Note: Descriptions are shown in the official language in which they were submitted.
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SPECIFICATION
BACKGROUND OF THE INVENTION
Field of the Invention: This invention relates to a method
for heat treating human plasma, plasma fractions and
comp~sitions thereof to render them substantially free of
infectious viruses, bacteria and retroviruses.
Descri~tion of the Prior Art: Many useful blood fractions
10 and blood proteins are obtained from human blood plasma by
fractionation according to known techniques.
Among such known techniques, to name but a few representa-
tive examples, there may be mentioned the alcohol fraction-
ation method of Cohn et al, U.S. Patent 2,390,074 (1945)
and the Journal of the American Chemlcal Society, 68, 459
(1946); polyethylene glycol fractionation methods of
Polson, U.S. Patent 3,415,804, Shanbrom et al, U.S. Patent
3,631,018 (Factor VIII), Schwarz et al, U.S. Patent
20 4,404,131, and the polyethylene glycol/glycine method of
Fekete et al, U.S. Patents 3,682,881 and Re.29,698; the
glycine fractionation method of Blomback et al, U.S. Patent
4,348,315; the aluminum hydroxide treatment of an aqueous
solution of cryoprecipitate with aluminum hydroxide
25 followed by ultrafiltration and, optionally, glycine
treatment of Mitra et al, U.S. Patent 4,386,068; the
isolation and purification of Factor IX disclosed in Wada
et al, U.S. Patent 3l717,708, and Mitra et al, U.S. Patents
4,36~1,510 and 4,404,132; the isolation and purification of
30 fibronectin disclosed in Wallace et al, U.S. Patent
4,455,300; the preparation of antithrombin-III disclosed in
Jordan, U.S. Patent 4,386,025; and the preparation of
alpha-l proteinase inhibitor disclosed in Coan et al, U.S.
Patents 4,379,087 and 4,439,358.
Therapeutic use of such plasma proteins and compositions
thereof to treat various disorders has been compromised due
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to the risk of contracting virus infection, particularly
hepatitis virus infection. Thus, numerous techniques to
reduce or eliminate infectious microorganisms an* to render
plasma protein compositions non-viral infective have been
reported.
For example, the preparations, in wet or dry state (that
is, as the liquid concentrate itself or freeze-dried), may
be heated at temperatures of about 60 to 85 C for a period
o of several minutes to several days as may be required,
generally in the presence of a heat stabilizing agent.
Suitable stabilizing agents include nonpolar anions with
molecular weights greater than 80, sugars, reduced sugars,
and amino acids.
Examples of suitable nonpolar anions include salts of
carboxylates; hydroxycarboxylates and amino acids such as a
sodium or potassium caprylate, caprate, oleate, laurate,
valerate, acetylphenylalaninate, acetyleucinate, and
~ acetyltryptophanate. Examples of suitable sugars include
glucose, sucrose and maltose to name but a fewJ and
examples of suitable reduced sugars include erythritol and
mannitol. Examples of suitable amino acids include lysine,
glysine, proline and glutamic acid to name but a few.
2s
By way of example without limitation, suitable conventional
known processes to reduce or eliminate infectious micro-
organisms and render the preparations non-viral infective
include those disclosed in U.S. Patents 3,041,242,
30 3,057,781, 3,227,626, 4,061,735, 4,137,307, 4,297,344,
2,705,230, 2,897,123, 3,284,301, 3,454,929, 4,379,085,
4,370,264, 4,440,679 and 4,424,206, and ~uropean Patent
Publications 0058993, 0077870 and 0094611, and in
references disclosed in the patents.
Rubinstein, U.S. Patent 4,456,590 and EP Patent Application
Publication 0,096,611, discloses the heat treatment of dry,
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for instance, lyophilized, plasma compositions containing
Factors VIII and IX at a temperature of at least about 60
C for a time sufficient to render hepatitis virus-present
in the composition non-infectious.
Since 1978, a disease syndrome has occurred which involves
a progressive reduction in cellular immunity leading to the
onset of opportunistic infections and cancers, particularly
~aposi's sarcoma and B cell lymphomas. This Acquired
o Immune Deficiency Syndrome (AIDS) has been observed in the
United States primarily in homosexual and bisexual men and
lV drug abusers. It has also been found in recipients of
blood transfusions, infants o~ individuals from one of the
risk groups, hemophiliacs, and individuals from Haiti and
15 Central Africa. Attempts have been made to isolate the
infectious agent responsible for this disease; the leading
candidates include herpes (cytomegalovirus) and retro-
viruses. Because AIDS has occurred in hemophiliacs who
have been treated with plasma products, for example, Factor
20 VIII and Factor IX concentrates, the infectious etiologic
agent can be assumed to be present in these preparations.
Therefore, one characteristic of the agent responsible for
AIDS would be its resistance to procedures employed in the
concentration and lyophilization of Factor VIII, Factor IX
25 and other plasma products.
Several suspect candidates for the infectious etiologic
agent causing the Acquired Immune Deficiency Syndrome have
recently been proposed by Jay A. Levy et al, Science, 225,
30 840 (1984) (ARV), M. Popovic et al, Science, 224, 437
(1984) (HTLV-III), and F. Barré-Sinoussi et al, Science,
2200 868 (1983) and Lancet, pages 753 - 757 (April, 1984)
(LAV). These suspect agen~s are all retroviruses isolated
from AIDS patients. The information now available
35 concerning properties of retroviruses demonstrates much
diversity among those retroviruses which have been studied
in response to exposure to the conditions used in processes
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to fractionate plasma and to inactivate viruses and
bacteria.
Levy et al, Lancet, pages 722 - 723 (September, 1984)
discloses that, upon heating lyophilized Factor VIII
concentrate containing a mouse xenotropic type C retrovirus
at 68 C for 12 - 48 hours, the infectious agent was still
present. However, upon heating the same sample at 68 C
for 72 hours, the retrovirus was completely inactivated.
Although the mouse retrovirus present in the dry Factor
VIII concentrate was shown to be inacti~ated by heat
treatment at 68 C and 72 hours, the effect of heat
treatment in the dry state on the suspect agents for human
AIDS has not heretofore been demonstrated.
DESCRIPTION OF T~E INVENTION
20 This invention is based on the discovery that the suspect
infectious etiologic agents causing Acquired Immune
~eficiency Syndrome (AIDS) in humans can be inactivated by
heating a lyophilized composition containing human plasma
or plasma fractions or products thereof at temperatures and
2s time periods in the range of about 60 - 90 C for about 10
to 120 hours, preferably about 60 - 80 C for about 24 to
96 hours, more preferably about 65 - 75 C for about 96
hours, and most preferably about 68 C for about 72 hours.
i
30 Preferably, the composition comprises a human plasma
fraction, or product thereof, consisting essentially of at
least one therapeutically active plasma protein selected
from Factor VIII, Factor IX, fibronectin, antithrombin-III
and alpha-l proteinase inhibitor. More preferably, the
3s composition contains one of Factor VIII or Factor IX. Most
preferably, the composition contains Factor VIII. The
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composition used in the method according to the
invention can be produced by an of the well-known
-techniques.
Thus in particular a heat s-tabilizing agent is added
to the composition prior to the lyophilization. Suit-
able stabilizing agents include nonpolar anions with
molecular weights greater than ~0, sugars, reduced
sugars and amino acids.
Examples of suitable nonpolar anions include salts of
carboxylates, hydroxycarboxylates and amino acids
such as a sodium or po-tassium caprylate, capra-te,
oleate, laurate, valerate, acetylphenylalaninate,
acetyleucinate and acetyltryptophanate. Examples of
suitable sugars include glucose, sucrose and maltose
to name bu-t a few, and examples of suitable reduced
sugars include erythritol and mannitol. Examples of
suitable amino acids include lysine, glysine, pro-
line and glutamic acid to name but a few.
Generally it is appropriate to select the temperature
and time of heating within the defined range in
accordance with experimental da-ta inactivation of at
leas-t 10 par-ticles of AIDS-associated retrovirus.
~he following examples illustrate but a few embodi-
ments of the present invention and are not to be con-
strued as limiting in scope. All parts and percent-
ages are by weight and all temperatures are in
degrees Celsius unless otherwise indicated.
,,, ~
EXAMPLE 1
The lymphocytopathic retrovirus called "AIDS-related
Virus" (ARV), which was recently isolated as
reported in Science, 225, 840 (1984) was added to
samples of AHF concentrate produced from normal
human plasma. The resulting mixtures were freeze-
dried and then heated for 48 to 72 hours. The pre-
sence of ARV was measured after culturing and con-
centrates in peripheral mononuclear cells and deter-
mining activity of the enzyme, reverse transcriptase(RT). The results showed tha-t a high level of RT
was detected in duplicate samples taken at 0 time
(0.5 - 1.5 x 106 CPM) whereas samples taken after
heating at 48 hours and d72 hours showed no signifi-
cantRT activity. These results indicate that the
heat -trea-tmen-t process used inactivated the ARV.
EXAMPLE 2
In this experiment, the samples of AHF concentrate
as ln Example 1 were inoculated with a different
retrovirus, the LAV retrovirus as disclosed in
Barre-Sinoussi et al, Science, 220, 868 (1983) and
Lancet, pages 753 - 757 (April 1984), and the pre-
sence of the LAV retrovirus was detected as
described in the reference.
The AHF concentrates were lyophilized and heated at
68C for varying periods of time and inoculated into
microculture plates containing human lymphocytes
stimulated with Interleukin-2 and phytohemoglutinin.
At 3-day intervals, there was removed some inoculum,
culture medium and the removed portion was replaced
wi-th fresh cell culture grow-th medium. Af-ter 9 days,
the inoculum was transferred to a microculture plate
coated with antibody to the LAV. Then, a standard
ELISA test was performed to determine the presence
of infectious particles. The results showed that at
0 time, there was present 104-27 infections particles
whereas at each period of 24 hours, 48 hours, 60
hours, 72 hours and 96 hours, there was present
less than 2 particles. (The limit of detection was
2 particles).
