 ml.) from Cohn Method 9, containing 166 mg protein/mL, was diluted in 0.3 M glycine- 0.005 M sodium citrate-0.1 percent sodium chloride buffer at pH 7.0 to a protein concentration of 22 mg./ml. The temperature was maintained at 25 C. The solution was adjusted by pH 3.5 by slowly adding 1 N hydrochloric acid, with mixing. Polyethylene glycol (PEG), 4000 molecular weight, was added to a final concentration of 20 grams per 100 ml. of solution. After minutes of mixing to dissolve the PEG and precipitate the HAA, the solution was centrifuged at room temperature for at least 10 minutes at a relative centrifugal force of 10,000, and the supernatant was separated cleanly from the HAA precipitate by vacuum aspiration and reserved.

12 EXAMPLE 9 The precipitate from Example 8 was reconstituted with three (10 ml.) volumes of saline solution, and the walls of the centrifuge bottle were washed with 5 ml. of saline bringing the volume to 35 ml.all of which was transferred to a small test tube. The pH was adjusted to 7.0 and the HAA was then reprecipitated from the saline solution with 20 percent PEG-4000. Since the precipitate contains the HAA, special care must be taken with this separation and the disposal of the precipitate after it has been reconstituted in 1.0 ml. saline solution and assayed.

EXAMPLE 10 The reserved supernatant from Example 8 containing gamma globulin was adjusted to pH 7.0 by slowly adding 1 N sodium hydroxide, with mixing. The precipitate was collected, washed with ethanol at 5 C. in buffer and dissolved in 500 ml. of 0.3 M glycine-0.005 M sodium citrate-0.1 percent butter, at pH 7.0.

EXAMPLE l1 Concentration of HAA from albumin and/ or preparation of albumin free of hepatitis-associated antigen (HAA) for clinical use Fraction V precipitate (2.5 grams) from Cohn Method 6 was diluted in 0.9 percent sodium chloride, to a final protein concentration of 50 mg./ml. and adjusted to pH 7.0 by slowly adding 1 N hydrochloric acid. The temperature of this solution was maintained at 25 C. Polyethylene glycol (PEG), molecular weight 4000, was added to a final concentration of 20 grams per ml. After 30 minutes of mixing to dissolve the PEG and precipitate the HAA, the solution was centrifuged at room temperature for 10 minutes at a relative centrifugal force of 10,000, and the supernatant was separated cleanly from the HAA precipitate by vacuum aspiration and reserved.

EXAMPLE 12 The precipitate from Example 11 was reconstituted with three (10 m1.) volumes of saline, and the walls of the centrifuge bottle were washed with 5 ml. bringing the total volume to 35 ml.all of which was then pooled in a small test tube. The pH was adjusted to 7.0 and the HAA was then reprecipitated from the saline with 20 percent PEG-4000 and centrifuged. Since the precipitate contains the HAA, special care must be taken in this separation and in the disposal of the precipitate after it has been reconstituted in 1.0 ml. of saline solution and assayed.

EXAMPLE 13 The pH of the reserved supernatant from Example 11 containing the albumin was adjusted to 4.8 by slowly adding 1 N hydrochloric acid, and the PEG concentration was raised to 30 grams per 100 ml. with PEG 4000. After 30 minutes of mixing to dissolve the PEG and precipitate the HTA, the solution was centrifuged at a RCF of 10,000 for at least 10 minutes at room temperature. The precipitate was collected, washed with 40 percent ethanol in 0.9 percent saline at 5 C., and dissolved in 500 ml. of 0.9 percent sodium chloride, at pH 7.0.

EXAMPLE l4 Concentration of HAA for assay from fibrinogen or AHF concentrates and/or preparation of fibrinogen or AHF concentrates free of hepatitis-associated antigen (HAA) for clinical use Lyophilized fibrinogen or fibrinogen-rich AHF concentrate prepared for clincial use (approximately l' /2-2 gm.) was reconstituted in 200 ml. of distilled water and diluted to a protein concentration of 2 mg./ml. with 0.02 M tris- 0.02 M citrate buifer, pH 7.0. Urea was added to a final concentration of 2.5 M.

High pH method: The pH of the solution was adjusted to 10.3 with 1 N NaOH. After the diluted fibrinogen or 13 AHF had been mixed for 30 minutes at room temperature, PEG-4000 was added to a final concentration of 30 grams per 100 ml. and mixing was continued for 30 mintues. The pH was maintained at 10.3 throughout this stage to prevent any precipitation of the fibrinogen or AHF, and to dissolve any that had already precipitated.

EXAMPLE 15 Low pH method: The procedure of Example 14 was followed, but the pH was adjusted to 3.5 using 1 N HCl. After the diluted fibrinogen or AHF had been mixed for 30 minutes at room temperature, PEG-4000 was added to a final concentration of 20 grams per 100 ml. and mixing was continued for 30 minutes at room temperature. The pH was maintained at 3.5 throughout this stage to prevent any precipitation of the fibrinogen or AHF, and to dissolve any that had already precipitated. Such precipitation may occur at a pH of 4.0 or more, but rarely below 4.2.

EXAMPLE 16 With both the high and low pH methods, of Examples 14 and 15, the mixture was then centrifuged for about one hour at 10,000 RCF at 20 C. to bring down the HAA, and the supernatant was aspirated carefully to avoid disturbing any minute quantity of precipitate visible at the bottom of the centrifuge cup. The precipitate was reconstituted with three successive 10 ml. volumes of saline, and the walls of the centrifuge bottle were washed with an additional ml. bringing the total volume to 35 ml.all of which was pooled in a small test tube. The HAA was then reprecipitated from the saline solution with 30 percent PEG-4000, at pH 7.0 and centrifuged. The final precipitate was dissolved in 1.0 ml. of 0.02 M tris-0.02 M citrate buffer for optimal concentration.

EXAMPLE 17 The fibrinogen or AI-IF in the aspirated supernatant from Example 16 was precipitated by adding sufiicient 1 N NaOH for the low pH method or 1 N HCl for the high pH method to bring the pH to 6.0. It was then centrifuged at an RCF of 10,000 for at least minutes at room temperature, the precipitate collected and washed with 10 percent ethanol in water at 2 C. and dissolved in 200 ml. of 0.4 percent citrate-0.9 percent NaCl, at pH 7.0.

EXAMPLE 18 Concentration of HAA from a plasma fraction containing prothrombin complex (Factors II, VH, IX and X) and extraction of the fraction free of hepatitis-associated antigen (HAA) for clinical use Precipitated or lyophilized partially purified prothrombin complex (500 mg), obtained by DEAE column chromatography, was dissolved in a 20 percent PEG- butfer solution of 0.03 M glycine-0.0005 M sodium citrate-0.01 percent sodium chloride, pH 7.0 (ionic strength of 0.003), to a protein concentration of 5.0 mg./ ml. The temperature of this solution was maintained at 25 C. After 30 minutes of mixing to extract the prothrombin complex and precipitate the HAA, the solution was centrifuged at room temperature for at least 10 minutes at a RCF of approximately 10,000, and the supernatant was separated cleanly from the HAA precipitate by vacuum aspiration and reserved.

EXAMPLE 19 The precipitate from Example 18 was reconstituted with three (10 ml.) volumes of saline, and the walls of the centrifuge bottle were washed with an additional 5 ml., bringing the total volume to 35 ml.all of which was pooled in a small test tube. The HAA was then reprecipitated from the saline at pH 7.0 with 20 percent PEG- 4000 and centrifuged. Since the precipitate contains the HAA, special care must be taken in this separation and in disposition of the precipitate after it has been reconstituted in 0.1-1.0 ml. normal saline and assayed.

14 EXAMPLE 20 The reserved supernatant from Example 18 containing Factors II, VII, IX and X was adjusted to a pH of 5.2 by adding 1 N hydrochloric acid, and the PEG concentration was raised to 30 grams per ml. with PEG-4000. After 30 minutes of mixing to dissolve the PEG and precipitate the HAA, the solution was centrifuged at a RCF of 10,000 for at least 10 minutes at room temperature. The precipitate was collected, washed with 30 percent ethanol at 5 C. in buffer (pH 5.2) and dissolved in 20 ml.- of 0.03 M glycine-0.005 M sodium citrate-0.1 percent sodium chloride buffer, at pH 7.0.

It should be understood that reference to the isoelectric point of a substance means the pH at which the net charge on a molecule in solution is 0. At this pH, amino acids exist almost entirely in the zwitterion state; that is, the positive and negative groups are equally ionized. A solution of proteins or amino acids at the isoelectric point exhibits minimum conductivity, osmotic pressure, and viscosity.

It is also understood that saline solution, unless otherwise indicated, refers to physiologic saline solution.

It should be apparent from the foregoing detailed description that the objects set forth hereinabove have been successfully achieved. Moreover, while there is shown and described a present preferred embodiment of the invention, it is to be distinctly understood that the invention is not limited thereto but may be otherwise variously embodied and practiced within the scope of the following claims.

What is claimed is:

1. A method of separating heptatitis-associated antigen from a solution comprising a solvent which is physiologically tolerable on injection, the hepatitis-associated antigen, and other proteins contained in a plasma fraction selected from the group consisting of albumin, gamma, globulin, coagulation Factors II, V, VII, IX, X, XI and XIII, fibrinogen, antihemophilic factor, plasminogen, cerloplasmin, transferrin, thyroxin-binding protein, antithrombin HI, a antitryspin, a macroglobulin, Ci inactivator, inter a-trypsin inhibitor and mixtures thereof, so as (1) to enable the remaining plasma fraction to be transfused with reduced likelihood of transmitting heptatitis to the recipient, or (2) to concentrate the antigen for assay with substantially increased antigen/other proteins ratio relative to the antigen/other proteins ratio in the original solution, or (3) to accomplish both (1) and (2) simultaneously, comprising the steps of:

(a) maintaining the pH of said solution within a range removed as far as possible from the isoelectric point of said other proteins without causing denaturation and still permitting precipitation of said antigen;

(b) adding polyethylene glycol having a molecular weight of from about 200 to about 6,000 to a concentration of from about 12 to about 30 grams per 100 milliliters of said solution to thereby obtain a precipitate consisting essentially of said antigen, said other proteins remaining in the supernatant substantially free of said antigen; and

(c) separating said precipitate from said supernatant.

2. A method of separating hepatitis-associated antigen from a solution comprising a solvent which is physiologically tolerable on injection, the hepatitis-associated antigen and other proteins contained in a plasma fraction selected from the group consisting of albumin, gamma globulin, coagulation Factors II, V, VII, IX, X, XI and XIII, fibrinogen, antihemophilic factor, plasminogen, ceruloplasmin, transferrin, thyroxin-binding protein, antithrombin IH, a antitryspin, a marcroglobulin, Ci inactivator, inter a-trypsin inhibitor and mixtures thereof, so as (l) to enable the remaining plasma fraction to be transfused with reduced likelihood of transmitting hepatitis to the recipient, or (2) to concentrate the antigen for assay with substantially increased antigen/other proteins ratio relative to the antigen/other proteins ratio in the original solution, or (3) to accomplish both (1) and (2) simultaneously, comprising the steps of:

(a) maintaining the pH of said solution within a range sulficiently close to the isoelectric point of at least some of said other proteins and said antigen to permit precipitation thereof;

(b) adding polyethylene glycol having a molecular weight of from about 200 to about 6,000 to a concentration of from about 12 to about 30 grams per 100 milliliters of said solution to thereby obtain a precipitate containing said antigen and at least some of said other proteins;

() separating said precipitate from its supernatant;

(d) extracting said other proteins from said antigen in said precipitate with a physiologically tolerable solvent having a pH removed from the isoelectric point of said other proteins to produce a supernatant substantially free of said antigen, said antigen remaining in the precipitate; and

(e) separating said precipitate consisting essentially of said antigen from said supernatant containing said other proteins.

3. A method as defined in claim 2, wherein said polyethylene glycol has a molecular weight of about 4,000 and said concentration is from about 20 to 30 grams per 100 milliliters of solution.

4. A method as defined in claim 2, wherein said steps are performed at a temperature of from about 15 to 25 C.

5. A method as defined in claim 4, wherein said steps are performed at room temperature.

6. A method as defined in claim 2, wherein said solvent has an ionic strength between 0.001 and 0.2.

7. A method as defined in claim 6, wherein said solvent is selected from the group consisting of glycine-citratesaline butler, tris(hydroxymethyl aminomethane-citrate =buifer, tris(hydroxymethyl aminomethane-citrate-urea buffer, phosphate bufier, phosphate-saline butler, ammonium or sodium acetate, sodium bicarbonate-CO an amino acid, physiologic saline solution, and water 8. A method as defined in claim'7, wherein said solvent is a buffer solution of a concentration suitable to provide a predetermined pH.

9. A method as defined in claim 2, wherein said antigen is removed from said protein by centrifugation or filtration.

10. A method as defined in claim 9, wherein said centrifugation varies from about 10,000 to 15,000 RCF for about 4 minutes to about 1 hour.

11. A method as defined in claim 9, wherein said separation is by filtration using a filter having a pore size below about 0.6 mg.

12. A method as defined in claim 11, wherein said pore size is from about 0.45 to about 0.2 m

13. A method as defined in claim 2, further comprising purifying and concentrating said antigen by the steps of:

(a) maintaining solubility of said antigen in a solvent at a pH of about 7;

(b) adding polyethylene glycol having a molecular weight of about 4,000 to a concentration of about 20 percent to reprecipitate said antigen; and

(c) removing said reprecipitated antigen from the resulting supernatant.

14. A method as defined in claim 2, further comprising concentrating said protein by:

(a) adjusting the pH of said supernatant to near the isoelectric point of said protein to thereby precipitate said protein; and

(b) collecting said precipitated protein.

15. A method as defined in claim 2 wherein said plasma fraction comprises prothrombin complex.

16. A method as defined in claim 1, wherein said polyethylene glycol has a molecular weight of about 4,000 and said concentration is from about 20 to 30 grams per milliliters of solution.

17. A method as defined in claim 1, wherein said steps are performed at a temperature of from about 15 to 25 C.

18. A method as defined in claim 17, wherein said steps are performed at room temperature.

19. A method as defined in claim 1, wherein said solvent has an ionic strength between 0.001 and 0.2.

20. A method as defined in claim 19, wherein said solvent is selected from the group consisting of glycinecitrate-saline buffer, tris(hydroxymethyl) aminomethanecitrate buffer, tris(hydroxymethyl) aminomethane-citrateurea buffer, phosphate buffer, phosphate-saline bufier, ammonium or sodium acetate, sodium bicarbonate-CO an amino acid, physiologic saline solution, and water.

21. A method as defined in claim 19, wherein said solvent is a butter solution of a concentration suitable to provide a predetermined pH.

22. A method as defined in claim 1, wherein said antigen is removed from said protein by centrifugation or filtration.

23. A method as defined in claim 22, wherein said centrifugation varies from about 10,000 to 15,000 RCF for about 4 minutes to about 1 hour.

24. A method as defined in claim 22, wherein said separation is by filtration using a filter having a pore size below about 0.6 mg.

25. A method as defined in claim 24, wherein said pore size is from about 0.45 to about 0.2 mg.

26. A method as defined in claim 1, further comprising purifying and concentrating said antigen by the steps of:

(a) maintaining solubility of said antigen in a solvent at a pH of about 7;

(c) removing said reprecipitated antigen from the resulting supernatant.

27. A method as defined in claim 1, further comprising concentrating said protein by:

(b) collecting said precipitated protein.

28. A method as defined in claim 1, wherein said plasma fraction comprises prothrombin complex.

References Cited UNITED STATES PATENTS 3,415,804 12/1968 Poison 260-112 3,652,530 4/1972 Johnson et al. 260-112 3,630,840 12/1971 Wagner 260-412 OTHER REFERENCES General Biochemistry, 1958, Fruton et al. pp. 101-102.

HOWARD E. SCHAIN, Primary Examiner US. Cl. X.R.