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CRISPR/Cas9-multiplexed editing of Chinese hamster ovary B4Gal-T1, 2, 3 and 4Tailors N-Glycan Profiles of Therapeutics and Secreted Host Cell Proteins
Amann, Thomas; Hansen, Anders Holmgaard; Kol, Stefan; Min Lee, Gyun; Andersen, Mikael Rørdam;Kildegaard, Helene FaustrupPublished in:Biotechnology Journal
Link to article, DOI:10.1002/biot.201800111
Publication date:2018
Document VersionPeer reviewed version
Link back to DTU Orbit
Citation (APA):Amann, T., Hansen, A. H., Kol, S., Min Lee, G., Andersen, M. R., & Kildegaard, H. F. (2018). CRISPR/Cas9-multiplexed editing of Chinese hamster ovary B4Gal-T1, 2, 3 and 4 Tailors N-Glycan Profiles of Therapeuticsand Secreted Host Cell Proteins. Biotechnology Journal, 13(10), [1800111 ].https://doi.org/10.1002/biot.201800111
1
CRISPR/Cas9-multiplexededitingofChinesehamsterovaryB4Gal-T1,2,3and4
tailorsN-glycanprofilesoftherapeuticsandsecretedhostcellproteins
ThomasAmann1*,AndersHolmgaardHansen1*,StefanKol1,GyunMinLee1,2,MikaelRørdam
Andersen3,HeleneFaustrupKildegaard1
1NovoNordiskFoundationCenterforBiosustainability,TechnicalUniversityofDenmark,
Kgs.Lyngby,Denmark
2DepartmentofBiologicalSciences,KAIST,Daejeon,RepublicofKorea
3Department of Biotechnology and Biomedicine, Technical University of Denmark, Kgs.
Lyngby,Denmark
*Theseauthorscontributedequallytothispublication
Correspondence: Helene Faustrup Kildegaard, Novo Nordisk Foundation Center for
Biosustainability,Kemitorvet,Building220,2800Kgs.Lyngby,Denmark
E-mail:hef@biosustain.dtu.dk
Keywords:Chinesehamsterovarycells,CRISPR/Cas9,N-glycosylation,Glycoengineering,
Multiplexing,Rituximab,Erythropoietin
Abbreviations:
Asn, Asparagine; AUC, area under curve; B4Gal-T, β-1,4-galactosyltransferase;
Cas9,CRISPR-associatedprotein9;CHO,Chinesehamsterovary;CRISPR,clustered
regularly interspaced short palindromic repeats; EPO, erythropoietin; FACS,
fluorescence-activatedcellsorting;Fc,fragmentcrystallizable;FcγRIIIa,Fc-gamma
2
receptorIIIa; FUT8,alpha-(1,6)-fucosyltransferase;G0,agalactosylated;GlcNAc,
N-Acetylglucosamine; HM, high–mannose; HPC4, human protein C4; IgG,
immunoglobulinG;indel,insertionordeletion;mAb,monoclonalantibody;sgRNA,
singleguideRNA;TSTA3, tissue-specific transplantationantigenP35B;UDP-Gal,
uridinediphosphategalactose;VCD,viablecelldensity;WT,wildtype
Abstract
In production of recombinant proteins for biopharmaceuticals, N-glycosylation is often
importantforproteinefficacyandpatientsafety.IgGwithagalactosylated(G0)-N-glycans
canimprovetheactivationofthecomplementsystemandbeadvantageousinthetherapy
of lupus and virus diseases. In this study, we aimed to engineer CHO-S cells for the
production of proteins with G0-N-glycans by targeting B4Gal-T isoform genes with
CRISPR/Cas9. Indel mutations in genes encoding B4Gal-T1, -T2, –T3 with and without
disrupted B4Gal–T4 sequence resulted in only ~1% galactosylated N-glycans on total
secretedproteinofthreeclonespergenotype.Inthetriple-KOclones,transientlyexpressed
erythropoietin(EPO)andtransientlyexpressedrituximabharboredonly~6%and~3%
galactosylatedN-glycans,respectively.However,simultaneousdisruptionofB4Gal-T1,-T2
and–T3was found todecrease cell growth.We furthermore revealedpossibleB4Gal-T
isoformbranchpreferenceswhereB4Gal-T2activityisrestrictedtoactonasingleN-glycan
branchandB4Gal-T4wasfoundtobeinactiveinN-glycangalactosylationinCHO-Scells.
Altogether,wepresent theadvantageofanalyzingtotalsecretedproteinN-glycansafter
disruptingglycosyltransferases followedbyexpressingrecombinantproteins inselected
clones with desired N-glycan profiles at a later stage. Furthermore, we provide a cell
platform that prevalently glycosylates proteins with G0-N-glycans to further study the
impact of agalactosylation on different in vitro and in vivo functions of recombinant
proteins.
3
1.Introduction
Chinesehamsterovary(CHO)-derivedcellsarethemajorworkhorseswithinmammalian
cell lines and represent the cell platform inwhich >50% of themarketed recombinant
proteins are produced[1]. Thereof, recombinantmonoclonal antibodies (mAbs) are the
main product subclass and are utilized for the treatment of cancer and various
inflammatorydiseases[2].Asaresultofpost-translationalproteinprocessing,mAbsharbor
twoN-glycans,oneoneachheavychainatAsparagine(Asn)297whereaserythropoietin
(EPO)hasthreeN-glycosylationsitesoccupiedbypredominantlytri-andtetra-antennary
structures[3].Ingeneral,N-glycosylationcanimpactproteinfolding,immuneregulation,
cellularhomeostasisandthebiologicalhalf-lifeofproteins[4,5].WithinmAbs,thefragment
crystallizable (Fc) N-glycans at Asn297 have a strong influence on anti-inflammatory
properties, antibody-dependent cell-mediated cytotoxicity and complement-dependent
cytotoxicity[6].
TheheterogeneousN-glycanprofileofglycoproteinsproducedinCHOisoneofthemain
factors that cause mAb heterogeneity and can be further optimized regarding core-
fucosylation,galactosylation,antennarityandterminalcappingbysialicacids.Rituximabis
an immunoglobulin G (IgG) 1-class molecule, one of the recombinant glycoproteins
producedinCHO,andexceedsannualrevenuesofUSD7billion[7].Rituximabtargetsthe
B-cell surfaceantigenCD20 inB-cell lymphomaand ispredominantlyN-glycosylatedby
A2FG0andA2FG1structureswhenproducedinnon-glyco-engineeredCHOcells[8].Since
severalstudiesrevealednon-fucosylatedIgGshavesignificantlyhigherbindingaffinityfor
the Fc-gamma receptor IIIa (FcγRIIIa) than fucosylated IgG versions[9, 10], different
approaches successfully removed the core-fucose by knockout of alpha-(1,6)-
fucosyltransferase(FUT8)ortissuespecifictransplantationantigenP35B(TSTA3)inIgG-
expressingCHOcelllines[11–14].
4
Additionally, agalactosylated IgG1 variantswith terminalN-Acetylglucosamine (GlcNAc)
(referredtoasG0glycoforms)canincreasethebindingtoFcRIIIa[15]andareaccessiblefor
themannose-bindingprotein.Theycan thereforepromoteactivationofthecomplement
system[16]without impacting in vivo clearance[17–19]. Furthermore,HIVpatientswith
high viral inhibition displayed an increased proportion of agalactosylated N-glycans on
global serum IgG, suggesting that agalactosylated IgG variants may have antiviral
activity[20]. Interestingly, Lupus patients showed improved disease symptoms after
treatmentwithagalactosylatedantibodies[21].TheseG0-IgGvariantscanbeobtainedby
sequential treatment of wild type (WT)-IgG with neuraminidase and galactosidase or
supplementingcultivationmediumwithgalactoseanaloguestoblockcellularB4Gal-Ts[22].
Nevertheless,fewercell-engineeringattemptswereinitiatedtoproduceG0-IgG1compared
toengineeringnon-fucosylatedIgG1variants.
Since the CHO genome sequence is publically available[23], CHO cell-engineering is no
longerperformedina“blackbox”,whichshortenscelllinedevelopmentandempowersa
targeted approach for the engineering of a G0 CHO cell line. The classes of
glycosyltransferases are made of homologous gene families, where the class of β-1,4-
galactosyltransferases (B4GalT) consists of seven members, B4Gal-T1–T7, which all
transfer galactose fromuridinediphosphate galactose (UDP-Gal) toGlcNAcandGlcNAc-
terminated oligosaccharides (EC 2.4.1.38)[24, 25]. The seven CHO B4Gal-Ts all share a
common WGXEDD sequence as part of their B4Gal-T motif[26] and have exclusive
specificity for the donor substrate UDP-Gal. B4Gal-T5 and -T6 are described tomainly
function in O-glycosylation[27, 28], whereas B4Gal–T7 transfers UDP-Gal within
glycosaminoglycan biosynthesis and therefore is not involved in the N-glycosylation of
proteins[29, 30]. A further study indicated that B4Gal-T1, -T2, -T3 and –T4 performN-
glycangalactosylationmoreefficientthanB4Gal-T5and-T6andsuggesteddifferentbranch
preferencesforthefamilymembersofβ-1,4-galactosyltransferases[31].Inaddition,B4Gal-
5
T4isreportedtoalsobeactiveinthegalactosylationofmucin-typecore2branchinginthe
O-glycosylation pathway[32]. Another study described B4Gal-T1-KO mutants to have
dramaticallyreducedgalactosylationonsecretedhostcellprotein(secretome)N-glycans
andreducedgrowthofmice[28,33].InapreviousstudyperformedwithCHO-K1derived
celllines,triple-KOofB4Gal-T1,-T2and–T3,double-KOofB4Gal-T1and-T3andsingle-KO
ofB4Gal-T1ledtoalmostfullyagalactosylatedEPOandrituximab[34].However,thatstudy
wasbasedononlyone cloneperKO-combinationand thereforedidnot consider clonal
variationanddidnotinvestigatetheimpactofB4Gal-Tdisruptionsoncellgrowth.Although
thissuggestsB4Gal-T1and–T3tobethemainplayersingalactosylationofrituximaband
EPON-glycansinCHO-K1cells,theN-glycosylationactivityofB4Gal-T1,-T2,-T3and–T4,
specifically in the industrially relevant CHO-S cell line, needs to be further explored to
generateafullyG0celllinewithinCHO-Scells.SincetheeffectofsingledisruptionsofB4Gal-
T1,-T2,-T3and-T4onN-glycosylationwasinvestigatedinpreviouswork[34],wedesigned
amultiplexingapproachtodisruptcombinationsofuptofourB4Gal-Ts.Thetargetdesign
forthemultiplexingapproachcontainedB4Gal-T1,B4Gal-T3orbothtargetsincombination
withB4Gal-T2and/orB4Gal-T4.Withthehelpofmultiplexing,theeffectofstackingB4Gal-
TdisruptionscouldbestudiedwithregardstocellgrowthandproteinN-glycosylationon
threeclonesforeachtriple-andquadruple-KOcombinationtoadditionallyexamineclonal
variation.
Inthiswork,weusedclusteredregularlyinterspacedshortpalindromicrepeats/CRISPR-
associatedprotein9(CRISPR/Cas9)asatoolformultiplexededitingofgenetargetswithin
thesametransfection[13].B4Gal-T1and–T3aswellasB4Gal–T2and–T4weredisrupted
simultaneouslytofacilitateinvestigationofthecombinatorialeffectofB4Gal-Tknockouts
onN-glycosylationandcellgrowth.N-glycosylationanalysisoftotalsecretedproteins,as
well as transiently expressed rituximab and EPO (representing dissimilar N-glycan
profiles),inB4Gal-TeditedCHO-Scelllineswasperformed.Theanalysisdemonstratesthat
6
N-glycanscanbetailoredforagreatervarietyofsecretedglycoproteins,asrepresentedby
morethan250proteinswithintheCHO-Ssecretome[35]inadditiontoEPOandrituximab.
Withthis,weinvestigatedifscreeningthesecretomeN-glycansofourengineeredclonesis
apromisingstrategytowardstheexpressionofrituximabandEPOwithG0N-glycansin
selectedclones.Thestrategywasbuilton(i)analyzingtheN-glycanprofileofallsecreted
proteins from selectedmultiplexed clones to then (ii) expressing rituximaband EPO in
selectedclonesforrituximab/EPON-glycananalysisand(iii)evaluatingoftheroleofeach
targetedB4Gal-TwithinthegalactosylationofN-glycans.EspeciallytheroleofB4Gal-T2
and–T4inCHO-SandtheeffectofB4Gal-Tindelsoncellgrowth,bothwithrespecttoclonal
variation,have toourknowledgenotbeen investigatedpreviouslyandwere thedriving
motivesofthiswork.
2.Materialsandmethods
2.1.sgRNAandGFP_2A_Cas9plasmiddesign
GFP_2A_Cas9 and single guide RNA (sgRNA) plasmids were constructed as previously
described[13].ThesgRNAtargetdesignforB4Gal-T1,B4Gal-T2,B4Gal-T3andB4Gal-T4
wasperformedusingCRISPy[36].Thetargetsitesforthementionedgenesandtheoligos
forsgRNAcloningarelistedinSupportingInformation,TableS1andTableS2,respectively.
2.2.Cellcultivationandtransfectionformultiplexedgenomeediting
CHO-S suspension cells (Life Technologies, Carlsbad, CA) were cultivated in CD CHO
medium supplemented with 8mM L-glutamine and 1 μL/mL anti-clumping agent (Life
Technologies).Cellswereincubatedinahumidifiedincubatorat120rpm,37°Cand5%CO2.
Cellpassagingwasconductedeverytwotothreedaysat3x105cells/mLaftermeasuring
viable cell densities (VCDs) andviabilitieswith theNucleoCounterNC-200Cell Counter
(ChemoMetec,Allerod,Denmark).OnedaypriortransfectionwithCRISPRreagents,anti-
7
clumpingagentwasremovedbycentrifugationand5-6x105cells/mLwereseededina
sixmulti-wellwellplate(BDBiosciences,SanJose,CA)foreachtransfection.Atthedayof
transfectioneachsamplewasseededat1x106cells/mLandatotalDNAloadof3.5μgwas
transfectedwithFuGENE®HDtransfectionreagent(Promega,Madison,WI)andOptiPRO
SFMmedium(LifeTechnologies)accordingtothemanufacturer´srecommendations.The
GFP_2A_Cas9/sgRNAplasmidratiosforeachsamplearepresentedinTableS3.Tomeasure
transfection efficiency, pmaxGFP® vector (Lonza, Basel, Switzerland) transfection was
performed.Cellswereharvestedforfluorescence-activatedcellsorting(FACS)48hafter
transfection.
2.3.SinglecellcloningusingFACS
BeforeFACS,cellswerefilteredthrougha40μmcellstrainerintoaFACS-compatibletube.
OperatingaFACSJazz(BDBiosciences)single fluorescent-positivecellsweresorted into
384-well plates (Corning, New York, NY) already containing 30 μL CD CHO medium
supplementedwith8mML-glutamine,1.5%HEPESbufferand1%Antibiotic-Antimycotic
(Gibco,Waltham,MA)perwell.Forcellsorting,fluorescent-positivecellpopulationswere
gatedbasedonnon-transfectedWTCHO-Scells.Twoweeksaftercell sortingtheclones
were moved to 96-well flat-bottom plates (BD Biosciences) and expanded for deep
sequencinganalysisandbatchcultivation.
2.4.Deepsequencinganalysis
Confluentcoloniesfrom96-wellflat-bottomreplicateplateswereharvestedforgenomic
DNAextraction.DNAextractionwasperformedusingQuickExtractDNAextractionsolution
(Epicentre,Illumina,Madison,WI)accordingtothemanufacturer´sinstruction.Thelibrary
preparationwasbasedonIllumina16SMetagenomicSequencingLibraryPreparationand
deepsequencingwascarriedoutonaMiSeqBenchtopSequencer(Illumina,SanDiego,CA).
8
The protocol for amplifying the targeted genomic sequences, amplicon purification,
adapter-PCRandfollowingqualityanalysiswasbasedonpreviouslypublishedwork[13].
PCRprimersarepresentedinSupportinginformation,TableS4.
2.5.BatchcultivationtostudycellgrowthandsecretomeN-glycans
Forbatchcultivationandsecretomeanalysis, cellswereseededat3.0x105cells/mL in
Corningventcapshakeflasks(Sigma-Aldrich,St.Louis,MI)asduplicatesin30mLCDCHO
medium supplemented with 8mM L-glutamine and 1 μL/mL anti-clumping agent (Life
Technologies).Cellswereincubatedinahumidifiedincubatorat120rpm,37°Cand5%CO2.
CelldensitiesandviabilitiesweredeterminedonceperdayusingtheNucleoCounterNC-
250CellCounter(ChemoMetec).Secretomesamplevolumewascalculatedtoharbor20x
106cellsandharvestedfivedaysafterseedingtobepooledwithinbiologicalreplicates.
2.6.Batchcultivationfortransientrituximab/EPOtransfectionandrituximab/EPO
N-glycananalysis
For transientexpressionofrituximab/EPO,cellswereseeded inCorningventcapshake
flasks(Sigma-Aldrich)asduplicateswithcelldensities~1x106cells/mLin60mLCDCHO
mediumsupplementedwith8mML-glutamine(LifeTechnologies).Cellswereincubatedin
ahumidifiedincubatorat120rpm,37°Cand5%CO2andtransfectedwith75μgofrituximab
or EPO encoding plasmid for each flask using FreeStyleTM MAX reagent together with
OptiPRO SFM medium (Life Technologies) according to the manufacturer´s
recommendations. 1μL/mL anti-clumping agent was added 24 h after transfection.
pmaxGFP®vector(Lonza)transfectionwasperformedtomeasuretransfectionefficiencies.
CelldensitiesandviabilitiesweredeterminedonceperdayusingtheNucleoCounterNC-
250 Cell Counter (ChemoMetec). To purify rituximab and EPO, the supernatants of the
9
transfected clones were harvested three days after transfection and pooled within
duplicates.
2.7.RituximabandEPOpurification
Forrituximabpurification,supernatantsampleswerecentrifuged(1000g,5minutes,4°C)
andafterwardsfiltered(~0.22μmporesize)toremovecellsandcelldebris.Rituximabwas
purifiedbyproteinAaffinitychromatography(MabSelect,GEHealthcare,Uppsala,Sweden)
according to the manufacturer´s protocol. Human protein C4 (HPC4)-tagged EPO was
purified from supernatants using Anti-Protein C Affinity Matrix from Roche (Basel,
Switzerland,Cat.Nr.11815024001)aspertheinstructionsofthemanufacturer.
2.8.N-Glycananalysis
SamplepreparationforN-glycananalysiswasperformedwithGlycoWorksRapiFluor-MS
N-Glycan Kit (Waters, Milford, MA) according to the manufacturer´s instruction. 12 μg
purified protein or 12 μl of 10x concentrated (Amicon Ultra-15, Merck, Darmstadt,
Germany)secretomesamplewereusedforeachsample.LabeledN-Glycanswereanalyzed
byaLC-MSsystemusingaThermoUltimate3000HPLCwithfluorescencedetectorcoupled
on-linetoaThermoVelosProIontrapMS,asdescribedpreviouslywithminormodifications
[13].Separationgradient30%to43%bufferandMSwasruninpositivemode.Amountof
N-Glycan was measured by integrating the areas under the normalized fluorescence
spectrumpeakswithThermoXcalibursoftware(ThermoFisherScientific,Waltham,MA)
givingthenormalized,relativeamountoftheglycans.
3.Results
10
3.1.GenerationofengineeredCHO-Scelllineswithcombinationsofindelsinmultiple
B4Gal-Tgenes
To investigate the exact impact of B4Gal-T1, -T2, -T3 and –T4-KO on N-glycan
galactosylation,weaimedtogeneratecloneswithinsertionordeletion(indel)mutationsin
oneorseveralofthegenes.Togetthesecombinationsinaminimalnumberofoperations,
weco-transfectedCas9(GFP_2A_Cas9)witheithersgRNAsagainstB4Gal-T1and–T3or
againstB4Gal-T1,-T2and–T3orsgRNAsagainstB4Gal-T1and–T2inthesametransfection
(SupportingInformation,TableS3).Aftertransfectionandsinglecellcloning,wecarriedout
deepsequencingtoidentifythegenomicchangesinthetargetedsequences.Weaimedto
identifycloneswithexclusivelyout-offrameindelsinoneormoreofthetargetsequences
leading to a potentially functional knockout of the targeted glycosyltransferase(s) to
investigatetheeffectonN-glycangalactosylation.Inasecondroundoftransfections,we
aimedtogeneratecloneswithindelsincombinationsofthreeorallfourtargetedB4Gal-Ts.
Thereforeweco-transfectedGFP_2A_Cas9witheithersgRNAsagainstB4Gal-T1and–T4or
againstB4Gal-T1intoaclonewithconfirmedindelsinB4Gal-T2and–T3(Table1).
Inour study, a total of 109potentialdeletion clonesweredeep sequenced for genomic
indelsinthetargetedregions(SupportingInformation,TableS5).Outofthese,23clones
revealedanuncleargenotypeforoneormoretargets(presenceofin-frameindelorindel-
frequencybetween5-98%).Thesewerediscarded.Weexpandedclearsingle-andmulti-KO
clonesof 1–4 targets (indel frequencies>98%).Next,we isolatedmultiple independent
clonesforeachgenotypetostudytruebiologicalreplicatesofthephenotypes,intotal17
clones(Table1).Oneclone(WTctr)whichshowednoinsertionordeletionwasadditionally
selectedtoserveasacontrolfortheanalysisofgrowthandN-glycanprofiles.Asecondclone
(T2-3-KOctr)whichdidnotrevealadditionalindelsafterthesecondroundoftransfection
wasalsocharacterizedtoinvestigatetheimpactoftransfectionandsubcloningongrowth
andN-glycanprofile.
11
3.2.EffectongrowthfromdifferentB4Gal-T-KO´sandindelcombinations
TheaimofourstudyistoprovideaCHOplatformtoproducerecombinantproteinswith
agalactosylatedN-glycans.ThroughengineeringcellstowardsG0-glycans,theN-glycansare
alterednotonlyontherecombinantprotein,butalsoonhostcellproteins.Ascellgrowth
performance is a substantial factor for industrialproteinproductionplatforms,we first
evaluated whether decreased N-glycan galactosylation influence CHO cell growth. We
carriedoutshakeflaskbatchexperimentswithselectedKOclones,theparentalCHO-SWT
aswellastwocontrolclones(Table1).SamplingforVCDsandviabilitieswasperformed
every24hforthetimecourseofsevendays.TheWTctrclonewasidentifiedtohavesimilar
growth and viability to CHO-SWT (Fig. 1). Double-KO of B4Gal-T1 and –T3 (T1-3-KO)
indicatedslightlydecreasedgrowthcomparedtoCHO-SWTandWTctr(Fig.1A).Thetwo
cloneswith frame-shifts in B4Gal-T3 (T3-KOA&T3-KOB)were not influenced in cell
growthandreachedslightlyhighermaximalVCDsthanCHO-SWT(Fig.1A).Furthermore,
the double-KO clone with indels in B4Gal-T2 and -T3 (T2-3-KO) revealed growth
comparable to CHO-SWT andWT ctr (Fig. 1B). The T2-3-KO ctr clone exhibited lower
growthcomparedto thegrowthcurveof theparentalT2-3-KOclone(Fig.1B).The four
triple-KOcloneswithframe-shiftsinB4Gal-T1,-T2and-T3(T1-2-3-KO)andthethreeT1-
2-3-4-KOmutantshaddecreasedgrowth compared toCHO-SWT (Fig. 1B, Fig. 1C).The
threeT2-3-4-KO cloneshadheterogeneous growthand comparedtoCHO-SWT, similar
maximalVCD(T2-3-4-KOC),lowermaximalVCD(T2-3-4-KOK)orincreasedmaximalVCD
(T2-3-4-KO H) was observed (Fig. 1D). Lastly, the three T1-2-KO clones exhibited also
heterogeneousgrowthbutcloneT1-2-KOBreachedsimilarmaximalviablecelldensities
comparedtoCHO-SWT(Fig.1E).
Altogether,theengineeredclonesrevealedanotablevariationingrowthwithingroupsof
cloneswithindelsinthesametargetgenes.WhereasT1-2-3-KOandT1-2-3-4-KOclones
12
haddecreasedgrowthcomparedtoCHO-SWT,cloneswithotherindelcombinationscould
growtosimilarmaximalviablecelldensitiesasCHO-SWT.
3.3.EffectsofB4Gal-T-KO´sonsecretomeN-glycanprofiles
Toinvestigatetheactivitiesofthetargetedβ-1,4-galactosyltransferaseswithinproteinN-
glycosylation,weanalyzed secretome samplesof CHO-SWT, the control clones and the
differentKO-cloneswithindelsin1-4sequencesforthetargetedB4Gal-T-genes(Table1).
ToexaminethecontributionofthetargetedB4Gal-Tswithingalactosylationofthedifferent
N-glycanbranches,westudiedtheremaininglevelsofN-glycangalactosylationinclones
withcombinatorialdisruptionofB4Gal-Ts.Toprobetheeffectofthegeneratedindelson
thesecretomeN-glycansoftheselectedclones,weanalyzedsupernatantsamplesharvested
fivedaysafterseeding.Secretomesampleswerecentrifugedandfilteredtoremovecells
andcelldebrisandup-concentratedbeforetotalN-glycanswerelabeledandanalyzedby
HPLC/MS.AspresentedinsupplementaryFigure1,thecomplexbi-antennarydi-sialylated
N-glycanstructure(A2FG2S2)wasthemajorstructurewithin theCHO-SWTsecretome.
Notably,intheCHO-SWTsecretome,onlyoneminorpeak(0.7%)ofG0-N-glycancouldbe
annotated(supplementaryFig.1).T3-KO,T2-3-KO,andT2-3-4-KOclonesshowedasimilar
N-glycanpattern toWTbutG0structureswereonlypresent inT2-3-KOandT2-3-4-KO
clones (Fig. 2). However, within the annotated N-glycan structures, the T1-3-KO clone
exhibitedatotalof~65%G0structures,butstilldisplayed~10%mono-galactosylatedN-
glycans in the secretome (Fig. 2). Compared to CHO-SWT, indels in B4Gal-T1 and –T2
resultedintheabsenceofG4 forms,reducedG3andG2 formsand increasedG1andG0
proportions(Fig.2)leadingtoincreasedoverallheterogeneityinN-glycangalactosylation
(supplementaryFig.1).T1-2-KOclonesoverallshoweddecreasedgalactosylatedN-glycans
compared to WT (~61% galactosylated structures) and revealed ~24% galactosylated
structures.
13
In contrast, we could only annotate ~1% galactosylated N-glycan structures in the
secretomesofT1-2-3-KOandT1-2-3-4-KOclones(Fig.2).ThemajorN-glycanstructuresof
T1-2-3-KOandT1-2-3-4-KOcloneswereA2FG0,A3FG0andA4FG0.Thebi-galactosylated
structures,whichwerethepredominantN-glycansinCHO-SWTandWTctrclone,werenot
presentanymore(seeFig.2andsupplementaryFig.1).Furthermore,theadditionalB4Gal-
T4indelinT1-2-3-4-KOclonesdidnotincreaseG0proportionsoreliminateG1N-glycans
whencomparedtoT1-2-3-KOcelllines(Fig.2).
Altogether,disruptionofB4Gal-T2 inconjunctionwithB4Gal-T1and–T3decreased the
galactosylatedsecretomeN-glycanproportionfrom~10%(T1-3-KO)downto~1%(T1-2-
3-KOs)andthesecretomeN-glycansofthetriple-andquadruple-KOsweredominatedby
agalactosylatedbi-,tri-andtetra-antennarystructureswithA2FG0asthedominatingN-
glycanstructure(supplementaryFig.1).
ToexaminetheroleofthefourtargetedB4Gal-TswithinproteinN-glycangalactosylation,
we studied the levels of agalactosylation in the secretome samples in a comparative
approach (Fig. 2). Confirming that B4Gal-T1 is the most active N-glycan β-1,4-
galactosyltransferase, we furthermore studied the presence of agalactosylationwithout
(cloneT3-KOAandT1-3-KO)andwithadditionalKOofB4Gal-T2(clonesT1-2-3-KOand
T2-3-KO) to investigate its contributing role inN-glycan galactosylation,which has not
previouslybeenstudied inexact terms.Therefore,wecompared twosetsof twoclones,
differingintheirgenotypebytheKOofB4Gal-T2.Inthefirstcomparison,thesingleKOof
B4Gal-T3exhibitednoG0-N-glycans,wherethedouble-KOofB4Gal-T2and–T3revealed
~6%G0-N-glycans (Fig. 2). Similarly, comparing theN-glycanproportionwith terminal
GlcNAcofcloneT1-3-KOandT1-2-3-KOclones,theadditionalKOofB4Gal-T2inthetriple-
KOcelllinesincreasedtheG0-N-glycanproportionby~10%.
14
3.4. TailoredRituximab and EPON-glycosylation after B4Gal-T-double and triple-
KO’s
ToprovethatengineeredsecretomeN-glycanswillalsoberepresentedonrituximaband
EPO which we used as model proteins, we transfected a rituximab- or EPO-encoding
plasmid intoCHO-SWTandKO-clonesT3-KOA,T2-3-KO,T1-3-KOandT1-2-3-KO.Cells
were transfected and rituximab- or EPO-containing supernatants were harvested after
threedays.Afterpurification,weanalyzedthecorrespondingN-glycanstructureswithin
thedifferentKOcelllines.
Clones T1-3-KO and T1-2-3-KO showed predominantly G0-N-glycans in the secretome
samplesandwereexpectedtoalsorevealpredominantlyG0structuresonthetransfected
rituximab. CHO-SWT, clone T3-KO A and T2-3-KO displayed comparable rituximab N-
glycanprofileswithG0andG1asprevalentstructureswithboth~40%oftotalrituximab
N-glycans (Fig. 3A). In contrast, rituximabpurified fromclonesT1-2-3-KOandT1-3-KO
cloneswasmostlyN-glycosylatedbybi-antennaryG0 structures,whereasG2structures
weremissing,whichisinlinewithsecretomeN-glycansoftheseclones.Notably,double-KO
ofB4Gal-T1and–T3inT1-3-KOresultedinhigherG0-N-glycanproportionsonrituximab
(~84%) than in clone T1-2-3-KO (~68%). Furthermore, triple-KO clone T1-2-3-KO had
increasedhigh-mannose(HM)structuresonrituximabwhencomparedto theothercell
lines.
Figure3BpresentsadetailedcomparisonofrituximabN-glycanspurified fromT1-3-KO
andT1-2-3-KOA.Herethebi-antennary,non-galactosylatedA2FG0wasclearlythemain
structure.However,wecouldalsoannotateHM,A2G0andA2FG1N-glycans.ThereofA2FG1
wasfoundinbothclonestocomparableamounts(~2-3%)buttotalHMproportionswere
higherinT1-2-3-KO(15%)thaninT1-3-KO(5.8%),representedbyMan5,Man7,Man8and
Man9 structures. Additionally, cell growth after rituximab transfectionwas comparable
15
betweenCHO-SWT,WTctr,T3-KOAandT1-3-KO(supplementaryFig.2)whereasclones
T2-3-KOandT1-2-3-KOrevealedincreasedviablecellconcentrationsondaythree.
FortransientlyexpressedEPO,theN-glycanprofilesofCHO-SWT,T3-KOAandT2-3-KO
are similar where annotated N-glycan structures predominantly harbor ≥4 galactose
residues;howeverG0formsarenotpresentinEPOfromCHO-SWT(Fig.3C).Incontrast,
double-KOofB4Gal-T1and–T3resultedinincreasedG0proportions(~72%)whereasG3-
andG4-glycanscouldnotbeidentifiedonEPOpurifiedfromT1-3-KO.AnalyzingN-glycan
structures of EPO from the triple-KO clone T1-2-3-KO A, we could only annotate
agalactosylatedandmono-galactosylatedN-glycans(supplementaryFig.3).
Overall,disruptionofB4Gal-T1and–T3withorwithoutadditionaldisruptionofB4Gal-T2
resultedinrituximab,whichonlyharbored~2-3%galactosylatedN-glycans.Ontheother
hand,singledisruptionofB4Gal-T3ordisruptionofbothB4Gal-T2and–T3,didnotchange
rituximabN-glycosylationcomparedtoCHO-SWT(Fig.3A).However,disruptionofB4Gal-
T2 in addition to indels in B4Gal-T1 and –T3 increased the G0 N-glycan proportion of
transientlyexpressedEPOfrom~72%to~91%(Fig.3C).
4.Discussion
Since recombinant proteins with agalactosylated N-glycans can be of interest for the
therapy of several diseases,we aimed to engineer CHO-S cells to revealpredominantly
agalactosylatedN-glycansonsecretedproteinsandontransientlyexpressedrituximaband
EPO.InapreviousstudyinCHO-K1derivedcelllines,disruptionofB4Gal-T1,-T2and–T3
resultedinprevalentlyagalactosylatedN-glycansonrituximabandEPOwhereastheeffects
ofB4Gal-Tdisruptionsoncellgrowthandtheglycosylationoftotalsecretedproteinswere
notaddressed[34].Withinthiswork,wealsoaimedtoassesstheimpactofB4Gal-Tindels
oncellgrowthandanalyzeN-glycansandcellgrowth ingroupsofcloneswith thesame
combination of indelswith respect to clonal variation.We investigated if disruption of
16
B4Gal-T1,-T2and–T3inCHO-Scellsissufficienttoproducepredominantlyagalactosylated
proteinsandifadditionaldisruptionofB4Gal–T4isofanybenefitwithregardstoN-glycan
agalactosylationanddecreasedN-glycanheterogeneity.Additionally,weanalyzedpossible
B4Gal-Tbranchpreferences after combinatorialKOofB4Gal-T-isoforms.Weperformed
CRISPR/Cas9-mediatedmultiplexingforallfourtargetsfollowedbysinglecellcloningand
genotype characterization via deep sequencing. Clones with different combinations of
B4Gal-T-indels were expanded and further characterized with regards to cell culture
performance, N-glycosylation of total secreted host cell proteins andN-glycosylation of
transientlyexpressedrituximabandEPO.
TargetingmultiplegenesinonetransfectionwithCRISPR/Cas9isatimesavingmethodto
generatecloneswithdifferentindel-combinationsinseveralgenes.However,clonesoften
havein-frameindelswhichmaynotdisruptthegene(s)[37].Sinceincreasingthenumber
ofco-transfectedsgRNAsmightincreasetheproportionoffunctionalin-frameindels(and
thereby render disrupting mutations in other genes unusable), we employed two
multiplexedtransfectionrounds.First,weco-transfectedwithsgRNAsagainstB4Gal-T1,-
T2and–T3orsgRNAsagainstB4Gal-T1and–T3orsgRNAsagainstB4Gal-T1and–T2.Ina
second round of transfection, we built up triple-KO (T1-2-3-KO and T2-3-4-KO) and
quadrupleKOclones(T1-2-3-4-KO)basedontransfectionsof theT2-3-KOcell linewith
sgRNAsagainstB4Gal-T3and–T4.Although it is faster,a limitationof thismultiplexing
methodisthatnotalldesiredKOcombinationsmightappearafterdeepsequencingofsingle
cellclones.Ideally,theselectionofcloneswouldincludeatleastthreecloneswiththesame
genedisruptionstoensurethattheresultingphenotypeisnotduetoclonalvariationupon
subcloning. The efficiency of indel-generation and clone survival are therefore critical
attributeswhenperformingmultiplexedexperimentswithCRISPR/Cas9.
17
BesidesinfluencingN-glycosylation,disruptingthefourtargetsalsoinfluencedcellculture
performancewherecloneswithindelsinB4Gal-T1,-T2and–T3revealeddecreasedgrowth
whencomparedtoCHO-SWT(Fig.1).ThereducedgrowthincloneswithT1-2-3-KOand
T1-2-3-4-KOcouldbeassociatedtothehighG0-N-glycanproportionsoftheirsecretome
(Fig.2)orbelinkedtoclonalvariationwhichisknowntobechallengingwhenworkingwith
CHOcells[38].However,glycosylationplaysamainroleincell-cellcommunicationviae.g.
endocytosis, receptor activation, and cell adhesion[39] and glycosylation engineering
thereforemightimpactcultivationperformance.Wealsoreportheterogeneouscellgrowth
ofcloneswithinthegeneratedindelcombinationgroups.Thiscanalsobearesultofclonal
variation after subcloning or due to off-target effects after sgRNA and GFP_2A_Cas9 co-
transfections.However,weusedthesgRNAdesignguidelinesandidenticalCas9-version
publishedinanearlierstudywhichdidnotshowsignificantoff-targetevents[13].While
subcloningdidnot influence growthof theWTctr clone, subcloningofT2-3-KO lead to
decreased growth of the T2-3-KO ctr (Fig. 1). Nonetheless, our results indicate that
subcloning hadno impact on secretomeN-glycosylation as theWT ctr and T2-3-KO ctr
clones showed comparable N-glycan structures to their parental cell lines in the batch
cultivation(Fig.2).
Incontrast toapreviousstudy,whichsuggestedB4Gal-T1-4toallbeactive inN-glycan
galactosylation[31], our results indicate thatB4Gal-T1, -T2 and–T3are themostactive
B4Gal-TsintheN-glycosylationpathwayofCHO-ScellsandthatB4Gal-T4hasverylittleor
nocontributiontogalactosylationofN-glycansinCHO-Scells.ThelackofN-glycosylation
activityofB4Gal-T4inourworksupportsanotherstudywhereB4Gal-T4wasreportedto
be active in the galactosylation of mucin-type core 2 branching in the O-glycosylation
pathway[32].
18
Furthermore,B4Gal-T5,-T6and–T7(andpotentiallyunknownB4Gal-Transferases)insum
contributeonlyupto~3%N-glycangalactosylationofthesecretomeasseeninFigure2.
ThesinglegalactosefoundonG1tetra-antennaryN-glycansindicatesthattheremaining
source for N-glycan galactosylation in the quadruple-KOs can only transfer a single
galactoseontheN-glycanstructure.However,peaksveryclosetothebaselinecouldnotbe
annotated andmight harbor little amounts of structureswithmore than one galactose.
WhetherthegalactosylationactivityinthequadrupleKOclonesiscarriedoutbyB4Gal-T5,
-T6,–T7oracombinationthereofhastobeinvestigatedfurther.
WeconcludethatB4Gal-T1isthemainactiveN-glycanB4Gal-TincloneT2-3-KO.Sincethe
T2-3-KOclonestillshowedupG1,G2,G3andG4structuresandallKOcelllineswithindels
inB4Gal-T1lackG4N-glycans,B4Gal-T1isverylikelycapableoftransferringgalactoseto
all four branches. Therefore we suggest that B4Gal-T1 is the most active N-glycan
processingB4Gal-Twithin the familyof β-1,4-galactosyltransferasesofCHO-S cells. The
predominantactivityofB4Gal-T1inN-glycangalactosylationwithinourstudyisinlinewith
previousworkinotherCHOcell lines[34].Moreover,weconcludethatB4Gal-T2activity
contributesto~5-10%ofN-glycangalactosylationsinceB4Gal-T2-KOinadditiontoKOof
B4Gal-T3orB4Gal-T1and-T3increasedG0structuresupto10%(Fig.2).
TheremaininglevelofrituximabgalactosylationoftheCHO-SderivedcloneT1-3-KO(~2-
3%) is comparable, yet slightly higher to another study where decreased rituximab
galactosylation(~1%)wasachievedbyknockingoutB4Gal-T1and–T3inCHO-K1derived
cell lines[34]. This difference in remaining N-glycan galactosylation could be due to
differencesintheN-glycanpathwaysofthecelllinesused(CHO-SversusCHO-K1)[34]or
duetoclonalvariation.AlthoughB4gal-T3-KOleadtodecreasedN-glycangalactosylation
activity when combined with B4Gal-T2-KO, single B4Gal-T3-KO did not decrease
galactosylationatall(Fig.2).ThissuggeststhatB4Gal-T3hasonlyaminorroleinCHO-SN-
19
glycosylationor that itsdisruptedN-glycan transferase functioncanbecompensatedby
B4Gal-T1and–T2activityintheT3-KOclone.
Forglycoproteinsharboringtri-ortetra-antennaryN-glycans,asisthecaseforEPO,KOof
B4Gal-T1and–T3isnotsufficienttoproducemainlyagalactosylatedglycoproteins(Fig.3B
with~20%EPOgalactosylationinT1-3-KO),whereasrituximabexpressedincloneT1-3-
KOresultedinonly~3%galactosylatedstructures(Fig.3A).Therefore,weproposethatbi-
antennary N-glycosylated proteins as rituximab can be produced with mostly
agalactosylatedN-glycansafterdouble-KOofB4Gal-T1and–T3buttri-andtetra-antennary
N-glycosylated secretome proteins as EPO additionally need KO of B4Gal-T2 to be
predominantlyagalactosylated.
FortransientlyexpressedEPOinCHO-K1derivedcellswithtriple-KOofB4Gal-T1,-T2and
–T3theproportionsofgalactosylatedN-glycanswerefoundtobe~4%inanearlierstudy
[34]. Inourstudyweannotated~6%galactosylatedN-glycanson transientlyexpressed
EPO from the CHO-S derived triple-KO T1-2-3-KO A (Fig. 3C). Although these results
indicate similar effects on galactosylation of EPO after disruption of two identical gene
targets,deviationscouldberelatedtodifferencesbetweenCHO-K1andCHO-Sexpression
levelsofnon-targetedB4Gal-Tisoforms.
Weconcludethatengineeringcellswithnon-galactosylatedN-glycansonasecretomelevel
inCHO-SWT isapromisingstrategy towardsproducingG0-IgG1andG0-EPOonalater
stage.DespitethedivergentgeneexpressionlevelsbetweendifferentCHOcelllines[40]this
engineeringstrategy issuitablenotonly forCHO-K1[34]butalso forCHO-Sderivedcell
linesasutilizedinourwork.Inthepresentedstudythetriple-KOwith~1%galactosylated
structures on the secretome also showed predominantly agalactosylated N-glycans on
transientlyexpressedrituximabwithonly~3%galactosylatedN-glycansandontransiently
expressedEPOwithremaining~6%galactosylatedN-glycanstructures.
20
Within our triple-KO cell line T1-2-3-KO A, we also noticed a significant amount of
hypermannosylated(HM)structuresontransientlyexpressedrituximab(Fig.3Aand3B).
HM structures are a critical quality attribute within biopharmaceutical protein
production[41]andcanaccumulateduringcellcultureperformance.Processdesignand
genetic engineering could be two possibilities to overcome accumulatedHM structures
whichmightrepresentproteinsaccumulatedintheGolgi-situatedN-glycanmachineryafter
disrupting Golgi-residing B4Gal-T1, -T2 and –T3.This disruptionmight cause increased
trafficandresidencetimeofsecretomeproteinsintheGolgilumenwithoutbeingfurther
processed by glycosyltransferases. Recent studies displayed increased processing of N-
glycans after overexpression of Mgat4 andMgat5 which could result in decreased HM
structures[42].
In N-glycan analysis of secretome, rituximab and EPO from T1-2-3-KO clones we still
detectedremaininggalactosylatedstructures.Here,addingKOofB4Gal-T5,-T6or–T7on
cloneT1-2-3-KOcouldhelptoinvestigatetheiractivitieswithinthegalactoslyationofN-
glycosylatedproteinstoe.g.removetheremaining~3%ofgalactosylatedN-glycansafter
transientrituximabexpression.
TheoutcomeofthisstudywiththegenerationofdifferentamountsofG0,G1,G2,G3andG4
formsafterdisruptionofthetargetedtransferasescouldbeastartingpointtoconstructa
N-glycangalactosylationmodel for thediscussionofpossiblebranch specificitieswithin
B4Gal-T1, -T2, -T3 and –T4 as conducted in an earlier study[31]. We suggest that the
functionofB4Gal-T1includesgalactosylationofallfourN-glycanbranches(Fig.2),although
itsbranchpreferenceneedstobeexploredfurther.IncloneT1-3-KO,B4Gal-T2issuggested
tobethemostactiveB4Gal-Tandonlymono-galactosylatedN-glycanswerefoundwithin
21
galactosylated structures (Fig. 2). This indicates that B4Gal-T2 N-glycosylation activity
includesonlythegalactosylationofasingleN-glycanbranch.
Studying the galactosylation levels ofT1-2-KO clones ina similar approach leads to the
assumption thatB4Gal-T3 isat least capableof transferring galactose toup to threeN-
glycanbranches(Fig.2).AspresentedinsupplementaryFigure1,cloneswithT1-2-KOwere
identifiedtonotproduceN-glycanswithdecreasedheterogeneityasfoundintheothersets
ofKO combinations.Theoriginof thisheterogeneity is the increasedpresenceofG1N-
glycanforms.However,G1formswithfurthermodificationscanbeofparticularinterest
since they are theplatform for glycoPEGylation, amethod to attachpolyethylene glycol
(PEG)onbiopharmaceuticalstoincreaseserumhalf-life[43].
Insummary,ourstudypresentsthenecessityofdisruptingthethreegenes,B4Gal-T1,-T2
and –T3, to produce not only predominantly G0 secretome proteins but also mainly
agalactosylatedrituximabandEPOinCHO-Scells.Further,weelucidateddifferentN-glycan
galactosylation activities within the four targeted genes where B4Gal-T1 is the most
contributingenzymetoN-glycangalactosylationandinvolvedinthegalactosylationofall
fourN-glycanbranches.Ourstudyconcludesthattargetingthepresentedtargetsdoesonly
interfere with cell growth if B4Gal-T1, -T2, and –T3 are disrupted simultaneously and
reveals the possibility to engineer tri- and tetra-antennary G0 N-glycans, which are
naturallynotproducedinCHO-SWTcells(supplementaryFig.1).Wealsoinvestigatedthe
B4Gal-T2activityinCHO-Scellsandconcludethatitsgalactosylationactivityisprevalent
tooneN-glycanbranchwhileB4Gal-T4hasnoN-glycangalactosylationactivityandB4Gal-
T3 can galactosylate up to three N-glycan branches. Prior engineering of secretome N-
glycansinaWTcellgivesrisetotheflexibilityofexpressingseveraldifferentmodelproteins
in the engineered cell line at a later stage. Such model proteinsmight include already
marketedantibodiesorothertherapeuticproteins.Withourcellplatformthatprevalently
22
glycosylatesproteinswithG0-N-glycanswedemonstrate an alternative to galactosidase
treatment of recombinant proteins to investigate further beneficial in vitro and in vivo
characteristicsbasedontailoredG0N-glycosylationprofiles.
Acknowledgement
TheauthorsthankSaraPetersenBjørn,BjørnVoldborg,JohnnyArnsdorf,YuzhouFanand
Patrice Menard for valuable guidance and support. The authors thank Karen Katrine
Brøndum, Nachon Charanyanonda Petersen, Karoline Schousboe Fremming and Zulfiya
Sukhova forexcellent technicalassistancewith theFACSandMiSeq librarypreparation,
HelleMunckPetersen for assistancewith theproteinpurification,AnnaKozaandMads
ValdemarAndersonforassistancewiththeMiSeqanalysis.TheNovoNordiskFoundation
(NNF10CC1016517)supportedthiswork.T.A.,H.F.K.andM.R.A.arereceivingfundingfrom
the EuropeanUnion’sHorizon 2020 research and innovationprogram under theMarie
Sklodowska-CuriegrantagreementNo.642663.
Authorcontributions
A.H.H.,M.R.A.,H.F.K.andT.A.plannedtheexperiments.T.A.performedtheexperimental
workandwrotethemanuscript.A.H.H.performedtheN-glycananalysisandS.K.conducted
the protein purifications. A.H.H., M.R.A., H.F.K., S.K. and G.M.L. guided the project,
contributedtoexperimentaldesign,andcommentedandcorrectedthemanuscript.
Conflictofinterest
Theauthorsdeclarenofinancialorcommercialconflictofinterest.
23
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26
Table1.OverviewofsgRNA/Cas9transfectionsandgeneratedcelllines.Thefirstroundof
transfectionswasperformedwithaCHO-SWT.TheT2-3-KOclonewasusedasparentalcell
lineforthesecondtransfectionround.Valuesinbracketsaregeneratedindelsinbpforeach
targetconfirmedbydeepsequencing.
27
Figure1. Growthprofiles of expanded clones inbatch cultivation. 30mLvolumebatch
cultivation with VCDs for the duration of seven days after sampling every 24 h (n=2).
Seedingwasperformedat3.0x105cells/mLanderrorbarsindicaterangeofshakeflask
duplicates.(A)T3-KO&T1-3-KO,(B)T1-2-3-KOs,(C)T1-2-3-4-KOs,(D)T2-3-KOs,(E)T1-
2-KOs.
28
Figure2.SecretomeN-glycanprofileofgeneratedB4Gal-T-KO-clones.N-glycansecretome
analysisfrombatchcultivationofparentalcelllinesandKOcelllinesharvestedafterfive
daysofcultivationandnormalizedtoareaunderthecurve(AUC)oftotalagalactosylated
(G0), mono-galactosylated (G1), bi-galactosylated (G2), tri-galactosylated (G3), tetra-
galactosylated(G4)andhigh-mannose(HM)N-glycanpeakspercell line.IncreaseofG0-
proportionisgivenin%afteradditionalB4Gal-T2-KOinT2-3-KOandT1-2-3-KOcompared
toT3-KOBandT1-3-KO,respectively.Wherepresent,errorbarsindicateSDofthree(T1-
2-3-KO,T2-3-4-KOandT1-2-KO)orfourreplicates(T1-2-3-KO).
29
Figure3.RituximabandEPON-glycosylationprofilesinWTandB4Gal-TKOcelllinesafter
transient transfection. (A) Comparison of rituximab N-glycans purified out of pooled
supernatantswithinshakeflaskduplicatesfromCHO-SWT,T3-KOA,T2-3-KO,T1-2-3-KO
A and T1-3-KOwithN-glycan proportions of agalactosylated (G0),mono-galactosylated
(G1),bi-galactosylated(G2)andhigh-mannosestructures(HM)normalizedtoAUCoftotal
N-glycanpeaksperclone.(B)DetailedN-glycanprofilesofrituximabpurifiedoutofpooled
supernatantswithinshakeflaskduplicatesfromT1-2-3-KOA(orangeline)andT1-3-KO
(black line) afterHPLC histogram annotation viaMS. (C) Comparison of EPON-glycans
purifiedoutofpooledsupernatantswithinshakeflaskduplicatesfromCHO-SWT,T3-KOA,
T2-3-KO, T1-2-3-KO A and T1-3-KOwith N-glycan proportions of agalactosylated (G0),
mono-(G1),bi-(G2),tri-(G3)andgreaterorequaltetra-galactosylatedstructures(≥G4)
normalizedtoAUCoftotalN-glycanpeaksperclone.(D)DetailedN-glycanprofileofEPO
purified out of pooled supernatants within shake flask duplicates from
T1-2-3-KOA.
30