WO1998055156A1 - Method of inactivating viruses in a cell-containing liquid - Google Patents

Method of inactivating viruses in a cell-containing liquid Download PDF

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Publication number
WO1998055156A1
WO1998055156A1 PCT/NL1998/000327 NL9800327W WO9855156A1 WO 1998055156 A1 WO1998055156 A1 WO 1998055156A1 NL 9800327 W NL9800327 W NL 9800327W WO 9855156 A1 WO9855156 A1 WO 9855156A1
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WIPO (PCT)
Prior art keywords
cell
containing liquid
virus
cells
concentration
Prior art date
Application number
PCT/NL1998/000327
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French (fr)
Dutch (nl)
Inventor
Johnny Van Steveninck
Thomas Marinus Albert Remko Dubbelman
Agatha Brand
Original Assignee
Rijksuniversiteit Leiden
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Application filed by Rijksuniversiteit Leiden filed Critical Rijksuniversiteit Leiden
Publication of WO1998055156A1 publication Critical patent/WO1998055156A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
    • A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
    • A61L2/0011—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using physical methods

Definitions

  • the present invention relates to a method of treating a cell-containing liquid in order to inactivate a virus present therein in the presence of dissolved molecular oxygen, which method comprises: i) introducing a sensitizer into the cell-containing liquid; and subsequently ii) exposing the sensitizer-containing liquid for a predetermined period of time to light of a chosen intensity and of a wavelength at which it is absorbed by the sensitizer, thus producing a quantity of reactive oxygen, which quantity suffices to substantially inactivate the viruses while the cells are substantially spared.
  • This quantity of reactive oxygen formed by the synthesizer depends on the light intensity of the light to which the liquid is exposed and the duration of exposure.
  • the Examples show that exposure lasts 10 minutes to 3 hours with a total light energy from 10 to 1000 J/cm 2 .
  • the known sensitizers exhibit little specificity and bind to both viruses and cells.
  • the object of the present invention is to improve the known method, and in particular, to improve the ratio of damaged cells in relation to inactivated viruses.
  • the method according to the invention is characterized in that the liquid is exposed to light of a chosen high intensity and for a chosen short period of time, thereby creating an environment surrounding the cell in which the concentration reactive oxygen is reduced in comparison with the concentration reactive oxygen surrounding the virus .
  • the total light energy used for the inactivation of the virus is kept constant whereas the duration is varied, in order to determine at what point of shortened duration the damage caused to the cells dimin- ishes, while the virus-inactivation remains the same.
  • the reactive oxygen concentration is reduced by at least 50%, preferably at least 90%. This reduction can be calculated on the basis of oxygen concentration measurements and the diffusion constant of oxygen. This has been described in the thesis by J.F. Keij ("The zapper", 1994, Leiden) .
  • the short duration will be less than 1 second, particularly less than 0.2 seconds and more particularly less than 0.04 seconds.
  • the high intensity will be higher than 20 kW/m J , particularly higher than 100 kW/m 2 and more particularly higher than 550 kW/m a .
  • the concentration dissolved molecular oxygen in the cell-containing, sensitizer-comprising liquid is preferably controlled.
  • the term controlled in the present invention is meant to indicate that said concentration is measured and/or adjusted.
  • the con- centration of dissolved oxygen is reduced in particular by, for instance, injecting a low-oxygen nitrogen-oxygen gas mixture.
  • the concentration dissolved molecular oxygen is reduced to less than 25%, preferably to less than 10% and most preferably to less than 3%.
  • the concentration of dissolved oxygen is reduced by admixing a photo-oxydi- zable compound to the liquid.
  • Such a photo-oxydizable compound is preferably a compound which selectively binds to the cells to be protected from damage.
  • the photo-oxydizable compound may, for instance, be a compound binding to the cell membranes.
  • An important application of the present invention concerns the treatment of a erythrocyte-containing liquid. It is preferred that said erythrocyte-containing liquid be treated at a lowered temperature, particularly at a temperature of from 4 - 10°C.
  • Haemoglobin present in the erythrocytes is able to retain oxygen better at a lower temperature, and so the damage caused to the erythrocytes is limited.
  • Another important application concerns the treatment of a stem cell-containing liquid.
  • erythrocyte-containing liquid and stem cell-containing liquid are important for transplantation purposes, and it is vital that the recipient does not become contaminated by viruses from the donor.
  • VSV Vesicular Stomatitis Virus
  • VSV Vesicular Stomatitis Virus
  • VSV is cultured in a manner known in itself on baby hamster kidney (BHK) cells and stored as stock solution at - 80°C. Based on the stock solution a virus-containing sol- ution is prepared of 10 4 - 5 infectious particles per ml phosphate buffer (PBS) . The suspension is incubated for 20 min. in the dark with 0.02 ⁇ g/ml dodecyl acridine orange (DAO) and subsequently introduced into a syringe. This syringe is placed into a perfusor pump which is adjustable to different pump velocities. b) Preparation of Kluyveromyces marxianus (CBS 397) .
  • PBS phosphate buffer
  • the yeast is cultured aerobically for 16 hours at 30°C in 2% glucose medium.
  • a yeast cell-containing liquid is prepared by suspending 1.10 6 yeast cells/ml in Dulbecco's PBS. The yeast is incubated for 20 min. in the dark with 0.25 ⁇ g/ml DAO. As described for a), this yeast cell-containing liquid is also placed in a syringe, into a perfusor pump.
  • the VSV- or yeast-containing liquid is passed through a glass capillary having an inside diameter of 1 mm and exposed to an Argon laser at 488 nm. The beam has a diameter of 1.5 mm. The light intensity and the flow rate are varied whereas the total amount of light energy is kept constant (21.5 kJ/m 2 ), as shown in Table I.

Abstract

The invention relates to a method of inactivating a virus in a cell-containing liquid with the aid of light. In accordance with the invention a sensitizer is introduced into the cell- and virus-containing liquid and the liquid is exposed for a short period of time to a high light intensity in the presence of oxygen. Surprisingly, it was found that by combining a high intensity and a short exposure time, viruses can be inactivated while the damage to the cells is significantly limited.

Description

Method of inactivating viruses in a cell-containing liquid
The present invention relates to a method of treating a cell-containing liquid in order to inactivate a virus present therein in the presence of dissolved molecular oxygen, which method comprises: i) introducing a sensitizer into the cell-containing liquid; and subsequently ii) exposing the sensitizer-containing liquid for a predetermined period of time to light of a chosen intensity and of a wavelength at which it is absorbed by the sensitizer, thus producing a quantity of reactive oxygen, which quantity suffices to substantially inactivate the viruses while the cells are substantially spared.
Such a method is known from the US patent specification 5,120,649. According to the method known from this publication photodynamic inactivation is applied to inactivate viruses in a blood cell-containing liquid. A photo- reactive compound, preferably a phthalocyanine, is brought into contact with a cell-containing liquid such as whole blood. For the destruction of a virus a quantity of reac- tive oxygen, in particular singlet oxygen, is required.
This quantity of reactive oxygen formed by the synthesizer depends on the light intensity of the light to which the liquid is exposed and the duration of exposure. The Examples show that exposure lasts 10 minutes to 3 hours with a total light energy from 10 to 1000 J/cm2. In general, the known sensitizers exhibit little specificity and bind to both viruses and cells.
The object of the present invention is to improve the known method, and in particular, to improve the ratio of damaged cells in relation to inactivated viruses.
To this end the method according to the invention is characterized in that the liquid is exposed to light of a chosen high intensity and for a chosen short period of time, thereby creating an environment surrounding the cell in which the concentration reactive oxygen is reduced in comparison with the concentration reactive oxygen surrounding the virus .
Surprisingly it was found, that it is possible to inactivate viruses just as effectively by exposure to light of high intensity for a short time, while causing less damage to the cells. Without wishing to be bound by any theory it is believed, that a diffusion limitation of dissolved molecular oxygen plays a role. It is thought that at short exposure with high intensity less molecular oxygen can be supplied from the vicinity of the cell than from the vicinity of a virus . A person skilled in the art can easily determine how short a time and how high an intensity are required in order to reduce the concentration reactive oxygen in the vicinity of the cell as com- pared with the concentration reactive oxygen around the virus. To do this, the total light energy used for the inactivation of the virus is kept constant whereas the duration is varied, in order to determine at what point of shortened duration the damage caused to the cells dimin- ishes, while the virus-inactivation remains the same. Preferably the reactive oxygen concentration is reduced by at least 50%, preferably at least 90%. This reduction can be calculated on the basis of oxygen concentration measurements and the diffusion constant of oxygen. This has been described in the thesis by J.F. Keij ("The zapper", 1994, Leiden) .
How short the period of time and how high the intensity has to be depends on factors such as the concentration of sensitizer, its effectiveness in generating oxygen and the concentration molecular of oxygen.
In a typical case the short duration will be less than 1 second, particularly less than 0.2 seconds and more particularly less than 0.04 seconds. Typically the high intensity will be higher than 20 kW/mJ , particularly higher than 100 kW/m2 and more particularly higher than 550 kW/ma .
The concentration dissolved molecular oxygen in the cell-containing, sensitizer-comprising liquid is preferably controlled. The term controlled in the present invention is meant to indicate that said concentration is measured and/or adjusted.
In the method according to the invention the con- centration of dissolved oxygen is reduced in particular by, for instance, injecting a low-oxygen nitrogen-oxygen gas mixture.
In particular, the concentration dissolved molecular oxygen is reduced to less than 25%, preferably to less than 10% and most preferably to less than 3%.
According to a simple embodiment the concentration of dissolved oxygen is reduced by admixing a photo-oxydi- zable compound to the liquid.
Such a photo-oxydizable compound is preferably a compound which selectively binds to the cells to be protected from damage. For the inactivation of membrane-less viruses the photo-oxydizable compound may, for instance, be a compound binding to the cell membranes.
An important application of the present invention concerns the treatment of a erythrocyte-containing liquid. It is preferred that said erythrocyte-containing liquid be treated at a lowered temperature, particularly at a temperature of from 4 - 10°C.
Haemoglobin present in the erythrocytes is able to retain oxygen better at a lower temperature, and so the damage caused to the erythrocytes is limited.
Another important application concerns the treatment of a stem cell-containing liquid.
The treatment of erythrocyte-containing liquid and stem cell-containing liquid is important for transplantation purposes, and it is vital that the recipient does not become contaminated by viruses from the donor.
The invention will now be elucidated with reference to examples and a drawing, in which the only Figure is a graph of the inactivation of Vesicular Stomatitis Virus (VSV) and yeast as function of the exposure intensity. Example I
Effect of duration and intensity on the inactiva- - tion of yeast cells and virus
EXPERIMENTAL SET-UP a) Preparation of Vesicular Stomatitis Virus (VSV) .
VSV is cultured in a manner known in itself on baby hamster kidney (BHK) cells and stored as stock solution at - 80°C. Based on the stock solution a virus-containing sol- ution is prepared of 104-5 infectious particles per ml phosphate buffer (PBS) . The suspension is incubated for 20 min. in the dark with 0.02 μg/ml dodecyl acridine orange (DAO) and subsequently introduced into a syringe. This syringe is placed into a perfusor pump which is adjustable to different pump velocities. b) Preparation of Kluyveromyces marxianus (CBS 397) .
The yeast is cultured aerobically for 16 hours at 30°C in 2% glucose medium. A yeast cell-containing liquid is prepared by suspending 1.106 yeast cells/ml in Dulbecco's PBS. The yeast is incubated for 20 min. in the dark with 0.25 μg/ml DAO. As described for a), this yeast cell-containing liquid is also placed in a syringe, into a perfusor pump. c) The VSV- or yeast-containing liquid is passed through a glass capillary having an inside diameter of 1 mm and exposed to an Argon laser at 488 nm. The beam has a diameter of 1.5 mm. The light intensity and the flow rate are varied whereas the total amount of light energy is kept constant (21.5 kJ/m2), as shown in Table I.

Claims

Table IAfter exposure a yeast cell- or virus-containing sample is taken and examined for inactivation. d) Inactivation of VSVThe virus-containing sample is serially diluted ten times and the various concentrations are used to inoculate the A549 cells. After incubating for three days at 37°C and 5% C02 the cytopathological effect of VSV on the A549 cells is determined. The virus titre was calculated with the Spearman-Karbermethod, and represents the amount of virus infecting 50% of the wells (TCID50) . e) inactivation of yeast CBS 397. 2% glucose/1% agar plates, pH 5.2 are inoculated with the yeast cell-containing sample, at 500 cells per plate. After incubation for two days in the dark at 30°C the number of cells is determined. The percentage of survival is calculated as 100 times the number of colonies formed divided by the number of yeast cells applied.RESULTSThe results of the inactivation of VSV and yeast by means of light are represented in the graph shown in Fig. l, in which the logarithm of the virus titre (left,*) , and the survival percentage of yeast (right ,o) respectively, are plotted against the intensity (I) given as kW/ms (note: the dose, i.e. the total light energy, is the same for all points of measuring, namely 21.5 kJ/m2 ) . CLAIMS
1. A method of treating a cell-containing liquid in order to inactivate a virus present therein in the presence of dissolved molecular oxygen, which method comprises: i) introducing a sensitizer into the cell-containing liquid; and subsequently ii) exposing the sensitizer-containing liquid for a predetermined period of time to light of a chosen intensity and of a wavelength at which it is absorbed by the sensitizer, thus producing a quantity of reactive oxygen, which quantity suffices to substantially inactivate the viruses while the cells are substantially spared, characterized in that the liquid is exposed to light of a chosen high intensity and for a chosen short period of time, thereby creating an environment surrounding the cell in which the concentration reactive oxygen is reduced in comparison with the concentration reactive oxygen surrounding the virus.
2. A method according to claim 1, characterized in that the short period of time is less than 1 minute.
3. A method according to claim 2 , characterized in that the short period of time is less than 0.2 minute.
4. A method according to claim 2, characterized in that the short period of time is less than 0.04 minute.
5. A method according to one of the preceding claims, characterized in that the high intensity is higher than 20 kW/m2.
6. A method according to claim 5, characterized in that the high intensity is higher than 100 kW/m2.
7. A method according to one of the preceding claims, characterized in that the concentration dissolved molecular oxygen in the cell-containing, sensitizer-comprising liquid is controlled.
8. A method according to claim 7 , characterized in that the concentration of dissolved oxygen is reduced.
9. A method according to claim 8 , characterized in that the concentration dissolved molecular oxygen is reduced to less than 25%, preferably to less than 10% and most preferably to less than 3%.
10. A method according to claim 8 or 9, characterized in that the concentration of dissolved oxygen is reduced by admixing a photo-oxydizable compound to the liquid.
11. A method according to one of the preceding claims, characterized in that an erythrocyte-containing liquid is treated.
12. A method according to one of the preceding claims, characterized in that said erythrocyte-containing liquid is treated at a lowered temperature.
13. A method according to claim 12 , characterized in that said erythrocyte-containing liquid is treated at a temperature from 4 - 10°C.
14. A method according to one of the claims 1 to 10, characterized in that a stem cell-containing liquid is treated.
PCT/NL1998/000327 1997-06-04 1998-06-04 Method of inactivating viruses in a cell-containing liquid WO1998055156A1 (en)

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Application Number Priority Date Filing Date Title
NL1006219A NL1006219C2 (en) 1997-06-04 1997-06-04 Method for lightly inactivating viruses in a cell-containing liquid.
NL1006219 1997-06-04

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0152072A2 (en) * 1984-02-16 1985-08-21 MOLECULAR BIOPHYSICS TECHNOLOGY, Inc. Pulsed light selective photolysis process for treatment of biological media and products made thereby
US4727027A (en) * 1983-05-02 1988-02-23 Diamond Scientific Co. Photochemical decontamination treatment of whole blood or blood components
US5120649A (en) * 1990-05-15 1992-06-09 New York Blood Center, Inc. Photodynamic inactivation of viruses in blood cell-containing compositions

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4727027A (en) * 1983-05-02 1988-02-23 Diamond Scientific Co. Photochemical decontamination treatment of whole blood or blood components
EP0152072A2 (en) * 1984-02-16 1985-08-21 MOLECULAR BIOPHYSICS TECHNOLOGY, Inc. Pulsed light selective photolysis process for treatment of biological media and products made thereby
US5120649A (en) * 1990-05-15 1992-06-09 New York Blood Center, Inc. Photodynamic inactivation of viruses in blood cell-containing compositions

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