WO1998010053A1

WO1998010053A1 - Compositions containing nucleases and chelators to enhance the recovery of cells during cell separating procedures

Info

Publication number: WO1998010053A1
Application number: PCT/US1997/015252
Authority: WO
Inventors: Joseph E. Curtis; John C. Brewer; Sean L. Macneil
Original assignee: Nexell Therapeutics Inc.
Priority date: 1996-09-04
Filing date: 1997-08-29
Publication date: 1998-03-12
Also published as: EP0938543A1; AU733602B2; CA2264885A1; EP0938543A4; AU4240797A

Abstract

The present invention provides compositions and methods which either prevent cell clumps from forming, or removing clumps which have formed, in cell separating devices. The present compositions contain a nuclease, divalent cations, and chelating agents. Surprisingly, nuclease activity is not decreased in the presence of chelating agents. During cell separation procedures using the present composition, cell yield, purity, and viability are enhanced. Also, kits for use with automated cell separating devices using these compositions are provided.

Description

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 e___, 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 e_£ al- (1974)) . Separation based on specific binding to receptors immobilized on surfaces such as fibers soon followed (Edelman ej_, al- (1974)) . Other solid phases, such as magnetic immunobeads, are also used in cell-separation techniques (Reynolds ej_. 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 e_£ al- (Carter ≤___ al- , in: Lasky et 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. Edelman e___. 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, Bon,e_. Marrow Processing and Purging; 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 fit al., in: Lasky ≤i. 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 Bl (June 18, 1986) ) . 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 ej_, al- Blood 85:275 (1995)), and mPBSC (Marolleau ≤£ 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 invention is 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. 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 self- contained kit for use in cell separating procedures.

Arrows indicate preferable sites where DNase may be provided. 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 MgCl₂.

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. 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 e_£_, al. , Methods of Enzymology 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 e___, 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 fiber- binding methods (Edelman e_L, 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 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 semi- automated 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 fit al- (1974), Carter fit al- n: Lasky_£ 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 above- described devices. 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 fi 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 a_L_, 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. 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 Bl (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 fit al. , PCT Application No. WO 96/06174 (February 29, 1996), herein incorporated by reference); DNase , β, and y (Tanu a, 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 fit 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 manganese- activated DNase I activity (Worthington Catalog Price List, Worthington Chemical Co., p. 67 (1996-1997)), 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 5 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 Biology. CRC Press, Boca Raton, FL (1989) (herein incorporated by reference) . Specific buffers include, for example, phosphate buffered saline, tris buffered 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, citrate ion provided as a salt at a concentration between about 1 and about 100 mM, magnesium ion provided as a salt at a concentration between about 0.1 and about 100 mM, albumin provided at a concentration between about 0.1 and about 10%, all in a buffer with a pKa between 6 and 8.

Preferably, the DNase is provided at about 10

KU/ml, the citrate ion at about 14 mM, the magnesium ion at about 2.5 mM, the albumin at about 1%, 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 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 pre- sterilized. 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 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 1. 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 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 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 μg in DNase (Pulmozyme^®, Genentech, Inc, South San Francisco, California) and 2.5mM of MgCl₂. DNase activity was measured using the DNA-methyl green substrate assay of Sinicropi fit 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 (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 2.5 mM MgCl₂ supported DNase activity whereas the Cell Separation Cell Bag Supernatant (as described in the above paragraph) plus 2.5 mM MgCl₂ 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 μg of DNase. For the following tables, "Standard" refers to a buffer containing 4 mM MgCl₂ and 4 mM CaCl₂ which has been determined to optimized DNase I activity (Sinicropi et al . (1994)) and "NA" means non-applicable. 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 KH₂P0₄ , 8.0 g/L of NaCl, 1.15 g/L of Na₂HP0„, and 2.16 g/L of Na₂HP0₄7H₂0.

Table 1-a Methyl Green Assay to Determine DNase Activity:

Varying [Mg++] mM TMg-r+l Relative Activity

0.0 4.4%

1.0 10.64%

2.5 17.91%

5.0 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 1-b. The linear range used to calculate the relative activity was 1 to 5 μg of DNase.

Table 1 -b

Methyl Green Assay to Determine DNase Activity:

Varying [Ca++]

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 1-c and 1-d. The linear range used to calculate the relative activity was 1 to 5 μg of DNase

Table 1 - c

Methyl Green Assay for Determining DNASE Activity: Varying [Ca++] at 2.5 mM [Mg++]

mM TMg++l

2.5 2.5 2.5 2.5 2.5

Standard

Methyl Green Assay for Determining DNASE Activity: Varying [Ca++] at 10 mM [Mg++]

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 MgCl₂, the following experiments were performed. DNA (0.2 mg/ l salmon milt DNA (CalBiochem, La Jolla, California)) in Cell Separation Buffer plus 2.5 mM MgCl₂) 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.

EXAMPLE IV

Efficacy of Cell Separation Using DNase in the

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 MgCl₂, or 3) 10 KU/ml of DNase + 10 mM MgCl₂. The resulting suspended cell populations were analyzed for 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 MgCl₂ was used rather than a concentration of 10 mM MgCl_2.

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°C) was incubated with 471 KU of DNase I (containing chymotrypsin activity) (Sigma Chemical Co., St. Louis, MO) in D-PBS without Mg++, Ca++, 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 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 g) , then the clump was divided into seven equal portions for the following treatments: PBS control, four different DNase I concentrations (with 5 mM MgCl₂) , 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 (Ila) (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 MgCl₂ (a required DNase I cofactor) greatly accelerated clump dissolution. Substantial cell lysis was observed for the DNase preparations of the instant Example; the preparations contained 33% chymotrypsin plus 66^ DNase in addition to MgCl₂.

As shown by the data for the second clump, immediate and complete dissolution of a fresh clump was observed at a concentration of lOOOKU 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.

TABLE 4 DEGRADATION OF CELL CLUMPS

Y Cell number and viability were determined after 1 hr (Clump #1 ) or 15 minutes (Clump #2). Ω Viability was determined by microscopy or calcien (Clump # 1 ) or by acridine orange/propidium iodide (Clump #2).

Claims

We Claim :

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.

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

10. 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 10%.

13. The composition of claim 12 wherein; a) the concentration of DNase is about 10 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.

15. 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.

20. 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.

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.