Heat treatment of lyophilized blood clotting factor VIII concentrate

A method for treating blood products to eliminate hepatitis virus present therein is disclosed. Blood products treated according to the method are first lyophilized and then subjected to heat for a period of time sufficient to inactivate the hepatitis virus. At the conclusion of the heating step the blood product can be reconstituted using sterile water or a like diluent. No adverse effects on blood product activity following treatment have been observed. The method has special applicability to blood plasma fractions such as Factor VIII, Factor IX and fibrinogen.

TECHNICAL FIELD 
The present invention relates generally to methods for heat treating plasma 
fractions and is particularly directed to a series of steps whereby 
various plasma fractions may be heated in lyophilized form for the purpose 
of inactivating hepatitis virus present in the fractions. 
BACKGROUND ART 
Utilization of clotting factor concentrates obtained from fractionated 
blood plasma for the purpose of intervening therapeutically with respect 
to inherited bleeding disorders such as hemophilia is severely compromised 
as a consequence of the inordinate risk posed to the hemophiliac patient 
by the presence of hepatitus virus in the concentrates. For example, 
commercial Factor VIII and IX concentrates are typically employed to 
increase the clotting ability of a hemophilia victim's blood, but these 
concentrates are prepared from pools of plasma contributed by thousands of 
donors and contain the inherent hepatitis risk of a like number of single 
unit transfusions. As McCullen and Zuckerman have shown, see Journal of 
Medical Virology, Vol. 8, No. 29 (1981), despite stringent screening of 
individual donors for hepatitis B surface antigens (HBsAg), such plasma 
pools clearly transmit both hepatitis B and non-A, non-B hepatitis. 
Hepatitis transmission by albumin and other heat-stable plasma components 
unrelated to blood coagulation has heretofore been prevented by heating 
the plasma components in solution at temperatures of 60.degree. C. for ten 
hours. Similar attempts to heat clotting factor concentrates in solution, 
by way of contrast, have been shown to markedly reduce or eliminate 
clotting factor activity in the concentrates and thus do not appear to 
offer a viable solution to the problem of hepatitis transmission 
associated with conventional hemophiliac therapy. More recently, highly 
purified Factor VIII precipitate has been dissolved in a solution of 
sucrose glycine and heated for ten hours at 60.degree. C. Although the 
Factor VIII concentrate subsequently derived from the heated precipitate 
does retain clotting factor activity, the yields obtained using this 
approach are very low, e.g., about 8%. See Heimburger, et al., Hemostasis, 
Vol. 10 (supplement 1), p. 204 (1981) and Heimburger, et al., Blut, Vol. 
20, p. 129 (1981). As a net result, the prior art to date does not furnish 
any means for effectively inactivating hepatitis virus present in clotting 
factor concentrates nor does the prior art teach a means for preventing 
the transmission of hepatitis virus to patients undergoing therapy with 
clotting factor concentrates. 
DISCLOSURE OF THE INVENTION 
It is therefore an object of the present invention to provide a method for 
heat treating clotting factor concentrates to inactivate any hepatitis 
virus present therein without reducing clotting factor activity. 
It is another object of the present invention to provide a method for heat 
treating clotting factor concentrates to inactivate any hepatitis virus 
present therein, which method results in substantial yields of concentrate 
without significant reduction of clotting factor activity in the 
concentrate. 
It is still another object of the present invention to provide a method for 
heat treating clotting factor concentrates wherein the concentrates are 
first prepared in lyophilized form to enhance the stability of the 
concentrates during the heating process. 
It is a further object of the present invention to provide a method for 
heat treating lyophilized plasma fractions to produce a vaccine effective 
against both hepatitis B virus and non-A, non-B hepatitus virus. 
These and other objects of the present invention are achieved by 
lyophilizing either whole plasma or plasma fractions such as Factor VIII 
concentrate, Factor IX concentrate, fibrinogen and cryoprecipitate and 
thereafter subjecting the lyophilized whole plasma or plasma fractions to 
elevated temperatures for varying periods of time.

BEST MODE FOR CARRYING OUT THE INVENTION 
The ability to isolate clotting factors present in human blood has been 
indispensable in understanding the pathology of hemophilia and other 
inherited bleeding disorders. Concommitantly, the discovery of plasma 
fractionation schemes for obtaining practical quantities of clotting 
factor concentrates has enabled medical science to utilize the clotting 
factor concentrates as therapeutic tools in treating bleeding disorders. 
Transfusion therapy employing Factor VIII and Factor IX concentrates in 
particular has proven quite successful in ministering to hemophiliac 
patients. Unfortunately, the risk of hepatitis transmission due to the 
large number of plasma donors required for commercial production of 
clotting factors concentrates remains as the one serious drawback 
associated with transfusion therapy. A typical plasma fractionation 
scheme, disclosed in Seminars in Thrombosis and Hemostasis, Vol. VI, No. 
1, p. 4 (1979), yields cryoprecipitate and supernate, the former fraction 
constituting a source of both Factor VIII concentrate and fibrinogen and 
the latter fraction constituting a source of Factor IX concentrate in 
addition to Factors II, VII, and X concentrates. As Gerety and Eyster have 
demonstrated in "Hepatitis Among Hemophiliacs", Non-A, Non-B Hepatitis, p. 
103-106 (1981), hepatitis B virus initially present in whole plasma is 
distributed to the Factor VIII and Factor IX derivatives during the plasma 
fractionation process. As also demonstrated by Maynard and Bradley, 
"Transmission by Blood Products", Non-A, Non-B Hepatitis, p. 78-79 (1981), 
non-A, non-B hepatitis exists in both Factor VIII and Factor IX 
derivatives. Previous attempts to heat-treat clotting factor concentrates 
in solution for the purpose of inactivating hepatitis virus have been 
ineffective. The development of techniques for lyophilizing clotting 
factor derivatives, however, has opened a new avenue of exploration with 
regard to stabilizing clotting factor derivatives during the heat treating 
process, in turn establishing a means for inactivating hepatitis virus 
present in the clotting factor derivatives without destroying clotting 
factor activity. 
TEST PROCEDURES FOR VERIFYING RETENTION OF CLOTTING FACTOR ACTIVITY 
Paired samples of various lyophilized plasma fractions, each such pair 
having identical lot numbers, were received from several manufacturers. 
The samples generally weighed less than 100 g and were packaged in vials 
having volumes of 60 ml to 90 ml. One sample in each pair was heated, 
either by placing the sample vial in a water bath or dry oven at a 
predetermined temperature under room pressure for a predetermined period 
of time, or by placing the lyophilized material itself in a dry oven 
without the vial present. The remaining sample in each pair served as a 
control and was refrigerated at 4.degree.-6.degree. C. during the 
heat-treating process. Following heat treatment, both the control and 
heat-treated lyophilized samples were reconstituted with sterile water. 
Reconstitution was generally carried out according to manufacturer's 
specifications, although the solubility of some heat-treated samples was 
markedly improved by increasing the amount of sterile water used during 
reconstitution over that recommended by the manufacturer. In vitro Factor 
VIII and Factor IX assays were performed using a one-stage manual 
fibrometer method at dilutions ranging between 1:40 and 1:400 to obtain a 
measure of Factor VIII and Factor IX clotting activity. In vitro recovery 
of fibrinogen following reconstitution of both the control and 
heat-treated lyophilized fibrinogen samples was measured in a similar 
fashion. In some of the experiments, reconstituted plasma fractions were 
observed for light transmission at 580 nm in a Beckman Model 25 
Spectrophotometer. Agarose gel electrophoresis with an ICL 
immunoelectrophoresis plate was carried out for several of the Factor VIII 
and Factor IX paired samples, using goat anti-human serum supplied by 
Hyland Diagnostics as a standard. The plates were specifically 
electrophoresed by a Buchler power supply set at 25 ma for 35 minutes. 
Upon completion of the electrophoresis, the plates were incubated in 
antisera for 18 to 24 hours and examined under indirect light. Panagell 
electrophoresis with a Worthington Diagnostics plate was carried out on 
additional paired samples of Factor VIII concentrate, using a Biorad 
water-cooled electrophoresis cell. 
Further in vitro experiments were performed by heating lyophilized samples 
of Factor VIII concentrate in a water bath at room pressure and at 
predetermined temperatures for predetermined periods of time. Factor 
VIII.sub.Ag was then determined using the method described by Laurell, 
"Electroimmuno Assay," The Scandinavian Journal of Clinical and Laboratory 
Investigation, Vol. 29, pp. 21-37 (1972). Factor VIII results were 
calculated for dilutions of 1:40, 1:80, 1:100 and 1:200 by plotting the 
height of the rockets of the Laurell standard curve against the percentage 
of dilution. Unknowns were expressed as a percentage of normal, based on 
the rocket heights of the unknowns in the standard curve. 
In vivo recovery of clotting factor activity for both heat-treated Factor 
VIII and Factor IX concentrates was measured by injecting reconstituted, 
heat-treated lyophilized Factor VIII and Factor IX concentrates 
respectively into hemophilia A and hemophilia B dogs. A heat-treated, 
lyophilized Factor IX concentrate was also injected into a control dog. 
Laboratory parameters including Hot, serum protein, WBC, platelet count, 
blood smear, respiration rate, body temperature, pulse and clotting factor 
activity were subsequently ascertained for each of the animals at various 
intervals following the injections. 
Results of the in vitro testing performed on Factor VIII concentrate are 
summarized in Tables I and II: 
TABLE I 
______________________________________ 
Measurements of Clotting Factor Activity Following 
Heat-Treatment of Lyophilized Factor VIII Concentrate 
Dil- 
Lot Temp. Time ution 
% Activity 
______________________________________ 
*A C-1081 
Control -- 1:20 Approximately 
" " -- 1:40 10% decrease in 
" " -- 1:80 activity was 
" 60.degree. C. 
10 hr. 1:20 observed for 
" " " 1:40 heat-treated 
" " " 1:80 samples relative 
to the control. 
A NC-8247 
Control -- 1:40 1438 
" " -- 1:80 1697 
" 62.degree.-64.degree. C. 
16.33 hr. 
1:40 1215 
" " " 1:80 1360 
B AHF-355 
Control -- 1:40 1912 
" " -- 1:80 1600 
" " -- 1:160 
1312 
" 64.degree. C. 
20 hr. 1:40 1080 
" " " 1:80 1072 
" 74.degree. C. 
17 hr. 1:40 1144 
" " " 1:80 1024 
" " " 1:160 
864 
" 76.degree. 17 hr. 1:40 1040 
" " " 1:80 976 
B 347 Control -- 1:100 
1180 
" " -- 1:200 
1000 
" 83.degree. C. 
24 hr. 1:100 
100 
" " " 1:200 
100 
" 85.degree. C. 
24 hr. 1:100 
&lt;1 
" 95.degree. C. 
7 hr. 1:100 
&lt;1 
" 97.degree. C. 
7.5 hr. 1:200 
&lt;1 
C Al-0470 
Control -- 1:100 
912 
" 75.degree. C. 
20 hr. 1:100 
2076 
C Al-1080 
Control -- 1:200 
2080 
" " -- 1:400 
1920 
" 80.degree. C. 
24 hr. 1:200 
1380 
" " " 1:400 
1360 
C Al-1120 
Control -- 1:40 2800 
" " -- 1:80 2032 
" 78.degree. C. 
21 hr. 1:40 1592 
" " " 1:80 1392 
" 80.degree. C. 
20 hr. 1:40 1176 
" " " 1:80 1600 
" 90.degree. C. 
12 hr. -- Clotted Specimen 
" 100.degree. C. 
1.5 hr. 1:40 1248 
" " " 1:80 1264 
C Al-1150 
Control -- 1:40 2176 
" " -- 1:80 2480 
" " -- 1:100 
1870 
" " -- 1:200 
2080 
" 65.degree. C.** 
26.33 hr. 
1:40 1592 
" 65.degree. C.** 
" 1:80 1520 
" 83.degree. C. 
24 hr. 1:100 
730 
" " " 1:200 
620 
" 85.degree. C. 
24 hr. 1:100 
1000 
" " " 1:200 
1160 
" 90.degree. C. 
10 hr. 1:40 1032 
" " " 1:80 1056 
" 95.degree. C. 
7 hr. -- Clotted Specimen 
" 97.degree. C. 
7.5 hr. 1:100 
80 
" " " 1:200 
100 
" 100.degree. C. 
10 hr. 1:40 88 
" " " 1:80 48 
C Al-1160 
Control -- 1:100 
3680 
" " -- 1:200 
3420 
" " -- 1:400 
3200 
" 78.degree. C. 
24 hr. 1:100 
2420 
" " " 1:200 
1520 
" " " 1:400 
1440 
" 78.degree. C. 
24 hr. 1:200 
1720 
" " " 1:400 
1680 
" 80.degree. C. 
22 hr. 1:200 
1400 
" " " 1:400 
1360 
" 100.degree. C. 
7 hr. 1:200 
1760 
" " " 1:400 
1760 
C Al-2120 
Control -- 1:100 
816 
" 110.degree. C.*** 
1.5 hr. 1:100 
18 
C Al-2531 
Control -- 1:200 
3500 
" " -- 1:400 
2700 
" 85.degree. C. 
20 hr. 1:200 
46 
" " " 1:400 
43 
______________________________________ 
Note: 
All times and temperatures are approximate. Following heat treatment at 
higher temperatures, amounts of sterile water in excess of manufacturer's 
recommendations were added to some concentrates until solubilization was 
visually confirmed. 
*A lots were manufactured by Cutter Laboratories; B lots were manufacture 
by Michigan Red Cross; C lots were manufactured by Alpha Therapeutics. 
**Heat-treated in a dry oven (sample removed from vial). 
***Heat-treated in a dry oven (sample contained in vial). 
TABLE II 
______________________________________ 
Determination of Factor VIII.sub.Ag Following 
Heat-Treatment of Lyophilized Factor VIII Concentrate 
Rocket 
Lot Temp Time Dilution 
Height (mm) 
% Ag 
______________________________________ 
B AHF-355 
Control -- 1:40 35 3720* 
" " -- 1:80 22 4080 
" 64.degree. C. 
20 hr. 1:40 39 4240 
" " " 1:80 26 5120 
" 74.degree. C. 
17 hr. 1:40 40 4400 
" " " 1:80 26 5120 
C Al-1120 
Control -- 1:100 15 4700 
" 90.degree. C. 
12 hr. 1:100 0 0 
C Al-1150 
Control -- 1:200 13 7200 
" 83.degree. C. 
24 hr. 1:200 12 6200 
" 85.degree. C. 
24 hr. 1:200 15 9400 
" 97.degree. C. 
7.5 hr. 1:200 0 0 
______________________________________ 
Note: 
All times and temperatures are approximate. Following heat treatment at 
higher temperatures, amounts of sterile water in excess of manufacturer's 
recommendations were added to some concentrates until solubilization was 
visually confirmed. 
*B lots were manufactured by Michigan Red Cross; C lots were manufactured 
by Alpha Therapeutics. 
Results from the testing of Factor IX concentrate are summarized in Table 
III: 
TABLE III 
______________________________________ 
Measurements of Clotting Factor Activity Following 
Heat-Treatment of Lyophilized Factor IX Concentrate 
Lot Temp. Time Dilution 
% Activity 
______________________________________ 
*A 9-C0044 
Control -- 1:40 616 
" " -- 1:80 1200 
" " -- 1:200 2400 
" " -- 1:400 3520 
" 100.degree. C. 
4 hr. 1:40 520 
" " " 1:80 1104 
" 100.degree. C. 
12 hr. 1:200 1680 
" " " 1:400 2480 
" 110.degree. C.** 
13 hr. 1:400 1640 
" 110.degree. C.** 
" 1:800 2560 
" 122.degree. C.** 
12 hr. 1:200 340 
" 122.degree. C.** 
" 1:400 480 
" 132.degree. C.** 
12 hr. 1:200 12 
" 132.degree. C.** 
" 1:400 24 
A NC9055 Control -- n/a 2600 
" 100.degree. C. 
0.5 hr. n/a 2350 
______________________________________ 
Note: 
All times and temperatures are approximate. Following heat treatment at 
higher temperatures, amounts of sterile water in excess of manufacturer's 
recommendations were added to some concentrates until solubilization was 
visually confirmed. 
*A lots manufactured by Cutter Laboratories. 
**Heat treated in a dry oven. 
Results of the testing performed on fibrinogen concentrate are summarized 
in Table IV: 
TABLE IV 
______________________________________ 
Recovery of Fibrinogen Following Heat-Treatment 
of Fibrinogen Concentrate in Lyophilized Form 
Dil- 
Lot Temp. Time ution 
Recovery (mg/dl) 
______________________________________ 
*D-003678 
Control -- 1:20 400 
" " -- 1:40 680 
" 60.degree. C. 
10-11 hr. 
1:20 660 
" " " 1:40 680 
" Control -- 1:10 195 
" " -- 1:20 760 
" " -- 1:40 700 
" 60.degree. C. 
10 hr. 1:10 105 
" " " 1:10 225 
" " " 1:20 250 
" 60.degree. C. 
17 hr. 1:10 190 
" " " 1:20 220 
*E Control -- 1:40 1280 
" " -- 1:80 1280 
" 60.degree. C. 
10 hr. 1:40 1520 
" " " 1:80 1320 
" 60.degree. C. 
10 hr. 1:40 1860 
" " " 1:80 1520 
" 65.degree. C. 
10 hr. 1:40 1640 
" " " 1:80 1400 
" " 23 hr. 1:40 1420 
" " " 1:80 1320 
" Control -- 1:40 1048 
" " -- 1:80 1064 
" 100.degree. C. 
3 hr. 1:40 788 
" " " 1:80 784 
" 254.degree. F.** 
3 hr. 1:5 133 
______________________________________ 
Note: 
All times and temperatures are approximate. Following heat treatment at 
higher temperatures, amounts of sterile water in excess of manufacturer's 
recommendations were added to some concentrates until solubilization was 
visually confirmed. 
*D lots manufactured by Cal Biochem; E lots manufactured by Kabi. 
**Heat-treated in a dry oven (sample contained in vial). 
DISCUSSION OF SELECTED TEST RESULTS 
The results summarized in Tables I-IV can be combined to provide a relative 
indication of clotting activity retention and fibrinogen recovery in 
lyophilized plasma fractions subjected to the heat-treatment process of 
the present invention. More particularly, the percentage activity or 
measured recovery at various dilutions of reconstituted Factor VIII, 
Factor IX and fibrinogen concentrates can be averaged for individual 
control samples and compared with similarly-averaged percentage activity 
or measured recovery in corresponding paired samples of heat-treated 
Factor VIII, Factor IX and fibrinogen concentrate. Where such comparisons 
are made, it can be seen, for example, that lyophilized Factor VIII 
concentrate obtained from one manufacturer (Lot No. A C-1081) and heated 
at 60.degree. C. for 10 hours retained greater than 90% of its in vitro 
Factor VIII clotting activity in comparison to an unheated control. The 
reconstituted, heat-treated Factor VIII concentrate further exhibited an 
absorbance of 0.30 at 580 nm in comparison to an absorbance of 0.20 for 
the unheated control, and showed no differences relative to the unheated 
control following immunoelectrophoresis with the goat anti-human serum. 
Reconstituted Factor VIII concentrates from a different lot (Lot. No. A 
NC-8247) of the same manufacturer, which had been heated in lyophilized 
form at 62.degree.-64.degree. C. for approximately 16 hours and then 
stored at 6.degree. C. for seven days, showed greater than 80% recovery of 
Factor VIII clotting activity in comparison to an unheated control. An 
overall increase in anodal migration relative to the unheated control was 
noted following immunoelectrophoresis against goat anti-human serum. 
In similar fashion, lyophilized Factor VIII concentrate obtained from a 
second manufacturer (Lot No. C Al-1120), when heated at approximately 
78.degree. C. for 21 hours, showed 62% in vitro retention of clotting 
activity upon reconstitution as compared to an unheated control. 
Reconstituted Factor VIII concentrate from the same lot of the second 
manufacturer, after heat treatment in lyophilized form for 20 hours at 
approximately 80.degree. C., still retained 57% in vitro clotting activity 
as compared to an unheated control, whereas reconstituted Factor VIII from 
the same lot of the second manufacturer, which had previously been heated 
in lyophilized form for one and one-half hour at approximately 100.degree. 
C., retained approximately 52% in vitro clotting activity as compared to 
an unheated control. When a different lot (Lot No. C Al-1150) of 
lyophilized Factor VIII concentrate from the second manufacturer was 
heat-treated in accordance with the present invention, in vitro recoveries 
of clotting activity in comparison to the unheated control ranged from 67% 
for 26 hours and 20 minutes of heat treatment at 65.degree. C. to 34% for 
24 hours of heat treatment at 83.degree. C. to 55% for 24 hours of heat 
treatment at 85.degree. C. Measurements of Factor VIII antigen for the two 
samples of Factor VIII concentrate heat-treated at 83.degree. C. and 
85.degree. C. respectively showed a 15% loss and no loss in antigen 
levels. It should also be noted that greatly improve solubilization of the 
latter sample of heat-treated Factor VIII concentrate was achieved by 
adding between 50 ml and 75 ml of sterile water to the sample rather than 
the 25 ml recommended by the manufacturer. 
Heat treatment of lyophilized Factor VIII samples obtained from a third 
manufacturer (Lot No. AHF-355) confirmed results observed for the first 
two manufacturers. That is, heat treatment of the third manufacturer's 
lyophilized Factor VIII concentrate for 20 hours at 64.degree. C. yielded 
clotting activity recovery of 61% in comparison to an unheated control, 
heat treatment of the same concentrate for 17 hours at 74.degree. C. 
yielded clotting activity recovery of 63% and heat treatment of the same 
concentrate for 17 hours at 76.degree. C. yielded clotting activity 
recovery of 57%. Factor VIII antigen levels in the reconstituted samples 
heated at 64.degree. C. and 74.degree. C. showed no decrease when compared 
to the unheated control level. 
A sample of lyophilized Factor IX concentrate obtained from the first 
manufacturer (Lot No. A 9-C0044) and immersed in a water bath at 
100.degree. C. for 20-30 minutes yielded essentially full in vitro 
recovery of clotting activity when compared to an unheated control. Factor 
II and Factor VII both appeared stable 2 hours following reconstitution of 
the heat-treated sample, while Factor X decreased approximately 20% within 
6 days of reconstitution. Absorbance measurements obtained 2 hours after 
reconstitution yielded values of 0.006 to 0.007 at 580 mm for both control 
and heat-treated samples. No visual difference could be detected between 
the heat-treated concentrate and the unheated control following 
immunoelectrophoresis of the Factor IX concentrate against goat anti-human 
serum. Additional samples of Factor IX concentrate from the first 
manufacturer, which were respectively heat-treated in lyophilized form at 
100.degree. C. for 12 hours and at 110.degree. C. for 13 hours, also 
showed full recovery of Factor IX clotting activity, although complete 
solubilization of the latter sample required 40 ml to 60 or greater ml of 
sterile water as opposed to the manufacturer's recommended 20 ml. The data 
from Table III thus suggests that Factor IX concentrate in lyophilized 
form is largely heat stable for 4 hours at temperatures between 
100.degree. C.-110.degree. C. 
A sample of lyophilized fibrinogen concentrate obtained from a fourth 
manufacturer (Lot No. D-003678) and heat-treated for 11 hours at 
60.degree. C. showed no in vitro loss of fibrinogen when compared with an 
unheated control. A sample of lyophilized fibrinogen concentrate obtained 
from the same manufacturer, when heat-treated for 17 hours at 60.degree. 
C., showed a fibrinogen recovery of 97% compared with the unheated 
control. Samples of lyophilized fibrinogen concentrates obtained from a 
fifth manufacturer (Lot E), when heated for 10 and 23 hours respectively 
at 60.degree. C. and 65.degree. C., showed no in vitro loss of fibrinogen 
relative to the unheated control, while a sample of lyophilized fibrinogen 
concentrate from the fifth manufacturer showed 74% fibrinogen recovery 
compared to the control following heat treatment for 3 hours at 
100.degree. C. 
As previously indicated, in vivo testing of heat-treated Factor VIII and 
Factor IX concentrates was carried out using hemophilia A or Factor VIII 
deficient and hemophilia B or Factor IX deficient dogs. The hemophilia A 
dog received reconstituted Factor VIII concentrate which had previously 
been heat-treated in lyophilized form at 60.degree. C. for 10 hours, while 
the hemophilia B dog received reconstituted Factor IX concentrate which 
had previously been heat-treated at 100.degree. C. for 3 to 4 hours. 
Results of the in vivo testing are reported in Tables V and VI below. 
TABLE V 
__________________________________________________________________________ 
F-VIII Deficient Dog Given 
Heat Treated Factor VIII Concentrate 
Protein 
WBC/ 
Platelets/ 
FVIII RA 
FVIII C 
Temp 
Respi- 
HCT % gm % 
mm.sup.3 
mm.sup.3 
%* %* .degree.F. 
ration 
Pulse 
__________________________________________________________________________ 
PRE 44 6.1 3,795 
330,000 
106 &lt;2 100.0 
48 132 
(1-2) 
Infusion 
20 ml given in 3.5 min 400.degree. 
15 min 
45 5.9 3,190 
110,000 
151 12 102.3 
42 138 
90 min 
47 6.0 6,050 
165,000 
n = 2 12 101.0 
pant 
108 
156, 159 
3 hours 
44 6.1 5,115 
231,000 
168 15 100.9 
pant 
108 
5 hours 
43 6.0 4,785 
165,000 
159 9 100.5 
30 114 
7.5 hours 
44 5.9 3,245 
198,000 
150 9 101.0 
42 108 
__________________________________________________________________________ 
TABLE VI 
__________________________________________________________________________ 
F-IX Deficient Dog Given 
Heat Treated Factor IX Concentrate 
Trombin 
Protein 
WBC/ 
Platelets/ 
clot time 
FVIII C 
F-IX 
Temp 
Respi- 
HCT % gm % 
mm.sup.3 
mm.sup.3 
sec %* %* .degree.F. 
ration 
Pulse 
__________________________________________________________________________ 
PRE 43 5.5 5,840 
187,000 
5.5 67 &lt;1 101.5 
36 150 
(0-5-1) 
Infusion 
20 ml given in 3 min. 5* 524* 
15 min 
42 5.6 5,005 
429,000 
5.5 47 9 101.8 
30 150 
90 min 
44 5.8 6,545 
253,000 
5.5 59 10 102.0 
48 144 
3 hours 
40 5.7 8,910 
429,000 
6.0 39 6 42 162 
5 hours 
41 5.8 5,610 
231,000 
6.0 64 6 100.3 
42 126 
7.5 hours 
44 5.7 6,985 
363,000 
5.5 95 4 101.0 
36 162 
__________________________________________________________________________ 
The results reported in Table V amply illustrate the marked increase in 
Factor VIII clotting activity for a hemophilia A dog following injection 
of heat-treated Factor VIII concentrate. Similarly, the results reported 
in Table VI amply illustrate the Factor IX recovery observed in a 
hemophilia B dog following injection of heat-treated Factor IX 
concentrate. The apparent absence of physiological stress or other adverse 
reaction as seen from the data in Tables V and VI suggests that Factor 
VIII and Factor IX concentrates which have been processed according to the 
steps of the present invention remain biologically acceptable. 
It has now been demonstrated that plasma fractions such as Factor VIII and 
Factor IX concentrates of varying purity can be safely heat-treated in 
lyophilized form at elevated temperatures for extended periods of time 
without significantly destroying the clotting activity of the 
concentrates. It has further been demonstrated that plasma fractions such 
as fibrinogen of varying purity can be safely heat-treated in lyophilized 
form at elevated temperatures for extended periods of time without 
destroying the recoverability of the fibrinogen. Visual observations 
confirm that the solubility of lyophilized Factor VIII, Factor IX and 
fibrinogen concentrates is not deleteriously affected by heat-treatment 
inasmuch as the amount of sterile water added to the lyophilized 
concentrate samples during reconstitution can simply be increased until 
complete solubility is achieved. Consequently, through suitable adjustment 
of the heating temperature, length of heating and purity levels involved, 
hepatitis virus of both the B type and the non-A, non-B type can be 
inactivated in plasma fractions while maintaining the viability and 
therapeutic integrity of the fractions. Given the additional fact that 
hepatitis B virus and probably non-A, non-B hepatitis virus are 
preferentially distributed in the clotting factor fractions, i.e., in 
Factor VIII and Factor IX concentrates, heat treatment of the clotting 
factor fractions in lyophilized form at suitable temperatures for suitable 
periods of time can also serve to render the hepatitis virus immunogenic 
as well as non-infectious. As a consequence, reconstituted heat-treated 
lyophilized Factor VIII and Factor IX concentrates can function as 
hepatitis vaccines while simultaneously providing the therapeutic benefits 
otherwise associated with clotting factor fractions. 
It should, moreover, be apparent from extrapolation of the test results 
reported in Tables I-VI that the method of the present invention can be 
performed at essentially any point during the plasma fractionation 
process. That is, at any point along the fractionation process where a 
plasma derivative can be lyophilized, heat treatment of the plasma 
derivative can be performed and the derivative resolubilized or 
reconstituted prior to continuation of the fractionation process. Thus, 
for example, where Factor VIII concentrate is ultimately derived from a 
plasma fractionation scheme such as that disclosed in Mammen, et al., 
"Treatment of Bleeding Disorders with Blood Components," Reviews of 
Hematology, Vol. 1, p. 144 (1980), cryoprecipitate obtained from fresh 
frozen plasma, clarified extract obtained from cryoprecipitate and 
supernatant obtained from clarified extract can all be lyophilized and 
heat-treated in the same manner as the Factor VIII concentrate itself. 
Selection of an appropriate point in the plasma fractionation scheme for 
applying the heat treatment can then be based on pragmatic considerations 
such as cost or convenience. 
Several embodiments of the present invention have been illustrated 
hereinabove. It is to be understood, however, that various modifications 
to the temperature ranges, heating periods and purity levels set forth in 
conjunction with the aforementioned embodiments can be made by those 
skilled in the art without departing from the scope and spirit of the 
present invention. It is therefore the intention of the inventor to be 
bounded only by the limits of the following claims.