Note: Descriptions are shown in the official language in which they were submitted.
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DEVICE AND METHODS OF INACTIVATING INFLUENZA VIRUS
AND ADVENTITIOUS AGENTS WITH ULTRAVIOLET LIGHT
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims priority from U.S. Provisional Patent
Application
Serial No. 60/827,014, filed on September 26, 2006, which is incorporated by
reference
herein in its entirety.
FIELD OF INVENTION
The present invention relates generally to a device and methods of
inactivating
infectious virus, for reducing microbial bioburden, and inactivating potential
adventitious
agents with ultraviolet (UV) light.
BACKGROUND
Vaccines are designed to stimulate the immune system through the use of
inactivated or weakened viruses and bacteria, so that live viruses and other
foreign
microbial organisms can be recognized quickly allowing the body to mount an
immune
response before infection can set in. Certain vaccines are produced from
naturally or
engineered live-attenuated, or non-pathogenic, strains of pathogen. In other
cases, a
virulent, infectious strain of pathogen is killed or inactivated to produce a
vaccine.
Viruses and other pathogenic microorganisms have been inactivated using a
variety of
methods, including heat, treatment with chemicals, such as formalin or
propiolactone,
gamma irradiation and ultraviolet irradiation, or combinations of such
methods.
Ultraviolet (UV) irradiation has become accepted, typically in combiriation
with
chemical (e.g., formalin) treatment, in the production of viral vaccines
because it is
effective for inactivating a wide variety of viral, as well as bacterial,
pathogens. Unlike
formalin, which targets proteins, UV light primarily targets nucleic acids,
leaving the
antigenic proteins relatively untouched. During UV inactivation, the
excitation energy of
the UV wavelength radiation disrupts the covalent bonds of the purine and
pyrimidin
bases, resulting in damage to target virus as well as adventitious agents and
bacterial
bioburden.
Although the process of UV inactivation has proven safe and effective in the
manufacture of vaccines, such as influenza vaccine, the devices currently
available for
inactivation are subject to numerous operating limitations that have limited
their
application in the industrial scale manufacture of vaccines. The present
disclosure
provides an improved UV irradiation device suitable for the manufacture of
vaccines in a
high-throughput industrial setting. These improvements render UV inactivation
feasible
in the context of an integrated industrial manufacturing process, suitable for
the
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production of vaccines from highly infectious viruses, including pandemic and
avian
strains of influenza.
SUMMARY OF THE INVENTION
This disclosure concerns the industrial production of safe and effective viral
vaccines, and provides a device for ultraviolet inactivation of live
infectious viruses in
biological fluids. This disclosure also provides operating parameters and
methods for
safely and effectively inactivating virus in a fluid, e.g., for the
manufacture of vaccines.
BRIEF DESCRIPTION OF THE FIGURES
The foregoing summary, as well as the following detailed description of
embodiments, will be better understood when read in conjunction with the
appended
drawings, in which are shown exemplary embodiments of the invention. It should
be
understood, however, that the invention is not limited to the precise
arrangements and
instrumentalities shown. In the drawings, which are not to scale, the same
reference
numerals are employed for designating the same elements throughout the several
figures. In the drawings:
FIG. 1 is a perspective view of an irradiator assembly according to an
exemplary
embodiment;
FIG. 2 is a side elevational view of the irradiator assembly shown in FIG. 1;
FIG. 3 is a side view, partially in section, of an irradiator unit mounted on
the
irradiator assembly of FIGS. 1 and 2;
FIG. 4 is a perspective view of an embodiment of an injector box mounted on an
injection end of the irradiator unit shown in FIG. 3;
FIG. 5 is a side elevational view of the injector box shown in FIG. 4;
FIG. 6 is a sectional view of the injector box taken along lines 6--6 of FIG.
5;
FIG. 7 is a sectional view of the injector box taken along lines 7--7 of FIG.
5;
FIG. 8 is a perspective view of an exemplary embodiment of an injection needle
that is inserted into the injection box of FIG. 4-7;
FIG. 9 is a bottom view of the injection needle shown in FIG. 8;
FIG. 10 is a side elevational view of the injection needle of FIG. 8;
FIG. 11 is a photograph of the injection box of FIGS. 4-7, with the injection
needle of FIGS. 8-10 and sensors inserted therein;
FIG. 12 is a photograph of the injection box of FIGS. 4-7, showing fluid being
injected from the injection needle of FIGS. 8-10 into the injection box and
irradiator unit;
FIG. 13 is an exploded perspective view of an exemplary embodiment of an
ultraviolet radiation source of the irradiator assembly of FIG. 1;
FIG. 14 is a side elevational view of the ultraviolet radiation source shown
in FIG.
13;
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FIG. 15 is an enlarged view of the discharge and of the irradiator unit;
FIG. 16 is a perspective view of an end cap of a collector hub assembly shown
in
FIG. 15;
FIG. 17 is a side elevational view of the end cap shown in FIG. 16;
FIG. 18 is a front elevational view of the end cap shown in FIG. 16;
FIG. 19 is an exploded perspective view of an exemplary embodiment of a
bearing assembly used in the irradiator assembly of FIGS. 1 and 2;
FIG.. 20 shows the UV inactivation results from at varying UV intensities for
each
of three flu strains; and
FIG. 21 shows the UV inactivation via a dose response curve for the 6
-~ .
adventitious agents.
DETAILED DESCRIPTION
Ultraviolet (UV) light acts on the DNA of viruses, such as influenza virus,
and
other microorganisms, rendering them incapable of replication, thereby making
the
viruses non-infectious. In the manufacturing processes disclosed herein, a
biological
fluid containing live virus is subjected to UV irradiation to inactivate the
virus and make
it safe for administration as a vaccine.
Overview of the Disclosure
The present invention provides a device for irradiating a fluid. The device
comprises an elongated tube having a fluid injection port and a fluid
discharge port,
wherein the elongated tube is rotatable about a longitudinal axis. The
longitudinal axis
extends at an angle oblique to the horizontal. A source of radiation extends
within the
elongated tube along the longitudinal axis. The source of radiation can be
one, or more
than one, ultraviolet light sources (e.g., lamps). In one embodiment of the
invention, a
sleeve extends within the elongated tube and surrounds a length of the source
of
radiation, thereby defining an airflow path between the source of radiation
and the
sleeve. An air flow source is in fluid communication with the airflow path
proximate to
the fluid injection port. An air flow discharge is in fluid communication with
the airflow
path proximate to the fluid discharge port. The fluid injection port and the
fluid discharge
port are in communication with the space between the tube and the sleeve.
In another embodiment, the present invention provides an injection box mounted
at the fluid injection end of the elongated tube. At least one sensor is
disposed within
the injection box upstream of the fluid injection end of the elongated tube
and is
targeted to obtain data from the elongated source of radiation. A fluid
injection needle is
coupled to the injection box and positioned to inject fluid downstream of the
sensor and
the elongated tube so as to avoid sensing fluid flowing through the elongated
tube. The
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discharge end of the fluid injection needle extends into the space between the
sleeve
and the elongated tube.
In still another aspect, the present disclosure provides a single bearing
assembly,
comprised of at least two axially spaced apart bearing members surrounding and
supporting the elongated tube between the fluid injection port and the fluid
discharge
port and permitting the rotational movement thereof.
The various features of the previously described embodiments may also be used
together in any combination thereof.
The present invention also provides a method for inactivating a virus
comprising
the steps of introducing a fluid comprising introducing a live virus into an
apparatus as
previously described, dispersing the fluid along an interior length of the
elongated tube;
irradiating the fluid with a radiation source disposed within the tube,
thereby
transforming the live virus to an inactivated virus while protecting the
radiation source
from direct contact with the live virus by providing a sleeve between the
radiation source
and the live virus along the length of the elongated tube. In this method, a
desired
temperature range of the radiation source is maintained by providing a flow of
air along
a length of the radiation source between the radiation source and the sleeve.
For example, the disclosed method is suitable for inactivating influenza
viruses
and potential adventitious agents present in allantoic fluid or other culture
media used to
grow and culture the influenza virus. The processes described herein yield
inactivated
influenza virus particles that can then be further treated (e.g., with a
chemical agent
such as formalin), purified and/or split for ultimate formulation into a
vaccine that can be
administered to patients.
In an exemplary embodiment, the method for inactivating a virus involves
introducing a fluid comprising introducing a live virus to an elongated tube
that is
inclined relative to the horizontal; rotating the elongated tube along its
longitudinal axis
to disperse the fluid and cause it to flow along the interior length of the
elongated tube;
and irradiating the fluid with a radiation source disposed within the tube
(and extending
along its longitudinal axis) to inactivate the virus. For virus inactivation,
the radiation
source typically emits ultraviolet (UV) light at a wavelength of about 254
nanometers.
Exposure of the fluid to the radiation source as it flows through the
elongated tube
transforms the live virus to an inactivated virus, which can be recovered by
causing the
fluid containing the inactivated virus to exit the elongated tube. In the
course of
operating the device, the radiation source is protected from direct contact
with the live
virus by providing a protective sleeve (such as a quartz sleeve) between the
radiation
source and the live virus along the length of the elongated tube. A desired
temperature
range of the radiation source is maintained within the protective sleeve by
providing a
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flow of air along a length of the radiation source between the radiation
source and the
sleeve.
In certain embodiments, the fluid is introduced and flowed through the device
where it is exposed to UV light emitted by the radiation source. The rate of
introduction
and flow is set to insure that the fluid remains exposed to the UV light for a
period
sufficient to fully eliminate bioburden and inactivate virus. For example, the
fluid is
typically introduced at a rate of at least 600 mI/min (0.6 L/min). The fluid
can be
introduced at rates up to approximately 3500 mI/min without compromising
inactivation.
For example, the fluid can be introduced and flowed through the device at
rates of up to
approximately 1700, or 1900 or 2200 or 2800 or 3500 ml/min, or at any
convenient rate
within this range. In certain embodiments, the fluid is introduced along an
interior wall
of the elongated tube at a flow rate of at least about 600 mI/min and no
greater than
about 900 ml/min. More typically, the fluid is introduced at a flow rate of at
least about
650ml/min, such as at least about 675ml/min. Usually the fluid is flowed into
the
elongated tube at a flow rate of at least about 680ml/min. Typically, the flow
rate does
not exceed about 850m1/min, more commonly, the flow rate is set below about
840m1/min, such as less than about 830m1/min. Favorably, the method involves
introducing the fluid containing live virus at a flow rate of about 755m1/min.
Following introduction into the device, the fluid is flowed through the
elongated
tube (e.g., at the rates indicated above). The elongated tube is typically in
the shape of
a cylinder. The fluid can be caused to flow through the elongated tube by way
of gravity
by inclining the elongated tube relative to the horizontal with the inflow
placed at a
higher elevation than the outflow. Typically, the elongated tube is inclined
at an angle of
at least 20 degrees relative to the horizontal. For example, the elongated
tube can be
inclined at an angle of between about 20 degrees and about 40 degrees relative
to the
horizontal. In an exemplary embodiment, the elongated tube is inclined about
30
degrees relative to the horizontal.
In an embodiment, the radiation source includes one or more (e.g., a plurality
of)
UV lamps. As the fluid flows through the rotating elongated tube, the
radiation source
irradiates the fluid with UV light the fluid at an intensity of at least about
10
mWatts/cm2. Typically, the intensity of UV light is maintained between 10 and
14
mWatts/cmz, such as at a UV light intensity of about 12 mWatts/cm2.
The disclosed method is suitable for inactivating a wide range of viruses,
including
both non-enveloped and enveloped viruses (such as orthomyxoviridae, e.g.,
influenza
virus), including highly pathogenic viruses. In one embodiment, the method
involves
inactivating a live influenza virus, such as a pandemic or avian strain of
influenza.
Likewise, the method is capable of inactivating virus in the various fluids in
which virus is
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grown, such as allantoic fluid (for example, from embryonic chicken or other
poultry
eggs), and from tissue or cell culture media. Along with the virus of
interest, the
methods disclosed herein inactivate adventitious viruses and bacteria
("bioburden")
present in the fluid.
Also disclosed are inactivated virus manufactured by the method disclosed
herein,
as well as pharmaceutical compositions, and methods of protecting a subject
against
viral infections by administering the inactivated virus.
UV Irradiation Device
Referring in general to FIGS. 1-19, there is shown an irradiator assembly 100
according to an exemplary embodiment of the present invention that may be used
to
inactivate virus in allantoic fluid, in accordance with another embodiment of
this
invention. More specifically, assembly 100 may be used to irradiate a virus
contained in
a fluid flowing through assembly 100 such that the virus is inactivated by the
UV light as
the virus flows through assembly 100. The fluid is injected into assembly 100
from a
virus supply (not shown), processed through assembly 100, and then discharged
from
assembly 100 for additional processing, such as for formalin processing.
Referring specifically to FIGS. 1 and 2, irradiator assembly 100 includes a
pair of
irradiator units 102 mounted on a support frame 104. Each of irradiator units
102 may
be a mirror image of the other irradiator unit 102. All of the elements in one
irradiator
unit 102 are also present in the other irradiator unit 102 and consequently,
only one
irradiator unit 102 will be discussed herein.
Irradiator unit 102 is mounted on a support frame 104. Irradiator unit 102
includes a cylindrically shaped elongated vessel or tube 106 having a fluid
injection end
or port 106a, a fluid discharge end or port 106b, and a longitudinal axis 107
extending
therethrough from the fluid injection end 106a to the fluid discharge end
106b. In an
exemplary embodiment, tube 106 is constructed from stainless steel in order to
minimize and chemical reaction between tube 106 and the fluid being
transmitted
through tube 106. In an exemplary embodiment, tube 106 has an inner diameter
of
about 2-3/4 inches (7 cm) and a length of about 28 inches (about 71 cm). An
injection
box assembly 108 is mounted at fluid injection end 106a of tube 106 and a
collector hub
assembly 110 is located at fluid discharge end 106b.
Support frame 104 is mounted on a plurality of castor wheels 112, which allow
irradiator assembly 100 to be maneuvered from one location to another, such as
by
pulling or pushing on a handle 114 extending from support frame 104.
Additionally, in
an exemplary embodiment, irradiator units 102 are each mounted on support
frame 104
at an oblique angle relative to the horizontal. Those skilled in the art will
recognize that
irradiator units 102 are angled relative to the horizontal at an angle
sufficient to impart
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gravity flow of a fluid along the length of tube 106 from fluid injection end
106a to fluid
discharge end 106b. In an exemplary embodiment, the oblique angle is between
about
25 degrees and about 35 degrees. In another exemplary embodiment, the oblique
angle
is about 30 degrees.
Referring now to FIG. 3, a partial sectional view of irradiator unit 102 is
shown. A
UV light source 116 is disposed within tube 106 and extends along a length of
tube 106
between fluid injection end 106a and fluid discharge end 106b, along
longitudinal axis
107. Tube 106 rotates about its longitudinal axis 107 to spread the fluid
containing the
virus into a thin film along the rotationally moving surface thereof as the
fluid flows
down from injection box assembly 108 to collection hub assembly 110. In an
exemplary
embodiment, tube 106 rotates at a speed of about 300 revolutions per minute.
The thin
film provides a sufficiently thin profile to allow UV light source 116 to
penetrate the fluid,
and thus to expose as much of the virus as possible for inactivation by UV
light source
116.
UV light source 116 also extends at least partially into injection box
assembly
108. UV light source 116 is used to inactivate the virus as the virus flows
through tube
106. UV light source 116 emits a light having a wavelength of about 254
nanometers,
which is the wavelength absorbed by nucleic acids. A photochemical process
within the
virus is generated, causing a rearrangement of the genetic information of the
virus,
thereby interfering with the cell's ability to reproduce. A virus that cannot
reproduce is
considered inactive because it is not able to multiply to infectious numbers
within a host.
Referring now to FIGS. 4-7, an injection box 118, which is part of injection
box
assembly 108, is shown, with the downstream end thereof to the left in FIG. 4
and to the
right in FIGS. 5 and 6. As shown in FIG. 5, a reducer 118a extends from the
downstream end of injection box 118 and is coupled over fluid injection end
106a of tube
106.
Injection box 118 is generally tubular in shape, with a generally circular
cross
section, and has a plurality of openings in the side wall to accommodate
various
features. A pair of sensor openings are diametrically opposed from each other
and
accommodate UV sensors (not shown in FIGS. 4-7) that measure the intensity of
UV
light source 116. Sensor openings are defined by bosses 120 that extend from
an
exterior of injection box 118 with a slight penetration into the interior of
injection box
118. The UV sensors are inserted into each respective boss 120. A cap (not
shown) is
threaded over each sensor and onto boss 120 to secure the sensor within boss
120.
Such configuration allows the sensor to be releasably coupled to injection box
118 such
that a sensor face of the sensor may be consistently located a predetermined
distance
from UV light source 116 in order to obtain consistent readings from the
sensors.
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Injection box 118 further includes a needle opening that is sized to allow
insertion
of an injection needle 126 (partially shown in phantom in FIGS. 5-6) into
injection box
118. The needle opening is defined by a needle boss 122 that extends outwardly
from
the cylindrical outer surface of injection box 118. Needle boss 122 is spaced
from
bosses 120 downstream from bosses 120 such that fluid flow that is discharged
from
injection needle 126 does not contact the sensors, which would reduce reading
obtained
from the sensors. As shown in FIG. 7, needle boss 122 is located below the
centerline of
injection box 118 and is pointed slightly downwardly so as to introduce fluid
more or less
tangentially to the lower surface of injection box 118. More specifically,
needle boss 122
is disposed at a declination angle between about 3 degrees and about 7 degrees
with
respect to the horizontal of a central plane of tube 106 and injection box
118. In an
exemplary embodiment, the declination angle is about 5 degrees. Such
declination
angle allows the fluid to be injected along an inside surface of tube 106 and
minimizes
splashing of the fluid. Injection box 118 also includes a view port 124 that
enables a
person, such as an a operator, to peer into view port 124 to observe fluid
flow from
injection needle 126.
A spill port boss 125 extends outwardly from the bottom of injector box 118
proximate to reducer 118a. Spill port boss 125 may be used to evacuate liquid
from
injection port 118 in the event of a spill. Alternatively, spill port boss 125
may be used
to insert a monitoring device, such as a temperature probe or spectrometer
(not shown)
into injection box 118.
Referring now to FIGS. 8-10, injection needle 126 is shown. Needle 126 is
constructed from a stainless steel tube having a supply end 128 and a
discharge end
130. Needle 126 extends into and through injection box 118 such that discharge
end
130 of needle 126 extends into tube 106 (shown in phantom in FIGS. 5 and 6 for
injection of fluid into elongated tube 106.
The supply end 128 is generally circular in cross section and includes a clamp
132
that is used to secure a fluid supply (not shown) containing active virus to
needle 126.
The body of needle 126 is curved proximate to clamp 132 in order to facilitate
the
coupling of the fluid supply to supply end 128 of'needle 126. Body 126 is
curved
proximate to discharge end 130 in order to angle discharge end 130 against the
inner
wall of tube 106. Such angling of discharge end 130 of needle 126, at about a
60
degree angle relative to longitudinal axis 107 in an exemplary embodiment,
assists in
maintaining the flow of fluid against the inner surface of tube 106, reducing
the
likelihood of splashing the fluid. Further, discharge end 130 includes a
generally oblong
opening 134 to spread out the fluid and to facilitate discharge of the fluid
along the inner
surface of tube 106. Discharge end 130 of fluid injection needle 126 extends
along a
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discharge axis 131, and generally oblong discharge opening 134 extends at an
angle "a"
oblique to the discharge axis (131). Such angle also assists in reducing the
likelihood of
splashing the fluid as the fluid is discharged from needle 126. In an
exemplary
embodiment, angle "a" may be between about 20 degrees and about 60 degrees.
A tightening cylinder 136 is slidably disposed about the body of needle 126
between supply end 128 and discharge end 130. During installation, needle 126
is
inserted into needle boss 122 a desired distance and tightening cylinder 136
is slid along
body of needle 126 until tightening cylinder 136 engages needle boss 122.
Screws (not
shown) in tightening cylinder 136 engage threaded openings 122a in needle boss
122
(shown in FIG. 7). As screws are tightened down, tightening cylinder 136
tightens
around needle 126, locking needle 126 into place relative to injection box
118.
FIG. 11 is a photograph of an internal view of injection box 118, showing a
pair of
sensors 138 diametrically opposed from each other within injection box 118.
Sensors
138 are each removeably installed within injection box 118 upstream from fluid
injection
port 106a a predetermined distance from UV light source 116 (not shown in FIG.
11) and
targeted to obtain data from UV light source 116. Sensors 138 are
electronically coupled
to UV light source 116 to regulate the amount of power emitted from UV light
source
116. In an exemplary embodiment, UV light source 116 emits light having an
intensity
of 12 mW/cmZ 2 mW/cm2 as measured at sensors 138. In an exemplary
embodiment,
the intensity may be between about 10 mW/cmz and 14 mW/cmz. In another
exemplary
embodiment, the intensity may be about 12 mW/cmZ. Each sensor 138 includes a
filter
(not shown) that ensures sensitivity of sensors 138 at 254 nanometers.
Additionally, needle 126 is shown extending into injection box 118. Discharge
end 130 of needle 126 is aimed into tube 106 such that fluid discharged from
needle 126
is expressed along the inside wall of tube 106. Discharge end 130 of needle
126 is
coupled to injection box 118 between sensors 138 and tube 106 such that
sensors 138
are not exposed to fluid and do not sense fluid flowing through tube 106. With
sensors
138 not being exposed to the fluid, sensors 138 are able to provide more
accurate
readings of the intensity of UV light source 116. FIG 12 shows fluid "F" being
discharged
along the inner wall of tube 106 as tube 106 rotates about its longitudinal
axis.
FIGS. 13 and 14 show an exemplary embodiment of UV light source 116 used in
assembly 100. UV light source 116 includes a plurality of UV light tubes 140
bundled in
a group and extending parallel to longitudinal axis 107. While FIG. 13 shows
four (4) UV
light tubes 140, those skilled in the art will recognize that other numbers of
UV light
tubes 140 may be used. UV light tubes 140 may be operated by either an
electromagnetic ballast or an electronic ballast (not shown).
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A support rod 142 extends within a perimeter of UV light tube 140 and extends
along a length of UV light source 116 to provide support to UV light source
116 within
tube 106. Each UV light tube 140 includes its own grounding wire 144, which
extends
along its respective UV light tube 140 parallel to support rod 142. Only one
grounding
wire 144 is shown in FIG. 13 for clarity. Grounding wires 144 are separate
from support
rod 142.
A quartz sleeve 146 extends within tube 106 and is disposed around and
surrounds UV light tubes 140. Quartz sleeve 146 protects UV light tubes 140 by
preventing any fluid travelling along the length of tube 106 from
inadvertently splashing
onto UV light tubes 140, which would potentially damage and/or contaminate UV
light
tubes 140. Sleeve 146 also defines a fluid flow path between sleeve 146 and
tube 106.
Fluid injection port 106a and fluid discharge port 106b are in communication
with the
space between tube 106 and sleeve 146. Discharge end 130 of needle 126 extends
into
the space between sleeve 146 and tube 106.
Sleeve 146 is constructed of quartz because quartz is a UV permeable barrier
and
allows the penetration of UV light with minimal losses. Sleeve 146 is coupled
to a socket
holder assembly 148 to support sleeve 146 at the fluid injection end of sleeve
146. A
socket plug 150 extends from a rear end of socket holder assembly 148 and
provides for
an electrical and structural connection (not shown) to UV light bulbs 140. A
mounting
sleeve 152 supports a wire cover 154 and a socket holder 156. Mounting sleeve
152 is
mounted at a rear of a top lamp holder assembly 158, which supports quartz
sleeve 146
and socket holder assembly 148. Gasket 160 seals the fluid injection end of
sleeve 146
with respect to top lamp holder assembly 158. A flange 162 couples top lamp
holder
assembly 158 to the upstream end of injection box 118. Top lamp holder
assembly 158
also includes an air flow supply 164, which provides a cooling air flow to UV
light tubes
140.
A downstream end of UV light source 116 includes a bottom lamp holder 166 that
supports the downstream end of UV light tubes 140. A helical spring 168 biases
the
downstream end of light tubes 140 away from bottom lamp holder 166. A
downstream
end of support rod 142 is inserted into and supported by bottom lamp holder
166. A
downstream end of quartz sleeve 146 is also inserted into bottom lamp holder
166, with
a gasket 170 sealing quartz sleeve 146 to bottom lamp holder 166. Gaskets 160,
170
seal each end of quartz sleeve 146, preventing any fluid flowing through tube
106 from
entering sleeve 146 and potentially contaminating UV light tubes 140.
Bottom lamp holder 166 also includes at least one opening 172 that allows for
air
flowing through sleeve 146 from air flow supply 164 to be discharged from
sleeve 146.
Air flow through sleeve 146 and around UV light tubes 140 draws away heat that
is
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generated by UV light tubes 140, maintaining UV light tubes 140 within a
predetermined
temperature range in order to reduce wear and extend the life of UV light
tubes 140.
Such temperature control also serves to regulate the intensity of UV light
discharged
from UV light tubes 140, thus maintaining a desired UV intensity to inactivate
viruses in
the fluid.
Referring now to FIG. 15, a discharge end of irradiator unit 102 is shown. A
shield 172 encircles tube 106 at the fluid discharge end 106b of tube 106 in
order to
prevent an operator from inadvertently engaging tube 106 as tube 106 rotates.
Shield
172 may be constructed from Lexan or another transparent material, so that
operator
may be able to view discharge end of tube 106.
Collection hub assembly 110 includes a collection cup 174 that is releasably
coupled to bottom lamp holder 166 to collect treated fluid as it is discharged
from tube
106. Fluid "F" is shown in FIG. 15 extending between tube 106 and quartz
sleeve 146,
then discharging into collection cup 174. Collection cup 174 includes a
generally angular
chamber which collects the fluid "F." The fluid "F" is then gravity drained
from collection
cup 174 away from irradiator unit 102 by a drain tube 176. Drain tube 176 may
be
coupled to a receiver (not shown), which collects the treated fluid discharged
from drain
tube 176 for further processing.
Collection hub assembly 110 also includes a center tube 178 that is inserted
into
and releasably coupled to bottom lamp holder 166 so that entire collection hub
assembly
110 may be removed from irradiator unit 102, such as for cleaning. Center tube
178
provides a passage for the air flowing through the space defined by quartz
sleeve 146
and UV light tubes 140 to exit irradiator unit 102. An air exhaust tube 180
fluidly
communicates with center tube 178 to allow the air flow to exhaust from
irradiator unit
102. Although not shown, exhaust tube 180 may be coupled to an air hose to
direct the
exhaust air away from the operator. A tang 182 extends from a flange 184 which
defines the discharge end of center tube 178. Tang 182 mates with a
corresponding
opening (not shown) in collection cup 174 in order to properly locate the
relative location
of drain tube 176 with respect to irradiator unit 102.
An airflow path is formed between UV light source 116 and sleeve 146 such that
air flow source 164 is in fluid communication with the airflow path proximate
to fluid
injection port 106a and air flow discharge, or exhaust tube 180, is in fluid
communication with the airflow path proximate to fluid discharge port 106b.
Referring back to FIG. 3, irradiator unit 102 and in particular, tube 106 is
rotated
by a motor assembly 186, which is shown in more detail in FIG. 19. As shown in
FIG.
19, motor assembly 186 is powered by a motor 188, which may be a brushiess DC
motor. A drive pulley 190, driven by motor 188, drives a timing belt 192.
Timing belt is
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drivingly coupled to a driven timing pulley 194, which is fixedly coupled to
tube 106 via
bolts 196 fixed to driven timing pulley and extending through a flange 198,
which is
circumferentially disposed around tube 106. Nuts 200 secure bolts 196 to
flange 198.
As shown in FIG. 19, tube 106 is rotatably mounted in a bearing assembly 202,
which concentrically surrounds and supports tube 106 approximately halfway
between
fluid injection port 106a of tube 106 and fluid discharge port 106b of tube
106 and
permits rotational movement of tube 106 about axis 107. Bearing assembly 202
includes a pair of ball bearing sleeves 204 that are fixedly mounted within a
housing 206
and axially separated from each other by an annular spacer 208. By mounting
bearing
sleeves 204 within housing 206, bearings 204 may be aligned with each other.
Alignment of bearing sleeves 204 with each other may offer multiple
advantages, such
as, for example, reduced noise and vibration, extended operating life of
bearing sleeves
204, and reduced maintenance cost. Furthermore, by containing bearing sleeves
204 in
housing 206 distanced from the inflow and outflow of the fluid, bearing
lubricant is
prevented from entering any portion of irradiator unit 102 that enters into
contact with
the fluid to be irradiated.
Support brackets 210, 212 support bearing housing 206 and are each mounted to
a mounting bracket 214. Gaskets 216, 218 seal bearing assembly 202 in order to
prevent the fluid flowing from tube 106 from coming into contact with bearing
sleeves
204 or any bearing grease that may be used to lubricate bearing sleeves 204.
Mounting
bracket 214 is fixably coupled to support frame 104 to support bearing
assembly 202
and tube 106 on support frame 104 (shown in FIG. 1). An inductive sensor 220
is
mounted to support bracket 210 to sense and regulate the rotational speed of
tube 106
during operation.
In operation, device 100 is used to irradiate a fluid, such as allantoic fluid
obtained from, for example, embryonic chicken eggs or a cell culture medium,
to
inactivate a live virus contained within the fluid. Fluid comprising the live
virus is placed
in fluid communication with device 100 from a fluid supply (not shown) by
coupling the
fluid supply to supply end 128 of injection needle 126. Additionally, an air
supply (not
shown) is coupled to air flow supply 164. UV light source 116 is activated by
energizing
UV light tubes 140. Sensors 138 regulate output power of UV light source 116
by
sensing output from UV light supply 116 and transmitting a signal to a
controller (not
shown), which in turn regulates power to UV light source 116. In an exemplary
embodiment, output power of an exemplary embodiment of UV light source 116 is
regulated to 12 mW/cmz 2 mW/cmZ. As an alternative to a threaded connection
between sensors 138 and their respective bosses 120, sensors 138 may be
inserted into
bosses 120 using a cap (not shown) to secure sensor 138 within boss 120.
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Referring back to FIG. 3, air flow is generated from an air supply, through
air flow
supply 164 and into the space defined between sleeve 146 and UV light tubes
140 to
cool UV light tubes 140 to within a predetermined temperature range such as,
for
example, between about 35 degrees Celsius and about 55 degrees Celsius. In an
alternative exemplary embodiment, the temperature range may be between about
39
degrees Celsius and about 49 degrees Celsius. In another alternative exemplary
embodiment, the temperature is maintained between about 42 degrees Celsius and
about 44 degrees Celsius. After flowing along UV light tubes 140, the air
flows out of air
exhaust tube 180, where the air is discharged from irradiator unit 102. Air
flow between
sleeve 146 and tube 106 and then exiting exhaust tube 180 is indicated by
arrows in
FIG. 15.
Elongated tube (106) is rotated along its longitudinal axis (107) by motor
188.
Motor 188 in turn drives timing belt 192, which in turn rotates tube 106 In an
exemplary embodiment, tube 106 is rotated at a rotational speed of 300 rpm.
Inductive
sensor 220 measures rotational speed of tube 106, and transmits a signal to a
controller
(not shown) to regulate rotational speed of tube 106.
When the device is operating at a steady state, regarding the rotation of tube
106, the power output of UV light source 116, and the air flow to cool UV
light source
116, the fluid to be treated is introduced to device 100 via needle 126. Fluid
may be
introduced into device 100 at a rate of between 600 and 900 liters per minute.
In an
exemplary embodiment of the present invention, the fluid may be introduced at
a rate
between about 600 and about 900 liters per minute. In another exemplary
embodiment,
the fluid may be introduced at a rate between about 700 and about 800 liters
per
minute. In yet another exemplary embodiment, the fluid may be introduced at a
rate of
about 755 liters per minute.
The oblong shape of needle 126 and its declination angle relative to the
centerline
of injection box 118 allow the fluid being discharged from needle 126 to be
dispersed in
a relatively thin film along fluid injection end 106a of tube 106 without
splashing fluid
onto sleeve 146, protecting UV light source 116 from direct contact with the
live virus
Such dispersion is shown in the photograph of FIG. 12.
The rotation of tube 106 about its longitudinal axis 107 serves to further
disperse
the fluid along the interior length of elongated tube 106. As the fluid
travels the length
of tube 106, the fluid is irradiated with light from UV light source 116,
thereby
transforming the live virus to an inactivated virus.
After the virus is inactivated within tube 106, the fluid containing the virus
flows
into collection cup 174, where the fluid then drains from collection cup 174
through drain
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tube 176. The fluid may be collected from drain tube 176 into a receiver (not
shown) for
further processing.
Process for UV Irradiation of fluids containing live virus
The methods disclosed herein are useful for inactivating viruses in the
production of immunogenic pharmaceutical compositions, including vaccines. The
methods are suitable for producing vaccines from highly pathogenic and
infectious
viruses, including highly pathogenic (pandemic and/or avian) influenza
strains. In one
embodiment, the device and methods disclosed herein are used in manufacturing
influenza vaccine to inactive live virus. In the production of influenza
vaccine, the virus
stock is typically grown and harvested from embryonic chicken eggs.
Alternatively, virus
stock can be grown in cultured cells, where it is harvested from the culture
media (for
example, as described in US Patent Publication 2004/0029251, 6,656,720,
6,344,3546,825,036, 6,951,752, which are incorporated herein by reference). In
the
course of producing a vaccine virus in a biological system such as chicken
eggs or
cultured cells, contaminating biological agents that are present in the eggs
and/or cells
can be present in the virus stock. Such biological contaminants must be
inactivated
along with the virus of interest in order to insure a safe, as well as
effective, vaccine. In
the context of this disclosure, biological contaminants include "adventitious
agents." The
term "adventitious agent" refers to a mammalian or avian virus, Mycoplasma, or
bacteria. These are biological contaminants that may be present in process
intermediates during the production of a finished vaccine suitable for
administration to
humans. Adventitious agents could be present in the starting materials due to
undetected disease in the hens or introduced as a foreign contaminant during
processing. Additionally, the term, "bioburden," as used herein, refers to the
population
of bacteria present in a fluid. Bioburden is typically expressed as Colony
Forming Units
(CFU) per milliliter (ml) of fluid tested. It is an important feature of the
method
disclosed herein that adventitious agents and bioburden are inactivated during
the UV
processing. For example, although formalin inactivation procedures are capable
of
inactivating adventitious viral agents, bacteria are relatively resistant to
treatment with
formalin. The procedures disclosed herein for inactivating virus,
advantageously also
inactive bacterial contaminants reducing bioburden early in the processing and
facilitating recovery of the virus for vaccine production.
Following harvesting from eggs, the allantoic fluid is usually clarified by
centrifugation to remove particulate matter prior to subsequent processing.
After
clarification, the virus-containing fluid is subjected to a UV irradiation
step. As disclosed
above, the UV irradiation unit consists of an elongated tube, such as a
stainless steel
cylinder, rotating at high speed, in the center of which are situated the UV
light source.
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Allantoic fluid is introduced into the elongated tube (e.g., along an interior
wall) from the
collection tank, and flows by gravity through the rotating tube, which is set
at an angle
of 300 (e.g., 200-400) relative to the horizontal. The rotation speed allows
the flowing
liquid to form a very thin layer along the length of the cylinder wall. As the
fluid travels
along the length of the interior wall of the tube, it is exposed to UV light
emitted by the
radiation source extending within the length of the elongated tube. Typically,
the
radiation source consists of one or more UV lamps. The UV radiation source is
protected
from direct contact with the live virus by encasing the lamps within a sleeve
made from
a UV permeable material, such as quartz. To ensure uniform radiation, the
lamps are
maintained within a desired temperature range by passing a current of air
within the
sleeve so that it passes over the lamps in the opposite direction from the
fluid flow).
Exposure to UV light as the fluid passes through the elongated tube
inactivates the virus,
as well as any adventitious agents, and reduces bioburden.
The UV-inactivated allantoic fluid is collected into sterile (e.g., autoclaved
stainless steel) tanks. In a continuous flow process, the allantoic fluid is
fed into and
collected from the UV inactivator device via silicon tubing connected under
laminar flow.
As a representative example, a harvest of one egg lot will yield approximately
1000L
(mean of lOmL/egg) and is collected in four to five tanks. Each tank has a
unique
identifying number in order to monitor the order in which they are filled.
Material is sampled in-line while filling each tank for measurement of HA
titer. After each use, the UV units are disassembled and the removable parts
(UV
cylinder and other machine parts) are machine washed and autoclaved. The UV
unit and
its pumping system is cleaned-in-place. Prior to processing each lot, the UV
units are re-
assembled, and connections are made under laminar flow. Non-autoclaved product-
contact surfaces are sanitized with a formaldehyde solution, followed by a PBS
rinse
prior to use.
The following tables provide exemplary Operating Parameters.
Table 1: UV Irradiation Parameters Example 1
Parameter Detail
Rotation Speed of Cylinder 300-340rpm
Flow Rate 680-830mL/min
Rotor Angle 30
UV Source 254nm lamp
UV Intensity 10-14mW/cm2
Collection of UV-Inactivated Allantoic Fluid 240L stainless steel tanks in
room 144
Harvest
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Table 2: UV Irradiation Parameters Example 2
Parameter Detail
Rotation Speed of Cylinder 320 20rpm
Flow Rate 755 75mL/min
Rotor Angle 300
UV Source 254nm lamp
UV Intensity 12f2mW/cmZ
Collection, of UV-inactivated Allantoic Fluid 3600L stainless steel tank
(described
Harvest below)
Although the preceding operating parameters have been proven effective for
inactivating virus, additional suitable parameters can be determined by one of
skill in the
art in accordance with the methods disclosed herein. Typically, the flow rate
is at least
about 600 ml/min (such as at least about 650 mI/min, or at least about 675
mi/min, or
at least about 680 ml/min). Usually, the flow rate does not exceed about 900
ml/min
(such as a rate of no more than about 850 ml/min, or 840 mI/min, or about 830
ml/min). Nonetheless, if desired to increase processing capacity, the flow
rate can be
modified to up to at least about 3500 ml/min. Similarly, UV light intensity
can be
increased, e.g., up to at least about 18 mW/cmZ.
Following UV treatment, the collected allantoic fluid is typically treated
with
formalin (formaldehyde), concentrated and further processed to produce
detergent split
antigen suitable for administration as a vaccine.
For example, methods for preparing and formulating antigen, such as influenza
HA and NA antigens for vaccines, are well known, and exemplary procedures are
described in, e.g., U. S. Patent Nos. 3,962,421, 4,064,232, 4,140,762,
4,158,054, and
6,743,900. These exemplary procedures for preparing antigens and formulating
vaccines are incorporated herein by reference. Such antigens are suitable for
administration to subjects, including human subjects for inducing an immune
response
against the virus. Typically, the antigens are formulated into pharmaceutical
compositions with a pharmaceutically acceptable excipient or carrier. Such
carriers are
well know in the art, and numerous examples are described, for example in
Gennaro:
Remington's Pharmaceutical Sciences, 18th Ed., Mack Pub. Co., Easton, PA
(1995).
Optionally, the pharmaceutical composition includes an adjuvant that further
enhances
the immune response to the viral antigen.
Accordingly, pharmaceutical compositions (e.g., vaccines), incorporating
inactivated viruses, and/or viral antigens produced from inactivated viruses,
according to
the methods disclosed herein are encompassed by this disclosure, as are
methods of
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protecting subjects from viral diseases by administering such pharmaceutical
compositions.
Examples
The following non-limiting examples illustrate operating parameters for UV
inactivation of virus in biological fluid.
Examgle 1: Inactivation and Reduction of Bioburden by UV inactivation
Inactivation and clearance studies were conducted to document the capacity of
the process to inactivate potential microbial agents. Reduction in infectious
virus and
Mycobacteria titres were analyzed as follows.
Samples (treated and untreated) are typically analyzed for viral/Mycoplasma
titer
by preparing serial dilutions of the test samples (referred to here as
titration) and
inoculation onto cell cultures (for analysis by TCID50) or agar plates (to
determine
Mycoplasma colony counts). In instances where it was expected that very low or
no
viral/Mycoplasma growth would be detected, a technique termed "Large Volume
Plating"
was used to decrease the assay limit of detection (LOD). The assay LOD is an
estimate
of the theoretical titer based on the sample size using the Poisson
distribution (the
probability of detecting a low titer in a small representative sample). The
LOD is
decreased by increasing the sample size that is analyzed. In samples where no
growth
was seen via titration, and large volume plating was performed, the log
reduction factor
was calculated using the large volume titer determination.
TCID50 endpoints were calculated according to the Spearman Karber formula, as
described in the Federal Gazette No. 84(4), May 1994, and by Schmidt, N.J. and
Emmons, R.W. in DIAGNOSTIC PROCEDURES FOR VIRAL, RICKETTSIAL AND
CHLAMYDIAL INFECTION, 6th Ed. (1989). Log reduction factors (LRF) are
calculated
using the following formula:
Logio (starting virus titer/final virus titer)
Where multiple log reduction factors result from replicate or additional
studies, the
antilogs of the LRFs are averaged using a standard mean calculation as
follows:
((LRFI + LRF2 + ... LRFõ)/n)
The 95% confidence limits are calculated by the square-root of the sum of the
squares
divided by the number of samples as follows:
(SQRT (((CL 12) + (CLZZ) + ... (CLR2))/n))
All of these studies were conducted by BioReliance Corp. at facilities in
Rockville,
MD, USA. UV inactivation studies were performed utilizing commercial scale
equipment
(a Dill UV unit was transferred to BioReliance).
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AII study designs and LRF calculations were based upon guidance and examples
provided in ICH document Q5A, "Viral Safety Evaluation of Biotechnology
Products
Derived from Cell Lines of Human or Animal Origin", March, 1997, found on the
World
Wide Web at: www.ich.org/LOB/media/MEDIA425.pdf.
Following preliminary experiments to confirm the qualification and calibration
of
the UV units, inactivation of influenza by UV irradiation was evaluated in
three full scale
lots of allantoic fluid using the A/New Caledonia/20/99 strain of virus.
Samples were
taken before and after irradiation for evaluation; these results are
summarized in Table
3.
Table 3: Exemplary UV Irradiation Results
Lot No. Infectious Titer Bioburden (CFU/ml) HA Titer
(EU/0.2mL)
Pre Post Pre Post Pre Post
10+8.3_ 10+0.3_ 2048-
SSESSAIISNC 10+9.a 10+0.7 100-600 <10 1536 4096
S5ESSAI16NC 10+7 4_ 10+0.4_ 1536- 4096-
10+9.7 10+3.7 100-200 <10 3072 6144
10+$'9- 10+0.1- 1024-
S5ESSAI17NC C 10+9,4 10+2.8 300-1400 <10 1536 768-1536
Example 2: UV inactivation of three strains of Influenza
UV inactivation studies were conducted with multiple strains of influenza,
using
manufacturing scale equipment, to demonstrate safety and efficacy. Three
strains of
influenza were selected, corresponding to those manufactured for the 2004-2005
season. Each lot was evaluated in two independent runs. Inactivation was
evaluated at
five UV intensity set-points (2, 6, 10, 12 and 14mW/cm2 ) a predetermined
production
target (12mW/cm2). All other conditions were representative of the exemplary
manufacturing process described above (e.g., flow rate 755m1/min, rotational
speed
320rpm, and cylinder angle 30 ).
For each run, the influenza virus containing allantoic fluid was harvested,
and
samples were taken prior to irradiation. The material was processed through
the
irradiator at the various UV intensity set-points listed above, and a second
set of
samples was taken. All samples were evaluated for the presence of infectious
influenza
virus by EID50 and for bioburden, samples were also analyzed for potential
damage to
the HA antigen. A summary of the results of the inactivation studies are shown
in FIG.
20 and Table 4.
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Table 4: Loglo Reduction Factors for UV Inactivation of Influenza Virus
Lot/Strain UV Intensity (mW/cmz)
0 2 6 10 12 14
Lot 3723 B/J/10/2003 -0.1 5.2 8.4 8.2 8.4 8.4
-0.2 4.2 7.8 7.6 7.6 7.6
Lot 3724 A/NC/20/99 0.0 3.3 6.1 9.0 8.8 8.8
-0.2 3.0 6.0 8.7 8.7 8.7
Lot 3725 A/W/3/2003 -0.2 3.4 6.9 9.1 9.0 8.9
-0.3 4.4 7.2 9.6 9.7 9.6
4
Process
Set-
point
The results illustrated in FIG. 20 demonstrate that inactivation for all three
strains
(New Caledonia, Wyoming, and Jiangsu) occurs at a minimum average set-point of
10mW/cmZ. The B/Jiangsu strain is inactivated at a set-point of 6mW/cm2. At an
irradiation intensity of 12mW/cmZ (process set-point), a mean of 9.1 logs (7.6
to 9.7
dependent on the strain) reduction of influenza virus was observed. Residual
infectivity
remaining after UV inactivation was eliminated by formalin treatment.
Additional studies were performed at higher flow rates and higher UV
intensities.
The Log Reduction Factor data is presented in the following table. The data,
shown in
Table 5, indicate the UV is highly effective in inactivating Influenza virus
even at high
flow rates. HA antigen integrity, as evaluated by total protein and HA titer,
did not show
any damage with increasing UV intensity levels.
Table 5: Loglo Reduction Factors for UV Inactivation of Influenza Virus at
High Flow
Rates & UV Intensity of 18 mW/cmz
Lot/Strain Flow Rate (L / min)
1.7 1.9 2.2 2.8 3.5
Lot 3723 B/J/10/2003 _8.2 _ 8.2
Lot 3724 A/NC/20/99 _8.66 _8.76
Lot 3725 A/W/3/2003 _9.51 ?9.51 _9.51 _8.85 6.98
UV irradiation of allantoic fluid containing A/New Caledonia influenza
resulted in a
decrease in viral infectivity. No bacterial growth was observed before or
after UV
irradiation, and UV irradiation had no effect on the hemagglutination
properties (HA
titer) of the virus.
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Example 3: Comparison of UV Inactivation for the A/New York/55/2004 and A/New
Caledonia strains
The inactivation steps used in influenza virus manufacturing processes of the
instant invention have been sufficient for all strains used to date.
Nevertheless, the
inactivation steps are typically re-validated for all new strains of influenza
virus on a
yearly basis to confirm suitability of the process. For example, in the 2005-
2006 season,
one new strain (A/New York/55/2004) was used. The ability of the manufacturing
process to provide complete influenza virus inactivation was assessed by the
following
experiments:
Allantoic fluid containing virus of the A/New York/55/2004 strain was
clarified by
low speed centrifugation (3000 rpm, 10 min., room temperature); this
clarification
procedure was previously shown not to alter viral concentration. Allantoic
fluid
containing the A/New Caledonia strain was clarified and used as a control for
this
experiment. Both samples were UV-irradiated using the same conditions
described
above, and aliquots were taken for analysis. Under manufacturing conditions,
the UV
irradiated fluid is subsequently treated with formaldehyde. The results for
the A/New
Caledonia and A/New York strains are shown in Table 6.
Table 6: Inactivation of A/New Caledonia/20/99 and A/New York/55/2004 by UV
Irradiation
Virus EID o/0.2 ml Bioburden CFU/ml HA titer
Before UV After UV Before UV After UV Before UV After UV
A/New 10-8.6 <100 <10 <10 131072 131072
Caledonia/20/99
A/New 10-8'8 <100 <100 <100 4096 4096
York/55/2004
UV irradiation of allantoic fluid containing A/New Caledonia influenza
resulted in a
decrease in viral infectivity of at least 8.6 loglo as measured by EID50
(Table 6). This
level of viral inactivation is similar to that previously observed with this
strain and serves
as a control for the experiment with the New York strain. No bacterial growth
was
observed before or after UV irradiation, and UV irradiation had no effect on
the
hemagglutination properties (HA titer) of the virus.
UV irradiation of the clarified allantoic fluid containing the A/New York
strain
produced an 8.8 log reduction in viral infectivity as measured by EIDso (Table
6). This
level of inactivation is comparable to that of A/New Caledonia. Again, the 8.8-
log
reduction of viral infectivity of A/New York by UV irradiation alone did not
result in full
inactivation, as live virus was detected by the viral inactivation assay in
the UV irradiated
sample. No bacterial growth was observed in the A/New York test samples,
either before
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or after irradiation, and UV irradiation had no effect on the hemagglutination
properties
(HA titer) of the virus.
The results illustrate the widespread applicability of the disclosed methods
for
inactivating influenza virus with UV light.
Example 4: Inactivation of Adventitious agents and Reduction of Bioburden by
UV Light
Adventitious viral or bacterial agents can originate in the chickens from
which
eggs are obtained (e.g., endogenous retrovirus) or be adventitiously
introduced during
production. Although inactivated influenza vaccines produced in eggs have not
been
implicated in the transmission of viruses or disease, a testing strategy to
ensure that
manufactured drug product is free of microbial contamination nonetheless
warranted. A
significant element in this strategy is the inclusion of steps in the
manufacturing process
with the capability to clear (by removal or inactivation) any contamination
that might
occur during the production process. A quantitative assessment of the
microbial
clearance capability of the process is part of the documentation of the
assurance of
safety of the vaccine. This assessment is carried out by experimentally
determining the
maximum amount of contaminant that can be inactivated by each individual step,
and
then calculating the total process inactivation capability as the sum of the
individual
steps.
Adventitious agent clearance in processes of the instant invention is
accomplished
by inactivation of infectivity, rather than removal. The inactivation capacity
of this step
has been assessed by spiking appropriate process samples with selected model
microbes
(two model viruses and four species of Mycoplasma) in laboratory studies that
replicate
the conditions of manufacturing operations.
In order to most accurately replicate the actual manufacturing conditions of
the
UV step during the inactivation studies, the test article to which the spiking
microorganism was added consisted of allantoic fluid harvest containing
influenza virus
(A/Wyoming strain). This strain of virus was chosen because it contained the
highest
virus titer among strains being produced at the time the studies were
performed (2004-
2005 strains), and thus offered the most potential to interfere with the UV
inactivation of
any adventitious agents present. In order to use this test article for these
studies, the
influenza virus itself was first inactivated, so that it would not interfere
with detection of
the spiking agents. Therefore, allantoic fluid containing influenza virus was
subjected to
UV irradiation, spiked with the specified adventitious agent, and then re-
irradiated as
part of the inactivation study.
The clearance studies were performed using two model viruses and four species
of Mycoplasma as spiking microorganisms (Table 7). The rationale for selection
of each
of these microorganisms is further described below.
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Table 7: Model Organisms Used in Spiking Studies
Organism Comment
Xenotropic Murine Leukemia Virus Enveloped RNA retrovirus
(XMuLV)
Human Adenovirus Type 2 (Ad-2) Non-enveloped DNA virus
Mycoplasma Species:
M. orale
M. gallisepticum Species known to infect and cause disease in
avian species and humans
M. pneumoniae
M. synoviae
An aliquot of the test material (UV-treated allantoic fluid) was spiked with a
known quantity of the model virus or Mycoplasma of interest. Aliquots were
taken to
serve as pretreatment samples which were analyzed immediately, and hold
samples,
which were not further processed, but were analyzed with the UV-irradiated
samples, to
serve as a control for any potential holding time effects. The remaining
volume of test
material was divided into two aliquots, and each aliquot was independently
processed at
the selected UV irradiation level. This procedure was repeated for each of the
five UV
doses tested.
All samples were run identically at a constant flow rate of 755 75 mI/min The
UV irradiation doses used were 2, 6, 10, 12 (manufacturing set-point), and
14mW/cm2.
After processing, each aliquot was analyzed to determine the titer of the
model agent by
the appropriate methods as described above, both titration and large volume
plating
were used. See the above discussion on titer determination by titration and
large
volume plating. When no challenge organism is detected in the titration
sample, the
large volume plating value is reported because of the improved limit of
detection. The
log-reduction factor (LRF) is calculated as the loglo of the ratio of the
virus load in the
starting material to that in the irradiated sample.
a. UV Inactivation of XMuLV and Adenovirus 2
XMuLV (Xenotropic Murine Leukemia Virus) is a model for avian leukosis virus
(ALV), which is an endogenous retrovirus of chickens that can be transmitted
vertically
and thus potentially appear in eggs. Hussain, et al., Emerg. Infect. Dis. 7:
66-72
(2001). ALV genomic RNA and reverse transcriptase have been detected in chick
cell-
grown vaccines, and endogenous ALV antigens and reverse transcriptase activity
can be
found in eggs, but evidence of human infection due to these vaccines is
lacking. XMuLV
is an enveloped RNA virus of 80-110 nm. in diameter that can be readily grown
in culture
to high titers and reliably assayed, making it suitable for use in such
spiking studies.
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XMuLV was chosen as a model for ALV because of the inability to grow ALV to
sufficiently
high enough titers to facilitate spiking studies. Inactivation of XMuLV is
measured by the
change in the 50% tissue culture infectious dose (TCID50) endpoint assay in PG-
4 cells.
Adenovirus 2 (Ad2) is a model for avian adenoviruses and other non-enveloped
DNA viruses. Adenoviruses are ubiquitous in nature and have been shown to
infect
many vertebrate species, including birds. The avian adenoviruses have a wide
range of
virulence in chickens, with infections ranging from sub-clinical to
symptomatic
outbreaks. Adenoviruses can be transmitted horizontally or vertically via the
egg. Virus
shedding may produce a potentially high titer in allantoic fluid, although no
shedding is
usually detectable following seroconversion. Avian adenoviruses are not
generally
believed to be of public health significance, as they are unable to undergo
productive
replication in human cells, and are more of an environmental concern. Ad2 is a
non-
enveloped DNA virus of 90nm in diameter that can be readily grown in culture
to high
titers and reliably assayed, making it suitable for use in such spiking
studies.
Inactivation of Ad2 is measured by the change in the 50% tissue culture
infectious dose
(TCID50) endpoint assay in A549 cells.
Samples were spiked with either XMuLV to an initial concentration of (loglo
TCID50/mL) of 8-9, or Ad-2 to an initial concentration of 7-8.8. The mean log
reduction
factors after UV irradiation are presented in FIG. 21. After UV treatment,
minimal
reduction in infectivity (1 log maximum) was observed for both viruses, even
at the
highest UV dose (14mW/cmZ) tested, demonstrating that this step does not
provide
significant inactivation for these organisms.
b. UV Inactivation of Mycoplasma Species
Various species of Mycoplasma are known to infect and cause disease in avian
and mammalian hosts. Some species of Mycoplasma, such as M. gallisepticum and
M.
synoviae, are likely to occur in chickens, but are not known to be mammalian
pathogens.
Other species, such as M. orale and M. pneumoniae, are of human origin and may
enter
the process stream via infected operators. These species can be grown to high
titers
(with the exception of M. synoviae) and reliably assayed, making them suitable
for
spiking studies. Inactivation of Mycoplasma is measured by the change in titer
determined from CFU counts on agar plates.
Samples were spiked with Mycoplasma species to achieve concentrations of
(loglo
CFU/ml) 6.3 to 9Ø In contrast to studies with the model virus, UV
irradiation results in
significant reduction in Mycoplasma titer for all four species tested. At all
UV irradiation
doses >10mW/cm2, the test Mycoplasma organism was reduced to undetectable
levels.
A dose-response curve presenting the data at all UV doses tested is presented
in FIG.
21, and the data are presented in tabular format in Table 8. The results
indicate that UV
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treatment is a robust Mycoplasma inactivation step. The log reduction factors
range
from at least 2.28 to 3.86 at a UV irradiation level of 12mW/cm2, however the
calculated
values were limited by the starting titers and assay limits of detection due
to sample
dilutions and statistical considerations.
Table 8: Mean Log Reduction Factors for UV Inactivation of Adventitious
Agentsa
UV Irradiation Dose
Model Agent
2mW/cm2 6mW/cm2 10mW/cm2 12mW/cm2 14mW/cmZ
XMuLV -0.43 0.53 0.06 0.49 0.44 0.63 0.62 0.56 0.73 0.60
Ad-2 0.25 0.47 0.61 0.55 -0.11f0.41 0.82 0.42 1.19 0.51
M. >.49t0.49b >_2.07f0.48 _2.90f0.40 >2.77f0.46 _2.68f0.40
gallisepticum
M. 0.57 0.58 2.59 0.12 _4.09f0.86 3.86 0.07 _4.12f0.43
pneumoniae
M. synoviae 1.36 0.69 _2.06f0.69 >2.47f0.25 _2.28t0.27 _2.44t0.45
M. orale _1.38f0.53 _1.48f1.26 >2.29f0.30 _2.33f0.50 _2.13f0.36
4
Process Set-
point
aAll values are shown as the average log reduction factor and 95% confidence
interval of
duplicate runs.
b'x >_"Reflects the fact that the clearance factor is a minimum value in that
no viable
microorganisms were detected in the sample tested and the reported value is
limited by
statistical considerations regarding the relative sample size and the assay's
limit of
detection.
Example 6: UV Process Capability for Inactivation of Adventitious Acients
The results for the process set-point of 12mW/cm2 of UV irradiation, are
summarized in Table 9 below in terms of the LRFs for each model microorganism.
It is
evident from the data that UV treatment is highly effective inactivating all
model
Mycoplasma species tested. Inactivation of the model viruses tested was not
100%
effective. Accordingly, the fluid can be further processed with a chemical
inactivating
agent, such as formaldehyde, to fully eliminate any risk of adventitious
agents in the
final drug product.
Table 9: Inactivation of adventitious agents and bioburden by UV light
V i ru s Mycoplasma
XMuLVb Adenovirus M. M. M. synoviae M. orale
2 gallisepticum pneumoniae
0.62 0.56 0.82 0.42 _2.77f0.46` 3.86 0.07 _2.28f0.27 _2.33f0.50
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AII publications and references, including but not limited to patents and
patent
applications, cited in this specification are herein incorporated by reference
in their
entirety, as if each individual publication or reference were specifically and
individually
indicated to be incorporated by reference herein as being fully set forth.
Although the invention is illustrated and described herein with reference to
specific embodiments, the invention is not intended to be limited to the
details shown.
Rather, various modifications may be made in the details within the scope and
range of
equivalents of the claims and without departing from the invention.
