Method of producing a factor VIII (AHF) containing fraction

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.

The invention relates to 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, at the therapeutic or 
prophylactic application of which the risk of transmission of viral or 
bacterial infections is avoided or largely 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. Pat. No. 3,973,002, a method is, furthermore, described, with which 
a cryoprecipitate obtained from blood plasma is macerated, the macerated 
product is suspended in a citrate-glucose buffer solution, is centrifuged, 
and the thus obtained buffer extract is adjusted to a pH in the range of 
6.0 to 6.8. Under these conditions, precipitation of undesired impurities 
takes place, whereupon the remaining Factor VIII containing residue is 
sterilized and lyophilized. However, a Factor 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 Factor VIII units, is undesiredly 
high. 
Similar methods are described in British Pat. No. 1,551,928 as well as in 
U.S. Pat. Nos. 4,170,639 and 4,104,266. There, likewisely by departing 
from plasma, a cryoprecipitate is recovered, which is dissolved in a 
buffer solution in the neutral pH range, with undesired proteins being 
separated, 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. Pat. 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 No. 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. Pat. No. 4,522,751 of Applicant, a method for producing a 
Factor VIII (AHF) containing preparation with an elevated specific 
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 VIII 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 yield products that, apart from 
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 activity. At 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 60.degree. C. This demand 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 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 
inactivation by heat treatment without lowering the Factor VIII activity 
in an undesired manner. 
In accordance with the invention, this object is achieved 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 in the neutral range, 
treating the thus purified Factor VIII containing solution with a protein 
precipitating agent, selected from ammonium sulfate, ammonium 
sulfate-glycine, sodium chloride-glycine, sodium sulfate, sodium 
sulfate-sodium citrate, ammonium sulfate-sodium citrate, citrate-glycine, 
in order to precipitate a Factor VIII containing precipitate, 
dissolving and lyophilizing the precipitated Factor VIII 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 precipitating agents 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 is the merit of the combination according to 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 possible and simultaneously being sufficiently heat-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 weight), and is treated in a closed container at a 
temperature ranging from 50.degree. to 121.degree. C. under elevation of 
the steam partial pressure. 
Suitably, the treatment of the lyophilisate with steam 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 embodiment, 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.degree. to 40.degree. C. 
An advantageous embodiment of the method according to the invention is 
characterized by the combination of the following measures: 
precipitating and separating undesired proteins from the solution of a 
cryoprecipitate in a citrated buffer optionally containing heparin, 
heparinoid, a complex compound of heparin and antithrombin III 
("Atheplex") and/or aprotinin, at a pH of 6.0 to 6.4 and a temperature of 
0.degree. to 25.degree. C., preferably 4.degree. to 8.degree. C., 
treating the purified supernatant containing Factor VIII with a solution 
containing ammonium sulfate, ammonium sulfate-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 the precipitated Factor VIII containing precipitate in a sodium 
chloride-sodium citrate buffer solution containing an 
antithrombin-heparinoid or antithrombin-III-heparinoid complex as well as 
albumin, ultrafiltering or dialyzing, lyophilizing and inactivating by 
heat 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 54" (Benechemie), 9 units of 
Atheplex as well as 27,000 units of aprotinin. The pH of the solution was 
adjusted to 6.25, the temperature was adjusted to 4.degree. C., undesired 
proteins being precipitated and separated by centrifugation. The 
supernatant was slowly admixed with glycine and ammonium sulfate under 
stirring at a pH 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 10 
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.degree. 
or 70.degree. C. for a period of 10 to 100 hours in closed containers 
under nitrogen atmosphere so as to inactivate possibly present viruses. 
Subsequently, the 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 
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Example 1 
according to invention 
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Factor VIII, lyophilisate, 
54.7 U/ml 100% 
not heated 
specific activity 4.31 U/mg 
content of IgG per 1000 
1.8 mg 
units Factor VIII 
Factor VIII, lyophilisate 
with moisture content 7.9% 
after 10 h heated at 60.degree. C. 
53.6 U/ml 98% 
after 70 h heated at 60.degree. C. 
42.5 U/ml 78% 
after 100 h heated at 60.degree. C. 
40.0 U/ml 73% 
after 10 h heated at 70.degree. C. 
43.3 U/ml 79% 
Factor VIII, lyophilisate 
45.0 U/ml 82% 
with moisture content 25.2% 
after 10 h heated at 60.degree. C. 
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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. Pat. No. 
4,522,751) with regard to thermal stability, the first part of Example 1 
was repeated; yet, instead of the precipitation with glycine and ammonium 
sulfate, 8% ethyl alcohol in the presence of 1.45 mol/l glycine was used 
at a pH of 6.0. 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 connection 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 being clearly lower, on the 
other hand. 
TABLE II 
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Comparative Example 
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Factor VIII, lyophilisate, 
54.3 U/ml 100% 
not heated 
specific activity 2.69 U/mg 
content of IgG per 1000 
30 mg 
units of Factor VIII 
Factor VIII, lyophilisate 
with moisture content 7.9% 
after 10 h heated at 60.degree. C. 
50.5 U/ml 93% 
after 70 h heated at 60.degree. C. 
24.4 U/ml 45% 
after 100 h heated at 60.degree. C. 
26.6 U/ml 49% 
after 10 h heated at 70.degree. C. 
29.3 U/ml 54% 
Factor VIII, lyophilisate 
24.4 U/ml 45% 
with moisture content 25.2% 
after 10 h heated at 60.degree. C. 
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EXAMPLE 3 
The first part of Example 1 was repeated; 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 1. The results are indicated in Table III. 
TABLE III 
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Factor VIII, lyophilisate, 
42.6 U/ml 100% 
not heated 
specific activity 3.18 U/mg 
content of IgG per 1000 
9 mg 
units Factor VIII 
Factor VIII, lyophilisate 
30 U/ml 71% 
with moisture content 8% 
after 10 h heated at 70.degree. C. 
Factor VIII, lyophilisate 
32 U/ml 75% 
with moisture content 24.6% 
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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% ammonium sulfate had been 
reached. Further processing, centrifugation, dissolution, dialyzation, 
lyophilization and moistening were effected as described in Example 1. The 
results are indicated in Table IV. 
TABLE IV 
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Factor VIII, lyophilisate, 
42.2 U/ml 100% 
not heated 
specific activity 3.49 U/mg 
content of IgG per 1000 
2.6 mg 
units Factor VIII 
Factor VIII, lyophilisate 
32.8 U/ml 78% 
with moisture content 8.3% 
after 10 h heated at 70.degree. C. 
Factor VIII, lyophilisate 
34.5 U/ml 82% 
with moisture content 27.1% 
after 10 h heated at 60.degree. C. 
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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% ammonium sulfate had been reached. 
Further processing, centrifugation, dissolution, dialyzation, 
lyophilization and moistening were effected as described in Example 1. The 
results are indicated in Table V. 
TABLE V 
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Factor VIII, lyophilisate, 
48.6 U/ml 100% 
not heated 
specific activity 5.70 U/mg 
content of IgG per 1000 
0.6 mg 
Factor VIII 
Factor VIII, lyophilisate 
28.9 U/ml 59% 
with moisture content 7.7% 
after 10 h heated at 70.degree. C. 
Factor VIII, lyophilisate 
39.2 U/ml 81% 
with moisture content 23.7% 
after 10 h heated 60.degree. C. 
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In all cases, the IgG content of the Factor VIII concentrate obtained is 
far below 10 mg and the residual activities still present upon heat 
treatment are more than 70%.