CA1164344A - Process for the preparation of highly purified antihemophilic factor - Google Patents
Process for the preparation of highly purified antihemophilic factorInfo
- Publication number
- CA1164344A CA1164344A CA000387782A CA387782A CA1164344A CA 1164344 A CA1164344 A CA 1164344A CA 000387782 A CA000387782 A CA 000387782A CA 387782 A CA387782 A CA 387782A CA 1164344 A CA1164344 A CA 1164344A
- Authority
- CA
- Canada
- Prior art keywords
- antihemophilic factor
- factor
- per
- antihemophilic
- activity
- 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
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
ABSTRACT
The present invention provides an improved process for preparing from aqueous compositions containing antihemophilic factor obtained from human plasma, antihemophilic factor compositions of high potency and purity and substantially free of denatured antihemophilic factor.
The present invention provides an improved process for preparing from aqueous compositions containing antihemophilic factor obtained from human plasma, antihemophilic factor compositions of high potency and purity and substantially free of denatured antihemophilic factor.
Description
`~ J ~4.~ ~
~ 3906 ANTIHEMOPHILIC FACTOR
This invention relates to antihemophilic factor present in human blood plasma. It particularly relates to compositions containing antihemophilic factor (AHF) of high potency and purity and substantially free of denatured AHF and to processes for the preparation thereof.
The isolation of AHF by fractionation from other proteins present in blood plasma has been achieved. The O prior art of AHF fractionation has demonstrated that AHF
can be separated in part or in toto from fibrinogen and other proteins by column chromatography, polyethylene glvcol (PEG) or polypropylene glycol ~PPG) pxecipitation, glycine precipitation (or with other amino acids), as well as alcohol precipitation. ~ritish patent no. 1,507,198 and U.S. patent no. 3,973,002 utilize pH and tempe~ature adjustments to result in some purification of AHF, but the procedure described in these patents uses a starting material different from what is used in the process described in this application, and results in low potency preparations of intermediate purity. The processes described in these patents utilize a fraction extracted from the cryoprecipi-tate rather than cryoprecipitate itself. Furthermore, fractionation stops after one cycle of pH and cooling adjustment. Others have also recognized the effect of using one cycle of pH and cooling adjustment (J.K. Smith et al., Transfus on 19, 299-306, (1979)).
The compositions of AHF obtained by the prior art processes discussed above are of relatively low concentra-tion ~of the order of about 5 to 15 units of AHF activity per ml) and low purity (less than 1 unit A~F activity per mg protein~, one of the undesirable impurities being denatured AHF.
.~
~,,~1 ~ :~ 6~3~ ~1 1 The present invention provides an improved process for preparing from aqueous compositions containing AHF
obtained from human plasma, A~F compositions of high potency and purity and substantially free of denaturecl AHF.
The present invention provides an aqueous composi-tion of high concentration (about 20 to 45 units of AHF
activity per ml) and high potency (at least 1 unit AHF
activity per mg of protein) and dried compositions having 1 to 10 units AHF per mg protein, such compositions being substantially free of denatured AHF, from human plasma by the process described below.
The process of the present invention may be applied to any aqueous composition of AHF having about 5 to 15 units AHF activity per ml, and a specific activity of about 0.3 to 1.0 unit AHF activity per mg of protein.
In accordance with the present invention such aqueous composition is subjected to the following steps:
(a~ effecting about a 2~ to 10-fold concentration of the solution, (b) adjusting the pH of said concentrated solution to about 5.0 to 6.0, (c) cooling resultant solution to about 0 to 14C, (d) removing the precipitate which forms at said pH and temperature, (e) further clarifying the solution by Eiltration or other equivalent means, and (f) adjusting the pH to about 7Ø
The resulting solution is subjected to sterile filtration and the filtrate obtained therefrom may be put 3o directly into sterile vials or freeze-dried to form a sterile dry preparation. The solutions and dry preparations may be marketed directly as such.
3 ~ 4 1 The present inven-tion will be illustrated in the descriptlon which follows, with reference to Figure 1 which is a flow sheet illustrating steps of a prior art process and the steps of the process of the present invention. This description is given by way of illustration and is not to be considered as limiting.
Human plasma is subjected to freezing, thawing, and centrifugation resulting in the removal of the cryopre-cipitate. At physiological pH and temperature (pH 7-7.4, room temperature to 37C), the cryoprecipitate (#4) can be dissol~ed in a buffer (such as tris hydrochloride or glycine with saline and sodium citrate~ to give a uniform solution. The cryoprecipitate, or a concentrated fraction from the cryoprecipitate contains a complex mixture of clotting proteins (including AHF), fibrinogen, albumin, and other proteins. By acidifying the resuspended cryoprecipi-tate (#6) (to a p~ 6.0-6.8) Istep #8) and cooling (to 5-15C) (step #9) some of the proteins (such as the fibrinogen and the cold-insoluble globulins can be made to precipitate (step #10). By carefully choosing the righ-t conditions, as described above ~pH 6-6.8, temperature 5-15C), AHF can be made to remain in the superna-tant (step #12).
If the pH is made more acidic than pH 6.4 and the solution cooled at this step, (steps 8-12), more proteins are precipitated and can be removed from the solution, but AHF is also precipitated resulting in no additional purification. The processes described in the prior art do not go beyond this stage, and this had led to the assumption that further purification is not possible by the continued 3o manipulation of p~ and temperature.
In accordance with the present invention a high purity concentrate of AHF is obtained by the additional ~,. . .
1 1 ~'1 3~
l steps described below, reference, as noted above, being made to tl~e flow sheet in Figure l.
The separated AI-IF solu-tion (~12) after Al(OH)3 absorbtion is concentrated or precipitated. The concen-trated AHF solution (#l~) has high potency (2~-45 units per ml), but intermediate purity (less than one AHF unit per mg protein). The concentrated AHF solution (#14~
(or concentrated resuspended AHF solution (#14) is then adjusted to pH 5.0-6.0 and cooled to 0.14C at which temperature a precipitate forms. The precipitate (~193 is removed and the AHF (#20) solution clarified, adjusted to pH 7, sterile filtered and bottled or bottled and freeze-~ried.
The sterile solution contains about 25 to ~5 units per ml, has a potency of about l to 10 units AHF
activity per mg of protein and is substantially ~ree of denatured AHF.
The freeze-dried preparation contains about l to 10 units AE~F activity per mg protein and is also sub-stantially free of denatured AHF.
The present invention teaches, therefore, that ifthe AHF solution from the acidified and cooled steps (steps 8-12) is read~usted to pH 7 and concentrated, that subse-quent acidification and cooling effect the removal by precipitation of additional large amounts of fibrinogen, plasminogen and plasmin, cold insoluble globulins, and other proteins without coprecipitation of AHF, and result in a gentle conversion from an AHF preparation of inter-mediate potency and purity to a preparation o~ high potency and purity.
Attempting to extend the first acidification and cooling (steps 8-12) without this sequence is fruitless.
3 4 ~1 1 An additional benefi-t of this purification is that it is a gentle procedure and results in an AHF
preparation which is native, that is, one in which the ratio of antigenic Factor VIII (FVIII Coagulant Antigen) to Factor VIII procoagulant activity is approximately one, indicating the absence of denatured AHF (one which shows substantial levels of inactive antigenic Factor VIII). No other purifica-tion scheme has been demonstrated to produce this unique result.
3o
~ 3906 ANTIHEMOPHILIC FACTOR
This invention relates to antihemophilic factor present in human blood plasma. It particularly relates to compositions containing antihemophilic factor (AHF) of high potency and purity and substantially free of denatured AHF and to processes for the preparation thereof.
The isolation of AHF by fractionation from other proteins present in blood plasma has been achieved. The O prior art of AHF fractionation has demonstrated that AHF
can be separated in part or in toto from fibrinogen and other proteins by column chromatography, polyethylene glvcol (PEG) or polypropylene glycol ~PPG) pxecipitation, glycine precipitation (or with other amino acids), as well as alcohol precipitation. ~ritish patent no. 1,507,198 and U.S. patent no. 3,973,002 utilize pH and tempe~ature adjustments to result in some purification of AHF, but the procedure described in these patents uses a starting material different from what is used in the process described in this application, and results in low potency preparations of intermediate purity. The processes described in these patents utilize a fraction extracted from the cryoprecipi-tate rather than cryoprecipitate itself. Furthermore, fractionation stops after one cycle of pH and cooling adjustment. Others have also recognized the effect of using one cycle of pH and cooling adjustment (J.K. Smith et al., Transfus on 19, 299-306, (1979)).
The compositions of AHF obtained by the prior art processes discussed above are of relatively low concentra-tion ~of the order of about 5 to 15 units of AHF activity per ml) and low purity (less than 1 unit A~F activity per mg protein~, one of the undesirable impurities being denatured AHF.
.~
~,,~1 ~ :~ 6~3~ ~1 1 The present invention provides an improved process for preparing from aqueous compositions containing AHF
obtained from human plasma, A~F compositions of high potency and purity and substantially free of denaturecl AHF.
The present invention provides an aqueous composi-tion of high concentration (about 20 to 45 units of AHF
activity per ml) and high potency (at least 1 unit AHF
activity per mg of protein) and dried compositions having 1 to 10 units AHF per mg protein, such compositions being substantially free of denatured AHF, from human plasma by the process described below.
The process of the present invention may be applied to any aqueous composition of AHF having about 5 to 15 units AHF activity per ml, and a specific activity of about 0.3 to 1.0 unit AHF activity per mg of protein.
In accordance with the present invention such aqueous composition is subjected to the following steps:
(a~ effecting about a 2~ to 10-fold concentration of the solution, (b) adjusting the pH of said concentrated solution to about 5.0 to 6.0, (c) cooling resultant solution to about 0 to 14C, (d) removing the precipitate which forms at said pH and temperature, (e) further clarifying the solution by Eiltration or other equivalent means, and (f) adjusting the pH to about 7Ø
The resulting solution is subjected to sterile filtration and the filtrate obtained therefrom may be put 3o directly into sterile vials or freeze-dried to form a sterile dry preparation. The solutions and dry preparations may be marketed directly as such.
3 ~ 4 1 The present inven-tion will be illustrated in the descriptlon which follows, with reference to Figure 1 which is a flow sheet illustrating steps of a prior art process and the steps of the process of the present invention. This description is given by way of illustration and is not to be considered as limiting.
Human plasma is subjected to freezing, thawing, and centrifugation resulting in the removal of the cryopre-cipitate. At physiological pH and temperature (pH 7-7.4, room temperature to 37C), the cryoprecipitate (#4) can be dissol~ed in a buffer (such as tris hydrochloride or glycine with saline and sodium citrate~ to give a uniform solution. The cryoprecipitate, or a concentrated fraction from the cryoprecipitate contains a complex mixture of clotting proteins (including AHF), fibrinogen, albumin, and other proteins. By acidifying the resuspended cryoprecipi-tate (#6) (to a p~ 6.0-6.8) Istep #8) and cooling (to 5-15C) (step #9) some of the proteins (such as the fibrinogen and the cold-insoluble globulins can be made to precipitate (step #10). By carefully choosing the righ-t conditions, as described above ~pH 6-6.8, temperature 5-15C), AHF can be made to remain in the superna-tant (step #12).
If the pH is made more acidic than pH 6.4 and the solution cooled at this step, (steps 8-12), more proteins are precipitated and can be removed from the solution, but AHF is also precipitated resulting in no additional purification. The processes described in the prior art do not go beyond this stage, and this had led to the assumption that further purification is not possible by the continued 3o manipulation of p~ and temperature.
In accordance with the present invention a high purity concentrate of AHF is obtained by the additional ~,. . .
1 1 ~'1 3~
l steps described below, reference, as noted above, being made to tl~e flow sheet in Figure l.
The separated AI-IF solu-tion (~12) after Al(OH)3 absorbtion is concentrated or precipitated. The concen-trated AHF solution (#l~) has high potency (2~-45 units per ml), but intermediate purity (less than one AHF unit per mg protein). The concentrated AHF solution (#14~
(or concentrated resuspended AHF solution (#14) is then adjusted to pH 5.0-6.0 and cooled to 0.14C at which temperature a precipitate forms. The precipitate (~193 is removed and the AHF (#20) solution clarified, adjusted to pH 7, sterile filtered and bottled or bottled and freeze-~ried.
The sterile solution contains about 25 to ~5 units per ml, has a potency of about l to 10 units AHF
activity per mg of protein and is substantially ~ree of denatured AHF.
The freeze-dried preparation contains about l to 10 units AE~F activity per mg protein and is also sub-stantially free of denatured AHF.
The present invention teaches, therefore, that ifthe AHF solution from the acidified and cooled steps (steps 8-12) is read~usted to pH 7 and concentrated, that subse-quent acidification and cooling effect the removal by precipitation of additional large amounts of fibrinogen, plasminogen and plasmin, cold insoluble globulins, and other proteins without coprecipitation of AHF, and result in a gentle conversion from an AHF preparation of inter-mediate potency and purity to a preparation o~ high potency and purity.
Attempting to extend the first acidification and cooling (steps 8-12) without this sequence is fruitless.
3 4 ~1 1 An additional benefi-t of this purification is that it is a gentle procedure and results in an AHF
preparation which is native, that is, one in which the ratio of antigenic Factor VIII (FVIII Coagulant Antigen) to Factor VIII procoagulant activity is approximately one, indicating the absence of denatured AHF (one which shows substantial levels of inactive antigenic Factor VIII). No other purifica-tion scheme has been demonstrated to produce this unique result.
3o
Claims (5)
1. A process for the preparation of an aqueous composition of antihemophilic factor, having a concentra-tion of about 20 to 45 units of antihemophilic factor per ml, a specific antihemophilic factor activity of at least 1 unit per mg protein, and being obtained from an aqueous solution of antihemophilic factor derived from human blood plasma, said aqueous solution having a concentration of about 5 to 15 units of antihemophilic factor per ml, and a specific antihemophilic factor activity of about 0.3 to 1.0 unit per mg protein, which comprises:
(a) effecting about a 2- to 10- fold concentra-tion of the low concentrate solution, (b) adjusting the pH of said concentrated solution to about 5.0 to 6.0, (c) cooling the resultant solution to about 0 to 14°C, (d) removing the precipitate which forms at said pH and temperature, (e) further clarifying the solution from step (d), and (f) adjusting the pH to about 7Ø
(a) effecting about a 2- to 10- fold concentra-tion of the low concentrate solution, (b) adjusting the pH of said concentrated solution to about 5.0 to 6.0, (c) cooling the resultant solution to about 0 to 14°C, (d) removing the precipitate which forms at said pH and temperature, (e) further clarifying the solution from step (d), and (f) adjusting the pH to about 7Ø
2. A process according to claim 1 wherein the starting aqueous solution of antihemophilic factor, having a concentration of about 5 to 15 units of antihemophilic factor per ml, said antihemophilic factor having a specific antihemophilic factor activity of about 0.3 to 1 unit per mg protein, is obtained from blood plasma by (a) cryoprecipitating the antihemophilic factor, (b) dissolving the cryoprecipitating in a buffer at about pH 7.0 to 7.4, (c) precipitating fibrinogen and cold insoluble globulen by adjusting the pH to about 6.0 to 6.8 and the temperature to about 5 to 15°C.
and removing the precipitate.
(d) removing the prothombin complex in the resulting solution by adsorbing on aluminum hydroxide, and (e) recovering the resulting solution.
and removing the precipitate.
(d) removing the prothombin complex in the resulting solution by adsorbing on aluminum hydroxide, and (e) recovering the resulting solution.
3. An aqueous composition of antihemophilic factor derived from human blood plasma, said composition having a concen-tration of about 20 to 45 units of antihemophilic factor per ml, said antihemophilic factor having a specific antihemophilic factor activity of at least 1 unit per mg protein, when prepared by a process according to claim 1 or 2 or their obvious chemical equivalents.
4. A feeze-dried composition of antihemophilic factor derived from human blood plasma, having about 1 to 10 units anti-hemophilic factor activity per mg protein with a ratio of antigenic Factor VIII to Factor VIII procoagulant activity of approximately one and being substantially free of denatured antihemophilic factor, when prepared by a process of claim 1 or 2 or their obvious chem-ical equivalents.
5. An aqueous composition of antihemophilic factor derived from human blood plasma, having a concentration of about 20 to 45 units antihemophilic factor activity per ml and at least 1 unit antihemophilic factor activity per mg protein, with a ratio of antigenic Factor VIII to Factor VIII procoagulant activity of approximately one and being substantially free of denatured anti-hemophilic factor, when prepared by a process of claim 1 or 2 or their obvious chemical equivalents.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA000387782A CA1164344A (en) | 1981-10-13 | 1981-10-13 | Process for the preparation of highly purified antihemophilic factor |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA000387782A CA1164344A (en) | 1981-10-13 | 1981-10-13 | Process for the preparation of highly purified antihemophilic factor |
Publications (1)
Publication Number | Publication Date |
---|---|
CA1164344A true CA1164344A (en) | 1984-03-27 |
Family
ID=4121150
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA000387782A Expired CA1164344A (en) | 1981-10-13 | 1981-10-13 | Process for the preparation of highly purified antihemophilic factor |
Country Status (1)
Country | Link |
---|---|
CA (1) | CA1164344A (en) |
-
1981
- 1981-10-13 CA CA000387782A patent/CA1164344A/en not_active Expired
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Legal Events
Date | Code | Title | Description |
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MKEX | Expiry | ||
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Effective date: 20010327 |