CA1297011C - Method of producing a factor viii (ahf) containing fraction - Google Patents
Method of producing a factor viii (ahf) containing fractionInfo
- Publication number
- CA1297011C CA1297011C CA000549552A CA549552A CA1297011C CA 1297011 C CA1297011 C CA 1297011C CA 000549552 A CA000549552 A CA 000549552A CA 549552 A CA549552 A CA 549552A CA 1297011 C CA1297011 C CA 1297011C
- Authority
- CA
- Canada
- Prior art keywords
- factor viii
- solution
- lyophilisate
- glycine
- precipitate
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
- Y10S530/827—Proteins from mammals or birds
- Y10S530/829—Blood
- Y10S530/83—Plasma; serum
Abstract
ABSTRACT OF THE DISCLOSURE:
There is disclosed a method of producing a Factor VIII
(AHF) containing fraction having a specific activity of at least 2.5 units of Factor VIII/mg protein as well as a portion of immunoglobulin G (IgG) of 10 mg/1000 units of Factor VIII at the most. Its risk of transmission of viral or bacterial infections is to be avoided or largely reduced. The method consists in that undesired proteins are at first precipitated from a Factor VIII (AHF) containing plasma fraction in the presence of SPS. The purified Factor VIII containing solution is treated with suitable salts or salt mixtures in order to obtain a Factor VIII containing precipitate. This precipitate is dissolved, lyophilized and finally heat-treated.
There is disclosed a method of producing a Factor VIII
(AHF) containing fraction having a specific activity of at least 2.5 units of Factor VIII/mg protein as well as a portion of immunoglobulin G (IgG) of 10 mg/1000 units of Factor VIII at the most. Its risk of transmission of viral or bacterial infections is to be avoided or largely reduced. The method consists in that undesired proteins are at first precipitated from a Factor VIII (AHF) containing plasma fraction in the presence of SPS. The purified Factor VIII containing solution is treated with suitable salts or salt mixtures in order to obtain a Factor VIII containing precipitate. This precipitate is dissolved, lyophilized and finally heat-treated.
Description
37~ ~
The invention relates to a method of produciny a Factor VïII (AHY) containing fractiol. having a specific activity of a-t least 2.5 units oE Factor VI~ ng protein as we~l as a portion of i~nunoglobulin G ~IgG) of 10 mg/1000 units of Factor VIII at the most, at the therapeutic or prophylactic application of which the risk of transmission OL viral or bacterial infections is avoided or largel~
reduced.
A plurality of methods for producing Factor VIII
concentrates are already known from the literature. As fractionation measures, these methods involve the treatment of plasma with ethanol, ether, polyethylene glycol and/or glycine. Also known is the cryoprecipitation of plasma according to Pool (1965, "The New England Journal of Medicine" 273, 1443) or the cryoethanol precipitation of plasma according to Johnson (Congr. Int. Soc. Blood Transf., Sydney, Australia, Abstracts of Paper, p. 1109 (1966).
In U.S. patent No. 3,973,002, a method is, fur~hermore, described, with which a cryoprecipitate obtained fro~ blood plasma is macerated, the macerated product lS suspended in a citrate-glucose buffer solution, is centrifuged, and the thus obtained buffer extract is adjusted to a pEI in the range of 6.0 to 6~o Under these conditions, precipitation of undesired impurities takes place, whereupon the remaining Factor VIII containing residue is sterilized and lyophilized. However, a ~actor VIII product obtained according to that method exhibits only a slight specific activity of Factor VIII units/mg of protein. In addition, the portion of immunoglobulin G
.
(IgG), based on the E'ac-tor VIII units, is undesiredly high.
Similar methods are described in British patent No.
1,551,928 as well as in U.S. patents Nos. 4,170,639 and 4,104,266. There, likewisely by departing from plasma, a cryoprecipitate is recovered, which is dissolved in a buf~er solution in the neutral pH range, with undesired proteins being separa-ted, the supernatant being treated with aluminum hydroxide in order to separate the prothrombin complex, and the Factor VIII containing solution subsequently being concentrated and lyophilized.
Even with this method, the specific activity of Factor VIII
units/mg protein is undesiredly low. Thus, the specific activity when operating according to U.S. patent No.
4,104,266 amounts to no more than 0.5 to 0.6 units/mg protein as indicated there.
According to PCT publication WO 82/04395, a process for the purification and concentration of a Factor VIII
complex is known, wherein the preparation is treated with a glycine solution and the supernatant is precipitated with a salt solution.
According to U.S. patent No. 4,522,751 of Applicant, a method for producing a Factor VIII (AHF) containing preparation with an elevated speci.fic activity (at least 1.5 units of Factor VIII/mg protein) has been proposed, wherein the precipitation of undesired proteins is carried out in the presence of sulfated polysaccharide in the neutral range and the Factor VIII concentrate is recovered from the supernatant by alcohol precipitation.
From tests carried out recently (Vox Sang. 49: 319-322 (1985)) it has become known that IgG complexes in Factor : .
~III concentrates induce adverse reactions, such as lymphocyte abnormalities as well as the changes of the T
helper/T suppressor cell ratio in hemophilia patients.
It has proved that the methods described ~ield products that, apart Erom the undesiredly high IgG content, are not sufficiently heat~stable in order to resist inactivation by thermal treatment without substantial loss of the Factor VIII activityO ~t present, there is the need to keep any coagulation factor preparations prepared from blood plasma and administered to patients in large amounts free from the risk of transmission of viral or bacterial infections, the applied inactivation methods, as a rule, involving several hour treatment at temperatures of above 60C. This delnand also holds for Factor VIII preparations, although - as known - the latter are relatively sensitive and unstable as compared to other coagulation factors.
The invention has as its object to avoid the difficulties pointed .out and to provide a method of ~ producing a Factor VIII (AHF) containing fraction in which - 20 the specific activity is raised to at least 2.5 units of Factor VIII and the portion of immunoglobulin G is as low : as possible. Moreover, the preparation obtained is to be thermally stable so as to enable inac-tivation by heat treatment without lowering the Factor VIII activity in an undesired manner.
In accordance with the invention, this object is achièved by the combination of the following measures: :
- precipitating and separating undesired proteins from a solution of a plasma fraction containing Factor VIII in the presence of sulfated polysaccharides at a pH approximately ::
The invention relates to a method of produciny a Factor VïII (AHY) containing fractiol. having a specific activity of a-t least 2.5 units oE Factor VI~ ng protein as we~l as a portion of i~nunoglobulin G ~IgG) of 10 mg/1000 units of Factor VIII at the most, at the therapeutic or prophylactic application of which the risk of transmission OL viral or bacterial infections is avoided or largel~
reduced.
A plurality of methods for producing Factor VIII
concentrates are already known from the literature. As fractionation measures, these methods involve the treatment of plasma with ethanol, ether, polyethylene glycol and/or glycine. Also known is the cryoprecipitation of plasma according to Pool (1965, "The New England Journal of Medicine" 273, 1443) or the cryoethanol precipitation of plasma according to Johnson (Congr. Int. Soc. Blood Transf., Sydney, Australia, Abstracts of Paper, p. 1109 (1966).
In U.S. patent No. 3,973,002, a method is, fur~hermore, described, with which a cryoprecipitate obtained fro~ blood plasma is macerated, the macerated product lS suspended in a citrate-glucose buffer solution, is centrifuged, and the thus obtained buffer extract is adjusted to a pEI in the range of 6.0 to 6~o Under these conditions, precipitation of undesired impurities takes place, whereupon the remaining Factor VIII containing residue is sterilized and lyophilized. However, a ~actor VIII product obtained according to that method exhibits only a slight specific activity of Factor VIII units/mg of protein. In addition, the portion of immunoglobulin G
.
(IgG), based on the E'ac-tor VIII units, is undesiredly high.
Similar methods are described in British patent No.
1,551,928 as well as in U.S. patents Nos. 4,170,639 and 4,104,266. There, likewisely by departing from plasma, a cryoprecipitate is recovered, which is dissolved in a buf~er solution in the neutral pH range, with undesired proteins being separa-ted, the supernatant being treated with aluminum hydroxide in order to separate the prothrombin complex, and the Factor VIII containing solution subsequently being concentrated and lyophilized.
Even with this method, the specific activity of Factor VIII
units/mg protein is undesiredly low. Thus, the specific activity when operating according to U.S. patent No.
4,104,266 amounts to no more than 0.5 to 0.6 units/mg protein as indicated there.
According to PCT publication WO 82/04395, a process for the purification and concentration of a Factor VIII
complex is known, wherein the preparation is treated with a glycine solution and the supernatant is precipitated with a salt solution.
According to U.S. patent No. 4,522,751 of Applicant, a method for producing a Factor VIII (AHF) containing preparation with an elevated speci.fic activity (at least 1.5 units of Factor VIII/mg protein) has been proposed, wherein the precipitation of undesired proteins is carried out in the presence of sulfated polysaccharide in the neutral range and the Factor VIII concentrate is recovered from the supernatant by alcohol precipitation.
From tests carried out recently (Vox Sang. 49: 319-322 (1985)) it has become known that IgG complexes in Factor : .
~III concentrates induce adverse reactions, such as lymphocyte abnormalities as well as the changes of the T
helper/T suppressor cell ratio in hemophilia patients.
It has proved that the methods described ~ield products that, apart Erom the undesiredly high IgG content, are not sufficiently heat~stable in order to resist inactivation by thermal treatment without substantial loss of the Factor VIII activityO ~t present, there is the need to keep any coagulation factor preparations prepared from blood plasma and administered to patients in large amounts free from the risk of transmission of viral or bacterial infections, the applied inactivation methods, as a rule, involving several hour treatment at temperatures of above 60C. This delnand also holds for Factor VIII preparations, although - as known - the latter are relatively sensitive and unstable as compared to other coagulation factors.
The invention has as its object to avoid the difficulties pointed .out and to provide a method of ~ producing a Factor VIII (AHF) containing fraction in which - 20 the specific activity is raised to at least 2.5 units of Factor VIII and the portion of immunoglobulin G is as low : as possible. Moreover, the preparation obtained is to be thermally stable so as to enable inac-tivation by heat treatment without lowering the Factor VIII activity in an undesired manner.
In accordance with the invention, this object is achièved by the combination of the following measures: :
- precipitating and separating undesired proteins from a solution of a plasma fraction containing Factor VIII in the presence of sulfated polysaccharides at a pH approximately ::
3~.
in tlle neutral range, - treating the thus purified Factor VIII containing solution with a protein precipitating agent, selected from an~onium sulEate, ammoni.um sulfate - glycine, sodium chloride - glycine, sodium sulfa-te, sodium sulfate - sodium citrate, ammonium sulfate - sodium citrate, citrate glycine, in order to precipitate a Facto.r VIII containing pr~cipitate, - dissolving and lyophilizing the precipitated Factor VII~ containing precipitate, and - thermally treating the lyophilisate at a temperature and for a period of time sufficient to inactivate possibly present viruses.
It is known per se that the indicated salts or salt-amino acid combinations constitute protein precipitatingagents and have already been used as such at the production of coagulation factor preparations, as has been illustrated by the introductory survey of the prior art.
~:~ Yet, it i5 the merit of the combination according to : 20 this invention to enable.the recovery of products that are superior to those known so far by exhibiting a high specific activity of Factor VIII at as low an IgG content as po~sible and simultaneously bei}lg sufficiently h~at-stable.
Preferably, the lyophilisate is adjusted to a water content of more than 0.05 ~5 ~ by weight~ and less than ~ 0.70 (70 ~ by weight), preferably less than 0.40 (40 % by ; ~ weightj, and is treated in a closed container at a temperature ranging from S0 to 121C under elevation of the steam partial pressure.
in tlle neutral range, - treating the thus purified Factor VIII containing solution with a protein precipitating agent, selected from an~onium sulEate, ammoni.um sulfate - glycine, sodium chloride - glycine, sodium sulfa-te, sodium sulfate - sodium citrate, ammonium sulfate - sodium citrate, citrate glycine, in order to precipitate a Facto.r VIII containing pr~cipitate, - dissolving and lyophilizing the precipitated Factor VII~ containing precipitate, and - thermally treating the lyophilisate at a temperature and for a period of time sufficient to inactivate possibly present viruses.
It is known per se that the indicated salts or salt-amino acid combinations constitute protein precipitatingagents and have already been used as such at the production of coagulation factor preparations, as has been illustrated by the introductory survey of the prior art.
~:~ Yet, it i5 the merit of the combination according to : 20 this invention to enable.the recovery of products that are superior to those known so far by exhibiting a high specific activity of Factor VIII at as low an IgG content as po~sible and simultaneously bei}lg sufficiently h~at-stable.
Preferably, the lyophilisate is adjusted to a water content of more than 0.05 ~5 ~ by weight~ and less than ~ 0.70 (70 ~ by weight), preferably less than 0.40 (40 % by ; ~ weightj, and is treated in a closed container at a temperature ranging from S0 to 121C under elevation of the steam partial pressure.
~:
:
Suitably, the treatment of the lyophillsate with s-team is carried out at a pressure of 0.01 to 2 bar for a period of up to 100 hours.
As the reproductive filterable pathogens, in particular, hepatitis viruses or HIV (human immune deficiency virus) are taken into consideration.
According to a preferred em:bodiment, the precipitation of the Factor VIII containing solution with the protein precipitating agent is effected at a pH of 5.6 to 6.8 and at a temperature of 1 to 40C.
An advantageous embodiment of the method according to the invention is characterized by the combination of the following measures:
- preeipitating and separating undesired proteins from the solution of a eryopreeipitate in a eitrated buffer optionally containing heparin, heparinoid, a eomplex eompound of heparin and antithrombin III ("Atheplex") and/or aprotinin, at a pH of 6.0 to 6.4 and a temperature of 0 to 25C, preferably 4 to 8C, - treating the puri~ied supernatant containing Factor VIII with a solution containing ammonium sulfate, ammonium sulfate - glycine, sodium sulfa-te, sodium sulfate - sodium eitrate, am~ionium sulfate - sodium eitrate, citrate glyeine at a eoncentration of 8 to 3S % and a pH of 5.6 to 6.8 so as to precipitate a Factor VIII containing preeipitate, - dissolvlng the precipitated Factor VIII containing precipitate in a sodium chloride-sodium citrate buffer solution containlng an antithrombin-heparinoid or antithlombin-III-heparinoid complex as well as albumin ~: - 5 -.: , ~,?,~7q3~ ~
ultrafilterin~ or dialyzing, lyophiliziny and inactivating by hea-t treatment.
The invention will now be explained in more detail by the following examples.
Example 1:
From 16.5 l plasma, 150 g cryoprecipitate were recovered by deepfreezing and rethawing. The latter was dissolved in 900 ml trisodium citrate buffer containing 90 mg sulfated polysaccharide "SP 5~" (Benechemie), 9 units of Atheplex as well as 27,000 units of aprotinin. The pH of the soiution was adjusted to 6.25, the temperature was adjusted to 4~C, undesired proteins being precipitated and separated by centrifugation. The supernatant was slowly admixed with glycine and ammonium sulfate under stirring at a p~ of 6.0 to 6.3 and at room temperature until a precipitation concentration of 120 g/l glycine and 85 g/l ammonium sulfate had been reached.
The precipitate formed was separated by centrifugation, dissolved in a sodium chloride - citrate buffer and dialyzed against the same buffer. The dialysate was admixed with glycine and albumin to a concentration of lO mg/ml glycine and 2 mg/ml albumin and the solution was ~; lyophilized. A portion of the powdery lyophilisate obtained was adjusted with steam to a moisture content of 8 w/w %
and another portion was adjusted to a moisture content of 24 to 26 w/w %.
These moistened preparations were subjected to heat treatment at 60 or 70C for a period of 10 to lO0 hours in closed containers under nitrogen atmosphere so as to inactivate possibly present viruses. Subsequently, the ~'7C~1~
specific Factor VIII activity was determined. The results as compared to the non heat-inactivated lyophilisate produced according to Example 1 are to be taken from the following Table I.
Table I
Example l according to invention Factor VIII, lyophilisate, not heated 54.7 U/ml 100 %
specific activity 4.31 U/mg content of IgG per 1000 units Factor VIII 1.8 mg Factor VIII, lyophilisate with moisture content 7.9 %
after 10 h heated at 60C 53.6 U/ml 98 %
after 70 h heated at 60C 42.5 U/ml 78 %
after 100 h heated at 60C 40.0 U/ml 73 %
after lO h heated at 70C 43.3 U/ml 79 %
Factor VIII, lyophilisate with moisture content 25.2 %
after 10 h heated at 60C 45.0 U/ml 82 %
Example 2:
In order to illustrate the superiority of the preparations produced according to the invention over known ones (e.g., known from U.S. patent No. 4,522,751) with .:
regard to thermal stabillty, the Eirst part of Example 1 was repeated; yet, instead of the precipitation with glycille and ammonium sulfate, 8 % ethyl alcohol in the presence of 1.45 mol/l glycine was used at a pH of 6Ø The precipitate was separated, dissolved in a citrated NaCl-glycine-albumin buffer, lyophilized and the lyophilisate was readjusted to a moisture content of 8 w/w % and 24 to 26 w/w %, respectively, with steam and, as described in connec-tion with Example 1, was heated under nitrogen atmosphere. The results are indicated in the following Table II, with the elevated IgG content (30 mg as compared to 1.8 mg) being significant on the one hand, and with the specific activities present after heat treatment, in particular after long-term heating, of the comparative prior art example bei.ng clearly lower, on the other hand.
_able II
~ Comparative Example Factor : 20 VIII, lyophilisate, not heated 54.3 U/ml 100 %
specific activity2.69 U/mg content of IgG per 1000 : units of Factor VIII 30 mg Factor VIII, lyophilisate with moisture content 7.9 %
after 10 h heated at 60C 50.5 U/ml 93 ~
after 70 h heated at 60C 24.4 U/ml 45 %
after 100 h heated at 60C26.6 U/ml 49 %
after 10 h heated a-t 70C29.3 U/ml 54 %
Factor VIII, lyophilisate with moisture content 25.2 %
after 10 h heated at 60C24.4 U/ml 45 %
xample 3:
The first part of Example l was repea~ed; yet a salt combination of NaCl and ammonium sulfate in an aqueous solution was used to precipitate the Factor VIII
concentrate until a precipitation concentration of 10 %
NaCl and 12 % ammonium sulfate had been reached. Further processing, centrifugation, dissolution, dialyzation, lyophilization and moistening were effected as in Example l. The results are indicated in Table III.
Table III
Factor VIII, lyophilisate, 20 not heated 42.6 U/ml 100 %
; specific activity3.18 U/mg content of IgG per 1000 units Factor VIII 9 mg Factor VIII, lyophilisate with moisture content 8 %
after 10 h heated at 70C 30 U/ml 71 %
Factor VIII, lyophilisate ~ 30 with moisture content 24.6 % 32 U/ml 75 %
: _ g _ : .
~ ~7~1~
Example 4:
The first part of Example 1 was repeated; yet, a salt combination of sodium citrate and ammonium sulfate in an aqueous solution was used to precipitate the Factor VIII
concentrate until a precipitation concentration of 5 sodium citrate and 12.5 ~ a:mmonium sulfate had been reached. Further processing, centrifugati.on, dissolution, dialyzation, lyophilization and moistening were effected as described in Example 1. The results are indicated in Table IV.
Table IV
Factor VIII, lyophilisate, not heated 42.2 U/ml 100 %
specific activity3.49 U/mg : content of IgG per 1000 units~:Factor VIII2.6 mg :~ ~
: ~ :
~` Factor VIII, lyophilisate with moisture content 8.3 after 10 h heated at 70C 32.8 U/ml 78 ~: Factor VIII, lyophilisate with moisture content 27.1 after 10 h heated at 60C 3~.:5 U/ml 82 ; Example 5:
The first part of Example 1 was repeated; yet, a salt :: : : : :
: ~
solution of ammonium sulfate was used to precipitate the Factor VIII concentrate until a precipitation concentration of 13.2 % a~nonium sulfate had been reached. Further processing, centrifugation, dissolution, dialyza-tion, lyophilization and moistening were effected as described in Example 1. The results are indicated in Table V.
Table V
10 Factor VIII, lyophilisate, not heated48.6 U/ml 100 %
specific activity5.70 U/mg content of IgG per 1000 Factor VIII 0.6 mg Factor VIII, lyophilisate with moisture content 7 7 ~ after 10 h heated at 70C 28.9 U/ml 59 ;: ~ 20 Factor VIII, lyophilisate with moisture content 23.7 %
.:
after 10 h heated 60C 39.2 U/ml 81 %
In all cases, the IgG content of the Factor VIII
.concentrate obtained is far below 10 mg and the residual activities sti.ll present upon heat treatment are more than 70 %.
.
:
Suitably, the treatment of the lyophillsate with s-team is carried out at a pressure of 0.01 to 2 bar for a period of up to 100 hours.
As the reproductive filterable pathogens, in particular, hepatitis viruses or HIV (human immune deficiency virus) are taken into consideration.
According to a preferred em:bodiment, the precipitation of the Factor VIII containing solution with the protein precipitating agent is effected at a pH of 5.6 to 6.8 and at a temperature of 1 to 40C.
An advantageous embodiment of the method according to the invention is characterized by the combination of the following measures:
- preeipitating and separating undesired proteins from the solution of a eryopreeipitate in a eitrated buffer optionally containing heparin, heparinoid, a eomplex eompound of heparin and antithrombin III ("Atheplex") and/or aprotinin, at a pH of 6.0 to 6.4 and a temperature of 0 to 25C, preferably 4 to 8C, - treating the puri~ied supernatant containing Factor VIII with a solution containing ammonium sulfate, ammonium sulfate - glycine, sodium sulfa-te, sodium sulfate - sodium eitrate, am~ionium sulfate - sodium eitrate, citrate glyeine at a eoncentration of 8 to 3S % and a pH of 5.6 to 6.8 so as to precipitate a Factor VIII containing preeipitate, - dissolvlng the precipitated Factor VIII containing precipitate in a sodium chloride-sodium citrate buffer solution containlng an antithrombin-heparinoid or antithlombin-III-heparinoid complex as well as albumin ~: - 5 -.: , ~,?,~7q3~ ~
ultrafilterin~ or dialyzing, lyophiliziny and inactivating by hea-t treatment.
The invention will now be explained in more detail by the following examples.
Example 1:
From 16.5 l plasma, 150 g cryoprecipitate were recovered by deepfreezing and rethawing. The latter was dissolved in 900 ml trisodium citrate buffer containing 90 mg sulfated polysaccharide "SP 5~" (Benechemie), 9 units of Atheplex as well as 27,000 units of aprotinin. The pH of the soiution was adjusted to 6.25, the temperature was adjusted to 4~C, undesired proteins being precipitated and separated by centrifugation. The supernatant was slowly admixed with glycine and ammonium sulfate under stirring at a p~ of 6.0 to 6.3 and at room temperature until a precipitation concentration of 120 g/l glycine and 85 g/l ammonium sulfate had been reached.
The precipitate formed was separated by centrifugation, dissolved in a sodium chloride - citrate buffer and dialyzed against the same buffer. The dialysate was admixed with glycine and albumin to a concentration of lO mg/ml glycine and 2 mg/ml albumin and the solution was ~; lyophilized. A portion of the powdery lyophilisate obtained was adjusted with steam to a moisture content of 8 w/w %
and another portion was adjusted to a moisture content of 24 to 26 w/w %.
These moistened preparations were subjected to heat treatment at 60 or 70C for a period of 10 to lO0 hours in closed containers under nitrogen atmosphere so as to inactivate possibly present viruses. Subsequently, the ~'7C~1~
specific Factor VIII activity was determined. The results as compared to the non heat-inactivated lyophilisate produced according to Example 1 are to be taken from the following Table I.
Table I
Example l according to invention Factor VIII, lyophilisate, not heated 54.7 U/ml 100 %
specific activity 4.31 U/mg content of IgG per 1000 units Factor VIII 1.8 mg Factor VIII, lyophilisate with moisture content 7.9 %
after 10 h heated at 60C 53.6 U/ml 98 %
after 70 h heated at 60C 42.5 U/ml 78 %
after 100 h heated at 60C 40.0 U/ml 73 %
after lO h heated at 70C 43.3 U/ml 79 %
Factor VIII, lyophilisate with moisture content 25.2 %
after 10 h heated at 60C 45.0 U/ml 82 %
Example 2:
In order to illustrate the superiority of the preparations produced according to the invention over known ones (e.g., known from U.S. patent No. 4,522,751) with .:
regard to thermal stabillty, the Eirst part of Example 1 was repeated; yet, instead of the precipitation with glycille and ammonium sulfate, 8 % ethyl alcohol in the presence of 1.45 mol/l glycine was used at a pH of 6Ø The precipitate was separated, dissolved in a citrated NaCl-glycine-albumin buffer, lyophilized and the lyophilisate was readjusted to a moisture content of 8 w/w % and 24 to 26 w/w %, respectively, with steam and, as described in connec-tion with Example 1, was heated under nitrogen atmosphere. The results are indicated in the following Table II, with the elevated IgG content (30 mg as compared to 1.8 mg) being significant on the one hand, and with the specific activities present after heat treatment, in particular after long-term heating, of the comparative prior art example bei.ng clearly lower, on the other hand.
_able II
~ Comparative Example Factor : 20 VIII, lyophilisate, not heated 54.3 U/ml 100 %
specific activity2.69 U/mg content of IgG per 1000 : units of Factor VIII 30 mg Factor VIII, lyophilisate with moisture content 7.9 %
after 10 h heated at 60C 50.5 U/ml 93 ~
after 70 h heated at 60C 24.4 U/ml 45 %
after 100 h heated at 60C26.6 U/ml 49 %
after 10 h heated a-t 70C29.3 U/ml 54 %
Factor VIII, lyophilisate with moisture content 25.2 %
after 10 h heated at 60C24.4 U/ml 45 %
xample 3:
The first part of Example l was repea~ed; yet a salt combination of NaCl and ammonium sulfate in an aqueous solution was used to precipitate the Factor VIII
concentrate until a precipitation concentration of 10 %
NaCl and 12 % ammonium sulfate had been reached. Further processing, centrifugation, dissolution, dialyzation, lyophilization and moistening were effected as in Example l. The results are indicated in Table III.
Table III
Factor VIII, lyophilisate, 20 not heated 42.6 U/ml 100 %
; specific activity3.18 U/mg content of IgG per 1000 units Factor VIII 9 mg Factor VIII, lyophilisate with moisture content 8 %
after 10 h heated at 70C 30 U/ml 71 %
Factor VIII, lyophilisate ~ 30 with moisture content 24.6 % 32 U/ml 75 %
: _ g _ : .
~ ~7~1~
Example 4:
The first part of Example 1 was repeated; yet, a salt combination of sodium citrate and ammonium sulfate in an aqueous solution was used to precipitate the Factor VIII
concentrate until a precipitation concentration of 5 sodium citrate and 12.5 ~ a:mmonium sulfate had been reached. Further processing, centrifugati.on, dissolution, dialyzation, lyophilization and moistening were effected as described in Example 1. The results are indicated in Table IV.
Table IV
Factor VIII, lyophilisate, not heated 42.2 U/ml 100 %
specific activity3.49 U/mg : content of IgG per 1000 units~:Factor VIII2.6 mg :~ ~
: ~ :
~` Factor VIII, lyophilisate with moisture content 8.3 after 10 h heated at 70C 32.8 U/ml 78 ~: Factor VIII, lyophilisate with moisture content 27.1 after 10 h heated at 60C 3~.:5 U/ml 82 ; Example 5:
The first part of Example 1 was repeated; yet, a salt :: : : : :
: ~
solution of ammonium sulfate was used to precipitate the Factor VIII concentrate until a precipitation concentration of 13.2 % a~nonium sulfate had been reached. Further processing, centrifugation, dissolution, dialyza-tion, lyophilization and moistening were effected as described in Example 1. The results are indicated in Table V.
Table V
10 Factor VIII, lyophilisate, not heated48.6 U/ml 100 %
specific activity5.70 U/mg content of IgG per 1000 Factor VIII 0.6 mg Factor VIII, lyophilisate with moisture content 7 7 ~ after 10 h heated at 70C 28.9 U/ml 59 ;: ~ 20 Factor VIII, lyophilisate with moisture content 23.7 %
.:
after 10 h heated 60C 39.2 U/ml 81 %
In all cases, the IgG content of the Factor VIII
.concentrate obtained is far below 10 mg and the residual activities sti.ll present upon heat treatment are more than 70 %.
.
Claims (9)
1. A method of producing a Factor VIII (AHF) containing fraction having a specific activity of at least 2.5 units of Factor VIII/mg protein as well as a portion of immunoglobulin G (IgG) of 10 mg/1000 units of Factor VIII
at the most, with the risk of transmission of viral or bacterial infections being avoided or largely reduced when applied therapeutically or prophylactically, which method comprises in combination:
- preparing a solution of a Factor VIII containing plasma fraction, - precipitating and separating undesired proteins from said solution in the presence of sulfated polysaccharides at a pH approximately in the neutral range so as to obtain a purified Factor VIII containing solution, - treating said purified Factor VIII containing solution with a protein precipitating agent selected from the group consisting of ammonium sulfate, ammonium sulfate - glycine, sodium chloride - glycine, sodium sulfate, sodium sulfate -sodium citrate, ammonium sulfate - sodium citrate, citrate - glycine, so as to precipitate a Factor VIII
containing precipitate, - dissolving and lyophilizing said Factor VIII
containing precipitate so as to obtain a lyophilisate, and - heat-treating said lyophilisate at a temperature and for a period of time sufficient to inactivate possibly present viruses.
at the most, with the risk of transmission of viral or bacterial infections being avoided or largely reduced when applied therapeutically or prophylactically, which method comprises in combination:
- preparing a solution of a Factor VIII containing plasma fraction, - precipitating and separating undesired proteins from said solution in the presence of sulfated polysaccharides at a pH approximately in the neutral range so as to obtain a purified Factor VIII containing solution, - treating said purified Factor VIII containing solution with a protein precipitating agent selected from the group consisting of ammonium sulfate, ammonium sulfate - glycine, sodium chloride - glycine, sodium sulfate, sodium sulfate -sodium citrate, ammonium sulfate - sodium citrate, citrate - glycine, so as to precipitate a Factor VIII
containing precipitate, - dissolving and lyophilizing said Factor VIII
containing precipitate so as to obtain a lyophilisate, and - heat-treating said lyophilisate at a temperature and for a period of time sufficient to inactivate possibly present viruses.
2. A method as set forth in claim 1, wherein said lyophilisate is adjusted to a water content of more than 0.05 (5 % by weight) and less than 0.70 (70 % by weight) and is treated in a closed container at a temperature ranging from 50 to 121°C under steam partial-pressure elevation.
3. A method as set forth in claim 2, wherein said water content is adjusted to less than 0.40 (40 % by weight).
4. A method as set forth in claim 1, wherein said lyophilisate is treated with steam at a pressure of 0.01 to 2 bar for a period of time up to 100 hours.
5. A method as set forth in claim 1, wherein said viruses are reproductive filterable pathogens selected from the group consisting of hepatitis viruses and HIV (human immune deficiency viruses).
6. A method as set forth in claim 1, wherein precipitating of said Factor VIII containing solution with said protein precipitating agent is carried out at a pH of 5.6 to 6.8 and at a temperature of 1 to 40°C.
7. A method of producing a Factor VIII (AHF) containing fraction having a specific activity of at least 2.5 units of Factor VIII/mg protein as well as a portion of immunoglobulin G (IgG) of 10 mg/1000 units of Factor VIII
at the most, with the risk of transmission of viral or bacterial infections being avoided or largely reduced when applied therapeutically or prophylactically, which method comprises in combination:
- preparing a first solution of a Factor VIII containing cryoprecipitate in a citrated buffer, - precipitating and separating undesired proteins from said first solution in the presence of sulfated polysaccharides at a pH of 6.0 to 6.4 and at a temperature of 0 to 25°C so as to obtain a purified Factor VIII
containing supernatant, - treating said purified Factor VIII containing supernatant with a protein precipitating agent selected from the group consisting of ammonium sulfate, ammonium sulfate - glycine, sodium chloride - glycine, sodium sulfate, sodium sulfate - sodium citrate, ammonium sulfate - sodium citrate, citrate - glycine at a concentration of 8 to 35 % and a pH of 5.6 to 6.8 so as to precipitate a Factor VIII containing precipitate, - dissolving said Factor VIII containing precipitate in a sodium chloride-sodium citrate buffer solution containing one of an antithrombin-heparinoid complex and an antithrombin-III-heparinoid complex as well as albumin so as to obtain a second solution, - one of ultrafiltering and dialyzing said second solution, and lyophilizing so as to obtain a lyophilisate, and - heat-treating said lyophilisate at a temperature and for a period of time sufficient to inactivate possibly present viruses.
at the most, with the risk of transmission of viral or bacterial infections being avoided or largely reduced when applied therapeutically or prophylactically, which method comprises in combination:
- preparing a first solution of a Factor VIII containing cryoprecipitate in a citrated buffer, - precipitating and separating undesired proteins from said first solution in the presence of sulfated polysaccharides at a pH of 6.0 to 6.4 and at a temperature of 0 to 25°C so as to obtain a purified Factor VIII
containing supernatant, - treating said purified Factor VIII containing supernatant with a protein precipitating agent selected from the group consisting of ammonium sulfate, ammonium sulfate - glycine, sodium chloride - glycine, sodium sulfate, sodium sulfate - sodium citrate, ammonium sulfate - sodium citrate, citrate - glycine at a concentration of 8 to 35 % and a pH of 5.6 to 6.8 so as to precipitate a Factor VIII containing precipitate, - dissolving said Factor VIII containing precipitate in a sodium chloride-sodium citrate buffer solution containing one of an antithrombin-heparinoid complex and an antithrombin-III-heparinoid complex as well as albumin so as to obtain a second solution, - one of ultrafiltering and dialyzing said second solution, and lyophilizing so as to obtain a lyophilisate, and - heat-treating said lyophilisate at a temperature and for a period of time sufficient to inactivate possibly present viruses.
8. A method as set forth in claim 7, wherein said citrated buffer contains at least one of the group consisting of heparin, heparinoid, a complex compound of heparin and antithrombin III ("Atheplex"), and aprotinin.
9. A method as set forth in claim 7, wherein precipitating and separating is effected at a temperature of 4 to 8°C.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ATA2923/86 | 1986-11-03 | ||
| AT0292386A AT391808B (en) | 1986-11-03 | 1986-11-03 | METHOD FOR PRODUCING A FACTOR VIII (AHF) CONTAINING FRACTION |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1297011C true CA1297011C (en) | 1992-03-10 |
Family
ID=3542434
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000549552A Expired - Lifetime CA1297011C (en) | 1986-11-03 | 1987-10-19 | Method of producing a factor viii (ahf) containing fraction |
Country Status (8)
| Country | Link |
|---|---|
| US (1) | US4814435A (en) |
| EP (1) | EP0270516B1 (en) |
| JP (1) | JPH0730117B2 (en) |
| AT (2) | AT391808B (en) |
| CA (1) | CA1297011C (en) |
| DE (1) | DE3769096D1 (en) |
| DK (1) | DK573587A (en) |
| ES (1) | ES2028913T3 (en) |
Families Citing this family (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1987005220A1 (en) * | 1986-03-10 | 1987-09-11 | Rubinstein Alan I | A method for treating gammaglobulin |
| DK162233C (en) * | 1989-11-09 | 1992-03-16 | Novo Nordisk As | PROCEDURE FOR INSULATING FACTOR VIII FROM BLOOD PLASMA AND PHARMACEUTICAL PREPARATION CONTAINING THE ASSOCIATED PHATAR VIII |
| AT402891B (en) * | 1991-06-20 | 1997-09-25 | Immuno Ag | METHOD FOR PRODUCING AN INACTIVATED BLOOD PRODUCT |
| 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 |
| AT404358B (en) | 1997-02-04 | 1998-11-25 | Immuno Ag | METHOD FOR CHROMATOGRAPHIC CLEANING OR FRACTIONATION OF VON WILLEBRAND FACTOR FROM A VWF-CONTAINING MATERIAL |
| AT407484B (en) * | 1997-11-12 | 2001-03-26 | Bio Prod & Bio Eng Ag | MEDICINES FOR PROMOTING Wound Healing |
| EP2921180B1 (en) | 1999-02-22 | 2019-08-14 | University of Connecticut | Albumin-free factor VIII formulations |
| US20020077276A1 (en) * | 1999-04-27 | 2002-06-20 | Fredeking Terry M. | Compositions and methods for treating hemorrhagic virus infections and other disorders |
| AT501088A2 (en) * | 2002-12-18 | 2006-06-15 | Bio Prod & Bio Eng Ag | STABLE THERAPEUTIC PROTEINS |
| US20060223988A1 (en) * | 2005-04-05 | 2006-10-05 | National Research Laboratories, Ltd. | High Resolution Methods and Precipitating Reagents for Isolating Proteins from Proteinaceous Material |
| WO2010054238A1 (en) | 2008-11-07 | 2010-05-14 | Baxter International Inc. | Factor viii formulations |
| JP6583766B2 (en) * | 2015-02-02 | 2019-10-02 | 学校法人北里研究所 | Protein separation method, protein analysis method, and protein separation kit |
| US10815270B1 (en) * | 2019-09-20 | 2020-10-27 | Plasma Technologies, Llc | Compositions and methods for high efficiency protein precipitation |
Family Cites Families (24)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4022758A (en) * | 1973-06-19 | 1977-05-10 | Ab Kabi | Isolation of coagulation factors I and VIII from biological material |
| US3973002A (en) * | 1974-04-12 | 1976-08-03 | E. R. Squibb & Sons, Inc. | Antihemophilic factor |
| US4188318A (en) * | 1975-06-16 | 1980-02-12 | Edward Shanbrom | Simplified method for preparation of high yield, high purity Factor VIII concentrate |
| JPS52125609A (en) * | 1976-04-09 | 1977-10-21 | Green Cross Corp:The | Purification of agglutination factor viii |
| CA1054052A (en) * | 1976-08-14 | 1979-05-08 | Edward Shanbrom | Simplified method for preparation of high yield, high purity factor viii concentrate |
| US4104266A (en) * | 1977-04-14 | 1978-08-01 | American National Red Cross | Method for preparation of antihemophilic factor |
| US4170639A (en) * | 1978-07-10 | 1979-10-09 | Warner-Lambert Company | Antihemophilic factor concentrate and its production |
| AT359646B (en) * | 1979-04-19 | 1980-11-25 | Immuno Ag | METHOD FOR PRODUCING SIDE-EFFECTIVE PLASMA FACTIONS |
| DE2916711A1 (en) * | 1979-04-25 | 1980-11-06 | Behringwerke Ag | Blood coagulation factors and process for their manufacture |
| US4440679A (en) * | 1980-03-05 | 1984-04-03 | Cutter Laboratories, Inc. | Pasteurized therapeutically active protein compositions |
| US4305871A (en) * | 1980-09-02 | 1981-12-15 | Edward Shanbrom | Method of selectively increasing yield and purity of certain cryoprecipitate proteins by heating |
| WO1982004395A1 (en) * | 1981-06-18 | 1982-12-23 | Erik Gustaf Birger Blombaeck | A process in purification and concentration of the factor viii complex |
| US4456590B2 (en) * | 1981-11-02 | 1989-05-30 | Heat treatment of lyphilized blood clotting factor viii concentrate | |
| US4495175A (en) * | 1982-08-05 | 1985-01-22 | University Of Rochester | Preparation of highly purified human antihemophilic factor |
| ZA838856B (en) * | 1982-12-02 | 1984-07-25 | Usv Pharma Corp | Hepatitis b and non-a-non-b-safe biological products |
| EP0124506B1 (en) * | 1983-05-02 | 1988-08-17 | IMMUNO Aktiengesellschaft für chemisch-medizinische Produkte | Method of inactivating pathogens |
| AT376884B (en) * | 1983-05-02 | 1985-01-10 | Immuno Ag | METHOD FOR INACTIVATING VARIABLE DISEASES |
| DE3318521A1 (en) * | 1983-05-20 | 1984-11-22 | Lentia GmbH Chem. u. pharm. Erzeugnisse - Industriebedarf, 8000 München | METHOD FOR PRODUCING AN ANTIHAEMOPHILIE FACTOR CONCENTRATE |
| AT379510B (en) * | 1983-05-20 | 1986-01-27 | Immuno Ag | METHOD FOR PRODUCING A FACTOR VIII (AHF) CONTAINING PRAEPARATION |
| AT379310B (en) * | 1983-05-20 | 1985-12-27 | Immuno Ag | METHOD FOR PRODUCING AN ANTITHROMBIN III-HEPARIN OR ANTITHROMBIN III HEPARINOID CONCENTRATE |
| US4486410A (en) * | 1984-02-09 | 1984-12-04 | Armour Pharmaceutical Company | Heat defibrinogenation of AHF preparation |
| AT389815B (en) * | 1984-03-09 | 1990-02-12 | Immuno Ag | METHOD FOR INACTIVATING VARIABLE FILTERABLE DISEASERS IN BLOOD PRODUCTS |
| US4543210A (en) * | 1984-10-04 | 1985-09-24 | Miles Laboratories, Inc. | Process for producing a high purity antihemophilic factor concentrate |
| GB8505882D0 (en) * | 1985-03-07 | 1985-04-11 | Central Blood Lab Authority | Purification of blood coagulation factor viii |
-
1986
- 1986-11-03 AT AT0292386A patent/AT391808B/en not_active IP Right Cessation
-
1987
- 1987-10-15 US US07/108,458 patent/US4814435A/en not_active Expired - Lifetime
- 1987-10-19 CA CA000549552A patent/CA1297011C/en not_active Expired - Lifetime
- 1987-10-29 DE DE8787890237T patent/DE3769096D1/en not_active Expired - Lifetime
- 1987-10-29 EP EP87890237A patent/EP0270516B1/en not_active Expired - Lifetime
- 1987-10-29 AT AT87890237T patent/ATE62133T1/en not_active IP Right Cessation
- 1987-10-29 ES ES198787890237T patent/ES2028913T3/en not_active Expired - Lifetime
- 1987-11-02 JP JP62278985A patent/JPH0730117B2/en not_active Expired - Lifetime
- 1987-11-02 DK DK573587A patent/DK573587A/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| ATA292386A (en) | 1990-06-15 |
| DK573587A (en) | 1988-05-04 |
| EP0270516B1 (en) | 1991-04-03 |
| EP0270516A3 (en) | 1988-06-22 |
| DK573587D0 (en) | 1987-11-02 |
| JPH0730117B2 (en) | 1995-04-05 |
| EP0270516A2 (en) | 1988-06-08 |
| ATE62133T1 (en) | 1991-04-15 |
| DE3769096D1 (en) | 1991-05-08 |
| AT391808B (en) | 1990-12-10 |
| JPS63132899A (en) | 1988-06-04 |
| US4814435A (en) | 1989-03-21 |
| ES2028913T3 (en) | 1992-07-16 |
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Legal Events
| Date | Code | Title | Description |
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| MKLA | Lapsed |