Download - 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] -
8/14/2019 Neowater in Biopharma arena
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
-
8/14/2019 Neowater in Biopharma arena
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.
-
8/14/2019 Neowater in Biopharma arena
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
-
8/14/2019 Neowater in Biopharma arena
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.
-
8/14/2019 Neowater in Biopharma arena
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
-
8/14/2019 Neowater in Biopharma arena
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
-
8/14/2019 Neowater in Biopharma arena
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
-
8/14/2019 Neowater in Biopharma arena
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.
-
8/14/2019 Neowater in Biopharma arena
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
-
8/14/2019 Neowater in Biopharma arena
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
-
8/14/2019 Neowater in Biopharma arena
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
-
8/14/2019 Neowater in Biopharma arena
13/30
-
8/14/2019 Neowater in Biopharma arena
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
-
8/14/2019 Neowater in Biopharma arena
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.
-
8/14/2019 Neowater in Biopharma arena
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
-
8/14/2019 Neowater in Biopharma arena
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.
-
8/14/2019 Neowater in Biopharma arena
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.
-
8/14/2019 Neowater in Biopharma arena
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.
-
8/14/2019 Neowater in Biopharma arena
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.
-
8/14/2019 Neowater in Biopharma arena
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.
-
8/14/2019 Neowater in Biopharma arena
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
-
8/14/2019 Neowater in Biopharma arena
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.
-
8/14/2019 Neowater in Biopharma arena
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
-
8/14/2019 Neowater in Biopharma arena
25/30
-
8/14/2019 Neowater in Biopharma arena
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
-
8/14/2019 Neowater in Biopharma arena
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
-
8/14/2019 Neowater in Biopharma arena
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
-
8/14/2019 Neowater in Biopharma arena
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
-
8/14/2019 Neowater in Biopharma arena
30/30