CA1158977A - Method of selectively increasing yield and purity of certain cryoprecipitate proteins - Google Patents
Method of selectively increasing yield and purity of certain cryoprecipitate proteinsInfo
- Publication number
- CA1158977A CA1158977A CA000384471A CA384471A CA1158977A CA 1158977 A CA1158977 A CA 1158977A CA 000384471 A CA000384471 A CA 000384471A CA 384471 A CA384471 A CA 384471A CA 1158977 A CA1158977 A CA 1158977A
- Authority
- CA
- Canada
- Prior art keywords
- plasma
- ahf
- cryoprecipitate
- cig
- purity
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/745—Blood coagulation or fibrinolysis factors
- C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/16—Blood plasma; Blood serum
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
- Y10S530/827—Proteins from mammals or birds
- Y10S530/829—Blood
- Y10S530/83—Plasma; serum
Abstract
METHOD OF SELECTIVELY INCREASING
YIELD AND PURITY OF CERTAIN
CRYOPRECIPITATE PROTEINS
Abstract of the disclosure AHF and CIg yield and purity in cryoprecipitate concentrate are improved by suspending said cryoprecipitate concentrate in aqueous solution and then subjecting to a heat treatment step at a temperature of from about 45°C
to about 56°C for a period of from about one minute to about sixty minutes to thereby selectively thermally precipitate the contaminating proteins without substantial loss of AHF or CIg activities and recovering the purified AHF or CIg from the precipitate of contaminating proteins.
YIELD AND PURITY OF CERTAIN
CRYOPRECIPITATE PROTEINS
Abstract of the disclosure AHF and CIg yield and purity in cryoprecipitate concentrate are improved by suspending said cryoprecipitate concentrate in aqueous solution and then subjecting to a heat treatment step at a temperature of from about 45°C
to about 56°C for a period of from about one minute to about sixty minutes to thereby selectively thermally precipitate the contaminating proteins without substantial loss of AHF or CIg activities and recovering the purified AHF or CIg from the precipitate of contaminating proteins.
Description
1 1~i8~7~
Back~roulld of tne Invention This invention relates to a method for the production of antihemophilic factor (AHF or Factor VIII) and cold-insoluble globulin (CIg or fibronectin).
For a number of years, cryoprecipitation as described by Pool et al. has been a principal method for the isolation and partial purification o~ AHF. Nature, London ~, 312 (1964)~ New Eng. J. Med. ~, 1443-7 (1965). AHF can thus be prepared from single units of plasma in routine blood banking procedures such as in closed bag collection system~ as illustrated, e.g., in U.S~ Patent~ 3,986,506 and 4,0259618, or concentrate~ can be prepared commercially on a large scale as described by 33rinkhous et al., J. Amer. illed Assn. ?~. 613-617 (1968) and in U.S. Patent 3,~31,018.
In order to obtain more highly purified concentrates o~
AHF, the cryoprecipitation procedure~ have been combined with fractionation methods that employ variou~ chemical agents .
such as~ e.g.~ ammonium sulfate, glycine, alcohol, heparin and polycondensed polyglycols such as polyethylene gly -ol (PEG)"
polypropylene glycol (PPG) and mixed polyethylene-polypropylene glycols, the latter being block 50polymers of ethylene oxide and polyoxypropylene which are available commercially under the trademark Pluronic from BASF Wyandotte Chemical Company~
~urther description o~ ~EG and its use in the production of AHF from cryoprecipitate can be had by reference to U.S. Patent 3,631,018, while similar disclosure of the Pluronic polyT~ers and their use in the production of AHF from cryoprecipitate can be had by reference to U.S. Patent 4,073,886. Disclosures o~ the use of heparin in the production of AHF from cryopre-cipitate can be had by re~erence to U.S. Patents 3,803,115, ~,0 4,203,891 and Re~ 29,698, i~-1 ~5~7~
It is kno~l~ that dur~ng the production of AHF ~rom cryoprecipitate there is an inevitable loss of AHF related to the fur-ther purification steps. Although these purification procedures may appear to be simple, they require great care to harvest an optimal amount of AHF. As noted recently by Johnson et al., of the New York University Medical Center, isolation o~ AHF by large scale techniques results in a maxi~u~ yield of about 30~ and most large scale producers have obtained a yield of ~nly 20~ to 25~. Vox San~ 36, 72-76 ~1979).
Thus it is desirable to develop some process which would allow removal of unwanted or u~lecessary proteins (such as fibrinogen and its denatured and degraded products) from an AHF concentrate without undue loss of the valuable AHF itself.
One approach to this ~oal is to produce a cryoprecipitate which contains increased amounts of AHF as described in recent U.S. Patents 4,086,218; 4,105,6505 4,137~223~ 4,189,425 and by Johnson et al., Vox San~ ~ , 72-76 (1979~o As disclosed in these patents and publication, small amounts of `PEG and/or Pluronic polymers with or without added heparin ~20 are emp1Gyed in the plasma prior to carrying out the freezin~
process to produce the cryoprecipitate~ These~procedures thereby result in a higher yield of cryoprecipitate and an associated greater amount of A;~F. However, other proteins (e.g., fibrinogen and its deriva~ives~ normally ~ound in the cryoprecipitate are also proportionally increased~ ;~ethods ; normally used to remove the unwanted proteins (such as the precipitation with glyc7ne, PEG and alcohol) unfortunately also remove a proportional amount of AHF by co-precipitation with no real net gain in the final yield of AHF.
. ~ ~ 5~
Another ~lood protein which preci~ tes ~ro.. plac.
in the cold is known as cold-insoluble globulin (CIg ~r fibronectin~. This is an ~psonic plasma factor now identified as ~2-surface binding globulin. Collection of the starting S plasma in the aforesaid polycondensed polyglycols and/or heparin prior to cryoprecipi-tation also results in precipitation of the CIg with the AHY. Recent comments and reports on the importance of CIg have been published by Rock and Palmer, ~hrombosis Res. 18, 551-556 (1980) and Anon., Hos~ital Pract.
4(7), 35-36 (1980). See also U.S. Patent 4,210,580 Description of the Invention In accordance with the present invention it has been unexpectedly found that fibrinogen and its denatured and degraded products, which are the prirnary contaminating proteins of cryoprecipitation, are also uniquely thermoreactive - whereby they can be selectively thermally removed from the cryoprecipitate without substantial loss of AHF or CIg activitiesc According to the method of the inven-tion, the cryoprecipitate is reconstituted in aqueous solution and then subjected to a heat treatment step at a temperature of from about 45 C to about 56 C, and preferably at about 50C, for a period of from about one to about SlXty rninutes, ; ~ollowed by removal of the resulting precipitate of contami-nating proteins and recovery of the desired AHF~ and CIg-containing supernatant.
- The aqueous solution for reconstitution can be, e.g., pyrogen-free water, physiolo~ically norm~l saline (0.9~ NaCl), c;trated saline solutior ~e.g., one par~ of 0.1 molar sodium citrate in four parts by weight normal saline), tris buffer at pH of about 7, or other physiologically asseptable aqueous solutions ~hich are cornpatible with AXF and CIg.
7 ~
As used herein, the cryoprecipitate refers to the precipi-tate obtained from the freezing and cold th~win~ of human or animal blood plasma, and separated from the supernatant fraction of the plasma, as well as concentrates of the cryoprecipitate.
5 The cryoprecipitate is preferably ob~ained by the rapid freezing of fresh plasma althou~h stored plasma can also be used. The freezing is usually carried out at temperatures of from about -20 C to about -80 C., followed by slow thawing at about 4C.
During the freezing and cold thawing of plas~a to form the cryoprecipitate, the starting plasma preferably is collected and processed with a polycondensed polyglycol such as PEG and/or Pluronic polymer and/or heparin as described in U.S. Patents 4,086,218; 4,105,650~ 4,137,223~ and 4,189,4250 In these procedures, preferably from about 0.1 .
to about 25 grams of said polyol and/or from about 10 to about 2000 units of heparin are used per ~iter of plasma. PEG 4000 is a preferred polyol but P~G having molecular weights of from about 2000 to about lOjOOO also can be used~ .Pluronic -20 F68 polyol having a polyoxypropylene base molecular weight of about 1750, a polyoxyethylene contant of ab~ut 80~o~ and a total molecular weight of about 8750 also is a preferred polyol, but similar such polyols having a polyoxyethylene content as low as about lO~o can be used provided that in such case the polyoxypropylene molecular weight is not greater than about 1750. Preferably, the polyoxye-thylene content is about 20~
or higher and the polyoxypropylene molecular weight is from about 900 to about 4000. The total molecular wei~ht of these block copolymers preferably lies ~ithin -the range of from about 2000 to about 10,000.
_4_ 1 1~8~77 , As used herein/ one unit of heparin is defined to rnsan one U.S.P~ (United States Pharmacopoeia) unit. The U.S.P.
unit of heparin is the quantity tha~ will prevent 1.0 ml of citrated sheep plasma from clotting for one hour after the addition of 0.2 ml of a 1l100 CaC12 solution. As used herein, the term "heparin" also is meant to include the sodium salt of heparin~ the latter substance being preferred due to its water solubility, , Following the heat treatment, the precipitatc of contaminatin~ proteins is removed such as by centrifugation, fiitration and the like separatlon procedurès, and the clari~ied supernatant is retained as a purified AXF- and CIg-containing concentrate. This concentrate can then be frozen and stored for later use, or further processed according to 1~ conventional procedures known in the art or used directiy for administration to a patient.
In those instanOes where a CIg fraction is desired without AHF, the AHF and Clg-cQntaining fraction can be heated to about 6Q C for at least about five minutes to inactivate the AHF without subsiantial loss of the CIg.
The starting material for recovery of a CIg concentrate can also be a cryoprecipitate concentrate from wnich a substantial portion of the fibrinogen has been precipitated .
in accordance with the method described in U.S. Patent 4,188,318.
- 25 Accordin~ to said method, AHF is separated from from fibrinogen and its denatured and degraded products by collectin~ the cryoprecipitate in lo~ ionic s~rength solution. After removal of lipids and prothrombin complex by adsorption, ~ibrinogen and its denatured and degraded products are then selectively 7 ~
precipitated in the cold while ~iF rem2ins in soiution.
This precipitate also contains CIg. The method of the present invention can then be used on this AHF-poor cryoprecipitate corlcentrate to inactivate the fibrinogen and its denatured and degraded products without substantial-destruction of the CIg.
Althou~h it is not intended to be bound by theory, it is believed that the presence of the polycondensed polyglycol and /or heparin enhances the heat denaturation o~ fibrinogen while preventing co~precipitation or destruction of AHF and Clg during the heating step.
The following exa~ples will further illustrate the inventîon although it should be understood that the invention i~ not limited to these specific examples.
~ ol~ I
Fresh human blood is collected in a donor blood bag containing a small amount of anticoagulant solution (ACD or CPD preservative solution~. The cells are spun dswn by centrifugation and the plasma supernatant is expressed into a satellite blood bag. Then 200 - 250 ml of whole plasma thus collected is admixed with PEG 4000 to a concen~ration of about 2~o by weight of the polyol ~Carbowax 4000, Union Carbide), After gentle agitation to ensure complete mixing, the treated plasma is subjected to a temperature between -20 and -50 C
to effect rapid free~ing. Once frozen, the plasma is subjected to a temperature between ~2 and ~4C for cold thawing to produce a cryoprecipitate. ~he supernatant plasma is then remo~ed from ~he cryoprecipitate by decantation. The retained cryoprecipitate can be frozen and stored for later l 1~8~7~
reconstitution and heat ~reat~ent or it can be reconstituted and heat treated i~ediately as follo-~sI
~ he cryoprecipitate is first reconstituted with about one to three volumes of pyrogen-free w~ter (or other physiolo~ically acceptable aqueous solution) per gram of cryoprecipitate. Although dissolution can be had at ambient temperature, warming to about 37 C is used to accelerate dissolution. The reconstituted cryoprecipitate is then subjected to the heat treatment step by heating in a water bath D although other apparatus such as incubating ovens, steam jacketed vessels and the like equipment can be used for larger scale amount of cryoprecipitate. The temperature during this heat treatment step is maintained from about to about 56 C~ and preferably at about 50 C for about one to about sixty minutes. In order to facilitate uniform and optimum heat transfer or distribution, constant mixing of thc solution is maintained dur1ng the heat treatment step by stirring or other such agitation. The one to sixty minute period of heat treatment can be calculated from thc time that opalescence or precipitation is first observer, thus signifying the beginning of fibrinogen denatura-tion.
, .
Following the aforesaid heat treatment step, the prepa-ration can be centrifuged for c1arification, or the supernatant can be directly aspirated into a syringe (which can contain a filter). The final prepara~ion can be administered to the patient directly or refrozen for later use.
When heated as above at about 50C for about thirty minutes, about 90~0 of the fibrinogen is denatured and only abDut 25~o Of the AHF activity is 10st. ;Ihen similar1y heated .
1 1~8~
for about forty minutes, 100% of the fibrinogen is denatured but still only about 25% of the AHF activity is lost. Thus the method of the invention is able to achieve a desirable selective separation of cryoprecipitate proteins.
`Example 2 The procedure of Example 1 is repeated except that an e~ual amount of Pluronic F-68 is substituted for the PEG
4000 in the starting plasma to produce substantially similar results.
Example 3 The procedure of Example 1 is repeated except that 125 to 250 units of heparin are added to the 200 to 250 ml of starting plasma with substantially similar results.
Example 4 The procedure of Example 2 is repeated except that 125 to 250 units of heparin are added to the 200 to 250 ml of starting plasma with substantially similar results.
Example 5 The procedure of Example 1 is repeated except that 125 to 250 units of heparin are added to the 200 to 250 ml of starting plasma without any PEG 4000 or other polycondensed polyglycol added to the plasma with substantially similar results.
Various other examples will be apparent to the person skilled in the art after reading the present disclosure without departing from the spirit and scope of the invention.
It is intended that all such further examples be included in the scope of the appended claims.
~f
Back~roulld of tne Invention This invention relates to a method for the production of antihemophilic factor (AHF or Factor VIII) and cold-insoluble globulin (CIg or fibronectin).
For a number of years, cryoprecipitation as described by Pool et al. has been a principal method for the isolation and partial purification o~ AHF. Nature, London ~, 312 (1964)~ New Eng. J. Med. ~, 1443-7 (1965). AHF can thus be prepared from single units of plasma in routine blood banking procedures such as in closed bag collection system~ as illustrated, e.g., in U.S~ Patent~ 3,986,506 and 4,0259618, or concentrate~ can be prepared commercially on a large scale as described by 33rinkhous et al., J. Amer. illed Assn. ?~. 613-617 (1968) and in U.S. Patent 3,~31,018.
In order to obtain more highly purified concentrates o~
AHF, the cryoprecipitation procedure~ have been combined with fractionation methods that employ variou~ chemical agents .
such as~ e.g.~ ammonium sulfate, glycine, alcohol, heparin and polycondensed polyglycols such as polyethylene gly -ol (PEG)"
polypropylene glycol (PPG) and mixed polyethylene-polypropylene glycols, the latter being block 50polymers of ethylene oxide and polyoxypropylene which are available commercially under the trademark Pluronic from BASF Wyandotte Chemical Company~
~urther description o~ ~EG and its use in the production of AHF from cryoprecipitate can be had by reference to U.S. Patent 3,631,018, while similar disclosure of the Pluronic polyT~ers and their use in the production of AHF from cryoprecipitate can be had by reference to U.S. Patent 4,073,886. Disclosures o~ the use of heparin in the production of AHF from cryopre-cipitate can be had by re~erence to U.S. Patents 3,803,115, ~,0 4,203,891 and Re~ 29,698, i~-1 ~5~7~
It is kno~l~ that dur~ng the production of AHF ~rom cryoprecipitate there is an inevitable loss of AHF related to the fur-ther purification steps. Although these purification procedures may appear to be simple, they require great care to harvest an optimal amount of AHF. As noted recently by Johnson et al., of the New York University Medical Center, isolation o~ AHF by large scale techniques results in a maxi~u~ yield of about 30~ and most large scale producers have obtained a yield of ~nly 20~ to 25~. Vox San~ 36, 72-76 ~1979).
Thus it is desirable to develop some process which would allow removal of unwanted or u~lecessary proteins (such as fibrinogen and its denatured and degraded products) from an AHF concentrate without undue loss of the valuable AHF itself.
One approach to this ~oal is to produce a cryoprecipitate which contains increased amounts of AHF as described in recent U.S. Patents 4,086,218; 4,105,6505 4,137~223~ 4,189,425 and by Johnson et al., Vox San~ ~ , 72-76 (1979~o As disclosed in these patents and publication, small amounts of `PEG and/or Pluronic polymers with or without added heparin ~20 are emp1Gyed in the plasma prior to carrying out the freezin~
process to produce the cryoprecipitate~ These~procedures thereby result in a higher yield of cryoprecipitate and an associated greater amount of A;~F. However, other proteins (e.g., fibrinogen and its deriva~ives~ normally ~ound in the cryoprecipitate are also proportionally increased~ ;~ethods ; normally used to remove the unwanted proteins (such as the precipitation with glyc7ne, PEG and alcohol) unfortunately also remove a proportional amount of AHF by co-precipitation with no real net gain in the final yield of AHF.
. ~ ~ 5~
Another ~lood protein which preci~ tes ~ro.. plac.
in the cold is known as cold-insoluble globulin (CIg ~r fibronectin~. This is an ~psonic plasma factor now identified as ~2-surface binding globulin. Collection of the starting S plasma in the aforesaid polycondensed polyglycols and/or heparin prior to cryoprecipi-tation also results in precipitation of the CIg with the AHY. Recent comments and reports on the importance of CIg have been published by Rock and Palmer, ~hrombosis Res. 18, 551-556 (1980) and Anon., Hos~ital Pract.
4(7), 35-36 (1980). See also U.S. Patent 4,210,580 Description of the Invention In accordance with the present invention it has been unexpectedly found that fibrinogen and its denatured and degraded products, which are the prirnary contaminating proteins of cryoprecipitation, are also uniquely thermoreactive - whereby they can be selectively thermally removed from the cryoprecipitate without substantial loss of AHF or CIg activitiesc According to the method of the inven-tion, the cryoprecipitate is reconstituted in aqueous solution and then subjected to a heat treatment step at a temperature of from about 45 C to about 56 C, and preferably at about 50C, for a period of from about one to about SlXty rninutes, ; ~ollowed by removal of the resulting precipitate of contami-nating proteins and recovery of the desired AHF~ and CIg-containing supernatant.
- The aqueous solution for reconstitution can be, e.g., pyrogen-free water, physiolo~ically norm~l saline (0.9~ NaCl), c;trated saline solutior ~e.g., one par~ of 0.1 molar sodium citrate in four parts by weight normal saline), tris buffer at pH of about 7, or other physiologically asseptable aqueous solutions ~hich are cornpatible with AXF and CIg.
7 ~
As used herein, the cryoprecipitate refers to the precipi-tate obtained from the freezing and cold th~win~ of human or animal blood plasma, and separated from the supernatant fraction of the plasma, as well as concentrates of the cryoprecipitate.
5 The cryoprecipitate is preferably ob~ained by the rapid freezing of fresh plasma althou~h stored plasma can also be used. The freezing is usually carried out at temperatures of from about -20 C to about -80 C., followed by slow thawing at about 4C.
During the freezing and cold thawing of plas~a to form the cryoprecipitate, the starting plasma preferably is collected and processed with a polycondensed polyglycol such as PEG and/or Pluronic polymer and/or heparin as described in U.S. Patents 4,086,218; 4,105,650~ 4,137,223~ and 4,189,4250 In these procedures, preferably from about 0.1 .
to about 25 grams of said polyol and/or from about 10 to about 2000 units of heparin are used per ~iter of plasma. PEG 4000 is a preferred polyol but P~G having molecular weights of from about 2000 to about lOjOOO also can be used~ .Pluronic -20 F68 polyol having a polyoxypropylene base molecular weight of about 1750, a polyoxyethylene contant of ab~ut 80~o~ and a total molecular weight of about 8750 also is a preferred polyol, but similar such polyols having a polyoxyethylene content as low as about lO~o can be used provided that in such case the polyoxypropylene molecular weight is not greater than about 1750. Preferably, the polyoxye-thylene content is about 20~
or higher and the polyoxypropylene molecular weight is from about 900 to about 4000. The total molecular wei~ht of these block copolymers preferably lies ~ithin -the range of from about 2000 to about 10,000.
_4_ 1 1~8~77 , As used herein/ one unit of heparin is defined to rnsan one U.S.P~ (United States Pharmacopoeia) unit. The U.S.P.
unit of heparin is the quantity tha~ will prevent 1.0 ml of citrated sheep plasma from clotting for one hour after the addition of 0.2 ml of a 1l100 CaC12 solution. As used herein, the term "heparin" also is meant to include the sodium salt of heparin~ the latter substance being preferred due to its water solubility, , Following the heat treatment, the precipitatc of contaminatin~ proteins is removed such as by centrifugation, fiitration and the like separatlon procedurès, and the clari~ied supernatant is retained as a purified AXF- and CIg-containing concentrate. This concentrate can then be frozen and stored for later use, or further processed according to 1~ conventional procedures known in the art or used directiy for administration to a patient.
In those instanOes where a CIg fraction is desired without AHF, the AHF and Clg-cQntaining fraction can be heated to about 6Q C for at least about five minutes to inactivate the AHF without subsiantial loss of the CIg.
The starting material for recovery of a CIg concentrate can also be a cryoprecipitate concentrate from wnich a substantial portion of the fibrinogen has been precipitated .
in accordance with the method described in U.S. Patent 4,188,318.
- 25 Accordin~ to said method, AHF is separated from from fibrinogen and its denatured and degraded products by collectin~ the cryoprecipitate in lo~ ionic s~rength solution. After removal of lipids and prothrombin complex by adsorption, ~ibrinogen and its denatured and degraded products are then selectively 7 ~
precipitated in the cold while ~iF rem2ins in soiution.
This precipitate also contains CIg. The method of the present invention can then be used on this AHF-poor cryoprecipitate corlcentrate to inactivate the fibrinogen and its denatured and degraded products without substantial-destruction of the CIg.
Althou~h it is not intended to be bound by theory, it is believed that the presence of the polycondensed polyglycol and /or heparin enhances the heat denaturation o~ fibrinogen while preventing co~precipitation or destruction of AHF and Clg during the heating step.
The following exa~ples will further illustrate the inventîon although it should be understood that the invention i~ not limited to these specific examples.
~ ol~ I
Fresh human blood is collected in a donor blood bag containing a small amount of anticoagulant solution (ACD or CPD preservative solution~. The cells are spun dswn by centrifugation and the plasma supernatant is expressed into a satellite blood bag. Then 200 - 250 ml of whole plasma thus collected is admixed with PEG 4000 to a concen~ration of about 2~o by weight of the polyol ~Carbowax 4000, Union Carbide), After gentle agitation to ensure complete mixing, the treated plasma is subjected to a temperature between -20 and -50 C
to effect rapid free~ing. Once frozen, the plasma is subjected to a temperature between ~2 and ~4C for cold thawing to produce a cryoprecipitate. ~he supernatant plasma is then remo~ed from ~he cryoprecipitate by decantation. The retained cryoprecipitate can be frozen and stored for later l 1~8~7~
reconstitution and heat ~reat~ent or it can be reconstituted and heat treated i~ediately as follo-~sI
~ he cryoprecipitate is first reconstituted with about one to three volumes of pyrogen-free w~ter (or other physiolo~ically acceptable aqueous solution) per gram of cryoprecipitate. Although dissolution can be had at ambient temperature, warming to about 37 C is used to accelerate dissolution. The reconstituted cryoprecipitate is then subjected to the heat treatment step by heating in a water bath D although other apparatus such as incubating ovens, steam jacketed vessels and the like equipment can be used for larger scale amount of cryoprecipitate. The temperature during this heat treatment step is maintained from about to about 56 C~ and preferably at about 50 C for about one to about sixty minutes. In order to facilitate uniform and optimum heat transfer or distribution, constant mixing of thc solution is maintained dur1ng the heat treatment step by stirring or other such agitation. The one to sixty minute period of heat treatment can be calculated from thc time that opalescence or precipitation is first observer, thus signifying the beginning of fibrinogen denatura-tion.
, .
Following the aforesaid heat treatment step, the prepa-ration can be centrifuged for c1arification, or the supernatant can be directly aspirated into a syringe (which can contain a filter). The final prepara~ion can be administered to the patient directly or refrozen for later use.
When heated as above at about 50C for about thirty minutes, about 90~0 of the fibrinogen is denatured and only abDut 25~o Of the AHF activity is 10st. ;Ihen similar1y heated .
1 1~8~
for about forty minutes, 100% of the fibrinogen is denatured but still only about 25% of the AHF activity is lost. Thus the method of the invention is able to achieve a desirable selective separation of cryoprecipitate proteins.
`Example 2 The procedure of Example 1 is repeated except that an e~ual amount of Pluronic F-68 is substituted for the PEG
4000 in the starting plasma to produce substantially similar results.
Example 3 The procedure of Example 1 is repeated except that 125 to 250 units of heparin are added to the 200 to 250 ml of starting plasma with substantially similar results.
Example 4 The procedure of Example 2 is repeated except that 125 to 250 units of heparin are added to the 200 to 250 ml of starting plasma with substantially similar results.
Example 5 The procedure of Example 1 is repeated except that 125 to 250 units of heparin are added to the 200 to 250 ml of starting plasma without any PEG 4000 or other polycondensed polyglycol added to the plasma with substantially similar results.
Various other examples will be apparent to the person skilled in the art after reading the present disclosure without departing from the spirit and scope of the invention.
It is intended that all such further examples be included in the scope of the appended claims.
~f
Claims (9)
1. The method of improving the yield and purity of a substance selected from the group consisting of AHF and CIg in a cryoprecipitate concentrate of said substance comprising suspending said cryoprecipitate concentrate in aqueous solution and subjecting to heat treatment at a temperature of from about 45°C to about 56°C for a period of from about one to about sixty minutes to thereby selectively thermally precipitate contaminating proteins without substantial loss of said AHF or CIg and recovering the purified AHF or CIg by separation from the precipitate of contaminating proteins.
2. The method of Claim 1 in which the temperature is maintained at about 50°C.
3. The method of Claim 1 in which the starting cryopre-cipitate is formed by freezing of plasma which contains from about 0.1 to about 25 grams of a polycondensed polyglycol per liter of said plasma.
4. The method of Claim 3 in which the polycondensed polyglycol is PEG having a molecular weight of from about 2000 to about 10,000.
5. The method of Claim 4 in which the PEG is PEG 4000.
6. The method of Claim 3 in which the polycondensed polyglycol is a mixed polyethylene-polypropylene glycol having a molecular weight of from about 2000 to about 10,000.
7. The method of Claim 6 in which the polycondensed polyglycol is a block copolymer containing about 80% of polyoxyethylene units in the molecule and the polyoxypropylene has a base Molecular weight of about 1750.
8. The method of Claim 1 in which the starting cryoprecipitate is formed by freezing plasma which contains from about 10 to about 2000 units of heparin per liter of plasma.
9. The method of Claim 3 in which said plasma contains additionally from about 10 to about 2000 units of heparin per liter of plasma.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US06/183,364 US4305871A (en) | 1980-09-02 | 1980-09-02 | Method of selectively increasing yield and purity of certain cryoprecipitate proteins by heating |
| US183,364 | 1980-09-02 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1158977A true CA1158977A (en) | 1983-12-20 |
Family
ID=22672507
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000384471A Expired CA1158977A (en) | 1980-09-02 | 1981-08-24 | Method of selectively increasing yield and purity of certain cryoprecipitate proteins |
Country Status (6)
| Country | Link |
|---|---|
| US (1) | US4305871A (en) |
| EP (1) | EP0047216B1 (en) |
| JP (1) | JPS57118520A (en) |
| AT (1) | ATE12504T1 (en) |
| CA (1) | CA1158977A (en) |
| DE (1) | DE3169689D1 (en) |
Families Citing this family (28)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS57140724A (en) * | 1981-02-25 | 1982-08-31 | Green Cross Corp:The | Heat-treatment of aqueous solution containing cold insoluble globulin |
| CA1178887A (en) * | 1981-10-01 | 1984-12-04 | Gail A. Rock | Factor viii concentrates prepared from heparinized plasma by the application of a cold precipitation technique |
| US4481189A (en) * | 1982-04-14 | 1984-11-06 | New York Blood Center Inc. | Process for preparing sterilized plasma and plasma derivatives |
| JPS5967228A (en) * | 1982-10-07 | 1984-04-16 | Green Cross Corp:The | Method for freeze-drying cold-insoluble globulin |
| ATE31023T1 (en) * | 1983-04-28 | 1987-12-15 | Armour Pharma | PHARMACEUTICAL PREPARATION CONTAINING PURIFIED FIBRINONECTIN. |
| US4820805A (en) * | 1983-07-14 | 1989-04-11 | New York Blood Center, Inc. | Undenatured virus-free trialkyl phosphate treated biologically active protein derivatives |
| US4764369A (en) * | 1983-07-14 | 1988-08-16 | New York Blood Center Inc. | Undenatured virus-free biologically active protein derivatives |
| US4486410A (en) * | 1984-02-09 | 1984-12-04 | Armour Pharmaceutical Company | Heat defibrinogenation of AHF preparation |
| US5149787A (en) * | 1984-05-22 | 1992-09-22 | The Blood Center Research Foundation | Method for maintaining intact, non-degraded factor VIII/von-Willebrand factor during blood processing |
| US4710381A (en) * | 1984-05-22 | 1987-12-01 | The Blood Center Of Southeastern Wisconsin | Method for maintaining intact, non-degraded factor VIII/von-Willebrand factor during blood processing |
| AT385658B (en) * | 1985-03-28 | 1988-05-10 | Serotherapeutisches Inst Wien | METHOD FOR PRODUCING A FIBRONEKTIN SOLUTION SUITABLE FOR HUMAN USE |
| US4749783A (en) * | 1986-07-11 | 1988-06-07 | Miles Laboratories, Inc. | Viral inactivation and purification of active proteins |
| AT391808B (en) * | 1986-11-03 | 1990-12-10 | Immuno Ag | METHOD FOR PRODUCING A FACTOR VIII (AHF) CONTAINING FRACTION |
| EP0399321B1 (en) * | 1989-05-24 | 1993-06-23 | Miles Inc. | Gel filtration of heat treated factor viii |
| USH1509H (en) * | 1989-06-09 | 1995-12-05 | Eran; Harutyun | Heparin enhanced process for separating antihemophilic factor (Factor VIII) and fibronectin from cryoprecipitate |
| US5139943A (en) * | 1989-06-13 | 1992-08-18 | Genencor International, Inc. | Processes for the recovery of microbially produced chymosin |
| US5151358A (en) * | 1989-06-13 | 1992-09-29 | Genencor International, Inc. | Processes for the recovery of naturally produced chymosin |
| US5770705A (en) * | 1996-11-01 | 1998-06-23 | Shanbrom Technologies Llc | Method for recovering proteins from plasma using insoluble, water-absorbing material |
| US6881731B1 (en) * | 2000-10-23 | 2005-04-19 | Shanbrom Technologies, Llc | Enhancers for microbiological disinfection |
| US7411006B2 (en) * | 2000-10-23 | 2008-08-12 | Shanbrom Technologies, Llc | Enhanced production of blood clotting factors and fibrin fabric |
| US8389687B2 (en) * | 2000-10-23 | 2013-03-05 | Shanbrom Technologies, Llc | Polyvinylpyrrolidone cryoprecipitate extraction of clotting factors |
| US7297716B2 (en) * | 2000-10-23 | 2007-11-20 | Shanbrom Technologies, Llc | Enhanced production of blood components, blood cells and plasma without freezing |
| MXPA03007069A (en) * | 2001-02-07 | 2004-10-15 | Shanbrom Tech Llc | Carboxylic acid such as citric acid for desinfecting or enhacing the production of blood products such as plasma, cryoprecipitate or/and platelet. |
| US6881573B2 (en) * | 2003-09-12 | 2005-04-19 | Allan L. Louderback | Augmented solvent/detergent method for inactivating enveloped and non-enveloped viruses |
| EP1950225A1 (en) * | 2007-01-25 | 2008-07-30 | Octapharma AG | Method for increasing protein yield |
| US20080306610A1 (en) * | 2007-06-07 | 2008-12-11 | Zimmer Orthobiologics, Inc. | Tissue processing for nonimmunogenic implants |
| US8435305B2 (en) | 2010-08-31 | 2013-05-07 | Zimmer, Inc. | Osteochondral graft delivery device and uses thereof |
| EP4013445A4 (en) * | 2019-09-20 | 2023-09-27 | Plasma Technologies, LLC | Therapeutic protein compositions and methods |
Family Cites Families (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3631018A (en) * | 1970-05-01 | 1971-12-28 | Baxter Laboratories Inc | Production of stable high-potency human ahf using polyethylene glycol and glycine to fractionate a cryoprecipitate of ahf concentrate |
| US3803115A (en) * | 1972-05-17 | 1974-04-09 | Baxter Laboratories Inc | Stabilization of ahf using heparin |
| US4073886A (en) * | 1973-01-30 | 1978-02-14 | Baxter Travenol Laboratories, Inc. | Blood fractionation process using block copolymers of ethylene oxide and polyoxypropylene |
| US4022758A (en) * | 1973-06-19 | 1977-05-10 | Ab Kabi | Isolation of coagulation factors I and VIII from biological material |
| CA1007568A (en) * | 1973-12-13 | 1977-03-29 | Michael C. Attwell | Bovine immunoglobulin isolation process |
| FR2279419A1 (en) * | 1974-07-25 | 1976-02-20 | Merieux Inst | Aggregate-free immunoglobulin prepns. - obtained by treatment with complement Clq factor or rheumatic factor |
| US3986506A (en) * | 1974-09-03 | 1976-10-19 | Baxter Travenol Laboratories, Inc. | Apparatus for separation of cryoprecipitate from blood plasma and method |
| US4025618A (en) * | 1974-09-03 | 1977-05-24 | Baxter Travenol Laboratories, Inc. | Method for separation of cryoprecipitate from blook plasma |
| US4105650A (en) * | 1975-04-11 | 1978-08-08 | Edward Shanbrom, Inc. | Method of preserving blood plasma i |
| US4189425A (en) * | 1975-04-11 | 1980-02-19 | Edward Shanbrom, Inc. | Method of preserving blood plasma I |
| DE2527064C3 (en) * | 1975-06-18 | 1979-11-15 | Biotest-Serum-Institut Gmbh, 6000 Frankfurt | Process for the production of an intravenous native human immunoglobulin preparation with a natural half-life and unchanged antibody activity compared to the starting material |
| NL7708005A (en) * | 1976-07-22 | 1978-01-24 | Monsanto Co | PROCESS FOR THE PREPARATION OF SERUM ALBUMIN. |
| DK25877A (en) * | 1977-01-21 | 1978-08-15 | Nordisk Insulinlab | PROCEDURE FOR EXTRACTING PURE ALBUMIN FROM BLOOD PLASMA |
| US4104266A (en) * | 1977-04-14 | 1978-08-01 | American National Red Cross | Method for preparation of antihemophilic factor |
| US4137223A (en) * | 1977-05-16 | 1979-01-30 | Edward Shanbrom, Inc. | Method of preserving blood plasma II |
| WO1979000299A1 (en) * | 1977-11-17 | 1979-05-31 | Atomic Energy Authority Uk | Purification of factor viii |
| CA1074698A (en) * | 1977-12-19 | 1980-04-01 | Gail A. Rock | Method of collecting anti-hemophilic factor viii from blood and blood plasma |
| DE2916711A1 (en) * | 1979-04-25 | 1980-11-06 | Behringwerke Ag | Blood coagulation factors and process for their manufacture |
-
1980
- 1980-09-02 US US06/183,364 patent/US4305871A/en not_active Expired - Lifetime
-
1981
- 1981-08-24 CA CA000384471A patent/CA1158977A/en not_active Expired
- 1981-09-01 AT AT81401370T patent/ATE12504T1/en not_active IP Right Cessation
- 1981-09-01 EP EP81401370A patent/EP0047216B1/en not_active Expired
- 1981-09-01 DE DE8181401370T patent/DE3169689D1/en not_active Expired
- 1981-09-02 JP JP56139207A patent/JPS57118520A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| EP0047216B1 (en) | 1985-04-03 |
| JPS57118520A (en) | 1982-07-23 |
| DE3169689D1 (en) | 1985-05-09 |
| ATE12504T1 (en) | 1985-04-15 |
| US4305871A (en) | 1981-12-15 |
| EP0047216A1 (en) | 1982-03-10 |
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Legal Events
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| MKEX | Expiry |