WO1988008004A1 - Extraction of factor viii - Google Patents

Extraction of factor viii Download PDF

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Publication number
WO1988008004A1
WO1988008004A1 PCT/GB1988/000267 GB8800267W WO8808004A1 WO 1988008004 A1 WO1988008004 A1 WO 1988008004A1 GB 8800267 W GB8800267 W GB 8800267W WO 8808004 A1 WO8808004 A1 WO 8808004A1
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Prior art keywords
plasma
heparin
factor viii
calcium
blood
Prior art date
Application number
PCT/GB1988/000267
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French (fr)
Inventor
Richard Wensley
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Richard Wensley
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Filing date
Publication date
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Publication of WO1988008004A1 publication Critical patent/WO1988008004A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—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)

Definitions

  • the present invention relates to tne extraction of Factor VIII from Dlood plasma.
  • Factor VIII is an important clotting protein used by haemophiliacs and is obtained either from whole blood or plasma given by donors.
  • blood from the donor is passed into a bag containing a solution of a calcium chelating anti-coagulant, e.g. a citrate based anti-coagulant.
  • the blood may be separated into two fractions, namely plasma reduced red cells and plasma, from the latter of which the Factor VIII is obtained.
  • the anti-coagulated plasma is subjected to a separation technique, e.g. cryoprecipitation in which the plasma is frozen rapidly (e.g.
  • Heparin is also known as an anti-coagulant for blood and in theory could be used in the above process instead of sodium citrate.
  • this has the disadvantage that the comparatively high levels of heparin which would be required may produce allergies or platelet deficiencies in patients to whom products derived from the blood donation are given.
  • EP-B-0 053 046 seeks to overcome the above problems by proposing a method of obtaining Factor VIII in which the whole blood or plasma is collected in a solution of a calcium chelating anti-coagulant (e.g. citrate based) and the collected product is mixed with dissolved calcium and heparin at a time not more than six hours after collection of the blood or plasma.
  • the Factor VIII is then extracted from the heparinised blood or plasma, e.g. by cryoprecipitation.
  • the effect of the dissolved calcium is to stabilize the Factor VIII by maintaining or restoring the calcium bridges removed by the chelating anti-coagulant, whilst the heparin provides a non-chelating anti-coagulant activity.
  • the stabilization ensures that there will be sufficient time available after collection of the blood or plasma to transport it to centres for Factor VIII extraction without the necessity of other preserving methods.
  • the technique is particularly applicable to plasmapheresis since the whole oiood may be mixed with citrate based anti-coagulant solution and, after centrifugation, it is only the plasma which is mixed with heparin and dissolved calcium.
  • the red cells, white cells, and platelets returned to the donor only contain the citrate based anti-coagulant and not heparin.
  • the amount of heparin required for optimum Factor VIII stabilization can easily be determined by routine experimentation but is generally in the range of 2 to 20 units heparin per ml plasma with best results generally being obtained with from 3 to 12, most preferably 4 to 8 units of heparin per ml plasma. It is also indicated that the amount of heparin is generally above 4 units per ml of plasma.
  • cryoprecipitate may be used in one of two ways. Firstly, it may be provided, with a small amount of plasma supernatont, to a haemophiliac for "on-demand" self-treatment of bleeding episodes. To effect such treatment, the cryoprecipitate and supernatant are warmed to ambient temperature to resolubilise the precipitate in the supernatant plasma. This dissolution should be as rapid as possible and require minimal manipulation.
  • cryoprecipitation is the most commonly performed first step in the preparation of high-pu ity Factor VIII concentrates.
  • the latter is firstly resolubilised, most commonly in a buffer solution, and may then be subject to number of possible purification steps.
  • High levels of fibrinogen and fibronectin delay the resolubilisation of the Factor VIII and it may be necessary to use a homogeniser to effect complete resolubilisation.
  • the use of a homogeniser may cause denaturation of the Factor VIII and reduction in yields.
  • a method of obtaining Factor VIII comprising taking blood from a donor, admixing the blood with a calcium chelating anti-coagulant, separating the plasma from the cellular components of the blood treating the plasma with 0.1 to less than 2 units per ml of plasma of heparin or heparin related compound
  • heparin SUBS (hereinafter referred to as heparin) and with calcium ions, and .subjecting the heparinised plasma to a fractionation technique for separation of Factor VIII in which the plasma is cooled below 4° c -
  • the amount of heparin to be used in accordance with the invention is 0.1 to less than 2 units (of heparin) per ml of plasma. Below 0.1 units of heparin per ml of plasma there may be insufficient anti-coagulation thus giving rise to clotting problems.
  • the number of units .of heparin used per ml of plasma is in the range of 0.3 to 1.8, more preferably 0.5 to 1.6, even more preferably 0.8 to 1.2.
  • Factor VIII obtained by the method of the invention thus contains comparatively low amounts of fibrinogen and fibronectin.
  • Factor VIII precipitates e.g. obtained by cryoprecipitation
  • Factor VIII I.U./mg total protein specific activities of precipitates (defined as Factor VIII I.U./mg total protein) are generally higher for precipitates obtained by the method of th invention than for comparable citrate controls.
  • donor blood is collected in a calcium chelating anti-coagulant.
  • anti-coagulants are well known and include citrate anticoagulants, e.g. sodium citrate, citrate phosphate dextrose, and acid citrate dextrose.
  • citrate anticoagulants e.g. sodium citrate, citrate phosphate dextrose, and acid citrate dextrose.
  • the amounts of these anti-coagulants used may be conventional.
  • the collection of the blood into the calcium chelating anti-coagulant has the advantage that the cellular component of the blood may, after separation of the plasma, be returned to the donor, and also that the amounts of heparin required are less than would be the case if heparin was used as the primary anti-coagulant.
  • Separation of the whole blood into plasma and cellular components may be by any conventional technique, e.g. centrifugation of the blood.
  • the invention is particularly applicable to the extraction of Factor VIII from plasma obtained by manual plasmapheresis or machine pheresis using conventional cell separators.
  • the obtained plasma should be admixed with the heparin and dissolved calcium ions as soon as possible so that the maximum possible amount of Factor VIII is stabilized by the calcium ions.
  • the plasma separated from the whole blood is passed into a vessel containing a solution (in sterile water) of calcium ions and heparin.
  • the amount of heparin used is in the range 0.1 to less than 2 units per ml of plasma.
  • the heparin may, for example, be any U.K., European or U.S. phar acopea heparin and may, for example, be synthetic heparin or low molecular weight heparin.
  • the heparin should have a minimum activity of 100 I.U./mg. The pharmacopea heparins quoted above exceed this level.
  • the amount of ionised calcium admixed with the plasma is preferably at least the amount required to restore physiological calcium levels. Typically these amounts will be 1.3 to 2.0 mM of ionised calcium as measured by a calcium electrode. However, the invention is not limited to the use of amounts of calcium which restore physiological levels since we believe that the use of higher amounts may also be beneficial.
  • the sources of calcium ions may be ionised organic or inorganic salts, particularly preferred examples being calcium chloride, calcium gluconate, and calcium lactate.
  • Extraction of the Factor VIII from the heparinised plasma may be by any of the conventional techniques involving cooling the plasma below 4oc. Typical examples of such techniques are cryoprecipitation, and alcohol precipitation (Cohn method). Other methods in use to separate Factor VIII (most commonly combined with cryoprecipitation as an initial preparative step) include glycine precipitation with polyethylene glycol precipitation of the contaminating fibrinogen, gel affinity column chromatography, and most recently affinity columns containing immobilised monoclonal antibodies against Factor VIII or van Willibrand protein.
  • Fig 1. is a graph representative of the Heparin Precipitable Fraction (HPF) of plasma at 0-lo c vs amount of Heparin.
  • absorbance (which is representative of HPF principally fibrinogen and fibronectin) begins to increase quite markedly above a heparin concentration of 1 unit/ml of plasma, and above 2 units the increase is almost linear.
  • Cryoprecipitates were routinely prepared. In ths manner, the donors received no heparin. Analysis of the cryoprecipitates. revealed no resolubilisation problems, a significant (p 0.02) gain in yield of VIIIrC compared with ACD A control cryoprecipitates, and a mean specific activity of 0.11 iu/mg (SD 0.03, 6 experiments) .
  • the two heparin/ C a 2+ reversed pools gave an average factor VIII yield of 576 I. ⁇ ./litre, and fibrinogen and fibronectin levels of 629 mg/Kg and 251 mg/Kg, the specific activity of the cryoprecipitate being 0.58.
  • the two control and heparin/Ca 2 + reversed pools were further treated by the fractionation methods in use at the Protein Fractionation Laboratory, Oxford, where a second purification stage is employed, namely a differential heparin cold precipitation step in which contaminating fibrinogen and fibronectin are precipitated at the temperature employed leaving Factor VIII in the supernatant solution.

Abstract

A method of obtaining Factor VIII comprises collecting blood from a donor into a calcium chelating anti-coagulant (e.g. citrate based), separating the plasma from the cellular components of the blood, and then treating the plasma with dissolved calcium (which stabilizes Factor VIII) and with heparin or heparin related compound (which acts as a non-chelating anti-coagulant). The amount of heparin used is from 0.1 to less than 2 units per ml of plasma. Factor VIII is extracted from the treated plasma by any of the conventional techniques involving cooling of the plasma below 4°C (e.g. by cryoprecipitation).

Description

EXTRACTION OF FACTOR VIII
The present invention relates to tne extraction of Factor VIII from Dlood plasma.
Factor VIII is an important clotting protein used by haemophiliacs and is obtained either from whole blood or plasma given by donors. In one known method of obtaining Factor VIII, blood from the donor is passed into a bag containing a solution of a calcium chelating anti-coagulant, e.g. a citrate based anti-coagulant. The blood may be separated into two fractions, namely plasma reduced red cells and plasma, from the latter of which the Factor VIII is obtained. To obtain Factor VIII, the anti-coagulated plasma is subjected to a separation technique, e.g. cryoprecipitation in which the plasma is frozen rapidly (e.g. to -70oc) and then thawed gently to a temperature of 0-2°C at which a precipitate containing proteins (including Factor VIII) is obtained. However, this process only yields about 50% of the Factor VIII present in the original plasma and this is partly due to the fact that calcium ions which stabilize Factor VIII have been removed by the calcium chelating anti-coagulant. There are also other losses of Factor VIII in this precipitation process.
Heparin is also known as an anti-coagulant for blood and in theory could be used in the above process instead of sodium citrate. However, this has the disadvantage that the comparatively high levels of heparin which would be required may produce allergies or platelet deficiencies in patients to whom products derived from the blood donation are given.
It is also possible to obtain blood plasma by the
SUBSTITUT plasmapheresis technique in which blood from εne donor is firstly mixed with the citrate oased anti-coagulant solution and then subject to centrifugation by a cell separator after which _he red cells, white cells and platelets are returned to the donor. The citrate returned to the donor is rapidly removed from the bloodstream and is non-toxic even on repeated administrations. The remaining plasma is used for obtaining Factor VIII (or other important proteins). However, this technique still has the disadvantage of a reduced yield of Factor VIII due to the use of a calcium chelating agent as the anti-coagulant.
EP-B-0 053 046 (G.A. Rock) seeks to overcome the above problems by proposing a method of obtaining Factor VIII in which the whole blood or plasma is collected in a solution of a calcium chelating anti-coagulant (e.g. citrate based) and the collected product is mixed with dissolved calcium and heparin at a time not more than six hours after collection of the blood or plasma. The Factor VIII is then extracted from the heparinised blood or plasma, e.g. by cryoprecipitation. The effect of the dissolved calcium is to stabilize the Factor VIII by maintaining or restoring the calcium bridges removed by the chelating anti-coagulant, whilst the heparin provides a non-chelating anti-coagulant activity. Due to the fact that Factor VIII has been stabilized, high yields of the product may be obtained from the blood or plasma and, furthermore, the stabilization ensures that there will be sufficient time available after collection of the blood or plasma to transport it to centres for Factor VIII extraction without the necessity of other preserving methods. Additionally, the technique is particularly applicable to plasmapheresis since the whole oiood may be mixed with citrate based anti-coagulant solution and, after centrifugation, it is only the plasma which is mixed with heparin and dissolved calcium. Thus, the red cells, white cells, and platelets returned to the donor only contain the citrate based anti-coagulant and not heparin.
It is stated in EP-B-0 053 046 that the amount of heparin required for optimum Factor VIII stabilization can easily be determined by routine experimentation but is generally in the range of 2 to 20 units heparin per ml plasma with best results generally being obtained with from 3 to 12, most preferably 4 to 8 units of heparin per ml plasma. It is also indicated that the amount of heparin is generally above 4 units per ml of plasma.
We do however have evidence that amounts of heparin as recommended in EP-B-0 053 046 lead to cryo- precipitates containing comparatively high amounts of the insoluble proteins fibrinogen and fibronectin in addition to the Factor VIII. These amounts are significantly higher than in cryoprecipitates obtained from conventional citrated plasma. The cryoprecipitate may be used in one of two ways. Firstly, it may be provided, with a small amount of plasma supernatont, to a haemophiliac for "on-demand" self-treatment of bleeding episodes. To effect such treatment, the cryoprecipitate and supernatant are warmed to ambient temperature to resolubilise the precipitate in the supernatant plasma. This dissolution should be as rapid as possible and require minimal manipulation. However the amounts of fibrinogen and fibronectin in the cryoprecipi ate occasioned by the use of the heparin levels quoted above delay dissolution and complete dissolution requires a greater degree of manipulation than a haemophiliac may be capable of, particularly since a haemophiliac may have badly crippled arthritic upper limbs. Furthermore, reconstituted Factor VIII solution requires filtration to remove any possible insoluble fibrils before injection and the presence of any insoluble fibrinogen and fibronectin will block filters and delay the administration of the Factor VIII vitally required by the haemophiliac to stop his bleeding.
Secondly, cryoprecipitation is the most commonly performed first step in the preparation of high-pu ity Factor VIII concentrates. In the purification of the cryoprecipitate the latter is firstly resolubilised, most commonly in a buffer solution, and may then be subject to number of possible purification steps. High levels of fibrinogen and fibronectin delay the resolubilisation of the Factor VIII and it may be necessary to use a homogeniser to effect complete resolubilisation. The use of a homogeniser may cause denaturation of the Factor VIII and reduction in yields.
The above problems do not arise with cryoprecipitates obtained from conventional citrated plasma.
It is therefore an object of the present invention to obviate or mitigate the abovementioned disadvantages.
According to the present invention there is provided a method of obtaining Factor VIII comprising taking blood from a donor, admixing the blood with a calcium chelating anti-coagulant, separating the plasma from the cellular components of the blood treating the plasma with 0.1 to less than 2 units per ml of plasma of heparin or heparin related compound
SUBS (hereinafter referred to as heparin) and with calcium ions, and .subjecting the heparinised plasma to a fractionation technique for separation of Factor VIII in which the plasma is cooled below 4°c-
Most, if not all of the fractionation techniques used for obtaining Factor VIII from plasma (e.g. the alcohol precipitation method (Cohn method) cryoprecipitation etc.) involve cooling the plasma to or below 4oc. The bulk of the fibronectin and fibrinogen precipitation occurs in the temperature range 0-4oc an(_ we have found that the use of amounts of heparin less than those taught in EP-B-0 053 046 causes significantly less precipitation of fibrinogen and fibronectin (thus avoiding the difficulties mentioned above) whilst not causing any significant reduction in the yield of' Factor VIII. More specifically the amount of heparin to be used in accordance with the invention is 0.1 to less than 2 units (of heparin) per ml of plasma. Below 0.1 units of heparin per ml of plasma there may be insufficient anti-coagulation thus giving rise to clotting problems. Preferably, the number of units .of heparin used per ml of plasma is in the range of 0.3 to 1.8, more preferably 0.5 to 1.6, even more preferably 0.8 to 1.2.
Factor VIII obtained by the method of the invention thus contains comparatively low amounts of fibrinogen and fibronectin. Factor VIII precipitates (e.g. obtained by cryoprecipitation) obtained by the method of the invention do not contain any significantly greater proportions of fibrinogen and fibronectin than precipitates produced from conventional citrated plasma. In fact, specific activities of precipitates (defined as Factor VIII I.U./mg total protein) are generally higher for precipitates obtained by the method of th invention than for comparable citrate controls.
In the first step of the method, donor blood is collected in a calcium chelating anti-coagulant. Such anti-coagulants are well known and include citrate anticoagulants, e.g. sodium citrate, citrate phosphate dextrose, and acid citrate dextrose. The amounts of these anti-coagulants used may be conventional. The collection of the blood into the calcium chelating anti-coagulant has the advantage that the cellular component of the blood may, after separation of the plasma, be returned to the donor, and also that the amounts of heparin required are less than would be the case if heparin was used as the primary anti-coagulant.
Separation of the whole blood into plasma and cellular components may be by any conventional technique, e.g. centrifugation of the blood. The invention is particularly applicable to the extraction of Factor VIII from plasma obtained by manual plasmapheresis or machine pheresis using conventional cell separators.
The obtained plasma should be admixed with the heparin and dissolved calcium ions as soon as possible so that the maximum possible amount of Factor VIII is stabilized by the calcium ions. Ideally, the plasma separated from the whole blood is passed into a vessel containing a solution (in sterile water) of calcium ions and heparin. As indicated above, the amount of heparin used is in the range 0.1 to less than 2 units per ml of plasma. The heparin may, for example, be any U.K., European or U.S. phar acopea heparin and may, for example, be synthetic heparin or low molecular weight heparin. Preferably, the heparin should have a minimum activity of 100 I.U./mg. The pharmacopea heparins quoted above exceed this level.
The amount of ionised calcium admixed with the plasma is preferably at least the amount required to restore physiological calcium levels. Typically these amounts will be 1.3 to 2.0 mM of ionised calcium as measured by a calcium electrode. However, the invention is not limited to the use of amounts of calcium which restore physiological levels since we believe that the use of higher amounts may also be beneficial. The sources of calcium ions may be ionised organic or inorganic salts, particularly preferred examples being calcium chloride, calcium gluconate, and calcium lactate.
Extraction of the Factor VIII from the heparinised plasma may be by any of the conventional techniques involving cooling the plasma below 4oc. Typical examples of such techniques are cryoprecipitation, and alcohol precipitation (Cohn method). Other methods in use to separate Factor VIII (most commonly combined with cryoprecipitation as an initial preparative step) include glycine precipitation with polyethylene glycol precipitation of the contaminating fibrinogen, gel affinity column chromatography, and most recently affinity columns containing immobilised monoclonal antibodies against Factor VIII or van Willibrand protein.
The invention will be further described by way of example only with reference to Fig 1. which is a graph representative of the Heparin Precipitable Fraction (HPF) of plasma at 0-loc vs amount of Heparin.
To obtain the results for Fig 1., individual samples (from a single donation) of ACD A-anticoagulated Autopheresis C plasma were - b -
recalcified to approximately 2 mM of ionised calcium (Ca^+) and further treated with amounts of heparin varying from 0.5 to 100 iu/ml plasma r as shown on che graph. The samples were incubated in crushed ice
(0-l°c) for 3 hours and their absorbance was then read at 350 M in a spectrometer calibrated with a plasma sample containing no heparin but recalcified as above. From the graph, it will be seen that absorbance (which is representative of HPF principally fibrinogen and fibronectin) begins to increase quite markedly above a heparin concentration of 1 unit/ml of plasma, and above 2 units the increase is almost linear.
The following non-limiting Examples illustrates the production of Factor VIII using the method of the invention.
Example 1
Donations of substantially platelet-free ACD A plasma were collected using the HemaSciences Autopheresis C plasmapheresis system. Samples of plasma were heparinized (using sodium heparin) to o.l iu/ml. Recalcification was achieved by the addition of CaCl2 until physiological levels of Ca2+ were restored. Low levels of FpA ( 5ng/ml) were measured in all cases, including following the incubation of plasma at 21θc for 24 hours.
Cryoprecipitates were routinely prepared. In ths manner, the donors received no heparin. Analysis of the cryoprecipitates. revealed no resolubilisation problems, a significant (p 0.02) gain in yield of VIIIrC compared with ACD A control cryoprecipitates, and a mean specific activity of 0.11 iu/mg (SD 0.03, 6 experiments) .
Example 2
The principle described above was also used to prepare small scale Factor 'JϋJ concentrates. Eight donors donated a half-litre of plasma on two occasions into ACD-A (controls! and ACD-A, which was subsequently added to heparin and CaCl2 to achieve heparin concentration of 1 i.U/ml and physiological calcium ion concentration. The procedure was repeated with a further eight donors. The two control pools gave an average factor VIII yield of 431 I.U./litre and fibrinogen and fibronectin levels of 474 mg/Kg and 237 g/Kg respectively, the specific activity of the cryoprecipitate being 0.45. The two heparin/Ca2+ reversed pools gave an average factor VIII yield of 576 I.ϋ./litre, and fibrinogen and fibronectin levels of 629 mg/Kg and 251 mg/Kg, the specific activity of the cryoprecipitate being 0.58. The two control and heparin/Ca 2+ reversed pools were further treated by the fractionation methods in use at the Protein Fractionation Laboratory, Oxford, where a second purification stage is employed, namely a differential heparin cold precipitation step in which contaminating fibrinogen and fibronectin are precipitated at the temperature employed leaving Factor VIII in the supernatant solution.
Final yields of factor VIII were 397 I.U. for the heparin/Ca2+ reversed pools and 326 I.U. for the controls. Specific activities were 8.63 for the heparin/Ca2+ pools and 7.09 for the controls. No difficulties with inadequate anticoagulation, or delayed or incomplete solubility were encountered.

Claims

CLAI MS
i. A method of obtain-in Factor VIII comprising taking blood from a donor, admixing the blood with a calcium chelating anti-coagulant, separating the plasma from the cellular components of the blood, treating the plasma with 0.1 to less than 2 units per ml of plasma of heparin or heparin related compounds and with calcium ions, and subjecting the heparinised plasma to a fractionation technique for separation of Factor VIII in which the plasma is cooled below 4oc.
2. A method as claimed in claim 1 wherein the fractionation technique is cryoprecipitation.
3. A method as claimed in claim 1 or 2 wherein the amount of heparin used is 0.3 to 1.8 units per ml of plasma.
4. A method as claimed in claim 3 wherein' the amount of heparin used is 0.5 to 1.6 units per ml of plasma.
5. A method as claimed in claim 4 wherein the amount of heparin used is 0.8 to 1.2 units per ml of plasma.
6. A method as claimed in any one of claims 1 to 5 wherein amount of ionised calcium used restores physiological calcium levels in the plasma.
7. A method as claimed in any of claims 1 to 6 wherein the calcium is provided by calcium chloride, calcium gluconate, or calcium lactate.
PCT/GB1988/000267 1987-04-06 1988-04-06 Extraction of factor viii WO1988008004A1 (en)

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GB878708181A GB8708181D0 (en) 1987-04-06 1987-04-06 Extraction of protein from blood
GB8708181 1987-04-06

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0357724A4 (en) * 1988-02-18 1990-02-20 Baxter Int Stabilized heparin solution.
WO1991016913A1 (en) * 1990-05-07 1991-11-14 Baxter International Inc. Stabilized heparin solution
AU648999B2 (en) * 1989-11-24 1994-05-12 Dade Behring Marburg Gmbh An agent for the storage and suspension of cells, especially erythrocytes
WO2000033853A1 (en) * 1998-12-07 2000-06-15 Global Hemostasis Institute Mgr Ab Anti-coagulation with calcium containing citrate solution

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0033582A2 (en) * 1980-01-18 1981-08-12 The Canadian Red Cross Society Method of obtaining factor VIII
EP0053046A1 (en) * 1980-11-26 1982-06-02 Gail Ann Rock Stabilisation of factor VIII activity in blood plasma
EP0098256A2 (en) * 1982-06-28 1984-01-11 Monsanto Company Production of blood coagulation factor VIII:C
WO1986005190A1 (en) * 1985-03-07 1986-09-12 Central Blood Laboratories Authority Purification of blood coagulation factor viii by precipitation

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0033582A2 (en) * 1980-01-18 1981-08-12 The Canadian Red Cross Society Method of obtaining factor VIII
EP0053046A1 (en) * 1980-11-26 1982-06-02 Gail Ann Rock Stabilisation of factor VIII activity in blood plasma
EP0098256A2 (en) * 1982-06-28 1984-01-11 Monsanto Company Production of blood coagulation factor VIII:C
WO1986005190A1 (en) * 1985-03-07 1986-09-12 Central Blood Laboratories Authority Purification of blood coagulation factor viii by precipitation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Chemical Abstracts, vol. 95, no. 17, 26 October 1981 (Columbus, Ohio, US) M.W. Mosesson et al.:"The potential of heparin as an agent for precipitation of plasma fibronectin (CIg) and certain components of the plasma factor VIII complex", see page 295, column 1, abstract no. 146586d, & Dev. Biochem. 1981, 12 (Chem. Biol. Heparin), 105-11 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0357724A4 (en) * 1988-02-18 1990-02-20 Baxter Int Stabilized heparin solution.
EP0357724A1 (en) * 1988-02-18 1990-03-14 Baxter Int Stabilized heparin solution.
AU648999B2 (en) * 1989-11-24 1994-05-12 Dade Behring Marburg Gmbh An agent for the storage and suspension of cells, especially erythrocytes
WO1991016913A1 (en) * 1990-05-07 1991-11-14 Baxter International Inc. Stabilized heparin solution
WO2000033853A1 (en) * 1998-12-07 2000-06-15 Global Hemostasis Institute Mgr Ab Anti-coagulation with calcium containing citrate solution

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GB8708181D0 (en) 1987-05-13

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