U.S. patent application number 10/196020 was filed with the patent office on 2003-03-13 for method of inactivating microorganisms in a fluid using ultraviolet radiation.
Invention is credited to Galloway, Cynthia J., Henzler, Hans-Juergen, Kaiser, Klaus, Kauling, Joerg, Remington, Kathryn, Treckmann, Rolf.
Application Number | 20030049809 10/196020 |
Document ID | / |
Family ID | 24859497 |
Filed Date | 2003-03-13 |
United States Patent
Application |
20030049809 |
Kind Code |
A1 |
Kaiser, Klaus ; et
al. |
March 13, 2003 |
Method of inactivating microorganisms in a fluid using ultraviolet
radiation
Abstract
A method of inactivating microorganisms such as viruses within a
fluid such as a biological fluid is disclosed. The method includes
the steps of providing a UV reactor, which may take the form of an
elongated generally annular reaction chamber surrounding at least
one elongated UV lamp, moving the fluid within the reaction chamber
in a primary flow directed along the length of the UV lamp, and
inducing a circulating secondary flow within the fluid with the
secondary flow being superimposed on the primary flow. As the fluid
moves through the reaction chamber in the primary flow, it is
circulated repeatedly toward and away from the UV lamp in the
circulating secondary flow to provide uniform and controllable
exposure of the entire volume of fluid to ultraviolet radiation.
Microorganisms such as viruses are thus inactivated while desirable
components in the fluid, such as proteins, are preserved without
the use of a free radical scavenger.
Inventors: |
Kaiser, Klaus; (Koeln,
DE) ; Henzler, Hans-Juergen; (Solingen, DE) ;
Kauling, Joerg; (Koeln, DE) ; Treckmann, Rolf;
(Cary, NC) ; Remington, Kathryn; (Cary, NC)
; Galloway, Cynthia J.; (Garner, NC) |
Correspondence
Address: |
STEVEN D. KERR, ESQ.
WOMBLE CARLYLE SANDRIDGE & RICE
POST OFFICE BOX 7037
ATLANTA
GA
30357-0037
US
|
Family ID: |
24859497 |
Appl. No.: |
10/196020 |
Filed: |
July 16, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10196020 |
Jul 16, 2002 |
|
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09711780 |
Nov 13, 2000 |
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Current U.S.
Class: |
435/173.1 ;
422/24 |
Current CPC
Class: |
A61M 2205/053 20130101;
C02F 2201/3223 20130101; A61L 2/10 20130101; B01J 2219/00164
20130101; C02F 2201/32 20130101; A61M 1/3683 20140204; B01J
2219/00168 20130101; C02F 2201/328 20130101; A23L 3/28 20130101;
A61L 2/0005 20130101; A61M 1/3681 20130101; B01J 2219/00139
20130101; Y10S 422/905 20130101; A61K 41/17 20200101; B01J 19/123
20130101; B01J 19/2405 20130101; C02F 1/325 20130101; A61L 2/0011
20130101 |
Class at
Publication: |
435/173.1 ;
422/24 |
International
Class: |
C12N 013/00; A61L
002/12 |
Claims
What is Claimed is:
1. A method of irradiating a fluid with ultraviolet radiation from
a UV source, said method comprising the steps of: (a) moving the
fluid in a primary flow along the UV source; and (b) inducing
within the fluid a circulating secondary flow superimposed on the
primary flow, the circulating secondary flow moving the fluid
toward and away from the UV source.
2. The method of claim 1 and wherein the fluid is a biological
fluid, the irradiation inactivating microorganisms within the
biological fluid.
3. The method of claim 2 and wherein the microorganisms are
viruses.
4. The method of claim 1 and wherein the fluid is a foodstuffs
fluid.
5. The method of claim 1 and wherein the UV source is at least one
elongated UV lamp and wherein step (a) includes defining a reaction
chamber surrounding the UV lamp and moving the fluid in a primary
flow along the reaction chamber.
6. The method of claim 5 and wherein step (b) comprises moving an
agitator within the reaction chamber.
7. The method of claim 5 and wherein step (b) comprises disposing
irregularities within the reaction chamber.
8. The method of claim 7 and wherein the step of defining a
reaction chamber around the UV lamp includes surrounding the UV
lamp with a housing having a wall, the reaction chamber being
defined between the UV lamp and the wall of the housing, and
wherein the step of disposing irregularities within the reaction
chamber comprises forming irregularities in the wall of the
housing.
9. The method of claim 1 and wherein the UV source is an elongated
UV lamp and wherein step (a) includes disposing a generally spiral
tubular conduit around the UV lamp and moving the fluid in a
primary flow direction through the tubular conduit, the interaction
of the fluid with the conduit inducing the circulating secondary
flow of step (b).
10. A method for inactivating microorganisms in a fluid, said
method comprising the steps of: (a) providing a reactor for
radiating ultraviolet light into the fluid, the reactor having a
reaction chamber connected to at least one inlet and one outlet for
the fluid, and an ultraviolet radiation source; (b) moving the
fluid through the reaction chamber in a primary flow generally
along the ultraviolet radiation source; and (c) inducing a
circulating secondary flow superimposed on the primary flow, the
secondary flow being oriented transversely relative to the
ultraviolet radiation source.
11. The method of claim 10, wherein the reaction chamber is
disposed around the ultraviolet radiation source.
12. The method of claim 10, wherein the ultraviolet radiation
source is disposed around the reaction chamber.
13. The method of claim 10, wherein the irradiation space of the
reactor is a ring-shaped tubular space defined by a housing and an
inner tube and having a longitudinal axis therein.
14. The method of claim 10, wherein the reaction chamber is formed
by a tubular conduit and wherein the tubular conduit spirals around
the ultraviolet radiation source.
15. The method of claim 10, wherein the reaction chamber is defined
between an outer cylindrical wall and a UV transparent inner
cylindrical wall, the ultraviolet source being surrounded by the
inner cylindrical wall.
16. The method of claim 10, wherein the microorganism to be
inactivated is a virus.
17. The method of claim 10, wherein the ultraviolet radiation is
UVC radiation.
18. The method of claim 10, wherein the wavelength of the
ultraviolet radiation is about 254 nm.
19. The method of claim 16, wherein the fluid is exposed to a viral
inactivating less than about 30 Joules/cm.sup.2.
20. The method of claim 16, wherein the fluid is exposed to a viral
inactivating fluency between about 0.01 Joules/cm.sup.2 and about
10 Joules/cm.sup.2.
21. The method of claim 16, wherein the fluid is exposed to a viral
inactivating fluency between about 0.02 Joules/cm.sup.2 and about 5
Joules/cm.sup.2.
Description
FIELD OF THE INVENTION
[0001] The present invention relates generally to the sterilization
of fluids such as biological fluids to inactivate undesired
microorganisms such as viruses in the fluids. More specifically,
the invention relates to sterilization of fluids by means of
controlled ultraviolet irradiation.
BACKGROUND
[0002] Sterilization of fluids is an essential step in the
manufacture of many pharmaceutical products and foodstuffs. Its
goal is the reliable elimination of microorganisms, including
viruses, while preserving, as intact as possible, the desirable
components of the products. Sterilization may be required of
biological fluids, such as nutrient media for fermentation, various
blood products, and fluids bearing active pharmaceutical proteins.
In the food industry, sterilization of fluid such as milk products
is common.
[0003] In terms of food sterilization, the selection of a
particular sterilization technique frequently is governed by how
the procedure will affect the shelf life or the palatability of the
food. While the greatest concern in the food industry is bacterial
or fungal contamination, dairy products also may carry the
additional risk of viral or prion contamination. Elimination or
inactivation of such microorganisms is a prerequisite to commercial
distribution of these products.
[0004] In contrast to the food industry, the choice and use of a
sterilization technique in the pharmaceutical industry is subject
to the strict demands and regulations imposed upon all
pharmaceutical agents that are to be directly administered to an
animal or human. There is particular concern about contamination of
biological fluids such as pharmaceutical products by viruses, which
may be co-isolated from a natural source or introduced during a
biotechnological process. For the sterilization of pharmaceutical
products, a multi-step process historically has been employed to
inactivate, or remove, or reduce viral contaminants. Each step in
the process is based on different operational principles to ensure
a reduction in the viral load within a fluid preferably by at least
four orders of magnitude while preserving the viability of proteins
and other desirable components of the fluid.
[0005] Irradiation of biological and other fluids with ultraviolet
(UV) light has been employed as a method for inactivating
undesirable microorganisms. Irradiating plasma and blood products,
for example, with UV-light to inactivate viruses was known during
WW II. UV-treatment of blood derivatives is especially useful for
treating uncoated, heat-stable viruses. Thus, Chin et al.,
Photochem. & Photobiol. 65, 432-435 (1997) teaches that
irradiation of plasma products with UV-light leads to inactivation
of the hepatitis A virus and parvoviruses.
[0006] UV-irradiation may inactivate microorganisms and/or viruses
by generating mutagenic alteration of their genetic material. Above
a minimum dose of radiation, the microorganisms lose their
reproductive capacity. UV-irradiation damages nucleic acid by
creating intrastrand nicks and inducing nucleotide
photodimerization, both of which disrupt nucleic acid replication.
Through such mechanisms, UV-irradiation can be an effective means
of inactivating undesirable microorganisms within biological and
other fluids. Unfortunately, the energy of short wavelength UV
light also can damage sulfur-containing cysteine bridges and
methionine peptide bonds and induce aromatic amino acid side
reactions, thereby disrupting the structural and functional
integrity of the very proteins that often are the desired
end-products of the irradiated fluid. Thus, an inherent problem in
the application of UV-irradiation techniques is controlling the
irradiation of a fluid so as to ensure sufficient radiation
exposure to inactivate undesirable microorganisms within a fluid
while at the same time minimizing or eliminating UV-radiation
damage to desirable proteins and other components within the
fluid.
[0007] Traditionally, UV reactors have been used for the UV
sterilization of biological fluids. Generally, a UV reactor
includes a source of UV radiation such as, for example, one or more
elongated tubular bulbs or lamps. In one configuration, an annular
reaction chamber with a predetermined width is formed around and
encloses the lamp and fluid to be irradiated is pumped or otherwise
moved through the chamber, where it is exposed to UV light from the
lamp. In another configuration, a UV source or sources may surround
and radiate inwardly into a central tubular reaction chamber. In
either case, flow rate, light intensity, chamber width or diameter,
and reactor length are selected for a particular fluid to ensure,
as much as possible, the most effective UV radiation dosage for
deactivating undesirable microorganisms while conserving the
viability of the desirable components of the fluid.
[0008] A problem with the use of UV reactors for irradiating fluid
with ultraviolet light results from the finite width of the
reaction chamber and the laminar nature of the fluid flow along the
chamber. More specifically, as the fluid flows along the chamber,
the UV radiation intensity in the treated fluid decreases
relatively rapidly as a function of distance from the radiation
source. This is due to many factors including the natural
inverse-square law of radiation intensity as a function of distance
from a source and the absorption characteristics of the fluid and
the proteinaceous material supporting the infectious particles. In
any event, microorganisms and viruses within layers of the fluid
that flow along the outside of the reaction chamber farther from
the radiation source receive no or a reduced dosage of radiation.
These microorganisms are, therefore, inactivated slowly or not at
all. On the other hand, microorganisms in layers of fluid that flow
along the inside of the reaction chamber closest to the radiation
source receive increased dosages, and in many cases overdoses, of
radiation, which, in some cases, is high enough to cause
significant damage to desirable proteins and other components in
these layers of the fluid. The result is unpredictable and
inefficient sterilization and higher levels of damage to desirable
components.
[0009] Attempts to address these limitations have led to the
development of thin-layer or thin film UV reactors in which the
width of the reaction chamber and thus the thickness of the fluid
layer adjacent the UV source is maintained relatively thin to
reduce the detrimental effects of radiation intensity gradients in
the fluid (see e.g. Kallenbach et al., Cur. Stud. Hematol. Blood
Transfus. Basel 56, 70-82, (1989); Habel et al., J. Immunol. 56,
273-279(1947); Milzer et al., J. Immunol 50, 331-340 (1945).
Oppenheimer et al., Am.,J. Pub. Health. 49, 903-923, (1959)). The
goal is to ensure that all of the fluid is constrained to a region
of relatively smaller radiation intensity change as it moves along
the radiation source. Thus, the difference in intensity at various
layers within the fluid flow is theoretically controlled.
[0010] While thin-film reactors have been somewhat successful on a
smaller scale, they are problematic in that they can only be scaled
up to industrial production throughput with difficulty. This is
because keeping the film thickness small and constant can only be
realized by increasing the diameter of the reactor and thereby
increasing the cross-sectional area of the film to accommodate the
desired higher throughput. On an industrial scale, this necessary
condition leads to unmanageably large reactors. One attempt to
circumvent this problem is suggested in U.S. Pat. No. 5,133,932
which discloses a cylindrical thin-film UV-irradiation reactor in
which the area of the film exposed to the UV-light is increased by
corrugating the surfaces of the reaction chamber. However, the
realized increase in throughput with such a device is marginal at
best and still insufficient to accommodate large scale industrial
production.
[0011] A further limitation of and problem with traditional
UV-irradiation reactors is the unfavorable flow profile and dynamic
conditions of fluid films when in laminar flow along the radiation
source. More specifically, in a laminar flow there is no or very
little fluid exchange normal to the flow direction. Thus, as
mentioned above, fluid layers farther from the source receive a
smaller radiation dose than fluid layers close to the source.
Furthermore, the flow velocity profile within a confined laminar
flow is such that the flow velocity is relatively low adjacent to
the walls of the reaction chamber and is substantially higher
intermediate the walls. Thus, fluid closest to the wall of the
reaction chamber adjacent the light source flows more slowly and is
exposed to the UV radiation substantially longer than fluid between
the walls of the reaction chamber. Accordingly, to produce the
minimum radiation dose necessary for inactivation of microbial
contaminants in the most rapidly flowing fluid layers, the average
residence time of the fluid in the reactor must be increased. This
leads, however, to increased radiation dosage in the slower moving
boundary layers of the fluid flow and consequent increased
probability of undesired damage to desirable components in these
layers. Thus, destruction of desirable components in the boundary
layers due to overexposure is virtually inevitable.
[0012] One adverse result of overexposure in some layers of the
fluid is the generation of free radicals, which become entrained in
the flow and which have adverse effects on desirable components of
the fluid. Attempts to minimize damage caused by free-radical
generation as a result of overexposure typically include the use of
free-radical scavengers in the fluid. Earlier studies have
suggested that the use of free-radical scavengers can reduce
indirect damage to proteins (Chin et al., Photochem. Photobiol. 65,
432 (1997). Chapman et al. in U.S. Pat. No. 5,922,278 discloses a
UV-irradiation sterilization of biological fluids wherein free
radicals are scavenged by a scavenging agent. Clark et al. in U.S.
Pat. No. 5,786,598 discloses high intensity pulses of short
wavelength light to deactivate microorganisms. Morgalis-Nunno et
al., U.S. Pat. No. 6,087,141, discloses the use of light in the
wavelength range of 340-400 nm (UVA) rather than short wavelengths
of about 280 nm or less. Protection of the desired functionality of
the fluid is afforded by adding a free-radical scavenger in the
form of psoralen. Morowitz et al., U.S. Pat. No. 5,981,163 teaches
the addition of quenching protective agents during irradiation
deactivation of viruses. While such techniques attempt to deal with
the free-radicals generated in the fluid, none address the
problems, such as overexposure, that result in the formation of
such free-radicals in the first place.
[0013] The disruption of the laminar fluid flow through UV reactors
has been proposed as a solution to some of the forgoing problems.
For example, tangential-flow ring-slot reactors have been proposed
as a means to disrupt and induce mixing within the laminar flow
layers of a UV reactor. EP 803472 A1 discloses a reactor for UV
irradiation of a fluid having an annular or ring-slot reaction
chamber surrounding a UV radiation source. The fluid inlet into the
reaction chamber is orientated so that the fluid enters
tangentially into the chamber in hopes of generating fluid
cross-mixing. U.S. Pat. No. 5,433,738 discloses an irradiation
reactor for the irradiation of water that includes a helical guide
with circular cross section in hopes of generating fluid
cross-mixing.
[0014] The tangential inflow solution has proven problematic in
that the fluid flow through the reaction chamber rapidly reverts,
due to wall friction and other hydrodynamic factors, to a fully
axial and laminar profile directed along the longitudinal axis of
the chamber. The Dean vortices, which are theoretically postulated
at least for the area of tangential inflow, and which are intended
to promote cross-exchange of the reaction medium within the
reaction chamber, are surprisingly not present according to visual
studies and CFD-investigations (flow simulation). Tangential entry
ring-slot reactors, therefore, afford only a limited solution to
the problems discussed above.
[0015] A need therefore exists for a method of sterilizing a fluid
such as a biological fluid with UV radiation that ensures adequate
exposure to inactivate undesirable microorganisms, while
simultaneously minimizing or eliminating damage to desirable
components in the fluid.
[0016] A further need exists for an improved method of inactivating
microorganisms in a fluid reaction medium with UV radiation that
eliminates the need to use free radical scavenging or quenching
agents.
[0017] There is also a need for a method of sterilizing biological
fluids that is effective at deactivating undesirable microorganisms
while preserving the viability of desirable components without the
use of scavengers and that is scalable to commercially viable
production throughput.
[0018] It is to the provision of a method that addresses these and
additional needs that the present invention is primarily
directed.
SUMMARY OF THE INVENTION
[0019] Briefly described, the present invention is a method of
inactivating microorganisms such as viruses suspended in a fluid by
irradiating the fluid with UV light. The method can be applied to
the sterilization of biological products and foodstuffs, including,
but not limited to, blood components, fermentation media from
recombinant technology, milk and milk products, drinking water,
fruit juices and other beverages like soft drinks, chemical and
pharmaceutical products, virus vaccines, genetically produced drugs
and proteins, drugs and proteins from transgenic animals and
plants, and blood plasma and products from blood plasma. In a best
mode of carrying out the invention, UV exposure is achieved in a
generally tubular reactor wherein the fluid flows through a
reaction chamber that surrounds an elongated tubular UV light
source.
[0020] In general, the method comprises the steps of establishing a
primary flow of the fluid in a first direction along the radiating
surface of a UV light source and superimposing on the primary flow
a circulating secondary flow of the fluid. The secondary flow
circulates in a direction substantially transverse to the radiating
surface of the UV source such that the entire volume of the fluid
circulates repeatedly toward and away from the UV source as the
primary flow carries it along the length of the source. As a
result, all of the fluid receives a constant average dosage of UV
radiation and the problems previously associated with laminar flows
in UV reactors, namely overexposure near the radiating surface and
underexposure farther from the radiating surface, are
eliminated.
[0021] Further, and in direct contrast to thin-film reactors, the
reaction chamber in a reactor for carrying out the method of the
present invention may be much wider than an effective "kill zone"
immediately adjacent the radiating surface of the UV light source
wherein the intensity of the radiation is always above the
inactivation threshold. This is because, as the fluid circulates
toward and away from the source in the circulating secondary flow,
all of the fluid moves successively into and out of the kill zone
adjacent the surface of the source. The average residence time of
the fluid in the kill zone and thus the radiation dosage received
is a function, among other things, of the thickness of the kill
zone in the particular fluid being treated, the intensity of the UV
light source, and the characteristics of the primary and secondary
flows. Significantly, these parameters can be controlled as needed,
according to the invention, to establish and maintain an average
kill zone residence time for the entire volume of fluid that
corresponds to a predetermined required dosage of UV radiation.
[0022] Further, since the reaction chamber can be much wider than
in thin-film reactors, reasonably sized high volume reactors that
are scalable to commercial production throughputs are possible.
Finally, since the average radiation dosage received by all of the
fluid is constant, i.e. no portions or layers of the fluid are
overexposed and none are underexposed, the formation of
free-radicals common in prior art UV reactors is virtually
eliminated. Thus, the method of the invention can be used to
sterilize biological or other fluids without the need to use
free-radical scavengers.
[0023] The methodology of the invention, including the
establishment and maintenance of a circulating secondary flow
superimposed on a primary flow, can be realized through a variety
of reactor and reaction chamber configurations. Several such
configurations are discussed in some depth in the detailed
description set forth. It will be understood, however, that the
method of the invention might well be carried out by other reactor
designs and configurations, but that the essence of the methodology
of the invention is substantially the same. Regardless of the
design of the apparatus for establishing and maintaining the
conditions of the invention, the method has been demonstrated to
provide controllable and predictable inactivation with minimum
damage to desirable components, without the need for free-radical
scavengers, and with the potential for commercially viable
throughput. Additional objects, features, and advantages of the
invention will become more apparent upon review of the detailed
description set forth below when taken in conjunction with the
accompanying drawing figures, which are briefly described as
follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 is a simplified schematic representation of a typical
ring-slot UV reactor illustrating the characteristics of a laminar
fluid flow.
[0025] FIG. 2 a simplified cross-sectional view of a portion of a
UV reactor illustrating fundamental principles of the present
invention.
[0026] FIGS. 3 through 7 are sectional views illustrating one
embodiment of a UV reactor with rotating agitator usable for
carrying out the methodology of the present invention.
[0027] FIGS. 8 and 9 are sectional views illustrating an alternate
embodiment of a UV reactor usable for carrying out the methodology
of the present invention.
[0028] FIGS. 10 and 11 are sectional views illustrating another
alternate embodiment of a UV reactor usable for carrying out the
methodology of the present invention.
[0029] FIGS. 12 and 13 are sectional views illustrating still
another alternate embodiment of a UV reactor usable for carrying
out the methodology of the present invention.
[0030] FIGS. 14 and 15 are sectional views illustrating still
another alternate embodiment of a UV reactor usable for carrying
out the methodology of the present invention.
[0031] FIGS. 16 and 17 are sectional views illustrating yet another
alternate embodiment of a UV reactor usable for carrying out the
methodology of the present invention.
[0032] FIG. 18 presents two graphs showing .alpha..sub.1PI potency
and porcine parvovirus (PPV) reduction as a function of fluency at
various .alpha..sub.1PI concentrations and illustrates the
determination of critical parameters in accessing UV sterilization
methodologies.
[0033] FIG. 19 is a graph showing PPV reduction in a solution of 5
mg/ml of .alpha..sub.1PI proteinase inhibitor as a function of time
and illustrates the results of a UVC inactivation of IVIG
experiment applying the methodology of the present invention.
[0034] FIG. 20 is a graph of PPV reduction in a solution of 5 mg/ml
of .alpha..sub.1PI proteinase inhibitor and percent .alpha..sub.1PI
activity as a function of fluency and illustrates the results of
another UVC inactivation experiment applying the methodology of the
present invention.
[0035] FIG. 21 is a graph of PPV reduction in a solution of 5 mg/ml
of .alpha..sub.1PI proteinase inhibitor and percent .alpha..sub.1PI
activity as a function of fluency and illustrates the results of
yet another UVC inactivation experiment applying the methodology of
the present invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0036] Referring now in more detail to the drawings, wherein like
numerals refer where appropriate to like parts throughout the
several views, FIG. 1 illustrates general principles of fluid flow
through a traditional prior art tubular or ring-slot UV reactor and
the problems and shortcomings associated therewith. The reactor 11,
which is shown in simplified schematic form for clarity, includes
an ultraviolet radiation source in the form of a centrally disposed
elongated tubular UV lamp 12. The UV lamp 12 is surrounded by a
cylindrical housing 13 having an outer wall 14 and an inner wall
16, which bound and define an annular or ring shaped reaction
chamber 17 surrounding the lamp 12. The inner wall 16 of the
housing is transparent to ultraviolet radiation such that UV light
from the lamp 12 radiates into the reaction chamber 17. The
reaction chamber 17 has a predetermined width defined by the
distance between its outer and inner walls 14 and 16 respectively.
A fluid inlet port 18 communicates with the reaction chamber 17 at
one end, the bottom end in FIG. 1, and a fluid outlet port
communicates with the reaction chamber 17 at the opposite end, the
top end in FIG. 1.
[0037] Fluid to be sterilized is pumped or otherwise fed to the
fluid inlet port 18 and flows upwardly through the reaction chamber
17 and along the length of the UV lamp as indicated by arrows 21
before exiting the reaction chamber through fluid outlet port 19.
As the fluid moves through the reaction chamber, it is exposed to
UV radiation from the UV lamp 12, which acts to sterilize the fluid
by inactivating undesirable components in the fluid. In the case of
the inactivation of viruses within a biological fluids such as
blood products, for example, the UV radiation theoretically
inactivates or "kills" the virus particles within the fluid as the
fluid flows through the reaction chamber.
[0038] The enlarged inset in FIG. 1 depicts in more detail the
fluid flow pattern through the reaction chamber 17 and its
relationship to the UV radiation intensity profile in the chamber
and also illustrates a fundamental cause of problems with prior art
reactors and UV inactivation techniques. More specifically, the
fluid moves through the reaction chamber and along the length of
the UV lamp 12 in a substantially laminar flow, meaning that there
is little if any fluid movement in a direction transverse to the
lamp. In other words, fluid layers within the reaction chamber tend
to retain their relative distances from the UV lamp as the fluid
moves along the entire length of the chamber. Thus, fluid layers
near the outer wall 14 tend to stay near the outer wall and fluid
layers near the inner wall 16 tend to stay near the inner wall.
Furthermore, as is true of confined laminar flows in general, the
boundary layers of fluid near the inner and outer walls of the
chamber move more slowly than fluid layers intermediate the walls,
as illustrated by the velocity profile arrows 21 in FIG. 1. Thus,
the residence time in the reaction chamber of fluid in the boundary
layers is greater than the residence time of fluid in intermediate
layers of the flow.
[0039] Curve 22 represents the radiation or light intensity within
the reaction chamber 17 as a function of distance from the UV lamp
12. The initial intensity immediately adjacent the UV lamp is
relatively high and essentially is the inherent surface intensity
of the lamp itself. However, as discussed in some detail above, the
light intensity falls off rapidly as a function of distance from
the lamp due to a variety of factors including the natural inverse
square law of radiation intensity and the light absorption
characteristics of the fluid. At some threshold distance from the
lamp, indicated at 23 in FIG. 1, the light intensity is equal to a
"critical" intensity, below which UV radiation levels are
insufficient to inactivate viruses within the fluid. This critical
distance defines the outer boundary of a "kill zone" 24 within
which viral inactivation occurs and outside of which viruses within
the fluid are substantially unaffected by the UV radiation. It will
thus be seen that with a traditional laminar fluid flow through the
reaction chamber 17, layers of fluid within the kill zone are
sterilized while layers of fluid outside the kill zone pass through
the reactor without being sterilized. As a result, reduction of
viral load in such a reactor is subject to natural limits imposed
by the fact that only a portion of the fluid is affected by the UV
radiation.
[0040] In an attempt to address this problem, thin-film reactors
have been developed wherein the width of the reaction chamber
itself is equal to or less than the width of the kill zone. The
theory is that with such a reactor, all of the fluid necessarily
will reside in the kill zone as it moves through the reactor and
thus will be subjected to sufficient doses of radiation to affect
sterilization. However, as mentioned above, such thin-film reactors
cannot be scaled up to accommodate commercially viable fluid
throughputs with a reasonably sized reactor. Furthermore, even if
practical upscaling were possible, a problem still exists with
thin-film reactors because of the fundamental laminar character of
fluid flow and the nature of the flow velocity profile across the
width of the reaction chamber. More specifically, even in a
thin-film reactor, layers of fluid adjacent the UV source are
exposed to substantially higher doses of radiation than layers of
fluid at the outer boundary of the reaction chamber. Furthermore,
because of the flow velocity profile of a confined laminar flow,
layers of fluid adjacent the UV source also experience a longer
residence time within the reaction chamber than layers of fluid
intermediate the walls of the chamber. As a consequence of these
conditions, fluid layers adjacent the UV source tend to be
overexposed, which results in a relatively high instance of damage
to desirable components such as proteins. The overexposure
increases the likelihood of the presence of free radicals within
the fluid, which themselves can result in further destruction of
desirable components of the fluid. Although the use of free radical
scavengers is commonly taught as a solution to this later problem,
this represents only an after-the-fact patch rather than a solution
and decreases the efficiency of the sterilization process.
[0041] With the forgoing background in mind, FIG. 2 illustrates, in
simplified schematic form, the unique methodology of the present
invention for addressing and eliminating the problems that plague
prior art UV reactors, including thin-film reactors. The invention
is illustrated in FIG. 2 within the context of a simplified UV
reactor 31 having an axially extending UV lamp 32 adapted to
radiate ultraviolet radiation in a predetermined frequency band. In
the preferred embodiment, the lamp 32 radiates UVC radiation; that
is, radiation having a wavelength between about 180 and 320 nm, or
more preferably between about 225 and 290 nm, and most preferably
about 254 nm. UVC radiation is preferred because it tends to cause
less detrimental effects on desirable components such as proteins
within a fluid being treated while retaining sufficient energy to
inactivate viruses and other target microorganisms within the
fluid. However, other types of UV radiation such as, for example,
UVA and UVB are contemplated and are within the scope of the
invention.
[0042] The UV lamp 32 is disposed along the central axis of a
generally tubular housing 33 having an outer wall 34 and an inner
wall 36 that bound and define an elongated annular reaction chamber
37. Obviously, the inner wall 36 of the housing is transparent to
UV radiation so that UV light from the lamp 32 radiates into the
chamber 37. A fluid, such as a biological fluid, to be treated is
pumped or otherwise moved through the annular reaction chamber 37
by an appropriate pump (not shown) so that the fluid progresses (in
the embodiment of FIG. 1) from the bottom of the reaction chamber
toward the top of the reaction chamber, where it exits the chamber
through an outlet port (not shown). Generally speaking, as the
fluid moves through the reaction chamber 37 and along the length of
the UV lamp 32, it is irradiated with UV radiation from the UV
source to inactivate microorganisms such as viruses contained
within the fluid.
[0043] As discussed above, an inactivation or kill zone 38 is
defined along the inner wall 36 of the reaction chamber. The width
of the kill zone is determined by many factors including the
intensity of the lamp, the composition and optical characteristics
of the fluid, and others; but generally represents the zone within
which the intensity of UV radiation is above a threshold required
to affect inactivation of microorganisms within the fluid. Outside
the kill zone 38, the radiation intensity generally is to low to
affect inactivation and this is the phenomenon that in the past has
led to the development of thin-film reactors as discussed
above.
[0044] In the method of the present invention the fluid to be
treated is moved in a primary flow 39 along the length of the
reaction chamber 37 and thus along the surface of the UV lamp 32 as
expected. However, and unlike prior art methods, a circulating
secondary flow 41 is established within the fluid and is
superimposed on the primary flow 39. The circulating secondary flow
41 preferably is generally radially or transversely relative to the
surface of the UV lamp. Thus, as the fluid moves along the UV lamp
in the general direction of the primary flow 39, it also circulates
repeatedly from the outer wall 34 toward the inner wall 36 of the
reaction chamber and back again in the circulating secondary flow
41. As a consequence, the fluid moves repeatedly from a region in
the reaction chamber outside the kill zone 38, into and through the
kill zone 38 to the inner wall 36 of the reaction chamber, and
thence away from the inner wall, back through the kill zone, and
back into the region outside the kill zone.
[0045] Imagine for a moment a droplet or particle of fluid
entrained within the fluid flowing through the reaction chamber.
The droplet may contain undesirable microorganisms such as viruses
as well as desirable components such as proteins. As the droplet
moves generally along the length of the reaction chamber in the
direction of the primary flow 39, it also circulates repeatedly
with the superimposed secondary flow first across the border of the
kill zone where it receives the threshold radiation intensity, then
through the kill zone 38 where it receives progressively increasing
radiation intensity until it reaches the inner wall 37 of the
reaction chamber, where it receives the maximum radiation
intensity. From the inner wall, the imaginary droplet continues to
move with the secondary flow away from the inner wall 36 and back
through the kill zone 38, receiving progressively less radiation
intensity, until it moves out of the kill zone and into the
inactive region of the reaction chamber outside the kill zone.
[0046] From the forgoing, it will be appreciated by skilled
artisans that, in each cycle through the kill zone, the imaginary
droplet of fluid experiences an average intensity or dosage of UV
radiation that is greater than the threshold intensity at the
boundary of the kill zone 38 and less than the maximum intensity at
the inner wall 36 of the kill zone. The total radiation "seen" by
the droplet during its residence in the reaction chamber is
therefore approximately equal to the average radiation experienced
in each cycle times the number of repetitive cycles within the
circulating secondary flow 41. The beneficial result is that each
droplet of the fluid, or, in other words, the entire volume of
fluid, experiences a constant average dosage of UV radiation as it
moves through the reaction chamber. Further, the dosage itself can
be controlled relatively easily by controlling the intensity of the
UV lamp 32, which effects the width of the kill zone, and the
characteristics of the primary flow 39 and the superimposed
circulating secondary flow 41. Therefore, not only is the entire
fluid exposed to a constant average dosage of radiation, but the
dosage is controllable and may be adjusted to achieve optimum
inactivation of undesirable microorganisms while preserving as
intact as possible the desirable components within the fluid.
[0047] The methodology of the invention as illustrated in FIG. 2
contrasts starkly with the processes within prior art laminar flow
UV reactors where, as mentioned above, fluid layers adjacent the
inner wall of the reaction chamber tend to be over-irradiated
resulting in unwanted damage to desirable components and the
creation of free radicals, while layers farthest from the inner
wall tend to be under-irradiated resulting in low microorganism
inactivation rates. Thus, it has been found that, with the method
of the present invention, high inactivation rates, on the order of
four orders of magnitude or more in viral inactivation of
biological fluids, can be obtained and consistently maintained.
Further, this level of inactivation is achieved without the need to
introduce free radical scavengers into the fluid. This is because
fewer free radicals are created when practicing the method of the
invention since no portion of the fluid is over-irradiated as is
the case in prior art UV reactors. Finally, and significantly,
since the circulating secondary flow of the present methodology
repeatedly moves into and out of the kill zone regardless of the
total width of the reaction chamber, the constraints that
previously gave rise to the development of thin-film reactors
simply are not present. Thus, the reaction chamber in a reactor for
carrying out the invention may be significantly wider than the
thickness of the kill zone itself, making such a reactor easily
scalable to commercial production throughput while maintaining a
reactor of reasonable size. It will thus be seen that the present
invention offers many significant advantages over prior art UV
inactivation methods and devices.
[0048] The methodology of the present invention will now be
described within the context of several exemplary reactor
configurations usable for carrying out the invention as it has
generally been described above. It will be appreciated, however,
that the invention is not limited to or constrained by the
illustrated reactor configurations, but that such are offered to
facilitate a better understanding of the invention and to provide
an enabling disclosure for its practice. In this regard, the
disclosure of German patent application serial no. ______ is hereby
incorporated by reference as if fully set forth herein.
[0049] FIGS. 3 through 5 illustrate a rotating agitator reactor
usable for carrying out the method of the invention. The reactor
includes an axially disposed elongated UV lamp 46 disposed within a
glass mantle or inner housing 47. A tubular housing 48 surrounds
the glass mantle 47 and a reaction chamber 49 through which fluid
may flow is defined between the inner wall of the tubular housing
and the glass mantle. The housing is capped and sealed at its top
end by a head cover 64 and associated O-rings 62 and at its bottom
end with a base cover 52 and associated O-rings 62. An inlet port
59 communicates with the bottom portion of the reaction chamber 49
for introduction of fluid into the reaction chamber and an outlet
port 61 communicates with the top portion of the reaction chamber
for egress of fluid therefrom.
[0050] A rotatable anchor agitator 51 is disposed within the
reaction chamber surrounding the glass mantle 47 and is formed with
from about 4 to about 10, and preferably about 8, vanes that
surround the glass mantle 47. The anchor agitator 51 is rotatably
journaled at its top end in a sleeve bearing 65 and is rotatable
supported and centered at its bottom end on an agitator shaft 54
that terminates in a tapered centering tip 53. The centering tip 53
sits and rides in an appropriately shaped depression in the bottom
of the base cover 52 so that the anchor agitator is rotatable about
the glass mantle 47 in such a way that its vanes repeatedly circle
the glass mantle within the reaction chamber 49.
[0051] A diametrically extending magnetic coupler arm 57 is
attached to the agitator shaft and is adapted to couple
magnetically with the magnetic coupler of a magnetic drive 58. It
will be appreciated that activation of the magnetic drive 58 causes
the anchor agitator 51 to rotate within the reaction chamber 49. A
centering pin 56 depends from the bottom of the glass mantle 47 and
is disposed in a corresponding seat in the bottom 55 of the anchor
agitator 51 to keep the mantle centered with respect to the anchor
agitator and to maintain the relatively small clearance between the
vanes of the agitator and the surface of the glass mantle.
Preferably, but not necessarily, an array of inwardly projecting
flow breakers 63 are disposed around the inner wall of the housing
48.
[0052] FIG. 4 illustrates use of the reactor 44 to carry out the
methodology of the present invention. Fluid to be irradiated is
pumped through the inlet port 59 and exits out the outlet port 61
establishing a primary flow 66 along the length of the UV lamp 46.
Thus, as the fluid flows upwardly along the length of the reaction
chamber 49, it is exposed to UV radiation through the glass mantle
47. At the same time, the anchor agitator 51 is rotated to move its
vanes around the glass mantle 47. The movement of the agitator
establishes a circulating secondary flow 67 of fluid that has a
major component oriented in a direction transverse to the UV lamp
47. The flow breakers 63 have been shown to weaken the tendency of
the secondary flow to establish tangential components in favor of a
more transverse or radial flow direction. Thus, the fluid moves
repeatedly toward and away from the UV source in the circulating
secondary flows 67 as it progresses along the length of the
reaction chamber with the primary flow to realize the benefits of
the invention as discussed above. Agitator rotation rate, lamp
intensity, and flow rate are all adjustable to obtain optimum
irradiation for a given fluid being treated in the reactor.
[0053] FIGS. 6 and 7 illustrate an alternative drive mechanism for
the anchor agitator of FIGS. 3 through 5. The sealless drive
mechanism 71 includes a drive housing 70 defining an internal
cylindrical impeller chamber 75 and an outer annular channel 78. An
array of tangentially oriented slots 77 communicate between the
outer channel 78 and the impeller chamber 75. An inlet port 73
communicates with the outer channel 78 and is oriented to direct
fluid tangentially into the outer channel as shown. With this
configuration, fluid moves around the outer channel and enters the
impeller chamber in a generally tangential direction as indicated
by the arrows in FIG. 7.
[0054] The stirrer shaft 54 of the anchor agitator 51 rests on its
tapered end in a corresponding depression in the bottom of the
drive housing 70 such that the anchor agitator is rotatable within
the reactor as described above. An array of arcuate vanes 72
project outwardly from the stirrer shaft 54 into the impeller
chamber 75 and together form an impeller.
[0055] As fluid to be treated moves tangentially into the outer
channel 78 and tangentially into the impeller chamber 75 through
slots 77, the fluid impinges the vanes 72, which imparts rotary
motion to the shaft 54, thus causing the anchor agitator 51 to
rotate. Since the motion of the fluid itself causes the rotation of
the anchor agitator, no ancillary drive mechanism, such as the
magnetic drive of FIG. 3, is required. As the fluid moves out of
the impeller chamber and into and through the reaction chamber of
the reactor, the rotating anchor agitator causes circulating
secondary flows superimposed on the primary flow as described above
relative to FIGS. 3 and 4.
[0056] FIGS. 8 and 9 illustrate an alternate embodiment of a UV
reactor usable to carry out the methodology of the present
invention. An elongated UV lamp 81 is surrounded by a UV
transparent (preferably quartz) spiral wound flow tube 82 defining
a plurality of individual windings 86. The spiral wound tube 82
terminates at its bottom end in an inlet port 83 that communicates
with the bottom end of the tube 82 and at its top end in an outlet
port 84 that communicates with the top end of the tube 82. As
indicated by the arrows in FIG. 8, fluid to be treated is pumped
into the inlet port 83 and thence moves through the spiral wound
tube 82 around and around the UV lamp 81, where it is exposed to UV
radiation from the lamp.
[0057] As best illustrated in FIG. 9, the windings 86 of the tube
82 are formed with a generally D-shaped cross section having a
generally rectilinear or flat surface adjacent the UV lamp and a
curved outer surface. As the fluid flows through the tube in the
general direction of a primary flow 87, the combination of surface
tension, wall friction, and the greater distance that the fluid
must transverse around the outer portion of the tube results in the
formation of circulating secondary flows 88, also known as Dean
vortices, within the tube. The circulating secondary flows 88
generally are oriented transversely with respect to and are
superimposed on the primary flow, and thus are oriented generally
transversely with respect to the UV lamp 81.
[0058] Thus, as the fluid moves along the surface of the UV lamp in
the primary flow direction, the circulating secondary flows carry
the fluid toward and away from the UV source according to the
methodology of the invention with the many benefits described
above. Obviously, an advantage to the reactor configuration of
FIGS. 8 and 9 is that it contains no moving parts or drive
mechanisms. The characteristics of the primary and secondary flows
87 and 88 respectively, and thus the UV radiation dosage
experienced by the fluid, may be controlled by controlling, where
feasible, the viscosity of the fluid, the dimensions of the spiral
wound tube 82, and the flow rate of the fluid through the tube.
[0059] FIGS. 10 and 11 illustrate a UV reactor configuration
similar to that of FIGS. 8 and 9, but with the spiral wound flow
tube of the reactor having a generally rectangular rather than a
D-shaped cross section. The elongated UV lamp 91 is disposed in and
surrounded by a spiral wound quartz tube 92 defining a plurality of
individual windings 93. An inlet port 94 communicates with the flow
tube 92 at its bottom end and an outlet port 96 communicates with
the flow tube 92 at its top end. Fluid to be treated is pumped into
the inlet port and moves through the spiral wound tube 92 and thus
in a spiral pattern along the surface of the UV lamp in the
direction of a primary flow 97 (FIG. 11), and is exposed to UV
radiation.
[0060] As with the embodiment of FIGS. 8 and 9, the surface
tension, friction, and path length gradients within the tube 92
combine to create Dean vortices that manifest themselves as
circulating secondary flows 98 superimposed on the primary flow 97.
The circulating secondary flows 98 are oriented substantially
transversely relative to the UV lamp and thus carry the fluid
toward and away from the lamp according to the methodology of the
invention and with the aforementioned benefits thereof. Again,
radiation dosage is controllable by controlling fluid
characteristics, lamp intensity, and flow rate through the
reactor.
[0061] FIGS. 12 and 13 illustrate still another UV reactor
configuration usable to carry out the methodology of the present
invention. The reactor 100 includes an elongated UV lamp 101
disposed within a tubular quartz (or other UV transparent material)
inner tube 102. An outer housing 103 surrounds the quartz tube 102
and, in conjunction therewith, defines a reaction chamber 102
extending along the length of the UV lamp 101. The housing 103 is
capped at its top end by a head cap 106 and at its bottom end with
a base cap 108, each of which is sealed to the housing 103 and
quartz tube 102 with appropriate O-ring seals 107.
[0062] The inner surface of the housing 103 is machined to define a
generally helical channel 109 that spirals continuously around the
quartz tube 102 from the bottom of the reactor to the top. The
helical channel approaches but does not engage the quartz tube 102
and thus defines a series of relatively narrow passages 111 between
each turn of the helical channel and the quartz tube 102. An inlet
port 112 communicates with the reaction chamber 104 at the bottom
of the reactor and an outlet port 113 communicates with the
reaction chamber 104 at the top of the reactor.
[0063] In use to carry out the methodology of the present
invention, fluid to be treated is pumped into the reactor through
the inlet port and flows generally around the helical channel and
along the surface of the UV lamp in a primary flow 114. This motion
of the primary flow generates circulating secondary flows 116 in
the form of Dean vortices as a result of fluid dynamical
interactions within the D-shaped channel. The circulating secondary
flows 116 are superimposed on the primary flow 114 and carry the
fluid toward and away from UV source according to the methodology
of the present invention.
[0064] At the same time, the spaces 111 permit a small volume of
the fluid to flow longitudinally along the length of the reactor in
a free jet flow 116 (FIG. 13). The fluid in the free jet flow 116
is directed almost perpendicularly onto the spiraling primary flow
114. The interaction between the two flows causes an enhancement of
the circulating motion of the secondary flows 116 as a result of
the fluid dynamical forces generated by the interacting flows.
This, in turn, leads to an improved and more even irradiation of
the fluid as it moves through the reactor. UV irradiation dosage
can be adjusted and controlled by controlling the dimensions of the
helical channel, the size of the spaces 111, the viscosity of the
fluid, the intensity of the lamp 101 and the fluid flow rates
through the reactor.
[0065] FIGS. 14 and 15 illustrate yet another embodiment of a UV
reactor usable to carry out the methodology of the present
invention. The reactor 119 is similar in some respects to the
reactor of FIGS. 12 and 13 and includes an elongated UV lamp 121
surrounded by a quartz tube 122. An outer housing 123 surrounds the
quartz tube 122 and in conjunction therewith defines a reaction
chamber 124 that extends along the length of the UV lamp 122. The
housing is capped at its top end by a head cap 126 and its bottom
end by a base cap 127, each of which is sealed to the housing and
the quartz tube with appropriate O-rings 128. An inlet port 129
communicates with the reaction chamber at the bottom thereof and an
outlet port communicates with the reaction chamber at its top
end.
[0066] The inner wall of the housing 123 is machined or otherwise
formed with a series of generally annular channels 132 separated by
inward protrusions 135. The inward protrusions 135 approach but do
not touch the quartz tube, thus defining relatively narrow passages
134 between the channels 132. An array of generally ring-shaped
baffles 133 project outwardly from the quartz tube 122 with each
baffle being disposed within a corresponding one of the annular
channels 132.
[0067] In use to carry out the methodology of the present
invention, fluid to be treated is pumped into the inlet port 129
and moves along the reactor 119 to be extracted at the outlet port
131. As best illustrated in FIG. 15, the fluid moves generally in a
primary flow 136 along the length of the UV lamp and through the
spaces 134, which confine the flow to a region close to the UV
source. However, when the primary flow encounters a baffle 133, it
is diverted toward the outside of the reaction chamber to a
location farther from the UV source. On the other side of the
baffle 133, the primary flow is again diverted back toward the UV
source, and then flows through the next space 134 to the next
succeeding channel and baffle combination.
[0068] Thus, it will be seen that the primary flow 136 itself moves
repeatedly toward and away from the UV source to obtain benefits of
the present invention. In addition, the movement and displacement
of the primary flow 136 within each chamber creates circulating
secondary flows 137 that are oriented generally transversely
relative the UV lamp and thus carry the fluid toward and away from
the UV source according to principles of the invention. The
circulating secondary flows therefore enhance the cross mixing that
characterizes the present invention and results in the benefits
thereof.
[0069] FIGS. 16 and 17 illustrate still another embodiment of a UV
reactor within which the methodology of the present invention may
be carried out. The reactor 140 is similar in many respects to the
reactor 119 of FIGS. 14 and 15 and includes an elongated UV lamp
141 disposed within a quartz tube 142. A housing 143 surrounds the
quartz tube 142 and in conjunction therewith defines a reaction
chamber 148. The housing is capped at its top end by a head cap 144
and at its bottom end by a base cap 146, each of which is sealed to
the housing and the quartz tube by appropriate O-rings 147. A fluid
inlet port 153 communicates with the bottom of the reaction chamber
148 and an outlet port 154 communicates with the top of the
reaction chamber for ingress and egress respectively of fluid to be
treated.
[0070] The inner wall of the housing 143 is machined or otherwise
formed with an array of generally annular chambers 149 separated by
respective partitions 151. The partitions extend toward but do not
engage the quartz tube 142 to define relatively narrow passages 152
between the partitions and the quartz tube. In use, fluid to be
treated is pumped through the inlet port 153 and moves upwardly
along the length of the UV lamp to be extracted through the outlet
port 154. As illustrated in FIG. 17, the fluid moves in a primary
flow 156 through the passageways 152 and along the length of the UV
lamp 142. The motion of the fluid in the primary flow past
successive ones of the annular channels 149 creates vortices that
result in circulating secondary flows 157 superimposed on the
primary flow within each of the annular chambers. The circulating
secondary flows are oriented substantially transversely relative to
the UV lamp so that the fluid moves with the secondary flows
repeatedly toward and away from the UV lamp according to the
methodology of the present invention. The result, again, is even
and constant irradiation of the entire volume of fluid with all the
attendant benefits thereof as discussed in detail above.
[0071] The invention will now be described and further
characterized within the context of various examples that represent
experiments and clinical trials conducted by the inventors. It will
be appreciated that the techniques of and the data presented in
conjunction with the examples are not intended to be limiting, but
are presented for a better understanding and more complete and
enabling disclosure of the methodology of the invention. Many
modifications might well be made to the examples presented herein
and other experiments not discussed below might be carried out, all
within the scope of the present invention.
EXAMPLE 1
[0072] Critical Parameters in a Process to Inactivate Virus
Particles by UV Radiation.
[0073] The goal of viral inactivation by UVC irradiation is to
inactivate high levels of virus without damaging the protein or
functionality of interest. Two parameters were found to be critical
to achieving this goal; namely protein concentration in the fluid,
and UV fluency. Fluency is dependent on the physical configuration
of the UV irradiator, since internal flow patterns significantly
affect the amount of UV light that is received by any given protein
molecule or virus panicle in suspension.
[0074] Since proteins absorb in the UV range, high protein
concentrations can serve to protect the bulk of the target protein
from UVC damage. The high protein concentration, however, will also
protect the virus. It is necessary therefore to independently
evaluate both protein integrity and viral inactivation at varying
protein concentrations, and then to select a concentration of
protein for the inactivation process that will maximize protection
of the integrity of the target protein as well as viral
reduction.
[0075] Thus, the UVC induced potency loss was determined as a
function of protein concentration, as shown in FIG. 18, chart A.
The UVC-induced potency loss was least at concentrations of 12.5
mg/ml .alpha..sub.1 proteinase inhibitor, but increased at protein
concentrations of 7.0, 5.0 and 4.0 mg/ml. The greatest effect on
potency was seen at the lowest protein concentration, 2.5 mg/ml. In
contrast, as shown in FIG. 9B, the smallest reduction in virus
infectivity was observed at the highest .alpha..sub.1 proteinase
inhibitor concentration of 12.5 mg/ml, and the highest level of
inactivation was observed at the lowest concentration, namely 2.5
mg/ml. Based on these data, 5 mg/ml of .alpha..sub.1 proteinase
inhibitor was used for UVC inactivation as a compromise between
acceptable protein potency and good viral inactivation.
[0076] Model Virus Studies
[0077] Virus Stocks. Porcine Parvovirus (PPV), strain Tennessee, a
non-enveloped, single-stranded DNA virus was used in these studies
as a model for human parvovirus B19. This virus has been shown to
be resistant to inactivation by several methods, including
pasteurization and dry heat.
[0078] Virus stocks were prepared by infection of porcine testicle
(PT) cells. Virus was propagated by infecting subconfluent
monolayers of PT cells at a low multiplicity of infection, adding
propagation medium and then incubating the cells at 37.degree. C.
in 5% C0.sub.2 until advanced cytopathology was observed. Virus
propagation media consisted of minimum essential medium, Earle's
salts supplemented with 7.5% fetal bovine serum and NHG. NHG was
added to prevent contamination and provide for the additional media
requirements of this cell line and consisted of 0.1 mM nonessential
amino acids, 10 mM HEPES (N-[2-Hydroxyethyl]piperazine-N'-[2-
-ethanesulfonic acid], 0.05 mg/ml gentamicin and fungizone (2.5
mg/ml Amphotericin B). Infected cells were disrupted by
freeze-thawing and the cell lysates were stored at about
-70.degree. C. until used. The virus spike for each experiment was
prepared by thawing the virus-infected cell lysate, centrifuging at
low speed (4000.times.g) to remove the cell debris and collecting
the clarified supernatants.
[0079] Virus Assay.
[0080] Viral inactivation by UVC was determined by endpoint
dilution in 96-well microtiter plates seeded with PT cells and
using MEM containing 7.5% FBS and NHG. Virus was diluted using
serial half log dilutions of the test sample or positive control in
Hank's Balanced Salt Solution (HBSS). Positive controls consisted
of the same lot of virus that was used as the virus spike. Unspiked
HBSS was used as a negative control. Each dilution was used to
inoculate 8 wells of a 96-well microtiter plate. After 7 days
incubation at 37.degree. C. in 5% CO.sub.2, cytopathology was
scored. Results were converted into a titer (log median tissue
culture infective dose per ml; TCID.sub.50/ml) by the method of
Spearman and Karber (Cavalli-Sfprza, L. Biometrie Grundzuge
biologisch-medizinischer Statistik [Biometry, the basics of
biological and medical statistics], Gustav Fischer Verlag
Stuttgart, 1974, p. 171-173.)
[0081] A variety of viral species were tested for their relative
inactivation susceptibilities.
1TABLE 1 Inactivation of virus with varying genome sizes and types
of nucleic acid. D.sub.4 is defined as the UV dose required to
reduce or inactivate the virus by 4 log magnitudes. genome genome
D.sub.4 virus size type envelope (Joules/cm.sup.2) PPV 5 kb DNA no
0.19 SV-40 5 kbp DNA no 0.14 polio 7.7 kb RNA no 1.125 HAV 7.5 kb
RNA no 2.25 FIV 10 kb RNA yes Sindbis 11.3 kb RNA yes 1.125 BVDV 12
kb RNA yes 2.25 Reo 23.5 kbp RNA no 2.25 Adeno 36 kbp DNA no 9 PRV
150 kbp RNA yes 9
[0082] As shown in Table 1, the processes of the present invention
inactivate PPV at a smaller fluency than other viruses, but all
were inactivated by at least four orders of magnitude when exposed
to fluencies within the range 0.014-9.0 Joules/cm.sup.2. Also, the
smaller the viral genome, typically the smaller the effective
fluency value.
EXAMPLE 2
[0083] Protein Integrity.
[0084] Following UVC exposure the retention of immunoglobulin
integrity was assessed by evaluating the extent of aggregation and
fragmentation of the molecule. This was done by size-exclusion HPLC
using a TSK-G3000 (Toso-Haas) column and 0.91 M Na.sub.2HPO.sub.4,
pH 5.2-0.2 M NaCl buffer. Immunoglobulin integrity was expressed as
the area percent monomeric protein.
[0085] For .alpha..sub.1PI, protein integrity was assessed by
determining the ability of the enzyme to inhibit porcine elastase.
Protein integrity was expressed as the percent of the activity
before UVC exposure.
[0086] Inactivation of PPV in IGIV.
[0087] Pre-formulation IGIV was diluted to 0.8% with water,
adjusted to pH 4.2 and spiked to 10% with PPV. To evaluate the
effect of UVC exposure on protein integrity, unspiked IGIV
solutions were used. Solutions of IGIV were pumped through a
tubular UV reactor with a peristaltic pump, calibrated to deliver
100 ml/min. The protein solution was pumped through the device and
re-circulated through a stirred reservoir containing the sample.
The protein solution was re-circulated though the entire assembly
for 5, 10, 15, 30 and 60 minutes, corresponding to fluencies of
2.8, 5.6, 8.4, 16.9 and 33.8 Joules/cm.sup.2, respectively. In this
case fluency was defined as the mean residence time (reactor volume
divided by volume flow rate) multiplied by the UV light intensity
at the surface of the reaction chamber nearest the UV source (which
may be the surface of a quartz sleeve surrounding the UV lamp). For
these calculations, ideal plug flow was assumed. As shown in FIG.
19, after 5 minutes of re-circulation, four logs of PPV reduction
was observed, and by 30 minutes, over seven logs of inactivation
was seen. After 60 minutes of UVC exposure, 95% monomeric IgG
remained.
EXAMPLE 3
[0088] Inactivation of PPV in Alpha.sub.1 Proteinase Inhibitor.
[0089] Alpha.sub.1 proteinase inhibitor (.alpha..sub.1PI) was
diluted to 5 mg/ml in 20 mM Na phosphate, pH 7.0 and 100 mM NaCl
and exposed to UVC in the same device as used in example 1. During
this experiment, however, the solution was pumped through the
device in a single pass at flow rates between 25 and 1200
ml/minutes, resulting in fluencies ranging from 0.19-18
Joules/cm.sup.2.
[0090] To evaluate virus reduction, the protein solution was spiked
to 10% with PPV and to evaluate protein integrity, unspiked
solutions were exposed to UVC. From FIG. 20 it can be seen that at
fluencies above 0.6 Joules/cm.sup.2 at least 4 logs of PPV was
inactivated. As is also shown in Table 2 at higher fluencies PPV
was reduced to a level below that of detection; variation in log
reduction was observed due to variation in starting titers of the
spiking virus. At least 95% of .alpha..sub.1PI activity remained
after exposure to fluencies less or equal to 2.3
Joules/cm.sup.2.
2TABLE 2 Fluency (J/cm.sup.2) Log.sub.10 PPV Reduction % Initial
.alpha..sub.1PI activity 18 4.2 76.2 n = 1 n = 1 9 4.8 .+-. 0.9
87.6 .+-. 2.1 n = 4 n = 4 4.5 5.3 .+-. 0.4 91.9 .+-. 4.5 n = 5 n =
7 2.3 5.4 .+-. 0.1 96.5 .+-. 1.2 n = 4 n = 2 1.5 5.4 .+-. 0.1 96.7
.+-. 3.4 n = 3 n = 3 1.1 5.2 .+-. 0.1 100 n = 3 n = 1 1.0 4.7 .+-.
0.4 100.0 .+-. 0.0 n = 2 n = 2 0.8 4.6 .+-. 0.4 98.9 .+-. 1.1 n = 2
n = 2 0.6 3.6 ND n = 1 0.5 2.9 .+-. 0.4 ND n = 2 0.38 2.6 ND n = 1
0.3 2.6 ND n = 1 0.49 2.1 ND n = 1
EXAMPLE 4
[0091] Inactivation of PPV in Alpha.sub.1 Proteinase Inhibitor.
[0092] Solutions of .alpha..sub.1PI that had been diluted to 5
mg/ml in 20 mM Na phosphate, pH 7.0-100 mM NaCl were exposed to UVC
in a second type of tubular reactor, wherein the inlet and outlet
ports are off-set. This produces a flow pattern that is primarily
tangential, but which also contains a radial component to the
annular flow in the reactor ("tangential flow reactor"). For
evaluation of virus reduction, the protein solutions were spiked to
10% with PPV. The data shown in FIG. 21 indicate that in this
reactor four logs of PPV inactivation can be inactivated at lower
fluencies than in the tubular reactor used in Examples 10 and 11.
At least 95% of the initial .alpha..sub.1PI activity was observed
at fluencies that were less than or equal to 2 Joules/cm.sup.2.
Since the same UV lamp and the same light intensity was used in all
of the experiments, this demonstrates that improved hydrodynamic
conditions (mixing), i.e. inducing a circulating secondary flow
within the primary flow, reduce the total residence time of protein
solution in the reactor that is necessary to gain adequate virus
inactivation.
[0093] Graph A shows the result of studies evaluating the
inactivation of porcine parvo virus (PPV) in a solution of 5 mg/ml
alpha.sub.1 proteinase inhibitor in three different reactor
configurations. It can be seen that a threshold of 4-log virus
reduction can be achieved at an approximate fluency of 0.7
J/cm.sup.2 in a simple tubular reactor, similar the prior art
reactor shown in FIG. 1. Improved hydrodynamic conditions,
especially an increase in radial flow components in a reactor with
tangential flow characteristics and a reactor with a spiral wound
reaction chamber (see FIG. 8) lead to a significant decrease in UV
light energy that is necessary to sterilize plasma solutions. These
data demonstrate that 4-logs of PPV inactivation can be achieved at
approximately 0.15 J/cm.sup.2 in a tubular reactor with tangential
inlet and outlet a. In a reactor with spiral wound reaction chamber
less than 0.1 J/cm.sup.2 are sufficient to inactivate 4-log of PPV.
It should be noted that log reduction values between 4.5 and 5 may
approach the detection limit of the virus assay and the actual
virus reduction may even be higher.
[0094] These results are consistent with data generated using a UV
photosensitive substance instead of viruses. In this case, the UV
induced formation of triiodide ions from iodide ions was used,
following an approach described by Rahn (Rahn, R. O.;
Photochemistry and Photobiology 58(1993)6, 874-880, ibid 66(1997)4,
450-455). Here, potassium iodide was used as a UV photosensitive
component to determine the UV light intensity at 254 nm, delivered
to the reaction medium in the same three reactors used in Graph A.
Comparison of the measured light intensity with the light intensity
that is emitted by the UV bulb gives a UV light yield. Since the
penetration depth of UV light into a potassium iodide solution is
extremely small (less than 1 mm) under the given conditions, it can
be approximated that iodide conversion only occurs directly at the
surface of the quartz sleeve that encapsulates the UV bulb. It is
obvious therefore that hydrodynamic conditions, especially radial
mixing as a result of circulating secondary flow patterns, should
determine the light yield. Data shown in Graph B clearly confirm
this. Due to superior hydrodynamic conditions the highest light
yield can be found in the reactor with a spiral wound reaction
chamber, compared to the other two reactors. Data in Graph B show
that radial mixing, i.e. an increase in the circulating secondary
flow, increases with increasing flow rate. In the reactor with a
spiral wound chamber,
[0095] however, the degree of mixing seems to level off at flow
rates higher than 1000 ml/min. Since radial mixing is slightly
better in the reactor with tangential inlet and outlet light yield
is higher compared to a simple tubular reactor.
EXAMPLE 6
[0096] Graph C shows the result of studies assessing the
inactivation of Reo-virus 3 in a solution of 5 mg/ml alpha.sub.1
proteinase inhibitor in the UV reactor with a spiral wound reaction
chamber. It can be seen from this that Reo inactivation increases
with increasing fluency and reaches a 4-log reduction at
approximately 0.15 J/cm.sup.2. At the same time protein activity is
not impacted, but it declines at fluencies above 0.15 J/cm.sup.2.
The fluency value of 0.15 J/cm.sup.2 corresponds to a flow rate of
1000 ml/min. As noted in Fig. YYY mixing apparently approaches a
limit at flow rates above 1000 ml/min in this device and plateaus.
Further increase of flow rate (decreasing of fluency) thus
decreases the overall residence time of the virus in the kill zone
in the reactor and therefore leads to a reduced virus inactivation.
At the same time protein activity declines with reduced flow rates
(increasing fluency). This example suggests that there is an
optimum flow rate where hydrodynamic conditions are appropriate to
assure proper mixing, but at the same time overall residence time
is still high enough to effectively kill virus and leave sufficient
high protein activity. This flow rate depends not only on reactor
design and configuration, as demonstrated, but also on the virus
and protein properties and their respective concentrations, as
described earlier. Therefore, optimal flow rates need to be
determined experimentally for each given system.
[0097] The invention has been described herein in terms of
preferred embodiments, configurations, methodologies, and examples.
It will be understood by those of skill in the art, however, that a
variety of additions, deletions, and modifications might well be
made to the illustrative embodiments without departing from the
spirit and scope of the invention as set forth in the claims.
* * * * *