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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