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.


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.


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.


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 


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; 

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.


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 

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 


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 
6. A method according to claim 2, wherein an aqueous solution of a 
   blood component is contacted with ozone.