HK1121975B - Method for irradiating thrombocyte concentrates in flexible containers with ultra-violet light - Google Patents

Description

The invention relates to a method for inactivating pathogens, such as bacteria and viruses, and/or leukocytes in platelet concentrate (PC) by irradiation with ultraviolet light.

US 4,952,812 teaches platelets - concentrates contaminated with leukocytes to be irradiated with wavelengths from 280 to 320 nm. The white blood cells essentially lose their ability to elicit an immune response. Flat flexible bags of platelets - concentrates are then strained into an apparatus and irradiated to form uniform layer thicknesses.

WO 20051089816 A1 reveals a method and device for the treatment of biological fluids in containers or flowing devices containing photoactive substances with electromagnetic radiation for the reduction of pathogens.

It is known that the therapeutic use of blood products carries a risk of infection of recipients of the blood product with viruses and bacteria, such as hepatitis B (HBV) and hepatitis C (HCV) viruses and the AIDS pathogens HIV-1 and HIV-2.

For the purposes of this application, UV light is defined as UVA of less than 400 to 320 nm, UVB of less than 320 to 280 nm and UVC of less than 280 to 200 nm. It is known that irradiation with shortwave ultraviolet (UV) light, i.e. in the wavelength range below about 320 nm (UVB and UVC), can inactivate both viruses and bacteria, for example in blood plasma or in cellular blood vessels. Above 320 nm, the energy of the radiation is too low to inactivate microorganisms and viruses. Compared with chemical, photochemical and photodynamic methods of pathogen activation, irradiation with UV light has the fundamental advantage of not being only photo-effective but also of being less effective than other chemical substances.

These additives, or their cleavage or photoproducts, often require subsequent removal because they are toxic or mutagenic. They can also cause the formation of neoantigenic structures in the treated preparation when they bind to plasma proteins and cell surfaces. As a rule, such additives are not completely removable, at least their removal requires additional effort.

UVC was used during and shortly after World War II to sterilize blood plasma and albumin solutions, especially to inactivate hepatitis viruses. The method was then to pass the solution in a flow apparatus as a thin film past a UVC light source. The method proved to be insufficiently safe and was abandoned (Kallenbach NR, Cornelius PA, Negus D, et al. Inactivation of viruses by ultraviolet light.

In all cases, the treatment involved larger volumes, i.e. plasma pools or protein solutions up to several hundred litres and even more (Hart H, Reid K, Hart W. Inactivation of viruses during ultraviolet light treatment of human intravenous immunoglobulin and albumin, Vox Sang 1993;642) (82-8) and Chin S, Williams B, Gottlieb P, et al. Virucidal short wavelength ultraviolet light treatment of plasma and factor VIII concentrate: protection of proteins by antioxidants; Blood865;1995;11:4331-6).

The above flow machines are not suitable for sterilising a large number of individual units of TC obtained from blood donations or by machine apheresis - up to several hundred ml in volume at most - but this is precisely what is needed in the daily practice of a blood bank.

UVB is also microbicide and virucide, although not to the same extent as UVC. It penetrates protein-containing solutions and cloudy suspensions slightly better than UVC, but its penetration depth, e.g. in plasma or CTs, can be detected even in the range of a few millimetres. UVB irradiation has been tested to inactivate T-lymphocytes in CTs, which are considerably more UV-sensitive than viruses or bacteria. This should prevent the recipients of the preparations from being immunized against foreign HLA antigens, which prevent the recipients from being refracted by other trans-Konservative trans-Konservative trans-Konservative trans-Konservative trans-Konservative trans-Konservative trans-Konservative trans-Konservative trans-Konservative trans-Konservative trans-Konservative trans-Konservative trans-Konservative trans-Konservative trans-Konservative trans-Konservative trans-Konservative trans-Konservative trans-Konservative trans-Konservative trans-Konservative trans-Konservative trans-Konservative trans-Konservative trans-Konservative trans-Konservative trans-Konservative trans-Konservative trans-Konservative trans-trans-trans-Konservative trans-trans-trans-trans-trans-trans-trans-Kons-trans-trans-trans-trans-trans-trans-Kons-trans-trans-trans-trans-trans-Kons-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans-trans

However, the method did not catch on because the leukocyte filtration developed almost simultaneously is a similarly effective but more cost-effective alternative (leukocyte reduction and ultraviolet B irradiation of platelets to prevent alloimmunization and refractoriness to platelet transfusions.

It was also described that viruses in thrombocyte suspensions can be inactivated by irradiation with monochromatic UVB light (wavelength 308 nm). An excimer laser was used, the sample volume was several ml (Produc KN, Fratantoni JC, Boone EJ, Bonner RF. Use of laser-UV for inactivation of virus in blood products. Blood 1987;70(2):589-92). This scale was apparently not exceeded.

The volume of the TCs obtained is generally between 200 and 350 ml. However, TCs are also produced from individual blood donations, with a correspondingly smaller volume (between 40 and 80 ml). Both pool and apheresis TCs have the thrombocytes suspended either in blood plasma or in special storage media with a residual content of about 30 to 40% in the stomach. The TCs are stored in flat, gas-permeable plastic bags at 20-24 °C.

It would be desirable to sterilize TCs in such bags with UV light, but the problem mentioned is that the preparations are almost impermeable to UV light. This is illustrated by the following calculation: if you have UVB for sterilization and assume a TC volume of about 300 ml, a penetration depth of 1 mm of UVB radiation and exposure of both sides of the bag, a suitable bag lighting surface area of at least 1500 cm2 would be required.

The pathogens are in particular viruses and/or bacteria.

The bags may be shaken with an orbital shaker, platform shaker, squat shaker or table shaker and are preferably moved for at least three quarters of the total exposure time.

The exposure bags typically have a volume of up to 5000 ml. When the exposure bags are placed on one side, the height of the exposure bag changes continuously over the entire upper surface of the exposure bag in contact with the contents of the bag during and by moving or shaking, in relation to the distance along the surface norm between the surface on which the exposure bag is placed and the intersection with the upper surface of the exposure bag.

It seems difficult, if not impossible, to process large quantities of bags of this size routinely, and the problem is even greater when sterilising the TCs with UVC instead of UVB, since its penetration depth is much lower.

Surprisingly, the above problem was found to be solved by a method according to claim 1.

The present invention requires that the TCs in their bags be moved in an appropriate manner, so that layers are formed in the bags which are thin enough to be penetrated by UV radiation. The movement must be simultaneous in such a way that the TC suspensions in the bags are effectively mixed. Both must be achieved if the following conditions are met: The bags are highly flexible and are not fixed during exposure, e.g. between quartz plates, so that they can adapt to any change in the shape of the TC suspension resulting from the movement of the bags.The bags move either horizontally (linearly in the back and forth direction or circularly or elliptically) or vertically (twiped).The bags are filled to a maximum of 30%, in particular to a maximum of 20% of their maximum filling volume.

In any case, the reversal of the direction of movement should be so abrupt that the greater part of the TK suspension moves in the original direction due to its inertia, and the remaining residue can form a thin layer that is permeable to UV radiation.

The bags are made of UV transparent plastic material. Suitable plastics are, for example, ethylene vinyl acetate and polyolefins with film thicknesses of 1 mm and less, especially film thicknesses of less than 0.5 mm. The bags are flattened and preferably do not have absorption peaks in the range of 200 to 320 nm. The bags are only a few mm thick when lying in the filled state, e.g. smaller than 10 mm and in particular 5 mm, preferably even smaller than 3 mm, and are designed to take sample volumes of, for example, up to 200 or up to 300 ml. The maximum capacity (volume) of the bag is however at least 3 times larger, i.e. at least 5 times larger, or at least 10 times larger, than the actual sample volume to be treated.

Experimental tests

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Platelet concentrate

The TCs were made from pools of 5 Buffy Coats each, which in turn were obtained from regular blood donations. The TCs had a volume of approximately 300 to 350 ml; the platelet concentration was approximately 109/ml. The platelets were suspended in the storage medium SSP+ (a product of MacoPharma). The residual plasma content was approximately 30 to 40%.

Bacteriological tests

The following bacterial strains were used in the inactivation trials: The following shall be considered as a risk factor for the use of the active substance:

Bacterial concentrations were determined by a colony formation assay and expressed as colony forming units (CFU)/ml. In the bacterial inactivation tests, complete TCs or TC aliquots were stained with 104 to 105 CFU/ml of one of the specified species and then irradiated with UV light.

Virological tests

TK aliquots were stabbed with Suid herpesvirus (SHV-1, Pseudorabiesvirus, Aujeszky strain) or Vesicular Stomatitis virus (VSV, Indiana strain). Viruses were determined by CPE assay (CPE = cytopath effect). They are indicated as TCID50 (TCID = tissue culture infective dose). Vero cells served as indicator cells. The initial virus concentration in the tests performed was approximately 105 to 107 TCID50.

The reporting

One of the two lighting systems used was equipped with tubes emitting UVB light. The irradiation was from both sides of the exposed bags, i.e. from above and below. The lighting system was equipped with a shaker that made horizontal movements at a frequency of 60 changes of direction/min. A second lighting system was also equipped with tubes emitting UVB light. The irradiation was also from both sides. A third system (similar to the second) was equipped with tubes emitting UVC light (wavelength: 254 nm).

Exposure bags

The exposure bags used were made of UV-permeable ethyl vinyl acetate (EVA) and were available in two sizes: The size of the bag is approximately 1.15 x 18.5 cm (outer bag area approximately 268 cm2) 2.22 x 38 cm (outer bag area approximately 855 cm2)

The small EVA bags tested had a sample volume of 80 ml, while the large bags tested about 300-350 ml (complete TCs were treated).

The test is performed on a single test tube. UVB inactivation of S. epidermidis with and without free movement of platelet suspension during shaking

The free movement of the platelet suspension during the shaking and thus the formation of a thin layer was prevented in one sample by the exposure bags being firmly wedged between two quartz plates. The resulting layer thickness was approximately 3 mm. In the second sample the spacing between the quartz plates was increased to allow the platelet suspension to move largely freely during the shaking. Both samples were irradiated with 1 J/cm2.

As shown in Table 1, the bacterial titer was reduced by about 2 log10 in the fixed samples, but by more than 4 log10 in the loosely placed samples. Other

Probenbezeichnung
Unbehandelte Kontrolle	4,1±0,03
Fest eingespannte Probe	1,4±1,29
Lose platzierte Probe	-0,40+0,35

The test is performed on a single test tube. UVB inactivation of S. epidermidis in complete CT scans, loose or tightly closed bags, with shaking

The TC volume in this experiment was 330 ml, the mean layer thickness in large EVA bags was therefore approximately 3.9 mm. The TCs were irradiated with 3 UVB doses (0.8, 1.0 and 1.2 J/cm2) under the following conditions: 1. without shaking, loosely placed between quartz plates2. with shaking, pressed between quartz plates.

As shown by the results of the experiment (Table 2), in the TCs which were tightly closed during shaking, UVB treatment reduced the bacterial titres by up to about 2 log10 compared with about 3.4 to more than 4 log10 depending on the dose in the loose samples. Other

Probenplatzierung
geschüttelt, fest eingespannt	0	4,11±0,00
geschüttelt, fest eingespannt	0,8	2,14±0,48
geschüttelt, fest eingespannt	1,0	1,95±0,03
geschüttelt, fest eingespannt	1,2	1,99±0,03
geschüttelt, lose	0	4,19±0,11
geschüttelt, lose	0,8	0,77±0,69
geschüttelt, lose	1,0	-0,14±0,05
geschüttelt, lose	1,2	-0,40±0,05

The test is performed on a single test tube. Inactivation of other bacteria in free-moving or fixed TK aliquots by UVB

The first two test cases showed that S. epidermidis is effectively inactivated in TCs provided that the TC suspension can move freely during UV irradiation.

The conditions were the same as in experimental example 1, with similar results in all three cases as with S. epidermidis: in the loosely placed TC samples the bacteria were inactivated by about 3.9 to 4.25 log10 while the titers in the fixed samples were reduced by only about 2 to 3.4 log10 (Table 3).

Bakterienstamm	Probenbezeichnung
unbehandelte Kontrolle	4,88±0,00
fest eingespannt, geschüttelt	1,49+1,30
lose platziert, geschüttelt	0,97±0,86
unbehandelte Kontrolle	4,99±0,09
fest eingespannt, geschüttelt	2,99±0,13
lose platziert, geschüttelt	0,74±0,68
unbehandelte Kontrolle	4,94±0,08
fest eingespannt, geschüttelt	2,34+0,24
lose platziert, geschüttelt	1,00±0,89

The test is performed on a single test tube. Inactivation of S. epidermidis in TK aliquots by UVB under various shaking conditions

The inactivation of S. epidermidis in loosely placed TK aliquots was investigated by using a horizontal shaker, which moved back and forth as mentioned in trials 1 to 3, and an orbital shaker, which moved in a circular motion (radius 3 cm, speed 50/min), and a swivel with 50 up and down movements per minute. The sample was recirculated with one of the two samples (80 ml) firmly fastened between quartz plates, the other loosely fastened.

Schüttler	Probenbezeichnung
---	unbehandelte Kontrolle	4,94±0,16
Orbital	fest eingespannt	4,23±0,00
Orbital	lose platziert	0,50±0,39
Wippe	fest eingespannt	4,02±0,17
Wippe	lose platziert	0,87±0,78

The test shall be repeated in the same way as the test. Inactivation of Suid herpes viruses by UVB, without or with free movement of TK aliquots during shaking

To test whether the increase in inactivation of pathogens in TCs not fixed during UV light exposure was not only for bacteria but also for viruses, the following experiment was carried out: 80 ml TC aliquots were stained with Suid herpes viruses (SHV-1) and treated with UVB as described in experimental example 1. Other

Probenbezeichnung
unbehandelte Kontrolle	4,4±0,2
fest eingespannte Probe	1,41±0,18
lose platzierte Probe	0,56±0,15

The test is performed on a single test tube. Inactivation of vesicular stomatitis viruses by UVB, without or with free movement of TK aliquots during shaking

The test was performed as described in experimental example 5, except that the TK aliquots were stabbed with VSV instead of SHV-1. Again, the level of virus inactivation in the loosely placed samples was greater than 6.46 log10 compared to the comparator samples fixed between quartz plates during exposure (Table 6). Other

Probenbezeichnung
unbehandelte Kontrolle	6,7±1,05
fest eingespannte Probe	1,29±1,05
lose platzierte Probe	≤ 0,24±0,00

The test is performed on a single test tube. Inactivation of S. epidermidis by UVC, with or without free movement of TK aliquots during shaking

In this experiment, UVC was used instead of UVB. The UV dose was 0.3 J/cm2 (exposure time: 60 sec). Otherwise, the conditions were as described in experimental example 1. As shown in Table 7, the bacterial titer in the fixed samples was reduced by only about 1 log10. This apparently reflects the low penetration depth of UVC into the platelet suspension. Other

Probenbezeichnung
unbehandelte Kontrolle	4,08±0,04
fest eingespannte Probe	2,99±0,13
lose platzierte Probe	≤ 0,24

The test shall be repeated in the same way as the test. UVC inactivation of VSV without or with free movement of TK aliquots during shaking

As in experimental example 6, VSV was used as the test virus, under the same conditions as in experimental example 7, and the results shown in Table 8 show that even here the degree of pathogen inactivation was much greater in the loosely placed samples than in the fixed ones: while the virustiter in these was reduced by only about 1.5 log10, in the unfixed samples it was about 6.2 log10. Other

Probenbezeichnung
unbehandelte Kontrolle	6,92±0,22
fest eingespannte Probe	5,47±0,21
lose platzierte Probe	0,74±0,76

Claims (15)

1. A method for inactivating pathogens and/or leucocytes in thrombocyte concentrates comprising the steps of:

   - providing thrombocyte concentrates obtained from donor blood and/or by machine aphaeresis, respectively located in flexible UV-permeable flat exposure bags, which thrombocyte concentrates consist of a multitude of individually manageable and separately stored units (TCs),

   - exposing said TCs to irradiation with ultra-violet (UV) light,

   characterized in that

   - said exposure bags are filled at less than 30 % of the maximum filling volume of said exposure bags, and

   - said exposure bags are moved during said irradiation with UV light, such that the exposure bag contents is circulated and with the movement, zones of varying layer thickness are formed, wherein said UV light used comprises UVC radiation of less than 280 nm to 200 nm.
2. The method according to any of the preceding claims, characterized in that, due to said movement, said exposure bags have zones of radiated areas, for which, at regular intervals, layer thicknesses of less than 1 mm result at times, wherein said exposure bags are moved by shaking, rocking or by rotation.
3. The method according to any of the preceding claims, characterized in that said movement, in particular the amplitude of said movement, takes place such that radiated areas are formed within the TCs, in which, at regular intervals, the layer thicknesses are less than 0.5 mm at times, wherein said exposure bags are moved by shaking, rocking or by rotation.
4. The method according to any of the preceding claims, characterized in that said exposure bags have a bottom side and a top side, and the sum of the areas of said bottom side and top side, which are or can be in contact, respectively, with the bag contents, constitutes more than 90 area percent, preferably more than 99 area percent of the overall inner area of the bag contents.
5. The method according to any of the preceding claims, characterized in that said irradiation furthermore comprises UVB radiation of less than 320 nm to 280 nm, and preferably exclusively consists of irradiation with wavelengths of less than 280 nm to 200 nm and less than 320 nm to 280 nm.
6. The method according to any of the preceding claims, characterized in that each unit is manufactured from samples of up to max. 8 donors, preferably up to max. 6 donors, and in particular each unit originates from one donor.
7. The method according to any of the preceding claims, characterized in that said TC contains at least 1x10⁸ thrombocytes per ml, preferably at least 5x10⁸ thrombocytes per ml.
8. The method according to claim 5, characterized in that said irradiation with UVB takes place with a light energy of 0.3 to 5 J/cm², preferably 0.5 to 2.5 J/cm² _.
9. The method according to any of the preceding claims, characterized in that said irradiation with UVC takes place with a light energy of 0.01 to 2 J/cm², preferably 0.1 to 1 J/cm².
10. The method according to any of the preceding claims, characterized in that said TCs

    (a) contain plasma and a suitable storage medium, wherein the plasma content preferably is higher than 20 wt/%, and/or

    (b) contain a buffered aqueous storage medium.
11. The method according to any of the preceding claims, characterized in that said TCs are free from photoactive substances.
12. The method according to any of the preceding claims, characterized in that said exposure bags are movably held in an apparatus, in which said exposure bags are moved and radiated, and in particular are not restrained between two surfaces, e.g. UV-permeable glass or plastic plates.
13. The method according to at least one of the preceding claims, characterized in that said exposure bags have an average filling level of less than 10 mm, preferably less than 5 mm, and with said movement, wave troughs are constantly created, which have layer thicknesses of less than half the average filling level, preferably layer thicknesses of less than 1 mm or even less than 0.1 mm.
14. The method according to at least one of the preceding claims, characterized in that during said irradiation said exposure bags are constantly moved with an amplitude of 0.2 to 8 cm at least in the x-direction and possibly also in the y-direction (y-direction perpendicular to the x-direction), and independent hereof, the frequency of the change in direction of the shaking movement is 0.5 to 10 Hz.
15. The method according to at least one of the preceding claims, characterized in that during said irradiation said exposure bags are filled at max. 20 % of their maximum filling volume.