US 3652530 A
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United States Patent 3,652,530 ANTIHEMOPHHLIC FACTOR PREPARED FROM BLOOD PLASMA USHJG POLYETHYLENE GLYCOL Alan .I. Johnson, New York, Jack Newman, Pelham, and Margaret Howell Karpatkin, New York, N.Y., assignors to American National Red Cross, Washington, DC. No Drawing. Filed Aug. 28, 1967, Ser. No. 663,507 Int. Cl. A23j 1/06; C08h 1/00 U.S. Cl. 260-]l12 B 11 Claims ABSTRACT OF THE DISCLOSURE Antihemophilic factor is separated as a precipitate from fresh-frozen human plasma with a low concentration of ethanol, preferably 3 percent, at low temperature, followed by extraction with a low ionic strength buffer, preferably a TRIS buffer solution. The resulting product of intermediate purity (ZS-60X purified) can be lyophilized and stored or can be further treated to obtain a high purity AHF (ZOO-400x purified). High purity AHF is produced by further treating the intermediate purity product with PEG, preferably of a molecular weight of 4,000, in two steps, the first at about 6 percent and the second at about 12 percent. The high purity product is lyophilized before storage. Both the intermediate, and the high, purity AHF are used in the treatment of classical hemophilia.
BACKGROUND OF THE INVENTION This invention relates to the separation of blood proteins. More particularly, this invention relates to largescale preparation of antihemophilic factor from human blood plasma.
It has been found that classical hemophilia is a deficiency disease caused by the absence of antihemophilic factor (AHF) in the blood. It has been further found that the AHF is concentrated in the blood plasma. Until recently, the only effective method of controlling bleeding in hemophiliacs was by the transfusion of fresh blood, fresh plasma or fresh-frozen plasma. The concentration of AHF in normal blood, or even plasma, however, is so low that the patients circulation may be overloaded before hemostasis can be achieved. Further, it is well known that AHF is susceptible to denaturation by heat, storage, freezing and thawing, thereby making the use of fresh whole blood or plasma extremely undesirable. This has led to increased research in recent years toward extracting AHF from the plasma for storage and subsequent use.
The earliest attempts to isolate AHF from normal human plasma, for widespread clinical use in hemophiliacs, were generally unsatisfactory. It was later found that fibrinogen prepared from human plasma by ethanol fractionation contained about X purified Other clinically useful AHF preparations were produced by various modifications of the ethanol fractionation procedure. These preparations were more potent, being l015 purified, and represented important therapeutic advance in hemophilia. Other methods were developed such as a recent method which utilizes cryoprecipitation from single units of fresh-frozen plasma, and produces a preparation about 10-20 purified. Although the above procedures are capable of being adapted to large scale use, the yield and purity of the products are relatively poor.
Thus, it can be seen that although considerable research has been performed in attempting to produce a commercially feasible high purity AHF preparation, there has been remarkably little success in this regard. The methods used to separate AH F from human plasma on a laboratory scale either cannot be performed on a large scale or can be performed only at prohibitive cost. In any event, these methods result in a product which is comparatively low in purity, ranging from 5 to about 20X purified. Other desirable characteristics of an AHF preparation, which have not hitherto been achieved, are that the products should be stable, of known potency, easily soluble to a low viscosity solution, and relatively inexpensive to produce.
It is, therefore, an object of this invention to provide a method of fractionating blood plasma, and particularly to provide an AHF preparation and a method for its production which is free of the aforementioned and other such disadvantages. More specifically, it is an object of this invention to provide an inexpensive highly purified AHF preparation of high stability and known potency.
It is yet another object of this invention to provide a method for separating AHF from human plasma on a large scale. Another specific object of this invention is to provide a relatively simple, inexpensive method for 0btaining AHF of intermediate purity from fresh-frozen blood plasma by treatment with a low concentration of ethanol at a low temperature and subsequent extraction with a low ionic strength buffer. Still another specific object of this invention is to provide a method for obtaining highly purified AHF from fresh-frozen blood plasma by treating the plasma with a low concentration of ethanol at low temperature, extracting with a low ionic strength buffer and further purifying the product of intermediate purity with polyethylene glycol.
These and other objects of this invention will in part be obvious and in part be pointed out as the description of the invention proceeds.
SUMMARY OF THE INVENTION Generally the inventive procedure utilizes large-scale ethanol cryoprecipitation of fresh-frozen human plasma and extraction of the AHF from the precipitate with tris- (hydroxymethyl) aminomethane buffer, hereinafter TRIS, to produce an intermediate purity AI-I F composition (25- 60 purified) followed by precipitation of contaminating proteins from the supernatant with polyethylene glycol, hereinafter PEG, resulting in a high purity AHF product (ZOO-400x purified). Both the intermediate and high purity AHF may be used to treat hemophiliacs, as well as to prepare AI-LF of still higher purity. AHF purity, as used herein, refers to the concentration of AHF, in units per milligram of protein in the concentrate related to the AHF concentration per milligram of protein in the starting plasma. The invention will be better understood by reference to the following flow-diagrams in which Diagram I represents the procedure for preparation of AHF of intermediate purity and Diagram II represents the procedure for preparation of AHF of high purity.
pre-filter is used to remove the residual aluminum hydroxide. While deprothrom-binization is preferable to insure against later untoward effects, it has been theorized that this step may not be necessary, and should not, therefore, be taken as unduly limiting. Trisodium citrate is added as an anti-coagulant to a final concentration of 0.02 M to prevent the formation of minute amounts of thrombin which might labilize the AHF. This solution can be rendered sterile by Millipore filtration and then lyophilized for a clinical product (intermediate purity), or used as the starting material for further purification.
Purification by polyethylene glycol The TRIS extracted AHF supernatant is brought to about pH 6.0 by the gradual addition of 0.02 M citric acid. The lower pH results in a significantly higher purification. The protein concentration is then approximately 10-12 mgm./ml. At this pH, a final concentration of 3 percent PEG-6,000 or 6 percent PEG-4,000 precipitates most of the fibrinogen without removing the AHF. The PEG may be added as a concentrated aqueous solution or as dry flakes. The operable concentration range of PEG-4,000 is about 3.5% to about 6.5%, with about 5.8% to about 6.0% being preferred. There is little increase in volume when dry flakes are used, which is a decided advantage with large-scale production. Vigorous mixing for at least 15 minutes, however, is required to insure complete solution of the dry PEG. Although the 6,000 molecular weight PEG may be used, we have found the PEG-4,000 is at least equally effective and has been tested extensively as a U.S.P. product: the latter is, therefore, preferred. The resulting large fiocculent precipitate is easily removed from the AHF-rich supernatant by centrifugation at 5,000- g. for 5 minutes at room temperature, and represents partially purified fibrinogen which may be fractionated further.
Concentration of AHF by PEG The concentration of PEG in the supernatant is increased to 10 percent if PEG-6,000 or 12 percent if PEG- 4,000, to precipitate the AHF and other residual proteins, and centrifuged for 10 minutes at 6,000 g. The PEG- rich supernatant is discarded, most of the remaining PEG is removed from the walls of the centrifuged container and the precipitate by 34 washes with ice cold 6 percent ethanol-TRIS solution, which selectively dissolves PEG, with agitation gentle enough not to dislodge the precipitate from the wall of the container. The precipitate may be redissolved in approximately 1,000th of the original plasma volume with 0.02 M TRIS-0.02 M citrate bulfer at pH 7.0. After sterile filtration through a Millipore filter, the AHF solution may be shell frozen and lyophilized. For the final filtration of either intermediate or high purity AHF it is important to use a Millipore GS filter which has a pore size of approximately 0.22 micron. It is also preferable to include an RA (approximately 1.2 microns) and an HA (approximately 0.45 micron) filter to prevent clogging of the GS by large molecular weight aggregates.
EXAMPLE I 211 packs of fresh-frozen human plasma weighing 45.6 kg. were warmed for 5 minutes on each side at 50 C. The tare weight of the packs was 5.82 kg., the plasma being 39.78 kg., or a volume of 38.7851.
The plasma was then crushed and added to a container in a bath at to 1 C. When half of the plasma was added, heating coils were placed into the ice layer, liquid at 6 C. was circulated through the coils, and the balance of the plasma was added. A liquid phase was allowed to form.
2 cc. of caprylic alcohol was added to reduce foaming. 2.311 1. of 53.3 percent ethanol at --5 C. were added with gentle mixing over a period of not more than minutes using a sterile burette. Liquid was circulated therethrough at 6 C. The bath temperature was maintained at 0 to 1 C. The coils were not raised out of the ice layer, and the liquid circulation to the bottom coils was stopped when only two inches of frozen plasma remained.
When the plasma had completely melted, the entire solution was transferred into the pressure tank of a Sharples Centrifuge #2 and centrifuged at a temperature of 0 to 1 C. using the /1 inch nozzle. At the start the small brine valve was fully opened, but was cut back A to /2 turn after the plasma surged out. The supernatant was discarded and the precipitate, or paste, was extracted with 4.875 1. of 0.02 M TRIS (pH 7) at 25 C. The pH of the solution was checked and was 7.0. The solution was stirred gently with a Vibromixer, model E 1, set at 1% turns from off for 30 minutes and then centrifuged in cups at 25 C., 4500 r.p.m. for 10 minutes. The supernatant was decanted and the precipitate was discarded. The supernatant was deprothrombinized at room temperature with 146.25 cc. of aluminum hydroxide. After the aluminum hydroxide was added the solution was stirred gently for 5 minutes and then centrifuged at 4500 r.p.m. for 5 minutes at 25 C. The supernatant was decanted and passed through a Millipore prefilter.
cc. of 0.5 M sodium citrate solution were then added and the solution poured into an 8 liter metal beaker using a 2 liter graduate to measure the amount which was 5.055 1. 0.02 M citric acid was added using a 250 cc. graduate to adjust the pH to 5.95-6.05, stirring continuously. The volume of citric acid solution added was 850 cc., bringing the total volume to 5,905 1.
Solid fla'lces of PEG 4,000 (CarboWax-4,000; Union Carbide Co.) were gradually added with the vibromixer in operation, to a final concentration of 5.8 percent, which required 343 grams. The mixing was continued for 15 minutes and then the solution was centrifuged for 5 minutes at 4,500 r.p.m. The supernatant was decanted into an 8 liter metal beaker and the paste was discarded.
With the vibromixer running, PEG 4,000 was gradually added to the final concentration of 12 percent, or 366 grams. Mixing was continued for 15 minutes and the solution was then centrifuged for 10 minutes at 4,000 r.p.m. in several portions. Each time the supernatant was decanted and discarded and the precipitates were allowed to collect in the four bottles of the centrifuge.
20 cc. of 0.02 M pH 7.0 TRIS-6 percent alcohol at 0 C. were added to each bottle and swirled gently to flush out all excess PEG solution without breaking up the precipitate. The wash solution was discarded, the mouth of each bottle was wiped with a gauze pad and the washing procedure was repeated twice more.
The precipitate was dissolved in 195 cc. of 0.02 M TRIS- CI'FRATE solution at 25 C. The diluent was added stepwise to each bottle and mixed gently until solution was complete. The pH was measured as 7.15.
The resulting solution was sterile filtered through a 142 mm. Millipore filter press, using the prefilter and RA, HA, and GS filters, and filled into 8 cc. amounts in vials. The vials of solution were then frozen rapidly in Dry Icealcohol and lyophilized. Both the intermediate and high purity AHF are extremely stable and have been kept at 37 C. for over three months.
EXAMPLE II Both the intermediate and the high purity AHF were assayed for AHF activity and for impurities (Al+++ in the intermediate purity AHF, Al+++ and PEG in the high purity AHF).
AHF was assayed by a modified thromboplastin generation test as described below. Serial dilution of a pooled normal human plasma control as well as a lyophilized AHF concentrate were used as references. Each dilution, made in 0.05 M imidazole bulfer at pH 7.3 was incubated in a generation mixture consisting of the following:
0.2 ml. of dilution, 0.2 ml. of 1:10 dilution serum in imidazole saline buffer (Celite activated), 0.2 ml. of deprothrombinized antihemophilic plasma diluted in imidazole buffer, 0.2 ml. of homolysate (prepared by freezethawing washed human red cells 6 diluted 1:20-1:40 in normal saline and 0.2 ml. of M/ 40 CaCl After 8-10 minutes incubation at 37 C., 0.1 ml. of this mixture was added along with 0.1 ml. of M/40 CaCl to 0.1 ml. of pooled human plasma pre-incubated at 37 C. and the clotting time recorded. The fastest clotting times obtained for each dilution were plotted on the ordinate of log-log paper and the inverse of the dilutions on the abscissa, and joined by a straight line. Samples of AHF were assayed in a similar way and a line of best fit drawn parallel to the control line. The activity of the unknown was determined by comparing the 15 second intercept dilution with that of the control.
Proteins were assayed by the biuret method (Colowick et al., Methods in Enzymology, Academic Press, N.Y., 3, p. 450, 1957) and protein nitrogen by gas chromatography.
The intermediate purity material had a specific activity of 0.2-0.4 units/mg. protein, and the high purity product had a specific activity of 36 units/mg. protein. 1 ml. of frozen pooled normal plasma usually contains 0.8 unit of AHF.
Aluminum assays of the intermediate purity AHF are shown in Table I.
TABLE 1 pg. Al lunit intermediate purity AHF #1;- #2.- AF' Lot No unit AHF Lot No. unit AHF N or1-:.l\1can=0.1116. Standard deviation=5.585
The PEG assay was performed by a modification of the method of Shatter et al. (Renal excretion and volume distribution of some polyethylene glycols in the dog, Anal. Chem., 19 p. 32, 1947). The PEG assay of the high purity AHF can be seen in Table II.
TABLE 11 PEG content of high purity AHF AHF activity, PEG, Mg: PEG] Lot No rug/unit mg./ml. unit AHF Mean 61. 87 2. 407 0. 056 Standard deviation. 44. 58 1.500 0. 04407 EXAMPLE III Twenty-seven patients have received intermediate purity AHF concentrate prepared by the ethanol cryoprecipitation-TRIS method, 23 with classical hemophilia and 4 with von Willebrands disease, during 29 hospital admissions. Three of these hemophiliacs took part in studies to estimate the in vivo recovery of AHF, and the other 24 were treated for spontaneous or traumatic hemorrhage or to ensure hemostasis during and after surgery.
The following report on these patients is given in Table III. Their hemostatic response to the drug was excellent. Apart from Patient G. C., who had a demonstrable AHF inhibitor, and Patient B. B., in whom none was demonstrated, the concentrate maintained satisfactory AHF levels 25% of normal following surgery or severe trauma and 15 of normal following minor bleeding episodes), with normal hemostasis.
Two patients complained of circumoral tingling during the actual infusion but it stopped immediately afterward. Their vital signs were unaltered. The temperature in one patient (R. W.) was elevated after infusion of one batch of AHF but other batches had no such efiect. Hemolysis, observed in one patient (T. D.), might have been due to very low levels of anti-A blood group antibodies present in the preparation. Antibody proteins have a relatively long half life in the circulation (variously reported as 14 to 30 days), and there may be a cumulative effect during prolonged treatment. This complication was also reported with AHF concentrates prepared by other methods.
No other untoward reactions were noted even in patients who habitually had had severe allergic reactions to freshfrozen plasma.
TABLE 111 Summary of clinical data on intermediate-purity AHF prepared according to the ethanol cryoprecipitation-TRIB method Highest Units plasma AHF AHF level Duration of adminrecorded, Patient Diagnosis treatment istered 2 percent T.S Severe AHF deficiency, recovery study. 30 min. approx.-. 3,150 65. W.C do d0 3,700 90. .d0 do 3,150 55. V.
1st admission. Severe AHF deficiency, eraniotomy and evacuation of subdural hemorrhage 200: 2d admission..- Traumatic cerebral hemorrhage 68. 111.0 Severe AHF deficiency, post-traumatic subdural hern0rrhage 71. \V.D Severe lien1ophMia-hemorrhage into spinal cord 10 days. .000 153. N .M Moderate hemophilia (12% AHF), thoracetoruy and biopsy of mediastinal mass and 12 days 30,250 Unknown.
TABLE III.Con tinued Highest Units plasma AHF AHF level Duration of adminrecorded, Patient Diagnosis treatment istered percent 1.0 Severe hemophilia, reduction and repair of strangulated hernia 8 days 13, 800 50. LR Severe hemophilia, spontaneous intraabdominal and intrathoi'acie hemorrhage, epis- 14 days 34, 118 Unknown.
taxis, GI bleeding. A.R Severe hemophilia, GI bleeding 9 days 20,000 Do. ILA Gangrenous bowel 3 weeks approx 3 48, 000 100. 1.D Removal of medial meniscus from knee 2 weeks 49,160 100. B.B Severe hemophilia, fractured leg- 3 days- 7,590 1. E.F Severe hemophilia, torn knee ligaments 8 day 25,000 Unknown. J11 Severe hemophilia, lip laceration; previous administration of sufiicient FFP to cause 7 days 9,200 Do.
pulmonary congestion failed to stop bleeding. W.l" Severe hemophilia, extraction of 8 permanent teeth ..do 9, 000 35. G.O Two-year old hemophiliac with AHF inhibitor, bleeding from cutdown site 4 days- 4, 984 1. AJ Severe hemophilia, hematuria; no response to previous administration of FFI and 1 infusion 2,013 106.
1st admission... Severe hemophilia, hematomas of buttocks 24 hours 2d admission.... Hematomas of arm and hand 1 infusion W.A Severe hemophilia, hemorrhage into forearm following vein-puncture. ..do. D.W- Extraction of teeth 4 days. S.R ..do--. J .M 7 days L.M. Recovery experiment 1 infusion S.C. Extraction of teeth 18 hours B.U. (von W.) Traumatic hemarthrosis of shoulder 1 infusion J.H. (von W.) G.I. bleeding do 1 Plasma AHF level: 1%. 3 Approximately. 1 1 unit AHF=AHF activity in 1.0 ml. of fresh pooled normal plasma. 4 Von Willebrands disease.
EXAMPLE IV thereafter. The total volume of the concentrate injected Nineteen patients with classical hemophilia have reper day ranged from 16 to 72 (equlvalent ceived the high purity AHF concentrate prepared by the 10004700 P of'plasma); After the fiIst post'operatlve PEG method: 11 participated in studies to estimate in f y a Smgl? lnlectlon Was glven every 12 hoursp vivo recovery after intravenous injection and 2 particinon, bleedlng was Comparable to that Observed 111 normal pated in intramuscular injection studies. The concentrate Chlldfen, I10 POSt-OPBIQUVG bleedlng Occurred, and rate was administered to the other six to ensure hemostasis of healing was normal. No toxic reactions were encounduring and after surgery; they were first given this AHF tered. for Pefiods p to one Week, and lmefmedlate p y mate- When the AHF was injected intravenously into volunrial vgas substituted for the remainder of the treatment tears with severe hemophilia, approximately 70 percent perio of the original dose was recovered. AHF levels exceeding The followmg report on the 18 patlents of a 30 percent (adequate to prevent bleeding due to severe marized table. The hemostatic response to intravenous injection of the concentrate was excellent: satisfactory f accldenta} were obtalned after AHF level was maintained 25% f normal), blood single 10-30 ml. in ection. Little AHF was recovered 1n loss and wound healing were normal. the plasma, however, after intramuscular injection.
TABLE IV Summary of clinical data on high-purity AHF prepared according to the PEG method Highest Units plasma AHF AHF level Duration of adminrecorded, Patient Diagnosis treatment istered 1 percent 'I.D Severe AHF deficiency, dental surgery, repair of herniadays 91 GN Severe AHF deficiency, excision of pilonidal sinus 7 days. ,000 150 G.F- Severe AHF deficiency, I.M. recovery study 1 minute 1,080 1 W.A ..do ..do 4,800 1 1 1 unit AHF=AHF activity in 1.0 ml. of fresh pooled normal plasma. 2 This material of intermediate purity.
It can be seen that several of the patients were children Thus, it can be seen that the method of this invention, wi h severe h p whose ages ranged from 7 as exemplified by the preferred embodiment but not When treated with the AHF of the instant invention they i i thereto, can be used efficienfly to prepare a human ai ed hgmostaisis fluring g after g i AHF concentrate of intermediate or high purity which is r c onso rimar an erman n ee 22 s ig g g l performed on clinically useful and extremely stable in storage. Certain each patient. Plasma AHF levels above 25 percent were modlficatlons and embodlments can be made to thls maintained during the operative period and for a week vention without departing from the spirit or scope thereof.
What is claimed is:
1. A method of preparing highly purified AHF comprising the steps of:
(a) mixing plasma with from about 2 percent to about 4 percent by volume of ethanol at a temperature between about and about +4 C. to obtain a precipitate containing AHF;
(b) extracting said precipitate with a low ionic strength butler solution comprising tris (hydroxymethyl) aminomethane at room temperature to obtain a solution comprising AHF and said buffer solution;
(0) deprothrombinizing said solution with aluminum hydroxide gel;
(d) buffering said solution to a pH of about 5.6 to
(e) adding polyethylene glycol to said solution to a concentration of from about 3.0 percent to about 6.5 percent to obtain a supernatant solution and a precipitate;
(f) adding polyethylene glycol to said supernatant to a concentration of from about 10 to about 12 percent to obtain a precipitate including AHF; and,
(g) recovering AHF of from about 200 to about 400x purity.
2. The method of claim 1, wherein said plasma is mixed with about 3 percent by volume of ethanol.
3. The method of claim 1, wherein said low ionic strength buffer solution has a concentration of from about 0.005 to about 0.02 M and a pH of about 7.0.
4. The method of claim 1, wherein said solution is buffered in step (d) to a pH of from about 5.8 to about 6.4.
5. The method of claim 4, wherein said solution is buffered in step (d) to a pH of about 6.0.
6. The method of claim 1, wherein said solution is buffered in step (d) with a buffer comprising sodium citrate and citric acid.
7. The method of claim 1, wherein said polyethylene glycol is added in step (e) to a concentration of about 5.8 percent.
8. The method of claim 1, wherein said precipitate from step (f) is dissolved in 0.02 M tris (hydroxymethyl) aminomethane-citrate solution and sterile filtered through a filter having a pore size of about 0.22 microns before recovering said AHF.
9. The method of claim 1, wherein step (g) is performed by lyophilization.
10. Highly purified AHF prepared by the method of claim 1 utilizing human blood plasma as the starting material.
11. A composition consisting essentially of highly purified AHF prepared by the method of claim 1 in aqueous solution utilizing human blood plasma as the starting material.
References Cited UNITED STATES PATENTS 2,390,074 12/1945 Cohn 260-122 2,867,567 1/1959 Bidwell 424-177 3,415,804 12/1968 Polson 2601 12 FOREIGN PATENTS 883,549 11/1961 Great Britain 16774 H OTHER REFERENCES Nature, vol. 203, 1964, p. 312, Pool et al. The Proteins, vol. III, 1965, p. 372, Neurath. Blood, vol. 28, p. 1011, Johnson et al., December 1966.
HOWARD E. SCHAIN, Primary Examiner US. Cl. X.R. 424-101, 177