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 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
Physiological buffers
2 mM cysteineReducing conditions
Physiological buffers
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
Product ID Description Digestion EUR USD
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
Product ID Description Digestion EUR USD
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®
Product ID Description Digestion EUR USD
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
Noneedforreducingagentsor co-factors
CPAPNLLG/GPSVF(belowthehinge)Digestion of Mouse IgG2a using FabRICATOR Z
Product ID Description Digestion EUR USD
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
Product ID Description Digestion EUR USD
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
Product ID Description Digestion EUR USD
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
Noneedforreducingagentsor co-factors
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
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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.
Product ID Description Digestion EUR USD
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.
Product ID Description Digestion EUR USD
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.
*ThermoScientific™CaptureSelect™resinfromThermoFisherScientificInc.anditssubsidiaries.ThermoScientificandCaptureSelectaretrademarksofThermoScientificInc.anditssubsidiaries.
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.
Product ID Description Digestion EUR USD
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
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40
60
80
100
24.740
27.214
min10 20 30 40 50
mAU
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20
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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
*ThermoScientific™CaptureSelect™resinfromThermoFisherScientificInc.anditssubsidiaries.ThermoScientificandCaptureSelectaretrademarksofThermoScientificInc.anditssubsidiaries.
GingisKHANFabkitconsistsof2000unitsoftheGingisKHANenzyme,5xlyophilizedreducingagentand 4xCaptureSelect™*CH1affinityspincolumnsforgenerationandpurificationofFabfragmentsfromhumanIgG1antibodies.
Product ID Description Digestion EUR USD
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.
Product ID Description Digestion EUR USD
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.
Product ID Description Digestion EUR USD
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
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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.
Product ID Description Digestion EUR USD
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]
EPO PNGaseF Smix LS 1-20_P1-A-1_01_160.d: UV Chromatogram, 280 nm
SAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGD
APPRLICDSRVLERYLLEAKEAEDITTGCAEHCSLDENITVPDTKVDFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAA
OpeRATOR + OglyZER + Pngase F
'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]
APPRLICDSRVLERYLLEAKEAEDITTGCAEHCSLDENITVPDTKVDFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAA
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
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]
'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)
0.0
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
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]
APPRLICDSRVLERYLLEAKEAEDITTGCAEHCSLDENITVPDTKVDFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAA
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
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]
'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)
0.0
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]
APPRLICDSRVLERYLLEAKEAEDITTGCAEHCSLDENITVPDTKVDFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAA
Teor:13714.0957DaMeas:13714.1199Da
Undigested,PngaseF treatedEPO:APPRLICDSRVLERYLLEAKEAEDITTGCAEHCSLDENITVPDTKVDFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDTeor:18596.6397DaMeas:18648.6797Da(unexplained+54Damassdifference)
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]
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]
EPO PNGaseF Smix LS 1-20_P1-A-1_01_160.d: UV Chromatogram, 280 nm
SAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGD
APPRLICDSRVLERYLLEAKEAEDITTGCAEHCSLDENITVPDTKVDFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAA
OpeRATOR + OglyZER + Pngase F
'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]
APPRLICDSRVLERYLLEAKEAEDITTGCAEHCSLDENITVPDTKVDFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAA
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
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]
'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)
0.0
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
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]
APPRLICDSRVLERYLLEAKEAEDITTGCAEHCSLDENITVPDTKVDFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAA
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
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]
'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)
0.0
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]
APPRLICDSRVLERYLLEAKEAEDITTGCAEHCSLDENITVPDTKVDFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAA
Teor:13714.0957DaMeas:13714.1199Da
Undigested,PngaseF treatedEPO:APPRLICDSRVLERYLLEAKEAEDITTGCAEHCSLDENITVPDTKVDFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDTeor:18596.6397DaMeas:18648.6797Da(unexplained+54Damassdifference)
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
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 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
5. + SialEXO and OglyZOR
1 2 3 4 5 6 7 8 91 2 3 4 5 6 7 8 9(kDa)
160
11080
60
50
40
6. + Endoglycosidase (E. faecalis) and sialidase (C. perfringens)
7. Fetuin
8. + SialEXO and OglyZOR
9. + Endoglycosidase (E. faecalis) and sialidase (C. perfringens)
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®
Enzymes for O-glycans
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.
Product ID Description Deglycosylation EUR USD
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.
Product ID Description Deglycosylation EUR USD
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.
Product ID Description Deglycosylation EUR USD
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
Product ID Description Deglycosylation EUR USD
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.
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Immobilized GlycINATOR
GalT(Y289L)UDP-GalNAz
DIBO alkyne label
AZIDE ACTIVATION CLICK REACTIONDEGLYCOSYLATION
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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).
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Antibody Conjugation
GlyCLICK®
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SmartEnzymes
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Legal and Disclaimers
Allrightsreserved.Genovisproductsarecoveredbyoneormorepatents,pendingpatentapplications,trademarksand/orcopyrightsownedor controlledbyGenovisAB.
Formoreinformationaboutcommercialrights,[email protected].
Genovisproductsareintendedforresearchuseonly.Theyarenotintendedtobeusedfortherapeuticordiagnosticpurposesinhumansoranimals.
FabRICATOR®
Thisproductisprovidedunderanexclusiveworld-wideintellectualpropertylicensefromHansaMedicalABderivedfrominternationalpublicationWO03051914,includinggrantedUSPatentNoUS7,666,582andgrantedEuropeanPatentNo1458861.ThelicenseencompassesIdeSfromStreptococcuspyogenesforbiotechnicalindustrialapplicationswhichareneithertherapeuticnordiagnostic,otherthanthefollowingexceptionwhichisincludedwithinthelicense:digestingIgGinvitroinclinicalsamplesfordiagnosticpurposes.
POROS® included in FabRICATOR® HPLCThisproductisprovidedunderanintellectualpropertylicensefromLifeTechnologiesCorporation.Thetransferofthisproductisconditionedonthebuyerusingthepurchasedproductsolelyinresearchorforanalytical
testingduringmanufacturing,allconductedbythebuyer,excludingcontractresearchoranyfeeforserviceresearch,andthebuyermustnot(1)usethisproductoritscomponentsfor(a)diagnostic,therapeuticorprophylacticpurposes;(b)testing,analysisorscreeningservices,orinformationinreturnforcompensationonaper-testbasis;and/or(c)sellortransferthisproductoritscomponentsforresale,whetherornotresoldforuseinresearch.Forinformationonpurchasingalicensetothisproductforpurposesotherthanasdescribedabove,contactThermoFisherScientific,5826NewtonDrive,Carlsbad,[email protected].
SiteClick® 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,contactLifeTechnologiesCorpo-ration,5791VanAllenWay,Carlsbad,CA92008USAor [email protected].
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SmartEnzymes
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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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