U.S. patent number RE29,698 [Application Number 05/674,270] was granted by the patent office on 1978-07-11 for stabilization of ahf using heparin.
This patent grant is currently assigned to Baxter Travenol Laboratories, Inc.. Invention is credited to Lajos F. Fekete, Stephen L. Holst.
United States Patent |
RE29,698 |
Fekete , et al. |
July 11, 1978 |
**Please see images for:
( Certificate of Correction ) ** |
Stabilization of AHF using heparin
Abstract
A method of improving the yield of antihemophilic Factor .[.A.].
.Iadd.VIII (AHF) .Iaddend.obtained from blood plasma and plasma
fractions .Iadd.by cryoprecipitation .Iaddend.comprising the
addition of from 0.01 to 10 units of heparin to .Iadd.a concentrate
of AHF obtained by cryoprecipitation from .Iaddend.said plasma or
plasma fraction per ml. of solution thereof.
Inventors: |
Fekete; Lajos F. (Costa Mesa,
CA), Holst; Stephen L. (Santa Ana, CA) |
Assignee: |
Baxter Travenol Laboratories,
Inc. (Deerfield, IL)
|
Family
ID: |
22963114 |
Appl.
No.: |
05/674,270 |
Filed: |
April 6, 1976 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
Reissue of: |
254148 |
May 17, 1972 |
03803115 |
Apr 9, 1974 |
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Current U.S.
Class: |
530/383; 424/530;
530/830 |
Current CPC
Class: |
C07K
14/755 (20130101); Y10S 530/83 (20130101); A61K
38/00 (20130101) |
Current International
Class: |
C07K
14/435 (20060101); C07K 14/755 (20060101); A61K
38/00 (20060101); C07G 007/00 () |
Field of
Search: |
;260/112B |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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1,949,314 |
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Apr 1970 |
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DE |
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2,029,455 |
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Feb 1971 |
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DE |
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1,178,958 |
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Jan 1970 |
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GB |
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Other References
Chem. Abstracts, vol. 55, 1961, 27584g, Engelberg. .
Am. J. of the Medical Sciences, 1965, pp. 643-650, Webster et al.
.
Circulation, vol. XXIII, 1961, pp. 578-581, Engelberg. .
Nature, vol. 203, 1964, Pool, p. 312. .
Dictionary of Organic Compounds, Fourth Ed., vol. 3, 1965, p. 1567.
.
Chem. Abstracts, vol. 56, 1962, 7847i, Mayer et al. .
Chem. Abstracts, vol. 78, 55900j, Wilms, 1971. .
Thrombosis et al., Diathesis Haemorrhagica, vol. XI, pp. 64-74,
(1964), Wagner et al. .
Human Blood Coagulation, Biggs, (1972), pp. 142-147, 272-285. .
"Plasma Transfusions in Hemophilia," Van Creveld et al., 1952.
.
Circulation Res., vol. 3, pp. 397-402, (1955), Monkhouse et al.
.
. . Klinische Medizin, J. Tagung der Sektion Inner Medizin . . .
10, bis 13, Oct. 1963, pp. 311-317, (English). .
Blood Clotting Enzymology, Seegers, (1967), pp. 113-115, 170-177.
.
"The Hemophilias," Brinkhous, Wagner, et al., pp. 81-86, 100-105.
.
"Hemophilia and New Hemorrhagic States," Brinkhous. pp. 67-76.
.
Plenary Session Papers, XII Congress International Society of
Hematology, 1968, pp. 315-320. .
Am. J. of the Medical Sciences, vol. 232, 1956, pp. 434-441 Penick
et al. .
"Hemophilia and Other Hemorrhagic States," Brinkhous, pp. 38-46,
Seegers et al. .
Nature, vol. 184, 1959, p. 450, Miller. .
Who Technical Report Series, 504, 1972, p. 19. .
British 3, Haemat, vol. 13, pp. 42-52 (1967), Preston. .
Preparation of Factor IX Concentrates . . . Josso et al., pp.
14-21. .
Thromb. Diath. Haemorrh., vol. 9, pp. 368-386 (1963), Blomback et
al. .
Compt. Rend. Soc. Biol., 155 (1961), pp. 1103-1106, Mayer et al.
.
Thromb. Diath. Haemorrhagia, vol. 17, pp. 381-387 (1967) Kisker.
.
British J. of Haematology, vol. 21 (1971), pp. 1-20, Newman et al.
.
. . Hamatologie Leipzif, 10-bis 13, 1963, pp. 311-317, Soulier et
al. .
Human Blood Coagulation, Denson et al., (1972), pp. 278-285. .
Lancet, 1963, pp. 70-73, von Francken et al. .
International Symposium, Brussels, 1976, pp. 27-29, 42-44, 50-60,
Verstraete et al. .
Proc. 7th Congr. Europ. Soc. Haemat., 1959, part II, pp. 587-593
(1960), Blomback et al..
|
Primary Examiner: Schain; Howard E.
Attorney, Agent or Firm: Flattery; Paul C. Flynn; Lawrence
W. Hensley; Max D.
Claims
What is claimed is:
1. The method of improving the yield of AHF obtained from blood
plasma and plasma fractions comprising admixing from about 0.01 to
about ten units of heparin to a concentrate of AHF obtained from
said plasma or plasma fraction by cyroprecipitate per ml. of
solution of said concentrate of AHF.
2. The method of claim 1 in which the plasma fraction is a
cryoprecipitate concentrate of AHF which is further fractionated
with both polyethylene glycol and glycine.
3. The method of claim 2 in which the polyethylene glycol has an
average molecular weight of about 4000.
4. The method of claim 2 in which the cryoprecipitate concentrate
is precipitated with from about 3% to about 4% polyethylene glycol,
the resulting supernant is precipitated with about 10% polyethylene
glycol, and the resulting supernate is then fractionated with
glycine.
5. The method of claim 4 in which the polyethylene glycol has an
average molecular weight of about 4000.
6. The method of claim 4 in which the glycine has a molarity of
about 1.8.
7. The method of claim 4 in which the polyethylene glycol has an
average molecular weight of about 4000 and the glycine has a
molarity of about 1.8. .Iadd.8. The method of claim 1 wherein
citrate in addition to heparin is admixed with said concentrate of
AHF. .Iaddend..Iadd. 9. The method of claim 1 wherein the heparin
admixed with said concentrate of AHF is a citrate solution of
heparin. .Iaddend. .Iadd. 10. The method of claim 1 further
including the steps of fractionating the concentrate of AHF with
which the heparin is admixed to increase the concentration of AHF,
and admixing additional heparin with the further fractionated
concentrate of AHF..Iaddend..Iadd. 11. The method of claim 10
wherein citrate in addition to heparin is admixed with said further
fractionated concentrate of AHF. .Iaddend..Iadd. 12. The method of
claim 11 wherein the additional heparin admixed with said further
fractionated concentrate of AHF is a citrate solution of heparin.
.Iaddend. .Iadd. 13. The method of improving the yield of AHF
comprising the addition of heparin and a citrate to a concentrate
of AHF obtained from plasma or a plasma fraction by
cryoprecipitation, said heparin being added in an amount of about
0.01 to about 10 units per ml. of solution of said concentrate of
AHF. .Iaddend..Iadd. 14. The method of claim 13 wherein about 1 to
10 units of heparin is added per ml. of solution of said
concentrate of AHF. .Iaddend..Iadd. 15. The method of claim 13
wherein the heparin containing concentrate of AHF is further
fractionated to increase the concentration of the AHF, and
additional heparin and citrate are added to the further
fractionated concentrate of AHF. .Iaddend. .Iadd. 16. The method of
improving the yield of AHF obtained from an AHF-rich
cryoprecipitate comprising forming a solution of said
cryoprecipitate and providing in said solution from an external
source heparin and citrate, said heparin being added in an amount
of about 0.01 to about 10 units per ml. of solution of said
cryoprecipitate. .Iaddend..Iadd. 17. The method of claim 16 wherein
said solution is provided from an external source with about 1 to
10 units of heparin per ml. .Iaddend..Iadd. 18. The method of
improving the yield of an AHF concentrate prepared from plasma and
plasma fractions comprising:
separating an AHF-rich cryoprecipitate from blood plasma or a blood
plasma fraction;
adding heparin and citrate to the cryoprecipitate, said heparin
being added in an amount of about 0.01 to about 10 units per ml. of
solution of said cryoprecipitate;
fractionating the heparin containing AHF cryoprecipitate with
polyethylene glycol to further concentrate the AHF; and
adding heparin and citrate to the further concentrated AHF, said
heparin being added in an amount of about 0.01 to about 10 units
per ml. of solution of said further concentrated AHF.
.Iaddend..Iadd. 19. The method of claim 18 wherein the
heparin-treated, further concentrated AHF is fractionated with
glycine. .Iaddend. .Iadd. 20. The method of improving the yield of
an AHF concentrate obtained from plasma by cryoprecipitation,
comprising forming a solution of said concentrate of AHF and
providing in said solution about 0.01 to about 10 units of
externally added heparin per ml. of solution. .Iaddend..Iadd. 21.
The method of claim 20 further including the step of providing in
said solution a citrate. .Iaddend.
Description
This invention relates to a method of making a concentrate of
Antihemophilic Factor (AHF, Factor VIII). More particularly, this
invention relates to a method for improving the yield of AHF
obtained from blood plasma and plasma fractions .Iadd.by a
cryoprecipitation technique. .Iaddend.
The process of blood coagulation is a complicated biological
activity and involves the interaction of several substances found
in normal whole blood. It is known that certain factors associated
with the blood coagulation mechanism are absent or seriously
deficient in certain individuals. Thus, it is known that classical
hemophilia (hemophilia A) is a deficiency disease caused by the
absence of AHF (Factor VIII). In individuals suffering from the
congential hemophilia known as hemophilia B, the blood is deficient
in plasma thromboplastin component (PTC, Factor IX). Several other
factors which are important in the coagulation mechanism are
Factors II, VII and X.
Until recent years, treatment of hemophiliacs consisted of
transfusing the patient with whole blood or blood plasma. Better
medical practice dictates that, whenever possible, the patient be
administered only those blood components in which he is deficient.
Due to the universal shortage of blood, it is also advantageous to
fractionate blood into its various components, whereby they can be
used for patient treatment as required.
Various methods of fractionating blood and blood plasma into its
separate components or concentrates thereof are known. The work of
Edwin Cohn and his associates at Harvard University in the
development of the alcohol fractionation method is particularly
noteworthy. With specific reference to the production of AHF,
recent U.S. Pats. .Badd..[.3,631,078.]. .Baddend..Iadd.3,361,018
.Iaddend.and 3,652,530 illustrate improved methods of obtaining a
highly purified concentrate of that factor.
In the investigational work of Wagner, Thelin, Brinkhouse and
others interested in the production of AHF, it was found that the
presence of prothrombin (Factor II) and its associated complex of
factors was detrimental to the stability of Factor VIII, both long
term and short term. The usual treatment to obviate this problem
was the removal of the prothrombin complex with various agents such
as aluminum hydroxide, magnesium hydroxide, barium carbonate,
barium sulfate, rivanol (6,9-diamino 2-ethoxyacridine lactate),
IRC-50 ion exchange resin (XE-64-Rivanol) and glycine ethyl
ester.
While the foregoing agents are generally effective, various serious
drawbacks to their use in clinical applications have become
apparent. Factor VIII is indeed stabilized by use of these agents
since the removal of prothrombin does not allow for thrombin
generation. Thrombin is chiefly responsible for the degradation of
Factor VIII, as reported by Kisker, Thromb, Diath. Haemorrhagica,
vol. 17, p. 381 (1967) and Penick and Brinkhous, Amer. J. Med.
Sciences, vol. 232, p. 434 (1956). Thus, in the preparation of
Factor VIII with aliphatic amino acids, the original work by Wagner
et al. employed aluminum hydroxide to (1) decrease degradation of
Factor VIII while concentrating manipulations were being employed,
and (2) increase the long term stability, both after lyophilization
and reconstitution. The Hemophilias, K. M. Brinkhous, Ed.,
International Symposium, Washington, D.C., Univ. of North Carolina
Press, p. 81 (1964). However, the inclusion of trace amounts of
aluminum was considered clinically unsafe.
The advent of cryoprecipitation and the one step production of
relatively high potency concentrates appeared to obviate the use of
any means of removing the prothrombin complex. Pool et al., Nature,
vol. 203, p. 312 (1964). However, it has been found in practice
that high variability existed in yields of AHF, up to and even
exceeding 50% variability of the mean values reported.
Accordingly, it is an object of this invention to provide an
improved method of making a .Iadd.cryoprecipitate
.Iaddend.concentrate of Antihemophilic Factor (AHF, Factor
VIII).
It is another object of this invention to provide a method for
improving the yield of AHF .Iadd.concentrates .Iaddend.obtained
.Iadd.by cryoprecipitation .Iaddend.from blood plasma and plasma
fractions.
Other objects and advantages of the present invention will be
apparent to those skilled in the art after reading the disclosure
hereof.
In brief, the present invention resides in the addition of heparin
to .[.the aqueous media containing the.]. .Iadd.an AHF concentrate
obtained from .Iaddend.blood plasma or .Iadd.a .Iaddend.plasma
fraction .Iadd.by cryoprecipitation, .Iaddend.which .[.is.].
.Iadd.may .Iaddend.then .Iadd.be further .Iaddend.fractionated to
obtain .[.a concentrate.]. .Iadd.an even more concentrated form
.Iaddend.of AHF.
.Iadd.Preferably, the heparin is added to the AHF-rich concentrate
in addition to citrate. A preferred method of conducting this
double anticoagulant addition is to add the heparin to the
cryoprecipitate in the form of a heparinized, citrated saline
solution. When the AHF-rich cryoprecipitate is further fractionated
to obtain a high purity, more concentrated form of AHF, heparin is
preferably added twice, once to the initial cryoprecipitate and
subsequently to the further fractionated AHF concentrate. In each
case, heparin is preferably added along with a citrate as a second
anticoagulant. .Iaddend.
It is believed that the invention will be better understood by
reference to the accompanying drawing. The FIGURE in the drawing
presents a diagram which represents the blood clotting mechanism.
The roman numerals represent the several blood clotting factors. It
will be understood, however, that this diagram represents only a
working hypothesis based on present knowledge, and the inventors do
not intend to be limited or bound by this hypothesis.
As has been stated above, high variability in the yield of AHF has
been obtained in practice in the production of AHF .Iadd.by
cryoprecipitation. .Iaddend.The longer the manipulations in
concentration take, the lower the yield of AHF. The losses in yield
have been somewhat ameliorated by scrupulous attention to the
removal of blood cells. These results may be explained by reference
to the clotting diagram of the accompanying drawing. Of particular
interest is the feedback mechanism shown by the dotted lines, which
indicate the action of Factor II.alpha. (thrombin) on Factor V
(proaccelerin), and Factor VII (AHF). The action consists of two
parts: the actions of VIII and V are (1) first enhanced by altered
tertiary structure and then (2) degraded to stop excessive
clotting, which could lead to circulatory embarrassment.
It is apparent, from the clotting diagram, that the removal of
Factors II, VII, X and IX (prothrombin complex) would eliminate the
feedback mechanism since there would be no II to produce II.alpha..
The observation, by the present inventors, that cell free plasma
allows concentration to proceed with higher yields of AHF being
produced also is explainable by the clotting diagram, since
thromboplastin is not produced and, therefore, the beginning of
clotting cannot start.
In accordance with the present invention, it has now been found
that the yield of AHF .Iadd.obtained by cryoprecipitation
.Iaddend.in production size amounts can be enhanced by the addition
of heparin during the fractionation. It has been observed, by the
inventors, that in contrast to prior practice without the use of
heparin, when the heparin is employed (1) cell contamination, such
as from the fractionation procedure, does not lead to lower yields,
(2) tissue contamination, such as occurs during intravenous
administration, does not produce lower yields, (3) longer
processing time does not reduce the yield, and (4) reconstituted
stability is increased over non-heparinized plasma concentrates by
at least an order of 30 times. Thus, it unnecessary in the practice
of this invention to remove the prothrombin complex to achieve
stability of Factor VIII concentrates and the usual losses in yield
from cell and tissue contamination or extended processing times are
overcome.
The amount of heparin employed in the practice of this invention
during the fractionation of AHF can vary within reasonable limits.
It has been found that a concentration of about one unit of heparin
per ml. of the plasma solution or plasma fraction is about optimum.
Concentrations greater than about 10 units per ml. are to be
avoided as unnecessary and dangerous. Concentrations of about 0.01
unit per ml. are also effective. The preferred range is from about
0.01 to about 10 units per ml. One unit of heparin is defined
herein to mean one U.S.P. (United States Pharmacopoeia) unit. The
U.S.P. unit of heparin is the quantity that will prevent 1.0 ml. of
citrated sheep plasma from clotting for one hour after the addition
of 0.2 ml. of a 1:100 CaCl.sub.2 solution. As used herein, the term
"heparin" also is meant to include the sodium salt of heparin, the
latter substance being preferred due to its water solubility.
The invention defined herein has been found useful in various
procedures for producing AHF concentrates .[.and is applicable to
any plasma or plasma fraction which contains AHF in admixture with
any of the prothrombin complex factors..]. .Iadd.by
cryoprecipitation. .Iaddend.Thus, it has been adapted to methods of
fractionating AHF (1) from plasma .Iadd.or plasma fractions
.Iaddend.by .[.glycine precipitation.].
.Iadd.cryoprecipitation.Iaddend., (2) from cryoprecipitate by
glycine precipitation, (3) from cryoprecipitate by polyethylene
glycol precipitation, (4) from cryoprecipitate by polyethylene
glycol and glycine precipitation, (5) from cryoprecipitate, .[.(6)
from plasma,.]. and .[.(7).]. .Iadd.(6) .Iaddend.from
cryoprecipitate by polyethylene glycol and glycine precipitation
followed by "Ecteola" chromatography.
A preferred method of producing AHF to which the present invention
is adapted is the method described in U.S. Pat. No. 3,631,018,
whereby polyethylene glycol (PEG) and glycine are used to
fractionate a cryoprecipitate of AHF concentrate. Thus, in Example
4 of said patent, at the step wherein the crypoprecipitate is
dissolved in glycine citrated saline, about one unit of heparin is
added per ml. of the solution. Following the precipitation with
polyethylene glycol at 10% concentration, the redissolved
precipitate is again treated to contain about one unit of heparin
per ml. of the solution due to the loss of heparin during the
fractionation with 10% polyethylene glycol. The heparin can
conveniently be added by incorporation in the citrated saline or
glycine citrated saline used to dissolve the respective
precipitates.
The following examples will further illustrate the present
invention, although it will be understood that the invention is not
limited to these specific examples.
EXAMPLE 1
A stable human AHF concentrate of high potency and in high yield is
produced in the following manner:
Reagents
Citrated saline.--One part 0.1 molar sodium citrate to four parts
by weight 0.9 percent saline.
Glycine citrated saline.--Sufficient glycine is added to the above
prepared citrated saline to make a 0.1 molar solution respective of
glycine.
Buffered wash water.--To distilled water is added 1/100 volume of
buffered citrate which is prepared by adjusting 0.5 molar sodium
citrate with 0.5 molar citric acid to pH 6.88.
Heparin.--U.S. Pharmacopoeia grade material is used
("Lipo-Hepin"-sodium heparin injection, aqueous).
Acetic acid.--Prepared in both 1.0 normal and 0.1 normal aqueous
solutions.
Glycine.--Prepared in both 1.3 and 1.8 molar aqueous solutions.
Procedure
Human blood plasma is received frozen (at below 4.degree. C,) from
a donor center. The plasma is pooled into Pfaudler kettles and,
while held at a .[.temperature at 20.degree. C. to -40.degree.
C.,.]. .Iadd.temperature sufficient to produce a cryoprecipitate
containing AHF, .Iaddend.it is centrifuged by continuous flow or
bucket centrifugation .Iadd.to recover the
cryoprecipitate.Iaddend.. To the cryoprecipitate, glycine citrated
saline solution containing one unit of heparin per ml. is added,
the amount being one-tenth the volume of plasma the cryoprecipitate
represents. Dissolution is brought about by mixing the
cryoprecipitate and glycine citrated saline solution in a warm
environment (normal room temperature, but not in excess of
30.degree. C.).
The dissolved cryoprecipitate is adjusted to pH 6.5 with 0.1 normal
acetic acid. Polyethylene glycol 4000 (molecular weight average
about 4000) is added to the solution to make the PEG concentration
about 3.5 percent. The mixture is gently agitated at room
temperature for ten minutes and then centrifuged for fifteen
minutes at 5000 r.p.m. The supernate is decanted and adjusted to pH
6.88 with 0.1 normal sodium hydroxide. Additional PEG 4000 is added
to the solution to make the final PEG concentration about 10
percent. The mixture is gently agitated at room temperature for
thirty minutes and centrifuged at 5000 r.p.m. for one-half hour.
The supernate is decanted and discarded. The precipitate is washed
in cold water (2.degree. C.) and spin washing is then carried out
for five minutes at 5000 r.p.m. at a temperature of -4.degree. C.
The supernate is decanted and the precipitate is redissolved in
glycine citrated saline solution containing one unit of heparin per
ml. Again, the amount of the redissolving solution is about
one-tenth the volume of plasma that the precipitate represents.
The redissolved precipitate is adjusted to pH 6.88 with 0.1 normal
acetic acid and reprecipitated with aqueous glycine having a
molarity of 1.8. Sufficient glycine is added during this
precipitation step to make the mixture 1.8 molal with respect to
glycine. The mixture is gently agitated for 45 to 60 minutes at a
temperature of from 2.degree. C. to 10.degree. C., and then
centrifuged by continuous flow or bucket centrifugation. The
resulting precipitate is collected and gently washed with buffered
wash water and redissolved in citrated saline. The solution is
clarified by filtration using a 293 mm. millipore filter (membranes
used: 1.2 microns, 0.45 micron, and 0.3 micron).
The liquid product is then frozen by shell freezing (-60.degree.
C.) and storing in a flash freezer (-20.degree. C. to -30.degree.
C.) for at least three hours.
The foregoing procedure was repeated except that no heparin was
added to the glycine citrated saline used to dissolve the initial
cryoprecipitate or to dissolve the dissolve from the fractionation
with 10% PEG or at any other point in the fractionation.
The foregoing procedures, both with and without the addition of
heparin, were both repeated.
The following table sets forth the results in these four
fractionation runs:
______________________________________ AHF YIELD Cryo Final yield,
product Percent Total percent yield AHF, recovery, volume AHF
percent percent of recovered recovered recovered starting from
start- from start- from cryo. plasma, Procedure used ing plasma ing
plasma step liters ______________________________________ Without
heparin 35 13.6 38 6,000 With heparin 35 17 49 300 Without heparin
32 12.5 39 6,000 With heparin 33 17 52 300
______________________________________
In the table, it is seen that the final AHF yield when heparin was
employed was 17%, whereas the yield without heparin averaged from
12.5% to 13.6%. The addition of heparin, therefore, increased the
yield by 25% to 35% over the non-heparinized procedure. When the
final AHF yield is divided by the cryoprecipitate AHF yield, it is
seen that the efficiency of the process was improved from 38% and
39% without the heparin addition to 49% and 52% with the heparin
addition.
When PEG 6000 is substituted for an equivalent amount of PEG 4000,
in the above example, substantially similar results are
obtained.
EXAMPLE 2
The procedure of Example 1 was repeated up to the step of
.[.resuspending.]. .Iadd.suspending .Iaddend.the initial
cryoprecipitate in glycine citrated saline both with and without
heparin in the solution. The final AHF yield in these two
cryoprecipitate concentrates of AHF was 34% when heparin was not
employed and 41% when heparin was used. This is equivalent to a 21%
improvement in yield.
EXAMPLE 3
The procedure of Example 2 was repeated except that the
cryoprecipitate concentrates, both with and without heparin, were
lyophilized and then reconstituted with water and allowed to stand
at room temperature (ca. 25.degree. C.) for 24 hours. The
heparinized sample retained 98% of the initial AHF activity prior
to the standing period whereas the non-heparinized sample retained
only 72% of the initial AHF activity.
Various other examples and modifications of the foregoing examples
will be apparent to the person skilled in the art after reading the
foregoing specification and the appended claims without departing
from the spirit and scope of the invention. All such further
examples and modifications are included within the scope of the
appended claims.
* * * * *