WO2008039494A1 - Device and methods of inactivating influenza virus and adventitious agents with ultraviolet light - Google Patents

Abstract

A device (100) for irradiating a fluid is disclosed. The device (100) includes an elongated tube (106) having a fluid injection port (106a) and a fluid discharge port (106b), wherein the elongated tube (106) is rotatable about a longitudinal axis (107). The longitudinal axis (107) extends at an angle oblique to the horizontal. A source of radiation (116) extends within the elongated tube (106) along the longitudinal axis (107). A sleeve (146) extends within the elongated tube (106) and surrounds a length of the source of radiation (116), thereby defining an airflow path between the source of radiation (116) and the sleeve (146). An air flow source (164) is in fluid communication with the airflow path proximate to the fluid injection port (106a). An air flow discharge (180) is in fluid communication with the airflow path proximate to the fluid discharge port (106b). The fluid injection port (106a) and the fluid discharge port (106b) are in communication with the space between the tube (106) and the sleeve ( 146). A method of inactivating a virus and an inactivated virus manufactured according to the method are also disclosed.

Description

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 combination 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 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; 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 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 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 ml/min (0.6 L/min). The fluid can be introduced at rates up to approximately 3500 ml/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 ml/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 850ml/min, more commonly, the flow rate is set below about 840ml/min, such as less than about 830ml/min. Favorably, the method involves introducing the fluid containing live virus at a flow rate of about 755ml/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/cm², 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 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 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. 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 pott 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 discharge axis 131, and generally oblong discharge opening 134 extends at an angle "α" 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 "α" 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/cm² ± 2 mW/cm² as measured at sensors 138. In an exemplary embodiment, the intensity may be between about 10 mW/cm² and 14 mW/cm². In another exemplary embodiment, the intensity may be about 12 mW/cm². 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). 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 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 brushless DC motor. A drive pulley 190, driven by motor 188, drives a timing belt 192. Timing belt is 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/cm² ± 2 mW/cm². 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. 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 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. 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 30° (e.g., 20°-40°) 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 IOOOL (mean of 10ml_/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 reassembled, 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

Table 2: UV Irradiation Parameters Example 2

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 ml/min, or at least about 675 ml/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 ml/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/cm².

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, 18^th 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 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.

Example 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 Ka^'rber 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, 6^th Ed. (1989). Log reduction factors (LRF) are calculated using the following formula:

Logi₀ (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 + ... LRF_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 (((CL₁ ²) + (CL₂ ²) + ... (CL_π ²))//7)) 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). All 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

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 755ml/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. Table 4: Logio Reduction Factors for UV Inactivation of Influenza Virus

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 lOmW/cm². The B/Jiangsu strain is inactivated at a set-point of 6mW/cm². At an irradiation intensity of 12mW/cm² (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: Logio Reduction Factors for UV Inactivation of Influenza Virus at High Flow

Rates & UV Intensity of 18 mW/cm²

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. 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

UV irradiation of allantoic fluid containing A/New Caledonia influenza resulted in a decrease in viral infectivity of at least 8.6 logi₀ 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 EID₅₀ (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 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. Table 7: Model Organisms Used in Spiking Studies

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 ml/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 logio 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 a/., 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. 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 (log₁₀ 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/cm²) 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 (log₁₀ CFU/ml) 6.3 to 9.0. 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 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 Agents^a

ϋ

Process Set- point aAII values are shown as the average log reduction factor and 95% confidence interval of duplicate runs. b"≥"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 Agents

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

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.

Claims

What is claimed: 1. A device (100) for irradiating a fluid, the device (100) comprising: an elongated tube (106) having a fluid injection port (106a)and a fluid discharge port (106b), wherein the elongated tube (106) is rotatable about a longitudinal axis (107), the longitudinal axis (107) extending at an angle oblique to the horizontal; a source of radiation (116) extending within the elongated tube (106) along the longitudinal axis (107); a sleeve (146) extending within the elongated tube (106) and surrounding a length of the source of radiation (116), thereby defining an airflow path between the source of radiation (116) and the sleeve (146); an air flow source (164) in fluid communication with the airflow path proximate to the fluid injection port (106a); and an air flow discharge (180) in fluid communication with the airflow path proximate to the fluid discharge port (106b), wherein the fluid injection port (106a) and the fluid discharge port (106b) are in communication with the space between the tube (106) and the sleeve (146). 2. The device according to claim 1, wherein the source of ultraviolet radiation (116) comprises a support member (142) extending along a length of the source of radiation (116) and a ground member (144) extending parallel to the support member (142). 3. The device according to claim 1, further comprising : an injection box (118) mounted at the fluid injection port (106a); at least one sensor (138) disposed within the injection box (118) upstream of the fluid injection port (106a) and targeted to obtain data from the source of radiation (116); and a fluid injection needle (126) coupled to the injection box (118) between the at least one sensor (138) and the elongated tube (106), wherein a discharge end (130) of the fluid injection needle (126) extends into the space between the sleeve (146) and the elongated tube (106). 4. The device according to claim 3, wherein each of the at least one sensor (138) is removably installed in the injection box (118) a predetermined distance from the source of ultraviolet radiation (116). 5. The device according to claim 3, wherein the fluid injection needle (126) comprises a generally oblong discharge opening (134), and wherein the discharge end (130) is coupled to the injection box at a declination angle between about 3 degrees and about 7 degrees from a center line of the elongated tube (106). 6. The device according to claim 1, further comprising a single bearing assembly (202) comprised of at least two axially spaced apart bearing members (204) surrounding and supporting the elongated tube (106) between the fluid injection port (106a) and the fluid discharge port (106b) and permitting the rotational movement thereof. 7. The device according to claim 1, wherein the source of radiation (116) comprises a plurality of sources (140) extending parallel to the longitudinal axis (107). 8. The device according to claim 1, wherein the source of irradiation (116) comprises an ultraviolet source of irradiation (140). 9. A device (100) for irradiating a fluid, the device comprising: an elongated tube (106) having a fluid injection end (106a), a fluid discharge end (106b), and a longitudinal axis (107) extending between the fluid injection end (106a) and the fluid discharge end (106b); an injection box (118) mounted at the fluid injection end (106a) of the elongated tube (106b); an elongated source of radiation (116) extending within the tube (106) and the injection box (118) along the longitudinal axis (107); at least one sensor (138) disposed within the injection box (118) upstream of the fluid injection end (106a) of the elongated tube (106) and targeted to obtain data from the elongated source of radiation (116), wherein the at least one sensor (138) is located to avoid sensing fluid flowing through the elongated tube (106); and a fluid injection needle (126) coupled to the injection box (118) between the at least one sensor (138) and the elongated tube (106), wherein a discharge end (130) of the fluid injection needle (126) extends into the space between the sleeve (146) and the elongated tube (106). 10. The device according to claim 9, wherein the injection box (118) comprises a generally circular cross section. 11. The device according to claim 9, wherein the fluid injection needle (126) comprises a generally oblong discharge opening (134), and wherein the discharge end (130) is coupled to the injection box (118) at a declination angle between about 3 degrees and about 7 degrees from a center line of the elongated tube (106). 12. The device according to claim 11, wherein the discharge end (130) of the fluid injection needle (126) extends along a discharge axis (131), and the generally oblong discharge opening (134) extends at an angle (α) oblique to the discharge axis (131). 13. The device according to claim 9, wherein the fluid injection needle (126) is positioned such that fluid discharged from the fluid injection needle (126) is discharged along the elongated tube (106). 14. The device according to claim 9, wherein each of the at least one sensor (138) is removably installed in the injection box (118) a predetermined distance from the source of radiation (116).

15. The device according to claim 9, wherein the source of radiation comprises a plurality of light sources extending parallel to the longitudinal axis (107). 16. The device according to claim 9, wherein the source of irradiation (116) comprises an ultraviolet source of irradiation (140). 17. In a device (100) for irradiating a fluid, the device (100) comprising: a rotatable elongated tube (106) having a fluid injection end (106a), a fluid discharge end (106b), and a longitudinal axis extending between the fluid injection end and the fluid discharge end; and an elongated source of radiation (116) extending through the tube (106) along the longitudinal axis (107) of the tube (106); an improvement comprising a single bearing assembly (202) comprised of at least two axially spaced apart bearing members (204) surrounding and supporting the elongated tube (106) between the fluid injection port (106a) and the fluid discharge port (106b) and permitting the rotational movement thereof. 18. The device according to claim 17, wherein the bearing assembly (202) is concentrically mounted to the elongated tube (106). 19. The device according to claim 17, wherein the source of irradiation (116) comprises an ultraviolet source of irradiation (140). 20. A device (100) for irradiating a fluid, the device comprising : an elongated tube (106) having a fluid injection port (106a), a fluid discharge port (106b), and a longitudinal axis (107) extending therethrough, wherein the tube (106) is rotatable about the longitudinal axis (107) and wherein the longitudinal axis (107) is angled at a sufficient angle relative to the horizontal to impart a gravity flow of a fluid along a length of the tube (106); an injection box (118) mounted at the fluid injection port (106a); a source of ultraviolet radiation (116) extending within the tube (106) along the longitudinal axis (107), wherein the source of ultraviolet radiation (116) comprises a plurality of ultraviolet light sources (140) extending longitudinally along the tube (106); a sleeve (146) surrounding and extending along a length of the source of ultraviolet radiation (116) defining an airflow path between the source of ultraviolet radiation (116) and the sleeve (146) and a fluid flow path between the sleeve (146) and the tube (106); an air flow source (164) in fluid communication with the airflow path proximate to the fluid injection port (106a); an air flow discharge (180) in fluid communication with the airflow path proximate to the fluid discharge port (106b); at least one sensor (138) disposed within the injection box (118) upstream of the fluid injection port (106a) and targeted to obtain data from the elongated source of ultraviolet radiation (116), wherein the at least one sensor (138) is located to avoid sensing fluid flowing through the elongated tube (106); a fluid injection needle (126) coupled to the injection box (118) between the at least one sensor (138) and the elongated tube (106), wherein a discharge end (130) of the fluid injection needle (126) extends into the elongated tube (106); and a single bearing assembly (202) comprised of at least two axially spaced apart bearing members (204) surrounding and supporting the elongated tube (106) between the fluid injection port (106a) and the fluid discharge port (106b) and permitting the rotational movement thereof. 21. A method for inactivating a virus comprising the steps of: introducing a fluid comprising a live virus to an elongated tube (106), wherein the elongated tube (106) is inclined relative to the horizontal; rotating the elongated tube (106) along its longitudinal axis (107), thereby dispersing the fluid along an interior length of the elongated tube (106); irradiating the fluid with a radiation source (116) disposed within the tube (106) and extending along the longitudinal axis (107) of the elongated tube (106), thereby transforming the live virus to an inactivated virus; protecting the radiation source (116) from direct contact with the live virus by providing a sleeve (146) between the radiation source (116) and the live virus along the length of the elongated tube (106); maintaining a desired temperature range of the radiation source (116) by providing a flow of air along a length of the radiation source (116) between the radiation source (116) and the sleeve (146); and causing the fluid containing the inactivated virus to exit the elongated tube (106). 22. The method according to claim 21, wherein the introducing step comprises introducing the fluid along an interior wall of the elongated tube (106). 23. The method according to claim 21, wherein the introducing step comprises introducing the fluid at a flow rate of between about 600 ml/min and about 900 ml/min. 24. The method according to claim 23, wherein the flow is from about 650ml/min to about 850ml/min. 25. The method according to claim 23, wherein the flow rate is from about 675ml/min to about 840ml/min. 26. The method according to claim 23, wherein the flow rate is from about 680ml/min to about 830ml/min.

27. The method according to claim 23, wherein the flow rate is about 755ml/min. 28. The method according to claim 21, wherein the elongated tube (106) is a cylinder. 29. The method according to claim 21, wherein the elongated tube (106) is inclined between about 20 degrees and about 40 degrees relative to the horizontal. 30. The method according to claim 21, wherein the elongated tube (106) is inclined about 30 degrees relative to the horizontal. 31. The method according to claim 21, wherein the radiation source (116) emits ultraviolet (UV) light at a wavelength of about 254 nanometers. 32. The method according to claim 21, wherein the radiation source (116) comprises a plurality of UV lamps (140). 33. The method according to claim 21, comprising irradiating the fluid with UV light at an intensity of at least about 10 mWatts/cm². 34. The method according to claim 21, comprising irradiating the fluid with UV light at an intensity of between about 10 and about 14 mWatts/cm². 35 The method according to claim 34, comprising irradiating the fluid with UV light at an intensity of about 12 mWatts/cm2. 36. The method according to claim 21, wherein the sleeve (146) is a quartz sleeve. 37. The method according to claim 21, comprising causing the fluid containing the inactivated virus to exit the elongated tube (106) by gravity. 38. The method according to claim 21, wherein the live virus is highly pathogenic in humans. 39. The method according to claim 21, wherein the live virus is influenza. 40. The method according to claim 21, wherein the live virus is a pandemic or avian strain of influenza 41 The method according to claim 21, wherein the fluid comprising live virus comprises allantoic fluid. 42. The method according to claim 41, wherein the allantoic fluid is obtained from embryonic chicken eggs. 43. The method according to claim 21, wherein the fluid comprising live virus comprises cell culture medium. 44. The method of claim 21, wherein irradiating the fluid comprising live virus inactivates adventitious virus and bacteria present in the fluid. 45. An inactivated virus manufactured by the method of claim 21. 46. A pharmaceutical composition comprising the inactivated virus of claim 45, formulated with a pharmaceutically acceptable excipient or carrier.

47. A pharmaceutical composition according to claim 46, further comprising an adjuvant. 48. A method of protecting a subject against a viral infection, comprising administering to the subject an immunologically effective dose of the pharmaceutical composition of claim 47.