SmartEnzymes€¦ · fragments with no enzyme in the final preparation. 90 MINUTES 60 Product ID Description Digestion EUR USD A2-FZ2-005 FragIT Z kit 0.5 mg IgG 380 520 A2-FZ2-025

1

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

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 IgG in the 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 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 hydrolyzes core 1 type O-glycans on native glycoproteins

Sialidases for removal of sialic acids from native glycoproteins

Sialidases for removal of sialic acids from native glycoproteins

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

FabRICATOR®Z 16FragIT™Z / FragIT™Z kit

FabALACTICA® 18Immobilized FabALACTICA® / FabALACTICA® Fab kit

GingisKHAN® 22GingisKHAN® Fab kit

GingisREX® 26

GlyCLICK® 42

FabULOUS® 24FabULOUS® Fab kit

GlycINATOR® 38Immobilized GlycINATOR®

OpeRATOR® 28

GlycOCATCH® 30

OglyZOR® 31

IgGZERO® 40deGlycIT™

SialEXO® 32SialEXO®23 / Immobilized SialEXO®

GalactEXO™ 36

IgG GLYCO

SIDASES

O-G

LYCAN

SEXO

GLYCO

SIDASES

CON

JUGATIO

NPRO

TEASESIgG PRO

TEASES

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

DigestsIgGinthehingeregionof severalspeciesandsubclassesGeneratesFabandFcfragments60minreactionRequiresreducingconditions

...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-proteins2-4hreactionRequirespre-removalofsialicacids

30MINUTES

30MINUTES

IgG GLYCOSIDASES O-GLYCANS

ImmobilizedGlycINATORenzymeonagarosebeadsHydrolyzesallFcglycoformsofIgGConvenientspincolumnformatNoenzymeinthefinalpreparation Enrichesmucin-typeO-glycosylated

proteinsandpeptides30min-2hbindingRequiresdesialylationApplicationsinglycomics

Site-specificconjugationofIgGActiveonhumanIgG1-4andseveralotherspeciesTheantibodyretainsantigenbindingQuantitativelabelingof2labelsperantibody

Alexa Fluor® 488 Alexa Fluor® 555 Alexa Fluor® 647

DFO

CONJUGATION

Biotin Azide Activation

EXOGLYCOSIDASES

SialEXOhydrolyzesallsialicacidsandSialEXO23specificallyhydrolyzes α2-3-linkedsialicacidsAvailableimmobilizedonagarosebeadsActiveonbothN-andO-linkedglycans1-2hreactionActiveonnativeglycoproteins

Hydrolyzesgalactcoseresidueson N-andO-glycosylatedproteinsEfficientremovalofβ1-3,4linkedgalactoseActiveonbothN-andO-linkedglycans2hreactionFornativeglycoproteinsandfreeglycans

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 IgG,

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,manydifferentqualityattributesofthelargeheterogeneousmoleculesneedtobecharacterized.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.42.

...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. FabRICATORisaconvenienttooltodeterminethedegreeofoxidationatthesubunitlevel(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-MSatthesubunitlevel.Ayoubandcolleagues(Ayoub,2013,p.42)usedthesubunitworkflowtodeterminetheglycanprofileoftheFabandFcdomainsofcetuximab(Fig. 4).

IgG Proteases

FabRICATO

R®

10

SmartEnzymes

Product ID Description Digestion EUR USD

A0-FR1-020 FabRICATOR, 2000 units 2 mg IgG 450 625

A0-FR1-050 FabRICATOR, 5000 units 5 mg IgG 875 935

A0-FR1-250FabRICATOR, 5 x 5000 units

25 mg IgG 3,395 3,740

A0-FR1-096 FabRICATOR, 96x100 units 96 x 100 μg IgG 1,735 2,255

A0-FR1-008 FabRICATOR, 8x100 units 8 x 100 μg IgG 305 390

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

2 mg IgG 500 680

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

5 mg IgG 965 1,020

Validation Kit


A0-FR4-060FabRICATOR, 3 x 2000 units

3 x 2 mg IgG 1,355 1,880

Anti-FabRICATOR™

Anti-FabRICATORisagoatpolyclonalantibodypurifiedonproteinGthatisusedfordetectionof FabRICATORwithwesternblotorELISA.

Product ID Description Concentration EUR USD

A3-AF1-010Anti-FabRICATOR 0.1 ml

4 mg/ml 260 365

ThreedifferentbatchesoflyophilizedFabRICATORareincludedintheFabRICATORValidationkitforvalidationofFabRICATOR-basedanalyticalmethods.

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

FabR

ICAT

OR®

11


A0-FR6-010 FragIT Microspin 2 x 0.5 mg IgG 335 465

A0-FR6-025 FragIT Microspin 5 x 0.5 mg IgG 765 1,060

A0-FR6-050 FragIT Microspin 10 x 0.5 mg IgG 1,270 1,765

A0-FR6-100 FragIT Midispin 1-10 mg IgG 1,020 1,415

A0-FR6-1000 FragIT Maxispin 10-100 mg IgG 3,045 4,255

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


A2-FR2-005 FragIT kit, Microspin 0.5 mg IgG 380 520

A2-FR2-025 FragIT kit, Microspin 5 x 0.5 mg IgG 1,020 1,415

A2-FR2-050 FragIT kit, Microspin 10 x 0.5 mg IgG 1,940 2,385

A2-FR2-100 FragIT kit, Midispin 10 mg IgG 1,275 1,775

A2-FR2-1000 FragIT kit, Maxispin 100 mg IgG 3,820 5,340

FabRICATO

R®

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 1,675 1,885

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

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

FabRICATO

R®

14

SmartEnzymes

Figure 1. FabRICATOR MagIC workfl ow on Thermo Scientifi c KingFisherTM Purifi cation System. IgG, FabRICATOR MagIC and PBS are dispensed into a 96-deepwell plate according to instructions for the KingFisher workfl ow. FabRICATOR MagIC beads are trans-ferred (1) to wells containing PBS for equilibration (2). The beads are collected (3), added to the antibody samples (4) and incubated with mixing for 10-20 min at 37°C (5). The beads are then collected (6) and transferred to waste wells and the pure F(ab’)2 and Fc are left in the sample wells (7). The trademark KingFisher is the property of Thermo Fisher Scientifi c.

FabRICATOR® MagIC enables parallel subunit generation of antibodies for automated middle-level workfl ows

FabRICATORMagICcontainsFabRICATOR(IdeS)enzymeimmobilizedonmagneticagarosebeadsfordigestionofantibodiesintoofF(ab’)2andFc/2subunits in20min.Themagnetic formatenablesparallelpreparationofsamplesfortheanalysisofmultipleantibodiesinmiddle-levelworkflowswithminimalhands-ontime.FabRICATORMagICcanbeusedinmanualorautomatedsetupsforrutineanalysisofIgG-basedbiopharmaceuticals(Fig.1).

EQUILIBRATION

1 2 3 4 5 6 7ANALYSISDIGESTION

�

��

Key Characteristics

Antibodysubunitgenerationin20min

Parallelprocessingofupto96samples

Optimizedforautomatedworkflows

Human IgG1-4, IgG from monkey, rabbit, sheep and rat IgG2b

CPAPELLG / GPSVF (below the hinge)

Over 95% digestion within 20 minutes

No need for reducing agents or co-factors

Digestandreduceinaone-potreaction

Onlyrequiresasinglepipettingstep

2

FabR

ICAT

OR®

153


A0-FRM-024

A0-FRM-096

FabRICATOR MagIC 2 mL 24 samples

FabRICATOR MagIC 4 x 2 mL96 samples

550

1,875

595

1,990

F(ab’)2

Fc/2

Retention time

LC

Fd’

Fc/2

IgG

Intact Antibody

+ Reduction

Figure 2. Analysis of a monoclonal antibody by RPLC-MS at the

intact level (top), after FabRICATOR MagIC digestion (middle) or after

treatment with FabRICATOR MagIC and 5 mM TCEP (bottom).

Automated Middle-Level Workfl ow

FabRICATOR MagIC generates intact F(ab’)2 and

Fc/2 subunits and the disulfi de bonds can easily be

reduced using 5 mM TCEP in a one-pot reaction. The

generated Fc/2, LC and Fd’ fragments allow detailed

analysis and monitoring of different anitbodies using

middle-level workfl ows resulting in a better resolution

compared to intact level approaches. Reverse phase

LC-MS analysis of the resulting subunits or fragments

from an antibody processed with FabRICATOR MagIC

demonstrates the complete digestion and subsequent

reduction of the generated subunits (Fig. 2).

Fast Automated Monitoring of CQAs

Using FabRICATOR MagIC for automated digestion of

multiple mAbs, different CQAs such as oxidation can

be analyzed. A mix of six different mAbs subjected to

forced degradtion was analyzed by LC-MS at the

intact or the subunit level using FabRICATOR MagIC.

The results show the ability to quickly detect and

monitor such modifi cations in a complex mixture of

mAbs using FabRICATOR MagIC (Fig. 3).

Figure 3. Analysis of tryptophan oxidation during forced degradation

studies. Six mAbs were analyzed by RPLC-MS at the intact level (top

panel) and after FabRICATOR MagIC with 5 mM TCEP (bottom panel).

Highlighted differences between control (blue) and sample (orange).

A detailed Fc-glycan profi ling was also performed on a

mAb by middle-level analysis after FabRICATOR MagIC

digestion and reduction with 5 mM TCEP (Fig. 4).

255002525025000

Man5 G0

G0F

G1F

G2F

GlycoformMan5G0G0FG1FG2F

Abundance2.5%4.7%

47.6%38.4%6.8%

LC Fd’Fc/2

Retention Time

m/z

Figure 4. Analysis of Fc glycosylation by middle-level LC-MS using

FabRICATOR MagIC (top) and quantifi cation of glycoforms from a

deconvoluted mass spectrum of the Fc/2 fragment (bottom).

Frozen sample (-80°C)Stability sample (25°C, 3 months)

mAb1

mAb2mAb3

mAb4

mAb5

mAb6

mAb

1 Fd

’

Fc/2

mAb

1 LC

mAb

4 LC

mAb

5 LC

mAb

6 LC

mAb

2 LC mAb

3 LC

mAb

3 Fd

’

mAb

2 Fd

’

mAb

4 Fd

’

mAb

6 Fd

’

mAb

5 Fd

’

mAb

3 LC

+0

Da

mAb

5 Fd

’ +29

Da

mAb

5 Fd

’ +14

Da

mAb

5 LC

-18

Da

Retention time

+ Reduction

Intact Antibodies

2

FabRICATO

R®

16

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 450 625

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

17

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

90MINUTES

60MINUTES


A2-FZ2-005 FragIT Z kit 0.5 mg IgG 380 520

A2-FZ2-025 FragIT Z kit 5 x 0.5 mg IgG 1,020 1,415


A0-FZ6-010 FragIT Z Mircospin 2 x 0.5 mg IgG 335 465

A0-FZ6-025 FragIT Z Mircospin 5 x 0.5 mg IgG 765 1,060

A0-FZ6-050 FragIT Z Mircospin 10 x 0.5 mg IgG 1,270 1,765

*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

FabRICATOR®Z

18

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.42),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®

19

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 535 655

FabALACTICAwasusedtodigestthethreetherapeuticmAbscetuximab,trastuzumabandadalimumab.Fig. 1showsthatFabALACTICA

generatesintactFabandFcfragmentsfromallthreeantibodies.

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

Fabfragmentcanbeanalyzedtostudymodificationsandlightandheavychainpairingforbispecificantibodies,orusedforcomparabilityassessment (Fig. 2b).

IgG Proteases

FabALACTICA®

20

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 365 510

A0-AG6-050 Immobilized FabALACTICA Microspin 10 x 0.5 mg hIgG1 1,310 1,835

A0-AG6-100 Immobilized FabALACTICA Midispin 5-10 mg hIgG1 1,100 1,520

A0-AG6-1000 Immobilized FabALACTICA Maxispin 10-100 mg hIgG1 3,305 4,600

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®

21

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 415 575

A2-AFK-025 FabALACTICA Fab kit 5 x 0.5 mg hIgG1 1,100 1,520

A2-AFK-100 FabALACTICA Fab kit 10 mg hIgG1 1,360 1,890

A2-AFK-1000 FabALACTICA Fab kit 100 mg hIgG1 4,145 5,825

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 Fabs

ThreetherapeuticmAbswereincubatedwithImmobilizedFabALACTICAinspincolumns.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®

22

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.42.

...KSCDK / THTCPPCP....

60MINUTE

60MINUTES

2000unitsofGingisKHANisprovidedasalyophilizedpowdertogetherwith5vialsoflyophilizedGingisKHANreducingagentfordigestionof2mghumanIgG1.


B0-GKH-020 GingisKHAN, 2000 units 2 mg hIgG1 450 625

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®

23

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 1,020 1,095

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

®

24

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.42.

Human IgG and IgG from mouse, rat, goat, sheep and rabbit.

60 min reaction

Requires reducing conditions (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 450 625

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

®

25

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 765 825

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®

26

SmartEnzymes

60MINUTES

GingisREX® (RgpB) 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 Sequence

Intact protein FVNQHLCGSHLVEALYLVCGERGFFYTPKA

1 GFFYTPKA

2 FVNQHLCGSHLVEALYLVCGER

3 GFFYTPK

4 FVNQHLCGSH

5 LVEALYLVCGER + Na

Ging

isR

EX®

27

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 - 1:200 475 530

TheGingisREXenzymecanbeusedtoanalyzeproteinsbymassspectrometryfortheuseinpeptidemapping,proteinfingerprintingandsequenceanalysis.Itgenerateslargerpeptideswithmorechargeperpeptide,whichisbeneficialformassspectrometricanalyses.Usingthisworkflow,themass-to-chargeoflongerpeptidescanberesolved,resultinginincreasedsequencecoverageandidentificationofparticularpost-translationalmodifications.

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

Applications of GingisREX

Proteases

GingisREX

®

28

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-glycans(core1)anddigeststheaminoacidbackboneN-terminallyoftheserineandthreonine(SandT)glycosylationsites(Fig. 2).ThisgeneratesglycopeptidescarryingO-glycansandenablesO-glycanprofiling,O-glycopeptidemappingandsiteoccupancydetermination,aswellasmiddle-levelapproachesusingmassspectrometry.TheOpeRATORenzymeismostactivetowardssiteswithasialylatedcore1glycans.Sialylatedsitesandsiteswithcore3O-glycansaredigestedwithsignificantlyreducedactivity.SialEXO®(p.30),asialidasemixforefficientandcompleteremovalofsialicacids,isprovidedwithOpeRATOR.

Digestsnativeproteinswithmucin-typeO-glycosylation

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 analyzed by SDS-PAGE.

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

1 2 3 4

TNFaR

TNFαR

+

(kDa)

50

40

30

OpeRATORdisplayssomeactivityonsialylatedO-glycoproteins,buttheactivityismuchhigherwhenthesialicacidsareremovedusingSialEXO(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®

29

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 880 985

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.29)wasusedtodemonstratetheO-glycan-dependentactivityofOpeRATOR.AscanbeseeninLane3inFig. 4,thereisnoOpeRATORdigestionofEPOwhentheO-glycanhasbeenremoved.

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

Etanercept,ahighlyO-glycosylatedFcfusionprotein,wasincubatedwithOpeRATORandtheresultingglycopeptideswereanalyzedusingLC-MS/MS.DuetotheheterogeneityintheO-glycanpatternoftheproteinandtheOpeRATORspecificityforO-glycan

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

structures,overlappingpeptideswereformed,makingitpossibletoacquireacompletemapoftheO-glycansites(Fig. 5).

Enzymes for O-glycans

OpeR

ATOR

®

30

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® enzymethathasbeenengineeredtobindcore1O-glycosylatedproteinsandpeptideswithhighaffinity.GlycOCATCHisprovidedinaspincolumnformattoalloweasy-to-useenrichmentofO-glycoproteins.DuetothestronginteractionbetweentheGlycOCATCHresinandO-glycoproteins/peptides,theelutionisperformedwith8Murea.Alternatively,theelutioncanbeperformedwiththeincludedOpeRATORenzyme.Foroptimalperformance,thesialicacidsoftheglycoproteinneedtoberemovedusingtheincludedSialEXO®sialidasemix(p.30).TheapplicationsofGlycOCATCHincludespecificenrichmentorremovalofO-glycoproteinsandpeptides,glycomics,studiesofcomplexsamplesandcharacterizationofbiopharmaceuticals.InFig. 1,theO-glycanspecificityofGlycOCATCHwasstudiedonthepeptidelevel.Aselectionofnon-glycosylatedandO-glycosylated(core1)peptideswasused.ThepeptideswereloadedontoGlycOCATCH,theresinwaswashedandtheboundpeptideswereelutedwith8Murea.Afterpurification,thesampleswereseparatedandanalyzedbyRP-

Bindsproteinsandpeptideswithmucin-typeO-glycosylation

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

®

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

TheGlycOCATCHboxcontains4microspincolumnsofGlycOCATCHaffinityresin,200unitsofSialEXOand200unitsofOpeRATORforenrichmentofupto200μgO-glycoprotein.

Product ID Description Enrichment EUR USD

G3-OC6-002 GlycOCATCH 200 μg O-glycoprotein 605 705

31

Enzymes for O-glycans

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 765 875

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®

32

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 545 655

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.

33

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)wasusedtoanalyzetwoglycanlibraries;onecontainingreleasedglycanswithα2-3-linkedsialicacidsandtheotherglycansmodifiedwithα2-6-linkedsialicacids.TreatmentwithSialEXOorSialEXO23canbecompared(Fig. 2).Theshiftinpeaksclearlyshowsthereleaseofsialicacidsonlylinkedbyα2-3withSialEXO23andofbothα2-3andα2-6linkedsialicacidsbySialEXO.


60minreaction

Requiresnoco-factorsα2-3-linkedsialicacids

TheSialEXO23enzymeconsistsof500unitsforhydrolysisofsialicacidson0.5mgglycoprotein. Theenzymeisprovidedasalyophilizedpowder.


G1-SD2-005 SialEXO 23, 500 units 0.5 mg glycoprotein 470 535

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- or α2-6-linked sialic acids after incubation with 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

EXOglycosidases

34

SmartEnzymes

Immobilized SialEXO® is a sialidase mix for complete removal of sialic acids from native glycoproteins within 30 minutes.

SialEXOhydrolyzessialicacidsonnativeglycoproteins,andisactiveonboth O-andN-linkedglycans.Itisacombinationoftwosialidasesactingonα2-3,α2-6andα2-8 linkages.ImmobilizedSialEXOisaresinwithamixtureofthetwosialidasescovalentlycoupledtoagarosebeadsforcompleteremovalofsialicacidswithnoenzymeinthefinalpreparation.0.5mgglycoproteinisdesialylatedin30minatroomtemperature.

Figure 1. Deconvoluted mass spectra of the Fd’ fragment of cetuximab, showing the Fab glycosylation profile. Sialylated structures (Neu5Gc, light blue diamond) are highlighted in purple and are absent in the lower spectrum. The asterisk marks peaks originating through neutral loss during ionization rather than remaining sialylated Fd’ fragments.

Fc/2 LC

Fd’

28000

27750

27250

27500

untr

eate

d

1 2 3 4 56 7 8 9 10 11 12

27000

26750

28250

Mass Spectrum Fd’ fragment

* *

10 11Fd´ Fd´

12Fd´

9Fd´

6 7Fd´Fd´

8Fd´

5Fd´

2 3 4Fd´Fd´Fd´

1Fd´

FabRICATOR® Reduction

Fc glycans

Fab glycans

Fc/2

F(ab’)2


30minreaction

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

Efficient Desialylation of a Monoclonal Antibody

AntibodieswithadditionalglycansintheirFabregionsareoftensialylatedtoasignificantextent.CetuximabcarriesaFabglycanwhichispartiallymodifiedwithN-Glycolylneuraminicacid(Neu5Gc).Thesesialic

acidscanberemovedrapidlyusingImmobilizedSialEXO(Fig.1),demonstratingtheutilityofthisenzymeforanalysisofmonoclonalantibodiesanditsactivitytowardsnon-humansialicacids.

30MINUTES

Sial

EXO

®

35

TheImmobilizedSialEXOMicrospincolumnscontainSialEXOcovalentlycoupledtoagarosebeads,fordesialylationofupto0.5mgnativeglycoproteinswithoutenzymeinthefinalpreparation.


G1-SM6-010 Immobilized SialEXO Microspin 2 x 0.5 mg 365 445

G1-SM6-025 Immobilized SialEXO Microspin 5 x 0.5 mg 775 995

G1-SM6-050 Immobilized SialEXO Microspin 10 x 0.5 mg 1,295 1,745

Figure 2. The C1 inhibitor was either treated with SialEXO in solution for 2 hours at 37°C, or with Immobilized SialEXO for 30 min at room temperature. N-glycans were released from the resulting desialylated protein using PNGase F and the resulting free glycans were labeled with 2-AB and analyzed by HILIC-FLD HPLC.

untreated

5Retention time (min)

10 15 20 25 30

sialylatedbiantennary

sialylatedtri- & tetra-antennary

non-sialylatedbiantennary

non-sialylated tri-& tetraantennary

Efficient Desialylation of a Complex Glycoprotein

WetestedtheperformanceofSialEXOandImmobilizedSialEXOonthehumanC1inhibitor.Thisglycoproteinisachallengingsubstratewith6N-andupto26O-glycans,modifiedwithbothα2,3andα2,6-linkedsialicacid.AnalysisofreleasedN-glycansdemonstratesacompletedesialylationbybothSialEXOinsolutionandImmobilizedSialEXO(Fig.2).

Simplified Charge Variant Analysis of Biologics

6 7 8 9 10

0

2,000

4,000

6,000

8,000

10,000

12,000

14,000

0

2,000

4,000

6,000

8,000

10,000

12,000

14,000

0

2,000

4,000

6,000

8,000

10,000

12,000

14,000

untreated

pI Marker 10.17pI Marker 5.85

Fluo

resc

ence

pI

Cetuximab

SialEXO

untreated

pI Marker 8.4pI Marker 4.05

Fluo

resc

ence

pI

Etanercept

4 5 6 7 8 9

0

5,000

10,000

15,000

20,000

25,000

30,000

0

5,000

10,000

15,000

20,000

25,000

30,000

0

5,000

10,000

15,000

20,000

25,000

30,000

SialEXO

Figure 3. Desialylation of cetuximab (a) and etanercept (b) using Immobilized SialEXO followed by imaged isoelectric focusing. Data obtained in collaboration with ProteinSimple.

Capillaryelectrophoresisiscommonlyusedtodeterminechargevariantsduringcharacterizationandqualitycontrolofbiologics.Theinherentchargeofsialicacidsmightcomplicatechargevariantprofiles,maskingotherimportantmodifications.Theremovalofsialicacidsthereforefacilitatesthestudyofunderlyingchargevariantsintheprotein.CetuximabandetanerceptweredesialylatedusingImmobilizedSialEXOandtheseparationwasimprovedinimagedisoelectricfocusing(icIEF;Fig.3),usingMauricefromProteinSimple.

A B

SialEXO®

EXOglycosidases

36

SmartEnzymes

GalactEXO™ is a β-galactosidase for effi cient hydrolysis of galactose from glycoproteins and released glycans

2

GalactEXOisaβ-galactosidasemixforcompletehydrolysisofgalactoseresiduesonN-andO-linkedglycansonnativeglycoproteins.TheenzymeswerediscoveredandcharacterizedfromAkkermansiamuciniphila forefficienthydrolysisofβ1-3andβ1-4linkedgalactoses.Thecombinedenzymaticactivitieswillcompletelyhydrolyzeallgalactoseson2mgofnativeglycoprotein.GalactEXOcanbeusedfortrimmingofthereleasedglycansinexo-glycosidasearraysequencingexperimentswhereβ1-3andβ1-4linkedgalactoseareremovedwithin1hofincubation.GalactEXOcanbeappliedtoobtain antibodieswith homogenousG0 glyco-sylationprofiles.

Hydrolysis of β1-4 Galactose on a mAb

Theβ-galactosidaseactivityofGalactEXOwasdemonstratedontrastuzumabcarryinggalactoseresiduesintheFcdomainoftheantibody.Aftera2hincubationwithGalaxtEXOorwithcompetingenzymesfromothersources,theantibodiesweredigestedintosubunitsusingFabRICATOR®andanalyzedusingLC-MS(Fig.1).TheshifttoG0FcanbeseenusingGalactEXOwhereasenzymesfromothersourcesresultsinincompletedigestion.

Hydrolyzes galactcose residues on N- and O-glycosylated proteins

β1-3 and β1-4 linked galactose

2 h incubation

No co-factors required

Figure1.DeconvolutedmassspectraoftheFc/2oftrastuzumabtreatedwiththreedifferentβ-galactosidases.TheantibodywasdigestedwithFabRICATOR,separatedby reversephaseHPLC(WatersBioResolveRP,2.1x100mm)andanalyzedbyESI-Q-TOFmassspectrometry(BrukerImpactII).

Man5 G0

G0FG1F

G2F

Streptococcus pneumoniaeβ1-4 galactosidase

Bovine testis (BTG) β1-3,4 galactosidase

24900 25000 25100 25200 25300 25400 25500 25600

mass (Da)

Undigestedtrastuzumab Fc/2

Key Characteristics

Efficientrevomalofβ1-3,4linkedgalactose

AcitvityonbothN-andO-glycanstructures

Fornativeglycoproteinsandfreeglycans

2HOURS

Gal

actE

XO®

373


G1-GM1-020 GalactEXO,2000units 750 845

GalactEXO for Released Glycan Trimming

Whenanalyzingreleasedglycanstructuresusingexoglycosidasesitiscrucialtoobtaincompletehydrolysistominimizeerrorsindatainterpretation.AlabeledN-glycanlibrarywasincubatedwiththeSialEXO®sialidaseandGalactEXOgalactosidasefor1handanalyzedforexoglycosidaseacitvities(Fig.3).TheHILIC-FLDseparationsshowcomplete

Figure3.HILIC-FLDUHPLCchromatogramsofa2-ABlabeledglycanlibraryanalyzedundigested(top),aftertreatmentwithSialEXO(middle)orbothSialEXOandGalactEXO(bottom).AnalysiswasperformedonaThermoScientificVanquishDuoUHPLCsystemequippedwithaThermoScientificAccurcore150AmideHILICcolumn(2.1x150mm).

Figure2.DeconvolutedmassspectraoftheTNFɑRfragmentofetanercepttreatedwiththreedifferentbeta-galactosidases.Theprotein was digested with FabRICATOR, separated by reversephaseHPLC(WatersBioResolveRP,2.1x100mm)andanalyzedbyESI-Q-TOFmassspectrometry(BrukerImpactII).

Activity on β1-3 Gal from Etanercept

GalactEXOalsoshowsexogalactosidaseactivityforβ1-3galactose,presentonO-glycosylatedbiopharmaceuticals.EtanerceptwasincubatedwithGalactEXOorgalactosidasesfromothersourcesandtheTNFɑRfragmentwasanalyzedusingLC-MSafterdigestionwithFabRICATORto removetheFcfragment(Fig.2).TheresultsshowefficientgalactosidaseactivityfromGalactEXOcomparedtotheenzymesfromothersources.

hydrolysisofboththesialylatedandgalactosylatedstructuresandtheremainingpeakscaneasilybeidentifiedasG0,G0ForG0FwithbisectingGlcNAc.

6 8 10 12 14 16 18 20 224Retention time (min)

1

24

4’

55’

66’

7

9

11

Undigested

galactosylated glycoforms

1

2

4

4’

5

5’

6

6’

78

8’

9

1

1

11 13

12

151416

1718

sialylated glycoforms

3

3

1

2

3

0

0’

1 2 3 4/4’ 5/5’ 6/6’ 7

8/8’

9

10/10’ 12

11

1413 15 16 17 18

sialylated glycoforms

galactosylated glycoformsG0/G0F glycoforms

8x

Xanthomonas manihotisβ1-3 galactosidase

Bovine testis (BTG) β1-3,4 galactosidase

29000 29500 30000 30500 31000mass (Da)

9x

10x

11x

TNFαR (PNGase F treated, desialylated)Undigested

28500 31500

EXOglycosidases

GalactEXO

®

38

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.42),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®

39

TheGlycINATORenzymeconsistsof2000unitsfordeglycosylationof2mgIgG.Theenzymeisprovidedasalyophilizedpowder.


A0-GL1-020 GlycINATOR, 2000 units 2 mg IgG 450 625

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

2 mg IgG 495 655

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


A0-GL6-010 Immobilized GlycINATOR Microspin 2 x 0.5 mg 335 465

A0-GL6-025 Immobilized GlycINATOR Microspin 5 x 0.5 mg 765 1,060

A0-GL6-050 Immobilized GlycINATOR Microspin 10 x 0.5 mg 1,270 1,765

A0-GL6-100 Immobilized GlycINATOR Midispin 1-10 mg 1,020 1,415

A0-GL6-1000 Immobilized GlycINATOR Maxispin 10-100 mg 3,045 4,255

30MIN

30MINUTE

30MINUTES

30MIN

IgG Glycosidases

GlycINATO

R®

40

SmartEnzymes

IgGZERO® (EndoS) is an IgG-specific endoglycosidase that hydrolyzes Fc glycans.

TheIgGZEROenzymehydrolyzesN-glycansspecifi-callyattheFcglycosylationsiteonIgG.IncontrasttoGlycINATOR,IgGZEROhaslimitedactivityonhigh-mannoseandhybrid-typeglycoforms.Thehydroly-sisofcomplexglycansisfastandcarriedoutundernativeandphysiologicalconditions,astheenzymerequiresnativeIgGfoldforactivity.TheIgGZERO enzymeisusedtorapidlyreducesamplecomplexity,inimmunoassaystoreduceantibodymediatedef-fectorfunctions,orasatooltoimproveimagingbyreducingFcinteractions(Gao,2014,p.42).

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.42).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

®

41

TheIgGZEROenzymeconsistsof2000unitsfordeglycosylationof2mgIgG.Theenzymeisprovidedasalyophilizedpowder.

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


A0-IZ1-010 IgGZERO, 1000 units 1 mg IgG 230 320

A0-IZ1-050 IgGZERO, 5000 units 5 mg IgG 915 1,270

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

2 mg IgG 500 655


A0-IZ6-010 deGlycIT Microspin 2 x 0.5 mg 335 465

A0-IZ6-025 deGlycIT Microspin 5 x 0.5 mg 765 1,060

A0-IZ6-050 deGlycIT Microspin 10 x 0.5 mg 1,270 1,765

A0-IZ6-100 deGlycIT Midispin 1-10 mg 1,020 1,415

A0-IZ6-1000 deGlycIT Maxispin 10-100 mg 3,045 4,255

deGlycIT™

30MIN

30MINUTE

30MINUTES

30MIN

IgG Glycosidases

IgGZERO

®

42

SmartEnzymes

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

*SiteClick™isprovidedunderanintellectualpropertylicensefromLifeTechnologiesCorporation.ThetrademarkSiteClick™isthepropertyofLifeTechnologiesCorporation. SeeLegalandDisclaimers,p.43.

GlyC

LICK

®

Immobilized GlycINATOR

GalT(Y289L)UDP-GalNAz

DIBO alkyne label

2. AZIDE ACTIVATION 3. CLICK REACTION1. DEGLYCOSYLATION

GlyCLICKisathree-stepconjugationtechnologyforsite-specificandquantitativeconjugationofIgGfromseveralspeciesandsubclasses(Fig.1).ThetechnologyutilizesFcglycanremodelingbytheGlycINATOR®endoglycosidaseforcompletedeglycosylationofallFcglycoformsfollowedbyazideactivation.Biocompatibleclick-chemistry(SPAAC)thenenablescompleteandrobustlabelingoftheantibodyusingacyclooctyne-functionalizedlabelofchoice.TheuniquecombinationofenzymesinGlyCLICKresultsinadegreeoflabeling(DOL)of2labelsperIgG,generatingstableandhomogenousconjugatessuitableforsensitiveandversatileapplications.TheGlyCLICKtechnologyisavailableinvariouskitformatstofacilitatetailoredandsite-specificlabelingofantibodies.

Available GlyCLICK Formats and sDIBO Labels

1. Deglycosylation TheIgG-specificendoglycosidaseGlycINATOR(EndoS2)digestsFcglycans,exposingthecoreGlcNAc. Theenzymeremovesallglycoforms,includinghigh-mannose,hybrid,complex,andbisectingtypeglycans.

2. Azide activation Azide-containingUPD-GalNAzisenzymaticallyattachedtotheexposedGlcNAcattheFcglycansitesbytheenzymeβ-1,4-Galactosyltransferase,GalT(Y289L),makingtheantibodyazide-activatedforconjugation.

3. CLICK reaction Azide-activatedantibodiesareconjugatedattheFcglycansitesusingaDBCOorsDIBO-labelthroughStrain-PromotedCopper-freeClick-Chemistry(SPAAC),resultinginastableattachmentofonelabelperFc/2.

GlyCLICK Conjugation Overview

Figure 1. Schematic presentation of the GlyCLICK conjugation process.

Kit format Kit size Name Examples of Applications

Fluorophore 250 μg, 2 mg AlexaFluor® 488Immunofluorescence Microscopy, Flow cytometry



Affinity 250 μg, 2 mg Biotin Immuno assays, ELISA, WB

Chelator 250 μg, 2 mg Deferoxamine In vivo immuno imaging

Azide Activation

250 μg, 2 mg, 10 mg

No label Custom conjugation

Human IgG1-4, IgG from mouse, rabbit, rat, monkey, sheep, goat, cow and horse

Labels: Alexa Fluor®, biotin and DFO. Azide activation kits for custom conjugation

GlycINATOR® hydrolyzes all Fc glycoforms

43

Antibody Conjugation

GlyCLICK®

GlyCLICK Characteristics

Site-specific and Modular Technology

GlyCLICKenablesquantitativeandcompleteconjugationwithaconstantdegreeoflabeling(DOL)ofonelabelperFc/2.TheuniquecombinationofenzymesallowsFcglycan-specificremodelingthatrestrictslabelingtotheFcglycansiteswithoutinterferencewiththeantigenbindingdomainoftheantibody.GlyCLICKisaversatileandscalabletoolforconjugationofseveralspeciesandsubclassesofIgGwithawiderangeoffunctionalizedlabels(Fig.2).Allconjugateswillhavethesamereproduciblemodeofincorporation,independentonthechoiceoflabelorpayload.

Figure 2. RP-HPLC analysis of panitumumab unmodified and conjugated with DFO, AlexaFluor488, Biotin or MMAE digested with FabRICATOR. RP-HPLC was performed on Agilent 1290 using Waters Acquity UPLC® BEH C4, 1.7 μm, 2.1 x 100 mm column.

Preserved Immunoreactivity

Site-specificconjugationattheFcglycansitesensuresintactimmunoreactivitybypreservingtheantigen-bindingcapabilityoftheantibody.SurfacePlasmonResonance(SPR)wasperformedontrastuzumabwithvariousGlyCLICKconjugatesandcomparedtorandomconjugates.TheresultsshowoverlappingcurvesbetweenGlyCLICK-conjugatesandnativetrastuzumab(Fig.3).Althoughtheaffinityisunchangedforrandomlyconjugatedmaterial,thelevelofbindingisseverelyimpared.

Figure 3. Affinity analysis of native and conjugated trasuzumab. Anti-human IgG (Fc) was used as the capturing molecule for trastuzumab, RandomDyLightTM488 (DOL=10) conjugates and GlyCLICK conjugates: AlexaFluor488, Biotin, DFO and MMAE (DOL=2).HER2 was injected at a range ensuring sufficient curvature. All data was fitted against a 1:1 mathematical model.

min4.5 5 5.5 6 6.5 7 7.5

mAU

0

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Fc/2

Fc/2 AlexaFluor®488

Fc/2 BiotinFc/2 DFO

Fc/2 MMAE

F(ab’)2F(ab’)2 of conjugates

0 200 400 600 8000

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Resp

onse

Uni

ts (R

U)

Trastuzumab

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200 400 600 8000

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onse

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U)

Trastuzumab DyLight488

20

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Trastuzumab DFO

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Trastuzumab Biotin

0 200 400 600 8000

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Resp

onse

Uni

ts (R

U)

Trastuzumab MMAE

0

Random GlyCLICK

Control GlyCLICK GlyCLICK

GlyCLICK

44

SmartEnzymes

Site-spectificconjugationofprimaryantibodiesusingGlyCLICKensuresquantitativelabelingforhigh-qualitydynamicimagingoftheantigendistributionincellsandtissuesections(Fig.4).TheconstantDOLof2enablesimprovedquantitationpossibilitiesatoptimalintensitylevelsforminimalsaturationorlossofsignalwithlessnoisefrombackgroundstainingandcross-reactivityascomparedtosecondarydetection.Themodular GlyCLICKtechnologyallowscustomconjugationusingarangeoffluorescentlabelsforimagingapplications.

“Thistechnologybenefitsthefieldsofbasicresearchanddrugdevelopmentandmayformthebasisforexcitingdiagnostictoolsinclinicalhistopathology.”

Immunofluorescent Microscopy

Radioactivelylabeledantibodiesareexcellenttracersforimmunoimaginginvivo.TheinvivoeffectofGlyCLICKconjugatedtrastuzumab-DFOlabeledwith89Zrwasanalyzedandcomparedtorandomlabelingatlysines(DOL=0-6).Tumor-bearingmiceinjectedwithGlyCLICK-conjugatesshowafivefoldincreaseintumoruptakeaswellasasignificantlylongercirculationtimecomparedtorandomlyconjugatedtrastuzumab(Fig.6).

Immunoimaging In Vivo

Figure 6. PET/CT imaging analysis of SK-OV-3 tumor-bearing mice showing mean tumor uptake over a time interval of 0-168 hours post-injection.

4h 24h 70h 120h 168h0

5

10

15

20

%ID

/g (m

ean)

Tumor PET uptake (mean)

89Zr-trastuzumab (Random)89Zr-trastuzumab (GlyCLICK)

Hours post-injection

GlyCLICK Applications

Flow Cytometry

Figure 4. HER2(+) cells or tissue. Left: PFA-fixed cells incubated with DAPI (0.2 uM), GlyCLICK-AlexaFluor647 (1 ug/ml). Middle: PFA-fixed cells incubated with DAPI (0.2 uM), GlyCLICK-AlexaFluor647(5 ug/ml), phallodinAF488. Right: paraffin embedded, BSA blocked tissue incubated with DAPI (0.2 uM), GlyCLICK-AlexaFluor647 (10 ug/ml).

Figure 5. Flow cytometry of Fc-blocked HER2(+) cells incubated with trastuzumab-GlyCLICK-AlexaFluor647 or Cy5 (3.7 ug/ml) 30 min or mouse primary antibody (3.7 ug/ml) 30 min and secondary donkey anti-mouse (1:200) 30 min. Cells were resuspended (PBS 1%BSA) and analyzed with CytoFlex flow cytometer.

Directlylabeledantibodiesforprimarydetectionofferadvantagesinflowcytometryapplicationsbuttherightcombinationofantibodyandlabelmaynotbeavailable.Theriskofunspecificbindingandcross-reactivityusingwidelyavailablesecondaryantibodiesoftenrequireintricateexperimentaldesignswithcross-adsorbedmaterialfromvariousspecies.GlyCLICKenablesrobustandspecificconjugationforincreasedflexibilityinprimaryantibodyselectionforsensitiveormultiplexedanalyses.PrimarydetectionwasevaluatedusingGlyCLICK-conjugatesandcomparedtoanoptimizedsecondarydetectionmethodusingflowcytometry.TheGlyCLICK-conjugatesdisplayedhigherseparationindexandreducedbackgroundcomparedtodetectionusingsecondaryantibodies(Fig.5).

Intens.

Count

1000

800

600

400

200

0102 103 104 105 106 107

Flow Cytometry (MDA-MB-453 cells)

Mouse anti-HER2 + Donkey anti-mouse-AF647T-GlyCLICK-AlexaFluor647 T-GlyCLICK-Cy5

Donkey anti-mouse-AF647 (Control)

GlyC

LICK

®

45

GlyCLICKcontainsallreagentsandmaterialsneededtoazideactivateorlabeltheIgG.

Antibody Conjugation

GlyCLICKenablescustomconjugationusingthewarheadofchoiceforhomogenousmaterialwithminimalbatch-to-batchvariation(Fig.8).

ADC Development

Figure 8. HIC separation of GlyCLICK conjugated trastuzumab-DM1, DBCO-PEG4-Ahx-DM1,and randomly conjugated trastuzumab-DM1 on TSKgel@Butyl-NPR column (Tosoh Biosciences) in 25 mM NaP, 1.5 M ammonium sulfate and eluted with decreasing salt gradient (20% isopropanol). DAR variants of T-DM1 were unresolved due to heterogeneity of hydrophobicity in the material.

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0,1 1 10 100

% v

ialbl

e ce

lls c

ompa

red

to c

ontro

l

Concentration T-GlyCLICK-DM1 (ug/mL)

GlyCLICK on SK-BR-3 cells

Antibody-drugconjugates(ADCs)arepotenttherapeuticagentscombiningthehighselectivityofantibodieswithcontrolleddosageofcytotoxicpayloads.Incontrasttoconventionalrandomlabelingataminoacidsites,site-specificlabelingoffersreliabledrug-antibody-ratio(DAR)andhavebeenobservedtoexhibitimprovedpharmacologicperformanceinvivo.Thesite-specificGlyCLICKconjugationpreservesfullantigen-bindingcapacityandexhibitsdose-responsecytotoxicity(Fig.7).

Figure 7. Cytotoxicity analysis of HER2(+) cells (SK-BR-3) by DM1-conjugated trastuzumab, DBCO-PEG4-Ahx-DM1, using GlyCLICK and compared to randomly conjugated trastuzumab-DM1. Cell viability was measured using CellTiter-Glo® Luminescent assay (Promega), 5000 cell/well incubated with 0-10 ug/ml GlyCLICK-conjugates at 37°C, 5% CO2 for 72 h prior to addition.

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mAU

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Trastuzumab

T-GlyCLICK-DM1

T-DM1

Brentuximab vedotin

Product ID Description Size EUR USD

L1-F01-025

L1-F01-200

GlyCLICK Alexa Fluor® 488


Conjugates 250 μg IgG

Conjugates 1 x 2 mg IgG

925

1,495

985

1,545

L1-F02-025

L1-F02-200





925

1,625

985

1,695

L1-F03-025

L1-F03-200





925

1,625

985

1,695

L1-C01-025

L1-C01-200

GlyCLICK DFO

GlyCLICK DFO



925

1,495

985

1,545

L1-A01-025

L1-A01-200

GlyCLICK Biotin

GlyCLICK Biotin



925

1,495

985

1,545

L1-AZ1-025

L1-AZ1-200

L1-AZ1-100

GlyCLICK Azide Activation



Activates 250 μg IgG

Activates 1 x 2 mg IgG

Activates 1 x 10 mg IgG

825

1,250

5,145

875

1,355

6,195

GlyCLICK®

46

SmartEnzymes

An,Y.etal.,2014.A new tool for monoclonal antibody analysis: application of IdeS proteolysis in IgG domain-specific characterization.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 electrophoresis. 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.

Gao,P.etal.,2014. Deglycosylation of mAb by EndoS for Improved Molecular Imaging. MolecularImagingandBiology,pp.1–9.Marcoux,J.etal.,2015.Native mass spectrometry and ion mobility characterization of trastuzumab emtansine, a lysine-

linked antibody drug conjugate. Proteinscience:apublicationoftheProteinSociety,24(8),pp.1210–1223.Sokolowska,I.etal.,2017.Subunit mass analysis for monitoring antibody oxidation. mAbs,9(3),pp.498–505.

Bobály,B.etal.,2017.Protocols for the analytical characterization of therapeutic monoclonal antibodies. II – Enzymatic and chemical sample preparation.JournalofChromatographyB,1060,pp.325–335.

Liu,S.&Zang,L.,2016.Rapid quantitation of monoclonal antibody N-glyco-occupancy and afucosylation using mass spectrometry. AnalyticalBiochemistry,509(C),pp.142–145.

Sjögren,J.etal.,2015. EndoS and EndoS2 hydrolyze Fc-glycans on therapeutic antibodies with different glycoform selectiv-ity and can be used for rapid quantification of high-mannose glycans. Glycobiology,25(10),pp.1053–1063.

Upton,R.etal.,2016.Orthogonal Assessment of Biotherapeutic Glycosylation: A Case Study Correlating N-Glycan Core Afucosylation of Herceptin with Mechanism of Action. AnalyticalChemistry,88(20),pp.10259–10265.

Wang,Y.etal.,2017.Monitoring Glycosylation Profile and Protein Titer in Cell Culture Samples Using ZipChip CE-MS. JournalofAnalytical&BioanalyticalTechniques,08(02).

Selected ReferencesFa

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47

Legal and Disclaimers

Allrightsreserved.Genovisproductsmaybecoveredbyoneormorepat-ents,trademarksandcopyrightsownedorcontrolledbyGenovisAB.

Formoreinformationaboutcommercialrights,[email protected].

Genovisproductsareintendedforresearchuseonly.Theyarenotintendedtobeusedfortherapeuticordiagnosticpurposesinhumansoranimals.

FabRICATOR®

Thisproductisprovidedunderanexclusiveworld-wideintellectualpropertylicensefromHansaBiopharmaABderivedfrominternationalpublica-tionWO03051914,includinggrantedUSPatentNoUS7,666,582andgrantedEuropeanPatentNo1458861.ThelicenseencompassesIdeSfromStreptococcuspyogenesforbiotechnicalindustrialapplicationswhichareneithertherapeuticnordiagnostic,otherthanthefollowingexceptionwhichisincludedwithinthelicense:digestingIgGinvitroinclinicalsamplesfordiagnosticpurposes.

POROS® included in FabRICATOR® HPLCThisproductisprovidedunderanintellectualpropertylicensefromLifeTechnologiesCorporation.Thetransferofthisproductisconditionedonthebuyerusingthepurchasedproductsolelyinresearchorforanalyticaltestingduringmanufacturing,allconductedbythebuyer,excludingcontract

researchoranyfeeforserviceresearch,andthebuyermustnot(1)usethisproductoritscomponentsfor(a)diagnostic,therapeuticorprophylacticpurposes;(b)testing,analysisorscreeningservices,orinformationinreturnforcompensationonaper-testbasis;and/or(c)sellortransferthisproductoritscomponentsforresale,whetherornotresoldforuseinresearch.Forinformationonpurchasingalicensetothisproductforpurposesotherthanasdescribedabove,contactThermoFisherScientific,5826NewtonDrive,Carlsbad,[email protected].

SiteClickTM included in GlyCLICK®

ThisproductisprovidedunderanintellectualpropertylicensefromLifeTechnologiesCorporation.Thetransferofthisproductisconditionedonthebuyerusingthepurchasedproductsolelyinresearchconductedbythebuyer,excludingcontractresearchoranyfeeforserviceresearch,andthebuyermustnot(1)usethisproductoritscomponentsfor(a)diagnostic,therapeuticorprophylacticpurposes;(b)testing,analysisorscreeningservices,orinformationinreturnforcompensationonaper-testbasis;or(c)manufacturingorqualityassuranceorqualitycontrol,and/or(2)sellortransferthisproductoritscomponentsforresale,whetherornotresoldforuseinresearch.Forinformationonpurchasingalicensetothisproductforpurposesotherthanasdescribedabove,contactLifeTechnologies Corporation,5781VanAllenWay,Carlsbad,CA92008USAor [email protected].

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48

SmartEnzymes

[email protected] | www.genovis.com

EMEA & Asia

Genovis AB Box 790 SE-220 07 Lund Sweden

Customer service: +46 46 10 12 30 Order phone: +46 46 10 12 30 Order fax: +46 46 12 80 20 Email: [email protected]

US & Canada

Genovis Inc. 245 First Street, Suite 1800 Cambridge, MA 02142 USA

Customer service: 617-444-8421 Order phone (toll free): 855-782-0084 Order fax: 858-524-3006 Email: [email protected]

PB

2020-3

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.

Documents

SmartEnzymes€¦ · fragments with no enzyme in the final preparation. 90 MINUTES 60 Product ID Description Digestion EUR USD A2-FZ2-005 FragIT Z kit 0.5 mg IgG 380 520 A2-FZ2-025