CA1115637A - Method of obtaining intermediate purity factor viii - Google Patents
Method of obtaining intermediate purity factor viiiInfo
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- CA1115637A CA1115637A CA344,000A CA344000A CA1115637A CA 1115637 A CA1115637 A CA 1115637A CA 344000 A CA344000 A CA 344000A CA 1115637 A CA1115637 A CA 1115637A
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- heparin
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Abstract
METHOD OF OBTAINING FACTOR VIII
ABSTRACT
A method of obtaining Factor VIII which comprises adding heparin to freshly obtained blood plasma collected into a calcium chelating anticoagulant or collecting blood plasma by plasma pheresis using heparin and a calcium chelating anticoagulant; freezing the plasma; resolubilizing the plasma;
isolating a cryoprecipitate from the plasma; resolubilizing the cryoprecipitate; adding a citrate saline heparin buffer to the resolubilized temperature of from about 0°C for a time in excess of about one hour, whereby Factor VIII present in the cryoprecipitate is insolubilized using heparin precipitable cold insoluble globulin; separating the Factor VIII rich precipitate; and isolating Factor VIII therefrom. The introduction of the cold insoluble globulin (CIg) step to Factor VIII
production results in markedly increased yields of Factor VIII in the cryoprecipitate and, as well, in the cold-insoluble globulin obtained from the cryoprecipitate. Using this procedure, 81% of the Factor VIII is recovered in the cryoprecipitate. The cold-insoluble globulin contains 62% of the starting Factor VIII activity. A final recovery of 666 units per litre of starting plasma is therefore obtained and the amount of protein is reduced to less than 1%. In addition, the procedure can be carried out in blood donor centers, although it is also envisaged to be useful in larger scale recovery of Factor VIII procedures.
ABSTRACT
A method of obtaining Factor VIII which comprises adding heparin to freshly obtained blood plasma collected into a calcium chelating anticoagulant or collecting blood plasma by plasma pheresis using heparin and a calcium chelating anticoagulant; freezing the plasma; resolubilizing the plasma;
isolating a cryoprecipitate from the plasma; resolubilizing the cryoprecipitate; adding a citrate saline heparin buffer to the resolubilized temperature of from about 0°C for a time in excess of about one hour, whereby Factor VIII present in the cryoprecipitate is insolubilized using heparin precipitable cold insoluble globulin; separating the Factor VIII rich precipitate; and isolating Factor VIII therefrom. The introduction of the cold insoluble globulin (CIg) step to Factor VIII
production results in markedly increased yields of Factor VIII in the cryoprecipitate and, as well, in the cold-insoluble globulin obtained from the cryoprecipitate. Using this procedure, 81% of the Factor VIII is recovered in the cryoprecipitate. The cold-insoluble globulin contains 62% of the starting Factor VIII activity. A final recovery of 666 units per litre of starting plasma is therefore obtained and the amount of protein is reduced to less than 1%. In addition, the procedure can be carried out in blood donor centers, although it is also envisaged to be useful in larger scale recovery of Factor VIII procedures.
Description
1~5~37 This invention is concerned with a method of obtalnlng Factor VIII without the use of chemical precipitating agents. The technique relies on the heparin-induced coprecipitation of Factor VIII with a plasma protein called cold-insoluble globulin (CIg).
In 1954, Thomas et al in a paper entitled "Cold precipitation by heparin of a protein in rabbit and human plasma" Proc. Soc. Exp. Biol. Med. 86:
813-819 reported the occurrence of heparin-induced cold or cryoprecipitate plasma from endotoxln-treated rabbits. This fraction has since come to be known as the heparin precipitable fraction of plasma. Subsequent studies reported in 1957 by Smith and Von Korff in a paper entitled "A
heparin-precipitable fraction of human plasma. I. Isolation and characterization of the fraction." J. Clin. Invest. 36: 596-604 and Smith in a paper entitled "A heparin-precipitable fraction of human plasma II.
Occurrence and significance of the fraction in normal individuals and in various disease states." J. Clin. Invest., 36: 605-616j showed that large amounts of this cold precipitable fraction were formed from plasmas of patients with certain inflamatory infectious and neo-plastic diseases when heparin was added to the plasma. In normal donors the amount produced was usually less. At this time it was established that fibrinogen was a major component of this fraction. A second minor component of the fraction was also ob~erved which was termed "cold-insoluble globulin". This cold-insoluble globulin (CIg) is known to be a normal plasma glycoprotein that is structurally and immunochemically distinct from fibrinogen and all other plasma proteins. CIg normally circulates at plasma levels of 0.33 -0.1g/litre. Cold-insoluble globulin in its various forms has been designated by a variety of terms, of which "fibronectin" has recently become most lllS~"37 accepted (The Structure and Biologic Activlties of Plasma Fibronectin, Mosesson, et al, ~lood, Volume 56, No. 2, pages 145-158).
In 1974, U.S. Patent No. 3,803,115 was issued to Fekete et al for a method of improving the yield of Factor VIII or A.H.F. obtained from blood plasma or plasma fractions which involved the addition of heparin to a concentrate of A.H.F. obtained from the plasma or plasma fraction by cryoprecipitation. This patent was reissued July 11, 1978 under Re 29698. In the reissue patent, Fekete et al indicate that the addition of heparin is made to an A.H.F.
concentrate obtained from blood plasma or a plasma fraction by cryoprecipitation which may then be further fractionated to obtain an even more concentrated form of A.H.F. In the reissue patent, column 2, lines 36 et al, it is indicated that the heparin is preferably added to the A.H.F. rich concentrate in addition to citrate. A preferred method of conducting the double anticoagulant addition is to add the heparin to the cryoprecipitate in the form of a heparinized, citrated-saline solution. When the A.H.F. rich cryoprecipitate is further fractionated to obtain a high purity, more concentrated form of A.H.F., heparin is preferably added twice, once to the lnltial cryoprecipitate and subsequently to the further fractionated A.H.F.
concentrate. In each case, heparin is preferably added along with citrate as a second anticoagulant.
It is especially important to note that in the original Fekete patent and the reissue patent, the plasma is received frozen from a donor centre and then a cryoprecipitate step is performed before the heparin is added. Heparin is added only to the cryoprecipitate or to the concentrate of A.H.F. after cryoprecipitation occurs. Reference should also be made to Col. 4 of the Fekete reissue patent, lines 46-49 wherein it is clear from the temperatures at which the cryoprecipitate is resolubilized, the CIg or fibronectin is lost and therefore unavailable to aid in the recovery of Factor VIII. According to 1~5~37 Fekete, the yield of Factor VIII is improved from 13.6 or 12.5~ in the non-heparinized procedure to 17% in the hepariDized procedure. Fekete indicates that this increase of approximately 4~ represents a 25~ increase over the non-heparinized procedure. However, as the Table found in column 5 of the patent, Example 1, would indicate, the total yield from the cryoprecipitate is low at 35~, representing only 350 units per litre of starting plasma. Therefore, the 17~ recovery in the final product represents only 170 units per litre of starting plasma. These calculations are based on the international reference standard of one unit per ml of plasma.
It will be clear from the above discussion that the cold precipitation of fibronectin and fibrinogen had been an established procedure for many years prior to the filing date of the original Fekete patent in 1972. It was generally known that addition of heparin or some other polysaccharide compound was necessary in order to effect precipitation of fibronectin in the cold.
However, at no point has the literature suggested or contained directions for the application of a cold-insoluble globulin or fibronectin step toward the production of Factor VIII. In fact, a 1975 paper by Mosher, entitled "Cross Linking of Cold-Insoluble Globulin by Fibrin-Stabilizing Factor", published in J.B.C. 250: 6614, 1975, indicated that cold-insoluble globulin was distinguished from antihemophilic factor (Factor VIII) by amino acid analysis and by the position of elution from 4~ agarose gels and by the electrophoretic mlgration in polyacrylamide gels.
Thus the present invention deals with the application of a procedure for production of fibronectin or cold-insoluble globulin (CIg) to the production of Factor VIII. The introduction of the cold-insoluble globulin cryoprecipitation step to Factor VIII production results in markedly increased yields of Factor VIII in the cryoprecipitate and, as well, in the cold-insoluble globulin obtained from the cryoprecipitate. Using this 1~15637 procedure, al% of the Factor VIII is recovered in the cryoprecipitate. The cold-insoluble globulin contains 62~ of the starting Factor VIII activity. A
final recovery of 666 units per litre of starting plasma is therefore obtained and the amount of protein is reduced to less than 1~. In addition the procedure can be carried out in blood donor centers, although it is also envisaged to be useful in larger scale recovery of Factor VIII procedures.
Thus, the present invention provides a method of obtaining Factor VIII
which comprises a) adding heparin to freshly obtained blood plasma collected into a calcium chelating anticoagulant or collecting blood plasma by plasmapheresis using heparin and a calcium chelating anticoagulant;
b) freezing the plasma;
c) resolubilizing the plasma;
d) isolating a cryoprecipitate from the plasma;
e) resolubilizing the cryoprecipitate;
f) adding a citrate saline heparin buffer to the resolubilized cryoprecipitate;
g) incubating the buffered, resolubilized cryoprecipitate at a temperature from about 0 to about 10C for a time in excess of about one hour, whereby Factor VIII present in the cryoprecipitate is insolubilized using heparin precipitable cold insoluble globulin and the resulting Factor VIII
rich precipitate also includes cold insoluble globulin;
h) separating the Factor VIII rich precipitate; and i) isolating Factor VIII therefrom The calcium chelating anticoagulant can be selected from any of the well known anticoagulants which function by lowering the physiological level of calcium in the blood or blood plasma. Preferred types of such anticoagulants are the citrate anticoagulants, ethylenediaminetetraacetic acid (EDTA), and .~
i637 ) oxalates. Of these, the citrate anticoagulants are preferred. The preferred citrate anticoagulants include acid-citrate-dextrose (ACD) and citrate-phosphate-dextrose (CPD).
The essential steps of the method of this invention are the addition of the citrate saline heparin (CSH) buffer to the resolubilized cryoprecipitate and the incubation of the cryoprecipitate.
The buffer is preferably added in amounts ranging from about 2 ~ about 10 ml per cryoprecipitate bag. More preferably, it is added in amounts from about 2 to about 4 ml per cryoprecipitate bag and most preferably, an addition of 2.5 ml per cryoprecipitate bag is made. The buffer preferably has a composition of 0.2 M sodium citrate, 0.9% by weight of sodium chloride and 1 unit per ml of buffer of sodium heparin. The pH of the buffer is maintained at 7.2.
The incubation, as stated before, is carried out at about 0 to about 10C for a time in excess of about one hour. Preferably, it is conducted at 0C for about two hours.
Cold insoluble globulin (CIg) is not insolubilized after treatment of CPD
plasma with heparin. The precipitate of the CIg is not formed until a specific set of conditions are met. Thus, while these conditions include introduction of heparin to fresh CPD plasma, this is not sufficient in itself to produce CIg. Indeed, visual observation indicates that this step alone does not produce the CIg since, when present, this material is seen as a white deposit at the bottom of a blood bag. These steps are the addition of the CSH
buffer to the cryoprecipitate and the cold incubation step which permits formation of the precipitating globulin. If these combined steps are not employed, cold insoluble globulin is not formed even in the presence of heparin.
More specifically, the invention may be characterized as a method of ' ~ -l~lSS~37 obtaining Factor VIII which comprises a) adding heparin to freshly obtained blood plasma collected into a citrate-phosphate-dextrose anticoagulant or collecting blood plasma by plasmapheresis using heparin and sodium citrate anticoagulant;
b) freezing the plasma to about -80C;
c) resolubiliæing the plasma at a temperature from about 0 to about 10C for from about 30 to about 120 minutes, preferably at about 4c for about 75 minutes;
d) centrifuging the resolubilized plasma to isolate a cryoprecipitate;
e) resolubilizing the cryoprecipitate at a temperature of about 37C for about 2-5 minutes;
f) adding from about 2 to about 10 ml citrate saline heparin buffer to each resolubilized cryoprecipitate bag;
g) incubating the buffered, resolubilized cryoprecipitate at about 0C
for about two hours, whereby Factor VIII present in the cryoprecipitate is insolubilized using heparin precipitable cold insoluble globulin, the resulting Factor VIII rich precipitate also including cold insoluble globulin;
h) separating the Factor VIII rich precipitate; and i) isolating the Factor VIII therefrom.
Pactor VIII may be isolated from the Factor VIII rich precipitate by wa~hing the insoluble precipitate in the cold at a temperature of from about 0C to about 10C, preferably at about 0C with citrate saline heparin buffer. The precipitate is then drained and resolubiliæed with further citrate saline heparin buffer, preferably at about 37C for 2 to 5 min. The Factor VIII rich solution can then be separated from the remaining debris, most easily by centrifugation. The Factor VIII rich solution obtained will be of intermediate purity when the starting plasma is obtained from whole ^
~15~3'7 blood from which the red blood cells have been separated. In the case where the starting plasma is pheresis plasma, the Factor VIII will have an activity which qualifies it as high purity.
Alternatively or when required, the Factor VIII rich precipitate can be subjected to conventional processes for obtaining high purity Factor VIII, examples of which are fractionation by precipitation, by glycine, ethanol, ethanol-glycine, polyethylene glycol, or glycine-polyethylene glycol precipitation and/or other known purification agents.
The heparin is preferably added at a concentration of 1 unit heparin per - 10 ml of plasma. Although a range of from about 1 to about 10 units heparin per ml of plasma is effective. Sodium heparin has proved effective in this method.
The method in precise terms involves freezing the fresh CPD plasma with the heparin added thereto at -80C and then reæolubilizing at 4C for 75 minutes, after which it is centrifuged (spin 7000 x g, 7 min. at 0C). The resulting cryoprecipitate is allowed to resolubilize at 37C for 2-5 minutes, after which 2.5 ml of CSH ~citrate saline heparin) buffer is added per cryoprecipitate bag. The resolubilized, pooled cryoprecipitate is then lncubated at 0C for two hours in a refrigerated water bath. The Factor VIII present in the cryoprecipitate pool now insolubilizes along with the cold-insoluble globulin, and the total insoluble precipitate is then separated from the Factor VIII poor supernatant by centrifugation (spin 7000 x g, 7 min.
at 0C). After draining off the supernatant, the cold-insoluble precipitate 1~ washed at 0C with 10 ml of cold CSH buffer, drained and allowed to resolubilize at 37C for five minutes with from 2-10 ml, preferably two ml of CSH buffer per original cryoprecipitate bag. A further centrifugation (~pln 3700 x g, 6 min.) is conducted and the Factor VIII rich solution is separated from the debris.
In the enclosed drawing which is used to illustrate the pres~nt invention l~S637 Figure 1 is a schematic drawing of the specific steps of the method of the invention, the particular steps being illustrated for a pool of six units of plasma. The previous specifically described method is illustrated by this drawing.
In the following Table 1, there is provided an analysis of the method of the invention. It will be seen that one unit of sodium heparin per ml of freshly obtained human blood plasma was added. Six bags or units of plasma were used to illustrate the method. The table sets out the total units of Factor VIII present in each of the fractions: starting plasma, cryoprecipitate pool and cold-insoluble precipitate as well as the total weight of protein, the specific activity of the Factor VIII units per milligram of protein, the purity of the Factor VIII and the volumes of each of the fractions.
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i 1~15637 In the following Table 2, the recovering of Factor VIII is expressed per litre of starting plasma. It will be seen that the cold-insoluble globulin precipitate provides 666 units per litre of Factor VIII or a 62~ recovery.
1 This can be compared with the recovery reported by Fekete et al in column 5, '' Example 1 of the reissue patent previously mentioned. The 17~ recovery in the final product obtained by Fekete represents only 170 units per litre of starting plasma.
Recovery of Factor VIII
Expressed per Litre of Starting Plasma~
Fraction Units/Litre ~ Recovery .
Plasma 1069 100 Cryoprecipitate868 81 Pool Cold Insoluble666 62 Globulin Precipitate -* Based on pools of 6 units of plasma In the following Table 3, there is found an analysis of the total contents of the starting plasma fraction, cryoprecipitate pool fraction and cold-insoluble precipitate fraction.
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1115~i37 Recovery of F.VIII:C/ml From Citrate and 1 Unit Heparin Per ml Pheresis Plasma by the Cold-Insoluble Cryoprecipitate Technique Sample ~ F.VIII: Volume Total % Recovery Unit/Liter C/ml Units Over Starting Plasma F.VIII:C Plasma _ Tubes:
CPD Plasma 60~/ml - - - -Citrate Plasma 60~/ml - - - -Pheresis:
Citrate Plasma 60~/ml 2032 mls 1219U 100~ 600U/L
and Heparin (1 U/ml) Cryoprecipitate 1120~/ml85 mls 1015U 83% 500U/L
Pool Cold-Insoluble 2520~/ml28.1 mls 755U 62~ 372U/L
Cryoprecipitate ~Washed) Four volunteerR (3 O's, lA) were plasmapheresed on a model 50 Haemonetics pheresis maahine with 1 part 4 g~ sodium citrate per 14 parts blood. They were also collected into CPD or citrate tubes as usual.
Specific Activity of citrate and heparin pheresis plasma fractions Fraction Total Total Specific Purity Over F.VIII:C Protein Activity Plasma (mg) Plasma 1219U195,0720.006U/mg (Citrate ~ 1 Unit Heparin/ml) Cryoprecipitate 1015U 5,032 0.201U/mg 33.5x Pool Cold-Insoluble 755.3U 786.8 0.96U/mg 160x Cryoprecipitate SUPPLEMENTARY DISCLOSURE
mis invention provides a method of obtaining Factor VIII which comprises a) adding heparin to freshly obtalned blood plasma collected into a citrate-phosphate-dextrose anticoagulant or collecting blood plasma by plasmapheresis using heparin and sodium citrate anticoagulant;
b) freezing the plasma to about -80C;
c) resolubilizing the plasma at a temperature of from about 0 to about 10C for from about 30 to about 120 minutes;
d) centrifuging the resolubilized plasma to isolate a cryoprecipitate;
e) resolubilizing the cryoprecipitate at a temperature of about 37C for about 2-5 minutes;
f) adding from about 2 to about 10 ml of citrate saline heparin buffer per bag of the resolubilized cryoprecipitate;
g) incubating the buffered, resolubilized cryoprecipitate at about 0C
for about two hours whereby Factor VIII present in the cryoprecipitate is insolubilized using heparin precipitable cold insoluble globulin and the resulting Factor VIII rich precipitate also includes cold insoluble globulin;
h) separating the Factor VIII rich precipitate; and i) isolating the Factor VIII therefrom.
The invention also provides a method of obtaining Factor VIII which comprises a) adding heparin to freshly obtained blood plasma collected into a cltrate-phosphate-dextrose anticoagulant or collecting blood plasma by plasmapheresis using heparin and sodium citrate anticoagulant;
b) freezing the plasma to about -80C;
c) resolubilizing the plasma at a temperature of from about 0 to about 10C for from about 30 to about 120 minutes;
~llS637 d) centrifuging the resolubilized plasma to isolate a cryoprecipitate;
e) resolubilizing the cryoprecipitate at a temperature of about 37C for about 2-5 minutes;
f) adding from about 2 to about 10 ml of citrate saline heparin buffer per bag of the resolubilized cryoprecipitate;
g) incubating the buffered, resolubilized cryoprecipitate at about 0C
for about two hours whereby Factor VIII present in the cryoprecipitate is insolubilized using heparin precipitable cold insoluble globulin and the resulting Factor VIII rich precipitate also includes cold insoluble globulin;
h) separating the Factor VIII rich precipitate: and i) isolating the Factor VIII therefrom.
Application of the cryo~recipitate-cold insoluble globulin procedure to citrate plasma obtained by plasmapheresis using the Haemonetics Model 50 maahine with the plasma made to 1 unit of sodium heparin per ml of plasma also produces a Factor VIII concentrate of considerable potency. This technique results in the recovery of 81-83~ of the initial plasma Factor VIII:C activity in the cryoprecipitate and an overall recovery in the final cold insoluble globulln precipitate of 56-62~ of the initial plasma Factor VIII activity.
The specific activity of the final product is 0.96-0.99 units/mg of protein with a 99-160 fold increase in purity compared to plasma. As indicated in Tables 4 and 5, the data indicate that, when using plasmapheresis plasma, it ls possible to increase the recovery to produce a concentrate with a specific activity which qualifies the compound as high purity tgreater than 1 unit/mg).
~ ,.
.
In 1954, Thomas et al in a paper entitled "Cold precipitation by heparin of a protein in rabbit and human plasma" Proc. Soc. Exp. Biol. Med. 86:
813-819 reported the occurrence of heparin-induced cold or cryoprecipitate plasma from endotoxln-treated rabbits. This fraction has since come to be known as the heparin precipitable fraction of plasma. Subsequent studies reported in 1957 by Smith and Von Korff in a paper entitled "A
heparin-precipitable fraction of human plasma. I. Isolation and characterization of the fraction." J. Clin. Invest. 36: 596-604 and Smith in a paper entitled "A heparin-precipitable fraction of human plasma II.
Occurrence and significance of the fraction in normal individuals and in various disease states." J. Clin. Invest., 36: 605-616j showed that large amounts of this cold precipitable fraction were formed from plasmas of patients with certain inflamatory infectious and neo-plastic diseases when heparin was added to the plasma. In normal donors the amount produced was usually less. At this time it was established that fibrinogen was a major component of this fraction. A second minor component of the fraction was also ob~erved which was termed "cold-insoluble globulin". This cold-insoluble globulin (CIg) is known to be a normal plasma glycoprotein that is structurally and immunochemically distinct from fibrinogen and all other plasma proteins. CIg normally circulates at plasma levels of 0.33 -0.1g/litre. Cold-insoluble globulin in its various forms has been designated by a variety of terms, of which "fibronectin" has recently become most lllS~"37 accepted (The Structure and Biologic Activlties of Plasma Fibronectin, Mosesson, et al, ~lood, Volume 56, No. 2, pages 145-158).
In 1974, U.S. Patent No. 3,803,115 was issued to Fekete et al for a method of improving the yield of Factor VIII or A.H.F. obtained from blood plasma or plasma fractions which involved the addition of heparin to a concentrate of A.H.F. obtained from the plasma or plasma fraction by cryoprecipitation. This patent was reissued July 11, 1978 under Re 29698. In the reissue patent, Fekete et al indicate that the addition of heparin is made to an A.H.F.
concentrate obtained from blood plasma or a plasma fraction by cryoprecipitation which may then be further fractionated to obtain an even more concentrated form of A.H.F. In the reissue patent, column 2, lines 36 et al, it is indicated that the heparin is preferably added to the A.H.F. rich concentrate in addition to citrate. A preferred method of conducting the double anticoagulant addition is to add the heparin to the cryoprecipitate in the form of a heparinized, citrated-saline solution. When the A.H.F. rich cryoprecipitate is further fractionated to obtain a high purity, more concentrated form of A.H.F., heparin is preferably added twice, once to the lnltial cryoprecipitate and subsequently to the further fractionated A.H.F.
concentrate. In each case, heparin is preferably added along with citrate as a second anticoagulant.
It is especially important to note that in the original Fekete patent and the reissue patent, the plasma is received frozen from a donor centre and then a cryoprecipitate step is performed before the heparin is added. Heparin is added only to the cryoprecipitate or to the concentrate of A.H.F. after cryoprecipitation occurs. Reference should also be made to Col. 4 of the Fekete reissue patent, lines 46-49 wherein it is clear from the temperatures at which the cryoprecipitate is resolubilized, the CIg or fibronectin is lost and therefore unavailable to aid in the recovery of Factor VIII. According to 1~5~37 Fekete, the yield of Factor VIII is improved from 13.6 or 12.5~ in the non-heparinized procedure to 17% in the hepariDized procedure. Fekete indicates that this increase of approximately 4~ represents a 25~ increase over the non-heparinized procedure. However, as the Table found in column 5 of the patent, Example 1, would indicate, the total yield from the cryoprecipitate is low at 35~, representing only 350 units per litre of starting plasma. Therefore, the 17~ recovery in the final product represents only 170 units per litre of starting plasma. These calculations are based on the international reference standard of one unit per ml of plasma.
It will be clear from the above discussion that the cold precipitation of fibronectin and fibrinogen had been an established procedure for many years prior to the filing date of the original Fekete patent in 1972. It was generally known that addition of heparin or some other polysaccharide compound was necessary in order to effect precipitation of fibronectin in the cold.
However, at no point has the literature suggested or contained directions for the application of a cold-insoluble globulin or fibronectin step toward the production of Factor VIII. In fact, a 1975 paper by Mosher, entitled "Cross Linking of Cold-Insoluble Globulin by Fibrin-Stabilizing Factor", published in J.B.C. 250: 6614, 1975, indicated that cold-insoluble globulin was distinguished from antihemophilic factor (Factor VIII) by amino acid analysis and by the position of elution from 4~ agarose gels and by the electrophoretic mlgration in polyacrylamide gels.
Thus the present invention deals with the application of a procedure for production of fibronectin or cold-insoluble globulin (CIg) to the production of Factor VIII. The introduction of the cold-insoluble globulin cryoprecipitation step to Factor VIII production results in markedly increased yields of Factor VIII in the cryoprecipitate and, as well, in the cold-insoluble globulin obtained from the cryoprecipitate. Using this 1~15637 procedure, al% of the Factor VIII is recovered in the cryoprecipitate. The cold-insoluble globulin contains 62~ of the starting Factor VIII activity. A
final recovery of 666 units per litre of starting plasma is therefore obtained and the amount of protein is reduced to less than 1~. In addition the procedure can be carried out in blood donor centers, although it is also envisaged to be useful in larger scale recovery of Factor VIII procedures.
Thus, the present invention provides a method of obtaining Factor VIII
which comprises a) adding heparin to freshly obtained blood plasma collected into a calcium chelating anticoagulant or collecting blood plasma by plasmapheresis using heparin and a calcium chelating anticoagulant;
b) freezing the plasma;
c) resolubilizing the plasma;
d) isolating a cryoprecipitate from the plasma;
e) resolubilizing the cryoprecipitate;
f) adding a citrate saline heparin buffer to the resolubilized cryoprecipitate;
g) incubating the buffered, resolubilized cryoprecipitate at a temperature from about 0 to about 10C for a time in excess of about one hour, whereby Factor VIII present in the cryoprecipitate is insolubilized using heparin precipitable cold insoluble globulin and the resulting Factor VIII
rich precipitate also includes cold insoluble globulin;
h) separating the Factor VIII rich precipitate; and i) isolating Factor VIII therefrom The calcium chelating anticoagulant can be selected from any of the well known anticoagulants which function by lowering the physiological level of calcium in the blood or blood plasma. Preferred types of such anticoagulants are the citrate anticoagulants, ethylenediaminetetraacetic acid (EDTA), and .~
i637 ) oxalates. Of these, the citrate anticoagulants are preferred. The preferred citrate anticoagulants include acid-citrate-dextrose (ACD) and citrate-phosphate-dextrose (CPD).
The essential steps of the method of this invention are the addition of the citrate saline heparin (CSH) buffer to the resolubilized cryoprecipitate and the incubation of the cryoprecipitate.
The buffer is preferably added in amounts ranging from about 2 ~ about 10 ml per cryoprecipitate bag. More preferably, it is added in amounts from about 2 to about 4 ml per cryoprecipitate bag and most preferably, an addition of 2.5 ml per cryoprecipitate bag is made. The buffer preferably has a composition of 0.2 M sodium citrate, 0.9% by weight of sodium chloride and 1 unit per ml of buffer of sodium heparin. The pH of the buffer is maintained at 7.2.
The incubation, as stated before, is carried out at about 0 to about 10C for a time in excess of about one hour. Preferably, it is conducted at 0C for about two hours.
Cold insoluble globulin (CIg) is not insolubilized after treatment of CPD
plasma with heparin. The precipitate of the CIg is not formed until a specific set of conditions are met. Thus, while these conditions include introduction of heparin to fresh CPD plasma, this is not sufficient in itself to produce CIg. Indeed, visual observation indicates that this step alone does not produce the CIg since, when present, this material is seen as a white deposit at the bottom of a blood bag. These steps are the addition of the CSH
buffer to the cryoprecipitate and the cold incubation step which permits formation of the precipitating globulin. If these combined steps are not employed, cold insoluble globulin is not formed even in the presence of heparin.
More specifically, the invention may be characterized as a method of ' ~ -l~lSS~37 obtaining Factor VIII which comprises a) adding heparin to freshly obtained blood plasma collected into a citrate-phosphate-dextrose anticoagulant or collecting blood plasma by plasmapheresis using heparin and sodium citrate anticoagulant;
b) freezing the plasma to about -80C;
c) resolubiliæing the plasma at a temperature from about 0 to about 10C for from about 30 to about 120 minutes, preferably at about 4c for about 75 minutes;
d) centrifuging the resolubilized plasma to isolate a cryoprecipitate;
e) resolubilizing the cryoprecipitate at a temperature of about 37C for about 2-5 minutes;
f) adding from about 2 to about 10 ml citrate saline heparin buffer to each resolubilized cryoprecipitate bag;
g) incubating the buffered, resolubilized cryoprecipitate at about 0C
for about two hours, whereby Factor VIII present in the cryoprecipitate is insolubilized using heparin precipitable cold insoluble globulin, the resulting Factor VIII rich precipitate also including cold insoluble globulin;
h) separating the Factor VIII rich precipitate; and i) isolating the Factor VIII therefrom.
Pactor VIII may be isolated from the Factor VIII rich precipitate by wa~hing the insoluble precipitate in the cold at a temperature of from about 0C to about 10C, preferably at about 0C with citrate saline heparin buffer. The precipitate is then drained and resolubiliæed with further citrate saline heparin buffer, preferably at about 37C for 2 to 5 min. The Factor VIII rich solution can then be separated from the remaining debris, most easily by centrifugation. The Factor VIII rich solution obtained will be of intermediate purity when the starting plasma is obtained from whole ^
~15~3'7 blood from which the red blood cells have been separated. In the case where the starting plasma is pheresis plasma, the Factor VIII will have an activity which qualifies it as high purity.
Alternatively or when required, the Factor VIII rich precipitate can be subjected to conventional processes for obtaining high purity Factor VIII, examples of which are fractionation by precipitation, by glycine, ethanol, ethanol-glycine, polyethylene glycol, or glycine-polyethylene glycol precipitation and/or other known purification agents.
The heparin is preferably added at a concentration of 1 unit heparin per - 10 ml of plasma. Although a range of from about 1 to about 10 units heparin per ml of plasma is effective. Sodium heparin has proved effective in this method.
The method in precise terms involves freezing the fresh CPD plasma with the heparin added thereto at -80C and then reæolubilizing at 4C for 75 minutes, after which it is centrifuged (spin 7000 x g, 7 min. at 0C). The resulting cryoprecipitate is allowed to resolubilize at 37C for 2-5 minutes, after which 2.5 ml of CSH ~citrate saline heparin) buffer is added per cryoprecipitate bag. The resolubilized, pooled cryoprecipitate is then lncubated at 0C for two hours in a refrigerated water bath. The Factor VIII present in the cryoprecipitate pool now insolubilizes along with the cold-insoluble globulin, and the total insoluble precipitate is then separated from the Factor VIII poor supernatant by centrifugation (spin 7000 x g, 7 min.
at 0C). After draining off the supernatant, the cold-insoluble precipitate 1~ washed at 0C with 10 ml of cold CSH buffer, drained and allowed to resolubilize at 37C for five minutes with from 2-10 ml, preferably two ml of CSH buffer per original cryoprecipitate bag. A further centrifugation (~pln 3700 x g, 6 min.) is conducted and the Factor VIII rich solution is separated from the debris.
In the enclosed drawing which is used to illustrate the pres~nt invention l~S637 Figure 1 is a schematic drawing of the specific steps of the method of the invention, the particular steps being illustrated for a pool of six units of plasma. The previous specifically described method is illustrated by this drawing.
In the following Table 1, there is provided an analysis of the method of the invention. It will be seen that one unit of sodium heparin per ml of freshly obtained human blood plasma was added. Six bags or units of plasma were used to illustrate the method. The table sets out the total units of Factor VIII present in each of the fractions: starting plasma, cryoprecipitate pool and cold-insoluble precipitate as well as the total weight of protein, the specific activity of the Factor VIII units per milligram of protein, the purity of the Factor VIII and the volumes of each of the fractions.
l~lS637 ~_ ~ o 3 li 1~ 1 o ~:
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_ 9 _ B
.
i 1~15637 In the following Table 2, the recovering of Factor VIII is expressed per litre of starting plasma. It will be seen that the cold-insoluble globulin precipitate provides 666 units per litre of Factor VIII or a 62~ recovery.
1 This can be compared with the recovery reported by Fekete et al in column 5, '' Example 1 of the reissue patent previously mentioned. The 17~ recovery in the final product obtained by Fekete represents only 170 units per litre of starting plasma.
Recovery of Factor VIII
Expressed per Litre of Starting Plasma~
Fraction Units/Litre ~ Recovery .
Plasma 1069 100 Cryoprecipitate868 81 Pool Cold Insoluble666 62 Globulin Precipitate -* Based on pools of 6 units of plasma In the following Table 3, there is found an analysis of the total contents of the starting plasma fraction, cryoprecipitate pool fraction and cold-insoluble precipitate fraction.
~1~15~;37 w ~- ~ a ~DP O ~ O ~ ~ ~ P~
8 P ~g ~ ~ r ~ ~ ~ a ~4 ~R o ~ ~ o ~
~ ~ o ~ ~ ~D ~d ~ ~ g ~ O O O N H _ S
æ ~ ? ~ o l ~ -- ^ I S I ~1~
. ~ . ~ O ~ ~ 3 ~ . ~- O
~ ~ a ? ~ ? 11~
~ O P' ~ N a~ ~ e r~
~ S 3 ~ ~ ~:1 _ _ _ ~ _ ~ t~ -~ ~ e o ~ ~ ~ J ~3 ~
~ 3 ~ 00 3~ 00 N 1~ ) ~t O
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P WdP (J~ dP N _ ~ 4 _ _ _ - ~D
1115~i37 Recovery of F.VIII:C/ml From Citrate and 1 Unit Heparin Per ml Pheresis Plasma by the Cold-Insoluble Cryoprecipitate Technique Sample ~ F.VIII: Volume Total % Recovery Unit/Liter C/ml Units Over Starting Plasma F.VIII:C Plasma _ Tubes:
CPD Plasma 60~/ml - - - -Citrate Plasma 60~/ml - - - -Pheresis:
Citrate Plasma 60~/ml 2032 mls 1219U 100~ 600U/L
and Heparin (1 U/ml) Cryoprecipitate 1120~/ml85 mls 1015U 83% 500U/L
Pool Cold-Insoluble 2520~/ml28.1 mls 755U 62~ 372U/L
Cryoprecipitate ~Washed) Four volunteerR (3 O's, lA) were plasmapheresed on a model 50 Haemonetics pheresis maahine with 1 part 4 g~ sodium citrate per 14 parts blood. They were also collected into CPD or citrate tubes as usual.
Specific Activity of citrate and heparin pheresis plasma fractions Fraction Total Total Specific Purity Over F.VIII:C Protein Activity Plasma (mg) Plasma 1219U195,0720.006U/mg (Citrate ~ 1 Unit Heparin/ml) Cryoprecipitate 1015U 5,032 0.201U/mg 33.5x Pool Cold-Insoluble 755.3U 786.8 0.96U/mg 160x Cryoprecipitate SUPPLEMENTARY DISCLOSURE
mis invention provides a method of obtaining Factor VIII which comprises a) adding heparin to freshly obtalned blood plasma collected into a citrate-phosphate-dextrose anticoagulant or collecting blood plasma by plasmapheresis using heparin and sodium citrate anticoagulant;
b) freezing the plasma to about -80C;
c) resolubilizing the plasma at a temperature of from about 0 to about 10C for from about 30 to about 120 minutes;
d) centrifuging the resolubilized plasma to isolate a cryoprecipitate;
e) resolubilizing the cryoprecipitate at a temperature of about 37C for about 2-5 minutes;
f) adding from about 2 to about 10 ml of citrate saline heparin buffer per bag of the resolubilized cryoprecipitate;
g) incubating the buffered, resolubilized cryoprecipitate at about 0C
for about two hours whereby Factor VIII present in the cryoprecipitate is insolubilized using heparin precipitable cold insoluble globulin and the resulting Factor VIII rich precipitate also includes cold insoluble globulin;
h) separating the Factor VIII rich precipitate; and i) isolating the Factor VIII therefrom.
The invention also provides a method of obtaining Factor VIII which comprises a) adding heparin to freshly obtained blood plasma collected into a cltrate-phosphate-dextrose anticoagulant or collecting blood plasma by plasmapheresis using heparin and sodium citrate anticoagulant;
b) freezing the plasma to about -80C;
c) resolubilizing the plasma at a temperature of from about 0 to about 10C for from about 30 to about 120 minutes;
~llS637 d) centrifuging the resolubilized plasma to isolate a cryoprecipitate;
e) resolubilizing the cryoprecipitate at a temperature of about 37C for about 2-5 minutes;
f) adding from about 2 to about 10 ml of citrate saline heparin buffer per bag of the resolubilized cryoprecipitate;
g) incubating the buffered, resolubilized cryoprecipitate at about 0C
for about two hours whereby Factor VIII present in the cryoprecipitate is insolubilized using heparin precipitable cold insoluble globulin and the resulting Factor VIII rich precipitate also includes cold insoluble globulin;
h) separating the Factor VIII rich precipitate: and i) isolating the Factor VIII therefrom.
Application of the cryo~recipitate-cold insoluble globulin procedure to citrate plasma obtained by plasmapheresis using the Haemonetics Model 50 maahine with the plasma made to 1 unit of sodium heparin per ml of plasma also produces a Factor VIII concentrate of considerable potency. This technique results in the recovery of 81-83~ of the initial plasma Factor VIII:C activity in the cryoprecipitate and an overall recovery in the final cold insoluble globulln precipitate of 56-62~ of the initial plasma Factor VIII activity.
The specific activity of the final product is 0.96-0.99 units/mg of protein with a 99-160 fold increase in purity compared to plasma. As indicated in Tables 4 and 5, the data indicate that, when using plasmapheresis plasma, it ls possible to increase the recovery to produce a concentrate with a specific activity which qualifies the compound as high purity tgreater than 1 unit/mg).
~ ,.
.
Claims (27)
PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A method of obtaining Factor VIII which comprises a) adding heparin to freshly obtained blood plasma collected into a calcium chelating anticoagulant or collecting blood plasma by plasma pheresis using heparin and a calcium chelating anticoagulant;
b) freezing the plasma;
c) resolubilizing the plasma;
d) isolating a cryoprecipitate from the plasma;
e) resolubilizing the cryoprecipitate;
f) adding a citrate saline heparin buffer to the resolubilized cryoprecipitate;
g) incubating the buffered, resolubilized cryoprecipitate at a temperature of from about 0° to about 10°C for a time in excess of about one hour, whereby Factor VIII present in the cryoprecipitate is insolubilized using heparin precipitable cold insoluble globulin and the resulting Factor VIII
rich precipitate also includes cold insoluble globulin;
h) separating the Factor VIII rich precipitate; and i) isolating Factor VIII therefrom.
b) freezing the plasma;
c) resolubilizing the plasma;
d) isolating a cryoprecipitate from the plasma;
e) resolubilizing the cryoprecipitate;
f) adding a citrate saline heparin buffer to the resolubilized cryoprecipitate;
g) incubating the buffered, resolubilized cryoprecipitate at a temperature of from about 0° to about 10°C for a time in excess of about one hour, whereby Factor VIII present in the cryoprecipitate is insolubilized using heparin precipitable cold insoluble globulin and the resulting Factor VIII
rich precipitate also includes cold insoluble globulin;
h) separating the Factor VIII rich precipitate; and i) isolating Factor VIII therefrom.
2. A method as claimed in claim 1 wherein the calcium chelating anticoagulant is selected from citrate anticoagulants,ethylenediaminetetraacetic acid and oxalates.
3. A method as claimed in claim 1 wherein the calcium chelating anticoagulant is a citrate anticoagulant.
4. A method as claimed in claim 3 wherein the anticoagulant is acid-citrate-dextrose.
5. A method as claimed in claim 3 wherein the anticoagulant is citrate-phosphate-dextrose.
6. A method as claimed in Claim 1 wherein the blood plasma is collected by plasmapheresis using heparin and sodium citrate.
7. A method of obtaining Factor VIII which comprises a) adding heparin to freshly obtained blood plasma collected into a citrate-phosphate-dextrose anticoagulant;
b) freezing the plasma to about -80°C;
c) resolubilizing the plasma at a temperature of from about 0° to about 10°C for from about 30 to about 120 minutes;
d) centrifuging the resolubilized plasma to isolate a cryoprecipitate;
e) resolubilizing the cryoprecipitate at a temperature of about 37°C for about 2-5 minutes;
f) adding from about 2 to about 10 ml of citrate saline heparin buffer per bag of the resolubilized cryoprecipitate;
g) incubating the buffered, resolubilized cryoprecipitate at about 0°C
for about two hours whereby Factor VIII present in the cryoprecipitate is insolubilized using heparin precipitable cold insoluble globulin and the resulting Factor VIII rich precipitate also includes cold insoluble globulin;
h) separating the Factor VIII rich precipitate; and i) isolating the Factor VIII therefrom.
b) freezing the plasma to about -80°C;
c) resolubilizing the plasma at a temperature of from about 0° to about 10°C for from about 30 to about 120 minutes;
d) centrifuging the resolubilized plasma to isolate a cryoprecipitate;
e) resolubilizing the cryoprecipitate at a temperature of about 37°C for about 2-5 minutes;
f) adding from about 2 to about 10 ml of citrate saline heparin buffer per bag of the resolubilized cryoprecipitate;
g) incubating the buffered, resolubilized cryoprecipitate at about 0°C
for about two hours whereby Factor VIII present in the cryoprecipitate is insolubilized using heparin precipitable cold insoluble globulin and the resulting Factor VIII rich precipitate also includes cold insoluble globulin;
h) separating the Factor VIII rich precipitate; and i) isolating the Factor VIII therefrom.
8. A method as claimed in claim 7 wherein the resolubilization of the plasma in step c) is carried out at about 4°C for about 75 minutes.
9. A method as claimed in claim 1, 7 or 8 wherein Factor VIII is isolated from the Factor VIII rich precipitate by washing the insoluble precipitate in the cold at a temperature of from about 0° to about 10°C with citrate saline heparin buffer; draining the precipitate and resolubilizing it in the presence of further citrate saline heparin buffer.
10. A method as claimed in claim 1, 7 or 8 wherein the Factor VIII is isolated from the Factor VIII rich precipitate by washing the insoluble precipitate in the cold at about 0°C with citrate saline heparin buffer; draining the precipitate and resolubilizing it at about 37°C in the presence of further citrate saline heparin buffer.
11. A method as claimed in claim 1, 7 or 8 wherein the isolated Factor VIII is subjected to further purification procedures.
12. A method as claimed in claim 1, 7 or 8 wherein the isolated Factor VIII is subjected to further purification procedures selected from fractionation by precipitation by glycine, ethanol, ethanol-glycine, polyethylene glycol and glycine-polyethylene glycol precipitation.
13. A method as claimed in claim 1, 7 or 8 wherein Factor VIII is isolated from the Factor VIII rich precipitate by washing the insoluble precipitate in the cold at a temperature of from about 0° to about 10°C with citrate saline heparin buffer; draining the precipitate and resolubilizing it in the presence of further citrate saline heparin buffer and the isolated Factor VIII
is subjected to further purification procedures.
is subjected to further purification procedures.
14. A method as claimed in claim 1, 7 or 8 wherein the Factor VIII is isolated from the Factor VIII rich precipitate by washing the insoluble precipitate in the cold at about 0°C with citrate saline heparin buffer; draining the precipitate and resolubilizing it at about 37°C in the presence of further citrate saline heparin buffer and the isolated Factor VIII is subjected to further purification procedures.
15. A method as claimed in claim 1, 7 or 8 wherein Factor VIII is isolated from the Factor VIII rich precipitate by washing the insoluble precipitate in the cold at a temperature of from about 0° to about 10°C with citrate saline heparin buffer; draining the precipitate and resolubilizing it in the presence of further citrate saline heparin buffer and the isolated Factor VIII
is subjected to further purification procedures selected from fractionation by precipitation by glycine, ethanol, ethanol-glycine, polyethylene glycol and glycine-polyethylene glycol precipitation.
is subjected to further purification procedures selected from fractionation by precipitation by glycine, ethanol, ethanol-glycine, polyethylene glycol and glycine-polyethylene glycol precipitation.
16. A method as claimed in claim 1, 7 or 8 wherein the Factor VIII is isolated from the Factor VIII rich precipitate by washing the insoluble precipitate in the cold at about 0°C with citrate saline heparin buffer; draining the precipitate and resolubilizing it at about 37°C in the presence of further citrate saline heparin buffer and the isolated Factor VIII is subjected to further purification procedures selected from fractionation by precipitation by glycine, ethanol, ethanol-glycine, polyethylene glycol and glycine-polyethylene glycol precipitation.
CLAIMS SUPPORTED BY SUPPLEMENTARY DISCLOSURE
CLAIMS SUPPORTED BY SUPPLEMENTARY DISCLOSURE
17. A method of obtaining Factor VIII which comprises a) adding heparin to freshly obtained blood plasma collected into a citrate-phosphate-dextrose anticoagulant or collecting blood plasma by plasmapheresis using heparin and sodium citrate anticoagulant;
b) freezing the plasma to about -80°C;
c) resolubilizing the plasma at a temperature of from about 0° to about 10°C for from about 30 to about 120 minutes;
d) centrifuging the resolubilized plasma to isolate a cryoprecipitate;
e) resolubilizing the cryoprecipitate at a temperature of about 37°C for about 2-5 minutes;
f) adding from about 2 to about 10 ml of citrate saline heparin buffer per bag of the resolubilized cryoprecipitate;
g) incubating the buffered, resolubilized cryoprecipitate at about 0°C
for about two hours whereby Factor VIII present in the cryoprecipitate is insolubilized using heparin precipitable cold insoluble globulin and the resulting Factor VIII rich precipitate also includes cold insoluble globulin;
h) separating the Factor VIII rich precipitate; and i) isolating the Factor VIII therefrom.
b) freezing the plasma to about -80°C;
c) resolubilizing the plasma at a temperature of from about 0° to about 10°C for from about 30 to about 120 minutes;
d) centrifuging the resolubilized plasma to isolate a cryoprecipitate;
e) resolubilizing the cryoprecipitate at a temperature of about 37°C for about 2-5 minutes;
f) adding from about 2 to about 10 ml of citrate saline heparin buffer per bag of the resolubilized cryoprecipitate;
g) incubating the buffered, resolubilized cryoprecipitate at about 0°C
for about two hours whereby Factor VIII present in the cryoprecipitate is insolubilized using heparin precipitable cold insoluble globulin and the resulting Factor VIII rich precipitate also includes cold insoluble globulin;
h) separating the Factor VIII rich precipitate; and i) isolating the Factor VIII therefrom.
18. A method as claimed in claim 17 wherein the resolubilization of the plasma in step c) is carried out at about 4°C for about 75 minutes.
19. A method of obtaining Factor VIII which comprises a) collecting blood plasma by plasmapheresis using sodium heparin and sodium citrate anticoagulant with 1 unit of sodium heparin per ml of plasma employed;
b) freezing the plasma to about -80°C;
c) resolubilizing the plasma at about 4°C for 75 minutes;
d) centrifuging the resolubilized plasma to isolate a cryoprecipitate;
e) resolubilizing the cryoprecipitate at a temperature of about 37°C for about 2-5 minutes;
f) adding about 2.5 ml of citrate saline heparin buffer per bag of the resolubilized cryoprecipitate;
g) incubating the buffered, resolubilized cryopercipitate at about 0°C
for about two hours whereby Factor VIII present in the cryoprecipitate is insolubilized using heparin cold insoluble globulin and the resulting Factor VIII rich precipitate also includes cold insoluble globulin;
h) separating by centrifuging the Factor VIII rich precipitate at 0°C
for 7 minutes; and i) isolating the Factor VIII therefrom by washing the insoluble precipitate in the cold at a temperature of about 0°C with cold citrate saline heparin buffer; draining the precipitate and resolubilizing it at 37° for 5 minutes in the presence of a further 2 ml citrate saline heparin buffer; centrifuging again for 6 minutes; and separating the Factor VIII rich solution from the debris..
b) freezing the plasma to about -80°C;
c) resolubilizing the plasma at about 4°C for 75 minutes;
d) centrifuging the resolubilized plasma to isolate a cryoprecipitate;
e) resolubilizing the cryoprecipitate at a temperature of about 37°C for about 2-5 minutes;
f) adding about 2.5 ml of citrate saline heparin buffer per bag of the resolubilized cryoprecipitate;
g) incubating the buffered, resolubilized cryopercipitate at about 0°C
for about two hours whereby Factor VIII present in the cryoprecipitate is insolubilized using heparin cold insoluble globulin and the resulting Factor VIII rich precipitate also includes cold insoluble globulin;
h) separating by centrifuging the Factor VIII rich precipitate at 0°C
for 7 minutes; and i) isolating the Factor VIII therefrom by washing the insoluble precipitate in the cold at a temperature of about 0°C with cold citrate saline heparin buffer; draining the precipitate and resolubilizing it at 37° for 5 minutes in the presence of a further 2 ml citrate saline heparin buffer; centrifuging again for 6 minutes; and separating the Factor VIII rich solution from the debris..
20. A method as claimed in claim 17 or 18 wherein Factor VIII is isolated from the Factor VIII rich precipitate by washing the insoluble precipitate in the cold at a temperature of from about 0° to about 10°C with citrate saline heparin buffer; draining the precipitate and resolubilizing it in the presence of further citrate saline heparin buffer.
21. A method as claimed in claim 17 or 18 wherein the Factor VIII is isolated from the Factor VIII rich precipitate by washing the insoluble precipitate in the cold at about 0°C with citrate saline heparin buffer; draining the precipitate and resolubilizing it at about 37°C in the presence of further citrate saline heparin buffer.
22. A method as claimed in claim 17 or 18 wherein the isolated Factor VIII is subjected to further purification procedures.
23. A method as claimed in claim 17 or 18 wherein the isolated Factor VIII is subjected to further purification procedures selected from fractionation by precipitation by glycine, ethanol, ethanol-glycine, polyethylene glycol and glycine-polyethylene glycol precipitation.
24. A method as claimed in claim 17 or 18 wherein Factor VIII is isolated from the Factor VIII rich precipitate by washing the insoluble precipitate in the cold at a temperature of from about 0° to about 10°C with citrate saline heparin buffer; draining the precipitate and resolubilizing it in the presence of further citrate saline heparin buffer and the isolated Factor VIII is subjected to further purification procedures.
25. A method as claimed in claim 17 or 18 wherein the Factor VIII is isolated from the Factor VIII rich precipitate by washing the insoluble precipitate in the cold at about 0°C with citrate saline heparin buffer; draining the precipitate and resolubilizing it at about 37°C in the presence of further citrate saline heparin buffer and the isolated Factor VIII is subjected to further purification procedures.
26. A method as claimed in claim 17 or 18 wherein Factor VIII is isolated from the Factor VIII rich precipitate by washing the insoluble precipitate in the cold at a temperature of from about 0° to about 10°C with citrate saline heparin buffer; draining the precipitate and resolubilizing it in the presence of further citrate saline heparin buffer and the isolated Factor VIII is subjected to further purification procedures selected from fractionation by precipitation by glycine, ethanol, ethanol-glycine, polyethylene glycol and glycine-polyethylene glycol precipitation.
27. A method as claimed in claim 17 or 18 wherein the Factor VIII is isolated from the Factor VIII rich precipitate by washing the insoluble precipitate in the cold at about 0°C with citrate saline heparin buffer; draining the precipitate and resolubilizing it at about 37°C in the presence of further citrate saline heparin buffer and the isolated Factor VIII is subjected to further purification procedures selected from fractionation by precipitation by glycine, ethanol, ethanol-glycine, polyethylene glycol and glycine-polyethylene glycol precipitation.
Priority Applications (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US06/210,385 US4289691A (en) | 1980-01-18 | 1980-11-26 | Method of obtaining intermediate purity factor VIII |
| DE8181300069T DE3163003D1 (en) | 1980-01-18 | 1981-01-08 | Method of obtaining factor viii |
| AT81300069T ATE7030T1 (en) | 1980-01-18 | 1981-01-08 | PROCEDURE FOR OBTAINING FACTOR VIII. |
| EP19810300069 EP0033582B1 (en) | 1980-01-18 | 1981-01-08 | Method of obtaining factor viii |
| JP56006248A JPS6059212B2 (en) | 1980-01-18 | 1981-01-19 | How to obtain factor 8 |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US06/210,385 US4289691A (en) | 1980-01-18 | 1980-11-26 | Method of obtaining intermediate purity factor VIII |
| US210,385 | 1980-11-26 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1115637A true CA1115637A (en) | 1982-01-05 |
Family
ID=22782697
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA344,000A Expired CA1115637A (en) | 1980-01-18 | 1980-01-18 | Method of obtaining intermediate purity factor viii |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1115637A (en) |
-
1980
- 1980-01-18 CA CA344,000A patent/CA1115637A/en not_active Expired
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