AU733602B2 - Compositions containing nucleases and chelators to enhance the recovery of cells during cell separating procedures - Google Patents

Description

WO 98/10053 PCT/US97/15252 1 COMPOSITIONS CONTAINING NUCLEASES AND CHELATORS TO ENHANCE THE RECOVERY OF CELLS DURING CELL SEPARATING PROCEDURES TECHNICAL FIELD OF THE INVENTION The general field of this invention is the separation of hemopoietic cells.

BACKGROUND OF THE INVENTION Specific cell fractionation has become of increased importance with the advent of cellular based therapies, diagnostics, and gene therapy regimes. The ability to specifically fractionate cells was questionable in the mid-1970's (Edelman at al., Methods of Enzymology, 34:195-225 (1974)). Early methods of cell fractionation included separation of cells based on their size, density, shape, non-specific adsorption, or charge (Edelman et al. (1974)). Separation based on specific binding to receptors immobilized on surfaces such as fibers soon followed (Edelman et al. (1974)).

Other solid phases, such as magnetic immunobeads, are also used in cell-separation techniques (Reynolds at al., Cancer Research, 46:5882-5886 (1986)).

A wide variety of automated devices are used to process cell populations, such as the ISOLEX® series (Baxter Healthcare Corp., Irvine, California) and those discussed by Carter et al. (Carter at al., in: Laskyat al., Marrow and Stem Cell Processing and Transplantation, American Association of Blood Banks, pp. 51-68 (1995)).

Cell clumping is particularly problematic in automated cell separating devices.

WO 98/10053 PCT/US97/15252 2 Edelman et al., (1974) also recognized that cell clumping was impeding the efficient operation of cell separation techniques and proposed the use of DNase in initial cell suspensions to prevent aggregation and nonspecific binding caused by the release of DNA from damaged cells. Others, however, argued that the use of DNase was unnecessary (Gee, Bone Marrow Processing and Purging: A Practical Guide, CRC Press, Boca Raton, pp.

294-295 (1991)).

The use of DNase to prevent cell clumping during cell separating techniques for a variety of cell sources has been proposed. Bone marrow cell preparations have been problematic because the cell preparations tend to clump, especially after freeze-thawing (Gee, Bone Marrow Processing and Purging: A Practical Guide, CRC Press, Boca Raton, pp. 137-142 (1991)). Others proposed the use of several additives to reduce clotting or clumping of marrow during processing, such as anticoagulants (heparin, citrate, ACD-A), enzymes (DNase), or solutions (medium 199, normal saline, Plasma- Lyte®-A) (Carter et al., in: Lasky et al., Marrow and Stem Cell Processing and Transplantation, American Association of Blood Banks, pp. 51-68 (1995)).

Additional reagents, such as proteases, have also been used to aid in cell separation procedures (Sharpe, Methods of Cell Separation, Elsevier, p. 182, (1988)).

DNase and RNase have been used in combination to increase the filterability of microbial polysaccharide broth (Drozd et al., European Patent No. 184 882 B1 (June 18, 1986)).

WO 98/10053 PCT/US97/15252 3 Cell separating techniques have also been used for a wide variety of sources, such as cord blood (Tseng- Law et al., Exp. Hematol., 22:20 (1994)), G-mPBSC (mobilized progenitor cells) (Lane et al., Blood 85:275 (1995)), and mPBSC (Marolleau at al., Blood;84:370 (1994)).

Currently, cell clumps formed during cell separation procedures remain problematic. With the advent of cellular-based therapy, there is a need for purified cellular preparations free of components which would be detrimental to a human subject injected with these cells. Therefore, methods which result in residual levels of proteases or heavy metal ions in these cell preparations would not be desirable. What is needed are compositions and methods to prevent or remove cell clumps from cell separating devices which do not leave these unwanted residues.

SUMMARY OF THE INVENTION One aspect of this invention is a composition capable of removing or preventing cell clumps from forming in a cell separating device comprising: a) one or more DNase enzymes; b) one or more divalent cations; c) one or more chelating agents; and d) one or more buffers having a pKa between about 5 and about 9.

A second aspect of this invention is the composition of the first embodiment with the addition of an RNase enzyme.

A third aspect of this inventionis a composition capable of removing or preventing cell clumps from forming in a cell separating device comprising: a) an RNase enzyme; b) one or more proteins; and c) one or more buffers with a pKa between about 5 and about 9.

WO 98/10053 PCT/US97/15252 4 A fourth aspect of this invention is a kit for use in a cell separating device comprising the compositions of the first, second, or third aspect of this invention wherein said composition is provided in a separate closed container or disposable container further comprising one or more outlet ports, wherein at least one of said ports is optionally provided with a septum.

A fifth aspect of this invention is a method of preventing cell clumping associated with an cell separation process comprising introducing the composition of the first, second, or third aspects of this invention into the initial cell population prior to cell separation in an amount sufficient to prevent cell clumping.

A sixth aspect of this invention is a method of preventing cell clumping associated with cell separation process comprising introducing the composition of the first, second, or third aspects of the present invention into the cell population to be separated during the cell separation process in an amount sufficient to prevent cell clumping.

A seventh aspect of this invention is a method of clearing a cell separation device of cell clumps following in the cell separation process comprising providing the composition of the first, second, or third aspects of the present invention; and subsequently introducing into the device said composition under conditions which allow said cell clumps to clear.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic diagram of a selfcontained kit for use in cell separating procedures.

Arrows indicate preferable sites where DNase may be provided.

WO 98/10053 PCT/US97/15252 Figure 2 compares the activity of DNase in Cell Separation Buffer and Cell Separation Cell Bag Supernatant, wherein both the Buffer and the Supernatant contain 2.5 mM MgC1 2 Figure 3 compares the activity of DNase in Cell Separation Buffer as a function of time and DNase concentration.

DETAILED DESCRIPTION OF THE INVENTION The present invention utilizes several different nucleases, such as DNase and RNase, one or more divalent cations, and one or more chelating agents to prevent or break up clumps of cells during or after a cell separation procedure. Surprisingly, this combination results in enhanced yield of the resulting cell populations. These results are particularly surprising because DNase requires divalent metal ions for activity and relatively high levels of chelators are present in the solutions of the present invention.

Therefore, down regulation of DNase activity would be expected. Surprisingly, as discussed below, DNase activity is increased in the presence of excess chelator under these conditions.

One aspect of this invention is a composition capable of removing or preventing cell clumps from forming in a cell separating device comprising: a) one or more DNase enzymes; b) one or more divalent cations; c) one or more chelating agents; and d) one or more buffers having a pKa between about 5 and about 9.

WO 98/10053 PCTIUS97/15252 6 Cell clumps form in cell separating devices during operation. They are problematic because they sequester cells from the efficient operation of a cell separating device which results in a decreased purity, yield, and viability of the final cell population.

Cell separating devices use a variety of means to separate cells. Cells have been separated based on their size, density, shape, non-specific adsorption, or charge using very simple or complex devices (Edelman et al., Methods of Enzyvmology 34:195-225 (1974), herein incorporated by reference). Specific-binding methods, which immobilize a specific binding member on a solid phase, have also been used in simple and complex devices such as, for example, the ISOLEX® series of cell sorters which utilize magnetic bead separation technology (Moubayad al al., PCT Application No. WO 95124969, published September 21, 1995, and Moubayad et al., US Patent Number 5,536,475, issued July 16, 1996, all of which are herein incorporated by reference) and fiberbinding methods (Edelman t al., (1974)). These immobilized binding members specifically bind to a cell type or cell population. The immobilized cells are then removed from the solid phase by chemical or competitive binding or by another ligand. Any solid phase may be used, such as, latex, magnetic surfaces, plastics, and a variety of cellulose derivatives, such as nitrocellulose.

The surfaces may take several forms, such as, for example, beads, wells, and sheets.

In the present invention, any cell populations can be used, for example, T-cells, B-cells, dendritic cells, neutrophils, granulocytes, bacteria, or any other suspension of cells. Also, non-nucleated cells such as platelts, for example, may be separated using the present invention. Preferably, the cell population to be purified are mononuclear hematopoietic cells (MNC), in WO 98/10053 PCT/US97/15252 7 particular the CD34+ cells. MNC can be derived from bone marrow, peripheral blood, or umbilical cord blood. Since obtaining bone marrow cells entails general anesthesia, it is preferable to obtain peripheral blood cells via leukapheresis, which is a non-invasive procedure performed without anesthesia. The patient or donor may undergo a treatment with cytokines and/or chemotherapeutic agents prior to cell collection, which agents mobilize CD34+ stem cells from the bone marrow into the peripheral blood. However, it is not absolutely necessary to administer mobilizing agents to the donor.

Many cell separation procedures may be practiced by hand using a variety of readily available devices, such as those described in the following documents, all are herein incorporated by reference; WO 95/34817, EP 438 520, WO 95/24969, WO 91/16116, US Patent Number 5,240,856, WO 92/07243, WO 95/05843, WO 95/02685, US Patent Number 5,411,863. However, automated cell separation devices allow increased speed and accuracy of the separation process while decreasing manual labor time and costs. Cells may be separated by such diverse semiautomated and automated procedures such as flow cytometry, magnetic immuno/ligand bead separation, and membrane-based separation techniques. A wide variety of cell separation methods are taught by Sharpe, Methods of Cell Separation, Elsevier, 182 (1988), Edelman et al.

(1974), Carter et al., in: LaskyZ et al., Marrow And Stem Cell Processing and Transplantation, American Association of Blood Banks 51-68 (1995), Hefton, U.S. Patent No.

4,769,317, issued September 6, 1988, and Hefton, U.S.

Patent No. 5,000,963, issued March 19, 1991, (all of which are herein incorporated by reference) and others are well-known in the art. The various aspects of this invention are useful in connection with all of the abovedescribed devices.

WO 98/10053 PCT/US97/15252 8 In the present invention the preferred cell separating device is the ISOLEX® cell separating series, such as the ISOLEX® 50, ISOLEX® 300 (also referred to as the ISOLEX® 300SA), and ISOLEX® 300i (Baxter Healthcare Corp., Irvine, California). The ISOLEX® 300i was used to perform the experiments reported below. This cell separator uses prepackaged buffers to suspend cell populations to be separated. A spinning membrane washes and concentrates cells. Magnetic beads coupled with antibodies specific for cell populations bind to the desired cell populations. A primary magnet is used to immobilize the magnetic beads and associated cells.

Cells are then released using a releasing agent which competitively binds to the specific antibody (Tseng-Law et al., PCT Application No. WO 95/34817, published December 21, 1995, herein incorporated by reference). A secondary magnet is used to remove any remaining magnetic beads. The released cells are then collected and concentrated using a spinning membrane. The ISOLEX® series of cell separators is described in the following materials, which are herein incorporated by reference: The ISOLEX® 300i Operators Manual (document 120-9123); ISOLEX® 300i Brochure (document IT 300i 3/96); ISOLEX® 300 Brochure (documents ITPR34 2/96 and BT/IT-3877.02.01- 2/96); and ISOLEX® 50 Brochure (document BT/IT- 336.3A.02.01-1/95). Other magnetic based cell separating devices and methods are known in the art and can be used with the instant invention. For example, see Miltenyi et al., German Patent No. DE 372084 C2 (January 5, 1989), Miltenyi, U.S. Patent No. 5,411,863 issued May 2, 1995; Miltenyi et al., U.S. Patent No. 5,385,707, issued January 31, 1995, Miltenyi, European Patent No. EP 0 452342131 (November 30, 1994), all of which are herein incorporated by reference.

WO 98/10053 PCT/US97/15252 9 DNase enzymes are a family of enzymes capable of degrading DNA. DNases are known to come in several different forms, such as DNase I, DNase II, Endo-DNase (Ando et al., European Patent No. 0 060 465 B1 (June 16, 1987) and Ando et al., U.S. Patent No. 4,430,432, issued February 7, 1984, herein incorporated by reference)); DNase B, (Adams e- al., PCT Application No. WO 96/06174 (February 29, 1996), herein incorporated by reference); DNase a, 3, and y (Tanuma, PCT Application No. WO 96/07735 (March 14, 1996), herein incorporated by reference). Any of these DNases, either alone or in combination, or any other form of DNase, are operable in the present invention. Preferably, DNase I and other divalent cation-dependent DNases, especially DNase I are contemplated.

Divalent cations are known to modulate DNase I activity (Campbell et al., J. Biol. Chem. 255(8):3726- 3735 (1980), herein incorporated by reference).

Preferably, magnesium and calcium ions are used to obtain high levels of DNase I activity, although other divalent cations such as zinc, manganese, and cobalt may also be used. DNase II activity does not require these divalent cations.

Chelating agents which bind divalent cations are well-known in the art. Any chelating agent which may bind divalent cations may be used in the present invention. Preferred chelating agents are those that would be acceptable for use with a cell sample that was to be reintroduced into the patient. Preferably, the present invention uses the bidentate chelators citrate ion and EDTA, although complex chelators such as heparin are also acceptable. Interestingly, citrate ions completely inhibit magnesium-activated but not manganeseactivated DNase I activity (Worthington Catalog Price List, Worthington Chemical Co., p. 67 (1996-1997)), 28/U3 Ul WED) 12:27 FAX 61 7 3221 1245 GKIFFITH HACK YATIXNT OFFICE lgU003 herein incorporated by reference). Therefore, it is unexpected that DNase I activity would be maintained when significant concentrations of citrate ion are present in the initial cell separation mixture as in the present invention.

Any buffer which is compatible with cellular viability is contemplated for the present invention.

Preferably, the buffer would have a pKa between about and about 9, more preferably between about 6 and about 8.

Specific buffers are numerous, and are reviewed in Fasman, Practical Handbook of Biochemistry and Molecular Biolo, CRC Press, Boca Raton, FL (1989) (herein incorporated by reference). Specific buffers include, 'for example, phosphate buffered saline, tris buffered 15 saline, HEPES, citrate/phosphate, phosphate, tris, boric .acid, MOPS, TES, PIPS, acetate, succinate, maleate, barbatol, glycine/HCl, carbonate/bicarbonate, HEPPS, MES, bis-tris, and MEM.

Ancillary reagents, such as proteins, are contemplated. Preferred proteins are albumin and other serum proteins such as immunoglobulins. Immunoglobulins may be provided as Gammagard® (Baxter Hyland, Glendale, California). Other useful reagents include gelatin or polyethylene glycol (PEG). Depending on the cell type, different albumins may be more preferable. When separating human cells, human serum albumin is preferable over albumin from other species.

In the above methods, the following ranges of concentration of the reagents are particularly useful: DNase between about 0.1 and about 100 KU/ml (KU=Kunitz Unit), citrate ion provided as a sale at a concentration between about I and about 100 mM, magnesium ion provided as a salt at a concentration between about 0.1 and about 100 mM, albumin 28/03 '01 WED 12:25 [TX/RX NO 8840] WO 98/10053 PCT/US97/15252 11 provided at a concentration between about 0.1 and about all in a buffer with a pKa between 6 and 8.

Preferably, the DNase is provided at about KU/ml, the citrate ion at about 14 mM, the magnesium ion at about 2.5 mM, the albumin at about in phosphate buffered saline.

Also, RNase may be added to this composition.

The addition of RNase to the composition containing DNase allows the degradation of DNA and RNA which further enhances the ability of both the components to prevent or dissolve cell clumps that form during operation of cell separating devices. This composition will therefore further enhance the ability of the present invention to reduce such cell clumping because cell clumps are held together by both DNA and RNA.

RNase is useful at a concentration range of 0.1 to about 1000 KU/ml. Preferably about 100 to about 400 KU/ml. RNase is a family of enzymes which degrades RNA and is available from a wide variety of biological sources and commercial suppliers. For example, RNase A, RNase B, RNase C, and RNase H are available from Sigma Chemical Co. (St. Louis, MO). RNases are generally inhibited by heavy metals, and are not dependent on the presence of divalent metal cations for its activity. The definitions of albumin and buffers suitable for use with RNase is as discussed above for the various DNase enzymes.

Another aspect of this invention is a composition capable of removing or preventing cell clumps from forming in a cell separating device comprising: a) one or more RNase enzymes; b) one or more proteins; and c) one or more buffers with a pKa between about 5 and about 9. An amount of the composition sufficient to WO 98/10053 PCTIUS97/15252 12 prevent cell clumping results in the increased efficiency in the operation of cell separation procedures, such as an increase in any one of cell purity, viability, or yield.

Yet another aspect of this invention is a kit for use in a cell separating device comprising the compositions of the first, second, or third aspects of this invention wherein said composition is provided in a disposable container further comprising one or more outlet ports, at least one of said ports provided with a septum. Because cell separating devices are routinely used to make cell populations for therapeutic uses, it is desirable to have reagents and ancillary hardware provided in a disposable format, preferably presterilized. Also, the solutions may be provided in collapsible plastic containers, such as those used to store blood. Such containers are routinely made from biocompatable polymers, for example, polyvinyl chloride (PVC), polycarbonate, and polypropylene, all of which are readily commercially available.

Because cell separation devices, such as the ISOLEX® 50, 300 (also referred to as ISOLEX® 300SA), and 300i series, come in a wide variety of mechanisms, shapes, and port size, number, and orientation, the particular components of the kit are linked to the particular cell separating device. Therefore, a wide range of disposable formats are envisioned and necessary.

Preferably, detachable inlet and outlet ports which carry fluids during operation of the cell separating device would be provided for use with the disposable container in order to minimize the possibility of contamination during operation of the cell separating device.

Preferably, these ports would contain a septum, such as a physical barrier through which a syringe needle may be inserted to inject various reagents. To minimize WO 98/10053 PCT/US97/15252 13 contamination further, a membrane filter capable of removing etiological agents such as bacteria, viruses, and parasites from solution, would preferably be included with at least one of the ports. The components of the present invention may be provided pre-mixed with reagents, or may be provided separately, preferably in a more concentrated form in a separate closed container, for example, a vial. Preferably, such a vial would contain DNase and/or RNase with or without divalent cations.

Often, air needs to be taken into the cell separating device during operation. Therefore, a septum may also be provided to the device and/or container to remove etiological agents such as bacteria, viruses, and parasites from air as it is taken into the cell separating device.

Preferably, the kit comprises all reagents and disposable hardware, such as reagent containers, tubing, filters, membranes, chambers, manifolds, and other ancillary elements necessary to perform a cell separation process using a particular cell separating device (such as primary and secondary chambers, and spinning membranes, for the ISOLEX 300i Cell Separating System), in a completely self-contained, sterile system. An example of such a self-contained system is provided in Figure i. Arrows indicate preferred sites of introducing the compositions of this invention. However, these compositions may be introduced at any site where the solution is to, at some time, pass through locations in the kit where cell clumps form.

Another aspect of this invention is a method of preventing cell clumping associated with a cell separation process comprising: a) providing the composition of the first, second, or third aspects of WO 98/10053 PCT/US97/15252 14 this invention into the mixture to be separated in an amount sufficient to prevent cell clumping during the cell separation process. An amount of the composition sufficient to prevent cell clumping results in the increased efficiency of the operation of cell separation procedures, such as an increase in any one of the parameters of cell purity, viability, or yield.

Another aspect of this invention is a method of preventing cell clumping associated with a cell separation process comprising: providing the composition of the first, second, or third aspects of the present invention during the cell separation process rather than prior to cell separation, in an amount sufficient to prevent cell clumping. This procedure reduces the amount of clumps which are formed and results in an increase in the yield of the final cell population obtained.

Increased purity and viability of resulting cell population are also envisioned. The compositions may be added at any point or time before or during the cell separation procedure, preferably prior to the first biochemical or immunological reaction.

Another aspect of this invention is a method of clearing a cell separation device of cell clumps following a cell separation procedure comprising: a) providing the composition of the first, second, or third embodiment of the present invention; and b) subsequently washing the device with said composition under conditions which allow said cell clumps to clear.

Frequently, cell separation devices become clogged from clumps formed during operation. Often, cell separating- devices comprise extensive lengths of small tubing and convoluted passages between plates which are kept very close together. The ability to clean this tubing and these passages is desirable to maintain WO 98/10053 PCT/US97/15252 efficient operation of the cell separating device.

Therefore, the compositions discussed in the previous embodiments may also be used to clear out existing clumps which interfere with the operation of such cell separating devices.

The instant invention is further described in the following Examples.

EXAMPLE I DNase Activity in Buffers Used in Cell Separation Procedures In order to determine DNase activity in buffers used in cell separation procedures, the following experiments were performed using an ISOLEX® 300i Magnetic Cell Separator (Baxter Healthcare Corp., Irvine, California).

Standard buffer provided in the ISOLEX® 300i cell separating device (Cell Separation Buffer) was used to determine DNase activity. Cell separation buffer is comprised of phosphate buffered saline (PBS), 14.6 mM citrate, and 1% human serum albumin. This buffer was made 0 to 12.5 Ag in DNase (Pulmozyme®, Genentech, Inc, South San Francisco, California) and 2.5mM of MgC1 2 DNase activity was measured using the DNA-methyl green substrate assay of Sinicropi ae al., Analytical Biochemistry, 222:351-358 (1994), herein incorporated by reference (see generally Garland et al., U.S. Patent No.

5,304,465, issued April 19, 1994, herein incorporated by reference). Briefly, this method allows spectrophotometric detection of methyl green free from DNA-methyl green complex (Sigma Chemical, St. Louis, MO).

DNase activity in Cell Separation Buffer with the addition and subsequent removal of cell populations WO 98/10053 PCT/US97/15252 16 (hereinafter "Cell Separation Cell Bag Supernatant") prior to the addition of DNA-methyl green complex (referred to herein as Cell Separation Cell Bag Supernatant) was also determined. Cell Separation Cell Bag Supernatant was made by adding greater than 1010 peripheral blood mononuclear cells (PBMNC) into Cell Separation Buffer and removing the cells by centrifugation.

The results of these studies are presented in Figure 2. Briefly, the Cell Separation Buffer plus mM MgC 2 1 supported DNase activity whereas the Cell Separation Cell Bag Supernatant (as described in the above paragraph) plus 2.5 mM MgC12 did not.

EXAMPLE II Effects of Divalent Cations on DNase Activity in Dulbecco's PBS Using the general procedure discussed above in Example I, the effects of varying the concentration of divalent cations was investigated. Specifically, magnesium ion concentration, in the absence of calcium ion, was investigated. The results are displayed in Table 1-a. The linear range used to calculate the relative activity was 1 to 7.5 Ag of DNase. For the following tables, "Standard" refers to a buffer containing 4 mM MgC12 and 4 mM CaCl 2 which has been determined to optimized DNase I activity (Sinicropi et al. (1994)) and "NA" means non-applicable.

WO 98/10053 PCT/US97/15252 17 All other activities for were determined in Dulbecco's PBS (D-PBS), the composition of which is set forth in the Gibco BRL Product Catalog and Reference Guide (1995-1996) (herein incorporated by reference) as; 0.2 g/L of KCL, 0.2 g/L KH 2

PO

4 8.0 g/L of NaCl, 1.15 g/L of Na 2

HPO

4 and 2.16 g/L of Na 2

HPO

4 7H 2

O.

Table 1-a Methyl Green Assay to Determine DNase Activity: Varying mM rMg++1 Relative Activity 0.0 4.4% 10.64% 17.91% 26.95% 7.5 34.22% 10.0 42.38% 20.0 82.98% Standard 100.0% Next, the effects of calcium ion concentration, in the absence of magnesium ion, was investigated. The results of the study are displayed in Table l-b. The linear range used to calculate the relative activity was 1 to 5 Ag of DNase.

WO 98/10053 PCT/US97/15252 18 Table 1-b Methyl Green Assay to Determine DNase Activity: Varying mM rCa++1 0.0 0.13 10.0 Standard Relative Activity 7.4% 10.7% 3.9% 23.8% 100.0% Next, while keeping the magnesium ion concentration at 2.5 or 10 mM, the concentration of calcium ion was varied from 0 mM to 10 mM. The results of these later experiments are depicted in Tables l-c and l-d. The linear range used to calculate the relative activity was 1 to 5 Ag of DNase WO 98/10053 WO 9810053PCT/US97/15252 1~9 Table 1-c Methyl Green Assay for Determining DNASE Activity: Varying at 2.5 mM [Mg++]I MM ±±J 2.5 Standard mM rIa±±I 0.0 0 .13 1.0 2.5 10. 0

NA

Relative-Activity 17. 8%; 21.7%6 9 .2% 17.4%6 15 .6%c 100 0% Table 1-d Methyl Green Assay for Determining DNASE Activity: Varying at 10 mM M.M g±±i 10 Standard 0.0 0 .13 1.0 2.5 10 .0

NA

Relative Activity 49.2% 45.716 63 .8! 68.4%- 61.3%- 100.0% WO 98/10053 PCT/US97/15252 EXAMPLE III Digestion of DNA in Cell Separation Buffer Using Different Concentrations of DNase In order to determine levels of DNase needed to maintain high levels of DNase activity in Cell Separation Buffer 2.5 mM MgC1 2 the following experiments were performed. DNA (0.2 mg/ml salmon milt DNA (CalBiochem, La Jolla, California)) in Cell Separation Buffer plus mM MgC1 2 was digested with DNase I (Pulmozyme®) at a concentrations of 20 KU/ml, 10 KU/ml, 5 KU/ml, 1 KU/ml, and 0.1 KU/ml. Samples were removed at various time points between 0.1 and 300 minutes and DNA activity was quenched by addition of a portion of 42 mM EDTA solution.

Samples were then separated by electrophoresis on a 1% agarose gel and DNA was visualized using ethidium bromide. The results are provided in Figure 3, with the digestion conditions provided below in Table 2.

WO 98/10053 PCT/US97/15252 TABLE 2 EFFECT OF TIME AND DNase I CONCENTRATION ON DEGREE OF DNA DEGRADATION TOP CONTENTS AND BOTTOM CONTENTS LANES REACTION CONDITIONS LANES 1 Standards 1 Standards 2 Standards 2 Standards 3 DNA: no DNase 3 DNA 5 KU/mL DNase: 15 minutes 4 DNA 20 KU/mL 4 DNA 5 KU/mL DNase: 0.1 minutes DNase: 30 minutes DNA 20 KU/mL 5 DNA 5 KU/mL DNase: 5 minutes DNase: 60 minutes 6 DNA 20 KU/mL 6 DNA 5 KU/mL DNase: 10 minutes DNase: 300 minutes 7 DNA 20 KU/mL 7 DNA 1 KU/mL DNase: 15 minutes DNase: 0.1 minutes 8 DNA 20 KU/mL 8 DNA 1 KU/mL DNase: 30 minutes DNase: 5 minutes 9 DNA 20 KU/mL 9 DNA 1 KU/mL DNase: 60 minutes DNase: 10 minutes DNA 20 KU/mL 10 DNA 1 KU/mL DNase: 300 minutes DNase: 15 minutes 11 DNA 10 KU/mL 11 DNA 1 KU/mL DNase: 0.1 minutes DNase: 30 minutes 12 DNA 10 KU/mL 12 DNA 1 KU/mL DNase: 5 minutes DNase: 60 minutes 13 DNA 10 KU/mL 13 DNA 1 KU/mL DNase: 10 minutes DNase: 300 minutes 14 DNA 10 KU/mL 14 DNA 0.1 KU/mL DNase: 15 minutes DNase: 0.1 minutes WO 98/10053 PCT/US97/15252 TABLE 2 EFFECT OF TIME AND DNase I CONCENTRATION ON DEGREE OF DNA DEGRADATION TOP CONTENTS AND BOTTOM CONTENTS LANES REACTION CONDITIONS LANES DNA 10 KU/mL 15 DNA 0.1 KU/mL DNase: 30 minutes DNase: 5 minutes 16 DNA 10 KU/mL 16 DNA 0.1 KU/mL DNase: 60 minutes DNase: 10 minutes 17 DNA 10 KU/mL 17 DNA 0.1 KU/mL DNase: 300 minutes DNase: 15 minutes 18 DNA 5 KU/mL 18 DNA 0.1 KU/mL DNase: 0.1 minutes DNase: 30 minutes 19 DNA 5 KU/mL 19 DNA 0.1 KU/mL DNase: 5 minutes DNase: 60 minutes DNA 5 KU/mL 20 DNA 0.1 KU/mL DNase: 10 minutes DNase: 300 minutes Efficacy of Cell SeParation TUsina nN~a in Initial Cell Population In order to determine the efficacy of cell separation methods using DNase in the initial cell populations, the following experiments were performed.

Populations of CD34+ cells from peripheral blood mobilized cells (Tseng-Law et al. (1994)) were separated using the ISOLEX® 300i Cell Separator in accordance with the manufacture's instructions. To D-PBS was added either 1) no DNase, 2) 10 KU/ml DNase 2.5 mM MgC1 2 or 3) 10 KU/ml of DNase 10 mM MgCl 2 The resulting suspended cell populations were analyzed for WO 98/10053 PCT/US97/15252 23 purity, yield, and viability. The results are provided in Table 3. Surprisingly, the recovery of cells was most enhanced when a concentration of 2.5 mM MgC12 was used rather than a concentration of 10 mM MgC1 2 TABLE 3 PURITY, YIELD, AND VIABILITY OF CD34+ CELLS SEPARATED USING DNase PLUS Mg++ Conditions Number Purity Yield Viability CD34+ of Runs Cells No DNase or 6 69 39 87 MgC12 of 6 68 57 85 2.1 DNase mM MgC1, KU/ml of 3 42 48 88 1.1 DNase MgC12 KU/ml of 1 67 56 97 1.6 DNase mM MgC1, EXAMPLE V Degradation of Clumps Previously Formed in a Cell Separation Device A small portion of a primary chamber clump from an ISOLEX® Cell Separator (previously stored for 2 weeks at 4 0 C) was incubated with 471 KU of DNase I (containing chymotrypsin activity) (Sigma Chemical Co., St. Louis, MO) in D-PBS without or citrate. Next, a fresh clump from an ISOLEX® 300i cell separation procedure of greater than 1010 PBMNC's was further tested.

The wet weight of the clump was measured as 1.841 g, then the clump was divided into eight equal portions for the following treatments: PBS control, mechanical WO 98/10053 PCT/US97/15252 24 trituration, a DNase I (with chymotrypsin; all previous examples used Pulmozyme® DNase I which does not contain chymotrypsin) (Sigma Chemical Co., St. Louis, MO) treatment in PBS, chymopapain (Baxter Healthcare, Irvine, California), plasmin (Sigma Chemical, St. Louis, MO), DNase I (with chymotrypsin)/plasmin mixture.

Using a second fresh clump from an ISOLEX® 300i cell separation procedure several more conditions were tested. The wet weight of the clump was measured (2.55 then the clump was divided into seven equal portions for the following treatments: PBS control, four different DNase I concentrations (with 5 mM MgCl 2 Polybrene® (Sigma Chemical, St. Louis, MO), DNase-free RNase (Sigma Chemical, St. Louis, MO), 9069N peptide (Baxter Healthcare, Irvine, California), and trypsin (Worthington, Freehold, New Jersey). Also, to investigate degradation of clots rather than cell clumps, trypsin was added to a physiological fibrin clot (PFC).

The PFC was generated by adding 5 units of thrombin (IIa) (Baxter Hyland, Glendale, California) to a 3 mg/ml solution of topical fibrinogen complex (TFC) (Baxter Hyland, Glendale, California).

The results of these experiments are presented in Table 4. Briefly, complete dissolution of the two week old chamber clump was achieved with treatment of 471 KU of DNase in 1 hour. As shown by the data for the first clump, after one hour, the combined treatment of the clump with DNase and plasmin released 13 million cells and dissolved most of the clump. Chymopapain and plasmin had relatively little effect after 24 hours of incubation. The addition of MgC12 (a required DNase I cofactor) greatly accelerated clump dissolution.

Substantial cell lysis was observed for the DNase preparations of the instant Example; the preparations WO 98/10053 PCT/US97/15252 contained 33% chymotrypsin plus 66% DNase in addition to MgCl 2 As shown by the data for the second clump, immediate and complete dissolution of a fresh clump was observed at a concentration of 1000KU of DNase I. RNase completely dissolved the clump in 24 hours. Treatment of clumps with Polybrene® or 9096N peptide had no effect.

Only minor degradation of the clumps was observed upon treatment with 5 mg/ml trypsin for 24 hours. As a control, a PFC clot was incubated with 5 mg/ml trypsin, wherein the clot completely dissolved in approximately five minutes.

WO 98/10053 WO 9810053PCTIUS97/15252 26 TABLE 4 DEGRADATION OF CELL CLUMPS Clump Treatment Quantity Clump Degredation No. of CelisY Visibility Source 0.25 hr 1 hr 24 hr 106)%)o 2 week old DNasel1 471 KU nd

OC)

Clump#lI PBS I ML -0.5 93 Mechanical I nML il-+ 22 nd DNascI1 235 KU -4I 0.4 Chymopapain 200 0.2 nd picokatals Plasmin I Unit -14 nd DNasel1+ 235 KU 235 13 82 Plasmin 1 Unit Clump #2 PBS 1 mL111, 0.5 87 DNase I+ 1000OKU 4 +ss+ 1.9 59 MM MgCI 2 100OKU +44+1.3 19 0.3 54 1KU 0.4 Polybrene4D I ug 0.4 58 RNase 405 KU 44 4--H nd nd 9069N I mg/mL nd nd Peptide Trypsin 5 mg/ml nd nd Clot Trypsin 5 mg/lL nd nd (PFC) Y Cell number and viability were determined after 1 hr (Clump or 15 minutes (Clump Q) Viability was determined by microscopy or calcien (Clump or by acridine orange/propidium iodide (Clump SUBSTITUTE SHEET (RULE 26) 26a EXAMPLE VI Cell Separation in Citrate-containing Buffer Three Isolex® 3001 procedures were done to determine the location(s) that Dnase I should be added to the process. Side by side matched Isolex® 3001 procedures were done with Dnase I added either to the apheresis bag plus chamber or chamber alone. In each case, 10 mM MgC1 2 (final concentration) was added to the chamber. As can be seen in Table 5, there were no significant differences in final product yield or purity. The purity of the final product is increased by lowering the MgC1 2 concentration to 2.5 mM. Therefore it is preferred that a final concentration of 2.5 mM MgC12 and 10 KU/ml Dnase I be added to the chamber prior to the introduction of the sensitized cells.

Table

S.

S

S

S

S

*SS*

S.

Yield Purity DNase I in Pre- DNase I in Pre- Process Process Dnase I in Apheresis Bag DNase I in Apheresis Bag Sample Chamber Only and Chamber Chamber Only and Chamber M297 48 44 47 51 M300 38 41 29 29 M303 60 57 40 46 Average 48 47 38 42 Standard 10 8 9 11 Deviation This study was performed to determine conditions for optimal DNase I activity in Isolex® buffer (D-PBS containing 1% human serum albumin and 14.6 mM sodium citrate). A colorimetric assay (Methyl Green) was adapted to measure relative DNase I activity in various solutions.

H PBe inc l Keep spec i \LB-C enclan\P33725. doc 26/02/01 26b The response was found to be linear between 1 to 5 pg DNase I. An agarose gel electrophoresis assay was developed to measure the distribution and content of DNA fragments. The Methyl Green and Agarose gel assays were then used to optimize divalent cation concentration.

Approximately 20% of maximum activity relative to published conditions was measured in Isolex® buffer with mM MgC1 2 Sufficient DNA hydrolysis was observed at KU/mL DNase I. DNase I activity was measured in preprocess and in-process samples using the Methyl Green assay. DNase I activity was found to be three-fold less in pre-process versus chamber samples. Recommended conditions for routine use of DNase I in Isolex® procedures are to add 10 Kunitz units/mL (final potency) of clinical grade DNase I and a final magnesium chloride concentration of 2.5 mM to the Isolex® chamber prior to the introduction of the sensitized cell product.

In the claims which follow and in the preceding description of the invention, except where the context o*o 20 requires otherwise due to express language or necessary implication, the word "comprising" is used in the sense of "including", i.e. the features specified may be associated with further features in various embodiments of the invention 25 It is to be understood that, if any prior art Spublication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Australia or in any other country.

*o H:\Be inda W\eep\sec i \Brendan\P33725 doc 26/02/01

Claims (37)

1. A composition capable of removing or preventing cell clumps from forming in a cell separating device comprising: a) one or more DNase enzymes; b) one or more divalent cations; c) one or more chelating agents; and d) one or more buffers having a pKa between about 5 and about 9.

2. The composition of claim 1 wherein the DNase is DNase I.

3. The composition of claim 2 wherein the divalent cations are selected from the group consisting of calcium ion, magnesium ion, cobalt ion, and zinc ion.

4. The composition of claim 3 wherein the chelating agent is heparin or a bidentate chelating agent.

5. The composition of claim 4 wherein the chelating agent is selected from the group consisting of EDTA and citrate ion.

6. The composition of claim 5 wherein the chelating agent is citrate ion.

7. The composition of claim 6 wherein the divalent cation is selected from the group consisting of magnesium ion, calcium ion, or combination of magnesium ion and calcium ion.

8. The composition of claim 7 wherein the divalent cation is magnesium ion. WO 98/10053 PCT/US97/15252 28

9. The composition of claim 8 wherein the buffer is selected from phosphate buffered saline, tris buffered saline, and HEPES. The composition of claim 9 wherein the buffer is phosphate buffered saline.

11. The composition of claim 10 further comprising one or more proteins.

12. The composition of claim 11 wherein: a) the concentration of DNase is between about 0.1 and about 100 KU/ml; b) the concentration of citrate ion as a salt is between about 1 and about 100 mM; c) the concentration of magnesium ion as a salt is between about 0.1 and about 100 mM; and d) the protein is albumin and is present at a concentration between about 0.1 and about

13. The composition of claim 12 wherein; a) the concentration of DNase is about KU/ml; b) the concentration of citrate ion as a salt is about 14 mM; c) the concentration of magnesium ion as a salt is about 2.5 mM; and d) the concentration of albumin is about 1%.

14. The composition of claim 12 further comprising an RNase enzyme. WO 98/10053 PCT/US97/15252 29 The composition of claim 14 wherein the concentration of RNase is about 0.1 to about 1000 KU/ml.

16. The composition of claim 15 wherein the concentration of RNase is about 400 KU/ml.

17. A kit for use in a cell separating device comprising the composition of claim 1 wherein said composition is provided in a separate closed container, or a disposable container further comprising one or more outlet ports, at least one of said ports optionally provided with a septum.

18. The kit of claim 17 wherein all reagents and disposable hardware are provided as a single, disposable unit.

19. A method of preventing cell clumping associated with a cell separation process comprising introducing the composition of claim 1 into the initial cell population to be separated prior to cell separation in an amount sufficient to prevent cell clumping. A method of preventing cell clumping associated with a cell separation process comprising introducing the composition of claim 1 into a cell separating device during the cell separation process in an amount sufficient to prevent cell clumping. WO 98/10053 PCT/US97/15252

21. A method of removing all clumps from a cell separation device of comprising: a) introducing the composition of claim 1; and b) subsequently washing the device with said composition under conditions which allow said cell clumps to clear. 31

22. agent is at a

23. concentration

24. concentration concentration 100 mM. The composition of claim 1, wherein said chelating higher concentration than said divalent cation. The composition of claim 2, wherein the of DNAse is between about 0.1 and about 100 KU/ml. The composition of claim 6, wherein the of citrate ion is between about 1 and about 100 mM. The composition of claim 8, wherein the of magnesium ion is between about 0.1 and about S.. S. S S. S S S S S S S S* S

26. The composition of claim 10 wherein: the concentration of DNase is between about 0.1 and about 100 KU/ml; the concentration of citrate ion is between about 1 and about 100 mM; and the concentration of magnesium ion is between about 0.1 and about 100 mM.

27. The composition of claim 26, wherein: the concentration of DNase is about 10 KU/ml; the concentration of citrate ion is about 15 mM; and the concentration of magnesium ion is about mM. 32

28. The composition of claim 12, wherein said concentration of magnesium ion is between about 0.1 and about mM.

29. The composition of claim 12, wherein said concentration of magnesium ion is between about 0.1 and about mM. The method of claims 19, 20 or 21, wherein said chelating agent is at a higher concentration than said divalent cation.

31. The method of claims 19, 20 or 21, wherein the DNase is DNase I. I 32. The method of claim 31, wherein the divalent cation is selected from the group consisting of calcium ion, magnesium ion, cobalt ion, and zinc ion.

33. The method of claim 32, wherein the chelating agent is heparin or a bidentate chelating agent.

34. The method of claim 33, wherein the chelating agent is selected from the group consisting of EDTA and citrate S ion. The method of claim 34, wherein the chelating agent is citrate ion. 33

36. The method of claim 35, wherein the divalent cation is selected from the group consisting of magnesium ion, calcium ion, or combination of magnesium ion and calcium ion.

37. The method of claim 36, wherein the divalent cation is magnesium ion.

38. The method of claim 37, wherein the buffer is selected from phosphate buffered saline, tris buffered saline, and HEPES.

39. The method of claim 38, wherein the buffer is phosphate buffered saline.

40. The method of claim 39, further comprising one or proteins. 00* o .41. The method of claim 40 wherein: the concentration of DNase is between about 0.1 and about 100 KU/ml; (b) S between about (c) between about (d) concentration the concentration of citrate ion as a 1 and about 100 mM; the concentration of magnesium ion as 0.1 and about 100 mM; and the protein is albumin and is present between about 0.1 and about salt is a salt is at a 34

42. The method of claim 41, wherein: the concentration of DNase is about 10 KU/ml; the concentration of citrate ion as a salt is about 14 mM; the concentration of magnesium ion as a salt is about 2.5 mM; and the concentration of albumin is about 1%.

43. The method of claim 41, further comprising an RNase enzyme.

44. The method of claim 43 wherein the concentration of RNase is about 0.1 to about 1000 KU/ml.

45. The method of claim 43 wherein the concentration of RNase is about 400 KU/ml. GRIFFITH HACK DFellows Institute of Patent and Fellows Institute of Patent and Trade Mark Attorneys of Australia 0 o