OxyFile #172
United States Patent 4632980
Filing Period: Jan. 1, 1979 - Dec. 31, 1986
Application Number: 719187
Application Type: Invention (Utility) Patent
Application Filing Date: April 3, 1985
Title of Invention: Ozone decontamination of blood and blood products
Issue Date: December 30, 1986
Primary Examiner: Schain; Howard E.
Inventor: Zee; Yuan C. (Davis, CA).
Bolton; David C. (Staten Island, NY).
Assignee: Immunologics (San Francisco, CA);
Assignee type: U.S. Company or Corporation.
U.S. Patent Documents:
4183962 (January, 1980; Asher)
4540573 (September, 1985; Neurath et al.)
4581231 (April, 1986; Purcell et al.)
Other References:
Chem. Abstracts, vol. 102, 1985, 126730f, Ignatenko et al.
Abstract
Blood and proteinaceous blood products employed for their
physiological and/or immunological properties are free of viable
enveloped viruses by treatment with low levels of ozone, levels at
which substantially all of the physiological and/or immunological
activity is retained.
BACKGROUND OF THE INVENTION
1. Field of the Invention
Human blood finds a wide variety of applications and uses, being used
not only in transfusions, but also as a source for individual
proteinaceous components. For many diseases, the human host is
defective in producing an essential factor, such as one of the factors
involved in the clotting cascade, where whole blood is used as a
source for such protein. The increasing needs for blood has encouraged
the employment of both domestic and foreign sources. In the case of
whole blood, usually a pint is from a single individual, so that any
contamination, as bad as it may be, will be limited to a single
individual. By contrast, where components are isolated and used,
frequently the blood will be pooled from a large number of different
donors. Thus, the presence of contamination from a single donor can
compromise the use of the entire batch. There appears to be an
increasing awareness of the incidence of viral diseases associated
with blood. Of recent date is the concern with the lymphadenopathy
virus or human T-cell lymphotropic virus-III (LAV/HTLV-III). While
there is an increasing effort to screen blood for the presence of the
AIDS viral agent as well as other viruses, such as hepatitis virus,
there is still the possibility for a significant number of false
negatives which could result in the transfer of the infectious agent,
particularly to an immunocompromised host. There is, therefore,
substantial interest in finding ways to ensure that none of the blood
or blood components which are to be administered to a human host have
any viable infectious agent.
2. Brief Description of the Prior Art
European Patent Application No. 0 086 071 describes the use of ozone
to inactivate enveloped viruses for use as a vaccine.
SUMMARY OF THE INVENTION
Blood and proteinaceous blood components are freed of infectious
enveloped viral agents by treating the blood under mild conditions for
a short period of time, where any virus is inactivated, while
retaining the physiological properties of the blood or blood
components.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
In accordance with the subject invention, methods and compositions are
provided involving the freeing of blood or blood components of
infectious enveloped viruses, while retaining the physiological
properties of the blood or blood component, to provide compositions
which may be introduced into a mammalian host without transmission of
such viral infection.
In freeing or decontaminating the blood or blood-derived biological
composition, an aqueous medium will be employed, which aqueous medium
includes the blood without modification or a blood product which is
less than whole blood, e.g, serum, clotting factors, or the like. The
blood component will be treated in an aqueous medium. The aqueous
medium is contacted with an enveloped virus deactivating amount of
ozone for a short time under mild conditions and the aqueous medium
removed from the ozone treatment, and may be subject to further
treatment such as contact with a reducing agent. The resulting
composition is freed of viable enveloped virus and may be used for
administration to a mammalian host, normally a human host.
The biological compositions which are employed in this invention will
be aqueous protein compositions involving blood or blood components.
Whole blood, packed red cells, platelets, and plasma (fresh or fresh
frozen) are exemplary. Other blood components of interest include
plasma protein portion, anti-hemophilic factor (Factor VIII); Factor
IX, and Factor IX complex (Factors II, VII, IX and X); fibrinogens,
Factor XIII, prothrombin and thrombin (Factors II and IIa);
immunoglobulins (IgA, -D, -E, -G, and -M); hyper-immune globulins;
cryoprecipitate; albumin; interferons; lymphokines; transfer factor;
etc.
In other than blood, the protein compositions in the aqueous media
will generally range from about 1 .mu.g/ml to about 500 mg/ml, usually
from about 1-100 mg/ml. The pH will normally be close to the
physiologic pH 7.4, usually being in the range of about 6-9, more
usually about 7-8. Other components which may be present in the
medium, include salts, additives, buffers, stabilizers or the like.
These components will be conventional to the use of the particular
blood product.
The subject method is effective with a wide variety of enveloped
viruses, both RNA and DNA. Illustrative viruses include hepatitis
virus, HTLV-I, -II, and -III, influenza virus, etc.
In decontaminating the blood or blood product, the medium may be
contacted with the ozone under a variety of conditions. Generally, a
bubbling of the ozone through the blood will not be employed,
particularly where red blood cells are present, since this may lead to
hemolysis. Various techniques for contacting the medium with the ozone
may include pumping the medium through a chamber containing the ozone,
employing a thin film, either by using a falling film or by using
rotating vessels, e.g., rotating bottles, by passing the blood through
porous fibers, such as hollow fibers, where the chamber containing the
fibers contains the ozone, or by using tubing such as Gore-Tex.RTM., P
T F E tubing, where the medium is in contact with an ozone atmosphere.
The concentration of ozone in the atmosphere will generally be from
about 1-100 ppm, usually from about 1-20 ppm, remaining gases may be
inert gases, such as nitrogen, or may be air. The temperature may be
maintained from about 4.degree.-37.degree. C., more usually from about
4.degree.-30.degree. C., and preferably from about 4.degree. to room
temperature. The time will vary widely, depending upon the nature of
the suspected contamination, generally employing about 0.5 hr and not
more than about 4 days, more usually from about 0.5 hr to about 1 day.
After the medium containing the blood or blood component has been
treated, it may be further treated with reducing agents to ensure the
absence of any active oxygen. Small amounts of ascorbic acid,
glutathione, sodium thionite, or the like, namely reducing agents
which are physiologically acceptable, may be employed. The amounts
will generally range from about 20 .mu.g to about 2 mg/ml.
After the blood or blood components has been decontaminated, it may
then be isolated and used directly for its intended purpose.
The following examples are offered by way of illustration and not by
way of limitation.
EXPERIMENTAL
Ozone exposure system:
The system accommodates two vessels for ozone exposure. To prevent the
reaction of ozone with non-biological components of the system, all
the system components which come into contact with ozone are made with
the inert materials glass, Teflon.RTM. or stainless steel. Compressed
air (3.5 kg/cm.sup.2 ) is introduced into the system through a
pressure regulator set at 141 g/cm.sup.2 and is filtered through two
ultrafilters (Mine Safety Appliances Co., Pittsburgh, PA) in series.
Each ultrafilter is rated at 99.99% efficiency for particles of 0.3
.mu.m diameter and contains a supplemental charcoal element.
Ozone at approximately 2 ppm is introduced into the system and mixed
with the filtered air using two regulating valves to adjust the ozone
concentrations. The main flow of the gas is directed into a room
temperature incubator which houses the humidifying bubblers, the
roller apparatus and the sequential sampler. The flow rate of gas
through each exposure vessel is held constant by diverting around the
sequential sampler a flow rate of gas equal to that sampled. The
biological material to be exposed is in sterile 11 cm.times.29 cm
borosilicate glass roller bottles (New Brunswick Scientific, New
Brunswick, N.J.) equipped with specially machined roller caps with
Viton (TM) seals. The bottles are rotated at a rate of 1.5 rpm during
exposure to permit the ozone to react with the samples with only a
thin film of fluid over most of the bottle surface.
Ozone generation and monitoring:
Ozone is generated in medical grade oxygen by silent electric arc
discharge with a Sander Model IV Ozoniser (Erwin Sander
Elektroapparatebau G.m.b.H. and Co., Am Osterberg, W. Germany)
regulated by a constant voltage power supply.
Exposure of blood to ozone:
Whole blood is used containing 10.sup.9 pfu/ml of hepatitis virus. 250
ml aliquots of the contaminated blood to be exposed is placed in two
11 cm.times.29 cm borosilicate glass roller culture bottles equipped
with specially designed caps. The bottles are connected to the
exposure system and rotated at 1.5 rpm at 20.degree. C. while the
humidified gas (2 ppm ozone) is flowed through the bottles at a rate
of 6 L/min. The blood is exposed for 48 hr.
The ozone-treated blood is then tested for the presence of viable
virus by inoculating susceptible chimpanzees.
The subject invention provides for a rapid, reliable, economic
procedure for decontaminating blood and blood products, freeing the
products of enveloped viruses, so that the products may be used
without transmission of viral infection. Furthermore, the blood and
blood products retain their physiological character, with minimal
lysis of red blood cells and substantially complete maintenance of
physiological activity of the protein components. The method is easy
to perform, large amounts of blood can be treated, and the system is
free of production of products which may have deleterious biological
effects.
Although the foregoing invention has been described in some detail by
way of illustration and example for purposes of clarity of
understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims.
Claims
What is claimed is:
1. A method for freeing blood and blood components of viable enveloped
viruses while retaining the physiological characteristics of the
blood or blood component, said method comprising:
contacting said blood or blood product in an aqueous medium
with an enveloped virus inactivating amount of ozone under mild
conditions for a sufficient time to inactivate all enveloped
viruses present; and isolating the blood or blood component
free of viable enveloped viruses.
2. A method according to claim 1, wherein said contacting is at a
temperature in the range of 4.degree. to 37.degree. C. and at an
ozone concentration of 1 to 100 ppm.
3. A method according to claim 2, wherein said blood or blood
component is contacted as a thin film.
4. A method according to claim 2, wherein the contacting is for a
duration of 0.5 hr to 4 days.
5. A method according to claim 2, wherein blood is contacted with
ozone.
6. A method according to claim 2, wherein an aqueous solution of a
blood component is contacted with ozone.