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

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

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US3790552A
US3790552A US00235211A US3790552DA US3790552A US 3790552 A US3790552 A US 3790552A US 00235211 A US00235211 A US 00235211A US 3790552D A US3790552D A US 3790552DA US 3790552 A US3790552 A US 3790552A
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haa
peg
precipitate
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hepatitis
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A Johnson
J Newman
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
    • Y10S530/806—Antigenic peptides or proteins
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
    • Y10S530/827—Proteins from mammals or birds
    • Y10S530/829—Blood
    • Y10S530/83—Plasma; serum
    • Y10S530/831—Cohn fractions

Definitions

  • 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.
  • Australia antigen 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.
  • HAA hepatitis-associated antigen
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • PEG polythylene glycol
  • 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.
  • PEG has also been used by virologists to concentrate and purify a number of plant viruses, bacteriophages, and some animal organisms:
  • WMV soil-borne wheat mosaic virus
  • HAA heptatitis-associated antigen
  • the primary object 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).
  • a method 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • HAA DESCRIPTION OF THE PREFERRED EMBODIMENT Concentration of HAA from fractions for assay
  • the added HAA was precipitated from the plasma fraction by PEG, reconstituted to its original volume in buffer and assayed (Table 1).
  • 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).
  • HAA hepatitis-associated antigen
  • 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.
  • PEG polyethylene glycol
  • 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.
  • RCF relative centrifugal force
  • 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.
  • 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.
  • 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.
  • a suitable buffer e.g., sodium citrate-sodium chloride.
  • 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.
  • 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.
  • acid e.g., acetic, hydrochloric or citric acid
  • PEG Polyethylene glycol
  • 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.
  • 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.
  • the reserved supernatant containing it is adjusted to about pH 7.0 by slowly adding a base, e.g., sodium hydroxide, with mixing.
  • a base e.g., sodium hydroxide
  • 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.
  • 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.
  • HAA hepatitis-associated antigen
  • 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.
  • 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.
  • 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.
  • 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.
  • a suitable solvent e.g., water or 0.9 percent sodium chloride.
  • the Fraction V precipitate can be further fractionated by Method 6 and the resulting albumin can then be processed as described above.
  • HAA hepatitis-associated antigen
  • 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.
  • 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
  • HAA hepatitis-associated antigen
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • HAA hepatitis-associated antigen
  • 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
  • HAA hepatitis-associated antigen
  • 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.
  • 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.
  • 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.
  • 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.
  • saline solution refers to physiologic saline solution.
  • 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.
  • said solvent is a buffer solution of a concentration suitable to provide a predetermined pH.
  • centrifugation varies from about 10,000 to 15,000 RCF for about 4 minutes to about 1 hour.
  • 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.

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US00235211A 1972-03-16 1972-03-16 Method of removing hepatitis-associated antigen from a protein fraction using polyethylene glycol Expired - Lifetime US3790552A (en)

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CA (1) CA1061251A (enrdf_load_stackoverflow)
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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3951937A (en) * 1973-12-20 1976-04-20 The Community Blood Council Of Greater New York, Inc. Large scale purification of hepatitis type B antigen using polyethylene glycol
DE2602892A1 (de) * 1976-01-27 1977-07-28 Community Blood Council Konzentriertes hepatitis-b-oberflaechenantigen enthaltende masse und verfahren zu ihrer herstellung
EP0001838A1 (de) * 1977-11-09 1979-05-16 BEHRINGWERKE Aktiengesellschaft Verfahren zur Entfernung von Detergentien aus Virusantigensuspensionen
US4164496A (en) * 1978-08-23 1979-08-14 American National Red Cross Preparation of albumin using PEG and EDTA
US4177188A (en) * 1977-01-21 1979-12-04 Nordisk Insulinlaboratorium Process for recovering purified albumin from blood plasma using PEG and caprylic acid
US4197238A (en) * 1977-04-12 1980-04-08 The Green Cross Corporation Method of preparation of human albumin using polyethylene glycol
US4344935A (en) * 1980-06-05 1982-08-17 Synthelabo Process for the isolation of viral glycoproteic antigens and its application to the preparation of vaccines
US4395395A (en) * 1979-05-21 1983-07-26 The United States Of America As Represented By The Department Of Health And Human Services Detection of non-A, non-B hepatitis associated antigen
US4683294A (en) * 1985-04-03 1987-07-28 Smith Kline Rit, S.A. Process for the extraction and purification of proteins from culture media producing them
US4684723A (en) * 1985-09-11 1987-08-04 Miles Laboratories, Inc. Method of separating proteins from aqueous solutions
US4833233A (en) * 1987-08-20 1989-05-23 The United States Of America As Represented By The Administrator Of The National Aeronautics And Space Administration Human serum albumin crystals and method of preparation
US4835257A (en) * 1984-07-07 1989-05-30 Armour Pharma Gmbh Process for preparing gamma globulin suitable for intravenous administration using peg and a citrate buffer
US5525519A (en) * 1992-01-07 1996-06-11 Middlesex Sciences, Inc. Method for isolating biomolecules from a biological sample with linear polymers
US20080187568A1 (en) * 2007-02-06 2008-08-07 Sawhney Amarpreet S Polymerization with precipitation of proteins for elution in physiological solution

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK144731C (da) 1976-07-30 1982-10-18 Nordisk Insulinlab Fremgangsmaade til isolering af proteinhormoner stammende fra humant hypofysevaev
US20080214795A1 (en) * 2007-02-14 2008-09-04 Amgen Inc. Method of isolating antibodies by precipitation

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3951937A (en) * 1973-12-20 1976-04-20 The Community Blood Council Of Greater New York, Inc. Large scale purification of hepatitis type B antigen using polyethylene glycol
DE2602892A1 (de) * 1976-01-27 1977-07-28 Community Blood Council Konzentriertes hepatitis-b-oberflaechenantigen enthaltende masse und verfahren zu ihrer herstellung
US4177188A (en) * 1977-01-21 1979-12-04 Nordisk Insulinlaboratorium Process for recovering purified albumin from blood plasma using PEG and caprylic acid
US4197238A (en) * 1977-04-12 1980-04-08 The Green Cross Corporation Method of preparation of human albumin using polyethylene glycol
EP0001838A1 (de) * 1977-11-09 1979-05-16 BEHRINGWERKE Aktiengesellschaft Verfahren zur Entfernung von Detergentien aus Virusantigensuspensionen
US4206014A (en) * 1977-11-09 1980-06-03 Behringwerke Aktiengesellschaft Process for removing detergents from virus-antigen suspensions
US4164496A (en) * 1978-08-23 1979-08-14 American National Red Cross Preparation of albumin using PEG and EDTA
US4395395A (en) * 1979-05-21 1983-07-26 The United States Of America As Represented By The Department Of Health And Human Services Detection of non-A, non-B hepatitis associated antigen
US4344935A (en) * 1980-06-05 1982-08-17 Synthelabo Process for the isolation of viral glycoproteic antigens and its application to the preparation of vaccines
US4835257A (en) * 1984-07-07 1989-05-30 Armour Pharma Gmbh Process for preparing gamma globulin suitable for intravenous administration using peg and a citrate buffer
US4683294A (en) * 1985-04-03 1987-07-28 Smith Kline Rit, S.A. Process for the extraction and purification of proteins from culture media producing them
US4684723A (en) * 1985-09-11 1987-08-04 Miles Laboratories, Inc. Method of separating proteins from aqueous solutions
US4833233A (en) * 1987-08-20 1989-05-23 The United States Of America As Represented By The Administrator Of The National Aeronautics And Space Administration Human serum albumin crystals and method of preparation
US5525519A (en) * 1992-01-07 1996-06-11 Middlesex Sciences, Inc. Method for isolating biomolecules from a biological sample with linear polymers
US5599719A (en) * 1992-01-07 1997-02-04 Middlesex Sciences, Inc. Method for isolating biomolecules from a biological sample with linear polymers
US20080187568A1 (en) * 2007-02-06 2008-08-07 Sawhney Amarpreet S Polymerization with precipitation of proteins for elution in physiological solution

Also Published As

Publication number Publication date
FR2175969B1 (enrdf_load_stackoverflow) 1977-08-12
DE2306107A1 (de) 1973-09-27
BE796770A (fr) 1973-07-02
CA1061251A (en) 1979-08-28
DK133292B (da) 1976-04-26
FR2175969A1 (enrdf_load_stackoverflow) 1973-10-26
GB1375811A (enrdf_load_stackoverflow) 1974-11-27
AT321465B (de) 1975-04-10
DK133292C (da) 1976-09-27

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