SmartEnzymes - Genovis · the digestion sites of the enzymes in human IgG1 is presented in the...

2

SmartEnzymes

Nature offers a vast source of enzymes, perfected through evolution to perform defined reactions. At Genovis, we believe that enzymes with unique properties can be used as biological tools to support the research and development of complex biopharmaceuticals to help bring safe and effective medicines to patients in need. Our task is to identify new enzymes and give them names. We call them SmartEnzymes.

3

SmartEnzymes

A cysteine protease that digests IgGand Fc-fusion proteins at a specific site below the hinge region

A cysteine protease that digests mouse IgG2a and IgG3 at a specific site below the hinge

A cysteine protease that digests human IgG1 at a specific site above the hinge

A cysteine protease that digestshuman IgG1 above the hinge

A cysteine protease that digests IgGin the upper hinge region

An arginine-specific protease that digests proteins C-terminally of arginine residues

A site-specific conjugation technology for IgG

An endoglycosidase that rapidly hydrolyzes the N-glycan structures of the Fc domain of IgG

An endoglycosidase that specifically hydrolyzes biantennary Fc glycans of IgG

An O-glycan-specific protease that digests mucin-type O-glycosylated proteins N-terminally of the O-glycosylation site

An enrichment resin for affinity purification of mucin-type O-glycosylated proteins and peptides

An O-glycosidase that specifically hydrolyzes core 1 and core 3 type O-glycans on native glycoproteins

Sialidases for removal of sialic acids from native glycoproteins

FabRICATOR® 8FabRICATOR®-HPLC / Validation Kit / Anti-FabRICATOR® / FragIT™ / FragIT™ kit

FabRICATOR®Z 14FragIT™Z / FragIT™Z kit

FabALACTICA® 16Immobilized FabALACTICA® / FabALACTICA® Fab kit

GingisKHAN® 20GingisKHAN® Fab kit

GingisREX® 24

GlyCLICK® 36

FabULOUS® 22FabULOUS® Fab kit

GlycINATOR® 32Immobilized GlycINATOR®

OpeRATOR® 26

GlycOCATCH® 28

OglyZOR® 29

IgGZERO® 34deGlycIT™

SialEXO® 30SialEXO®23

IgG GLYCO

SIDASES

O-G

LYCAN

SCO

NJUG

ATION

PROTEASES

IgG PROTEASES

Specific-oneprecisedigestion sitebelowthehingeofIgGF(ab’)2andFc/2fragmentsin30minNeedsnoreducingagentsorco-factorsAvailableinanHPLCcolumnformatforfaston-columdigestion

Specific-onedigestionsiteabove thehingeofhumanIgG1GeneratesintactFabandFcfragmentsOvernightdigestionreactionNeedsnoreducingagentsorco-factors

ImmobilizedFabRICATORenzymeonagarosebeadsGeneratesF(ab’)2andFc/2fromIgGConvenientspincolumnformatNoenzymeinthefinalpreparationAvailableasFragITkitforpurificationofF(ab’)2andFc/2fragments

ImmobilizedFabRICATORZenzymeonagarosebeadsGeneratesF(ab’)2andFc/2from mouseIgG2aandIgG3ConvenientspincolumnformatNoenzymeinthefinalpreparationAvailableasFragITZkitforpurificationofF(ab’)2andFc/2fragments

Specific-oneprecisedigestionsitebe-lowthehingeofmouseIgG2aandIgG3GeneratesF(ab’)2andFc/2fragments2hreactionNeedsnoreducingagentsorco-factors

ImmobilizedFabALACTICAenzymeonagarosebeadsGeneratesintactFabandFcfragmentsConvenientspincolumnformatNoenzymeinthefinalpreparationAvailableasFabALACTICAFabkitforpurificationofFabandFcfragments

OnedigestionsiteabovethehingeofhumanIgG1GeneratesintactFabandFcfragments60minreactionRequiresmildreducingagents (included)

...CPAPNLLG / GPSVF....

...KSCDKT / HTCPPCP....

30MINUTES

...CPPCPAPELLG / GPSVF...

Arginine-specificproteaseDigestsproteinsandpeptides C-terminallyofarginineresidues60minreactionActivein6Mureaand0.1%SDS

DigestsIgGintheupperhingeregionofseveralspeciesandsubclassesGeneratesFabandFcfragments60minreactionRequiresreducingconditions

...KTHT / CPPCPAPEL....

60MINUTES

60MINUTES

...KSCDK / THTCPPCP....

IgG PROTEASES PROTEASES

HydrolyzestheN-glycanstructureof theIgGFcdomain30minreactionRequiresnativeIgGfoldHydrolyzesallFcglycoformsofIgG

HydrolyzestheN-glycanstructureoftheIgGFcdomain30minreactionRequiresnativeIgGfoldLimitedactivityonhigh-mannoseandhybrid-typeFcglycans

ImmobilizedIgGZEROenzymeon agarosebeadsHydrolyzesIgGFcN-glycansConvenientspincolumnformatNoenzymeinthefinalpreparation

Digestsmucin-typeO-glycosylatedpro-teinsN-terminallyoftheO-glycosylationsite2htoovernight(16-18h)reactionMapsO-glycosylationsiteoccupancy

O-glycosidaseactingonO-glycansHydrolyzescore1andtosomeextentcore3typeO-glycansonnativeglyco-proteins2-4hreactionRequiresremovalofsialicacids

Site-specificconjugationofIgGActiveonhumanIgG1-4andseveralotherspeciesTheantibodyretainsantigenbindingQuantitativelabelingof2labelsperantibody

30MINUTES

30MINUTES

Alexa Fluor® 488

DFO

IgG GLYCOSIDASES CONJUGATIONO-GLYCANS

ImmobilizedGlycINATORenzymeonagarosebeadsHydrolyzesallFcglycoformsofIgGConvenientspincolumnformatNoenzymeinthefinalpreparation

Biotin Azide Activation

Enrichesmucin-typeO-glycosylatedproteinsandpeptides30min-2hbindingRequiresdesialylationApplicationsinglycomics

SialEXOhydrolyzesallsialicacidsandSialEXO23specificallyhydrolyzesα2-3sialicacidsActiveonbothN-andO-linkedglycans1-2hreactionActiveonnativeglycoproteins

6

SmartEnzymes

7

Enzyme

IgG species and subclasses

Human IgG1-4, mouse IgG2a

and IgG3, some classes of rat, monkey, rabbit

and sheep

Human IgG1 Human IgG1Human IgG1-4,

mouse, rat, goat, sheep and rabbit

Human IgG1-4, Mouse IgG2a

and IgG3, some classes of

monkey, rabbit and sheep

Digestion site(human IgG1) LLG / GPS DKT / HTC CDK / THT THT / CPP LLG / GPS

Above / below hinge (human IgG1)

Below Above Above Above Below

Reaction requirements

Physiological buffers


2 mM cysteineReducing conditions


Reaction time 30 min O/N 1 h 1 h 2 h

pH 5.5 - 8 6 - 8 8 6.5 - 8 5.5 - 8

Table 1. Comparison of the Genovis IgG proteases.

IgG Proteases

Genovis IgG ProteasesGenovisprovidesuniqueenzymesandtechnologiesusedincharacterizationandconjugationofbiopharmaceuticalssuchasmonoclonalantibodies(mAbs),Fc-fusionproteins,biosimilarsandantibody-drugconjugates(ADCs).Theriseofmonoclonalantibodiesandotherbiomoleculesintobiotherapeuticshas increasedtheanalyticalchallengessignificantly.Toensuresafeandpotentdrugs,manydifferentqualityattributesofthelargeheterogeniousmoleculesneedtobecharacterized.Forthisreason,theanalysisofantibodysubunitssuchasFab,F(ab’)2andFc/2usingliquidchromatographyandhighresolutionmassspectrometry(LC-MS)hasemergedasanewplatformmethodforcharacterization.Traditionaltechniquesareoftentimeconsumingandmayinduceartefactsinthesample,whereasthemiddle-levelapproachisfasterandgeneratesdatathatiseasiertointerpret.TheIgGproteasesfromGenovisareagroupofpoteolyticenzymesthatdigestantibodiesfromseveral speciesandsubclassesintosubunits.TheenzymesFabRICATOR®(IdeS)andFabALACTICA®(IgdE)arespecificproteases,digestingIgGatasinglesitebeloworabovethehinge,respectively.OtherproteasesfordigestionofIgGincludeFabRICATOR®Z(IdeZ),FabULOUS®(SpeB)andGingisKHAN®(Kgp).AnoverviewofthedigestionsitesoftheenzymesinhumanIgG1ispresentedinthefigureonpage6,andacomparisonoftheIgGproteasesisgiveninthetablebelow.

8

SmartEnzymes

FabRICATOR® (IdeS) is a unique cysteine protease that digests IgG at a specific site below the hinge, enabling antibody subunit analysis.

FabRICATORisanIgG-specificcysteineproteasethatdigestsantibodiesatasingleaminoacidsitebelowthehingeregion,generatingahomogenouspoolofF(ab’)2andFc/2fragmentswithin30minu-tes.NeutralpHandnorequirementsforco-factorsmaketheenzymeeasytouseandenableplatformanalyticalworkflowsbasedonFabRICATORwithouttheneedforoptimization.FabRICATORiswidelyusedincharacterization,qualitycontrol,stabilitytesting,productionmonitoringandcloneselectionofantibody-basedtherapeutics,suchasmAbs,ADCs,biosimilarsandFc-fusionproteins.Aselec-tionofpublicationsusingFabRICATORisavailableonp.38.

...CPPCPAPELLG / GPSVF....

30MINUTES

HumanIgG1-4,Fc-fusionproteins,ADCs,mouseIgG2aandIgG3*,IgGofsomeclassesfrommonkey,rat,rabbitandsheep

30minreaction

Noneedforreducingagentsor co-factors

CPAPELLG/GPSVF(belowthehinge)

FabRICATOR® Reduction

Antibody Subunit Workflow

Figure 1. FabRICATOR digestion of IgG results in F(ab’)2 and Fc/2 fragments that can be further reduced to antibody subunits.

*FabRICATORhaslimitedactivityonmouseIgG2aandIgG3.Fordigestionoftheseantibodies,FabRICATOR®Zisrecommended.

TheFabRICATORsamplepreparationofantibodiesisacommonworkflowforsubunit LC-MSanalysis(Fig. 1).IgGisdigestedusingFabRICATORat37°Cfor30minutestogenerateF(ab’)2andFc/2frag-mentsfollowedbyreductionanddenaturation.The

generated~25kDasubunitsallowincreasedmassresolutionusingLC-MSinstrumentationandenablefastandaccurateanalysisofIgGglycansandotherqualityattributessuchasoxidation,deamidationandpyroglutamination.

FabR

ICAT

OR®

9

Enzyme FabRICATOR Papain/Ficin Pepsin

Digestion site

Specificity IgG specific/ One digestion site

Unspecific/ Several digestion sites

Unspecific/ Several digestion sites

Selectivity IgG (only known substrate) Several proteins Several proteins

Reaction conditions No optimization Requires optimization Requires optimization

Reaction time 30 min 1-24 h 1-24 h

Comparison to Other Common Enzymes

Figure 3. Reversed-phase chromatogram of bevacizumab exposed to oxi-dizing reaction conditions (0, 1 or 6 hours) before FabRICATOR digestion.

Determining the Degree of Oxidation

1

2

2

3

3

Glycan Profiling of Cetuximab

Figure 4. Relative abun-dance of glycans on the Fc domain of cetuximab. 11 glycans were quantified and no additional digestion or labeling were needed for similarity assessment.

High Resolution LC-MS for Amino Acid Sequence Verification

36 38 40 42 44 Time (min)

MassspectrometerswithhighresolvingpowerallowforaminoacidverificationofmAbs. FabRICATORgeneratesthepreciseantibodysubunitfragmentsFc/2,LCandFdthatcanbemono-isotopicallyresolvedandanalyzedusingLC-MS(Fig. 2).

Figure 2. LC-MS on adalimumab

Oxidationofantibodiesisakeyqualityatt-ributethatmayaffecttherapeuticantibodyfunctionality. FabRICATORisaconvenienttooltodeterminethedegreeofoxidationonthesubunitlevel(Fig. 3).Theshiftinretentiontimeinthechro-matogramshowsthattheamountofoxidizedantibodyincreasesastheoxidationtimeisprolonged.

Non-oxidized Fc1 h oxidation6 h oxidation

1 Met 252 & Met 428 ox

2 Met 252 ox

3 Non-oxidized Fc

Byanalyzingantibodysubunitsgeneratedby FabRICATOR,themassresolutionissignificantlyincreased.ThisallowsforfastandaccurateglycanprofilingofantibodiesusingLC-MSonthesubunitlevel.Ayoubandcolleagues(Ayoub,2013,p.38)usedthesubunitworkflowtodeterminetheglycanprofileoftheFabandFcdomainsofcetuximab(Fig. 4).

IgG Proteases

FabRICATO

R®

10

SmartEnzymes

Product ID Description Digestion EUR USD

A0-FR1-020 FabRICATOR, 2000 units 2 mg IgG 430 599

A0-FR1-050 FabRICATOR, 5000 units 5 mg IgG 835 895

A0-FR1-250FabRICATOR, 5 x 5000 units

25 mg IgG 3,195 3,495

A0-FR1-096 FabRICATOR, 96x100 units 96 x 100 μg IgG 1,735 2,225

A0-FR1-008 FabRICATOR, 8x100 units 8 x 100 μg IgG 295 375

A0-FR8-020 FabRICATOR LE (low endotoxin), 2000 units

2 mg IgG 475 650

A0-FR8-050FabRICATOR LE (low endotoxin), 5000 units

5 mg IgG 920 975

Validation Kit


A0-FR4-060FabRICATOR, 3 x 2000 units

3 x 2 mg IgG 1,295 1,795

Anti-FabRICATOR™

Anti-FabRICATORisagoatpolyclonalantibodypurifiedonproteinGthatisusedfordetectionof FabRICATORwithwesternblotorELISA.

Product ID Description Concentration EUR USD

A3-AF1-010Anti-FabRICATOR 0.1 ml

4 mg/ml 250 350

ThreedifferentbatchesoflyophilizedFabRICATORareincludedintheFabRICATORValidationkitforvalidationofFabRICATOR-basedanalyticalmethods.

LyophilizedFabRICATORforrapidantibodysubunitgenerationisavailableindifferentsizes.FabRICATORLEisalowendotoxinpreparationandissuitableforcell/tissue-basedassays,andtheplates8x100and96x100unitsallowforarapidantibodysubunitgenerationinahigh-throughputformat.

FabR

ICAT

OR®

FabR

ICAT

OR®

11


A0-FR6-010 FragIT Microspin 2 x 0.5 mg IgG 320 445

A0-FR6-025 FragIT Microspin 5 x 0.5 mg IgG 730 1,010

A0-FR6-050 FragIT Microspin 10 x 0.5 mg IgG 1,210 1,685

A0-FR6-100 FragIT Midispin 1-10 mg IgG 975 1,350

A0-FR6-1000 FragIT Maxispin 10-100 mg IgG 2,900 4,055

TheFabRICATORenzymeisimmobilizedonagarosebeads,andthespincolumnsareprovidedwithim-mobilizedenzymefordigestionof0.5mgupto100mgofantibodyorFc-fusionprotein.FragITgeneratesantibodysubunitswithnoenzymeinthefinalprepara-tion.

FragIT ™

FragIT™ (immobilized FabRICATOR®) digests IgG from several species and subclasses and generates F(ab’)2 and Fc/2 fragments.

FragITkitconsistsofspincolumnsofFragITforanti-bodydigestionandspincolumnsofCaptureSelect™*FcresinforaffinitybindingoftheFcfragments.Afterdigestion,theFcfragmentsarecapturedintheaffinityspincolumnandpureF(ab’)2fragmentsareobtainedintheflowthrough.

FragIT ™kit

60MIN

60MINUTE

60MINUTES

30MIN

*Thermo Scientific™ CaptureSelect™ resin from Thermo Fisher Scientific Inc. and its subsidiaries. Thermo Scientific and CaptureSelect are trademarks of Thermo Scientific Inc. and its subsidiaries.

FragIT™ kit easily generates and purifies F(ab’)2 and Fc/2 fragments from IgG.

30MINUTES

IgG Proteases

FabRICATO

R®


A2-FR2-005 FragIT kit, Microspin 0.5 mg IgG 360 499

A2-FR2-025 FragIT kit, Microspin 5 x 0.5 mg IgG 975 1,350

A2-FR2-050 FragIT kit, Microspin 10 x 0.5 mg IgG 1,850 2,273

A2-FR2-100 FragIT kit, Midispin 10 mg IgG 1,215 1,695

A2-FR2-1000 FragIT kit, Maxispin 100 mg IgG 3,640 5,090

12

SmartEnzymes

Figure 1. Potential set-up for an automated middle-level workflow using FabRICATOR-HPLC. FabRICATOR digests IgGs to F(ab’)2 and Fc, which is well suited for high resolution MS analysis.

FabRICATOR-HPLC offers on-column digestion of monoclonal antibodies for rapid subunit generation in automated middle-level workflows.

FabRICATOR-HPLCcontainstheFabRICATOR(IdeS) enzymeimmobilizedonanHPLCcompatible resin,generatingF(ab’)2andFcfragments withoutriskofover-digestion.In-housetesting(Fig. 2and3)highlightsthestabilityandreproducibilitywiththecolumndeliveringconsistentdigestionfor14daysat37°C.Morethan450injectionsofmAbweremadeduringtestingandnocarry-overwasobserved.

FabRICATOR-HPLCcanbeusedinastandardLC-MSsetupforroutineanalysis(Fig. 1).Butmore advancedconfigurationsarepossible,for examplewith2D-LC.Ultimately,abioreactorcanbeconnecteddirectlytotheMSinanautomatedonlinemiddle-levelworkflow.Thiswouldsignificantlyreduceoperatortime,samplehandlingerrorsandincreasethroughput.

WASTE

PUMP

WASTE

Mass Spectrometer

Step 1: Digestion

to WASTE to WASTEto RP column

Abs

280

nm

retention time(min)

0 2 4 6 8 10 12 14

F(ab’)2

Fc/2

Step 3: Separation

Abs

280

nm

retention time(min)

1614 18 20

*

Fc/2 LC Fd’

Step 4: Analysis

Sign

al in

tens

ity

m/z25800256002540025200250002480024600

G2F

G1FG0F

G00.1% FA, ACN gradient150 mM ammonium acetate, pH 7

Digestion Reduction Desalt Separation PurgePurge

Analysis: 1 h 20 min

10 min 10 min10 min20 min 20 min 20 min 30 minRP column wash

Column wash: 40 min

AutosamplermAb

1-2 ug0.025-2 mg/ml

RP-HPLC UV

LC Fc/2

Fd’

TCEP

Step 2:on-column Reduction

FabRICATOR-HPLC

Reduction

FabR

ICAT

OR®

13

Product ID Description EUR USD

A0-FRC-050 FabRICATOR-HPLC 1,599 1,799

FabRICATOR FabRICATOR-HPLC

Fc/2 LC Fd’ Fc/2 LC Fd’

*UV

chro

mat

ogra

m

100

75

50

25

0

% d

iges

tion

0 4 14

>95%

Operation (days)10 12862

5Retention time (min)

10 15 5Retention time (min)

10 15

Figure 3. a) Deconvoluted mass spectra of the trastuzumab Fc/2 fragment from in-solution FabRICATOR digestion (orange), or FabRICATOR-HPLC (blue). b) Glycosyla-tion profiles of trastuzumab generated by in-solution FabRICATOR digestion (orange, n=10) or FabRICATOR-HPLC (blue, n=28, 2 samples per day).

Figure 2. a) Comparison between digestion of trastuzumab using standard in-solution FabRICATOR protocol (left) and digestion using the automated FabRICATOR-HPLC workflow (right). The asterisk marks LC fragments that are not completely reduced with one intramolecular disulfide bridge intact. b) Quan-tification of digestion performance over a period of 14 days.

Robust On-column Performance

DigestionefficiencyismaintainedbyFabRICATOR-HPLCduringcontinuousoperationat37°Cforupto14days.Inourtests,2μgtrastuzumabsampleswereinjectedevery4hundernativeconditionsin150mMammoniumacetate,pH7ataflowrateof25μl/min.Theresultingantibodyfragmentswerereducedon-columnandanalyzedusingtheauto-matedsubunitanalysisworkflowdescribedinFig. 1 (Fig. 2a).Morethan95%oftheantibodywasdigestedduringtheentire14-dayperiod.(Fig. 2b).

Reproducible Glycan Analysis

TheperformanceandoperationalstabilityoftheFabRICATOR-HPLCcolumnaredemonstratedhereusinganalysisoftrastuzumabFcglycosylationasanexample.Automatedmiddle-levelanalysisusingFabRICATOR-HPLCyieldedmassspectravirtu-allyindistinguishablefromtheoneobtainedfromastandardin-solutionFabRICATORdigestionworkflow(Fig. 3a).TheresultingFcglycosylationprofileswerestableandreproducibleduring14daysofcontinuousoperationwithstandarddeviationsoflessthan0.5%forallglycoforms(Fig. 3b).

Product Overview

Column hardware: PEEK/biocompatibleColumn dimensions:2.1mmDx50mmLSupport resin:POROS®(seeLegaland Disclaimers,p.39)Typical flow rate:0.025-0.05mL/minMaximum Pressure:100bar

Operating pH:6.5-8.0Operating temperature:37°CStorage conditions:+4-8°C(Donotfreeze!)Number of days of continuous operation:>10*Injections per column:>200*Start material:HumanIgG1-4,Fc-fusionproteins

*Depending on specific application

FabRICATO

R®

IgG Proteases

FabRICATOR-HPLCcontainstheFabRICATORenzymeimmobilizedinanHPLCcolumnforfaston-columndigestionofmonoclonalantibodies.

A

B

25600

25400

25000

G0

G0F G1F

G2F

25200

25600

25400

25000

G0

G0F G1F

G2F

25200

Fc/2

MS

spec

trum

G0 G0F G1F G2F0

20

40

% o

f tot

al

FabRICATOR-H

PLCFabRICATO

R

A

B

14

SmartEnzymes

FabRICATOR® Z (IdeZ) is a cysteine protease that digests mouse IgG2a and IgG3 at a specific site below the hinge.

FabRICATORZdigestsmouseIgG2aandIgG3andgeneratesahomogenouspoolofF(ab’)2andFc/2fragments.SomemouseIgG2athatFabRICATORfailstodigest,arereadilydigestedbyFabRICATORZ,butlongerincubationtimesmayberequired.Thereisnoriskofoverdigestionbecauseofthehighspecificityoftheenzyme.

MouseIgG2aandIgG3,humanIgG1-4,IgGofsomeclassesfrommonkey,rabbitandsheep

2hreaction


CPAPNLLG/GPSVF(belowthehinge)Digestion of Mouse IgG2a using FabRICATOR Z


A0-FRZ-020 FabRICATOR Z, 2000 units 2 mg IgG 430 599

TheFabRICATORZenzymeconsistsof2000unitsfordigestionof2mgmouseIgG2aandIgG3.Theen-zymeisprovidedasalyophilizedpowder.

2h

...CPAPNLLG / GPSVF....

Figure 1. Digestion of mouse IgG2a using FabRICATOR Z and FabRICATOR. F(ab’)2 is de-tected at approximately 110 kDa and Fc fragments at approximately 30 kDa. The enzymes are detected at approximately 37 kDa.

Lane 1-3: Mouse IgG2a digested with three different concentrations of FabRICATOR Z (IdeZ)

Lane 4-6: Mouse IgG2a digested with three different concentrations of FabRICATOR (IdeS)

Lane 7: Non-digested mouse IgG2a

ThreedifferentconcentrationsofFabRICATORZ(IdeZ)andFabRICATOR(IdeS)wereusedtodigestmouseIgG2a(Fig. 1).After2hoursofincubation,FabRICATORZ(IdeZ)readilydigestsmouseIgG2a,whereasFabRICATORonlydigestsasmallamountoftheantibody.

FabR

ICAT

OR®

Z

15

TheFabRICATORZenzymeisimmobilizedonagarosebeads,andthespincolumnsareprovidedwithimmobilizedenzymefordigestionof0.5mgmouseIgG2aorIgG3.FragITZgeneratesF(ab’)2andFc/2fragmentswithnoenzymeinthefinalpreparation.

90MINUTES

60MINUTES


A2-FZ6-005 FragIT Z kit 0.5 mg IgG 360 499

A2-FZ6-025 FragIT Z kit 5 x 0.5 mg IgG 975 1,350


A0-FZ6-010 FragIT Z Mircospin 2 x 0.5 mg IgG 320 445

A0-FZ6-025 FragIT Z Mircospin 5 x 0.5 mg IgG 730 1,010

A0-FZ6-050 FragIT Z Mircospin 10 x 0.5 mg IgG 1,210 1,685

*ThermoScientific™CaptureSelect™resinfromThermoFisherScientificInc.anditssubsidiaries.ThermoScientificandCaptureSelectaretrademarksofThermoScientificInc.anditssubsidiaries.

FragIT™ Z (immobilized FabRICATOR® Z) digests mouse IgG2a and IgG3 and generates F(ab’)2 and Fc/2 fragments.

FragIT™ Z kit easily generates and purifies F(ab’)2 and Fc/2 fragments from mouse IgG2a and IgG3.

FragITZkitconsistsofspincolumnsofFragITZforantibodydigestionandspincolumnsofCaptureSelect™*FcresinforaffinitybindingoftheFcfragments.Afterdigestion,theFcfragmentsarecapturedintheaffinityspincolumnandpureF(ab’)2fragmentsareobtainedintheflowthrough.

IgG Proteases

FabRICATO

R®Z

16

SmartEnzymes

FabALACTICA® (IgdE) digests human IgG1 at a specific site above the hinge without the need for reducing conditions.

FabALACTICAisacysteineproteasethatspecificallydigestshumanIgG1abovethehingewithouttheneedforreducingconditionsorco-factors.FabALACTICAisusedtogenerateintactandhomogenousFabandFcfragmentsfromhumanIgG1.TheuseofproteaseswithhighspecificityforIgGhasallowedforsubunitprofilingofantibody-basedtherapeuticsandstudiesofkeyqualityattributesusingLC-MS.TheFabALACTICAenzymecanbeusedtocharacterizeintactandpairedFcglycosylation,bi-ormultispecificantibodies,monovalentbinding,higherorderstructures,disulphidescrambling(Faid,2017,p.38),andforsubunitworkflowsonantibodieswithmutatedhingeregions.

HumanIgG1

Overnight(O/N)reaction


KSCDKT/HTCPPCP(abovethehinge)

...KSCDKT / HTCPPCP…

Enzyme FabALACTICA GingisKHAN Papain Lys-C

Digestion site

Specificity IgG specific/ One digestion site

One digestion siteUnspecific/

Several digestion sites

Unspecific/ Several digestion

sites

Selectivity Human IgG1 Human IgG1 Several proteins Several proteins

Reducing conditions No Yes, 2mM cysteine Yes No

Reaction time O/N (16-18 h) 1 h 1-24 h 1-24 h

FabA

LACT

ICA®

17

Intact Fab and Fc Fragments from Therapeutic mAbs using FabALACTICA

Paired Glycan and Intact Fab Analysis using FabALACTICA and LC-MS

Figure 1. Digestion of cetuximab, trastuzumab, and adalimumab using FabALACTICA O/N at 37°C. a) Non-reduced SDS-PAGE. b) Separation of intact Fc and Fab fragments on RP-HPLC.

Figure 2. Trastuzumab was digested using FabALACTICA O/N at 37°C and intact Fc and Fab fragments were studied using LC-MS. a) Paired glycan analysis of trastuzumab Fc fragments. b) LC-MS of the intact Fab fragment of trastuzumab.

IgG

FabALACTICA

Fc

Fab

Ladder Adalimumab Trastuzumab Cetuximab

+MS, 9.51-9.61min, Smoothed (0.20,1,SG), Baseline subtracted(0.80), Deconvoluted (MaxEnt, 977.21-2379.18, *1.66667, 3000)

0.0

0.2

0.4

0.6

0.8

4x10Intens.

52600 52800 53000 53200 53400 53600 53800 m/z

Hi

CH

2C

H3

CH

3C

H2

Hi

Hi

CH

2C

H3

CH

3C

H2

Hi

Hi

CH

2C

H3

CH

3C

H2

Hi

Hi

CH

2C

H3

CH

3C

H2

Hi

Hi

CH

2C

H3

CH

3C

H2

Hi

Hi

CH

2C

H3

CH

3C

H2

Hi

Assignment Theoretical (Da) Experimental (Da)

Fc G0 / G0F

Fc G0F / G0F

Fc G0F / G1F

Fc G1F / G1F

Fc G1F / G2F

Fc G2F / G2F

52946.8

53092.9

53255.1

53417.2

53579.4

53741.5

52949.3

53093.7

53255.2

53417.2

53580.7

53741.7

+MS, 9.51-9.61min #571-577, Smoothed (0.20,1,SG), Baseline subtracted(0.80)

0

50

100

150

1000 1200 1400 1600 1800 2000 2200 m/z

1406.8

+MS, 9.73-9.91min, Smoothed (0.20,1,SG), Baseline subtracted(0.80), Deconvoluted (MaxEnt, 999.02-2537.68, *1.66667, 3000)

0

1

2

3

4

5

5x10Intens.

47350 47400 47450 47500 47550 47600 47650 47700 m/z

Assignment Theoretical (Da) Experimental (Da)

Fab 47499.5 47499.6VH

CH1

CL

VL

min12.5 15 17.5 20 22.5 25 27.5 30

mAU

0

50

100

150

200

min12.5 15 17.5 20 22.5 25 27.5 30

mAU

0

50

100

150

200

min12.5 15 17.5 20 22.5 25 27.5 30

mAU

0

50

100

150

200

250

Cetuximab

Trastuzumab

Adalimumab

Fc

Fc

Fc

Fab

Fab

Fab

18.870

18.833

18.980

22.874

20.646

22.196

A

A B

B

TheFabALACTICAenzymeconsistsof2000unitsfordigestionof2mghumanIgG1.Theenzymeisprovi-dedasalyophilizedpowder.


A0-AG1-020 FabALACTICA, 2000 units 2 mg hIgG1 510 625

FabALACTICAwasusedtodigestthethreetherapeuticmAbscetuximab,trastuzumabandadalimumab.Fig. 1showsthatFabALACTICA

generatesintactFabandFcfragmentsfromallthreeantibodies.

TheintactFcfragmentof~53kDaenablescharacterizationofthetwoconservedFc-glycosylationsitessimultaneouslywithhighmassaccuracy(Fig. 2a).AfterFabALACTICAdigestion,themassoftheintact

Fabfragmentcanbeanalyzedtostudymodificationsandlightandheavychainpairingforbispecificantibodies,orusedforcomparabilityassessment (Fig. 2b).

IgG Proteases

FabALACTICA®

18

SmartEnzymes

Immobilized FabALACTICA® digests human IgG1 and generates intact Fab and Fc fragments.

ImmobilizedFabALACTICAspincolumnsareprovidedfordigestionof0.5mgupto100mgofhumanIgG1antibody.


A0-AG6-010 Immobilized FabALACTICA Microspin 2 x 0.5 mg hIgG1 350 490

A0-AG6-050 Immobilized FabALACTICA Microspin 10 x 0.5 mg hIgG1 1,250 1,750

A0-AG6-100 Immobilized FabALACTICA Midispin 1-10 mg hIgG1 1,050 1,450

A0-AG6-1000 Immobilized FabALACTICA Maxispin 10-100 mg hIgG1 3,150 4,400

Load & Incubate

Spin & Collect

FabALACTICA®

Sample Preparation Workflow

Figure 1. The FabALACTICA sample preparation workflow.

TheFabALACTICAenzymeisimmobilizedonagarosebeads,andthespincolumnsareprovidedwith immobilizedenzymefordigestionof0.5mgupto100mgofantibody.ImmobilizedFabALACTICAgeneratesantibodysubunitswithnoenzymeinthefinalpreparation.

TheFabALACTICAenzymecanbeusedtogeneratesubunitsofhumanIgG1.TheantibodyisdigestedusingtheImmobilizedFabALACTICAenzymeatroomtemperatureovernighttogenerateintactFabandFcfragments.

FabA

LACT

ICA®

19

FabALACTICA® Fab kit easily generates and purifies intact Fab fragments from human IgG1.

TheFabALACTICAFabkitconsistsofspincolumnsofImmobilizedFabALACTICAforantibodydigestion,andspincolumnsofCaptureSelect™*FcresinforaffinitybindingoftheFcfragments.Afterdigestion,theFcfragmentsarecapturedintheaffinityspincolumnandintact,pureFabfragmentsareobtainedintheflowthrough.

TheFabALACTICAFabkitconsistsofImmobilizedFabALACTICAspincolumnsandCaptureSelect™*FcaffinityspincolumnsforeasygenerationandpurificationofFabfragmentsfromhumanIgG1antibodies.

Product ID Description Digestion & Purification EUR USD

A2-AFK-005 FabALACTICA Fab kit 0.5 mg hIgG1 395 550

A2-AFK-025 FabALACTICA Fab kit 5 x 0.5 mg hIgG1 1,050 1,450

A2-AFK-100 FabALACTICA Fab kit 10 mg hIgG1 1,295 1,800

A2-AFK-1000 FabALACTICA Fab kit 100 mg hIgG1 3,950 5,550

Load & Incubate

Spin & Collect

Spin & Collect

kDa

40

50

60

80110

160

260

Adalimumab

Undigested

DigestedPurifie

d Fab

30

20

kDa

Trastuzumab

Undigested

DigestedPurifie

d Fab

40

50

60

80110

160

260

30

20

kDa

Cetuximab

Undigested

DigestedPurifie

d Fab

40

50

60

80110

160

260

30

20

Preparation of Pure FabsThreetherapeuticmAbswereincubatedwithImmobilizedFabALACTICAinspincolumns.TheFcfragmentswerecapturedintheCaptureSelect™*FcspincolumnsandtheFabscouldeasilybeelutedbycentrifugation(Fig. 2).TheresultingFabpreparationishomogenousandpure.

Figure 2. Adalimumab, trastuzumab and cetuximab digested by Immobilized FabALACTICA. Pure Fab fragments were obtained in a high yield from all three mAbs using the FabALACTICA Fab kit.


IgG Proteases

FabALACTICA®

20

SmartEnzymes

HumanIgG1

60minreaction

2mMcysteine(included)

KSCDK/THTCPPCP(abovethehinge)

GingisKHAN® (Kgp) is a cysteine protease that digests human IgG1 at a specific site above the hinge.

DigestionofhumanIgG1usingGingisKHANgener-atesahomogenouspoolofintactFabandFcfrag-ments.Mildreducingreactionconditions,2mMcys-teine,arerequiredandready-to-usereducingagentisprovidedtogetherwiththeenzyme.GingisKHANisusedtocharacterizeantibody-basedbiotherapeu-ticsusingLC-MS,andtostudyFcglycananalysis,bispecificantibodies,affinityandavidityeffectsandgeneralPTMidentification.TheactivityonIgG1hingeregionsallowsdigestionofbothmonoclonalantibodies(trastuzumabandadalimumab)aswellasFc-fusionproteins(etanercept)carryinganIgG1hingeregion(Fig. 1).Onotherantibodysubclasses,additionaldigestionsatexposedlysinesmayoccur.PublicationsusingGingisKHANtostudybispecificantibodiesarelistedonp.38.

...KSCDK / THTCPPCP....

60MINUTE

60MINUTES

2000unitsofGingisKHANisprovidedasalyophilizedpowdertogetherwith5vialsoflyophilizedGingisKHANreducingagentfordigestionof2mghumanIgG1.


B0-GKH-020 GingisKHAN, 2000 units 2 mg hIgG1 430 599

min10 20 30 40 50

mAU

0

20

40

60

80

100

24.630

min10 20 30 40 50

mAU

0

20

40

60

80

100

24.740

27.214

min10 20 30 40 50

mAU

0

20

40

60

80

100

16.025

24.700

Trastuzumab

Adalimumab

Etanercept

Fc

Fab

Fab

Fc

Fc

TNFR

Figure 1. GingisKHAN digestion of trastuzumab, adalimumab and etanercept.

Ging

isKH

AN®

21

CL

VLVH

CH1

Hi

CH2

CH3

CH3

CH2

Hi

VH

CH1

VL

CL

VH

CH1CL

VLVH

CH1

CL

VL

Hi

CH2

CH3

CH3

CH2

Hi

Hi

CH2

CH3

CH3

CH2

Hi

VH

CH1CL

VLVH

CH1

CL

VL

A

B

C

D

GingisKHAN® Fab kit generates and purifies Fab fragments from human IgG1.

Figure 3. SDS-PAGE analysis of purified Fab fragments from trastuzumab using GingisKHAN Fab kit. Lane 1 and 6: MW marker Lane 2: Intact human IgG1 Lane 3: Fab and Fc fragments after GingisKHAN digestion Lane 4: Flowthrough Fc fragments Lane 5: Eluted Fab fragments


GingisKHANFabkitconsistsof2000unitsoftheGingisKHANenzyme,5xlyophilizedreducingagentand 4xCaptureSelect™*CH1affinityspincolumnsforgenerationandpurificationofFabfragmentsfromhumanIgG1antibodies.


B0-GFK-020 GingisKHAN Fab kit, 2000 units 2 mg hIgG1 975 1,045

TheGingisKHANFabkitconsistsoflyophilizedGingisKHANenzymeforantibodydigestion,andspincolumnsofCaptureSelect™*CH1resinforaffinity

Figure 2. Digestion and purification of Fab fragments using GingisKHAN Fab kit. a) Intact antibody (human IgG1). b) Analysis of antibody fragments after GingisKHAN digestion. c) The flowthrough Fc. d) Elution of the purified Fab fragments.

bindingoftheFabCH1domains.Afterdigestion,theFabfragmentsarecapturedintheaffinityspincolumnandcaneasilybeeluted.

60MINUTE

60MINUTES

IgG Proteases

GingisKHAN

®

22

SmartEnzymes

FabULOUS® (SpeB) is a cysteine protease that digests in the hinge region of IgG from several different species and subclasses.

FabULOUSdigestsIgGandgeneratesFabandFcfragments.TheprimarydigestionsiteonhumanIgG1isbetweentheaminoacidsT225andC226.TheFabULOUSenzymewillalsodigestIgGfrommouse,rat,goat,sheepandrabbitandcanbeusedtogenerateintactFabfragmentsfrommouseIgG1,forinstance.Theenzymerequiresreducingconditionsforoptimalactivity,andifstrongerreducingconditionsareused,itislikelythatinterchainthiolswillbereduced.PublicationsusingFabULOUSarelistedonp.38.

Human IgG1-4, IgG from mouse, rat, goat, sheep and rabbit.

60 min reaction

Requires reducing conditions (1-5 mM DTT, 1-5 mM TCEP, 50-100 mM cysteine, 50-100 mM cysteamine, 50-100 mM 2-mercaptoethanol (not included))

Human IgG1: KTHT / CPPCPAP (above the hinge)

60MIN

60MINUTE

60MINUTES

30MIN

...KTHT / CPPCPAPELLG....

TheFabULOUSenzymeconsistsof2000unitsfordigestionof2mgIgG.Theenzymeisprovidedasalyophilizedpowder.


A0-PU1-020 FabULOUS, 2000 units 2 mg IgG 430 599

1 MW marker

2 mouse IgG1 FabULOUS

3 mouse IgG1

4 mouse IgG2a FabULOUS

5 mouse IgG2a

1 MW marker

2 hIgG1 FabULOUS

3 hIgG1

4 hIgG2 FabULOUS

5 hIgG2

Human IgG

260

1601108060

5040

30

20

15

10

1 2 3 4 5

hIgG1 hIgG2

Mouse IgG

260

1601108060

5040

30

20

15

10

1 2 3 4 5

mIgG1 mIgG2a

Figure 1. FabULOUS digestion of human (IgG1 and IgG2) and mouse (IgG1 and IgG2a) antibodies.

FabU

LOUS

®

23

FabULOUS® Fab kit generates and purifies Fab fragments from mouse IgG.

TheFabULOUSFabkitisdesignedtogenerateandpurifyFabfragmentsfrommouseIgG.TheFabULOUSFabkitconsistsoflyophilizedFabULOUSenzymeforantibodydigestionandCaptureSelect™*LC-Kappa(mur)affinityspincolumnsforeasypurificationofthepreparedFabfragmentsfrommouseIgG.CaptureSelect™*LC-Lambda(mouse)affinitycolumnsareavailableuponrequest.Theantibodyisdigestedwithin60minutesusingthe

*ThermoScientific™CaptureSelect™resinfromThermoFisherScientificInc.anditssubsidiaries.ThermoFisherandCaptureSelectaretrademarksofThermoFisherScientificInc.anditssubsidiaries.

FabULOUSFabkitconsistsof2000unitsoftheFabULOUSenzymeand4xCaptureSelect™*LC-kappa(mur)affinityspincolumnsforgenerationandpurificationofFabfragmentsfrommouseIgG.


A1-PFK-020 FabULOUS Fab kit mouse, 2000 units 2 mg mIgG 730 790

Figure 4. Non-reducing SDS-PAGE analysis of purified Fab fragments from monoclonal mIgG1 using FabULOUS Fab kit. Lane 1: Intact mIgG1 Lane 2: Digested mIgG1 Lane 3: Flowthrough from CaptureSelect™* column Lane 4: Eluted Fab fragmentsLane 5: MW marker

Figure 2. a) Intact monoclonal mouse IgG1 antibody. b) Analysis of the fragments after FabULOUS Fab kit digestion. c) Eluted Fab fragments from the CaptureSelect™* LC-Kappa (mur) column.

min20 22 24 26 28 30 32 34

mAU

0

200

400

600

800

1000

1200

min20 22 24 26 28 30 32 34

mAU

0

200

400

600

800

1000

min20 22 24 26 28 30 32 34

mAU

0

200

400

600

800

1000

1200

CL

VLVH

CH1

Hi

CH2

CH3

CH3

CH2

Hi

VH

CH1

VL

CL

VH

CH1CL

VLVH

CH1

CL

VL

Hi

CH2

CH3

CH3

CH2

Hi

VH

CH1CL

VLVH

CH1

CL

VL

lyophilizedFabULOUSenzyme,andthepreparedFabfragmentsbindtotheCaptureSelect™*affinityspincolumnsandareeasilyeluted(Fig. 3).ThepreparedFabfragmentsfrommouseIgG1aredemonstratedinFig. 2 and 4andcanbeusedinaffinitystudies,studiesofFabglycosylationandstructuralstudies.

60MINUTES

Figure 3. Schematic overview of the generation and purification of Fab fragments from mouse IgG using FabULOUS Fab kit.

A

B

C

IgG Proteases

FabULO

US®

24

SmartEnzymes

60MINUTES

GingisREX® (Rgp) is a protease that digests proteins C-terminally of arginine residues.

GingisREXspecificallydigestspeptidesandproteinsC-terminallyofarginineresidues.Theproteasedoesnothaveactivityatlysines,ascommonlyobservedusingArg-C(Fig. 1 andTable1).TheenzymaticactivityofGingisREXincludesdigestionofArg-Prolinkagesthataredifficulttodigestwithotherenzymes.TheenzymeisactiveatabroadpHrangeof5.5-9.0,butisinhibitedbyguanidinehydrochloride.

Digestsanypeptideorproteincontain-ingarginine.SpecificforArg-Xmotifs

60minreaction

Activein6Mureaand0.1%SDS

C-terminallyofarginineresidues

Unique Specificity for Arginine Residues

Figure 1. Oxidized insulin β-chain digested O/N at 37°C with GingisREX or Arg-C. The resulting sequences are presented in Table 1.

Table 1. Sequences of oxidized insulin β-chain digested by GingisREX or Arg-C, as indicated in Fig. 1. Green color indicates arginine residues and red color indicates lysine residues.

Peptide No. Amino Acid SequenceIntact protein FVNQHLCGSHLVEALYLVCGERGFFYTPKA

1 GFFYTPKA

2 FVNQHLCGSHLVEALYLVCGER

3 GFFYTPK

4 FVNQHLCGSH

5 LVEALYLVCGER + Na

Ging

isR

EX®

25

Peptide Mapping of Trastuzumab

Figure 2. Peptide maps of trastuzumab after digestion using GingisREX or Arg-C.

GingisREXisanarginine-specificproteasethatdigestsproteinsC-terminallyofarginineresidues.Theenzymeisprovidedasalyophilizedpowderinvialsof5μgenzyme.

Product ID Description Enzyme:Protein ratio EUR USD

B0-GRX-005 GingisREX, 5 μg enzyme 1:20 - 1:200 470 520

TheGingisREXenzymecanbeusedtoanalyzeproteinsbymassspectrometryfortheuseinpeptidemapping,proteinfingerprintingandsequenceanalysis.Itgenerateslargerpeptideswithmorechargeperpeptide,whichisbeneficialformass

spectrometricanalyses.Usingthisworkflow,themass-to-chargeoflongerpeptidescanberesolved,resultinginincreasedsequencecoverageandidentificationofparticularpost-translationalmodifications.

Onalargeandcomplexsample,suchasatherapeuticantibody,thedigestionatarginineresiduesgiveslargerpeptidesandresultsinfewerpeaksandalesscomplicatedpeptidemap.Thisisbeneficialfore.g.datainterpretationinmassfingerprintanalyses.Asanexample,theGingisREXandArg-CdigestionprofilesoftrastuzumabarepresentedinFig. 2.

Applications of GingisREX

Proteases

GingisREX

®

26

SmartEnzymes

N-terminus C-terminus

+ OpeRATOR+ SialEXO

OpeRATOR® is an O-glycan-specific protease that digests proteins carrying mucin-type O-glycans N-terminally of Ser and Thr glycosylation sites.

TheOpeRATORenzymeisanoveltoolforanalysisofmucin-typeO-glycansonglycoproteinsandglycopeptides.TheenzymebindstoO-glycansanddigeststheaminoacidbackboneN-terminallyoftheserineandthreonine(SandT)glycosylationsites(Fig. 2).ThisgeneratesglycopeptidescarryingO-glycansandenablesO-glycanprofiling,O-glycopeptidemappingandsiteoccupancydetermination,aswellasmiddle-levelapproachesusingmassspectrometry.TheOpeRATORenzymeisactiveonnativeglycoproteinswithorwithoutthepresenceofsialicacids.However,theactivityishigherwhensialicacidsareremoved.Therefore,SialEXO®(p.30),asialidasemixforefficientandcompleteremovalofsialicacids,isincludedwithOpeRATOR.

Digestsnativemucin-type O-glycosylatedproteins

2htoovernight(16-18h)reaction

DesialylationusingSialEXO®(included)increasesperformance

N-terminallyofO-glycosylatedserineandthreonineresidues

Effect of SialEXO Pretreatment on OpeRATOR Enzymatic Activity

Figure 1. The native O-glycosylated TNF receptor (TNFR) was incu-bated with OpeRATOR with or without the addition of SialEXO, and the reactions were separated on SDS-PAGE.

1. Control TNFR2. + OpeRATOR3. + SialEXO4. + OpeRATOR + SialEXO

1 2 3 4

TNFaR

TNFαR

+

(kDa)

50

40

30

OpeRATORisactiveonsialylatedO-glycoproteins,buttheactivityishigherwhenthesialicacidsareremovedusingSialEXO(Fig. 1).

Figure 2. a) Schematic overview of OpeRATOR activity. O-glycans are required for OpeRATOR activity and the enzyme is not active at N-glycans. b) With OpeRATOR and SialEXO treatment, the O-glycosylated protein is digested into peptides carrying O-glycans. The digestion occurs N-terminally of the O-glycosylation sites.

A

B

Ope

RAT

OR®

27

O-glycan Site-specific Digestion of EPO using OpeRATOR

LC-MS Analysis of Etanercept using OpeRATOR Maps O-glycan Sites

Figure 4. EPO was incubated with SialEXO to remove sialic acids (Lane 1), and with OpeRATOR to digest the protein N-terminally of the O-glycans (Lane 2). EPO was incubated with OglyZOR™ to remove the O-glycan before OpeRATOR incubation (Lane 3). No OpeRATOR activity was observed in the absence of O-glycans.

Figure 3. N-glycans were removed from EPO using PNGaseF and sialic acids were removed using SialEXO. In parallel, OpeRATOR hydro-lyzed the protein N-terminally of the serine O-glycan (core 1) site. After reduction of disulfide bridges with DTT, the resulting two fragments were separated on a RP C4 column and intact mass was analyzed with a Bruker Impact II ESI QTOF MS. a) UV trace and b) QTOF MS.

T S P T R S M A P G A V H L P Q P V S T R S Q H T Q P T P E P S T A P S T S F L L P M G P S P P A E G S T G D E P K S C D K T H T184 186 199 200 202 208 212 216 217 226 243 245

O-glycosylated region

205

MS/

MS

on p

eptid

es

( ) ( )

TheOpeRATORenzymeconsistsof2000unitsfordigestionof2mgO-glycoprotein.Theenzymeis providedtogetherwith2000unitsofSialEXOforoptionalsialicacidremoval.Bothenzymesareprovidedaslyophilizedpowders.


G2-OP1-020 OpeRATOR, 2000 units 2 mg O-glycoprotein 840 940

Figure 5. OpeRATOR digestion of the O-glycosylated hinge region of etanercept. OpeRATOR generates overlapping peptides, making it possible to map the O-glycan sites.

1 2 3

1 2 31 2 3

(kDa)

2015

10

3.5

Erythropoietin(EPO)isa~30kDaglycoproteinwithasingleO-glycosylationsite.TheproteinwasusedasasubstratetodemonstratethespecificactivityofOpeRATOR(Fig. 3 and4).TheO-glycosidaseOglyZOR®(p.26)wasusedtodemonstratetheO-glycan-dependentactivityofOpeRATOR.AscanbeseeninLane3inFig. 4,thereisnoOpeRATORdigestionofEPOwhentheO-glycanhasbeenremoved.

1. EPO + SialEXO2. EPO + SialEXO + OpeRATOR3. EPO + SialEXO + OglyZOR, followed by incubation with OpeRATOR

EtanerceptisanFc-fusionproteinwithahighlyO-glycosylatedhingeregion.EtanerceptwasincubatedwithOpeRATORandtheresultingglycopeptideswereanalyzedusingLC-MS/MS.DuetotheheterogeneityintheO-glycanpatternofthe

25 30 35 40 45 50 55 60 Time [min]

0

10

20

30

Intens.[mAU]

EPO PNGaseF Smix LS 1-20_P1-A-1_01_160.d: UV Chromatogram, 280 nm

SAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDAPPRLICDSRVLERYLLEAKEAEDITTGCAEHCSLDENITVPDTKVDFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAA

OpeRATOR + OglyZER + Pngase F

25 30 35 40 45 50 55 60 Time [min]

0

10

20

30

Intens.[mAU]


SAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGD

APPRLICDSRVLERYLLEAKEAEDITTGCAEHCSLDENITVPDTKVDFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAA


'13763.1111Mr

'18648.6797Mr

2. EPOPNGaseFSmixLS1-20_P1-A-1_01_160.d:+MS,46.3-47.9min,-PeakBkgrnd,Deconvoluted(MaxEnt,659.96-1833.90,0.1,50000)

0.0

0.2

0.4

0.6

0.8

1.0

6x10Intens.

5000 10000 15000 20000 25000 30000 35000 40000 45000 Mass [Da]


Teor:13714.0957DaMeas:13714.1199Da

Undigested,PngaseF treatedEPO:APPRLICDSRVLERYLLEAKEAEDITTGCAEHCSLDENITVPDTKVDFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDTeor:18596.6397DaMeas:18648.6797Da(unexplained+54Damassdifference)

Intens. x106

'13714.1199Mr2. EPOPNGaseFSmixLS1-20_P1-A-1_01_160.d:+MS,46.3-47.9min,-PeakBkgrnd,Deconvoluted(MaxEnt,659.96-1833.90,0.1,50000)

0.0

0.2

0.4

0.6

0.8

1.0

6x10Intens.

-20000 0 20000 40000 60000 80000 Mass [Da]

'13763.1111Mr

'18648.6797Mr


0.0

0.2

0.4

0.6

0.8

1.0

6x10Intens.

5000 10000 15000 20000 25000 30000 35000 40000 45000 Mass [Da]

'4738.5187Mr


0.0

0.2

0.4

0.6

0.8

7x10Intens.

3750 4000 4250 4500 4750 5000 5250 5500 5750 6000 Mass [Da]

'4738.5187Mr

'4900.5868Mr1.EPOPNGaseFSmixLS1-20_P1-A-1_01_160.d:+MS,36.7-37.5min,-PeakBkgrnd,Deconvoluted(MaxEnt,577.17-1554.57,0.1,50000)

0

2

4

6

8

6x10Intens.

3750 4000 4250 4500 4750 5000 5250 5500 5750 Mass [Da]

SAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDTeor:4900.5546DaMeas.:4900.5868Da

SAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDTeor:4738.5018Meas.:4738.5187Da

Intens. x106

'13763.1111Mr

'18648.6797Mr


0.0

0.2

0.4

0.6

0.8

1.0

6x10Intens.

5000 10000 15000 20000 25000 30000 35000 40000 45000 Mass [Da]


Teor:13714.0957DaMeas:13714.1199Da


Intens. x106


0.0

0.2

0.4

0.6

0.8

1.0

6x10Intens.

-20000 0 20000 40000 60000 80000 Mass [Da]

'13763.1111Mr

'18648.6797Mr


0.0

0.2

0.4

0.6

0.8

1.0

6x10Intens.

5000 10000 15000 20000 25000 30000 35000 40000 45000 Mass [Da]

'4738.5187Mr


0.0

0.2

0.4

0.6

0.8

7x10Intens.

3750 4000 4250 4500 4750 5000 5250 5500 5750 6000 Mass [Da]

'4738.5187Mr


0

2

4

6

8

6x10Intens.

3750 4000 4250 4500 4750 5000 5250 5500 5750 Mass [Da]


Teor:13714.0957DaMeas:13714.1199Da


Intens. x106

SAAPLR….ACRTGDAPPRLI…..SPPDAA

SAAPLR….ACRTGD

SAAPLR….ACRTGD

APPRLI…..SPPDAA

APPRLI…..SPPDAASAAPLR…..ACRTGD

Theor: 4900.5546 DaMeas: 4900.5868 Da

Theor: 4738.5018 DaMeas: 4738.5187 Da

Theor: 13714.0957 DaMeas: 13714.1199 Da

Undigested EPO

A

B

125

126

126

126

1251

1

1651165

165

25 30 35 40 45 50 55 60 Time [min]

0

10

20

30

Intens.[mAU]


SAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDAPPRLICDSRVLERYLLEAKEAEDITTGCAEHCSLDENITVPDTKVDFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAA


25 30 35 40 45 50 55 60 Time [min]

0

10

20

30

Intens.[mAU]


SAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGD



'13763.1111Mr

'18648.6797Mr


0.0

0.2

0.4

0.6

0.8

1.0

6x10Intens.

5000 10000 15000 20000 25000 30000 35000 40000 45000 Mass [Da]


Teor:13714.0957DaMeas:13714.1199Da


Intens. x106


0.0

0.2

0.4

0.6

0.8

1.0

6x10Intens.

-20000 0 20000 40000 60000 80000 Mass [Da]

'13763.1111Mr

'18648.6797Mr


0.0

0.2

0.4

0.6

0.8

1.0

6x10Intens.

5000 10000 15000 20000 25000 30000 35000 40000 45000 Mass [Da]

'4738.5187Mr


0.0

0.2

0.4

0.6

0.8

7x10Intens.

3750 4000 4250 4500 4750 5000 5250 5500 5750 6000 Mass [Da]

'4738.5187Mr


0

2

4

6

8

6x10Intens.

3750 4000 4250 4500 4750 5000 5250 5500 5750 Mass [Da]

SAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDTeor:4900.5546DaMeas.:4900.5868Da

SAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDTeor:4738.5018Meas.:4738.5187Da

Intens. x106

'13763.1111Mr

'18648.6797Mr


0.0

0.2

0.4

0.6

0.8

1.0

6x10Intens.

5000 10000 15000 20000 25000 30000 35000 40000 45000 Mass [Da]


Teor:13714.0957DaMeas:13714.1199Da


Intens. x106


0.0

0.2

0.4

0.6

0.8

1.0

6x10Intens.

-20000 0 20000 40000 60000 80000 Mass [Da]

'13763.1111Mr

'18648.6797Mr


0.0

0.2

0.4

0.6

0.8

1.0

6x10Intens.

5000 10000 15000 20000 25000 30000 35000 40000 45000 Mass [Da]

'4738.5187Mr


0.0

0.2

0.4

0.6

0.8

7x10Intens.

3750 4000 4250 4500 4750 5000 5250 5500 5750 6000 Mass [Da]

'4738.5187Mr


0

2

4

6

8

6x10Intens.

3750 4000 4250 4500 4750 5000 5250 5500 5750 Mass [Da]


Teor:13714.0957DaMeas:13714.1199Da


Intens. x106

SAAPLR….ACRTGDAPPRLI…..SPPDAA

SAAPLR….ACRTGD

SAAPLR….ACRTGD

APPRLI…..SPPDAA

APPRLI…..SPPDAASAAPLR…..ACRTGD

Theor: 4900.5546 DaMeas: 4900.5868 Da

Theor: 4738.5018 DaMeas: 4738.5187 Da

Theor: 13714.0957 DaMeas: 13714.1199 Da

Undigested EPO

A

B

125

126

126

126

1251

1

1651165

165

A B

proteinandtheOpeRATORspecificityforO-glycanstructures,overlappingpeptideswereformed,makingitpossibletoacquireacompletemapoftheO-glycansites(Fig. 5).

Enzymes for O-glycans

OpeR

ATOR

®

28

SmartEnzymes

0.0

0.5

1.0

1.5

2.08x10

Intens.

0.0

0.5

1.0

1.5

8x10Intens.

0.0

0.5

1.0

1.5

10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 Time [min]

8x10Intens.

Load

Flowthrough

Eluate

Glycodrosocin H2686H4062 IOB

SialEXO

H8390

TiC

GlycOCATCH® is an enrichment resin for affinity purification of mucin-type O-glycosylated proteins and peptides. GlycOCATCHisdesignedtospecificallybindmucin-typeO-glycosylatedproteinsandpeptides.TheaffinityresinisbasedoninactiveOpeRATOR® enzyme(p.26)thathasbeenengineeredtobindO-glycosylatedproteinsandpeptideswithhighaffinity.GlycOCATCHisprovidedinaspincolumnformattoalloweasy-to-useenrichmentofO-glycoproteins.DuetothestronginteractionbetweentheGlycOCATCHresinandO-glycoproteins/peptides,theelutionisperformedwith8Murea.Alternatively,theelutioncanbeperformedwiththeincludedOpeRATORenzyme.Foroptimalperformance,thesialicacidsoftheglycoproteinwillneedtoberemovedusingtheincludedSialEXO®sialidasemix(p.30).TheapplicationsofGlycOCATCHincludespecificenrichmentorremovalofO-glycoproteinsandpeptides,glycomics,studiesofcomplexsamplesandcharacterizationofbiopharmaceuticals.InFig. 1,theO-glycanspecificityofGlycOCATCHwasstudiedonthepeptidelevel.Aselectionofnon-glycosylatedandO-glycosylatedpeptideswasused.ThepeptideswereloadedontoGlycOCATCH,theresinwaswashedandtheboundpeptideswereelutedwith8Murea.Afterpurification,thesamples

Bindsmucin-typeO-glycosylated proteinsandpeptides

30min-2hbinding

DesialylationusingSialEXO®(included)increasesbinding

Glycoproteinsandpeptidescarrying mucin-typeO-glycans

Figure 1. Selective purification of an O-glycosylated peptide (glycodrosocin) carrying a core 1 O-glycan using GlycOCATCH.

Glyc

OCA

TCH

®

wereseparatedandanalyzedonRP-LC-MS.Thedatashowsselectivepurificationoftheglycodrosocinpeptide,carryingacore1O-glycan(Fig. 1).

TheGlycOCATCHboxcontains4microspincolumnsofGlycOCATCHaffinityresin,200unitsofSialEXOand200unitsofOpeRATORforenrichmentofupto200μgO-glycoprotein.

Product ID Description Enrichment EUR USD

G3-OC6-002 GlycOCATCH 200 μg O-glycoprotein 595 695

29


OglyZOR® is an O-glycosidase that specifically hydrolyzes core 1 type O-glycans on native glycoproteins.

OglyZORisanO-glycosidasethatcatalyzestheremovalofcore1andtosomeextentcore3typeO-linkeddisaccharidesfromnativeglycoproteins.Theenzymedoesusuallynotrequiredenaturationofthesubstrate.ThesialicacidsoftheO-glycansneedtoberemovedforOglyZORactivity.Therefore,theOglyZORenzymeisprovidedtogetherwiththesialidaseSialEXO®(p.30).OglyZORisusedforremovalofO-glycansforglycananalysis,confirmationofO-glycanpresenceandreductionofsampleheterogeneity.

HydrolyzesO-glycansonglycoproteins

2-4hreaction

Requiresremovalofsialicacidsusing SialEXO®(included)

Core1typeO-glycandisaccharides

OglyZOR and SialEXO Compared to Other Enzymes

TheOglyZORenzymeconsistsof2000unitsforhydrolysisofO-glycanson2mgglycoprotein.Theenzymeisprovidedtogetherwith2000unitsofSialEXOforsialicacidremoval.Bothenzymesareprovidedas lyophilizedpowders.

Product ID Description Deglycosylation EUR USD

G2-OG1-020 OglyZOR, 2000 units 2 mg glycoprotein 730 835

Figure 1. Comparison of the enzymatic activities of OglyZOR and SialEXO to commercially available endoglycosidases and sialidases. All incubations (4 h) were performed according to the manufacturers instructions, and the samples were all separated on SDS-PAGE.

1. TNF receptor

2. + SialEXO and OglyZOR

3. + Endoglycosidase (E. faecalis) and sialidase (C. perfringens)

4. Etanercept


1 2 3 4 5 6 7 8 91 2 3 4 5 6 7 8 9(kDa)

160

11080

60

50

40


7. Fetuin



InFig. 1,theenzymaticactivitiesofOglyZORandSialEXOarecomparedtoothercommerciallyavailableendoglycosidasesandsialidases.

OglyZO

R®

30

SmartEnzymes

Sial

EXO

®

SialEXO® is a sialidase mix for complete removal of sialic acids from native glycoproteins.SialEXOisusedforremovalofsialicacids onnativeglycoproteins,anditworksonboth O-andN-linkedglycans.Itisacombinationoftwosialidasesactingonα2-3,α2-6andα2-8 linkages(Fig. 1).SialEXOcanbeusedtopretreatanO-glycosylatedproteinpriortodigestionwithOpeRATOR®(p.26),orpriortodeglycosylationwithOglyZOR®(p.29).ByusingSialEXOincombination withtheabove-mentionedenzymes,theactivitiesoftheenzymesareenhanced.ApplicationsofSialEXOincluderemovalofallsialicacidsanditcanalsobeusedinexoglycosidasearrays.

ByquantifyingliberatedsialicacidsafterSialEXOtreatmentofthesyntheticsubstrates3’-sialyllactose(α2-3bonds),6’-sialyllactose(α2-6bonds)andcolominicacid(α2-8bonds),thespecificityofSialEXOwasdetermined.AscanbeseeninFig. 1,SialEXOisactiveonallsialicacidlinkages.

HydrolyzessialicacidsonN-andO-linkedglycans

2hreaction

Requiresnoco-factorsα2-3,α2-6andα2-8-linkedsialicacids

TheSialEXOmixconsistsof2000unitsforhydrolysisofsialicacidson2mgglycoprotein.Themixis providedasalyophilizedpowder.

Product ID Description Desialylation EUR USD

G1-SM1-020 SialEXO, 2000 units 2 mg glycoprotein 520 625

Figure 1. SialEXO activity on sialic acid linkages from the substrates 3’-sialyllactose (a2-3 bonds), 6’-sialyllactose (a2-6 bonds), and colominic acid (a2-8 bonds).

!

"!!!!

#!!!!

$!!!!

%!!!!

&!!!!

'!!!!

! "#$ ! "%$ ! "&$

()*+),*-./-0.1)2)1)34

5.+*

3)6.

/7+8

9:.-1

.;1.

<;3.

;-)34

Sialidase Specificity

Rel

ativ

e Fl

uore

scen

ce In

tens

ity

60000

50000

40000

30000

20000

10000

02-3 2-6 2-8

Species Akkermansia muciniphila Arthrobacter ureafaciens Arthrobacter ureafaciens

Reaction time 2 h 1 h or O/N 1 h or more if branched

pH range (optimal) 6.5 to 9.0 (6.8) (6.0) 4.5 to 8.0 (6.0)

Molecular weight (kDa) 43 + 66 100 51 - 88

Other Sialidase 1 Other Sialidase 2

Table 1. Summary of key attributes for general α2-3, 6, 8 sialidases, including SialEXO.

31

SialEXO®


SialEXO®23 is an α2-3-specific sialidase, allowing targeted analysis of α2-3 linked sialic acids.IncontrasttoSialEXOwhichisasialidasemix,SialEXO23isanα2-3specificsialidase,allowingtargetedanalysisofα2-3linkedsialicacidsonly.Efficientα2-3desialylationofO-andN-glycosylatedproteinscanbeachievedwithinonehour.Hydrophilicinteractionliquidchromatography(HILIC)wasusedtoseparatetwoglycanlibraries;onecontainingreleasedglycanswithα2-3sialicacidsandtheotherglycansmodifiedwithα2-6sialicacids.TreatmentwithSialEXOandSialEXO23canbecompared(Fig. 2).Thedataclearlyshowstheshiftofthepeaksastheα2-3butnottheα2-6linkedsialicacidsarereleasedbySialEXO23.Incontrast,SialEXOreleasesallsialicacidsirrespectiveoflinkage.

HydrolyzessialicacidsonN-andO-linkedglycans

60minreaction

Requiresnoco-factorsα2-3-linkedsialicacids

TheSialEXO23enzymeconsistsof500unitsforhydrolysisofsialicacidson0.5mgglycoprotein. Theenzymeisprovidedasalyophilizedpowder.

Product ID Description Desialylation EUR USD

G1-SD2-005 SialEXO 23, 500 units 0.5 mg glycoprotein 450 510

α(2-3) sialylated biantennary N-glycans

5 10 15 20 25Retention time (min)

SialEXO 23

SialEXO

α(2-6) sialylated biantennary N-glycans

undigested

5 10 15 20Retention time (min)

Figure 2. HILIC analysis of released glycans with α2-3 and α2-3/α2-6 sialic acids released by SialEXO 23 and SialEXO respectively. Sialylated glycan structures are shaded in purple.

Table 2. Summary of key attributes for specific α2-3 sialidases, including SialEXO 23.

SpeciesAkkermansia muciniphila

Streptococcus pneumoniae

Streptococcus pneumoniae

Reaction time 1 h 1 h 1 h

pH range (optimal)

7.0 to 9.0 (7.5) (5.5) (6.0)

Molecular weight (kDa)

66 74 75

Other Sialidase 3

Other Sialidase 4

32

SmartEnzymes

GlycINATOR® (EndoS2) is an IgG-specific endoglycosidase that rapidly hydrolyzes all Fc glycans. TheGlycINATORenzymeisanFc-specificendoglycosidasethateffectivelyhydrolyzesallIgGglycoforms,includingcomplex,high-mannose,hybridandbisectedglycans.TheenzymeactsonthechitobiosecoreandallFc-glycansaredigestedaftertheinnerGlcNAc.Thereactionisfastandoptimalatphysiologicalconditions.ThespecificityfortheFcglycosylationsiteinvolvesaprotein-proteininteraction,andforthisreason,thenativefoldoftheFcisrequired.TheFcglycosylationsiteisconservedamongmanyspeciesandGlycINATORdeglycosylatesantibodiesfromarangeofspecies.TheapplicationsofGlycINATORincluderapidassessmentofafucosylationlevelsbyLC-MS(Liu,2016,p.38),reductionofsamplecomplexity,inactivationofantibodiesinimmunoassaysandsite-specificconjugationusingGlyCLICK®.

HumanIgG1-4,Fc-fusionproteins,IgGfrommouse,rabbit,rat,monkey,sheep,goat,cowandhorse

30minreaction

RequiresnativeIgGfold

AllFcglycoformsofIgG

30MIN

30MINUTE

30MINUTES

30MIN

Enzyme GlycINATOR IgGZERO PNGase F Endo H

Digestion site

Works on native IgG Yes Yes Yes/No Yes/No

pH optimum 7.4 7.4 7.5 5-6

Reaction time 30 min 30 min 6-24 h 1-16 h

IgG specific Yes* Yes No No

Glycoform specificity All Fc glycoforms

Complex Fc glyco-forms, limited activity on high-mannose and hybrid-type glycans

All glycoforms except core 1,3 alpha-fucose

High-mannose and some hybrid-type

glycans

* GlycINATOR has one more known substrate, alpha-1-acid glycoprotein.

Glyc

INAT

OR®

33

TheGlycINATORenzymeconsistsof2000unitsfordeglycosylationof2mgIgG.Theenzymeisprovidedasalyophilizedpowder.


A0-GL1-020 GlycINATOR, 2000 units 2 mg IgG 430 599

A0-GL8-020GlycINATOR LE (low endotoxin), 2000 units

2 mg IgG 475 625

TheGlycINATORenzymeisimmobilizedonagarosebeads,andthespincolumnsareprovidedwith immobilizedenzymefordeglycosylationofFc glycanson0.5mgupto100mgofantibodyor Fc-fusionprotein.ImmobilizedGlycINATORdeglycosylatestheFc domainwithnoenzymeinthefinalpreparation.


A0-GL6-010 Immobilized GlycINATOR Microspin 2 x 0.5 mg 320 445

A0-GL6-025 Immobilized GlycINATOR Microspin 5 x 0.5 mg 730 1,010

A0-GL6-050 Immobilized GlycINATOR Microspin 10 x 0.5 mg 1,210 1,685

A0-GL6-100 Immobilized GlycINATOR Midispin 1-10 mg 975 1,350

A0-GL6-1000 Immobilized GlycINATOR Maxispin 10-100 mg 2,900 4,055

30MIN

30MINUTE

30MINUTES

30MIN

IgG Glycosidases

GlycINATO

R®

34

SmartEnzymes

IgGZERO® (EndoS) is an IgG-specific endoglycosidase that hydrolyzes biantennary Fc glycans. TheIgGZEROenzymehydrolyzesbiantennaryN-glycansspecificallyattheFcglycosylationsiteonIgG.IncontrasttoGlycINATOR,IgGZEROhaslimitedactivityonhigh-mannoseandhybrid-typeglycoforms.Thehydrolysisofcomplexglycansisfastandcarriedoutundernativeandphysiologicalconditions,astheenzymerequiresnativeIgGfoldforactivity.The IgGZEROenzymeisusedtorapidlyreducesamplecomplexity,inimmunoassaystoreduceantibodymediatedeffectorfunctions,orasatooltoimproveimagingbyreducingFcinteractions(Gao,2014,p.38).

HumanIgG1-4,Fc-fusionproteins,IgGfrommouse,rat,monkey,sheep,goat,cowandhorse

30minreaction

RequiresnativeIgGfold

ComplexFcglycoforms,limitedactivity onhigh-mannoseandhybrid-type glycans

30MIN

30MINUTE

30MINUTES

30MIN

IgGZERO® (Endo S) + Cetuximab

GlycINATOR® (Endo S2) + Cetuximab

IgGZERO and GlycINATOR Deglycosylation of Cetuximab Fc glycans

TheglycoformspecificityofIgGZEROand GlycINATORwascomparedbymonitoringthereleasedglycansafterincubationwithcetuximab,atherapeuticantibodycontaininghigh-mannosegly-

cans(Sjögren,2015,p.38).ThedatashowsspecificreleaseofFc-glycans,andGlycINATOReffectivelyhydrolyzedhigh-mannoseglycansaftera30minreactiontimewhereasIgGZEROdidnot(Fig. 1).

Figure 1. The N-glycans released by IgGZERO (A-E) and GlycINATOR (1-8) were analyzed by MALDI-TOF. High-mannose structures (2, 4 and 7) were readily hydrolyzed by GlycINATOR, but not by IgGZERO.

IgG

ZERO

®

35

TheIgGZEROenzymeconsistsof2000unitsfordeglycosylationof2mgIgG.Theenzymeisprovidedasalyophilizedpowder.

TheIgGZEROenzymeisimmobilizedonagarosebeads,andthespincolumnsareprovidedwith immobilizedenzymefordeglycosylationofFc glycanson0.5mgupto100mgofantibodyor Fc-fusionprotein. deGlycITdeglycosylatestheFcdomainwithno enzymeinthefinalpreparation.


A0-IZ1-010 IgGZERO, 1000 units 1 mg IgG 220 305

A0-IZ1-050 IgGZERO, 5000 units 5 mg IgG 875 1,210

A0-IZ8-020IgGZERO LE (low endotoxin), 2000 units

2 mg IgG 475 625


A0-IZ6-010 deGlycIT Microspin 2 x 0.5 mg 320 445

A0-IZ6-025 deGlycIT Microspin 5 x 0.5 mg 730 1,010

A0-IZ6-050 deGlycIT Microspin 10 x 0.5 mg 1,210 1,685

A0-IZ6-100 deGlycIT Midispin 1-10 mg 975 1,350

A0-IZ6-1000 deGlycIT Maxispin 10-100 mg 2,900 4,055

deGlycIT™

30MIN

30MINUTE

30MINUTES

30MIN

IgG Glycosidases

IgGZERO

®

36

SmartEnzymes

GlyCLICK® is a site-specific conjugation technology for IgG.

GlyCLICKenablesquantitativeconjugationoffunctionalgroupssite-specificiallytotheFcdomainofIgG.TheGlyCLICKtechnologyutilizestheFc-specificendoglycosidaseactivityofGlycINATORandtheGalNAztransferandclick-chemistryofSiteClick®*forrobustandpreciseattachmentoflabelstoIgG.Theresultingantibodyconjugatehas2.0labelsperantibodyandallowssensitivequantitativeapplications.ConjugationattheFcdomainensuresunaffectedantigenbindingafterIgGlabeling.ThefunctionalgroupsavailableasGlyCLICKkitsincludeAlexaFluor®488,BiotinandDFO.TheGlyCLICKAzideActivationkitallowssite-specificconjugationofacustomlabelofchoice.TheGlyCLICKtechnologyisappliedinboth in vitro and in vivoimagingapplicationsaswellasADCandtherapeuticantibodydevelopment.

GlyCLICK Conjugation Overview

Available DIBO-Functionalized Labels

Label Name Examples of Applications

Fluorophore AlexaFluor® 488FACS, IHC, in vitro cell

imaging

Affinity BiotinImmuno assays, ELISA,

western blot

Chelator Deferoxamine (DFO) Immuno imaging, in vivo

HumanIgG1-4,Fc-fusionproteins,IgGfrommouse,rabbit,rat,monkey,sheep,goat,cowandhorse

~3dayprotocol

Availableconjugates:AlexaFluor®488,biotinandDFO.Azideactivationkitsareavailableforcustomconjugation

*SiteClick®isprovidedunderanintellectualpropertylicensefromLifeTechnologiesCorporation.ThetrademarkSiteClick®isthepropertyofLifeTechnologiesCorporation. SeeLegalandDisclaimers,p.39.

TheGlyCLICKtechnologyinvolvestwoenzymaticstepsandclick-chemistrytoobtainasite-specificantibodyconjugate.TheprocessofGlyCLICKconjugationisschematicallyillustratedinFig. 1.

Figure 1. Schematic presentation of the GlyCLICK conjugation process.

1. ImmobilizedGlycINATORhydrolyzestheFc-glycansofIgGandexposesthecoreGlcNAc.

2. Theenzymeβ-1,4-galactosyltransferaseGalT(Y289L)transfersGalNAztotheexposedGlcNAc,renderingtheantibodyazide-activated.

3. Throughastrain-promotedcopper-freeclickreactionusingaDIBO-alkynelabel(cyclooctyne),thefunctionalgroupisconjugatedtothetwoazidesofGalNAzattheFcdomainsoftheantibody.

1

2

3

Immobilized GlycINATOR

GalT(Y289L)UDP-GalNAz

DIBO alkyne label

AZIDE ACTIVATION CLICK REACTIONDEGLYCOSYLATION

1 2 3

GlyC

LICK

®

37

min2 4 6 8 10

LU

0

5

10

15

20

25

30

35

40

45

mAU

0

20

40

60

80

mAU

0

20

40

60

80

A

B

C

Figure 2. RP-HPLC analysis of trastuzumab. a) Trastuzumab digested with FabRICATOR into F(ab’)2 and Fc/2 fragments. b) Trastuzumab conjugated with Alexa Fluor®488 prior to FabRICATOR digestion into F(ab’)2 and Fc/2 fragments. c) Fluorescence signal of b).

Figure 3. LC/MS analysis of trastuzumab conjugated with MMAE (monomethyl auristatin E) using GlyCLICK, showing the Fc-specific attachment of one toxin per Fc/2 fragment. a) Native, b) deglycosylated, c) conjugated trastuzumab.

Fc-specific and Complete GlyCLICK Conjugation

GlyCLICKcontainsallreagentsandmaterialsneededtoazideactivateorlabeltheIgG.

Product ID Description Size EUR USD

L1-F01-025 GlyCLICK Alexa Fluor® 488 Conjugates 250 μg IgG 885 940

L1-C01-025 GlyCLICK DFO Conjugates 250 μg IgG 885 940

L1-A01-025 GlyCLICK Biotin Conjugates 250 μg IgG 885 940

L1-AZ1-025 GlyCLICK Azide Activation Activates 250 μg IgG 790 835

L1-AZ1-100 GlyCLICK Azide Activation Activates 1 x 10 mg IgG 4,900 5,900

TheGlyCLICKconjugationofthedesiredlabeloccursonlyattheazide-activatedsitesontheFcregion,ensuringsite-specificconjugation.TrastuzumabwasconjugatedwithAlexaFluor®488usingGlyCLICKanddigestedusingFabRICATOR,resultinginF(ab’)2andFc/2fragments.ThefluorescencesignalwasdetectedonlyfromtheFcdomain,indicatingsite-specificconjugation(Fig. 2).

TheefficacyoftheGlyCLICKprocesswasstudiedbyconjugatingmonomethylauristatinE(MMAE)totrastuzumabusingGlyCLICKandanalyzetheFc/2fragmentusingLC-MS.Thenative(Fig. 3a)anddeglycosylated(Fig. 3b)Fc/2showthehydrolysisoftrastuzumab.AfterconjugationofMMAE,theFc/2displaysamassshiftofonetoxinandlinkerindicatingacompleteconjugationtotheFc/2andadrugtoantibodyratioof2.0(Fig. 3c).

25393.7216

24136.2511

26162.3000

0

1

2

3

4

5x10Intens.

0.0

0.2

0.4

0.6

0.8

6x10

0.0

0.5

1.0

1.5

2.0

2.5

3.0

5x10

15000 17500 20000 22500 25000 27500 30000

A.

B.

C.

25379.6674

25379.6880

25379.7692

0

1

2

3

4

55x10

Intens.

0

1

2

3

4

5

5x10

0

1

2

3

4

5

65x10

15000 17500 20000 22500 25000 27500

Hi

VH

CH1

Hi

VH

CH1

23439.6567

23439.6944

23438.7549

0.0

0.2

0.4

0.6

0.8

1.06x10

Intens.

0.0

0.2

0.4

0.6

0.8

1.0

6x10

0.0

0.2

0.4

0.6

0.8

1.0

6x10

15000 17500 20000 22500 25000 m/z

VL

CL

VL

CL

VL

CLHi

VH

CH1

2026.05

A

B

C

Antibody Conjugation

GlyCLICK®

38

SmartEnzymes

An,Y.etal.,2014.A new tool for monoclonal antibody analysis: application of IdeS proteolysis in IgG domain-specific charac-terization.mAbs,6(4),pp.879–893.

Ayoub,D.etal.,2013.Correct primary structure assessment and extensive glyco-profiling of cetuximab by a combination of intact, middle-up, middle-down and bottom-up ESI and MALDI mass spectrometry techniques. mAbs,5(5),pp.699–710.

Beck,A.,Diemer,H.,etal.,2013. Analytical characterization of biosimilar antibodies and Fc-fusion proteins. TrACTrendsinAnalyticalChemistry,48,pp.81–95.

Beck,A.,Wagner-Rousset,E.,etal.,2013.Characterization of therapeutic antibodies and related products.AnalyticalChemis-try,85(2),pp.715–736.

Botzanowski,T.etal.,2017. Insights from native mass spectrometry approaches for top- and middle- level characterization of site-specific antibody-drug conjugates. mAbs,9(5),pp.801–811.

Faid,V.etal.,2017. Middle-up analysis of monoclonal antibodies after combined IgdE and IdeS hinge proteolysis: Investiga-tion of free sulfhydryls.JournalofPharmaceuticalandBiomedicalAnalysis,149,pp.541–546.

Lynaugh,H.,Li,H.&Gong,B.,2013.Rapid Fc glycosylation analysis of Fc fusions with IdeS and liquid chromatography mass spectrometry. mAbs,5(5),pp.641–645.

Sjögren,J.,Olsson,F.&Beck,A.,2016. Rapid and improved characterization of therapeutic antibodies and antibody related products using IdeS digestion and subunit analysis. TheAnalyst,141(11),pp.3114–3125.

Sokolowska,I.etal.,2017. Subunit mass analysis for monitoring antibody oxidation.mAbs,9(3),pp.498–505.Wagner-Rousset,E.etal.,2014.Antibody-drug conjugate model fast characterization by LC-MS following IdeS proteolytic

digestion.mAbs,6(1),pp.173–184.Yang,R.etal.,2017.Rapid assessment of oxidation via middle-down LCMS correlates with methionine side-chain solvent-

accessible surface area for 121 clinical stage monoclonal antibodies.mAbs,9(4),pp.646–653.

Faid,V.etal.,2017.Middle-up analysis of monoclonal antibodies after combined IgdE and IdeS hinge proteolysis: Investiga-tion of free sulfhydryls.JournalofPharmaceuticalandBiomedicalAnalysis,149,pp.541–546.

SpoerryC,etal.,2016. Novel IgG-Degrading Enzymes of the IgdE Protease Family Link Substrate Speci city to Host Tropism of Streptococcus Species.PLoSONE11(10):e0164809.doi:10.1371/journal.pone.0164809

Leblanc,Y.etal.,2017. Charge variants characterization of a monoclonal antibody by ion exchange chromatography coupled on-line to native mass spectrometry: Case study after a long-term storage at +5°C.JournalofChromatographyB,1048,pp.130–139.

Moelleken,J.etal.,2017. GingisKHAN™ protease cleavage allows a high-throughput antibody to Fab conversion enabling direct functional assessment during lead identification of human monoclonal and bispecific IgG1 antibodies. mAbs,131(6),pp.1–12.

Sjögren,J.etal.,2017. Generating and Purifying Fab Fragments from Human and Mouse IgG Using the Bacterial Enzymes IdeS, SpeB and Kgp. MethodsinMolecularBiology,1535,pp.319–329.

vandenBremer,E.T.J.etal.,2017.Cysteine-SILAC Mass Spectrometry Enabling the Identification and Quantitation of Scrambled Interchain Disulfide Bonds: Preservation of Native Heavy-Light Chain Pairing in Bispecific IgGs Generated by Controlled Fab-arm Exchange.AnalyticalChemistry,89(20),pp.10873–10882.

Mahan,A.E.etal.,2015. A method for high-throughput, sensitive analysis of IgG Fc and Fab glycosylation by capillary electro-phoresis. JournalofImmunologicalMethods,417,pp.34–44.

Zhang,Z.etal.,2016.SpeB Proteolysis with Imaged Capillary Isoelectric Focusing for the Characterization of Domain-Specific Charge Heterogeneities of Reference and Biosimilar Rituximab. JournalofChromatographyB,1020,pp.148–157.

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FabRICATOR®

Thisproductisprovidedunderanexclusiveworld-wideintellectualpropertylicensefromHansaMedicalABderivedfrominternationalpublicationWO03051914,includinggrantedUSPatentNoUS7,666,582andgrantedEuropeanPatentNo1458861.ThelicenseencompassesIdeSfromStreptococcuspyogenesforbiotechnicalindustrialapplicationswhichareneithertherapeuticnordiagnostic,otherthanthefollowingexceptionwhichisincludedwithinthelicense:digestingIgGinvitroinclinicalsamplesfordiagnosticpurposes.

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Genovis provides SmartEnzymes™ used in characterization and conjugation of biopharmaceuticals such as monoclonal antibodies (mAbs), Fc-fusion proteins, biosimilars and antibody-drug conjugates (ADCs). The enzymes and technologies we offer are IgG-specific proteases, general proteases, IgG-specific glycosidases, enzymes and technologies for O-glycan analysis and a site-specific conjugation technology for antibodies.

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