US4289691A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Method of obtaining intermediate purity factor VIII
Assignee: Canadian Red Cross Society
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Filing: 1980-11-26
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Granted: 1981-09-15
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Expires: 2000-11-26
Abstract
Abstract 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 cryoprecipiate; (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. 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 liter 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.
Inventors
- 👤 Gail A. Rock
- 👤 Douglas S. Palmer
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 10:48
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/435; C07K14/745; C07K14/755; A; A61; A61K; A61K38/00
Claims Analysis
Total Claims: 18
Claim 1
Independent Claim
132 words
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.
Claim 2
Independent Claim
132 words
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.
Claim 3
Dependent Claim
21 words
A method as claimed in claim 1 wherein the calcium chelating anticoagulant is selected from citrate anticoagulants, ethylenediaminetetraacetic acid and oxalates.
Claim 4
Dependent Claim
16 words
A method as claimed in claim 1 wherein the calcium chelating anticoagulant is a citrate anticoagulant.
Claim 5
Dependent Claim
12 words
A method as claimed in claim 3 wherein the anticoagulant is acid-citrate-dextrose.
Claim 6
Dependent Claim
12 words
A method as claimed in claim 3 wherein the anticoagulant is citrate-phosphate-dextrose.
Claim 7
Dependent Claim
20 words
A method as claimed in claim 1 wherein the blood plasma is collected by plasmapheresis using heparin and sodium citrate.
Claim 8
Independent Claim
163 words
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 precipitate 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.
Claim 9
Independent Claim
163 words
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 precipitate 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.
Claim 10
Dependent Claim
27 words
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.
Claim 11
Dependent Claim
60 words
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.
Claim 12
Dependent Claim
58 words
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.
Claim 13
Dependent Claim
21 words
A method as claimed in claim 1, 7 or 8 wherein the isolated Factor VIII is subjected to further purification procedures.
Claim 14
Dependent Claim
36 words
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.
Claim 15
Dependent Claim
71 words
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.
Claim 16
Dependent Claim
69 words
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.
Claim 17
Dependent Claim
86 words
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.
Claim 18
Dependent Claim
84 words
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.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1984-09-14 | 1985-03-16 | 1985-09-15 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1988-09-14 | 1989-03-16 | 1989-09-15 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1992-09-14 | 1993-03-16 | 1993-09-15 | $7700.00 | Window Closed / Lapsed |