CA2264885A1 - Compositions containing nucleases and chelators to enhance the recovery of cells during cell separating procedures - Google Patents

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

1015202530CA 02264885 1999-03-03wo 98/10053 PCT/US97/15252COMPOSITIONS CONTAINING NUCLEASES AND CHELATORS TOENHANCE THE RECOVERY OF CELLS DURINGCELL SEPARATING PROCEDURESIEQHNIQAL_ElELD_QE_IHE_lNMENIlQNThe general field of this invention is theseparation of hemopoietic cells.BAQKGEQUND_QE_IHE_lNMENIlQNofincreased importance with the advent of cellular basedTheSpecific cell fractionation has becometherapies, diagnostics, and gene therapy regimes.ability to specifically fractionate cells wasquestionable in the mid—1970's (Edelman gt gl., Mgthgdggfi_Enzymglggy, 34:195—225 (1974)). Early methods ofcell fractionation included separation of cells based ontheir size, density, shape, non—specific adsorption, or(Edelman gt gl. (1974)).specific binding to receptors immobilized on surfaces(Edelman gt 31. (1974)).such as magnetic immunobeads, arecharge Separation based onsuch as fibers soon followedOther solid phases,also used in cell—separation techniques (Reynolds gt gl.,Qgnggr_Rgggg1gh, 46:5882—5886 (1986)).A wide variety of automated devices are used tosuch as the ISOLEX® series(Baxter Healthcare Corp., Irvine, California) and thosediscussed by Carter gt gl. (Carter gt gl., in: Lasky_gtgig, Marrow and Stem Cell Processing and Transplantation,process cell populations,American Association of Blood Banks, pp. 51-68 (1995)).Cell clumping is particularly problematic in automatedcell separating devices.1015202530W0 98/ 10053CA 02264885 1999-03-03PCT/US97/152522Edelman et al., (1974)cell clumping was impeding the efficient operation ofalso recognized thatcell separation techniques and proposed the use of DNasein initial cell suspensions to prevent aggregation andnonspecific binding caused by the release of DNA fromdamaged cells. Others, however, argued that the use ofDNase was unnecessary (Gee, CRC Press, Boca Raton, pp-294-295 (1991)).The use of DNase to prevent cell clumpingduring cell separating techniques for a variety of cellsources has been proposed. Bone marrow cell preparationshave been problematic because the cell preparations tendto clump, especially after freeze—thawing (Gee, BoneMarrow Processing and Purging: A Practical Guide, CRC(1991)). Othersthe use of several additives to reduce clotting orPress, Boca Raton, pp. 137-142 proposedclumping of marrow during processing, such asanticoagulants (heparin, citrate, ACD-A),(DNase), or solutions (medium 199, normal saline, Plasma-Lyte®—A) (Carter et 31., in: Lasky et al., MaxrQw_andStem Cell Processing and Transplantation, AmericanAssociation of Blood Banks, pp. 51-68 (1995)).Additional reagents, such as proteases, have also beenenzymesused to aid in cell separation procedures (Sharpe,MethQds_gf_§ell_§epara;iQn, Elsevier, p. 182, (1988)).DNase and RNase have been used in combination to increasethe filterability of microbial polysaccharide broth(Drozd et_al+, European Patent No. 184 882 B1 (June 18,1986)).1015202530W0 98/10053CA 02264885 1999-03-03PCT/US97/152523Cell separating techniques have also been usedfor a wide variety of sources, such as cord blood (Tseng—Law gt a1., Expo_HomaLolo, 22 20 (1994)), G—mPBSC(mobilized progenitor cells) (Lane gt al., Blood 85:275(1995)), and mPBSC (Marolleau on al., Blood 84:37O(1994)).Currently, cell clumps formed during cellwith theadvent of cellular—based therapy, there is a need forseparation procedures remain problematic.purified cellular preparations free of components whichwould be detrimental to a human subject injected withthese cells. Therefore, methods which result in residuallevels of proteases or heavy metal ions in these cellpreparations would not be desirable. What is needed arecompositions and methods to prevent or remove cell clumpsfrom cell separating devices which do not leave theseunwanted residues.SHMMARI_QE_IHE_INÂ¥ENIlQNOne aspect of this invention is a compositioncapable of removing or preventing cell clumps fromforming in a cell separating device comprising: a) one ormore DNase enzymes; b) one or more divalent cations; c)one or more chelating agents; and d) one or more buffershaving a pKa between about 5 and about 9.A second aspect of this invention is thecomposition of the first embodiment with the addition ofan RNase enzyme.A third aspect of this invention is acomposition capable of removing or preventing cell clumpsfrom forming in a cell separating device comprising: a)and c) one oran RNase enzyme; b) one or more proteins;more buffers with a pKa between about 5 and about 9.l015202530CA 02264885 1999-03-03W0 98/ 10053 PCT/US97/152524A fourth aspect of this invention is a kit foruse in a cell separating device comprising thecompositions of the first, second, or third aspect ofthis invention wherein said composition is provided in aseparate closed container or disposable container furthercomprising one or more outlet ports, wherein at least oneof said ports is optionally provided with a septum.A fifth aspect of this invention is a method ofpreventing cell clumping associated with an cellseparation process comprising introducing the compositionof the first, second, or third aspects of this inventioninto the initial cell population prior to cell separationin an amount sufficient to prevent cell clumping.A sixth aspect of this invention is a method ofpreventing cell clumping associated with cell separationprocess comprising introducing the composition of thefirst, second, or third aspects of the present inventioninto the cell population to be separated during the cellseparation process in an amount sufficient to preventcell clumping.A seventh aspect of this invention is a methodof clearing a cell separation device of cell clumpsfollowing in the cell separation process comprisingproviding the composition of the first, second, or thirdaspects of the present invention; and subsequentlyintroducing into the device said composition underconditions which allow said cell clumps to clear.ERIEE_DES£ElEIlQH_QE_IHE_DBAfllNQSFigure 1 is a schematic diagram of a self-contained kit for use in cell separating procedures.Arrows indicate preferable sites where DNase may beprovided.10152025CA 02264885 1999-03-03W0 98/ 10053 PCT/US97/ 152525Figure 2 compares the activity of DNase in CellSeparation Buffer and Cell Separation Cell BagSupernatant, wherein both the Buffer and the Supernatantcontain 2.5 mM MgCl,Figure 3 compares the activity of DNase in CellSeparation Buffer as a function of time and DNaseconcentration.DEIAILED_DESQBlEII£EL£ELIHE_lNMENIlQNThe present invention utilizes severaldifferent nucleases, such as DNase and RNase, one or moredivalent cations, and one or more chelating agents toprevent or break up clumps of cells during or after athiscombination results in enhanced yield of the resultingcell separation procedure. Surprisingly,cell populations. These results are particularlysurprising because DNase requires divalent metal ions foractivity and relatively high levels of chelators arepresent in the solutions of the present invention.Therefore, down regulation of DNase activity would beexpected. Surprisingly, as discussed below, DNaseactivity is increased in the presence of excess chelatorunder these conditions.One aspect of this invention is a compositioncapable of removing or preventing cell clumps fromforming in a cell separating device comprising: a) one ormore DNase enzymes; b) one or more divalent cations; c)one or more chelating agents; and d) one or more buffershaving a pKa between about 5 and about 9. 101520253035CA 02264885 1999-03-03W0 98/ 10053 PCT/US97/152526Cell clumps form in cell separating devicesduring operation. They are problematic because theysequester cells from the efficient operation of a cellseparating device which results in a decreased purity,yield, and viability of the final cell population.Cell separating devices use a variety of meansto separate cells. Cells have been separated based ontheir size, density, shape, non—specific adsorption, orcharge using very simple or complex devices (Edelman gtaJ_., 34:195-225 (1974), hereinincorporated by reference). Specific—binding methods,which immobilize a specific binding member on a solidphase, have also been used in simple and complex devicessuch as, for example, the ISOLEX® series of cell sorterswhich utilize magnetic bead separation technology(Moubayad et 31., PCT Application No. WO 95124969,published September 21, 1995, and Moubayad e;_al+, USPatent Number 5,536,475, 1996, all ofwhich are herein incorporated by reference) and fiber-binding methods (Edelman et 31., (1974)). Theseimmobilized binding members specifically bind to a cellissued July 16,type or cell population. The immobilized cells are thenremoved from the solid phase by chemical or competitivebinding or by another ligand. Any solid phase may beused, such as, latex, magnetic surfaces, plastics, and avariety of cellulose derivatives, such as nitrocellulose.The surfaces may take several forms, such as, forexample, beads, wells, and sheets.In the present invention, any cell populationscan be used, for example, T—cells, B-cells, dendriticcells, neutrophils, granulocytes, bacteria, or any otherAlso,platelts, for example, may be separated using the presentsuspension of cells. non-nucleated cells such asinvention. Preferably, the cell population to bepurified are mononuclear hematopoietic cells (MNC), in101520253035................................. . . . . . . . . . . . .CA 02264885 1999-03-03WO 98/10053 PCT/US97/152527particular the CD34+ cells. MNC can be derived from bonemarrow, peripheral blood, or umbilical cord blood. Sinceobtaining bone marrow cells entails general anesthesia,it is preferable to obtain peripheral blood cells vialeukapheresis, which is a non—invasive procedureperformed without anesthesia. The patient or donor mayundergo a treatment with cytokines and/orchemotherapeutic agents prior to cell collection, whichagents mobilize CD34+ stem cells from the bone marrowinto the peripheral blood. However, it is not absolutelynecessary to administer mobilizing agents to the donor.Many cell separation procedures may bepracticed by hand using a variety of readily availabledevices, such as those described in the followingdocuments, all are herein incorporated by reference; W095/34817, EP 438 520, WO 95/24969, WO 91/16116, US PatentNumber 5,240,856, WO 92/07243, WO 95/05843, WO 95/02685,US Patent Number 5,411,863.separation devices allow increased speed and accuracy ofHowever, automated cellthe separation process while decreasing manual labor timeand costs. Cells may be separated by such diverse semi-automated and automated procedures such as flowcytometry, magnetic immuno/ligand bead separation, andmembrane—based separation techniques. A wide variety ofcell separation methods are taught by Sharpe, M§thQds_Qfigell_§eparatign, Elsevier, 182 (1988), Edelman et al.(1974), Carter er. al., in: Lasky_e1; a2L., Ma.rr9.u_And_fl:_e.mCell Processing and Transplantation, American Associationof 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 ofwhich are herein incorporated by reference) and othersare well—known in the art. The various aspects of thisinvention are useful in connection with all of the above-described devices.l015202530CA 02264885 1999-03-03W0 98/ 10053 PCT/US97/152528In the present invention the preferred cellseparating device is the ISOLEX® cell separating series,such as the ISOLEX® 50, ISOLEX® 300the IsoLEx® 300SA), and IsoLEx® 3001The ISOLEX® 3001 was used toThis cellseparator uses prepackaged buffers to suspend cell(also referred to as(Baxter HealthcareCalifornia).Corp., Irvine,perform the experiments reported below.populations to be separated. A spinning membrane washesand concentrates cells. Magnetic beads coupled withantibodies specific for cell populations bind to thedesired cell populations. A primary magnet is used toimmobilize the magnetic beads and associated cells.Cells are then released using a releasing agent whichcompetitively binds to the specific antibody (Tseng—Lawgt gl., PCT Application No. WO 95/34817, publishedDecember 21, 1995, herein incorporated by reference). Asecondary magnet is used to remove any remaining magneticThe released cells are then collected andThe IsoLEx@series of cell separators is described in the followingbeads.concentrated using a spinning membrane.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.O2.01—1/95). Other magnetic based cell separatingdevices and methods are known in the art and can be usedwith the instant invention. For example, see Miltenyi gtalt, German Patent No. DE 372084 C2 1989),Miltenyi, U.S. Patent No. 5,411,863 issued May 2, 1995;Miltenyi gt al., U.S. Patent No. 5,385,707,January 31, 1995, Miltenyi, European Patent No.452342131 (November 30, 1994),incorporated by reference.(January 5,issuedEP 0all of which are herein101520253035CA 02264885 1999-03-03W0 98/10053 PCT/US97l152529DNase enzymes are a family of enzymes capableof degrading DNA. DNases are known to come in severaldifferent 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, issuedFebruary 7, 1984, herein incorporated by reference));DNase B, (Adams et al., PCT Application No. WO 96/06174(February 29, 1996), herein incorporated by reference);DNase d, B, PCT Application No. WO96/07735 (March 14, 1996), herein incorporated byAny of these DNases,or any other form of DNase,and v (Tanuma,reference). either alone or incombination, are operable inthe present invention. Preferably, DNase I and otherdivalent cation—dependent DNases, especially DNase I arecontemplated.Divalent cations are known to modulate DNase Iactivity (Campbell et al., Q*_Bigl*_Qhem. 255(8):3726—3735 (1980), herein incorporated by reference).Preferably, magnesium and calcium ions are used to obtainhigh levels of DNase I activity, although other divalentcations such as zinc, manganese, and cobalt may also beused. DNase II activity does not require these divalentcations.Chelating agents which bind divalent cationsare well—known in the art. Any Chelating agent which maybind divalent cations may be used in the presentinvention. Preferred Chelating agents are those thatwould be acceptable for use with a cell sample that wasthepresent invention uses the bidentate chelators citrateto be reintroduced into the patient. Preferably,ion and EDTA, although complex chelators such as heparinare also acceptable. Interestingly, citrate ionscompletely inhibit magnesium—activated but not manganese-activated DNase I activity (Worthington Catalog PriceList, Worthington Chemical Co., p. 67 (1996-1997)),l015202530CA 02264885 1999-03-03%}«i“;'T.«“ ~l0herein incorporated by reference). Therefore, it isunexpected that DNase I activity would be maintained whensignificant concentrations of citrate ion are present inthe initial cell separation mixture as in the presentinvention.Any buffer which is compatible with cellularviability is contemplated for the present invention.Preferably, the buffer would have a pKa between about 5and about 9, more preferably between about 6 and about 8.Specific buffers are numerous, and are reviewed inFasman, Eiglggy, CRC Press, Boca Raton, FL (1989) (hereinincorporated by reference). Specific buffers include,for example, phosphate buffered saline, tris bufferedsaline, HEPES, citrate/phosphate, phosphate, tris, boricacid, MOPS, TES, PIPS,barbatol, glycine/HCl, carbonate/bicarbonate, HEPPS, MES,acetate, succinate, maleate,bis-tris, and MEM.Ancillary reagents, such as proteins, arecontemplated. Preferred proteins are albumin and otherserum proteins such as immunoglobulins. Immunoglobulinsmay be provided as Gammagard® (Baxter Hyland, Glendale,Other useful reagents include gelatin or(PEG) .different albumins may be more preferable.California).Depending on the cell type,Whenpolyethylene glycolseparating human cells, human serum albumin is preferableover albumin from other species.In the above methods, the following ranges ofconcentration of the reagents are particularly useful:DNase between about 0.1 and about 100 KU/ml (KU=KunitzUnit),between about 1 and about 100 mM, magnesium ion providedcitrate ion provided as a salt at a concentrationas a salt at a concentration between about 0.1 and about100 mM, albumin provided at a concentration between about.uI\;‘ -.fikl.)CA 02264885 1999-03-03 9 {V/, 0 "0 0CT1993lO/l0.1 and about 10%, all in a buffer with a pKa between 6and 8..:;.r.:NoEo SHEET1015202530CA 02264885 1999-03-03wo 93/10053 PCT/US97/15252iG%7waEPeH+ib£m£éer>wiEh—a—pKa—be%ween—64M%L4h~-Preferably, the DNase is provided at about 10KU/ml, the citrate ion at about 14 mM, the magnesium ionat about 2.5 mM, the albumin at about 1%, in phosphatebuffered saline.Also, RNase may be added to this composition.The addition of RNase to the composition containing DNaseallows the degradation of DNA and RNA which furtherenhances the ability of both the components to prevent ordissolve cell clumps that form during operation of cellseparating devices. This composition will thereforefurther enhance the ability of the present invention toreduce such cell clumping because cell clumps are heldtogether by both DNA and RNA.RNase is useful at a concentration range of 0.1to about 1000 KU/ml. Preferably about 100 to about 400KU/ml. RNase is a family of enzymes which degrades RNAand is available from a wide variety of biologicalRNase A,RNase B, RNase C, and RNase H are available from Sigmasources and commercial suppliers. For example,Chemical Co. (St. Louis, MO). RNases are generallyinhibited by heavy metals, and are not dependent on thepresence of divalent metal cations for its activity. Thedefinitions of albumin and buffers suitable for use withRNase is as discussed above for the various DNaseenzymes.Another aspect of this invention is acomposition capable of removing or preventing cell clumpsfrom forming in a cell separating device comprising: a)one or more RNase enzymes; b) one or more proteins; andc) one or more buffers with a pKa between about 5 andabout 9. An amount of the composition sufficient to101520253035CA 02264885 1999-03-03WO 98/10053 PCT/US97/1525212prevent cell clumping results in the increased efficiencyin the operation of cell separation procedures, such asan increase in any one of cell purity, viability, oryield.Yet another aspect of this invention is a kitfor use in a cell separating device comprising thecompositions of the first, second, or third aspects ofthis invention wherein said composition is provided in adisposable container further comprising one or moreoutlet ports, at least one of said ports provided with aseptum. Because cell separating devices are routinelyused to make cell populations for therapeutic uses, it isdesirable to have reagents and ancillary hardwareprovided in a disposable format, preferably pre-sterilized. Also, the solutions may be provided incollapsible plastic containers, such as those used tostore blood. Such containers are routinely made frombiocompatable polymers, for example, polyvinyl chloride(PVC), polycarbonate, and polypropylene, all of which arereadily commercially available.Because cell separation devices, such as theIsoLEx® so, 300 (also referred to as IsoLEx® 3oosA), and300i series, come in a wide variety of mechanisms,theparticular components of the kit are linked to theshapes, and port size, number, and orientation,particular cell separating device. Therefore, a widerange of disposable formats are envisioned and necessary.Preferably, detachable inlet and outlet ports which carryfluids during operation of the cell separating devicewould be provided for use with the disposable containerin order to minimize the possibility of contaminationduring operation of the cell separating device.Preferably, these ports would contain a septum, such as aphysical barrier through which a syringe needle may beinserted to inject various reagents. To minimizel015202530CA 02264885 1999-03-03wo 93/10053 PCT/US97/1525213contamination further, a membrane filter capable ofremoving etiological agents such as bacteria, viruses,and parasites from solution, would preferably be includedwith at least one of the ports. The components of thepresent invention may be provided pre~mixed withreagents, or may be provided separately, preferably in amore concentrated form in a separate closed container,for example, a vial. Preferably, such a vial wouldcontain DNase and/or RNase with or without divalentcations.Often, air needs to be taken into the cellseparating device during operation. Therefore, a septummay also be provided to the device and/or container toremove etiological agents such as bacteria, viruses, andparasites from air as it is taken into the cellseparating device.Preferably, the kit comprises all reagents anddisposable hardware, such as reagent containers, tubing,filters, membranes, chambers, manifolds, and otherancillary elements necessary to perform a cell separationprocess using a particular cell separating device (suchas primary and secondary chambers, and spinningmembranes, for the ISOLEX® 300i Cell Separating System),Anexample of such a self—contained system is provided inin a completely self—contained, sterile system.Figure 1. Arrows indicate preferred sites of introducingthe compositions of this invention. However, thesecompositions may be introduced at any site where thesolution is to,at some time, pass through locations inthe kit where cell clumps form.Another aspect of this invention is a method ofpreventing cell clumping associated with a cellseparation process comprising: a) providing thecomposition of the first, second, or third aspects of 1015202530CA 02264885 1999-03-03W0 98/10053 PCT/US97/1525214this invention into the mixture to be separated in anamount sufficient to prevent cell clumping during thecell separation process. An amount of the compositionsufficient to prevent cell clumping results in theincreased efficiency of the operation of cell separationprocedures, such as an increase in any one of theparameters of cell purity, viability, or yield.Another aspect of this invention is a method ofpreventing cell clumping associated with a cellseparation process comprising: providing the compositionof the first, second, or third aspects of the presentinvention during the cell separation process rather thanprior to cell separation, in an amount sufficient toprevent cell clumping. This procedure reduces the amountof clumps which are formed and results in an increase inthe yield of the final cell population obtained.Increased purity and viability of resulting cellpopulation are also envisioned. The compositions may beadded at any point or time before or during the cellseparation procedure, preferably prior to the firstbiochemical or immunological reaction.Another aspect of this invention is a method ofclearing a cell separation device of cell clumpsfollowing a cell separation procedure comprising: a)providing the composition of the first, second, or thirdembodiment of the present invention; and b) subsequentlywashing the device with said composition under conditionswhich allow said cell clumps to clear.cell separation devices becomeOften, cellseparating devices comprise extensive lengths of smallFrequently,clogged from clumps formed during operation.tubing and convoluted passages between plates which areThe ability to clean thistubing and these passages is desirable to maintainkept very close together.1015202530CA 02264885 1999-03-03W0 98/ 10053 PCT/US97/ 1525215efficient operation of the cell separating device.Therefore, the compositions discussed in the previousembodiments may also be used to clear out existing clumpswhich interfere with the operation of such cellseparating devices.The instant invention is further described inthe following Examples.EXAEEEJl;IDNase Activity in Buffers Used in Cell SeparationP_r_o_c_e_du1:esIn order to determine DNase activity in buffersused in cell separation procedures, the followingexperiments were performed using an ISOLEX® 300i MagneticCell Separator (Baxter Healthcare Corp., Irvine,California).Standard buffer provided in the ISOLEX® 300icell separating device (Cell Separation Buffer) was usedto determine DNase activity. Cell separation buffer is(PBS), 14.6 mMThis buffer wascomprised of phosphate buffered salinecitrate, and 1% human serum albumin.made 0 to 12.5 pg in DNase (Pulmozyme®, Genentech, Inc,and 2.5mM of MgclpDNase activity was measured using the DNA—methyl greensubstrate assay of Sinicropi et 31., AalytigalBiochemistry, 222:35l-358 (1994), herein incorporated by(see generally Garland et al,, U.S. Patent No.5,304,465, issued April 19, 1994, herein incorporated bythis method allowsspectrophotometric detection of methyl green free fromSouth San Francisco, California)referencereference).Briefly,DNA—methyl green complex (Sigma Chemical, St. Louis, MO).DNase activity in Cell Separation Buffer with theaddition and subsequent removal of cell populations10152025CA 02264885 1999-03-03W0 98/ 10053 PCT/U S97! 1525216(hereinafter "Cell separation Cell Bag Supernatant")prior to the addition of DNA-methyl green complex(referred to herein as Cell Separation Cell BagSupernatant) was also determined. Cell Separation CellBag Supernatant was made by adding greater than 10”(PBMNC) into CellSeparation Buffer and removing the cells byperipheral blood mononuclear cellscentrifugation.The results of these studies are presented inFigure 2. Briefly, the Cell Separation Buffer plus 2.5mM MgCl2 supported DNase activity whereas the CellSeparation Cell Bag Supernatant (as described in theabove paragraph) plus 2.5 mM MgCl2 did not.EXAMELE_llEffects of Divalent Cations on DNase Activity inD_ul.besss2'_s_EB_SUsing the general procedure discussed above inExample I, the effects of varying the concentration ofdivalent cations was investigated. Specifically,magnesium ion concentration, in the absence of calciumion, was investigated. The results are displayed inTable 1-a.relative activity was 1 to 7.5 pg of DNase.following tables, "Standard"containing 4 mM MgCl2 and 4 mM CaCl2 which has beendetermined to optimized DNase I activity (Sinicropi eta1; (1994)) and "NA" means non—applicable.The linear range used to calculate theFor therefers to a bufferCA 02264885 1999-03-03W0 98/ 10053 PCT/U S97/ 1525217All other activities for were determined inDulbecco's PBS (D—PBS), the composition of which is setforth in the Gibco BRL Product Catalog and ReferenceGuide (1995-1996) (herein incorporated by reference) as;5 0.2 g/L of KCL, 0.2 g/L KHZPO4, 8.0 g/L of NaCl, 1.15 g/Lof Na2HPO.,, and 2.16 g/L of Na2HPO,,7H2O.Table 1-aMethyl Green Assay to Determine DNase Activity:Varying [Mg++]10 mM___[M9:._L+ 0.0 4.4%1.0 10.64%2.5 17.91%5.0 26.95%15 7.5 34.22%10.0 42.38%20.0 82.98%Standard 100.0%Next, the effects of calcium ion20 concentration, in the absence of magnesium ion, wasinvestigated. The results of the study are displayed inTable 1—b. The linear range used to calculate therelative activity was 1 to 5 pg of DNase. CA 02264885 1999-03-03wo 9s/10053 PCT/US97/1525218Table 1-bMethyl Green Assay to Determine DNase Activity:Varying [Ca++]mM TCa++1 5 0.0 7.4%0.13 10.7%1.0 A 2.5%2.5 3.9%10.0 23.8%10 Standard 100.0%Next, while keeping the magnesium ionconcentration at 2.5 or 10 mM, the concentration ofcalcium ion was varied from 0 mM to 10 mM, The resultsof these later experiments are depicted in Tables 1-c and15 1-d. The linear range used to calculate the relativeactivity was 1 to 5 pg of DNaseCA 02264885 1999-03-03W0 98/ 1005319Table 1—cMethyl Green Assay for Determining DNASE Activity:Varying [Ca++] at 2.5 mM [Mg++]5 mM_lMg::l mM_iQa::l EelaLize_A2tiMiLÂ¥2.5 0.0 17.8%2.5 0.13 21.7%2.5 1.0 9.2%2.5 2. 17.4%10 2.5 10. 15.6%Standard NA 100.0%Table l—dMethyl Green Assay for Determining DNASE Activity:Varying [Ca++] at 10 mM [Mg++]15mM_lMg::l mM_lQa::l BslsLiy:_A&tiMitx10 0.0 49.2%10 ‘0.13 45.7%10 1.0 63.8%20 10 2.5 68.4%10 10.0 61.3%Standard NA 100.0%PCT/US97/152521015WO 98/10053CA 02264885 1999-03-03PCT/US97ll 525220EXAMBLE_lllDigestion of DNA in Cell Separation Buffer UsingDifferent Concentrations of DNaseIn order to determine levels of DNase needed tomaintain high levels of DNase activity in Cell SeparationBuffer + 2.5 mM MgCl2, the following experiments wereDNA (0.2 mg/ml salmon milt DNA (CalBiochem,California)) in Cell Separation Buffer plus 2.5performed.La Jolla,mM MgCl2) was digested with DNase I (Pulmozyme®) at aconcentrations of 20 KU/ml, 10 KU/ml, 5 KU/ml, 1 KU/ml,and 0.1 KU/ml.points between 0.1 and 300 minutes and DNA activity wasquenched by addition of a portion of 42 mM EDTA solution.Samples were then separated by electrophoresis on a 1%Samples were removed at various timeagarose gel and DNA was visualized using ethidiumbromide. The results are provided in Figure 3, with thedigestion conditions provided below in Table 2.CA 02264885 1999-03-03W0 98,1003 PCT/US97/1525221TABLE 2EFFECT OF TIME AND DNase I CONCENTRATION ON DEGREE OFDNA DEGRADATIONTOP CONTENTS AND BOTTOM CONTENTSLANES REACTION CONDITIONS LANES1 Standards 1 Standards2 Standards 2 Standards3 DNA: no DNase 3 DNA + 5 KU/mLDNase: 15 minutes4 DNA + 20 KU/mL 4 DNA + 5 KU/mLDNase: 0.1 minutes DNase: 30 minutes5 DNA + 20 KU/mL 5 DNA + 5 KU/mLDNase: 5 minutes DNase: 60 minutes6 DNA + 20 KU/mL 6 DNA + 5 KU/mLDNase: 10 minutes DNase: 300 minutes7 DNA + 20 KU/mL 7 DNA + 1 KU/mLDNase: 15 minutes DNase: 0.1 minutes8 DNA + 20 KU/mL 8 DNA + 1 KU/mLDNase: 30 minutes DNase: 5 minutes9 DNA + 20 KU/mL 9 DNA + 1 KU/mLDNase: 60 minutes DNase: 10 minutes10 DNA + 20 KU/mL 10 DNA + 1 KU/mLDNase: 300 minutes DNase: 15 minutes11 DNA + 10 KU/mL 11 DNA + 1 KU/mLDNase: 0.1 minutes DNase: 30 minutes12 DNA + 10 KU/mL 12 DNA + 1 KU/mLDNase: 5 minutes DNase: 60 minutes13 DNA + 10 KU/mL 13 DNA + 1 KU/mLDNase: 10 minutes DNase: 300 minutes14 DNA + 10 KU/mL 14 DNA + 0.1 KU/mLDNase: 15 minutes DNase: 0.1 minutes10CA 02264885 1999-03-03wo 98/10053 PCT/US97/1525222TABLE 2EFFECT OF TIME AND DNase I CONCENTRATION ON DEGREE OFDNA DEGRADATIONTOP CONTENTS AND BOTTOM CONTENTSLANES REACTION CONDITIONS LANES15 DNA + 10 KU/mL 15 DNA + 0.1 KU/mLDNase: 30 minutes DNase: 5 minutes16 DNA + 10 KU/mL 16 DNA + 0.1 KU/mLDNase: 60 minutes DNase: 10 minutes17 DNA + 10 KU/mL 17 DNA + 0.1 KU/mLDNase: 300 minutes DNase: 15 minutes18 DNA + 5 KU/mL 18 DNA + 0.1 KU/mLDNase: 0.1 minutes DNase: 30 minutes19 DNA + 5 KU/mL 19 DNA + 0.1 KU/mLDNase: 5 minutes DNase: 60 minutes20 DNA + 5 KU/mL 20 DNA + 0.1 KU/mLDNase: 10 minutes DNase: 300 minutesEKAMELE_IMEfficacv of Cell Separation Using DNase in the..]n J.In order to determine the efficacy of cellseparation methods using DNase in the initial cellpopulations, the following experiments were performed.Populations of CD34+ cells from peripheralblood mobilized cells (Tseng—Law et al. (1994)) wereseparated using the ISOLEX® 300i Cell Separator inaccordance with the manufacture's instructions. To D—PBSwas added either 1)MgCl2, or 3) 10 KU/ml of DNase + 10 mM MgCl2.no DNase,2)10 KU/ml DNase + 2.5 mMTheresulting suspended cell populations were analyzed forCA 02264885 1999-03-03W0 98/1005323purity, yield, and viability.in Table 3.enhanced when a concentration of 2.5 mM MgCl2 was usedrather than a concentration of 10 mM MgcllPCT/US97ll5252The results are providedSurprisingly, the recovery of cells was mostTABLE 3PURITY, YIELD, AND VIABILITY OF CD34+ CELLS SEPARATED USINGDNase PLUS Mg++Conditions Number Purity Yield Viability CD34+of Runs (%) (%) (%) Cells<10“)No DNase or 6 69 39 87 1.5Mgclzl0KU/ml Of 6 68 57 85 2.1DNase + 2.5mM MgCl210 KU/ml of 3 42 48 88 1.1DNase + 10mMMgCl220 KU/ml of l 67 56 97 1.6DNase + 2.5mM MgCl2EXAM2LE_MDegradation of Clumps Previouslv Formed in a11 E . I .10A small portion of a primary chamber clump froman ISOLEX® Cell Separator (previously stored for 2 weeksat 4°C) was incubated with 471 KU of DNase I (containingchymotrypsin activity) (Sigma Chemical Co., St. Louis,MO) in D—PBS without Mg++, Ca++, or citrate. Next, afresh clump from an ISOLEX® 300i cell separationprocedure of greater than 10” PBMNC's was further tested.The wet weight of the clump was measured as 1.841 g, thenthe clump was divided into eight equal portions for thefollowing treatments: PBS control, mechanical1015202530CA 02264885 1999-03-03W0 98/ 10053 PCT/US97/1525224trituration, a DNase I (with chymotrypsin; all previousexamples used Pulmozyme® DNase I which does not containMO)treatment in PBS, chymopapain (Baxter Healthcare, Irvine,chymotrypsin) (Sigma Chemical Co., St. Louis,California),DNase Iplasmin (Sigma Chemical, St. Louis, MO),(with chymotrypsin)/plasmin mixture.Using a second fresh clump from an ISOLEX® 300icell separation procedure several more conditions weretested. The wet weight of the clump was measured (2.55g), then the clump was divided into seven equal portionsfor the following treatments: PBS control, four differentDNase I concentrations (with 5 mM MgCl2), Polybrene®(Sigma Chemical, St. Louis, MO), DNase-free RNase (SigmaChemical, St. Louis, MO), 9069N peptide (BaxterHealthcare, Irvine, California), and trypsin(Worthington, Freehold, New Jersey). Also, toinvestigate 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/mlsolution of topical fibrinogen complex (TFC) (BaxterHyland, Glendale, California).The results of these experiments are presentedin Table 4. Briefly, complete dissolution of the twoweek old chamber clump was achieved with treatment of 471As shown by the data for thefirst clump, after one hour, the combined treatment ofKU of DNase in 1 hour.the clump with DNase and plasmin released 13 millioncells and dissolved most of the clump. Chymopapain andplasmin had relatively little effect after 24 hours ofThe addition of Mgclzcofactor) greatly accelerated clump dissolution.incubation. (a required DNase ISubstantial cell lysis was observed for the DNasepreparations of the instant Example; the preparations10......—_—......w.........._,.... Ø 0 .WO 98/10053CA 02264885 1999-03-03PCT/US97/1525225contained 33% chymotrypsin plus 66% DNase in addition toMgCl2.As shown by the data for the second clump,immediate and complete dissolution of a fresh clump wasobserved at a concentration of l0OOKU of DNase I. RNaseTreatment ofclumps with Polybrene® or 9096N peptide had no effect.Only minor degradation of the clumps was observed upontreatment with 5 mg/ml trypsin for 24 hours.completely dissolved the clump in 24 hours.As acontrol, a PFC clot was incubated with 5 mg/ml trypsin,wherein the clot completely dissolved in approximatelyfive minutes.CA 02264885 1999-03-03W0 98/ 10053 PCT/US97l1525226TABLE 4DEGRADATION OF CELL CLUMPSClump Treatment Quantity Clump Degredation No. of CellsY VisibilitySource (*10"-6) (%) YQ0.25 hr 1 hr 24 hr2 week old DNase I 471 KU + ++++ ++++ nd nd(+4 °C)Clump #1 PBS 1 mL - - - 0.5 93Mechanical 1 mL +-H—+ ++-+-+ +-H-+ 22 ndDNase I 235 KU + -H- ++++ 0.4 90Chymopapain 200 - - - 0.2 ndpicokatalsPlasmin 1 Unit - -H- ++ ndDNase 1+ 235 KU + 235 -+++ -H—-H- 13 82Plasmin 1 UnitClump #2 PBS 1 mL - - - 0.5 87DNase I + 1000 KU -H-1-+ +9-H «H-4+ 1.9 595 mM MgCl,100 KU + H 4-+—++ 1.3 1910 KU - - + 0.3 541 KU - - + 0.4 60Polybrene® 1 ug - - - 0.4 58RN&se 405 KU + -++ +-H-+ nd nd9069N lmg/mL - - - nd ndPeptideTrypsin 5 mg/mL - - ++ nd ndClot Trypsin 5 mg/mL ++++ +-H-+ ++++ nd nd(PFC)Y Cell number and viability were determined after 1 hr (Clump #1) or 15 minutes (Clump #2).Q Viability was determined by microscopy or calcien (Clump #1) or by acridine orange/propidiumiodide (Clump #2).SUBSTIT UTE SHEET (RULE 26)

Claims (21)

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 bindentate chelating agent is selected from the group consisting of EDTA and citrate ion.

6. The composition of claim 5 wherein the bidentate 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, and combinations thereof.

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 the group consisting of 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 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 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 100 to 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 a11 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.