WO1998035079A1

WO1998035079A1 - Fiber pad

Info

Publication number: WO1998035079A1
Application number: PCT/US1998/002560
Authority: WO
Inventors: Lawrence J. Fama; Donald Lamons; Stephen Schoenberg
Original assignee: Rorer Pharmaceutical Products Inc
Current assignee: Aventis Pharmaceuticals Inc
Priority date: 1997-02-10
Filing date: 1998-02-10
Publication date: 1998-08-13
Anticipated expiration: 1999-08-10
Also published as: AU6154998A; ZA981072B

Abstract

This invention relates to fiber pads (10) which can be used in various application, for example, as carrier substrates for selective separation of one or more types of biological particles, for example, cells or virus particles, from a medium containing a mixture of different types of biological particles. In particular, the present invention concerns a fiber pad (10) and method for using the fiber pad for the separation of different types of biological particles based on proteins present on the surfaces of the biological particles.

Description

FIBER PAD

Field of the Invention

This invention relates to the manufacture and use of fiber pads. More specifically, this invention relates to fiber pads which can be used in various applications, for example, as carrier substrates for selective separation of one or more types of biological particles, for example, cells or virus particles, from a medium containing a mixture of different types of biological particles. In particular, the present invention concerns a fiber pad and method for using the fiber pad for the separation of different types of biological particles based on proteins presert on the surfaces of the biological particles.

The term "receptor," as used herein, refers to a chemical group which is capable of binding specifically to a complementary molecule comprising a biologicε 1 particle. The term "carrier substrate," as used herein, refers to the base or support material on which the receptor is carried.

The present invention will be described initially in connection with the use of polystyrene fibers formed into a pad which can be used to separate one or more types of cells from a mixture comprising different cell types. However, there are other applications in which the imention can be used, as described below.

Cell depletion is a well known process by which one or more types of cells are removed or separated from a mixture of different cell types. There are two types of embodiments associated with cell depletion. One type is referred to as"positive selection" and the other type is referred to as "negative selection".

In an application involving positive selection, one or more "desired" cell types are isolated on a biologically active receptor (receptor) from a mixture of different cell types which include "unwanted" cells. The isolated cells are recovered from the receptor for use as desired and the mixture, depleted of the isolated cells and comprising principally the unwanted cells, is disposed of.

In an application involving negative selection, the isolated cells are the "unwanted" cells and the receptor with the unwanted cells is disposed of. The mixture of desired cells, depleted of the isolated "unwanted" cells, is used for its desired purpose.

The applications involving the use of cell depletion are. many. For example, allogeneic transplantation in a patient of hematopoietic stem cells is accompanied frequently by serious patient complications due to the presence of immunoco petent T-cells in the transplant materials. The T- cells can attack host cells and cause Graft-vs-Host-Disease (GVHD) . The incidence and severity of GVHD κ.ay be reduced by careful tissue matching of host and donor, bit this reduces the likelihood of locating a successful donor by limiting the field of potential donors. Rather than attempting to find a perfect host-donor match, cell depletion base:d on negative selection may be used to pre-treat donated cellular materials to remove those cellular components associated with GVHD. Clinical end animal research related to allocenic transplantation of hematopoietic ste cells suggests that T- lymphocytes with CD5 and CD8 cell-surface protein markers are responsible for GVHD and that reduction of those cells will prevent or minimize GVHD.

It has also been found to be desirable to remove GVHD- causing cells from peripheral blood leukocyte collections intended for treatment of leukemic or lymphoruatous relapse. In this type of application, it is believed that removal of cells with CD8 markers from the donor cells ill prevent severe GVHD while allowing the remaining doncr cells to aid in elimination of the malignant cells.

Reported Developments

Various techniques and apparatus are known for accomplishing cell depletion. Examples of such techniques include fluorescence cell sorting, the use of magnetic beads covered with antibodies, complement-mediated lysis, affinity chromatography, and centrifugal elutriation. There are disadvantages associated with the use of each of these techniques, for example, contamination by antibody or cell lysis products, limited depletion of target cells, and adverse effects on non-target cells.

Certain techniques for effecting cell depletion involve the use of a biologically active receptor which is capable of binding specifically to a complementary molecule and then exposing to the receptor a biological medium comprising a mixture of different cell types, including a cell type comprising the complementary molecule. The biologically active receptor is typically affixed to a carrier substrate by conventional derivitization methods in which a linking group is attached to the carrier substrate. In effect, the receptor is affixed to the carrier substrate through the linking group. For example, if the carrier substrate is polystyrene, the surface can be derivitized by substitution of the benzene ring of the polystyrene with ε.n electrophilic reagent, particularly by a Friedel-Crafts reε.ction in a solvent which does not soften or dissolve the polystyrene. For this purpose, sulfolane finds particular application. Relatively mild conditions may be employed and the benzene may be derivatized with a variety of agents, such as nitro, which may be reduced to amino, hydroxy, or thiol group, which function as the linking group.

When the biological medium is exposed to the receptor, a cell type comprising the complementary molecule binds to the receptor and is separated from the other cells comprising the biological medium. A few examples of receptors include a ligand which includes both haptens and antigens and a steroid-binding protein.

A widely studied technique for cell depletion involves the use of polystyrene. Polystyrene panning of cells was developed originally by Wysocki and Sato, P. .A.S.. 75:2844 (1978), utilizing passively adsorbed antibody on polystyrene. Unfortunately, only low recoveries can be achieved in the use of this technique and the process suffers from lack of specificity and contamination of the separated cells with antibody.

Other techniques which involve the use of polystyrene have been developed also to aid in cell depletion. They include the use of containers which house stacked receptor- coated solid polystyrene plates or receptor-coated polystyrene membranes. The surface of the receptor-coated polystyrene is flooded with a biological medium and then rinsed with a flow of rinse medium to draw a ay any unbound cells. Such techniques require significant operator intervention which involves rotation or inversion of the device during use.

It is also generally known that specific: receptor cells can be bound to fibers such as, for example, nylon fibers. The present invention provides new and efficient means for separating one or more types of biological particles from a medium containing different types of bioloc ical particles.

Summary of the Invention In accordance with the present inventior , there is provided a fiber mat in the form of a parallelepiped comprising fibers in tangled form. In a preferred form the fibers are derivitized to form a linking grovp attached to the fibers, and in more preferred form receptors are attached to the linking group.

In the most preferred construction, the fibers are between approximately 40/xM and 160μM in diameter, and the fiber pad has a fiber-to-air ratio of between about 20% fiber/80% air and about 40% fiber and 60% air. The preferred shape for the pad is a parallelepiped.

A preferred process for creating the pads comprises air- laying fibers of between 40μM and 160μM diameter into an air- lay box to form a web; needle punching the web to form a mat; and heat pressing the mat to a thickness between 0.50 inches and 0.54 inches to form said fiber pad. In creating the preferred parallelepiped shape preferred process further comprises cutting the fiber pad into the shape of a parallelepiped and heat pressing the cut fiber pad to a thickness of between 0.50 inches and 0.54 inches.

A preferred method for using the pads of the present invention to selectively deplete specific types of biological particles from a biological medium containinc a plurality of biological particle types, wherein the specific types of biological particles are bound to a biologically active receptor upon exposure thereto and wherein the biologically active receptor is bound to a carrier substrate comprising a polystyrene fiber pad having a length, a width, and a thickness, comprises: contacting the biological medium with the fiber pad at one end of its length; pushing the biological medium through the thickness of the fiber pad along the length and leaving the biological medium in contact with the fiber pad for a predetermined time period; and removing the biological medium from contact vith the fiber pad, whereby the specific types of biological particle types remain bound to the biologically active receptor. There are numerous advantages associatec with the practice of the present invention. An increased level of cell depletion is achieved over the methods ε.nd devices of the prior art. These and other advantages will be readily apparent iy reference to the accompanying specification, drawings and appended claims.

Brief Description of the Drawings FIG. 1 is a perspective view of a fiber pad produced according to the present invention.

FIG. 2 is a block diagram (front view) .howing the use of the fiber pad of the present invention is a cell separator- system.

Detailed Description of the Preferred ≤mbodiment The present invention is particularly useful as a receptor substrate for effecting cell depletion or separation of other biological particles.

The type of receptor used will vary according to the nature of the purification being performed. Antibodies, especially monoclonal antibodies, are particularly useful as receptors in the practice of cell separation. Examples of other receptors include: cell surface membrane proteins which bind specifically to complementary molecules such as, for example, T-cells and hormones, including, for example, insulin; molecules which are found intracellularly such as, for example, steroid-binding proteins; molecules which are found in tody fluids such as, for example, t yroxine-binding globulin or lipoproteins; and ligands such as, for example, haptens and antigens.

In preferred form, the carrier substrate comprises a fiber pad which is made in the following way. A suitable fiber such as pre-made polystyrene fiber (DOT* 685D, Dow Chemical Co. , Midland, Michigan) is shot into a box by a process called "airlaying") . The air-layed fiber is then formed into a mass of tangled fiber, for example, by needle- punching in a conventional manner to form a mass of tangled fiber (the mat) . Any number of fibers may be utilized to form the mat. The procedure can be performed with as few as one continuous fiber. Once the needle-punching process is completed, the fiber mat is then compressed in a hot press to form a pad of the desired thickness, cut to ε desired shape, and then hot-pressed again.

As noted above, it is contemplated that a pad formed according to this process will be used as a carrier substrate in a cell separation process. Based on extensive testing it has been determined that, to achieve optimal hydrodynamic flow for cell separation, the fiber pad should be constructed most preferably from polystyrene fibers having a diameter in a range from about 40μM to about 160μM, with a diameter of about 75μM to 85μM being the preferred diameter.

Another preferred parameter of the present invention is the distance between the fibers which comprise the pad. This distance is related to the spaces or "pores" through which fluid flows through the pad and can be characterized by a "fiber-to-air" ratio (the ratio of solid fibious material to the air spaces between the fibers) . A preferred fiber-to-air ratio comprises about 20% fiber/80% air to about 40% fiber/60% air, with the 20/80 fiber/air ratio being particularly preferred. At a 20/80 fiber/air ratio, the average pore size of the pad is approximately 100-140μM, which provides for particularly good hydrodynamic flow.

Before needle punching, the fibers should be distributed evenly into a "web" that then is input into ε needle loom.

One way of preparing such a web is to use an airlay machine, which provides means for unwinding a spool ox spools of fiber yarn onto a support mat. The support mat is placed into an airlay box and air is blown into the box as the yarn is unwound from spools. The air helps distribute the yarn evenly across the support mat.

A fiber pad having the above parameters can be produced, for example, as follows. First, a spool of J.OμM polystyrene fiber is loaded onto the creel of an airlay n.achine and threaded through the creel eyelets. A support mat is positioned over the airlay blower screen in the airlay box. The scale should be tared to 0.0 ounce and the air pressure of the airlay machine is set to 20 psi. The unwinding mechanism of the airlay machine should be set for 20 fpm and then turned on. This causes the fiber to be unwound into the box. This can be continued until a desired rmount of fiber (e.g., 4.9 ounces) has been airlayed onto the; support mat.

The airlayed fiber is removed from the Ϊ irlay machine using the support mat as a carrier. Next, tre support mat with the airlayed fiber thereon is placed on the input side of a needle loom with the support mat threade d through the loom to the output side. A 12" down-acting James Hunter needle loom is an example of one type of needle loom that can be used for the needle-punching process. The needle gap of the loom should be set to the desired specification, e.g. , 1.5 to 1.75 inches. The needle rate should fca set for approximately 200 strokes per minute (+/- 5 :-tokes per minute) . When the needle loom is turned on, the support mat will pull the airlayed fiber through the neec'.le loom, creating a needle punched mat of airlayed fi er. The needle punched mat is then flipped over and the support mat is removed. The needle punched mat is fed through the loom again, needle punching the other side. If desired, the needle punched mat can be sent through the lcom multiple times to further needle punch the mat.

Once the mat has been needle punched, it is heat pressed for approximately two minutes at approximately 155°F (+/- 15 °F) . This firms up the mat and compresses it to the specified thickness. The heat-pressed pad is then cut to shape using a die press. After being cut to shape, the pad is subjected to a second identical heat pressing step to complete the production of the pad. The second heat pressing step compresses the cut edges of the pad, which are sometimes expanded during the die-cutting process. This assures that the pad is compressed to the specified thickness.

The above process can be used to form a pad approximately 0.25 inches thick. In the preferred embodiment the pad should be thicker, for example, approximately 0.5 inches thick. To produce a 0.5 inch thick pε.d, the above process is carried out twice, with one excep ion. After the first pad has been fed through the needle loom the first time, the support pad is removed and the needle punched pad (punched on one side) is set aside while a second pad is made in identical fashion. Next, the two once needle-punched pads are placed together, with the non-needle punched sides abutting. The two mats are then fed into the needle loom to complete the needle punching process. The heat pressing step is then carried out as set forth above. Thiε produces a double-thick pad as desired.

If the fiber pad is to be used for cell separation, the pad can be subjected to conventional derivati.zation procedures to affix thereto linking groups which in turn are bonded to receptors.

When a mixture of different types of biological particles is exposed to receptors bound to the pad (carrier substrate) , complementary molecules present in the mixture bind with the receptors and are retained. Ir an application involving protein retention, the bound separated particles can be released from the receptors by means available in the art and put to their intended use. In an application involving negative selection, the unbound molecules can be collected and utilized for their intended purpose and the bound particles can be disposed of.

A method of using the pads for selective' cell separation is described hereafter. As seen in FIGS. 1 ε nd 2 , a fiber pad 10 is placed in a cell separation chamber 20. In a preferred embodiment the pad is cut to the shape of a parallelepiped as shown in FIGS. 1 and 2. This shape provides efficiency of manufacture. There iε very little waste material left over when cutting the pads to this shape. The shape provides also efficiency of use. hen fluid is introduced to a rectangular shaped pad, the area near the corners of the pad nearest to the point of fluid introduction are not exposed to much, if any, fluid. By introducing the fluid to one of the points of the parallelepiped (e.g., point 12 in FIG. 2) the highly efficient use of the- pad area is achieved.

Referring to FIG. 2, a source of cell material 22 and a source of rinse fluid 24 are connected to an input 28 of cell separation chamber 20 via input tube 26. Alt.hough not shown, valves should be placed in the input line 26 so that the source of liquid cell material 22 and source of rinse liquid 24 can be selectively turned on and off as desired.

Liquid introduced to input 28 of cell separation chamber 20 is coupled directly to the lower corner of fiber pad 10 at point 12. An output 30 is provided to connect the top of the fiber pad 10, at point 14, to the outside of the cell separation chamber 20. An output line 32 allows fluid leaving the cell separation 20 to enter a cell collection container 34. A valve (not shown) can be pieced in the output line 32 to allow the output of the cell separation chamber 2C. to be started or stopped as desired. For example, it would normally be appropriate to prevent the flow of fluid from the cell separation chamber 20 to the cell collection chamber 34 during the "incubation" period when the cell material is sitting in the cell collection chamber 20.

To achieve maximum cell separation from the fiber pad, it is preferable to house the fiber pad in a chamber in which the pad snugly fits. When the pad is form-fitted into the chamber, there is less likelihood of the fluid channeling to the edge of the pad and a greater likelihood that the fluid will flow through the pad and, therefore, cortact more of the receptor bound to the fibers of the pad.

Because the polystyrene is formed as a porous pad rather than a plate, the fluid input to the separation chamber is pushed through the pad rather than over the polystyrene as occurs with polystyrene plates. Although the pad can be oriented in almost any direction, the preferred orientation is to stand the pad on one end and force the fluid through the pad lengthwise as shown in FIG. 2. This cannot be done with prior art polystyrene plates because it is not possible to push the fluid through the plate. Because the fluid surrounds the fiber and, therefore, the receptor attached to the fiber, there is no need to flip or to otherwise agitate the chamber as is required with polystyrene p ate separation. The fluid flows around the fibers and the desired cells bind easily with the receptors that are affixed tc the pad.

It should be appreciated that the pad of the present invention provides advantages over prior art carrier substrates. A significant advantage of the fiber pad of the present invention is the increased surface area resulting from the use of the fibers to form the pad. Because of the increased surface area, a much greater amount of cell separation can be achieved without increasing the size of the cell separation device. Thus, a device in which a receptor- coated polystyrene fiber pad is installed will separate significantly more cells than a device of equal size containing polystyrene plates. Although the above description makes specific reference to the use of polystyrene fiber for producing the fiber pads, the present invention is not limited to polystyrene fiber. Any fiber material that can be produced to the diameters specified herein and which can have receptor materials bound thereto (directly or indirectly) can be used. For example, nylon fibers can be lightly hydrolysed and then formed into fiber pads having the same physical parameters specified above (e.g. , fiber diameter of between 40 microns and 160 microns and with a fiber-to-air ratio of bet een 20% fiber/80% air and 40% fiber/60% air) . Similarly, glass fibers can be silanized and then formed into a pad having the same specifications. Receptors can be coval>£ntly bound to the nylon or glass pads and the pads could be used according to the method disclosed herein.

Although the foregoing invention has be>. n described in some detail by way of illustration and example for purposes of clarity and understanding, it will be reac ily apparent to those of ordinary skill in the art in light : f the teachings of this invention that certain changes and m: difications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A fiber mat in the form of a parallelepiped comprising fibers in tangled form.

2. A fiber mat as set forth in claim 1, wherein said fibers are derivitized to form a linking group attached to said fibers.

3. A fiber mat as set forth in claim 2, wherein receptors are attached to said linking group.

4. A fiber mat as set forth in claim 1, wherein said fibers are between approximately 40μM and 16CμM in diameter.

5. A fiber mat as set forth in claim 1, wherein said fiber pad has a fiber-to-air ratio of betweer. about 20% fiber/80% air and about 40% fiber and 60% air.

6. A process for the preparation of a fiber pad which comprises the steps of: air-laying fibers of between 40μM and 160μM diameter into an air-lay box to form a web; needle punching said web to form a mat; and heat pressing said mat to a thickness between 0.50 inches and 0.54 inches to form said fiber pad.

7. A process as set forth in claim 6, wherein said process further comprises cutting said fiber pad into the shape of a parallelepiped.

8. A process as set forth in claim 7, wherein said process further comprises heat pressing said cut fiber pad to a thickness of between 0.50 inches and 0.54 inches.

9. A process as set forth in claim 6, wherein said fiber comprises polystyrene fiber.

10. A process as set forth in claim 6, wherein said fiber comprises nylon fiber.

11. A process as set forth in claim 6, wherein said fiber comprises glass fiber.

12. A process as set forth in claim 6 , wherein said fiber pad has a fiber-to-air ratio of between about 80% fiber/20% air to about 60% fiber/40% air.

13. A process for preparation of fiber pads for use in cell separation which comprises the steps of: air-laying fibers into an air-lay box to form a web; needle punching said web to form a fiber mat; heat pressing said fiber mat to form a liber pad; cutting said fiber pad into a predetermined shape; heat pressing said fiber pad; and binding a receptor to the resultant fibf.r pad.

14. A process as set forth in claim 13, wherein said receptor is covalently bound to said resultart fiber pad.

15. A process as set forth in claim 13, wherein said receptor is bound to said resultant fiber pa by adsorption.

16. A method for selective depletion of specific types of biological particles from a biological medium containing a plurality of biological particle types, wherein said specific types of biological particles are bound to a biologically active receptor upon exposure thereto and wherein said biologically active receptor is bound to a carrier substrate comprising a fiber pad having a length, a wicth, and a thickness, said method comprising: contacting said biological medium with said fiber pad at one end of its length; pushing said biological medium through t e thickness of said fiber pad along said length and leaving said biological medium in contact with said fiber pad for a predetermined time period; and removing said biological medium from contact with said fiber pad, whereby said specific types of biological particle types will remain bound to said biologically active receptor.

17. A fiber pad comprising tangled fibers of between 40μM and 160μM diameter needle punched and heat compressed so that the distance between the individual fibers of the pad is approximately 100 - 140μM.