neowater in biopharma arena

8/14/2019 Neowater in Biopharma arena

1/30

This Provisional PDF corresponds to the article as it appeared upon acceptance. Fully formattedPDF and full text (HTML) versions will be made available soon.

Enhancement of hybridoma formation, clonability and cell proliferation in ananoparticle-doped aqueous environment

BMC Biotechnology2008, 8:3 doi:10.1186/1472-6750-8-3

Natalie Gavrilov-Yusim ([email protected])Ekaterina Hahiashvili ([email protected])

Marina Tashker ([email protected])Victoria Yavelsky ([email protected])

Ohad Karnieli ([email protected])Leslie Lobel ([email protected])

ISSN 1472-6750

Article type Research article

Submission date 8 August 2007

Acceptance date 14 January 2008

Publication date 14 January 2008

Article URL http://www.biomedcentral.com/1472-6750/8/3

Like all articles in BMC journals, this peer-reviewed article was published immediately uponacceptance. It can be downloaded, printed and distributed freely for any purposes (see copyright

notice below).

Articles in BMC journals are listed in PubMed and archived at PubMed Central.

For information about publishing your research in BMC journals or any BioMed Central journal, go to

http://www.biomedcentral.com/info/authors/

BMC Biotechnology

2008 Gavrilov-Yusim et al., licensee BioMed Central Ltd.This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),

which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
mailto:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]://www.biomedcentral.com/1472-6750/8/3http://www.biomedcentral.com/info/authors/http://creativecommons.org/licenses/by/2.0http://creativecommons.org/licenses/by/2.0http://www.biomedcentral.com/info/authors/http://www.biomedcentral.com/1472-6750/8/3mailto:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]


2/30

1

Enhancementofhybridomaformation,clonabilityandcellproliferationinananoparticle-

dopedaqueousenvironment

NatalieGavrilov-Yusim1,EkaterinaHahiashvili

1,MarinaTashker

1,VictoriaYavelsky

1,Ohad

Karnieli

2

andLeslieLobel

1*

1Department ofVirology andDevelopmental Genetics, Ben GurionUniversity of theNegev,

Beersheva84105,Israel

2DepartmentofHumanMolecularGeneticsandBiochemistry,SacklerSchoolofMedicine,Tel

AvivUniversity,TelAviv69978,Israel

*Correspondingauthor:

LeslieLobel

DepartmentofVirologyandDevelopmentalGeneticsBenGurionUniversityoftheNegev

Beersheva84105,Israel

E-mail:[email protected]

Phone:+972-8-6479941

Mobile:+972-54-2156235


3/30

2

Abstract:

Background: The isolation and production ofhumanmonoclonal antibodies isbecomingan

increasinglyimportantpursuitasbiopharmaceuticalcompaniesmigratetheirdrugpipelinesaway

from small organicmolecules.As such, optimization ofmonoclonal antibody technologies is

important,asthisisbecomingthenewrate-limitingstepfordiscoveryanddevelopmentofnew

pharmaceuticals.Themajorlimitationsofthissystemaretheefficiencyofisolatinghybridoma

clones, the process of stabilizing these clones and optimization of hybridoma cell secretion,

especiallyforlarge-scaleproduction.

Manypreviousstudieshavedemonstratedhowperturbationsintheaqueousenvironment

can impactupon cell biology. Inparticular, radio frequency (RF) irradiationof solutions can

have dramatic effects on behavior of solutions, cells and in particular membrane proteins,

although this effect decays following removal of the RF. Recently, it was shown that

nanoparticledopingofRFirradiatedwater(NPDwater)producedastabilizedaqueousmedium

thatmaintainedthecharacteristicpropertiesofRFirradiatedwaterforextendedperiodsoftime.

Therefore,theorderingeffectinwateroftheRFirradiationcannowbestudiedinsystemsthat

requiredprolongedperiodsforanalysis,suchaseukaryoticcellculture.Sincetheformationof

hybridoma cellsinvolves the formationof anewmembrane, aprocessthat isaffected bythe

surrounding aqueous environment, we tested these nanoparticle doped aqueous media

formulationsonhybridomacellproduction.

Results: In this study, we tested the entire process of isolation and production of human

monoclonal antibodies in NPD water as a means for further enhancing human monoclonal

antibody isolation andproduction.Our results indicate an overallenhancementof hybridoma

yield,viability,clonabilityandsecretion.Furthermore,wehavedemonstratedthatimmortalcells

proliferatefasterwhereasprimaryhumanfibroblastsproliferateslowerinNPDwater.

Conclusions: Overall, these studies indicate that NPD water can enhance cell proliferation,

clonability and secretion. Furthermore, the results support the hypothesis that NPD water is

effectivelycomposedofstablemicroenvironments.


4/30

3

Background:

Emerging therapeutic strategies are becoming increasingly dependent on

immunotherapeuticapproaches.Inparticular,thisconsistsofhumanizedorhumanmonoclonal

antibody production,which is largely dependent on cell-based technologies.Developmentof

humanmonoclonalantibodiesis achievedwith acoupleof different techniques,oneofwhich

involves theformation ofhumanhybridomacells.Our laboratory isolates humanmonoclonal

antibodies from peripheral blood lymphocytes with the hybridoma technique, using both

humanizedandfullyhumanfusionpartnercelllines[1,2].Hybridomacells,likeprimarycell

cultures,areexquisitelysensitivetotheirenvironment[3-5]andrequireconditionedmediathat

contains various stimulatory and growth factors for stabilization [6]. Indeed, like non-

transformedcells,theycloneverypoorly,andcombinedwithpoorstability,thishasbecomethe

limitingfactorintheabilitytocontinuallyproduceanyhumanmonoclonalantibody(andmurine

forthatmattertoo),whichisidentifiedinaprimaryhybridomaculture.

The formation of hybridoma cells involves the fusion of two cells and therefore the

productionofanewlipidbilayermembranesurroundingthecontentsoftwocells.Similartothe

formation of artificial lipid vesicles in an aqueous buffer system in vitro [7], the aqueous

environmentlikelyhasanimpactonthesesimilaryetdistinctprocesses.Biologicalsystems,in

general,aredependentontheaqueousenvironment,aslifeitselfhasevolvedasafunctionofthe

propertiesofwater.Biologicalprocessesfromdivisionoflivingcellstoenzymaticreactionsand

DNAreplicationareintimatelyassociatedwith,anddependentupon,thepropertiesofwater[8].

However,theaqueousfoundationoflifeis,forthemostpart,takenforgrantedandmostassume

thatitisauniformmedium[9].Assuch,questionsconcerningtherolethatwaterplaysinthe in

vitroprocessesofexperimentalbiologyhave toa largeextentbeenavoided inmostbiological

studies. Thus, much of experimental biology rests upon the assumption that the aqueous

environmentoflifeisnotanimportantfactorintheoutcomeofmostexperimentation.Thishas

resulted, thus far, in little investigation into the effect of the physicalproperties ofwater on

biologicalsystems.

Previousstudiesofwaterhavedemonstratedthatirradiationinthemicrowaverangeof

frequencies changes certain physical properties of water, likely generating water of a higher

orderedstructure[10,11].However,thischange isephemeralandlastsonlya coupleofhours

followingremovalof theirradiation.Recently, itwasdiscoveredthatnanoparticle-dopingof


5/30

4

this irradiated (RF)water (NPD water) could stabilize the altered environment for extended

periodsof time[12].Assuch,it isnowpossibletoexamine the effect of thisnovel aqueous

environmenton systems that requireprolonged periods, such as days, for analysis. Thisnew

aqueous formulation has several unique properties including a probable higher ordered

structure[12].Inmanyways itis akintodopingofsilicon inthesemiconductorindustry for

productionofsiliconwaferswithuniqueconductivities[13].

Recentstudiesinelectrochemistry[12]haverevealedsomeinterestingpropertiesofNPD

water. These experiments suggest that our conventional view of watermay have overlooked

hidden properties that become apparent under unique conditions. Specifically, patterns of

electrochemicaldepositionarealtered[12],providingadramaticdemonstrationoftheeffectof

environmentalorderonchemistry.Inthefieldofbiology,studieshavedemonstratedsignificant

changesintheopeningfrequencyofpotassiumchannelsincellsgrowninRFtreatedmedium

[14-16]andthatbacteriahaveanincreasedgrowthratewhengrowninRFtreatedmedium[17].

Sinceisolationofhumanmonoclonalantibodiesisbecomingpivotalfordevelopingnew

immunotherapies,evaluationoftechnologiesfor enhancingtheprocessof thechemicalfusion

technique[18]andoptimizationofitsefficiencytoyieldalargernumberofhybridomacellshas

beenanongoingpursuit.Thesestudiesexaminewhetheralterationsintheaqueousenvironment

impactonhybridomacellformationandantibodysecretion.Theydemonstrate thatNPDwater

enhances the entire process of human monoclonal antibody isolation and production.

Furthermore, proliferation studies on immortal and primary cells support the hypothesis that

NPDwateriseffectivelycomposedofstabilizedmicroenvironments.


6/30

5

Methods:

Materials

RFtreatednanoparticledopedwater

The effectsof RF-treatment of water can be amplified and stabilized by doping with

smallquantitiesofcrystallinenanoparticles(nanoparticledopedwater(NPD)).ThisNPDwater

waspreparedaccordingtopatent#PCT/IL2005/000198(FDADMFfilenumber20503),andas

described byKatsir et. al. [12], and kindly provided by Docoop Technologies (Or Yehuda,

Israel).TheprocessutilizesultrapureDIwaterthatiskeptbelowthedensityanomalypoint

(i.e.justbelow4C)andisradiofrequencyirradiated(RF)at915MHzwithapowerof60watts.

After10minutesofRFirradiation,sub-micronsizedpowderofbariumtitanate,whichisheated

to900C, isdropped fromafurnaceinto thewaterandtheRF-irradiationiscontinuedforan

additionalfiveminutes.Thetreatedwateristhenmaintainedatroomtemperaturefortwodays,

atwhichpoint ithas clarifiedasmostof the sourcepowder (thatcontains largerparticles) is

separated at the bottom by gravity. The clarified treated water is then filtered through 0.22

micrometerfilterstoremovesourceparticlesoflargesize.Whereasthesourcepowderforthis

processcontains large agglomerates composed of small particleswithrectangularandfaceted

shapes,afterthedopingprocessmostoftheparticlesthatremainsuspendedinthewaterhave

almostperfectsphericalshape.Theseobservationsindicate thatduringtheproductionsomeofthelargeagglomeratesdisintegrateandthatsomeoftheindividualparticlesaltertheirshapeand

becomespherical.This effect is reminiscentof thephenomenaobservedduringsonochemical

synthesisofnanoparticlesusingcavitation[12,19,20].

The nanoparticle doped water that was produced by the above process for these

experiments,wasdopedwithsphericalnanoparticles(approximately10-50nmsize)ofbarium

titanateat1015particlesperliter,whichstabilizestheeffectofRFonwater[12].Asacontrolwe

used18.2megaohmultrapuredeionizedwater(DIwater,UHQPS,ELGALabwater)thatisthestartingmaterial for preparation of the nanoparticle stabilizedRF treatedwater. Asa further

control,wealsotestedDIwaterthatwasdopedwithnanoparticleswithoutirradiation.Allwater

was filtered through a 0.22 m filter prior to use. Experiments with DI water doped with

nanoparticleswithoutirradiationyieldedsimilarresultstoDIwaterinallexperiments(datanot

shown) andwe thereforepresentonlyexperimental resultsofDI water versusNPDwater in


7/30

6

thesestudiesforsimplicity.

Reagentsforcellgrowth

AllthemediaandsupplementsforcellgrowthwerepurchasedfromGIBCOBRL,Life

Technologies.RPMI1640andDMEMwerepurchasedinpowderformandreconstitutedeither

in NPD or in DI water. After reconstitution sodium bicarbonate was added to the media

according to themanufacturersrecommendation,andtherewasnofurther adjustmentofpH.

Prior touse, all themedia were filter-sterilized through a0.22 m filter (Millipore).For the

growthofhybridomacells,RPMIwassupplementedwith10%fetalcalfserum,L-glutamine(4

mM),penicillin(100U/mL),streptomycin(0.1mg/mL),MEM-vitamins(0.1mM),non-essential

aminoacids(0.1mM)andsodiumpyruvate(1mM).Allthesupplementsmentionedabovewere

boughtinaliquidformandusedasisfromthemanufacturer(meaning,theyweredilutedinto

the NPD or DI based media). 8-Azaguanine, HTandHAT were purchased fromSigma and

reconstituted from powder form with NPD or DI RPMI. DMEM used for human primary

fibroblastsandCHOcellsgrowthwassupplementedwith10%fetalcalfserum,L-glutamine(4

mM),penicillin(100U/mL),streptomycin(0.1mg/mL).Hybridomacloningfactorwasbought

fromBioVeris.

Chemicalreagents

PowderedPBSwasobtainedfromGIBCOBRL,LifeTechnologies.PEG-1450(P5402,

Sigma)waspurchasedfromSigmaandreconstitutedwithsterilePBSbasedonNPDoronDI

water (50%w/v).Thepreparationwasadjusted topH7.2,DMSO (v/v)(Sigma)wasaddedto

10% followed by sterile filtration of the PEG solution through a 0.45 m filter (Millipore).

Hanksbalancedsaltsolutionwasbought fromBiologicalIndustriesBeit-HaEmekLTD, Israel

and used as is for NPD and DI based experiments. Carbonate-bicarbonate buffer (0.05 M,

pH=9.6)forELISAplate-coating,OPD(usedin0.4mg/mL)andphosphate-citratebuffer(0.05

M,pH=5.0)wereboughtfromSigma.

Antibodies

Goat anti-human IgM/IgG and HRP-conjugated goat anti-human IgM/IgG were

purchasedfromJacksonImmunoResearch.StandardhumanIgM/IgGwereboughtfromSigma.

Cells

Allcellsusedintheseexperiments(MFP-2,CHOandprimaryhumanfibroblasts)were


8/30

7

maintained for aweekin eitherNPDorDIbasedmedia sothat the cellswereadapted tothe

media prior to experimentation. In addition, the fusion partner cell line MFP-2 [1] was

maintainedinRPMI1640withtheadditionoffetalbovineserumandadditivesaspreviously

described[1]alongwith8-azaguaninetomaintaintheHGPRTminusphenotype.Primaryhuman

fibroblastswere obtainedfrom theATCCandmaintainedinDMEM.TheCHO cell linewas

maintained inDMEM.All cell culturewas performed in completemedia, which consists of

culturemediawith theadditionoffetalcalf serum,glutamineandpenicillin/streptomycin.For

the MFP-2 cell line vitamins, nonessential amino acids and pyruvate were also added in

completemedium.

Methods

CellFusion

Weemploythechemicalfusiontechnique[18]withPEG1450,whichactsasafusogen,

forcreationofhybridomacellswithhumanperipheralbloodlymphocytes.PEG1450istypically

preparedinPBSwiththeadditionof10%DMSO.Fortheseexperiments,NPDwaterwasused

topreparePBS,whichwasthenusedtomakeaPEG/DMSOsolution;asa controlpreparation

weusedPEGpreparedinDIbasedPBS.ForallfusionexperimentscomparingNPDtoDIwater,

all reagents were prepared in either NPD or DI water except for fetal bovine serum and

concentratesofsupplements. Inaddition,dilutionofcellsinHanksbalancedsalts(HBSS)(see

below),followingfusionwithPEG-1450,wasperformedwithapurchasedliquidformofHBSS

(BeitHaEmek,Israel)andusedas isfrom themanufacturer.Wepreferrednottoprepare this

mixture ourselves as a minor deviation from its salt composition can introduce error when

comparingfusioninNPDandDIwater.

Forproductionofhybridomacells,humanperipheralbloodmononuclearcells (PBMC)

wereisolatedfrom40mLoffreshlydrawnwholeblood,purifiedwithHistopaque1077(Sigma)

aspreviouslydescribed,andwashed4 times inDIbasedculturemediumwithoutserum.The

MFP-2fusionpartnercellswereeithergrowninNPDorDIbasedmediaandthenwashedwith

therespectivemedia4timeswithoutserum.ForeachexperimentasinglebatchofPBMCwas

dividedintotwoequalfractions,oneofwhichwasusedforNPDandtheotherforDIfusions.

Next,MFP-2 and PBMC were mixed in either NPD or DI basedmediawithout serum and

pelleted.PEG-1450pre-warmedto37Cwas thenadded at300L for 10-200x106 ofmixed


9/30

8

cells.ThecellmixturewasincubatedwithPEGfor3minuteswithconstantshaking.PEGwas

then diluted out of the cell mixture with Hanks balanced salt solution and complete RPMI

(prepared ineitherNPD orDIwater).Tothe resultant cellsuspensionwere added: fetalcalf

serum(10%)andHT(x2).Thehybridomacellsthatweregeneratedinthisprocesswerecultured

in96-wellplates(celldensity-2x106lymphocytes/well)incompleteRPMIwithHATselection.

The screening of the supernatants for immunoglobulin production was performed after the

hybridomacellsoccupiedapproximatelyofthewell.

SandwichELISA

AsandwichELISAwasusedto screenhybridomasupernatantsforIgM/IgG.Briefly,a

capturingantibody (goatanti-humanIgM/IgG)wasprepared inacarbonate/bicarbonatebuffer

andappliedona 96-wellplateina concentration of100ng/100L/well. Theplatewas then

incubatedovernightat4C.Allthefollowingstepswereperformedatroomtemperature.After1

hourofblockingwith0.3%drymilkinPBS,thesupernatantsfromthehybridomacellswere

appliedfor1.5hours.Humanserumdiluted1:500inPBSwasusedasapositivecontrol.Fora

background and as a negative control hybridoma growth medium was used. The secondary

antibody (HRP-conjugatedgoat anti-human IgM/IgG)wasprepared in blocking solution at a

concentrationof1:5000andincubatedfor1hour.Toproduceacolorimetricreactiontheplates

wereincubatedwithOPDinphosphate-citratebuffer,containing0.03%H2O2.Thecolorreaction

wasstoppedwith10%HClafter15minutes.Thereadingandtherecordingofthereactionwere

performed with a Multiscan-Ascent (Thermo Scientific) ELISA reader using the 492 nm

wavelength filter.All reagentsusedwere standardwith the exception of the sandwich layer,

whichconsistedoftheNPDorDIbasedhybridomasupernatant.

Cloning

Twohundredcellsofachosencloneweredilutedinavolumeof10mLofmediaand

seededina96-wellplate(100L/well),sothatonaveragethewellscontained1-2cells.The

cells were incubated and periodically fed and microscopically monitored for clonal growth.

When a clone occupied 1/4-1/2 of the well, its supernatant was analyzed. The efficiency of

cloningwas expressed ina number of viable clonesper plate. Ten percentHCF (hybridoma

cloningfactor)wasaddedaccordingtotheexperimentaldesign.


10/30

9

Cellgrowthassay

Growthofprimaryandimmortalizedcelllineswasmonitoredwithacrystalvioletdye

retention assay aspreviouslydescribed [21].A fixed number ofcells was seeded in96-well

platesinmultiplerepeats.Cellgrowthwasstoppedbyfixationin4%formaldehyde.Fixedcells

were then stained with 0.5% crystal violet followed by extensive washing with water. The

retaineddyewas extractedin100L/wellof0.1Msodiumcitratein50%ethanol(v/v).The

absorbanceofthewellswasthenreadat550nmwithaMultiscan-Ascentmicroplatereaderand

theappropriatefilter.

Primaryhumanfibroblastculture

Starting at passagetwenty,humanfibroblastswere cultured andpassed everyweek as

longasthecellsdisplayedtypicalfibroblastmorphologyandtheirnumberdidnotdropbelow

theinitiallyseededamount.Thenumberofpassagesandcalculatedpopulationdoublingswere

recorded.Themorphologyand viability of thecellsweremonitoredmicroscopically. Human

fibroblastsusedintheseexperimentsweregenerallyatapopulationdoublingof25.

Dataanalysis

Thestatisticalsignificanceofdifferenceintheefficiencyoffusionandcloningbetween

NPDandDIbasedexperimentswasdeterminedbytheChi-squaretest.Theresultsofthegrowth

testwithprimaryhumanfibroblastswereanalyzedbyanunpairedStudents't-test.Statisticalp-

values


11/30

10

Results:

Nanoparticle doped RF treated water (NPD water) enhances efficiency of hybridoma

formationforproductionofhumanmonoclonalantibodies

ResultsofchemicalfusionexperimentsarepresentedinFigure1.Fortheseexperiments

PBMCs fromasingleindividualwere divided intotwogroupsafterpurification forfusionin

either a NPD or DI based environment. In our experiments we witnessed a statistically

significantdifferenceintheyieldofhybridomacellsbetweenNPDandDIenvironments.There

wasacleartendencyforagreateryieldofhybridomacellsintheNPDbasedfusionexperiments

as compared to the parallel fusions in DI based media. The percent of enhancement was

calculatedby theformula[(number ofhybridomacells inNPDfusion/numberofhybridoma

cells in DI fusion) x100%-100%] and these results are depicted in Figure 1. The extent of

enhancementisvariable,andwithinaseriesofeightfusionexperimentsvariedfrom22to227

percent.AlthoughtheincreasedefficiencyoffusioninNPDisvariable,thisisnotunexpectedas

eachfusionwasperformedwithlymphocytesfromadifferentdonor.Assuch,magnitudeofthe

effectofaNPDaqueousenvironmentonhybridomaformationisafunctiontosomeextentofthe

geneticbackground.

IncreasedyieldofhybridomasubclonesinNPDwater

Oneofthecrucialstepsintheprocessofmonoclonalantibodyproductionistheisolation

ofastablesubclonefromaprimaryhybridomapopulationfoundtobepositiveforsecretionofa

specificmonoclonal antibody. This is typically achieved by serially subcloning of a specific

primaryhybridomaclone.Thepurposeofsubcloning,whichinvolvesseeding1-2cellsperwell,

is to produce clones of a single origin, which are genetically stable and produce a unique

monoclonalantibody.Duringthisprocess,hybridomacellscandieduetogeneticinstabilityor

proliferatebutlose theircapacity toproduceantibodies.Toovercomethesedifficultiesweuse

hybridoma cloning factor (HCF), which consists of macrophage conditioned media [6]

containingavarietyof factorsthatfacilitatecloneoutgrowthandstabilization.However,since

the fusion partner cell line we use is of myeloma origin [1], the hybridoma cells that are

producedwithitlikelysecreteautocrinefactorsthatpromotetheirownclonalexpansion[22-24].

Theautocrineactionofthesefactors,however,isnotapparentinstandardinvitroculturedueto

their relatively low concentration.We therefore tested the hypothesis that NPD based media

enhances the bioavailability, and hence autocrine activity, of these secreted factors, through


12/30

11

increaseinthecell-localizedconcentration.Thiswasbestachievedthroughsubcloningprimary

hybridomacellsinDIversusNPDbasedmediaandalsoobservingtheeffectofaddingHCFto

bothcloningmedias.

Following fusionof PBMCwithMFP-2 and outgrowth of primary hybridoma clones,

antibody-producinghybridomapopulationswere identifiedandsubclonedineitherNPDorDI

based media with supplements. The results of these experiments are displayed in Figure 2.

Overall, for primary hybridoma populations tested, we observed greater clonal outgrowth of

antibodysecretinghybridomacellsinNPDbasedmediaascomparedtoDIbasedmedia.When

HCFwasaddedtobothNPDandDIbasedmediawesawasimilarpercentageincreaseinthe

number of antibody producing clones in both formulations. Figure 2 panel A illustrates a

representative cloningexperiment fora primaryhybridomapopulation from five independent

cloningexperiments.AlldisplayedthesametrendwherethenumberofclonesinDIbasedmedia

withHCFwasstatisticallythesameasthatobservedinNPDbasedmediawithoutaddedHCF.

As primary hybridoma populations are highly unstable and each is different, data cannot be

combined frommultiple experimentsnormultiple primary hybridomapopulationsublconings.

Finally, as shown in Figure 2 panel B, clonability of cells from a semi-stable clone is also

enhancedinNPDbasedmedia.

Increasedsecretionofmonoclonalantibodies fromhybridomacellsgrowninNPDwater

TostudytheeffectofaNPDaqueousenvironmentonsecretionofmonoclonalantibodies

westudiedtheproductionofhumanmonoclonalantibodiesfromseveralstabilizedhybridoma

clones.Hybridoma clones from ourcollection that have been stablyproducingantibodies for

over5yearsweregrowninDIbasedmediumandthentwoparallelcultureswerepreparedfrom

it, one in NPD and the other in DI based medium. Following a period of several days of

adaptation,cellswere seededatequivalentdensities inreplicateandafter fivedaysofgrowth

supernatantswereharvestedandantibodyconcentrationsweremeasuredbystandardsandwich

ELISA.TheresultsofoneoftheseexperimentsarepresentedinFigure3(panelA),althoughall

showedsimilarresults.Asisevidentfromthegraph,althoughtheyieldsfromthereplicateNPD

based cultures were somewhat variable (NPD culture concentrations ranged from101 to 40

g/mL,whereasinDItherangewasmuchnarrower:30-32g/mL),therewasoverallagreater

yieldofmonoclonalantibodyintheNPDbasedmedia.However,somecellsgrowfasterinNPD

basedmedia(seebelow).Thusthisresultmightnotreflectgreatersecretionpercellbutrather


13/30


14/30

13

Figure5,whichdemonstratesthatinNPDmediumthecellsgrewfasterbyanaverageofnearly

30%.ToexaminetheeffectofserumdepletiononCHOcellgrowth,cellswereseededinparallel

NPDandDI based cultures inreplicatewitheither5%or1%FCS. In these experiments cell

masswas quantitated bymeans of crystal violet dye retention assay[21].The resultsof this

experiment,illustratedinFigure5indicatethatunderserumreducedconditionscellsgrowfaster

inNPDbasedmediaascomparedtoaDIbasedmedia.

PrimaryhumanfibroblastsgrowslowerinNPDwater

Primary human fibroblasts at a relatively low passage (twenty population doublings)

were firstcultured inDI and NPD based media toadapt the cells to their respective growth

media. Since primaryfibroblastsare sensitive tocelldensity,weassessed the effect ofNPD

versusDIbasedmediaoncellproliferationwithdifferentinitialseedingdensity.Ina96-well

platetwocelldensitieswereseededinreplicatewellsinbothNPDandDIbasedmedia,fiveand

tenthousandcellsperwell.Afteranovernightgrowththeplateswereanalyzedwithacrystal

violetdyeretentionassay.TheresultsofthisassayaredepictedinFigure6,panelA.Atbothcell

densities,fibroblastsgrowninDIbasedmediaproliferatedfasterthaninNPDbasedmedia.This

differencewas found to behighly statistically significant (p


15/30

14

Discussion:

Theprocessof isolatinghumanmonoclonal antibodies by thehybridoma techniqueis

laborious andweare constantly seekingways tooptimize this processfor increased yield of

viableantibodysecretinghybridomaclones,aswellasforincreasedsecretionofantibodiesfrom

the nascent clones for screening. To increase the efficiency of hybridoma isolation and

monoclonalantibodyproductionwetestedNPDwaterasanalternativeaqueousenvironmentfor

cell culture. Itdemonstrated a positive effecton thehybridomayield,whichwasstatistically

significant,andthefusionefficiency(portrayedbythenumberofviablehybridomacellsineach

experiment)roseseveralfoldinsomecases.Thevariabilityoftheenhancement(10-230%)was

expected, as the fusion efficiency for human hybridoma formation typically differs between

experiments,anddependsonmanyfactorsthatarelargelyhostspecific[1].

Forproductionandtherapeuticuseofhumanmonoclonalantibodies,efficientisolationof

hybridomaclonesandlarge-scaleproductionandpurificationisrequired.Wethereforetestedthe

effectofNPDbasedmediaonoutgrowthofhybridomaclonesduringtheprocessofsubcloning

the primary hybridoma population and secretion of human monoclonal antibodies from

hybridoma cells. Our results demonstrate a marked enhancement of NPD based media on

efficiency of hybridoma clonal outgrowth and significantly increased antibody yield inNPD

culture.Thebasisoftheseeffectsislikelymultifactorial,howeveradirecteffectofNPDbased

mediaonhybridomaclonabilityandsomepartofantibodysynthesisand/orsecretionislikely.

Overall, there is an enhancement of antibody yield from a hybridoma culture regardless of

whetherit'scausedbyahigherpercellsecretionrateorbyahighernumberofhybridomacells.

TostudytheeffectofNPDbasedmediaoncellularproductionandsecretionalone,we

decidedtoseeifthepicturewouldchangeifweslowedproliferationoftheNPDandDIcultures

through reduction of serum in the culture. The differences between antibody concentrations

measuredinculturesgrownin3%FCSwereverydramatic.Whiletheproductionofantibodies

droppedinculturesgrowninaNPDenvironment,theDIculturedidnotproduceanymeasurable

concentrations of antibody whatsoever. Interestingly, the number of cells in the cultures at

varioustimespostinoculationwassimilar.Therefore,itappearsthatNPDbasedmediadirectly

promotesproductionandsecretionofantibody.Wearecurrentlystudyingthisinmoredetailto

determineifincreasedyieldsofantibodymightresultfromadirectimpactofNPDbasedmedia

onsecretionalone.


16/30

15

The propagation and growth of hybridoma cells under conditions of reduced serum

diminishes antibody secretion by hybridoma cells [25-27]. However, secretion of human

monoclonal antibodies in NPD based culture, under reduced serum conditions, suggests that

productionoftheseantibodiesmightbeachieved inamore economicalway.Both serumand

serum free media formulations that contain growth factors are expensive and also require

extensivepurification.UseofNPDbasedmediaproducts,therefore,mightfacilitatepurification

andlowertotalcostoflarge-scaleproduction.

Following the observation of a faster proliferation rate of hybridoma cells in a NPD

environment, testing of standard cells in laboratory use was performed. As a model of an

immortalizedcellline,CHOcellswerestudiedastheyarewidelyusedforproductionofprotein

productsinbothacademicandcommercialsettings.SimilartohybridomacellstheCHOcells

proliferatebetterinNPDbasedmediaascomparedtoDImedia.Initself,thiscanbeusefulto

enhanceproductionofbiopharmaceuticals,however,testingisunderwaytodetermineifthereis

asimilareffectofNPDbasedmediaonsecretioninCHOcelllines.

Unlikeimmortalcells,studiesofprimaryhumanfibroblastgrowthinNPDbasedmedia

revealed dramatically different results.Primary fibroblastsproliferated reproducibly slower in

NPDbasedmedia.Thiseffectontherateofpopulationdoublingofthecellsappearstohaveno

impactoncellviabilityortheproliferativecapacityoftheprimaryfibroblaststosenescence(data

not shown). Indeed, fibroblastsgrown inNPD basedmedia senesced at the same numberof

populationdoublingsasfibroblastsfromthesamestarterculturegrowninDIbasedmedia.Since

thecellsinNPDmediagrewslower,theysurvivedlongerinculturechronologically.

ThereverseeffectofNPDbasedmediaonimmortalcellsandprimaryhumanfibroblasts

isintriguing.ExperimentsperformedhereindemonstratethatprimaryhumanfibroblastsinNPD

mediasensealowercelldensityascomparedtoDIbasedmedia.Ontheotherhand,ourfusion

partner cell line,MFP-2, is of myeloma origin [1] and likely secretes autocrine factors that

promoteitsowncolonyformationfromasinglecell.Thelocalconcentrationofthesesubstances

is typically quite low in standard culture media, necessitating addition of exogenous growth

factors frommacrophage conditioned media (HCF) topromote clonal expansion. To explain

these phenomena, we propose that the order imposed in a NPD based environment [12]

establishesmicroenvironmentsinthebulkenvironmentthatdisruptcross-talkorinter-cellular

communication. These microenvironments might effectively shield cells leading to a higher


17/30

16

localized concentration of autocrine growth factors. Thismay explain the growth promoting

propertiesofNPDbasedmediaonimmortalcells,andatthesametimethegrowthinhibitory

effectonprimaryhumanfibroblasts.

Overall,ourinvestigationoftheimpactoftheaqueousenvironmentonvariousaspectsof

cellbiologysuggeststhat invitrobiologymightyielddifferentresultsinastructuredoraltered

aqueous environment, as opposed to the standard reverse osmosis water used in most

laboratories. It supports the notion that environment should be considered in biological

experimentationandapplications[9,28].OurstudiessupporttheworkinghypothesisthatNPD

water can play a significant role in cell biology and dramatically enhance the efficiency of

processes that are of importance to bioprocessing and the biopharmaceutical industries. All

together,ourexperimentssuggestthatamoredetailedinvestigationofwaterstructure,andits

impactinallscientificdisciplines,iswarranted.

Conclusions:Wehave demonstrated thatNPDwater canenhance hybridoma formation, cell

proliferation,clonabilityandsecretion.Inaddition,NPDbasedmediumcanenhancetheviability

of cultures under serum-reduced conditions. Finally, primary human fibroblasts proliferate

poorly in NPD based medium and appear to sense a lower effective cell density in this

environment. Taken together with the enhanced proliferation of hybridoma cells that likely

secrete autocrine factors, these results support the hypothesis that NPD water is effectively

composedofstabilizedmicroenvironments.


18/30

17

Authorscontributions:

N.G-Y.coordinatedtheworkofthisproject,performedmuchofthecellcultureexperimentsand

helped write the manuscript. E.H. & M.T. performed experiments relating to hybridoma

production,subcloningandantibodysecretionstudies.V.Y.,O.K.&L.L.designedallofthese

studies,participatedinanalysisofalldataandparticipatedinwritingthismanuscript.Allauthors

readandapprovedthefinalmanuscript.

Acknowledgements:

The authors gratefully acknowledge Docoop Technologies (OrYehuda, Israel) for supplying

NPDwaterforthesestudies.WealsothankProf.YakirAharonov,Prof.EshelBenJacoband

EranGabbaifortheirstimulatingdiscussions,infiniteinsight,wisdomandloveofnature.This

workwassupportedbyaScienceforPeacegrantfromNATO(SfP981812)forproductionof

humanmonoclonalantiviralantibodiesandbyagrantfromtheIsraelCancerAssociation.


19/30

18

References:

1. KalantarovGF,RudchenkoSA,LobelL,TrakhtI:Developmentofa fusionpartner

celllineforefficientproductionofhumanmonoclonalantibodiesfromperipheral

bloodlymphocytes.HumAntibodies2002,11(3):85-96.

2. Kirman I,KalantarovGF,LobelLI,HibshooshH,EstabrookA,CanfieldR,Trakht I:

Isolation of native human monoclonal autoantibodies to breast cancer. Hybrid

Hybridomics2002,21(6):405-414.

3. ReuvenyS,VelezD,MacmillanJD,MillerL:Factorsaffectingmonoclonalantibody

productioninculture.Developmentsinbiologicalstandardization1987,66:169-175.

4. Reuveny S, Velez D,Riske F, MacMillan JD, Miller L: Production ofmonoclonal

antibodiesinculture.Developmentsinbiologicalstandardization1985,60:185-197.

5. SeifertDB,PhillipsJA:Theproductionofmonoclonalantibodyingrowth-arrested

hybridomascultivatedinsuspensionandimmobilizedmodes.Biotechnologyprogress

1999,15(4):655-666.

6. Bazin R, Lemieux R: Increased proportion of B cell hybridomas secreting

monoclonal antibodies of desired specificity in cultures containing macrophage-

derivedhybridomagrowthfactor(IL-6).JImmunolMethods1989,116(2):245-249.

7. MalininVS,FrederikP,LentzBR:OsmoticandcurvaturestressaffectPEG-induced

fusion of lipid vesicles but not mixingof their lipids.Biophys J 2002, 82(4):2090-

2100.

8. LevyY,OnuchicJN:Watermediationinproteinfoldingandmolecularrecognition.

Annualreviewofbiophysicsandbiomolecularstructure2006,35:389-415.

9. ChaplinM:Doweunderestimate the importance ofwater in cell biology?Nature

reviews2006,7(11):861-866.

10. Colic M, Morse D: Mechanism of the Long-Term Effects of Electromagnetic

RadiationonSolutionsandSuspendedColloids.Langmuir1998,14(4):783-787.

11. Colic M, Morse D: Effects of Amplitude of the Radiofrequency Electromagnetic

RadiationonAqueousSuspensionsandSolutions.Journal ofColloidand Interface

Science1998,200(2):265-272.


20/30

19

12. Yael Katsir LM, YakirAharonov, EshelBenJacob:The effect of rf-irradiation on

electrochemicaldepositionand itsstabilizationbynanoparticledoping.Journal of

TheElectrochemicalSociety2007,154(4):D249-D259.

13. Chen X, Lou Y, Dayal S, Qiu X, Krolicki R, Burda C, Zhao C, Becker J: Doped

semiconductor nanomaterials. Journal of nanoscience and nanotechnology 2005,

5(9):1408-1420.

14. Fesenko EE, Geletyuk VI, Kazachenko VN, Chemeris NK:Preliminarymicrowave

irradiationofwatersolutionschangestheirchannel-modifyingactivity .FEBSletters

1995,366(1):49-52.

15. FesenkoEE,GluvsteinA:Changesinthestateofwater,inducedbyradiofrequency

electromagneticfields.FEBSletters1995,367(1):53-55.

16. GeletyukVI,KazachenkoVN,ChemerisNK,FesenkoEE:Dualeffectsofmicrowaves

onsingleCa(2+)-activatedK+channelsinculturedkidneycellsVero .FEBSletters

1995,359(1):85-88.

17. BenJacobE,AharonovY,ShapiraY:Bacteriaharnessingcomplexity .Biofilms2004,

1(4):239-263.

18. Kohler G, Milstein C: Continuous cultures of fused cells secreting antibody of

predefinedspecificity.Nature1975,256(5517):495-497.

19. NagataY,MizukoshiY,OkitsuK,MaedaY:Sonochemicalformationofgoldparticles

inaqueoussolution.Radiationresearch1996,146(3):333-338.

20. MizukoshiY, Takagi E, OkunoH, Oshima R,Maeda Y,Nagata Y: Preparation of

platinum nanoparticles by sonochemical reduction of the Pt(IV) ions: role of

surfactants.Ultrasonicssonochemistry2001,8(1):1-6.

21. SenaratneSG,PirianovG,MansiJL,ArnettTR,ColstonKW:Bisphosphonatesinduce

apoptosisinhumanbreastcancercelllines.BrJCancer2000,82(8):1459-1468.

22. JourdanM,MahtoukK,VeyruneJL,CoudercG,FiolG,RedalN,DuperrayC,DeVosJ,

KleinB:Delineationoftherolesofparacrineandautocrineinterleukin-6(IL-6)in

myelomacelllinesinsurvivalversuscellcycle.Apossiblemodelforthecooperation

ofmyelomacellgrowthfactors.EurCytokineNetw2005,16(1):57-64.


21/30

20

23. VillungerA,EgleA,KosM,HittmairA,MalyK,GreilR:Constituentsofautocrine

IL-6 loops inmyeloma cell linesand their targeting for suppressionofneoplastic

growthbyantibodystrategies.IntJCancer1996,65(4):498-505.

24. Demartis A, Bernassola F, Savino R,Melino G, CilibertoG: Interleukin 6 receptor

superantagonistsarepotentinducersofhumanmultiplemyelomacelldeath .Cancer

Res1996,56(18):4213-4218.

25. PannellR,MilsteinC:Anoscillatingbubblechamberforlaboratoryscaleproduction

ofmonoclonal antibodiesasanalternative toascitic tumours.J ImmunolMethods

1992,146(1):43-48.

26. LowK,HarbourC:Growthkineticsof hybridomacells: (1)The effectsofvarying

foetalcalfserumlevels.Developmentsinbiologicalstandardization1985,60:17-24.

27. Jaspert R, Geske T, Teichmann A, Kassner YM, Kretzschmar K, L'Age-Stehr J:

Laboratory scale production ofmonoclonal antibodies in a tumbling chamber.J

ImmunolMethods1995,178(1):77-87.

28. Chaplin MF:Aproposal for the structuring of water.Biophysical chemistry 2000,

83(3):211-221.


22/30

21

FigureLegends

Figure1:Fusionefficiencyenhancement

Thefusionswereperformedaccordingtoastandardprotocol,wheretheculturemediaandPEG

were reconstituted frompowder formswitheitherNPD orDIwater. Foreachfusion,PBMC

from a single batch were divided into two equal fractures and used to prepare two parallel

experiments, inNPDorDIbasedreagents.Thefigurepresentspercent ofhybridoma-positive

wells in each fusion experiment. The percent was calculated as the number of hybridoma-

positive wells from 96-well plates where the cells were seeded and grown after the fusion

process. The differencebetween theNPD-andDI-fusion resultswasfound tobestatistically

significantbyChi-squareanalysis(p


23/30

22

Figure3:IgMproductionbyastablehybridomaclonein10%FCS

Two parallel cultures were prepared in replicates from a stable hybridoma clone from our

collection.OnewasgrowninNPDandtheotherinDImediumandbothwerekeptinstandard

cultureconditions. Afteraweekofgrowth the supernatantswere collected,and the antibody

concentrationsweremeasuredbyastandardsandwichELISA.Eachcolumnrepresentsthemean

antibody concentration thatwasmeasured inNPDandDIcultures.The errorbars denote the

standarderrorofthemeans.Wehaveobservedincreasedsecretionofmonoclonalantibodywith

aseriesofstablehybridomaclonesandpresentedadetailedanalysiswithoneoftheminthis

manuscript.

PanelA:Totalantibodyconcentrationmeasuredintheculturesupernatants;PanelB:Antibody

concentrationnormalizedpercell.

Figure4:IgMproductionbyastablehybridomaclonegrownin3%FCS

Two culturesderived from the same culture of a stable hybridoma clonewere grown,one in

NPDandtheotherinDIbasedmediumsupplementedwith3%FCS.Beforeseedingthecells

werewashedinserum-freemediatoverifytheremovalofanyresidualserum.Duringaperiodof

twoweeksthesupernatantswerecollectedasindicatedandthecellswerecountedonthesame

day.Thecultureswerefedonthe4thand10

thdayandmediumwasplacedintheculturesonday

6.AlthoughthecellsinDIcultureproliferatednormallyundertheseconditions,theyfailedto

producemeasurablequantitiesofantibody.

PanelA:IgMproductionperhybridomacellin3%FCS;PanelB:Numberoflivecellsateach

antibodytitration.

Figure5:EffectofserumreductiononCHOcellgrowthinNPDmedium

PanelA:CHOcellgrowthincompletemedium:

Cellswereseededat aninitialdensity of 1.5x106 per 10-cmPetri dish inNPDandDIbased

mediumintriplicates.Afterovernightgrowththeyweredetachedbytrypsinizationandcounted.

Theresultsaregivenasthenumberofviablecells.Eachcolumnrepresentsameannumberof

cells ineach treatment.The errorbarsdenote thestandarderrorofthemeans.The difference

between the treatments is 30%.The graph provides a representative result of an experiment,

whichwasconductedwithreplicatesandrepeatedthreetimes.


24/30

23

PanelB&C:CHOcellgrowthinreduced-serummedium:

Cellswereseededin96-wellplatesinmultiplereplicates(18wellspertreatment)inNPDorDI

mediumsupplementedwith5%(PanelB)or1%(PanelC)FCS.Theresultswerequantifiedand

analyzedbymeansofcrystalvioletdyeretentionassay.Eachcolumnrepresentsthemeancell

densityfollowingagiventreatmentinO.D.units.Theerrorbarsdenotethestandarderrorofthe

mean.*SignificantdifferencebetweenNPDandDIgrowncellsp=0.0006,totaldifference7%.

**SignificantdifferencebetweenNPDandDIgrowncellsp=0.0001,totaldifference14%.

Figure6:PrimaryhumanfibroblastcultureinNPDmedium

PanelA:Primaryhumanfibroblastproliferationaccordingtoinitialcelldensity:

Primaryhumanfibroblastswereseededinreplicateina96-wellplateattwoinitialcelldensities:

fiveandtenthousandcellsperwell.Afteranovernightgrowththecellswerefixedandassayed

bymeansofcrystalvioletdyeretentionmethod.TheresultsarepresentedinO.D.values.Each

columnrepresentsameanO.D.ofagivengrowthcondition;theerrorbarsdenotethestandard

error of the mean. *Significant difference between DI and NPD for cell density of 5000

cells/well(p


25/30


26/3010

20

30

40

50

60

70

0

10

20

30

40

50

60

DI+HCF NPD+HCF DI NPD

ercentofhybridoma-positive

antibodys

ecretingwells

Growth medium

Percentofhybridoma-p

ositive

antibodysecretingw

ells

Panel A

Panel B


27/30

Panel B

Panel A

0

10

20

30

40

50

60

70

80

90

NPD DI

Growth medium

nti

0.800

1.000

1.200

1.400

1.600

1.800

2.000


28/30

Panel A

0.000

0.005

0.010

0.015

0.020

0.025

0 1 2 3 4 5 6 7 8 9 10 11 12 13

Days

IgMconcentration(ng/cell)

NPD

DI

0

1

2

3

4

5

6

7

8

9

0 1 2 3 4 5 6 7 8 9 10 11 12 13

Days

Numberoflivecells

x

105

NPD

DI

Panel B


29/30

0

1

2

3

4

5

6

7

8

9

NPD DI

Growth medium

0.25

0.26

0.27

0.28

0.29

0.3

0.31

0.32

0.33

NPD DI

**

0.140

0.145

0.150

0.155

0.160

0.165

NPD DI

Growth medium Growth medium

Panel A

Panel B Panel C

*

gure 5


30/30

neowater in biopharma arena

Documents