WO1998009513A1

WO1998009513A1 - Method and apparatus for the sterilization of biological substrates

Info

Publication number: WO1998009513A1
Application number: PCT/IB1997/001081
Authority: WO
Inventors: Walter J. Dermott; Peter Lea
Original assignee: Lifetech Corp
Current assignee: Lifetech Corp
Priority date: 1996-09-06
Filing date: 1997-09-08
Publication date: 1998-03-12
Anticipated expiration: 1999-03-06
Also published as: AU4027997A

Abstract

An apparatus and method for the treatment of biological materials such as blood cells are disclosed which comprise passing the materials cells through a first passageway, and directing them into contact with a solution of a treating agent such as ozone disposed in an intermediary carrier solution such as phosphate buffered saline. The treatment agent solution surrounds the cells contained in the biological sample, and by the principle of hydrodynamic focussing, segregates the cells into a virtually linear procession that is optimally exposed to the ozone-saturated buffer solution. Greater efficiency of treatment with minimal cell lysis is achieved. Multiple apparatus designs are included.

Description

METHOD AND APPARATUS FOR THE STERILIZATION OF BIOLOGICAL SUBSTRATES

The present invention relates to the sterilization of biological substrates such as fluids, cells, proteins or other organelles, especially red blood cells, platelets and stem cells by ozonation, and particularly, to the treatment of blood by contacting the red blood cells e.g. with an ozone-infused intermediary carrier such as an isotonic solution of phosphate buffered saline (PBS). More particularly, the present invention directs itself to such a method that is believed to make use of the principle of hydrodynamic focussing to maximize the speed, thoroughness and overall efficiency of such treatment.

BACKGROUND OF THE INVENTION

The treatment of blood with ozone for purposes of sterilization and for the more general application as an inactivation strategy against viral and bacterial infections is well-known. Numerous apparatus and corresponding methods have been developed over the years to achieve the desired objectives of ozone treatment. Typically, such treatment involves the exposure of whole blood to ozone for a predetermined period of time sufficient or necessary for the appropriate treatment of the blood.

One of the traditional problems that plagues the performance of such treatments is the tendency on the part of the apparatus and corresponding method to cause excessive damage to fragile substrates such as proteins and red blood cells. Unwanted damage to proteins and hemolysis result frequently from the excessive turbulence arising from the concurrent flow of blood and the ozone composition and from the outgassing of ozone from the treated substrate which results in bubble formation with subsequent protein damage and cell lysis. Apparatus has been described which is claimed in patents and patent applications assigned to the assignee of this invention; for example, U.S. Patent No. 5,366,696 to Williams and U.S. Patent No. 5,094,822 to Dunder, which remedy these shortcomings and by doing so, improve efficiency and reduce costs of such blood treatments. These patents and applications are incorporated herein by reference.

The invention described herein is an improvement in the methods and apparatus of the aforesaid patents and applications which makes it possible to directly treat cells such as tissue culture cells in single cell suspension, blood cells, and particularly red blood cells which have been fractionated from whole blood as well as proteins placed in suspension.

SUMMARY OF THE INVENTION

In accordance with the present invention, a method for the optimal treatment of a biological fluid or a particle containing substrate, such as proteins or cells, and including for example, tissue culture cells, liver cells, blood cells, platelets, or other organelles, and more particularly red blood cells, with e.g. a sterilizing agent such as ozone is disclosed which comprises passing the substrate comprising the cells through a first passageway, and directing them into an annular, displaced coaxial flow of an ozone-infused intermediary carrier, said ozone-infused intermediary carrier comprising ozone at a selected concentration in an intermediary carrier fluid such as an isotonic solution, e.g. , phosphate buffered saline. Other intermediary carriers for ozone such as Hank's balanced solution, and hydrocarbons such as perfluorocarbons, may be employed.

Preferably, the blood cells are passed through a first conduit that may have a relatively smaller internal diameter. The first conduit is joined by a second conduit that may also have a larger internal diameter, that carries the ozone-infused intermediary carrier, and the first and second conduits define in combination, and at their junction, a third inlet conduit for the coaxial transport and flow therethrough of the co-axially segregated streams of the cells and the ozone-infused intermediary carrier. As contemplated and theorized, the annular, displaced ozone-infused intermediary carrier envelops the cells and by the principle of hydrodynamic focussing, segregates the blood cells into a virtually linear procession that is optimally exposed to the ozone- infused intermediary carrier.

For example, a first conduit conveying the cells, and the red blood cells in particular, may be up to 10 mm in diameter, and may then join with a conduit of up to 20 mm in diameter carrying the ozone intermediary carrier or buffer solution, so that both conduits as joined merge into a single larger conduit or passage for the conjoint transport of both fluid streams. The cells are maintained at the axial center of the passage and are bathed and sterilized by the ozone-saturated buffer solution which is generally disposed coaxial thereto and radially outward therefrom and thereby flows concurrently with the cells under treatment.

A further aspect of the invention resides in the ability to remove gas bubbles and outgassing in the substrate being treated.

According to the invention, the biological substrate, for example, blood cells may be treated as a whole blood preparation, as packed blood cells, or as isolated blood cells, eg., red blood cells, leukocytes, and platelets. A further aspect of the invention resides in the ability to prepare and circulate a saturated isotonic buffered solution of ozone. In this way, a controlled optimal quantity of ozone can be delivered for intimate contact with the blood cells and can thereby optimize the treatment of same during flow through the second conduit.

Control of the time of exposure of the cells to ozone is maintained by controlling the flow rate and distance in the third conduit. In a further aspect of the present invention, an apparatus for the treatment of biological substrates such as red blood cells with a treatment agent such as ozone is disclosed which comprises a combination of tubes or conduits, which tubes or conduits comprise a first conduit of a lesser diameter for the transport of the red blood cells, at least one second conduit of larger diameter for the transport of the ozone-infused intermediary carrier, which second conduit joins with said first conduit at a junction, and a third conduit proceeding from the junction of said first and said second conduits, said third conduit also of larger diameter for the co-axial transport of the combined cell and ozone-infused intermediary carrier. In a particular embodiment, the third conduit is disposed for annular engagement with an outgassing conduit in the first conduit and integration with the first and the second conduits.

Accordingly, it is a principal object of the present invention to provide a method and corresponding apparatus for the treatment of biological substrates with an agent such as ozone that is effective and safe.

It is a further object of the present invention to provide a method and corresponding apparatus as aforesaid that is capable of treating blood cells with little or no damage thereto.

Other objects and advantages will become apparent to those skilled in the art from a consideration of the ensuing detailed description to be taken in conjunction with the following illustrative drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1 is a schematic sectional view of a conduit assembly in accordance with the present invention.

FIGURE 2 is a schematic sectional view of a further device serving as a conduit assembly in accordance with the present invention. DETAILED DESCRIPTION

As described earlier, the present invention is based on the discovery that individual cells can be treated by immersion in a liquid such as phosphate buffered saline (PBS) or another gas infused intermediary fluid, which contains a quantity of ozone effective to stress the cells and to achieve viral inactivation in the substrate, without damaging and destroying the fragile cells. While the immersion of the cells in the ozone- containing bath is itself an improvement in induced oxidative stress technique, the ability to safely and efficiently treat and process such cells in an automated, continuous fashion by application of the principle of hydrodynamic focussing represents an advance having far reaching implications for cell treatment and sterilization.

Referring to the Figures wherein like numerals depict like parts, an apparatus is schematically illustrated which comprises a conduit assembly 10, comprising a first conduit 12 that may be of smaller internal diameter as illustrated in Figure 1. Conduit 12 is intended to transport a stream 14 of a biological substrate, e.g. a particle containing substrate containing proteins and/or cells such as tissue cells, liver cells, organelles, and platelets or red blood cells. Conduit assembly 10 may be prepared from a variety of bioinert materials currently conventionally employed in the manufacture of cardiac or urethral catheters and like equipment. The invention is not limited to the fabrication of the assembly 10 from materials of specific composition or properties other than those broadly stated herein.

Conduit 12 is joined with at least one second conduit 16, which conduit provides for the transport of the ozone-infused intermediary carrier. While a single second conduit 16 is illustrated, it is to be appreciated that a plurality of conduits 16 may extend radially toward a junction 18 with first conduit 12, and accordingly such a design and construction is contemplated by the present invention. Naturally, the converse, e.g. a plurality of conduits 12, is likewise included herein. More generally, and with reference to Figure 2, conduits 12 and 16 are positioned so that ozone flow sweeps past the tip of conduit 12 and effectively bathes the biological substrates emerging therefrom. Further, the converging streams resulting from the conduits 12 and 16 are stabilized by directing them against an inner side wall of conduit 10. This creates a "sheeting" action of the stream that reduces turbulence, and that assists in achieving the desired outgassing of ozone.

Referring further to the Figures, conduit assembly 10 includes a third conduit 20 that is seen to extend from fluid registry with junction 18 and that has as its function to provide for the coaxial transport of the biological substrate and the ozone-infused intermediary carrier 16. As illustrated, fluid is seen to be traveling at the axial center of conduit 20 about to be surrounded on all sides with the ozone-infused intermediary carrier, so that the solution appears to serve as a sheath around the cells. While not wishing to be bound to a particular theory of operation, it is believed that the introduction of the ozone-infused intermediary carrier 16 and its convergence with the protein or cell-containing substrate 14 causes the proteins or cells to assume an almost linear dispersion within third conduit 24 that maximizes the surface area exposure of individual cells, such as erythrocytes to the beneficial effects of the ozone 22, and thereby potentiates the thoroughness and completeness of their sterilization. The effect just described is in turn, believed to result from the principle of hydrodynamic focussing, as the outer stream impinges 16 and contains the inner stream 20 and thereby controls its shape and flow characteristics.

The following non-limiting examples are presented to illustrate and confirm the principles of the invention. Where appearing, all percentages are understood to be by weight. EXAMPLE 1

The invention is illustrated herein by the treatment of suspensions of human red blood cells (RBC) with ozone to inactivate Bovine Viral Diarrhea Virus (BVDV). Accordingly, 10 ml of human RBC at 10% hematocrit were processed through a device such as that illustrated in Figure 2 (also referred to herein by the designation HDF-6), using phosphate buffered saline (PBS) as the intermediary carrier fluid for ozone, at a concentration of 4 μg/ml.

Briefly, the experiment was set up as follows. A 10.0 ml sample of red blood cells (RBC) at 10% hematocrit were treated with a solution at an ozone concentration of 4μg O₃/ml PBS. The treatment was preformed in a single pass. Two additional single pass runs were performed, each with different samples of RBC. Two further runs were performed, in this instance, with three passes of the RBC through the ozone- infused intermediary carrier.

All of the cells were washed at a ratio of 1 :4 and the results of this treatment are set forth in Table 1 , below.

TABLE 1

Bovine Viral Diarrhea Virus Inactivation in RBC using HDF-6 and 4μg/O₃ Setting

No. Processing

O,(μg) RBC: PBS Passes Sample ID Sample # TV. Time

4μglN 1:4 Single P-l T 1 49 2.17

S 2

4μg IN Single P-l T 3 49 2.18

S 4

4μg IN 1:4 Triple P-l T 5 50 2.23

S 6

P-2T 7 50 2.28

S 8

P-3T 9 50 2.34

S 10

4μg IN 1:4 Triple P-l T 11 50 2.29 s 12

P-2 T 13 52 2.40 s 14

P-3T 15 51 2.50

S 16

Substrate: 10 ml RBC 10% Hct Virus: B.D.V.D.

Following the above treatments, the hemolysis levels of the treated samples were measured, and the results are set forth in Table 2, below. TABLE 2 Hemolysis Measurements

Nu ber of Dilution % Plasma Treatments Factor Abs Abs Abs Hemolysis

2 49 0.0224 0.0964 00139 035

4 49 0.0630 0.2776 0.0372 493

6 50 0.0596 0.2585 00350 458

8 50 0.0494 0.1937 00303 340

10 5.0 0.1060 04583 00628 8.27

12 50 0.1101 04766 00647 861

14 50 00475 0.2112 00281 384

16 5.2 0.1065 04716 00629 8.90

10 51 01136 04803 00677 879

The extent of the inactivation of BVDV in the various samples was determined by plaque assays to show the plaque forming units remaining as a result of treatment. The results of this assay are set forth in Table 3, below.

TABLE 3 Results of Plaque Assay (PFU/ml)

Control Samples NT-1 NT-2 CC(cell controls) TC(toxicιty controls)

Total (RBC) 1.1 xlO⁶ 1.3 xlO⁶ 0

Supernatant 12x 10⁶ 1.8 x 10⁶ 0

Samples S1&S2 S3&S4 S5&S6 S7&S8 S9&S10 S11&S12 S13&S14 S15&S16

Total (RBC) 20x10" 775x10" 35x10" <25 <25 325x10" <25 <25

Supernatant 275x10" 95x10" 65x10" 25 <25 725x10" <25 <25

4μg 4μgPI-P3 4μgPl-P3 From the above, it can be seen that there was significant deactivation of the virus population with minimal destruction of the red blood cells as measured by the extent of hemolysis.

EXAMPLES 2 AND 3

Additional samples were prepared and processed in accordance with the procedure set forth in Example 1 , and as set forth in Tables 4 - 6B, corresponding measurements were taken of extent of hemolysis and inactivation of virus via plaque assay. The results, presented below, correspond with and are cumulative in their support of the conclusions drawn from the experiments set forth in Example 1.

TABLE 4

Bovine Viral Diarrhea Virus Inactivation in 10% Hct RBC using 4μg/ml O₃/PBS in HDF-6

Processing

O,(μg) RBC:PBS Sample ID Sample # TV. Time

4μglN 1:4 NT-1

P-l T 1 50.0 2.59

S 2

P-2T 3 56.0 3.11

S 4

P-3T 5 49.0 2.47

S 6

4μglN 1:4 P-l T 7 50.0 2.40

S 8

P-2T 9 50.0 2.55

S 10

P-3T 11 50.0 3.10

S 12

NT-2

Substrate: 10 ml 10% Hct Virus: B.V.D.

TABLE 5 Hemolysis Measurements umber of Dilution % Plasma

Sample No. Treatments Abs Abs Abs Factor Hemolysis

1 Neg Control 0.0402 0.1884 0.0227 0.7

2 2s 0.0261 0.0861 0.0086 4 1.2

3 4s 0.0670 0.2482 0.0331 4.6 4.0

4 6s 0.0753 0.2898 0.0383 3.9 4.0

5 8s 0.0258 0.1009 0.0101 4 1.5

6 10s 0.0794 0.3240 0.0406 4 4.7

7 12s 0.0851 0.3498 0.0438 4 5.1

8 Virus Control 0.0327 0.1687 0.0164 0.6

Total Hb = 34 g/

Hct = 0.100

1 -Hct = 0.900

Plasma Hb = 83.6 (2A₄|₅-A₄₅₀-Aj₈₀)

% Hemolysis = Plasma Hb/Total Hb x ( 1 -Hct)

TABLE 6A Results of Plaque Assay (PFU/ml)

Control Samples NT-1 NT-2 VPC-1 VPC-2 RBCC UICC

Total (RBC) 1.5 x 10" 3.8 x 10" 1.5 x lO⁷ 1.9 x 10⁷ 0 0

Supernatant 1.9 x 10⁶ 2.3 x 10⁶ - - 0 0

Samples S1&S2 S3&S4 S5&S6 S7&S8 S9&S10 S1 1-S12

Total (RBC) 5.8x10" 25 25 7.3x10" 25 <25

Supernatant 8.5x10" <25 <25 5.5x10" <25 <25 TABLE 6B Results of Plaque Assay (PFU/ml)

Control Samples NT- 1 NT-2 VPC- 1 VPC-2 RBCC UICC

Total (RBC) 1.8 x lO⁶ 3.8 x 10⁶ 1 .9 x 10⁷ 1 .6 x l 0⁷ 0 0

Supernatant 1.7 x 10⁶ 2.3 x 10⁵ - - 0 0

Samples S1&S2 S3&S4 S5&S6 S7&S8 S9&S10 S1 I-S12

Total (RBC) 4.2x 10" 25 25 7.4x10" 25 <25

Supernatant 6.8x10" <25 <25 6.5x 10" <25 <25

With regard to Tables 6 A and 6B, the results in the respective tables represent calculations performed according to two different methods, which are notable however, in the observation that virus titer reduction was dramatically reduced in both cases. This further supports the efficacy of the method and apparatus of the present invention.

More generally, the above data further illustrates and confirms the conclusions reached in Example 1 , that the virus was inactivated with minimal hemolysis of red blood cells. It can also be seen that the application of a washing technique using ozonated PBS to directly treat red blood cells can be practiced and will yield an efficient and efficacious method for sterilization that minimizes or avoids unwanted hemolysis. This further supports the application of the technique of hydrodynamic focussing disclosed herein to red blood cell sterilization, and the particular enhancements in safety and economy that this technique and the corresponding equipment and materials confer.

It is to be understood that the invention is not limited to the illustrations described and shown herein, which are deemed to be merely illustrative of the best modes of carrying out the invention, and which are susceptible of modification of form, size, arrangement of parts and details of operation. The invention rather is intended to encompass all such modifications which are within its spirit and scope as defined by the claims.

Claims

WHAT IS CLAIMED IS:

1. A method for the treatment of biological substrates with ozone which comprises passing said substrates through a first passageway, and directing them into an annularly displaced coaxial flow of an ozone containing solution, said ozone containing solution comprising ozone at a selected concentration in an intermediary carrier fluid solution.

2. The method of Claim 1 , wherein said biological substrates are passed through a first conduit having a relatively smaller internal diameter to define a first fluid stream, said first fluid stream is directed into contact with a second fluid stream, said second fluid stream emanating from a second conduit having a larger internal diameter and comprising an ozone-infused intermediary carrier, wherein said first and said second conduits converge and define in combination and at their junction, a third inlet conduit, and said first fluid stream flows in coaxial segregation within said second fluid stream.

3. The method of Claim 1 , wherein said biological substrates are selected from the group consisting of tissue cells, organ cells, organelles, blood cells, platelets, proteins, and mixtures thereof.

4. The method of Claim 3, wherein said blood cells are red blood cells.

5. An apparatus for the controlled treatment of a biological substrate, such as proteins or cells with a treatment agent such as ozone, which apparatus comprises a combination of tubes or conduits, which tubes or conduits comprise at least one first conduit for the transport of said substrate, at least one second conduit of larger diameter for the transport of said treatment agent dispersed in an intermediary carrier solution such as a buffer solution, said second conduit joining with said first conduit at a junction, and a third conduit located at the junction of said first and said second conduits and proceeding therefrom to define an outlet port, said third conduit of larger diameter for the transport of the combined substrate and ozone-containing buffer solution.

6. The apparatus of Claim 5, wherein said third conduit defines a chamber having an inlet end and said outlet port, and said first and said second conduits extend from said inlet end and into said chamber.

7. The apparatus of Claim 5, further including an outgassing conduit for the release of unwanted or extraneous gaseous effluent from the combination of said biological substrate and said treatment solution.

8. The apparatus of Claim 7, wherein said third conduit defines a chamber having an inlet end and said outlet port, and said outgassing conduit extends from said inlet end.

9. An apparatus for the controlled treatment of biological substrates with ozone which comprises a combination of tubes or conduits, which tubes or conduits comprise a first conduit of a lesser diameter for the transport of said substrates, at least one second conduit of larger diameter for the transport of an ozone-infused intermediary carrier, which second conduit joins with said first conduit at a junction, and a third conduit proceeding from the junction of said first and said second conduits, said third conduit also of larger diameter for the co-axial transport of the combined substrate and ozone-infused intermediary carrier.