CA1265051A - Pasteurized, isoagglutinin-free factor viii preparation and a process for its production - Google Patents
Pasteurized, isoagglutinin-free factor viii preparation and a process for its productionInfo
- Publication number
- CA1265051A CA1265051A CA000489738A CA489738A CA1265051A CA 1265051 A CA1265051 A CA 1265051A CA 000489738 A CA000489738 A CA 000489738A CA 489738 A CA489738 A CA 489738A CA 1265051 A CA1265051 A CA 1265051A
- Authority
- CA
- Canada
- Prior art keywords
- factor viii
- solution
- viii
- pasteurized
- anion exchanger
- 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 - Lifetime
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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
Abstract of the disclosure:
A process for the production of a preparation of blood coagulation factor VIII which makes it possible to obtain a pasteurized product which is virtually free of immunoglobulins, isoagglutinins, fibronectin and coag-ulable fibrinogen is described.
A product of this type can be used for the treat-ment of blood coagulation disturbances.
A process for the production of a preparation of blood coagulation factor VIII which makes it possible to obtain a pasteurized product which is virtually free of immunoglobulins, isoagglutinins, fibronectin and coag-ulable fibrinogen is described.
A product of this type can be used for the treat-ment of blood coagulation disturbances.
Description
r~
~he invention rela~es to a process for the pro-duction of a pasteurized and purified preparation of blood coayulation factor V}II o This preparation can be used for the treatment of blood coagula~ion disturbances.
S ~lood coagulation is a complex process which takes place via several reaction steps and which involves at least 13 coagulation ~actors (F~ ~hich are identified by roman numerals. The coagulation factors are predomi nantly proteins and, in particular, proteins equipped ~ith the properties of proteases or accelerators~ The only actual substra~e is fibrinogen ~hich, in cases of injury, is converted by thrombin into its insoluble form, fibrin, ~hich forms ~he primary wound closure. If one of the 13 coagulation factors is missing, the formation of thrombin and fibrin does not take place; the conse-quence is hemorrhage. An instance of this is hemophilia A
which is the mos~ ~idespread disease with a tendency to hemorrhage and which is due ~o a deficiency of factor VIII. ~oth hemophilia A and 8 (F IX deficiency) can only be effectively treated by replacement of the factor which is lacking~
The problem of obtaining F VIII from human plasma in good yield and high purity, as is necessary, in par-ticular9 for the self-treatment of patients, still has no opti~al solution. T-his particularly applies to pas-teuri2ed F VIII concentrates which have increasingly displaced the conventional commercial product because it has been possible with them to eliminate the risk of transmission of hepatitis.
The isolation of a highly purified F ~iII in good yield is made difficult by, in particular, the fact that although F YIII is enriched in the cryoprecipitate, it is associated ~ith fibrinogen and fibronectin, which are two proteins which have relatively high molecular weights and are sparingly soluble and have similar physicochemical properties to F VIII.
, , :. : ' ' ' ' '~
::
A factor VIII concentrate should be as pure as possible~ that is to say free of undesired conco~itant proteins and, in particular, of immunoglobulins, includ-ing isoagglutinins; this is because there are ;ndications that administration of non-specific proteins leads to overstrain of the reticuloendothelial system (RES) and to impairment of the ;mmune defenses manifes~ed by a change in the composition of the lymphocy~e population and of the immunoglobulins~ The significance of this becomes more evident ~hen conn~cted with the fact that hemophiliacs have to undergo life-long treat~en~ with such F VI~I concentrates. This resuL~s in ~he demand for a native, highly purified, pasteurized F VIII con-centrate, i.e~ a product ~hich rules ou~ transmission of hepa~itis viruses and other infectious material and rules ou~ sensitization to isoantigens.
This invention relates to a product of this type which rules out the transmission of hepatitis and is free of isosgglutinins, and to a process for its production.
2û I~ has been found that a solution containing F VIII but ~hich is virtually free of factors of the prothrombin complex ~F II, VII, IX and X~ can be pasteur-ized in a manner known per se after the addition of stabilizers to protect against thermal inactivation, the heated solution can be treated with an anion exchanger in the pH range 5 - 6.5, and the adsorbed F VIII can be washed free of concomitant pro~eins, especially fibrino-gen~ fibronectin and immunoglobulins, including isoagglu-tinins, eluted with a concentrated solution of Na, K or Ca salts ~ith a halogen, and obtained by, for example, precipitation from the eluate.
Ecteola-cellulose and an anion exchanger bearing ~AE ~quaternary aminoethyl) groups have already been used for the purification of F VIII tvan Creveld, S. et al.~ Thromb. Diath. Haem. t1961) VI, No. Z/3~ 282 and Baugh, R. et al., Bioch. Biophys. Acta (1974) 371. 360).
However~ ~ransfer to pilot-plant or manufacturing scale has not been possible. It has merely been possible to use ion exchange chromatography for the final purifica-.
~l~&~ 'a~
~ion of prefractionated material (Fay, Ph. J. et al.~Proc. ~atl. Acad. Sci. (1982) 79, 7200). Adsorption with high specifi~ty takes place only at a pH around 5.5;
however, at this most of ~he proteins contained ;n the cryoprecipitate~ in particular human fibrinogen, preci~
pi~ate ou~, especially on contact with the adsorbent in the column~ Moreover, according to the papers quo ted, relatively large amounts of adsorben~ are neces-sary. This in turn appears to have had an enormous effect on ehe yield, ~hich was tiny, apparenely o~ing to non-spe~ific adsorption of F VIlI - i~s physiologi-cal ~ask is, after all~ to adhere to non-physiological surfaces - to the large amounts of adsorbent~ It was also found that the ma~erial finally purified on a QAE
exchanger rapidly lost activity; for this reason, anion exchangers have not been used for the manufacture of F VIII.
However, 1~ has now been found~ surpris1ngly, that chroma~ography on bas~c ion exchangers is p~rfectly suitable for the manufacture of F VIII preparations.
It ~as surprising that, starting from a pasteurized cryo-precipitate~ 1t is possible to obtain a highly purified F YIII preparation in one step by using anion exchange chromatography~
Thus the invention relates to a process for the productîon of a factor VIII preparation, in ~hich a solu-tion containing factor VIII is, in the presence of stabilizers, pasteurized and purified, ~hich process comprises the solution being treated wi~h ~n anion exchanger ~hich 1s based on carbohydraees~ the exchanger being ~ashed, and the ~ VIII being eluted~
In one embodiment of the invention, a process for the production of a factor VIII preparation is provided which comprises comprises treating a solution containing factor VIII with an anion exchanger based on carbohydrates in the pH
range 5.0 to 6.5, washing the exchanger; eluting the factor VIII with chaotropic solutions or CaC12, and pasteurizing the sol.ution containing factor VIII in the presence of stabil.izers prior to treating with the anion exchanger or pasteurizing the eluted Eactor VIII in the presence.of stabilizers.
~e~
': ~ . '. ' ' .' :, ~ ' ~: ' .: .
: ` : ' '': ~ : , .
5~3~.
- 4~ -Suitable s~arting materials are a cryoprecipitate obtained by the method of Pool et al., Nature (1964) 203 312, Çohn fract10n I (Minot, GoR~ et al. J. Cl;n. I -est.
(1945) Z4, 7047, plasma, F VIII :C-containing cell culture ~
medium and side fractions containing F VIII obtalned from the latter~ Ho~ever, it is advantageous to use directly cryoprecipitate or Cohn I fraction.
It is possible to use as the stabilizer, for ::
.
,, P~
protection against thermal inactivation of F VIII during pasteurization, carbohydrates and amino acids, preferably 35~60 9 of sucrose per 100 9 of solution and 1-3 mol of glycine per li~er of solution and~ whera appropriate, calcium ions. The method can be that of, for example~
German Offenlegungsschrift Z,916~711 or 3,237,512.
Examples of suitable anion exchangers for the adsorption of F VIII are exchangers bearing DEAE, QAE
or ecteola groups~ specifically those based on~ in par-ticular, cellulose, sephadex or sepharose, but DEAE-sepharose is preferred.
In contradistinction to van Creveld and Baugh ~see above) these exchangers are in fact suitable for purification of F VIIIo but under conditions which have not hitherto been disclosed and are described belo~.
The adsorption conditions have proved to be vital.
This is because it has been found, like~ise surprisingly, that in a physiological saline medium, preferably at pH 5.5, F VIII is substantially selectively bound to DEAE-sepharose, whtle concomitant proteins, such as fibrinogen and fibronectin, remain in the supernatant ~batch process) or pass through a column. Finally, it has been foun~, again surprisingly, that a pasteurized solu~ion of the cryoglobulins can in fact be chromato-graphed a~ pH 5.5 in a physiological saline medium,since certain cryoglobulins precipitate under these condi-tions, especially fibrinogen. The adsorption and, in particular, the specificity with ~hich F VIII is bound to the anion exchangers decrease greatly as neutrality is approached. Ho~ever, under normal chromatography conditions, DEAE is only loaded when the pH exceeds 7Ø Nevertheless, precipitation does not take place in the process described here, because the carbohydrates which have been present in the cryosolutions since the ~5 pasteurization keep the fibrinogen and cig in solution in a slightly acid medium~
Thus an advantage of the process described is that on adsorption of pasteurized cryoglobulin onto the anion exchanger the fibrinogen and fibronectin remain . '` ~ . ..
.
in the supernatan~ or effluent and can be obtained from them as pasteurized products. An example of a method for fibronectin is in German Offenlegungsschrift 2,8480529.
The chromatography on, for example, DEAE- or QAE-exchangers is carried ou~ in a slightly acid medium (pH 5.5) and on exchangers which have been appropria~ely equiLibrated, for exa~ple with 0.1 mol/l Na acetate buffer containing 0~1 moL/L Lysine~ It is aLso possible to use this buffer to diLute the pasteurized solu~ion contain-ing F VIII to ~wice the voLume before it is treated with~he exchanger in a batch or column process. Batch adsorp-tionO ~hich is preferably used, has the advan~age that i~ is possible to foLLow the binding of F VIII to the exchanger during adsorption by functional determination 15 of F VIII in the supernatant, and, after the activity has disappeared, it is possibLe to separate the non-adsorbed factors from the ion exchanger by sedimentation or centrifugation and, immediateLy thereaf~erO to start with the washing of the exchanger loaded with F VIII.
~hereas the washing is advantageously carried out as a batch process, for example on a suction filter~ it is advisable to transfer the exchanger to a column for the solution since eLut;on by coLumn chromatography has the advan~age that the F VIII is obtained in a ~elatively ~5 concentrated form - of the order of SD - 100 IU F VIII /mL
under experimental conditions.
The buffers which are suitable for ~ashing the exchanger which is loaded with F VIII are, in particular, those which, on the one hand, do not dissolve off the 30 F VIII but, on the other hand, are suitable for removaL, ~hich is as nearly quantitative as possible, of non-specific proteins, such as the immunoglobulins, and thus also the isoagglutinins~ Surprisingly, an appropriate buffer has proved to be a buffer in ~hich the starting 35 material can be dissolved for the adsorption and which contains 0.1 mol/l Na acetate, 0.1 mol/l lysine and 1 g/l NaCl at pH 5.5. The ion exchanger loaded with F VIII was ~ashed with this buf~er until the eluate has free of isoagglutinins~ The highly sensitive Coombs , ~ 7 -test was used for testing, this also indicating incom-pLe~e antibodiesO
Concentra~ed salt solutions, for example those containing NaCl, are suitable for the desorption of the 5 F VIII from the anion exchangers.
However, other salts of halogens with Na, K or Ca have proved ~o be more advantageous: KBr~ ~a~r and CaCl2.
They have ~he advantage that the F VIII is eluted as a relatively sharp peak with a high activity per unit volume, and this is an important advantage for the precipitation.
The concentrations are in the range 0.05 mol/l up to the satura~ion limit.
Finally, the yield also depends quite significantly on the rate of elution~ which should be of the order of 1-10 ml/cm2/h, preferably 5~7 ml/cm2/hO
The elua~e containing F VIII can be concentra~ed by the customary me~hods, that is to say by precipita~ion with neutral salts, such as ammonium sulfate or NaCl, advantageously with NaCl which can be diaLyzed out more rapidly than ammonium sulfate and, moreover, need not be removed completeLyO
The fur~her processing of the precipitate contain-ing F VIII is carried out in such a manner that the residue from the precipitation is dissolved in, for e~ample, a buff~r of p~ 6.9 which contains Na citrate ~0.02 mol/l~, NaCl tO.06 mol/l), glycine ~20 g/l) and albumin (5 g/l) ~dialysis buffer) and is dialyzed to equilibrium against the same buffer without albumin. The dialyzsd solu~ion is diluted wi~h the albumin-containing dialysis buffer to an activity of 25 to 30 IU F VIII/ml and, after sterilization by filtration, is dispensed into containers and, ~here appropriate, freeze-drieq. The final product is a white lyophilizate ~hich dissolves in Less than one minute~ has a F VIII activity of 5 10 IU/mg protein~ and is pasteurized and free of isoagglutinins. Compared with products of the state of the art, it has ~he advantage that it contains no undesired proteins, in particular those which, as isoantigens~ might lead to sensitization.
..
1~5(~51 The product has not given rise to blood-group incompati-bilities. Transmission of viral diseases, in particular the various types of hepatitis, appears to be ruled out.
The advantages of the process include the facts that it is relatively straightforward and thus can be transferred without difficulty to an industrial scale and that it comprises few ~orking steps. The small number of working steps is reflected by the good yield, since experience has shown that every purification step is associated with loss of activity to a greater or lesser extent.
Thus the invention also relates to a highly puri-fied, pasteurized fac~or VIII preparation which is virtualLy free of immunoglobulins, isoagglutinins, fibronectin and coagulable fibrinogen and which has a specific clotting ~C) activity (F YIII :C~ of about 100 U/mg protein and a ratio of F VIII :C to F VIII R:Ag (related antigen) of > 1.
F VIII can be determined by the following proce-dure: One part, for example 0.1 ml of partial thromboplastin, for example that prepared by the method of German Offen-ZO legungsschrift 2,316,43û, is mixed with one part of F VIII-deficient plasma and one part of diluted normal plasma.
This mixture is ~aintained at 37C for 6 minutes. After the addition of one part of a 0.025 molar calcium chloride solution prewarmed to 37C, the time ~hich eLapses be-ZS tween addition of the calcium chloride solution and theappearance of a clot is determined. A calibration curve dra~n up using serial dilutions of normal plasma is used for quantitation.
1 international unit ~= 1 IU) of F VIII is equiva-lent to the F VIII activity of 1 ml of normal plasma asa substandard to the 3rd International WHO Standard.
The process for obtaining a pasteurized F VIII
preparation ~hich is free of isoagglutinins is illustrated below~
Example 1. Starting material 250 9 of crude cryoprecipitate were dissolved by heating at 30-37C in 750 ml of an NaCl solution (0008 mol/L), which contained glycine ~0.25 mol/l) and 9 _ heparin (1 25 USP-U/ml). rhis resulted in 1,000 ~l of a solution ~ith a concentration of 0~06 mol/l NaCl, 0.2 mol/l glycine and about 1 USP-U heparin/ml~ The pH
of the solution was adjusted with 1 N HCl ~o 6.5.
~he invention rela~es to a process for the pro-duction of a pasteurized and purified preparation of blood coayulation factor V}II o This preparation can be used for the treatment of blood coagula~ion disturbances.
S ~lood coagulation is a complex process which takes place via several reaction steps and which involves at least 13 coagulation ~actors (F~ ~hich are identified by roman numerals. The coagulation factors are predomi nantly proteins and, in particular, proteins equipped ~ith the properties of proteases or accelerators~ The only actual substra~e is fibrinogen ~hich, in cases of injury, is converted by thrombin into its insoluble form, fibrin, ~hich forms ~he primary wound closure. If one of the 13 coagulation factors is missing, the formation of thrombin and fibrin does not take place; the conse-quence is hemorrhage. An instance of this is hemophilia A
which is the mos~ ~idespread disease with a tendency to hemorrhage and which is due ~o a deficiency of factor VIII. ~oth hemophilia A and 8 (F IX deficiency) can only be effectively treated by replacement of the factor which is lacking~
The problem of obtaining F VIII from human plasma in good yield and high purity, as is necessary, in par-ticular9 for the self-treatment of patients, still has no opti~al solution. T-his particularly applies to pas-teuri2ed F VIII concentrates which have increasingly displaced the conventional commercial product because it has been possible with them to eliminate the risk of transmission of hepatitis.
The isolation of a highly purified F ~iII in good yield is made difficult by, in particular, the fact that although F YIII is enriched in the cryoprecipitate, it is associated ~ith fibrinogen and fibronectin, which are two proteins which have relatively high molecular weights and are sparingly soluble and have similar physicochemical properties to F VIII.
, , :. : ' ' ' ' '~
::
A factor VIII concentrate should be as pure as possible~ that is to say free of undesired conco~itant proteins and, in particular, of immunoglobulins, includ-ing isoagglutinins; this is because there are ;ndications that administration of non-specific proteins leads to overstrain of the reticuloendothelial system (RES) and to impairment of the ;mmune defenses manifes~ed by a change in the composition of the lymphocy~e population and of the immunoglobulins~ The significance of this becomes more evident ~hen conn~cted with the fact that hemophiliacs have to undergo life-long treat~en~ with such F VI~I concentrates. This resuL~s in ~he demand for a native, highly purified, pasteurized F VIII con-centrate, i.e~ a product ~hich rules ou~ transmission of hepa~itis viruses and other infectious material and rules ou~ sensitization to isoantigens.
This invention relates to a product of this type which rules out the transmission of hepatitis and is free of isosgglutinins, and to a process for its production.
2û I~ has been found that a solution containing F VIII but ~hich is virtually free of factors of the prothrombin complex ~F II, VII, IX and X~ can be pasteur-ized in a manner known per se after the addition of stabilizers to protect against thermal inactivation, the heated solution can be treated with an anion exchanger in the pH range 5 - 6.5, and the adsorbed F VIII can be washed free of concomitant pro~eins, especially fibrino-gen~ fibronectin and immunoglobulins, including isoagglu-tinins, eluted with a concentrated solution of Na, K or Ca salts ~ith a halogen, and obtained by, for example, precipitation from the eluate.
Ecteola-cellulose and an anion exchanger bearing ~AE ~quaternary aminoethyl) groups have already been used for the purification of F VIII tvan Creveld, S. et al.~ Thromb. Diath. Haem. t1961) VI, No. Z/3~ 282 and Baugh, R. et al., Bioch. Biophys. Acta (1974) 371. 360).
However~ ~ransfer to pilot-plant or manufacturing scale has not been possible. It has merely been possible to use ion exchange chromatography for the final purifica-.
~l~&~ 'a~
~ion of prefractionated material (Fay, Ph. J. et al.~Proc. ~atl. Acad. Sci. (1982) 79, 7200). Adsorption with high specifi~ty takes place only at a pH around 5.5;
however, at this most of ~he proteins contained ;n the cryoprecipitate~ in particular human fibrinogen, preci~
pi~ate ou~, especially on contact with the adsorbent in the column~ Moreover, according to the papers quo ted, relatively large amounts of adsorben~ are neces-sary. This in turn appears to have had an enormous effect on ehe yield, ~hich was tiny, apparenely o~ing to non-spe~ific adsorption of F VIlI - i~s physiologi-cal ~ask is, after all~ to adhere to non-physiological surfaces - to the large amounts of adsorbent~ It was also found that the ma~erial finally purified on a QAE
exchanger rapidly lost activity; for this reason, anion exchangers have not been used for the manufacture of F VIII.
However, 1~ has now been found~ surpris1ngly, that chroma~ography on bas~c ion exchangers is p~rfectly suitable for the manufacture of F VIII preparations.
It ~as surprising that, starting from a pasteurized cryo-precipitate~ 1t is possible to obtain a highly purified F YIII preparation in one step by using anion exchange chromatography~
Thus the invention relates to a process for the productîon of a factor VIII preparation, in ~hich a solu-tion containing factor VIII is, in the presence of stabilizers, pasteurized and purified, ~hich process comprises the solution being treated wi~h ~n anion exchanger ~hich 1s based on carbohydraees~ the exchanger being ~ashed, and the ~ VIII being eluted~
In one embodiment of the invention, a process for the production of a factor VIII preparation is provided which comprises comprises treating a solution containing factor VIII with an anion exchanger based on carbohydrates in the pH
range 5.0 to 6.5, washing the exchanger; eluting the factor VIII with chaotropic solutions or CaC12, and pasteurizing the sol.ution containing factor VIII in the presence of stabil.izers prior to treating with the anion exchanger or pasteurizing the eluted Eactor VIII in the presence.of stabilizers.
~e~
': ~ . '. ' ' .' :, ~ ' ~: ' .: .
: ` : ' '': ~ : , .
5~3~.
- 4~ -Suitable s~arting materials are a cryoprecipitate obtained by the method of Pool et al., Nature (1964) 203 312, Çohn fract10n I (Minot, GoR~ et al. J. Cl;n. I -est.
(1945) Z4, 7047, plasma, F VIII :C-containing cell culture ~
medium and side fractions containing F VIII obtalned from the latter~ Ho~ever, it is advantageous to use directly cryoprecipitate or Cohn I fraction.
It is possible to use as the stabilizer, for ::
.
,, P~
protection against thermal inactivation of F VIII during pasteurization, carbohydrates and amino acids, preferably 35~60 9 of sucrose per 100 9 of solution and 1-3 mol of glycine per li~er of solution and~ whera appropriate, calcium ions. The method can be that of, for example~
German Offenlegungsschrift Z,916~711 or 3,237,512.
Examples of suitable anion exchangers for the adsorption of F VIII are exchangers bearing DEAE, QAE
or ecteola groups~ specifically those based on~ in par-ticular, cellulose, sephadex or sepharose, but DEAE-sepharose is preferred.
In contradistinction to van Creveld and Baugh ~see above) these exchangers are in fact suitable for purification of F VIIIo but under conditions which have not hitherto been disclosed and are described belo~.
The adsorption conditions have proved to be vital.
This is because it has been found, like~ise surprisingly, that in a physiological saline medium, preferably at pH 5.5, F VIII is substantially selectively bound to DEAE-sepharose, whtle concomitant proteins, such as fibrinogen and fibronectin, remain in the supernatant ~batch process) or pass through a column. Finally, it has been foun~, again surprisingly, that a pasteurized solu~ion of the cryoglobulins can in fact be chromato-graphed a~ pH 5.5 in a physiological saline medium,since certain cryoglobulins precipitate under these condi-tions, especially fibrinogen. The adsorption and, in particular, the specificity with ~hich F VIII is bound to the anion exchangers decrease greatly as neutrality is approached. Ho~ever, under normal chromatography conditions, DEAE is only loaded when the pH exceeds 7Ø Nevertheless, precipitation does not take place in the process described here, because the carbohydrates which have been present in the cryosolutions since the ~5 pasteurization keep the fibrinogen and cig in solution in a slightly acid medium~
Thus an advantage of the process described is that on adsorption of pasteurized cryoglobulin onto the anion exchanger the fibrinogen and fibronectin remain . '` ~ . ..
.
in the supernatan~ or effluent and can be obtained from them as pasteurized products. An example of a method for fibronectin is in German Offenlegungsschrift 2,8480529.
The chromatography on, for example, DEAE- or QAE-exchangers is carried ou~ in a slightly acid medium (pH 5.5) and on exchangers which have been appropria~ely equiLibrated, for exa~ple with 0.1 mol/l Na acetate buffer containing 0~1 moL/L Lysine~ It is aLso possible to use this buffer to diLute the pasteurized solu~ion contain-ing F VIII to ~wice the voLume before it is treated with~he exchanger in a batch or column process. Batch adsorp-tionO ~hich is preferably used, has the advan~age that i~ is possible to foLLow the binding of F VIII to the exchanger during adsorption by functional determination 15 of F VIII in the supernatant, and, after the activity has disappeared, it is possibLe to separate the non-adsorbed factors from the ion exchanger by sedimentation or centrifugation and, immediateLy thereaf~erO to start with the washing of the exchanger loaded with F VIII.
~hereas the washing is advantageously carried out as a batch process, for example on a suction filter~ it is advisable to transfer the exchanger to a column for the solution since eLut;on by coLumn chromatography has the advan~age that the F VIII is obtained in a ~elatively ~5 concentrated form - of the order of SD - 100 IU F VIII /mL
under experimental conditions.
The buffers which are suitable for ~ashing the exchanger which is loaded with F VIII are, in particular, those which, on the one hand, do not dissolve off the 30 F VIII but, on the other hand, are suitable for removaL, ~hich is as nearly quantitative as possible, of non-specific proteins, such as the immunoglobulins, and thus also the isoagglutinins~ Surprisingly, an appropriate buffer has proved to be a buffer in ~hich the starting 35 material can be dissolved for the adsorption and which contains 0.1 mol/l Na acetate, 0.1 mol/l lysine and 1 g/l NaCl at pH 5.5. The ion exchanger loaded with F VIII was ~ashed with this buf~er until the eluate has free of isoagglutinins~ The highly sensitive Coombs , ~ 7 -test was used for testing, this also indicating incom-pLe~e antibodiesO
Concentra~ed salt solutions, for example those containing NaCl, are suitable for the desorption of the 5 F VIII from the anion exchangers.
However, other salts of halogens with Na, K or Ca have proved ~o be more advantageous: KBr~ ~a~r and CaCl2.
They have ~he advantage that the F VIII is eluted as a relatively sharp peak with a high activity per unit volume, and this is an important advantage for the precipitation.
The concentrations are in the range 0.05 mol/l up to the satura~ion limit.
Finally, the yield also depends quite significantly on the rate of elution~ which should be of the order of 1-10 ml/cm2/h, preferably 5~7 ml/cm2/hO
The elua~e containing F VIII can be concentra~ed by the customary me~hods, that is to say by precipita~ion with neutral salts, such as ammonium sulfate or NaCl, advantageously with NaCl which can be diaLyzed out more rapidly than ammonium sulfate and, moreover, need not be removed completeLyO
The fur~her processing of the precipitate contain-ing F VIII is carried out in such a manner that the residue from the precipitation is dissolved in, for e~ample, a buff~r of p~ 6.9 which contains Na citrate ~0.02 mol/l~, NaCl tO.06 mol/l), glycine ~20 g/l) and albumin (5 g/l) ~dialysis buffer) and is dialyzed to equilibrium against the same buffer without albumin. The dialyzsd solu~ion is diluted wi~h the albumin-containing dialysis buffer to an activity of 25 to 30 IU F VIII/ml and, after sterilization by filtration, is dispensed into containers and, ~here appropriate, freeze-drieq. The final product is a white lyophilizate ~hich dissolves in Less than one minute~ has a F VIII activity of 5 10 IU/mg protein~ and is pasteurized and free of isoagglutinins. Compared with products of the state of the art, it has ~he advantage that it contains no undesired proteins, in particular those which, as isoantigens~ might lead to sensitization.
..
1~5(~51 The product has not given rise to blood-group incompati-bilities. Transmission of viral diseases, in particular the various types of hepatitis, appears to be ruled out.
The advantages of the process include the facts that it is relatively straightforward and thus can be transferred without difficulty to an industrial scale and that it comprises few ~orking steps. The small number of working steps is reflected by the good yield, since experience has shown that every purification step is associated with loss of activity to a greater or lesser extent.
Thus the invention also relates to a highly puri-fied, pasteurized fac~or VIII preparation which is virtualLy free of immunoglobulins, isoagglutinins, fibronectin and coagulable fibrinogen and which has a specific clotting ~C) activity (F YIII :C~ of about 100 U/mg protein and a ratio of F VIII :C to F VIII R:Ag (related antigen) of > 1.
F VIII can be determined by the following proce-dure: One part, for example 0.1 ml of partial thromboplastin, for example that prepared by the method of German Offen-ZO legungsschrift 2,316,43û, is mixed with one part of F VIII-deficient plasma and one part of diluted normal plasma.
This mixture is ~aintained at 37C for 6 minutes. After the addition of one part of a 0.025 molar calcium chloride solution prewarmed to 37C, the time ~hich eLapses be-ZS tween addition of the calcium chloride solution and theappearance of a clot is determined. A calibration curve dra~n up using serial dilutions of normal plasma is used for quantitation.
1 international unit ~= 1 IU) of F VIII is equiva-lent to the F VIII activity of 1 ml of normal plasma asa substandard to the 3rd International WHO Standard.
The process for obtaining a pasteurized F VIII
preparation ~hich is free of isoagglutinins is illustrated below~
Example 1. Starting material 250 9 of crude cryoprecipitate were dissolved by heating at 30-37C in 750 ml of an NaCl solution (0008 mol/L), which contained glycine ~0.25 mol/l) and 9 _ heparin (1 25 USP-U/ml). rhis resulted in 1,000 ~l of a solution ~ith a concentration of 0~06 mol/l NaCl, 0.2 mol/l glycine and about 1 USP-U heparin/ml~ The pH
of the solution was adjusted with 1 N HCl ~o 6.5.
2. Aluminium hydroxide adsorption 80 ml of a suspension containing 10 g/l aluminium hydroxide tBehring~erke Marburg) were added to 10000 ml of solution from 1. and ~he mixture was stirred for 15 minutes (temperature abou~ 30C). It was then centrifuged at 3,000 x g for 15 min, the residue was discarded~ and the supernatant ras pas~eurized after the addition of stabilizers.
3. Pas~eurization The follo~ing stabilizers were added~ in this sequence, to 1~000 mL of supernatant from 20 5 ml of CaCl2 so~ution, 1 mol/~ t5 mmol/l) 1,000 9 of sucrose (SOO g/kg solution) 15û 9 of glycine (2 mol in 1 l of solution)~
The pH was adjusted wi~h 2 N NaOH to 7~3. The volume increased to 1,700 ml o~ing to the additions.
The solution was kept at 60C in a water bath for 10 hours.
The pH was adjusted wi~h 2 N NaOH to 7~3. The volume increased to 1,700 ml o~ing to the additions.
The solution was kept at 60C in a water bath for 10 hours.
4. Ion exchanger treatment 1,700 ml of solution from 3. ~ere diluted ~ith 1~700 ml of a solution which contained 0.2 mol/l Na acetate, pH S.5, and 0.2 mol/l lysine. The pH ~as adjusted , with dilute acetic acid to 5.5, and 70 ml of DEAE-sepharose ?
6 B Cl~ equilibrated with a solution containing ~a acetate ~0~1 mol/l), pH 5.50 lysine (0.1 mol/l) and NaCl (1 g/l)~
were added. The mixture was stirred at room tem-perature for 2-3 hours, and the binding of the F YIII
~as deter~ined by determination of the activity in the supernatant. When the F VIII activity had decreased from 4 IU to 0.1 TU/ml, the adsorbent was re~oved by centri-fugation.
The supernatant was poured off and the adsor-bent was separated from the remainder of the supernatent~
Entrapped protein ~as remo~ed from the sepharose by wash-ing with a solution containing 0.1 mol/l Na acetate, de~ e~ ~r~ n~
, ':
S
pH 5.5, 0.1 mol/l lysine and 1 g/l NaCl~ and it was then ~ransferred as a slurry in the same buffer to a column (dimensions: 10x3 cm).
The exchanger was washed in the column un~il there ~as no longer any measurable absorp~ion of light at 280 nm and the isoagglutinin values in the Coombs test were at the detection limit.
6 B Cl~ equilibrated with a solution containing ~a acetate ~0~1 mol/l), pH 5.50 lysine (0.1 mol/l) and NaCl (1 g/l)~
were added. The mixture was stirred at room tem-perature for 2-3 hours, and the binding of the F YIII
~as deter~ined by determination of the activity in the supernatant. When the F VIII activity had decreased from 4 IU to 0.1 TU/ml, the adsorbent was re~oved by centri-fugation.
The supernatant was poured off and the adsor-bent was separated from the remainder of the supernatent~
Entrapped protein ~as remo~ed from the sepharose by wash-ing with a solution containing 0.1 mol/l Na acetate, de~ e~ ~r~ n~
, ':
S
pH 5.5, 0.1 mol/l lysine and 1 g/l NaCl~ and it was then ~ransferred as a slurry in the same buffer to a column (dimensions: 10x3 cm).
The exchanger was washed in the column un~il there ~as no longer any measurable absorp~ion of light at 280 nm and the isoagglutinin values in the Coombs test were at the detection limit.
5. Elution The exchanger from 4O ~as eluted with a solu~ion, of pH 5.5~ containing 0.1 mol/l Na acetate, 0.1 mol/l lysine and 0.3 mol/l CaCl2. This resul~ed in the appearance of a peak measurable at a wavelength of 280 nm. The corresponding fractions were collected~ and a volume of 180 ml con~aining 40 IU F VIII/ml was obtained.
Precipitation of F VIII
2.2 mol/l glycine and 15û g/l NaCl wer~ added to 180 ml of eluate from 5., and the mixture was stirred at room temperature for 30 min.
The precipitation ~as recognizable by a marked opalescence. The precipitate was separated off by centrifugation at 30,000 x g in an ultracentrifuge for 30 min and, after the supernatant had been poured off~
it was kept at 4C overnight~
7. ~orking up The precipitate from 6. was taken up in 145 ml of buffer, of pH 7.0, ~hich contained 0~02 mol/l ~ri-Na citrate~ 0.06 mol/l NaCl, 10 g/l glycine and S g/l human albumin tdissolving buffer). The activity de~er-mined in the solution was 40 IU F VIII :Ctml. It ~as dialyzed against the abovementioned buffer containing no albumin, at room temperature for 3 hours, and the dialyzate was warmed to 30C and centrifuged at 3û,000 x 9 and 25C for 30 minutes~ After determination of F VIII, which showed 36 IU/ml, the solution ~as adjusted to 30 IU/ml with dissolving buffer, and the solution was warmed to 37C and sterilized by filtration through a membrane filter.
It was dispensed into vials, ~rozen and freeze-dried.
, ' ~ .
, : .
Precipitation of F VIII
2.2 mol/l glycine and 15û g/l NaCl wer~ added to 180 ml of eluate from 5., and the mixture was stirred at room temperature for 30 min.
The precipitation ~as recognizable by a marked opalescence. The precipitate was separated off by centrifugation at 30,000 x g in an ultracentrifuge for 30 min and, after the supernatant had been poured off~
it was kept at 4C overnight~
7. ~orking up The precipitate from 6. was taken up in 145 ml of buffer, of pH 7.0, ~hich contained 0~02 mol/l ~ri-Na citrate~ 0.06 mol/l NaCl, 10 g/l glycine and S g/l human albumin tdissolving buffer). The activity de~er-mined in the solution was 40 IU F VIII :Ctml. It ~as dialyzed against the abovementioned buffer containing no albumin, at room temperature for 3 hours, and the dialyzate was warmed to 30C and centrifuged at 3û,000 x 9 and 25C for 30 minutes~ After determination of F VIII, which showed 36 IU/ml, the solution ~as adjusted to 30 IU/ml with dissolving buffer, and the solution was warmed to 37C and sterilized by filtration through a membrane filter.
It was dispensed into vials, ~rozen and freeze-dried.
, ' ~ .
, : .
Claims (13)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for the production of a factor VIII
preparation which comprises treating a solution containing factor VIII with an anion exchanger based on carbohydrates in the pH range 5.0 to 6.5, washing the exchanger, eluting the factor VIII with chaotropic solutions or CaCl2, and pasteurizing the solution containing factor VIII in the presence of stabilizers prior to treating with the anion exchanger or pasteurizing the eluted factor VIII in the presence of stabilizers.
preparation which comprises treating a solution containing factor VIII with an anion exchanger based on carbohydrates in the pH range 5.0 to 6.5, washing the exchanger, eluting the factor VIII with chaotropic solutions or CaCl2, and pasteurizing the solution containing factor VIII in the presence of stabilizers prior to treating with the anion exchanger or pasteurizing the eluted factor VIII in the presence of stabilizers.
2. The process as claimed in claim 1, wherein the solution containing factor VIII is pasteurized in the presence of stabilizers prior to treating with the anion exchanger.
3. The process as claimed in claim 1, wherein the eluted factor VIII is pasteurized in the presence of stabilizers.
4. The process as claimed in claim 1 or 2, wherein the solution containing factor VIII is a solution of cryoprecipitate.
5. The process as claimed in claim 1, 2 or 3, wherein the anion exchanger is DEAE, QAE, an amine resin or ecteola derivatives of cellulose, sephadex or sepharose.
6. The process as claimed in claim 1, 2 or 3, wherein the solution containing factor VIII is plasma, Cohn fraction I or culture medium from cells synthesizing F VIII.
7. The process as claimed in claim 1, 2 or 3, wherein the anion exchanger is washed with a dilute buffered salt solution.
8. The process as claimed in claim 1, 2 or 3, wherein the factor VIII is eluted with a concentrated buffered aqueous solution of a salt of sodium, potassium or calcium with halogens.
9. The process as claimed in claim 2, wherein the solution is pasteurized in the presence of amino acids, carbohydrates and calcium.
10. The process as claimed in claim 3, wherein the eluted factor VIII is pasteurized in the presence of amino acids, carbohydrates and calcium.
11. A process for the production of a factor VIII
preparation which comprises pasteurizing a solution of cryoprecipitate containing sucrose, glycine and calcium ions, cooling and diluting the solution, treating the solution with an anion exchanger based on carbohydrates at pH 5.5, washing the anion exchanger with a dilute buffered salt solution until it is free of foreign proteins including isoagglutinins, eluting the factor VIII with a concentrated buffered salt solution and dialyzing and freeze-drying the eluted factor VIII.
preparation which comprises pasteurizing a solution of cryoprecipitate containing sucrose, glycine and calcium ions, cooling and diluting the solution, treating the solution with an anion exchanger based on carbohydrates at pH 5.5, washing the anion exchanger with a dilute buffered salt solution until it is free of foreign proteins including isoagglutinins, eluting the factor VIII with a concentrated buffered salt solution and dialyzing and freeze-drying the eluted factor VIII.
12. The process as claimed in claim 11, wherein the solution of cryoprecipitate contains 35-60g of sucrose/100 ml, 1-3 mol of glycine/1, and 1-20 mmol/l calcium ions.
13. A pasteurized, highly purified factor VIII
preparation which is virtually free of immunoglobulins, isoagglutinins, fibronectin and coagulable fibrinogen and having a specific clotting (C) activitiy (F VIII:C) of about 100 U/mg of protein and a ratio of F VIII:C to F VIII R:Ag (related antigen) of > 1.
preparation which is virtually free of immunoglobulins, isoagglutinins, fibronectin and coagulable fibrinogen and having a specific clotting (C) activitiy (F VIII:C) of about 100 U/mg of protein and a ratio of F VIII:C to F VIII R:Ag (related antigen) of > 1.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP3432083.0 | 1984-08-31 | ||
| DE19843432083 DE3432083A1 (en) | 1984-08-31 | 1984-08-31 | PASTEURIZED, ISOAGGLUTININ-FREE FACTOR VIII PREPARATION AND METHOD FOR THE PRODUCTION THEREOF |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1265051A true CA1265051A (en) | 1990-01-30 |
Family
ID=6244387
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000489738A Expired - Lifetime CA1265051A (en) | 1984-08-31 | 1985-08-30 | Pasteurized, isoagglutinin-free factor viii preparation and a process for its production |
Country Status (8)
| Country | Link |
|---|---|
| EP (1) | EP0173242B2 (en) |
| JP (1) | JPS6160614A (en) |
| AT (1) | ATE73666T1 (en) |
| CA (1) | CA1265051A (en) |
| DE (2) | DE3432083A1 (en) |
| DK (1) | DK174066B1 (en) |
| ES (1) | ES8702146A1 (en) |
| IL (1) | IL76260A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5424401A (en) * | 1989-08-07 | 1995-06-13 | Behringwerke Aktiengeseschaft | Process for the preparation of a stable factor VIII |
Families Citing this family (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4847362A (en) * | 1985-02-01 | 1989-07-11 | New York University | Method for purifying antihemophilic factor |
| US4952675A (en) * | 1985-02-01 | 1990-08-28 | New York University | Method for purifying antihemophilic factor |
| US4743680A (en) * | 1985-02-01 | 1988-05-10 | New York University | Method for purifying antihemophilic factor |
| CA1339946C (en) * | 1987-03-31 | 1998-07-07 | Michael J. Griffith | Ultrapurification process for polypeptides |
| ES2053619T3 (en) * | 1988-05-27 | 1994-08-01 | Centre Regional De Transfusion | PROCEDURE FOR THE MANUFACTURE OF AN ANTIHEMOPHILIC FACTOR OF GREAT PURITY AND FREE OF VIRUSES THROUGH CHROMATOGRAPHY. |
| AU2684488A (en) | 1988-06-27 | 1990-01-04 | Carter-Wallace, Inc. | Test device and method for colored particle immunoassay |
| ATE85221T1 (en) | 1988-11-05 | 1993-02-15 | Octapharma Ag | PROCESS FOR THE PREPARATION OF A HIGH PURITY, NON-INFECTIOUS ANTIHAEMOPHILIA FACTOR USING CHROMATOGRAPHY. |
| DE4001451A1 (en) * | 1990-01-19 | 1991-08-01 | Octapharma Ag | STABLE INJECTABLE SOLUTIONS OF FACTOR VIII AND FACTOR IX |
| DE4143678B4 (en) * | 1991-02-27 | 2005-03-10 | Holland Letz Felo Werkzeug | Holder for screwdriver bits |
| AT402891B (en) * | 1991-06-20 | 1997-09-25 | Immuno Ag | METHOD FOR PRODUCING AN INACTIVATED BLOOD PRODUCT |
| FR2681867B1 (en) | 1991-09-26 | 1993-12-31 | Pasteur Merieux Serums Vaccins | FACTOR VIII PURIFICATION PROCESS AND PREPARATIONS OBTAINED. |
| AT399818B (en) * | 1992-04-24 | 1995-07-25 | Immuno Ag | METHOD FOR PRODUCING A HIGH PURIFIED VIRUS-SAFE FACTOR VIII PREPARATION |
| AU6653094A (en) * | 1992-12-16 | 1994-07-04 | Immuno Aktiengesellschaft | Process for preparing a virus-safe biological composition |
| DE4320294A1 (en) * | 1993-06-18 | 1994-12-22 | Immuno Ag | Use of human protein C to prevent and treat platelet deposits |
| DE4337573C1 (en) * | 1993-11-04 | 1995-05-18 | Octapharma Ag | Process for the preparation of a virus-inactivated factor VIII-containing fraction by means of chromatographic methods |
| DE19802007C1 (en) * | 1998-01-20 | 1999-11-11 | Wacker Werke Kg | Safety arrangement for the operating elements of a hand-held soil compaction roller |
| DE102004009400A1 (en) | 2004-02-24 | 2005-09-08 | Zlb Behring Gmbh | Fibrinogen purification |
| CN102295696B (en) * | 2011-08-16 | 2013-06-26 | 山东泰邦生物制品有限公司 | Method for preparing coagulation factor VIII, fibrinogen and fibronectin from cryoprecitation |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE2653534C2 (en) * | 1975-12-22 | 1986-08-28 | Baxter Travenol Laboratories, Inc., Deerfield, Ill. | Solid antihemophil factor preparation and process for its manufacture |
| DE2635894A1 (en) * | 1976-08-10 | 1978-02-16 | Biotest Serum Institut Gmbh | Prepn. of concentrates contg. anti-haemophilic globulin:A - by adsorption onto di:ethylamino-ethyl-cellulose from plasma etc., then elution with buffer |
| JPS57136526A (en) * | 1981-02-17 | 1982-08-23 | Green Cross Corp:The | Preparation of blood coagulation factor 8 |
| AU555305B2 (en) * | 1982-09-29 | 1986-09-18 | Bayer Corporation | Antihemophilic factor concentrate |
| DE3237512A1 (en) * | 1982-10-09 | 1984-04-12 | Behringwerke Ag, 3550 Marburg | METHOD FOR PASTEURIZING ANTIHAEMOPHILIC CRYOPRAEZIPITATE (AHK) AND ANTIHAEMOPHILE CRYOPRAECIPITATE PRODUCED THEREOF |
| US4508709A (en) * | 1983-12-05 | 1985-04-02 | Armour Pharmaceutical Company | Process for purifying factor VIII:C |
-
1984
- 1984-08-31 DE DE19843432083 patent/DE3432083A1/en not_active Withdrawn
-
1985
- 1985-08-21 EP EP85110499A patent/EP0173242B2/en not_active Expired - Lifetime
- 1985-08-21 AT AT85110499T patent/ATE73666T1/en not_active IP Right Cessation
- 1985-08-21 DE DE8585110499T patent/DE3585649D1/en not_active Expired - Fee Related
- 1985-08-29 ES ES546526A patent/ES8702146A1/en not_active Expired
- 1985-08-30 DK DK198503981A patent/DK174066B1/en not_active IP Right Cessation
- 1985-08-30 IL IL76260A patent/IL76260A/en not_active IP Right Cessation
- 1985-08-30 JP JP60189997A patent/JPS6160614A/en active Granted
- 1985-08-30 CA CA000489738A patent/CA1265051A/en not_active Expired - Lifetime
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5424401A (en) * | 1989-08-07 | 1995-06-13 | Behringwerke Aktiengeseschaft | Process for the preparation of a stable factor VIII |
Also Published As
| Publication number | Publication date |
|---|---|
| ES546526A0 (en) | 1986-12-16 |
| DK398185A (en) | 1986-03-01 |
| EP0173242A2 (en) | 1986-03-05 |
| JPH0580455B2 (en) | 1993-11-09 |
| ES8702146A1 (en) | 1986-12-16 |
| DK174066B1 (en) | 2002-05-21 |
| EP0173242B2 (en) | 1996-07-24 |
| ATE73666T1 (en) | 1992-04-15 |
| IL76260A (en) | 1992-02-16 |
| JPS6160614A (en) | 1986-03-28 |
| DE3585649D1 (en) | 1992-04-23 |
| EP0173242A3 (en) | 1987-10-07 |
| DE3432083A1 (en) | 1986-03-06 |
| DK398185D0 (en) | 1985-08-30 |
| EP0173242B1 (en) | 1992-03-18 |
| IL76260A0 (en) | 1986-01-31 |
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