USP WORKSHOP ON GLYCOSYLATION ANALYSIS FOR ... · 8/25/2015 · USP Workshop on Glycosylation...

USP Workshop on Glycosylation Analysis for Biopharmaceuticals Workshop August 25-26, 2015 USP Meetings Center, Rockville, Maryland USA

USP WORKSHOP ON GLYCOSYLATION ANALYSIS FOR BIOPHARMACEUTICALS

August 25-26, 2015 USP Meetings Center, Rockville, MD USA

Final Agenda

DAY ONE: Tuesday, August 25, 2015 8:00 – 8:30 a.m.

Registration

8:30 – 8:40 a.m.

USP Welcome & Workshop Introduction Tina Morris

Senior VP, Science Global Biologics, USP

SESSION 1 Opening Keynote Presentation Introduction: Chris Jones, Ph.D.

8:40 – 9:20 a.m.

The Critical Features of Glycosylation for the Therapeutic Products

Pauline M. Rudd, Ph.D. National Institute for BioProcessing Research and Training in Ireland (NIBRT), University College, Dublin (Ireland)

SESSION 2

The Role of Glycosylation for Therapeutic Proteins Moderator: Michael DeFelippis, Ph.D.

9:20 – 9:45 a.m.

The Use of Alternative Methods as Surrogate Measures of Biological Activity

Guoying Jiang, Ph.D. Genentech

9:45 – 10:10 a.m.

Break

10:10 – 10:35 a.m.

Characterization and Impact of Glycosylation in an IgG fusion protein

Christopher Barton, Ph.D. MedImmune

10:35 – 11:00 a.m.

Case Study: Influence of Fab Glycosylation on The Pharmacological Properties of a Monoclonal Antibody

Bryan Harmon, Ph.D. Eli Lilly and Company


11:00 a.m. – 11:30 a.m.

Panel Discussion

11:30 a.m. – 12:30 p.m.

Lunch & Poster Session

SESSION 3 Current Technologies Moderator: Trish Li, Ph.D.

12:30 – 12:55 p.m.

Common Analytical Methods for Oligosaccharide and Monosaccharide Analysis

Parastoo Azadi, Ph.D. Member, USP Glycoproteins and Glycan Analysis Expert Panel

12:55 – 1:20 p.m.

The Common Challenges Faced Today When Performing Glycosylation Analysis

Jeffrey S. Rohrer, Ph.D. Member, USP Glycoproteins and Glycan Analysis Expert Panel

1:20 – 1:45 p.m.

Practical Glycoprofiling Approach for Measurement and Control of Glycosylation

Tapan Das, Ph.D. Bristol-Myers Squibb

1:45 – 2:15 p.m.

Panel discussion

2:15 – 2:35 p.m.

Break

SESSION 4 Standardization Moderator: Chris Jones, Ph.D.

2:35 – 2:55 p.m.

Standard procedures: USP <1084>, <212> , <210>, and <129> Chris Jones, Ph.D.

Chair, USP Glycoproteins and Glycan Analysis Expert Panel

2:55 – 3:05 p.m.

USP Reference Standards Edith Chang, Ph.D.

USP

3:05 – 3:30 p.m.

The NIST mAb: A Reference Material to Supplement Biopharmaceutical Characterization

John Schiel, Ph.D. National Institute of Standards and Technology (NIST)

3:30 – 4:00 p.m.

Panel discussion

4:00 – 5:00 p.m.

Networking Reception & Poster Session


DAY TWO: Wednesday, August 26, 2015 8:00 – 8:30 a.m.

Registration

8:30 – 9:10 a.m.

SESSION 5 Plenary Session: Regulatory Expectations

Christopher Downey, Ph.D. U.S. Food and Drug Administration (FDA)

SESSION 6

Emerging Technologies Moderator: Parastoo Azadi, Ph.D.

9:10 – 9:35 a.m.

In Vitro Glycoengineering – A Useful Tool for Protein Characterization and Optimization

Marco Thomann Roche Diagnostics GmbH

9:35 – 10:00 a.m.

Bacterial Glycoengineering: from enzymes and pathways to human therapeutics and vaccines

Matt DeLisa, Ph.D. Department of Chemical and Biomolecular Engineering, Cornell University

10:00 – 10:25 a.m.

Break

10:25 – 10:50 a.m.

Relative and Absolute Quantitation of Glycoprotein Glycans Using Isotopically Labeled Standard Glycoproteins

Ron Orlando, Ph.D. Complex Carbohydrate Research Center, University of Georgia

10:50 11:15 a.m. MAb Glycan Profiling by LC/MS Peptide Mapping Bhavana Shah, Ph.D.

Amgen 11:15 11:45 a.m. Panel discussion

11:45 a.m. – 12:45 p.m.

Lunch & Poster Session

SESSION 7 Regulatory Approaches on Glycosylation Moderator: John Cipollo, Ph.D.

12:45 – 1:10 p.m.

What are the Expectations from the Regulatory Agencies John Mark, Ph.D.

Health Canada


1:10 – 1:35 p.m.

Expectations for Glycosylated Biosimilars (manufacturer perspective)

Catherine Srebalus Barnes, Ph.D. Hospira

1:35 – 2:00 p.m.

Expectations for Glycosylated Biosimilars (regulatory perspective) Birgit Schmauser, Ph.D.

Federal Institute for Drugs and Medical Devices (BfArM) (Germany)

2:00 – 2:30 p.m.

Break

SESSION 8 Post-approval changes registration Moderator: John Schiel, Ph.D.

2:30 – 2:55 p.m.

Modulation of Glycosylation Patterns by Bioprocess Techniques José M. Gomes, Ph.D.

Pfizer

2:55 – 3:20 p.m.

Post-approval Changes – Regulatory Perspective Maria-Teresa Gutierrez-Lugo, Ph.D.

OBP/CDER/U.S. Food and Drug Administration

3:20 – 3:50 p.m.

Panel discussion

3:50 – 4:10 p.m.

What Was Said & Next Steps Trish Li, Ph.D.

USP

4:10 – 4:30 p.m.

Closing Remarks Chris Jones, Ph.D.

Chair, USP Glycoproteins and Glycan Analysis Expert Panel

4:30 p.m. Workshop Adjourns


USP WORKSHOP ON GLYCOSYLATION ANALYSIS FOR BIOPHARMACEUTICALS

August 25-26, 2015 USP Meetings Center, Rockville, MD USA

Poster Presentations

1. Tomasz Baginski et al. Genetech

End-to-End HILIC UHPLC Glycoprofiling Method for Development, Process Characterization/Validation and Quality Control of Therapeutic Monoclonal Antibodies

2. Kudrat Goswami et al. Merck

Advanced analytical Technologies for N-Glycan Profiling: Assessment of InstantABTM AssayMap® Automation and GlycoworksTM Rapifluor-MSTM

3. András Guttman et al. Sciex Separations

Multi-Site N-Glycan Mapping by Capillary Electrophoresis

4. Akira Harazono National Institute of Health Sciences

New Japanese Pharmacopoeia General Test and General Information for Glycosylation Analysis of Glycoprotein

5. Jenifer L. Hendel et al. Ludger Ltd

A Fluorescent Labeling and Enrichment System for Glycopeptides Generated from Proteolytic Digestion of IgG mAbs; A System That Can Be Used as Part of the Peptide Mapping Workflow

6. Anne Kroll Kristensen et al. Novo Nordisk

Implementation of QbD Principles in Designing a Fast, Robust Method for Profiling of N-linked Carbohydrates in Antibodies

7. Jeremy Kunkel et al. Health Canada

Comparison of Orthogonal Chromatographic and Lectin-Affinity Microarray Methods for Glycan Profiling of a Therapeutic Monoclonal Antibody

8. Matthew A. Lauber et al. Waters Corporation

Wide-Pore Amide HILIC Separations for Assaying the Domain-Specific Glycosylation of mAbs

9. Matthew A. Lauber et al. Waters Corporation

Rapid Preparation of N-Glycans Using a Novel Fluorescence and MS Active Labeling Reagent

10. Leitão, O.L. et al. National Institute for Health Quality

New methods for Oligosaccharide Analysis from Recombinant Human Erythropoietin by UPLC/ESI/QTOF/MS in Brazil

11. Mark Lies et al. Sciex Separations

Rapid Level-3 Characterization of Glycoprotein Therapeutics BY CESI-MS

12. Paula Magnelli et al. New England Biolabs

Improving Sample Workflow: Rapid PNGase F for Accurate Antibody Characterization

13. Loretta Yang et al Glycosensors and Diagnostics

Development of a Glycoprofiling Method Using Multiplex Flow Cytometry

14. Jia Zhao et al. Merck

An effective LC-MS Based Method for On-line Characterization of N-linked Glycans in Biological Therapeutics

USP WORKSHOP ON GLYCOSYLATION ANALYSIS

FOR BIOPHARMACEUTICALS

Speaker / Moderator / Planning Committee Information (alpha order)

U . S . P H A R M A C O P E I A L C O N V E N T I O N

ParastooAzadi,Ph.D.USPAffiliation:Member,USPGlycoproteinsandGlycanAnalysisExpertPanelTechnicalDirectorUniversityofGeorgiaAthens,GA,USADr.AzadireceivedherB.Sc.inChemistryin1987fromUniversityofNorthLondon,UKandherPh.D.degreeinbiochemistryin1991fromImperialCollegeofScienceandTechnology,UniversityofLondon,studyingstructuralcharacterizationofcarbohydratesandglycoproteinsbymassspectrometryunderthesupervisionofProfs.A.DellandH.R.Morris.In1990throughto1994shewastheseniorscientistandthestudydirectoratM‐Scanlimited,anAnalyticalMassSpectrometryConsultancyinUKwhereshewasresponsibleforcompletestructuralcharacterizationofnativeandrecombinantproteinsandglycoproteinsusingmassspectrometryasaservicetothepharmaceuticalindustry.In1994shejoinedtheComplexCarbohydrateResearchCenterasapostdoctoralfellowandstudiedtheeffectoftheenzymesendohyrolaseandendolyaseonrhamnogalacturonanI,andcharacterizationofthefragmentsproducedbytheseenzymesbyESI‐MSandESIMS/MS.In1996shebecametheAssociateTechnicalDirectorofplantandmicrobialAnalyticalServicesattheComplexCarbohydrateResearchCenterwhereshewasresponsibleforplantandmicrobialserviceprogramwherepolysaccharidesandlipopolysaccharideswereanalyzedforotherinstitutes.Since2001,Dr.AzadihasbeentheTechnicalDirectorofAnalyticalServiceandTrainingattheComplexCarbohydrateResearchCenter.AstheTechnicalDirector,thesheoverseesandmanagesallanalyticalservicesandtrainingconductedattheCCRC,whicharesupportedbythreefederalresourcecentersofexcellencethatCCRChasbeenawarded:TheDepartmentofEnergy‐fundedCenterforPlantandMicrobialComplexCarbohydrates,theNationalInstitutesofHealthResourcesCenterforIntegratedGlycotechnology,andtheNationalInstitutesofHealthforBiomedicalGlycomics.Theanalyticalserviceprogramofferstwomainareasofservice:standardanalysesandcontractanalyses.Thesamplessubmittedforthesetypesofanalysescomefromacademic,government,non‐profitorganizationsandprivatecompanies,throughouttheUnitedStatesandinternationally.Dr.Azadiworkscloselywiththeresearchscientistsinindustryondevelopingcarbohydratebaseddrugsandtheneedforout‐sourcingpriortophaseIandphaseIIclinicaltrials.

USPWorkshoponGlycosylationAnalysisforBiopharmaceuticalsWorkshopAugust25‐26,2015,USPMeetingsCenter,Rockville,Maryland,USA

Herlaboratoryalsoconductsresearchinareasofstructuralcharacterizationofplant,bacterialandanimalpolysaccharides,glycoproteinsandglycolipidsusingMSandNMRtechniques.

PlanningCommitteeMemberModeratorSession6:EmergingTechnologiesWednesday,August26,2015,9:10a.m.–11:45a.m.PresentationAbstractCommonAnalyticalMethodsforOligosaccharideandMonosaccharideAnalysisTuesday,August25,2015,12:30p.m.–12:55p.m.Productionofhigh‐qualitypharmaceuticalrecombinanttherapeuticglycoproteinwithconsistencyinglycanqualityisstillchallenging.Sinceglycansareresponsibleforbioactivity,solubility,immunogenicity,andclearanceratefromcirculation,itisvitaltohavedetailedmapofglycansinrecombinanttherapeuticglycoprotein.However,duetotheenormousdiversityofcarbohydratestructuresandtheirheterogeneity,thisstillremainsoneofthebottlenecksoffullstructuralcharacterization.Detailedglycoproteinstructuralanalysishastobeabletoidentifythepeptidesequencewheretheglycansareattached,aswellasthestructureoftheglycanportion,includingoligosaccharidesequenceandglycosyllinkages.Wewilldetailmethodsformassspectrometry(MS)experimentsonbothreleasedglycans(“glycomics”),aswellasonintactglycopeptides(“glycoproteomics”)usingEDT,HCDandCIDfragmentationpathwaysthatareneededtofullyelucidatethestructureofglycoproteins.Additionalprotocolswillbeshownwhereacombinationofglycosylcompositionandglycosyllinkageanalysisusingacombinationofmethylationanalysis,MSnandexoglycosidasedigestionwillprovideinformationontheglycantopologyaswellasdetectionmethodsforpotentialnon‐humanmodificationsthatcouldarisefrommammalianexpressionsystemssuchasGalα1‐3GalandN‐glycolylneuraminicacid(NeuGc).Ourconsolidatedexperimentswilloutlineallthenecessaryinformationpertainingtotheglycoprotein,includingglycanfinestructure,attachmentsite,andglycosylationdegreetobeobtainedpharmaceuticalrecombinantglycoproteins.


ChristopherBarton,Ph.D.SeniorScientistMedImmuneWashington,DC,USAChrisisaSeniorScientistatMedImmune,leadingthenovelmoleculesgroupwithinanalyticalbiotechnology.HestartedworkingoncarbohydrateanalyticsduringaPhDonpolysaccharidesandglycoproteinsynthesisattheUniversityofCambridge.Hecompletedpost‐doctoraltrainingworkinginaUKanti‐dopinglaboratory,anddevelopedexpertiseinmassspectrometryinthecontractresearchsector.HisroleatMedImmuneisfocusedonthedevelopmentofanalyticalapproachesforthetestingandcharacterizationofnon‐monoclonalantibodyproducts.Hisresearchinterestsincludedevelopingtoolsfortheanalysisandunderstandingofproteinglycosylationandpost‐translationalmodificationincellculture.

PresentationAbstractCaseStudy:CharacterizationandImpactofGlycosylationonPKofanIgGfusionproteinTuesday,August25,2015,10:10a.m.–10:35a.m.Non‐monocloncalantibodybiotherapeuticsoftenhavemultipleglycosylationsitesoccupiedwitharangeofgalactosylatedandsialylatedglycans.Thenatureandextentofthesespeciescanimpacttheprotein’spharmacokineticandpharmacodynamicprofile,andisanimportantfeaturetomonitorduringbiotherapeuticdevelopment.WepresentacasestudyofthecharacterizationofN‐glycosylationinadimericIgGfusionproteinwithtwoFcglycosylationsitesandfournon‐Fcsites.Theglycosylationoftheproteinwascharacterizedbyaseriesoftoolsofincreasingdetail.Thefirststagewascompositionalanalysisofthelevelofcomponentneutralmonosaccharidesandsialicacidspecies.Oligosaccharideprofilingofenzymaticallyreleasedglycanswasthenundertakentocharacterizetheglobalglycosylationprofileoftheprotein.Finallyanalysisbypeptidemappingwasusedtodeterminethesite‐specificglycosylationpattern.ThemodelproteinwasdeterminedtobedecoratedwithdistinctglycansateachoftheN‐glycosylationsites.TheFcwasoccupiedprimarilybyagalactosylatedbi‐antennaryglycans.Thenon‐Fcglycanswereoccupiedbyabroaderrangeofspecies,withbi‐antennaryandtri‐antennarycoresbeingobserved,decoratedwithvariablelevelsofgalactosylationandsialylation.Wefurtherpresentanapproachtounderstandtheimpactofglycosylationonproteinfunctionatanearlystageindevelopment,byproducingsamplesofproteinwithspecificglycosylationpatternsandstudyingtheseininvitroandinvivomodelsystems,includinganon‐humanprimatemodeltostudytheimpactof


glycosylationonPK.Thisinformationcanbecombinedwiththeglycosylationanalysistoolsdescribedabovetotargetprocessdevelopmentonaproteinwiththemostdesirableglycosylationstate.


EdithChang,Ph.D.ScientificLiaison,Global BiologicsU.S. Pharmacopeia (USP)Rockville,MD,USADr.ChangisaScientificLiaisonofGlobalBiologicswithUSP.SheservesasaleadliaisonforUSPProteinsExpertCommittee.Dr.ChangreceivedherM.S.andPh.D.inBiochemistrywithaminorinMolecularGeneticsfromUniversityofCalifornia,Riverside.PriortojoiningUSP,sheworkedmostrecentlyatKansasCityUniversityofMedicineandBiosciences,KansasCity,MO,whereshewasAssistantProfessorofBiochemistryandPrincipleInvestigatorforresearchprojectstoinvestigatetheroleofcyclooxygenase2inmalignanttransformationandcancerinvasion.EarlieremploymentwasatThermoFisherPiercewhereshewasaResearchScientistatR&Ddepartment.Dr.Changistheauthororco‐authorofmanypeer‐reviewedpublicationsinthefieldsofbiochemistry,molecularbiologyandcancer,andhasservedasanad‐hockreviewerforseveralpeer‐reviewedjournalsincludingCancerResearch,MolecularCancerTherapeutics,ClinicalCancerResearch,andTheInternationalJournalofBiochemistry&CellBiology.

PlanningCommitteeMemberPresentationAbstractUSPReferenceStandardsTuesday,August25,2015,2:55p.m.–3:05p.m.USP Reference Standards (RSs) are highly-characterized physical specimens used in testing by pharmaceutical and related industries, and are closely tied with the documentary standards published in the USP–NF, Food Chemicals Codex, and Dietary Supplements Compendium. This presentation will provide an overview of USP reference standards, and discuss the biologics RSs that are associated with compendial procedures for analysis of protein glycosylation.


JohnCipollo,Ph.D.USPAffiliation:GovernmentLiaison,VaccinesforHumanUse–ViralVaccinesSeniorStaffFellowU.S.FoodandDrugAdministrationBethesda,MD,USADr.CipolloreceivedhisPh.D.in2000fromtheStateUniversityofNewYorkatAlbany.Heperformedhispost‐doctoralworkatBostonUniversitySchoolofDentalMedicineunderProfessorsCatherineCostelloinmassspectrometryandCarlosHirschberginbiochemistry.Hehaspublishedoverthirtyscientificarticlesintheareasofcarbohydratestructuralanalysisandglycomicswithstrongemphasisincarbohydratemassspectrometry.Dr.Cipollohasworkedextensivelyintheglycomicsofaseriesoforganismsincludinghuman,Caenorhabditiselegans,Entamoebainvadens,Entamoebahistolytica,andseveralyeastspecies.Hiscurrentinterestsincludethefunctionofglycosylationinproteincarbohydrateinteractionsinadaptiveandinnateimmunityandtheimpactofthosefunctionsinvaccinedevelopmentandperformance.Otherinterestsincludenovelchemistriesforimprovementofpolysaccharideconjugateandotherglycoconjugatebasedvaccines.AstherearefewbroadlyacceptedinformaticsplatformsforglycomicsanalysistheCipollogrouphas,andiscurrentlydeveloping,inhouseglycomicssoftwareforprocessingofmassspectrometryandglycanarrayglycomicsdataDr.CipolloservesasaProductSpecialistforCBERFDAprimarilyasaproductreviewerforbacterialpolysaccharidesandpolysaccharideconjugatevaccines.PlanningCommitteeMemberModeratorSession7:RegulatoryApproachesonGlycosylationWednesday,August26,2015,12:45p.m.–2:00p.m.


TapanDas,Ph.D.Director,BiologicsDevelopmentBristol‐MyersSquibbNewYork,USATapanisaDirectorintheAnalyticalDevelopmentgroupofBristol‐MyersSquibb.HeleadstheMassSpectrometryandBiophysicsCenterofExcellenceengagedinadvancedcharacterizationandanalyticsforbiologicsdevelopment.PriortojoiningBMS,hewasatPfizerBiotherapeuticsR&Dandbuiltaworldclassstate‐of‐the‐artfacilityforBiophysicalCenterofExcellence.TapanisamemberofAAPS(AmercianAssociationofPharmaceuticalScientists)andservedAAPSinvariousroles,mostrecentlyservedastheChairoftheBiotechnologySection.

PresentationAbstractPracticalGlycoprofilingApproachforMeasurementandControlofGlycosylation)Tuesday,August25,2015,1:20p.m.–1:45p.m.Glycancompositionisakeyqualityattributeformanyproteintherapeutics.Glycanscaninfluencethemodeofaction,pharmacokinetics,andsafety.Robustanalyticalmethodsarecriticalformeasurementandcontrolofglycansduringprocessdevelopmentandforprocesscontrolinmanufacturing.Choiceofglycanassaymaydependondevelopmentphase,neededthroughput,andcharacterizationdatasought.Thispresentationwillincludebiotherapeuticcasestudiesfor(a)applicationinprocessdevelopmentand(b)forextendedcharacterization.


MichaelDeFelippis,PhDUSPAffiliation:Member,Monographs–BiologicsandBiotechnology1ExpertCommittee;Member,GlycoproteinsandGlycanAnalysisExpertPanel;andChair,TherapeuticPeptidesSeniorResearchFellowEliLilly&CompanyIndianapolis,IN,USAMichaelR.DeFelippis,PhDjoinedtheLillyResearchLaboratoriesofEliLillyandCompanyin1990aftercompletinghisdoctorateinbiochemistry.HeiscurrentlyaSeniorResearchFellowworkingintheBioproductResearchandDevelopmentdivision.Hisworkisfocusedondevelopmentofproteinandpeptidebiopharmaceuticalproductswithparticularemphasisoncharacterizingphysicochemicalproperties,definingdeliveryoptions,developingcontrolstrategies,executingtechnologytransfersandpreparingdatapackagestosupportworldwideregulatorysubmissionsandpost‐launchregistrations.Dr.DeFelippishaspublishedmanuscripts,reviewarticlesandbookchaptersonthesubjectsofproteinandpeptidestructuralcharacterizationandformulationdesign/deliverystrategies.Hehasgivennumerouspresentationsonthesetopicsandisanamedinventoronseveralpatentsrelatedtotheseareas.

PlanningCommitteeMemberModeratorSession2:TheRoleofGlycosylationforTherapeuticProteinsTuesday,August25,2015,9:20a.m.–11:30a.m.


MattDeLisa,Ph.D.WilliamL.LewisProfessorofEngineeringDepartmentofChemicalandBiomolecularEngineering,CornellUniversityIthaca,NewYork,USAProfessorDeLisareceivedaB.S.inChemicalEngineering fromtheUniversityofConnecticut in1996;aPh.D.inChemicalEngineering fromtheUniversityofMarylandin2001;anddidpostdoctoralworkattheUniversityofTexas‐Austin,DepartmentofChemicalEngineering.DeLisajoinedtheDepartmentofChemicalandBiomolecularEngineeringatCornellUniversityasanassistantprofessorin2003.Hewaspromotedtoassociateprofessorin2009andtofullprofessorin2013.Inaddition,herecentlyservedasaGastprofessur attheSwissFederalInstituteofTechnology (ETHZürich)intheInstitutfürMikrobiologie.DeLisahasreceivedseveralawardsforhisworkincludinganNSFCAREERaward(2005),aNYSTARWatsonYoungInvestigatoraward(2004),aBeckmanFoundationYoungInvestigatoraward(2005),anOfficeofNavalResearchYoungInvestigatoraward(2006),aNYSTARDistinguishedFacultyAward(2007),aCornellProvost'sAwardforDistinguishedScholarship(2009),andtheAmericanChemicalSocietyBIOTdivisionYoungInvestigatoraward(2010).Hewasalsonamedasoneofthetop35younginnovators(TR35)byMIT'sTechnologyReview(2005),wasselectedastheAllanP.ColburnMemorialLecturerattheUniversityofDelaware(2009),andwaschosenastheinauguralrecipientoftheWiley‐Blackwell Biotechnology andBioengineeringDanielI.C.Wangaward(2008),whichhonorsadistinguishedyoungresearcherinthisfield.Mostrecently,hewasselectedtotheIDA/DARPADefenseScienceStudyGroup(2014‐15)andelectedtotheAmericanInstituteforMedicalandBiologicalEngineering (2014).ProfessorDeLisa'sresearchfocusesonunderstanding andcontrollingthemolecularmechanismsunderlyingproteinbiogenesis ‐‐foldingandassembly,membranetranslocationandpost‐translationalmodifications ‐‐inthecomplexenvironmentofalivingcell.Hiscontributions toscienceandengineeringincludetheinventionofnumerouscommerciallyimportanttechnologies forfacilitatingthediscovery,designandmanufacturing ofhumandrugsandseminaldiscoveries intheareasofcellularproteinfoldingandproteintranslocation. PresentationAbstractInterestinusingnon‐MammaliancellsWednesday,August26,2015,9:35a.m.–10:00a.m.Carbohydratesaddalevelofdiversityacrossallformsoflifethatisunparalleledbytheinformationcontentofnucleicacidsandproteins.Thelackofasimpletemplatetotranslateaglycancodeintodefinedsugarstructurescontributestothis


complexityandimpedeseffortsaimedattheproductionofbiologicallyimportantglycansandglycoconjugates.Withthediscoveryofglycoproteinsynthesisinbacteriaandfunctionaltransferofglycosylationpathwaysbetweenspecies,Escherichiacolicellshavebecomeatractablehostforunderstandingglycosylationandtheunderlyingglycancodeoflivingcells.Moreover,effortstomanipulatethepathwaysfromsugarnucleotidestoglycolipidstoglycoproteinshavetransformedE.coliintoalivingfactoryforscalable,bottom‐upproductionofcomplexglycoconjugatesbydesign.Here,IwilldiscussoureffortstodevelopE.coliforthebiosynthesisofadiversearrayofglycanstructures,whichcanbeusedtotailortheactivity,stability,half‐life,andimmunogenicityofanarrayofproteintargetsrangingfrombiopharmaceuticalstoindustrialenzymes.IwillalsodiscussoureffortstounifyproteinglycosylationinE.coliwiththeadvancedtoolsofproteinengineeringsuchascellsurfaceandphagedisplaytechnologies.Theresultisapowerfulnewwaytoengineertheenzymes,pathways,end‐products,andgenomesofglycoengineeredbacteriaforcreatingthenextgenerationofproteintherapeuticsandvaccines.


ChrisopherD.Downey,Ph.D.LeadChemistU.S.FoodandDrugAdministration(FDA)/CDEROfficeofBiotechnologyProductsDr.DowneyreceivedhisPhDin2006fromtheUniversityofColoradoatBoulder,wherehestudiedtheconformationaldynamicsofRNAtertiarystructures.From2007to2011,heperformedhispostdoctoralworkattheUniversityofColoradoatBoulder,studyingthemolecularmechanismsofgenomicDNAreplication.Dr.DowneyservedasaProductQualityreviewerinFDA/CDER’sOfficeofBiotechnologyProducts(OBP)beginningin2012.Hehasextensiveexperienceinthereviewofenzymereplacementtherapies,hematologicgrowthfactors,anddigestiveenzymesandintheinspectionoffacilitiesmanufacturingtherapeuticbiotechnologyproducts.Since2014,Dr.DowneyhasservedasaLeadChemistinOBP’sDivisionofBiotechnologyReviewandResearch‐II,providingtraining,technicalandregulatoryguidance,andsecondaryreviewforhisteamofproductqualityreviewers.

PresentationAbstractPlenarySession:RegulatoryExpectationsWednesday,August26,2015,8:30a.m.–9:10a.m.Proteinglycosylationaffectsawide‐rangeofattributesthatcontributetothesafetyandefficacyofproteintherapeutics.Theseattributesincludephysicochemicalstability,immunogenicity,bioavailability,andbiodistribution.Thecomplexityofthebiologicalfunctionstowhichglycosylationcontributesandtheheterogeneousnatureofglycoformsrepresentadistinctchallengeinproductdevelopmentandinregulatoryreview.Thispresentationwillprovideageneraloverview,fromareviewer’sperspective,ofregulatoryexpectationsforcharacterizingglycosylation,linkingglycosylationattributestofunction,anddevelopingacontrolstrategy.Thetalkwillalsodiscussgeneralconceptsandexpectationsforestablishingcomparabilitybeforeandaftermanufacturingchangesandforanalyzinganalyticalsimilaritydataforglycosylationinthecontextofbiosimilars.


JoséM.GomesPrincipalScientist,CultureProcessDevelopmentPfizer,Inc.Andover,MA,USAJoséisabiochemist&biophysicistwithconsiderableexperienceincellculture,chemicalengineering,andmolecularbiologyincluding18yearsofindustryexperience.HejoinedPfizerthroughtheacquisitionofWyethandcurrentlyholdsthepositionofPrincipalScientistwithintheCultureProcessDevelopmentgroupinBiotherapeuticsR&D.Hisworkfocusesondevelopmentandsupportofupstreamprocessesforawidegamutofprojectsfromearly‐stagetolate‐stageclinicalproducts,biosimilars,commercialproducts,andnext‐generationproducts.HeiscurrentlyleadingtheupstreamprocessteamforPfizer’s1stbiosimilarprogram.Hisparticularareasofinterestincludeunderstandingeffectsofprocessparametersonproductquality,utilizationofdesignofexperiments&qualityriskmanagement,applicationofQbD,andunderstandingfactorsaffectingprocesstechtransfer&scale‐up(includingagitationandaerationeffects).Priortojoiningindustry,JoséwasinvolvedinstudyinglipidandhormonemetabolismatTuftsMedicalSchoolandtheU.S.D.A.

PresentationAbstractModulationofGlycosylationPatternsbyBioprocessTechniquesWednesday,August26,2015,2:30p.m.–2:55p.m.Theglycosylationofrecombinantproteinsmaybeacriticalqualityattributedependentonmodeofaction.Variabilityinglycosylationpatternsmayalteranantibody’seffectorfunctionsinpatients,suchasantibody‐dependentcellmediatedcytotoxicity(ADCC),complement‐dependentcytotoxicity(CDC),andFc‐receptorbinding.Therefore,theabilitytomodulateglycosylationpatternscanenablestructure/functionstudiestofurtherproductunderstandingandenablethedevelopmentofoptimizedmanufacturingprocessescapableofconsistentlydeliveringmaterialofdesiredquality.Thispresentationwilldiscusstheapproachesandstrategiesofmodulatingglycosylationpatternsthroughuseofvariousbioprocesstechniquessuchasprocessparametercontrol,cellculturemediasupplements,enzymaticinhibitors,andcelllineselection.


Maria‐TeresaGutierrez‐Lugo,Ph.D.U.S.FoodandDrugAdministrationSilverSpring,MD,USADr.Gutierrez‐LugoisaProductQualityTeamLeaderintheOfficeofBiotechnologyProducts(OBP),CDER,FDA.PriortojoiningtheFDAin2008,Dr.Gutierrez‐LugoconductedpostdoctoralresearchattheNIHandattheUniversityofArizona.SheholdsaPh.D.inChemicalSciences(Pharmacy)fromtheNationalAutonomousUniversityofMexico

PresentationAbstractRegulatoryPerspectiveWednesday,August26,2015,2:55p.m.–3:20p.m.Post‐approvalmanufacturingchangesofbiopharmaceuticalscouldresultinchangesinproductqualityattributeswhichcouldimpactsafetyandefficacyofthedrugproduct.Comparabilityofpre‐changeandpost‐changeproductisneededtodemonstratethatthemanufacturingchangedidnotadverselyimpactthesafetyandefficacyofthedrugproduct.Thispresentationwillprovideageneralbackgroundontheexpectationsofcomparabilityandwillpresentcasestudiesofpost‐approvalmanufacturingchangesofglycosylatedproducts.


BryanHarmon,Ph.D.ResearchFellowEliLillyandCompanyIndianapolis,IN,USAResearchFellow,EliLillyandCompany,Dr.HarmonreceivedhisB.S.inChemistryfromRose‐HulmanInstituteofTechnologyandhisPh.D.inAnalyticalChemistryfromPurdueUniversityunderProf.FredRegnier.Followingapost‐doctoralpositionunderProf.DanielI.C.WangattheBiotechnologyProcessEngineeringCenteratMIT,heworkedinthevaccinedivisionofMerckResearchLaboratoriesfor3years.SincejoiningLillyin1999,Dr.HarmonhasservedasagroupleaderfortheBioproductStructuralCharacterizationTeam.Histeamisresponsiblefordetailedstructuralcharacterizationofallpeptide,protein,andmonoclonalantibodycandidatesinLilly'sdevelopmentpipeline,includingtheelucidationofpost‐translationalmodifications,suchasglycosylation,aswellasdegradationproductsandmechanisms.AtLilly,Dr.HarmonhasalsoplayedaleadingroleinestablishingcomparabilitystrategiesandinthedevelopmentandapplicationofQualitybyDesignprinciplesforbioproducts.Hehasalsoservedastheleaddevelopmentscientistforseverallatephaseprojects.

PresentationAbstractCaseStudy:InfluenceofFabGlycosylationonThePharmacologicalPropertiesofaMonoclonalAntibodyTuesday,August25,2015,10:35a.m.–11:00a.m.Recombinantmonoclonalantibodies(mAbs)intendedforuseastherapeuticagentstypicallycontainaconservedN‐linkedglycosylationsiteintheFcregionthatcanplayanimportantroleinthepharmacologicalpropertiesofthemAb,includingmediatingeffectorfunctionsforIgG1mAbs.ThiscasestudyinvolvesamAbthatcontainsanadditionalN‐linkedglycosylationsiteinitsFabregion.InordertounderstandtheimpactoftheFabglycosylationonthepharmacokineticsofthemAb,themAbwasisolatedfromtimepointsamplesobtainedfromahumanclinicalstudy,andLC‐MSglycosylationanalysiswasperformedinordertoidentifytheoligosaccharidestructuresthatareclearedmorerapidly.ThisinformationwasfurtherutilizedtoestablishacontrolstrategyforensuringconsistentFabglycosylationprofilesand,thus,consistentpharmacokinetics,includingthevalidationoftheLC‐MSmethodasaGMPspecificationtest.


GuoyingJiang,Ph.D.SeniorScientist|BiologicalTechnologiesGenentechSanFrancisco,CA,USAGuoyingJiang,Ph.D.,isaSeniorScientistandGroupLeaderintheBiologicalTechnologiesgroupwithintheAnalyticalDepartment&QualityControlDepartmentatGenentech,aMemberoftheRocheGroup.Guoyingisleadingateamofscientistsandresearchassociatesfocusingonpotencyassaydevelopment,optimizationandvalidationinsupportofGMPlotreleaseandstabilitytesting.HergroupisalsosupportingpotencymethodstransferanddevelopingcharacterizationmethodsforFceffectorfunction.GuoyingobtainedherB.SdegreefromPekingUniversityinChina,andPh.D.degreefromColumbiaUniversityinNewYork.ShewasapostdoctoralscientistinMountSinaischoolofMedicineinNewYorkbeforejoininginGenentechin2008.

PresentationAbstractTheUseofAlternativeMethodsasSurrogateMeasuresofBiologicalActivityTuesday,August25,2015,9:20a.m.–9:45a.m.Antibodiesrecognizingcellsurfaceexpressedantigens,areabletoengageFcγreceptorsoneffectorcellssuchasmonocytes,macrophages,naturalkillercells,neutrophils,orbindtoC1q,andelicitimmuneeffectorfunctionssuchasADCP,ADCC,andCDC.Glycosylationrepresentsoneofthemainsourcesofheterogeneityofantibodyglycoformsandcangreatlyinfluenceeffectorfunctionwithpotentialimpacttosafetyorefficacy.Casestudieswillbepresenteddescribingthecorrelationbetweenglycosylationandbioactivity,andthepotentialuseofphysicochemicalorbindingassaysasasurrogatemeasuresofCDCandADCCactivity.


ChrisJones,Ph.D.USPAffiliation:Chair,USPGlycoproteinsandGlycanAnalysisExpertPanelHeadofDivision,LaboratoryofMolecularStructureNationalInstituteforBiologicalStandardsandControlPottersBar,Hertfordshire,UnitedKingdom

Dr. Chris Jones has worked at NIBSC since 1982, with 23 years as a Head of Division responsible for developing, implementing and validating sophisticated physico-chemical methods for the characterisation of complex biological products. He uses Nuclear Magnetic Resonance (NMR) spectroscopy, circular dichroism and other optical spectroscopy approaches, mass spectrometry and specialised HPLC approaches for glycan and peptide mapping.

Dr. Jones is a world expert on the physico-chemical characterisation of polysaccharide and glycoconjugate vaccines, widely used in infants to protect against meningitis, acute respiratory infections and invasive pneumococcal infections, and typhoid. He is also closely involved in writing regulatory guidance for these products and teaches courses.

PlanningCommitteeMemberModeratorSession4:StandardizationTuesday,August25,2015,2:35p.m.–4:00p.m.PresentationAbstractStandardprocedures:USP<1084>,<212>,<210>,and<129>Tuesday,August25,2015,2:35p.m.–2:55p.m.TheUnitedStatesPharmacopeiaaredevelopingaseriesofgeneralchaptersandreferencestandardstosupporttheroutinetestingofglycoproteins,called<1084>Glycoproteinandglycananalysis–generalconsiderations,<210>MonosaccharideAnalysisand<212>OligosaccharideAnalysis.Thistalkwilldescribetheaims,scope,structureandevolutionofthesechapters,theassociatedreferencestandardsandrelationshiptoproductmonographsandtheproductclasschapter<129>AnalyticalProceduresforRecombinantTherapeuticMonoclonalAntibodies.ClosingRemarksWednesday,August26,2015,4:10p.m.–4:30p.m.


TrishLi,Ph.D.SeniorScienceandStandardsLiaisonU.S. Pharmacopeia (USP)Rockville,MD,USATrishLi,Ph.D.joinedUSPin2006.SheiscurrentlyaSr.ScienceandStandardsLiaisonworkingatBiologics&BiotechnologyDepartment.Shemanagesaportfolioofreferencestandardsandmonographs.PriortoUSP,Dr.LiworkedforPfizer’sGlobalManufacturingDivision,QualityOperationDepartmentatGroton,Connecticut.SheobtainedBScinChemistryfromPekingUniversity,andPh.D.inOrganicChemistryfromUniversityofMiami.

PlanningCommitteeMemberModeratorSession3:CurrentTechnologiesTuesday,August25,2015,12:30p.m.–2:15p.m.PresentationAbstractWhatWasSaid&NextStepsWednesday,August26,2015,3:50p.m.–4:10p.m.Thissessionwillbeasummaryoftheworkshop’sdiscussions.


JohnMark, MSc,PhDHealthCanadaJohnMarkreceivedhisMScinPhysiologyattheUniversityofManitobadevelopingmagneticresonance‐based,non‐invasivediagnosticmethodsattheNationalResearchCouncilCanada‐InstituteforBiodiagnostics.Subsequently,hecompletedaPhDinBiochemistryattheUniversityofOttawawherehestudiedproteinstructure‐functionrelationshipsintherapeutictargetproteins.JohneventuallyjoinedHealthCanadain2008asaResearchChemistintheCenterforVaccinesEvaluation’sProteinChemistrylaboratorywherehisworkfocusedonthedevelopmentofnovelin‐vitromethodstoassessproteincomparability,withaspecificfocusonSubsequentEntryBiologics.Currently,JohnisaSeniorBiologist/EvaluatorwiththeCMCQualitygroupinHealthCanada’sCenterfortheEvaluationofRadiopharmaceuticalsandBiologics,MonoclonalAntibodiesDivision.

PresentationAbstractWhataretheExpectationsfromtheRegulatoryAgenciesWednesday,August26,2015,12:45p.m.–1:10p.m.Proteinglycosylationisoneofnature’smostwidespreadpost‐translationalmodifications.Itisalsooneofthemoredifficultattributestocharacterizebytraditionalanalyticalmethods.Nevertheless,duetothepotentialeffectsthatglycosylationcanhaveonabiologic’spotency,immunogenicity,andcirculatoryhalf‐life;acomprehensivedeterminationoftheglycosylationisrequiredinmajorregulatoryfilings.Butwhataretheexpectations?TherecanbesomeconfusionsincetherequirementsdescribedinICHQ5EandQ6Baregeneral,butthedetailsrequestedbyregulatorsareoftenmuchmorespecificandvaryfromregiontoregionorevenreviewertoreviewer.Toprovidesomeinsightintowhatisexpected,thistalkwillfocusontheCMCQualityreviewprocessatHealthCanadaandtheexpectationsfromaCanadianperspective.Topicswillincludethetheoreticalrisksrelatedtochangesinglycosylation,thechallengesassociatedwithproducingproductwithacceptableglycanvariability,anddiscussionoftheregulatoryexpectationsforglycancharacterizationandanalysis.


RonOrlando,Ph.D.ProfessorofBiochemistryandMolecularBiologyComplexCarbohydrateResearchCenter,UniversityofGeorgiaAthens,GA,USADr.RonOrlandoreceivedhisB.S.innaturalsciencein1983fromSt.Mary'sCollegeofMaryland,hisPh.D.inchemistryin1988fromtheUniversityofDelaware,andservedasapost‐doctoralfellowattheUniversityofMarylandBaltimoreCountyfrom1988until1990.AfterservingfortwoyearsasaseniorscientistattheSuntoryInstituteofBioOrganicResearch(Osaka,Japan),hejoinedthefacultyoftheComplexCarbohydrateResearchCenterattheUniversityofGeorgiainJanuary1993,whereheiscurrentlyaProfessorofBiochemistry&MolecularBiologyandChemistry.RonOrlandohasover27yearsofexperiencewithmassspectrometry,23oftheseyearsfocusedontheidentification,characterization,andquantitationofproteinsandtheirpost‐translationalmodifications.Hehasco‐authoredover120publicationsinpeerreviewedjournals,hasgivenover100invitedlecturesatvariousconferences,andhasservedonover40NIHreviewpanels.Dr.OrlandoisthecurrentEditor‐in‐ChieffortheJournalofBiomolecularTechniques(JBT),andservesonanumberofeditorialboards.Hehasorganizedandtaughtshort‐coursesontheAnalysisofGlycoproteinsbyMassSpectrometryatmeetingsoftheAmericanSocietyforMassSpectrometry(ASMS),theAssociationofBiomolecularResourceFacilities(ABRF)andtheInternationalSymposiumandExhibitontheSeparationofProteins,Peptides&Polynucleotides(ISPPP),andisthefounderandpast‐chairoftheABRFGlycoproteinResearchGroup.Dr.Orlandoisalsoaserialentrepreneurhavingfounded3spin‐off/start‐upcompanies:BioInquirein2007,GlycoScientificin2009andPhotochemTechnologiesin2012.Hewasgiventheawardof"EntrepreneuroftheYear"fromtheGeorgiaBioBusinessCenter,andwasnamedaleaderoftomorrowbySpectroscopymagazine.PresentationAbstractRelativeandAbsoluteQuantitationofGlycoproteinGlycansUsingIsotopicallyLabeledStandardGlycoproteinsWednesday,August26,2015,10:25a.m.–10:50a.m.Theabilitytoaccuratelyquantitatetheglycanchainsattachedtoglycoproteinshaswide‐rangingimplications.Numerousstudiesoverthepast40yearshavedemonstratedthatabnormalglycosylationoccursinvirtuallyalltypesofhumancancers,anddemonstratethepotentialofusingglycanmarkersineitheradiagnosticoraprognosticmanner.Theglycosylationonrecombinantproteintherapeuticsisalsoknowntohaveprofoundeffects,withoneofthebetterknownexamplesbeingtheincreasedserumhalf‐lifeoferythropoietin(EPO)resultingfromglycoengineering.Hence,thequantificationofglycoproteinglycansplayimportantrolesfromthediscoveryofnewdiagnostic/prognosticmarkerstothedevelopmentoftherapeuticagents.


Acurrentimpedimentforperformingquantitativeglycomicsistheshortageofstandardglycoproteinsandisotopicallylabeledreagentstoenableaccuratequantitation.Thedifficultywithglycanquantitationhasbeenhighlightedbyinter‐laboratorystudiesconductedbytheHumanProteomeOrganization(HUPO)andtheAssociationofBiomolecularResourceFacilities(ABRF),whichdemonstratedthatquantitativemeasurementoftheglycansinasinglesampledeviatedbymorethan100%acrossparticipatinglaboratories.ThefocusofthispresentationistheevaluationofanIgGwithisotopicallylabeledglycansasaninternalstandardforboththerelativeandabsolutequantitationofN‐linkedglycansreleasedfrombothhumanserumIgGsandwholehumanserum.Thesestudiesdemonstratedthattheuseoftheinternalstandardleadtoasignificantlyimprovedaccuracy,reducedthecoefficientsofvariation(CVs)byover10fold,droppingthestandarddeviationtolessthan10%fromtheover100%deviationobtainedwithoutthelabeledstandard,andgreatlyreducedtheinter‐laboratoryvariabilitydespiteexperimentsbeingperformedbydifferentresearchersindifferentlocations.TheimprovementsassociatedwiththeisotopicallylabeledIgGleadustopredictthatthiswillbeavaluablestandardfortheanalysisofglycoproteinglycans.


JeffRohrer,Ph.D.DirectorofApplicationsDevelopmentDionexProductsforThermoFisherScientific,Inc.Sunnyvale,CADr.RohreristheDirectorofApplicationsDevelopment,DionexProductsforThermoFisherScientific,.InthispositionhedirectstheworkofthecorporateapplicationslaboratoryinSunnyvaleCalifornia.HealsoadvisesandreviewstheworkofotherchromatographylabsatThermoFisherScientific.TheselabsdevelopHPLC,IC,HPAE‐PAD,andotherLC‐basedassays.Dr.Rohrerisanauthorof69peer‐reviewedpublicationsandisamemberoftheUnitedStatesPharmacopeia(USP)ExpertCommitteeonMonographDevelopmentforSmallMolecules#1,amemberoftheUSPExpertPanelonModernizationofIdentificationTests,andamemberoftheUSPExpertPanelonGlycoproteinandGlycanAnalysis.Between2005and2010hewasamemberoftheUSPExpertCommitteeforantibioticsmonographdevelopment.Heisalsotheco‐editorofabooktitledApplicationofIonChromatographyforPharmaceuticalandBiologicalProductsthatwaspublishedin2012.Dr.RohrerjoinedThermoFisher(thenDionex)in1989andhasheldpositionsthatinclude,MarketingFieldChemist,SeniorBiochemistinR&D,andApplicationsLabManager.PriortojoiningThermoFisherhespenttwoyearsasapost‐doctoralassociateinDr.ElizabethTheil'slabatNorthCarolinaStateUniversitystudyingthemechanismofirondepositionintotheproteinferritin.JeffreceivedhisBSinChemistryfromFranklinandMarshallCollegein1981andhisPh.D.inChemistryfromtheUniversityofDelawareworkingwithDr.HaroldWhiteIIIstudyingtheroleofglycosylationinthetransportofchickenserumriboflavin‐bindingproteinacrosstheoocytemembrane.

PresentationAbstractTheCommonChallengesFacedTodayWhenPerformingGlycosylationAnalysisTuesday,August25,2015,12:55p.m.–1:20p.m.Whiletherearenumerousmethods/techniquesforcharacterizingaprotein’sglycosylation,nosingleonecanprovidealltheinformationdesired,andnomethodiswithoutitsliabilities.Forthesereasons,fewifanylabsrelyonjustonemethodofglycosylationanalysis.Threecommonglycosylationassaysaremonosaccharidecomposition,sialicacidcomposition,andoligosaccharideprofiling.Thispresentationwilllookatsomeofthechallengesfacedforeachofthesethreeassays,withafocusonliquidchromatographictechniques.Thechemicalandenzymaticsamplepreparationrequiredforeachassaywillbeamajorpartofthediscussion.Forexample,whataretheproblems/challengesofacidhydrolysisformonosaccharideanalysis,shouldIchooseacidhydrolysisorneuraminidasetreatmentforsialicacidanalysis,andshouldIanalyzenativeorlabeledoligosaccharides?Accuracy,reproducibility,sensitivity,analysistime,andotherfactorswillbediscussedastheypertaintoeachmethodandtypicalanalysisneeds.


PaulineRudd,Ph.D.ResearchProfessorofGlycobiologyNationalInstituteforBioProcessingResearchandTraining(NIBRT),UniversityCollege,DublinDublin,IrelandProfessorPaulineM.RuddBSc,LRIC,MA(Oxon),PhDistheResearchProfessorofGlycobiologyatUniversityCollege,Dublin.SheheadstheGlycoSciencesResearchGroupattheNationalInstituteforBioProcessingResearchandTraininginIreland(NIBRT)wheresheisaPIandconsultant.Inadditiontoherbasicresearchinterests,shehasmanylinkswithpharmaceuticalcompaniesacrosstheworldbecauseGlycoScienceisamajorareaofspecialisedexpertiserequiredtoensurethesafetyandefficacyofbiotherapeuticdrugs,suchasmonoclonalantibodiesforcancerandautoimmunedisorders.TheopenaccessdatabasesandbioinformaticsprogrammesdevelopedinherlabhaverecentlybeenincorporatedintotheWatersUNIFIsoftwarewheretheyaretailoredtowardstheneedofBioPharma.ProfessorRuddobtainedaBScinChemistryattheUniversityofLondonandaPhDinGlycobiologyattheOpenUniversity,UK.ShewasaFoundingScientistofWessexBiochemicals(laterSigmaLondon),VisitingResearchAssociateatTheScrippsResearchInstitute,CA,VisitingProfessorofBiochemistryatShanghaiMedicalUniversityPRC,VisitingScientistatBenGurionUniversityoftheNegev,IsraelandanErskineVisitingFellow,CanterburyUniversity,Christchurch,NewZealand.SheisaFellowoftheRoyalSocietyofMedicine,London,visitingProfessoratSt.George’sHospital,LondonandanAdjunctProfessoratNorthEasternUniversity,Boston,NUIGalway,TrinityCollegeDublinandatUniversityCollege,Dublin.SheisaVIatBTI,AStar,Singapore.In2010shewasawardedtheJamesGregoryMedalandanAgilentThoughtLeaderawardandin2012shereceivedaWatersGlobalInnovationaward.In2014shewasawardedanHonoraryDoctorateatGothenburgUniversity,Sweden.ProfessorRuddhasmorethan280scientificpublicationsandhasgivenover350lecturesandseminarsatinternationalmeetings.BeforemovinghergrouptoDublinin2006,ProfessorRuddwasamemberoftheOxfordGlycobiologyInstitutefor25years,whereshewasaSeniorResearchFellowandaUniversityReaderinGlycobiologyattheUniversityofOxford.PresentationAbstractTheCriticalFeaturesofGlycosylationfortheTherapeuticProductsTuesday,August25,2015,8:40a.m.–9:20a.m.AlterationsinglycosylationarecommoninphysiologicalandpathologicalprocessesaswellasinthebioprocessingofproductssuchasMAbsandEtanercept..GlycansgiverisetocriticalfeaturesorGCQAs(GlycosylatedCriticalQualityAttributes)thatcanaffectsafetyandefficacy,suchashalf‐life,Fc


dependentdownstreamfunctionandimmunogenicity.Glycanstructuresare,inthefirstinstance,controlledbygenes,howevertheprocessingpathwaysalsodependonthebioprocessingconditionsandcellspecificpathways.Theseregulateglycoproteinexpressionandprovidefurthermechanismsforfinetuninganddiversifyingtheglycansandthefunctionsoftheproteinstowhichtheyareattached.Theprofiling,characterisation,anddetailedanalysisofreleasedN‐andO‐glycansandglycopeptidesischallenging,thereforeaplatformhasbeendesignedtosimplifythetechnologywhilepreservingtheintegrityofthedata.Aroboticplatformtoreleaseandlabelglycansfromglycoproteinsina96/384wellplateformathasbeendevelopedasafrontendtoglycanseparationstechnologiesincludingonline‐HILIC/MS/MSandcapillaryelectrophoresis.DatabasesforthesetechnologiesareopensourceandalsoincorporatedintoWatersUNIFIsoftwareaswllasbeingavailableontheNIBRTwebsite(http://glycobase.nibrt.ie/tools.html).Asensitivelabel(ADC)hasrecentlybeendevelopedbyNIBRTandWatersalsohavedevelopedRapifluor.Theplatformhasbeenoptimisedfortherigorous,detailedandquantitativeanalysisofbiopharmaceuticalsateachstageofthemanufacturingprocessandisalsoapplicabletobasicresearch.Theplateformatmakesitconvenientforlargesamplesets;itisrelativelycheap,robustandquantitative.Thebioinformaticsprogrammesenablethelessexperiencedresearchertohandlethedataandthepossibilityofintegratingtheprogrammeswithother–omicsdataisnowonthehorizon.ReferencesAutomated,high‐throughputIgG‐antibodyglycoprofilingplatform.StöckmannH,AdamczykB,HayesJ,RuddPM.AnalChem.2013Sep17;85(18):8841‐9.Genomicsmeetsglycomics‐thefirstGWASstudyofhumanN‐GlycomeidentifiesHNF1αasamasterregulatorofplasmaproteinfucosylation.LaucG,EssafiA,HuffmanJE,RuddPM,RudanI.etal.,PLoSGenet.2010;6(12).Automated,high‐throughputserumglycoprofilingplatform.StöckmannH,O'FlahertyR,AdamczykB,SaldovaR,RuddPM.IntegrBiol(Camb).2015Jul20.[Epubaheadofprint]UltrahighThroughput,Ultrafiltration‐BasedN‐GlycomicsPlatformforUltraperformanceLiquidChromatography(ULTRA3).StöckmannH,DukeRM,MillánMartínS,RuddPM.AnalChem.2015Jul31.[Epubaheadofprint]Inpress:JustinM.Prien,HenningStöckmann,SimoneAlbrecht,SilviaM.Martin,M.Varatta,M.Furtado,S.Hosselet,MeiyaoWang3TrinaFormolo,PaulineM.Rudd,and,JohnE.SchielACSpublicationsCh8.TheElucidationofCoandPost‐TranslationalModificationsOrthogonalTechnologiesforNISTmAbN‐GlycanStructureElucidationandQuantitation


JohnSchiel,Ph.D.ResearchChemistNationalInstituteofStandardsandTechnology(NIST)Gaithersburg,MD,USADr.SchielreceivedhisBS(2004)andPh.D.(2009)inchemistryfromtheUniversityofNebraska‐Lincoln.Dr.SchieljoinedtheNationalInstituteofStandardsandTechnologyin2009tobeginworkonglycoanalyticalmethoddevelopment,andiscurrentlyaresearchchemistintheNISTBiomolecularMeasurementDivisionwithanappointmentattheInstituteforBioscienceandBiotechnologyResearch.HeisleadingLC‐andMS‐basedbiomanufacturingresearcheffortsatNIST/IBBR;developingasuiteoffundamentalmeasurementscience,standards,andreferencedatatoenablemoreaccurateandconfidentcharacterizationofproductqualityattributes.Dr.SchielisalsotheprojectcoordinatorfortherecombinantIgG1κNISTmonoclonalantibodyReferenceMaterial(NISTmAb)programandaco‐editoroftheassociatedACSbookseries“State‐of‐the‐ArtandEmergingTechnologiesforTherapeuticMonoclonalAntibodyCharacterization”.Heisanauthorofover20peerreviewedpublications,4bookchapters,andrecipientofnumerousAwards,includingtheACSDivisionofAnalyticalChemistryFellowship,BioanalysisYoungInvestigatorAward,andUNLEarlyAchieverAward.

ModeratorSession8:Post‐approvalchangesregistrationWednesday,August26,2015,2:30p.m.–3:50p.m.PresentationAbstractProvidingWell‐characterizationStandards(NIST)Tuesday,August25,2015,3:05p.m.–3:30p.m.Monoclonalantibodies(mAbs)arethefastedgrowingclassoftherapeutics,whosesafetyandefficacyhavebeendirectlylinkedtoglycoprofilecontent.Thecomplex,multi‐antennarynatureofglycanshasresultedindevelopmentofatiered,lifecyclestage‐appropriatetoolboxofanalyticalapproachesforglycoanalysis.Asaruleofthumb,moreinformation‐richmethodsrequiremorecomplexsamplehandlingandanalysis.Evaluationofevolvingglycoanalyticalmethodshasbeensupplementedbyawidelyavailablerepresentativetestmaterial.ThispresentationwilldiscusstheestablishmentofanIgG1kReferenceMaterialexpectedtomorefirmlyunderpinregulatorydecisionsandfacilitatethedevelopmentoforiginatorandfollow‐onbiologics.TheRMisintendedforavarietyofusesincluding,butnotnecessarilylimitedto:systemsuitabilitytests,establishingmethodorinstrumentperformanceandvariability,comparingchanginganalyticalmethods,assistinginmethodqualification,etc.TheNISTmAbglycoanalyticalcharacterizationandcertificationwillbediscussed.Conclusionsdrawnfromtheunprecedentedindustry‐widebookcollaboration“State‐of‐the‐ArtandEmergingTechnologiesforTherapeuticMonoclonalAntibodyCharacterization”willalsobepresented.


BirgitSchmauser,Ph.D.FederalInstituteforDrugsandMedicalDevices(BfarM)Bonn,GermanyDr.BirgitSchmauserisaseniorassessorofpharmaceuticalqualityattheGermanRegulatoryAuthority(BfArM)intheUnit“PharmaceuticalBiotechnology,Biologicals,Inspections”.ShestudiedpharmacyattheUniversityofMunichandholdsaPhDfromBerlinFreeUniversity.Shehasscientificandregulatoryexperienceofmanyyears.Herexpertiseandprofoundknowledgeinglycobiologyoptimallyapplyinevaluationofbiotechnology‐derivedmedicinalproductsincludinginvestigationalmedicinalproducts.ForseveralyearsshehasbeenatemporaryadviserfortheWorldHealthOrganisation(WHO)inaprequalificationprogramme.

PresentationAbstractExpectationsforGlycosylatedBiosimilars(regulatoryperspective)Wednesday,August26,2015,1:35p.m.–2:00p.m.Asimilarbiologicalmedicinalproductisdevelopedbaseditsown,uniquemanufacturingprocess.Atthesametimetheclaimtobebiosimilartoanexistingbiologicalmedicinalproductrequiresadaptationsofprocessdesignandperformanceinsuchawaythatthebiosimilar´squalitytargetproductprofileaswellasitsqualityattributesandcharacteristicsarehighlysimilartothereferencemedicinalproduct.Glycosylationofabiologicalsubstancemayexhibitahighlevelofcomplexityandinherentmolecularvariabilityperseincludingdiversityintypeandsiteofglycosidiclinkages,intypeofmonosaccharideswithsite‐specificmodificationsandintheextentofbranchingandbranchelongation.Themanufacturerofabiosimilarisconsequentlychallengedtodemonstratethatthereareconsistentrangesofsimilaritybetweentheglycostructuresofthebiosimilarandreferenceproducttoasufficientextentandthattheseconsistentsimilarityrangesallowreducingthepanelofnon‐clinicalandclinicalstudiestobeperformedwiththebiosimilar.Thelevelofdetailthatcanbeachieveduponanalysisofglycosylationprofileshastremendouslyincreasedinthepastyears.Thusitisoneofthemainregulatoryexpectationsthatmanufacturersofbiosimilarsprovideathorough,comprehensivecomparisonofbiosimilarandreferenceproductwiththenecessarylevelofstructuraldetailtopicturetheglycosylationprofilesofbothproductsandderivethesimilarityclaimthereof.Evaluationofsimilarityshouldincludeathoroughdiscussionofallstructuralresultsobtainedaswellasasoundjustificationfortherepresentativenessofresultsbasedonnumberandchoiceofbatchesforcomparison.Itisconsideredessentialtodistinguishacceptableandunacceptabledifferencestakingintoaccountthecriticalqualityattributesdefined.Neverthelessaregulatorydecisionwillalwaysincludethequalityofdataprovidedaswellasinterpretationandjustificationprovidedtoexcludethatpotentialdifferencesobservedmayhaveanunexpectedimpactonsafetyandefficacy.


BhavanaShahSeniorScientistAmgenInc.,ThousandOaks,CA,USABhavanaisaseniorscientistintheproductattributedsciencegroupoftheprocessdevelopmentdepartmentatAmgenInc.ShehasworkedasananalyticalchemistinvariousAmgendepartmentsincludingresearch,quality,andprocessdevelopmentformorethan25years.ShehasdevelopedamethodofglycanprofilingforglycoproteinandantibodyproductsbyLC‐MSMSpeptidemapping.Shehasextensiveexperienceinsite‐specificglycancharacterizationaswellasreferencestandardcharacterizationsofglycoproteinandmonoclonalantibodies,usingvariousLCandmassspectrometrytechniques.Asapartofqualitydepartment,shedeveloped,validated,andimplementedroutinelotreleasemethods,suchasglycanprofilingandsialicacidestimationforglycoproteins.Sheisalsoinvolvedinmethoddevelopmentfortheestimationofpost‐translationmodificationsandmisincorporationofproteins,aswellasmonoclonalantibodies.BhavanahasanM.S.inAnalyticalandMedicinalChemistryfromtheUniversityofBombay,India.Shehasauthoredseveraltechnicalresearcharticlesinpeer‐reviewedpublications.PresentationAbstractMAbGlycanProfilingbyLC/MSPeptiteMappingWednesday,August26,2015,10:50a.m.–11:15a.m.TraditionallyN‐glycananalysisofaglycoproteinisdonebyhighpHanionexchangechromatography(HPAEC),reversedphaseliquidchromatography(RPLC)orrecentlydevelopedhydrophilicinteractionliquidchromatography(HILIC).ThesemethodsrequirereleaseofN‐glycansbyenzymefollowedbyfluorescentlabelingofN‐glycans.Theprotocolislaborintensiveandtimeconsuming,anddoesnotprovidesite‐specificglycosylationinformation.Liquidchromatography‐tandemmassspectrometry(LC‐MS/MS)peptidemappingisroutinelyusedforproteinstructuralcharacterizationand,asabonus,canpotentiallyprovideglycanprofileoneachindividualglycosylationsite.Inthiswork,arecentlydevelopedglycopeptidefragmentationmodelisusedforautomatedidentificationofglycan,basedontheirMS/MS(fragmentationpattern)ofN‐glycopeptidesfromproteolyticdigestionofmonoclonalantibodies(mAbs).LC‐MSconditionswereoptimizedforaccurateglycanprofiling.GlycanprofilesobtainedfromLC‐MS/MSwerecomparedwiththeprofilesobtainedbyconventionalmethodslikeHPAEC,RPLCandHILIC.RobustnessoftheLC‐MS/MSmethodisalsoevaluatedforaccuracy,reproducibility,andlinearity.Basedonthesameconceptafullyautomatedfasthighthroughputmethodisalsodevelopedforat‐lineprofilingofmAbglycansdirectlyfromcellculturemedia.TheLC‐MS/MSpeptidemappingmethodwithfullyautomateddataanalysisrequireslesssample


preparationandalsoprovidessite‐specificinformation.ItcanserveasanalternativemethodforroutineprofilingofN‐glycansonimmunoglobulinsaswellasotherglycoproteinswithsimpleN‐glycans.


CatherineSrebalusBarnes,Ph.D.SeniorDirector,GlobalBiologics–AnalyticalSciencesHospiraGlobalBiologicsR&DLakeForest,IL,USADr.SrebalusBarnesiscurrentlytheSeniorDirectorforHospiraGlobalBiologics,BioanalyticalSciences.HercurrentresponsibilitiesincludemanagementofHospira’sGlobalBioanalyticalSciencesgroupslocatedintheU.S,AsiaandAustralia.Thesegroupsareresponsibleforbioanalyticalandbioassaymethoddevelopmentandvalidation,characterizationofHospiraandoriginatorbiologicproductsaspartofanalyticalbiosimilarityassessments,bioanalyticalsupportforcelllineandprocessdevelopment/optimizationandtestingoroversightforoutsourcedrelease/stabilitytestingofproductsforuseinclinicaltrials.Inherrole,CathyisresponsiblefordefinitionandimplementationofstrategiesfordemonstrationofanalyticalbiosimilaritybetweenHospiraandoriginatorproductsandprovidingoversightfordevelopmentandimplementationofanalyticalcontrolstrategiesforHospira’sbiologicproducts.Sheactivelyparticipatesinauthoring/reviewofRegulatorysubmissionsandmeetingswithRegulatoryagenciesinsupportofHospira’sbiologicsprograms.PriortojoiningHospirain2012,CathyheldanumberofleadershiproleswithincreasinglevelsofresponsibilityintheBioproductDevelopmentorganizationatEliLillyandCompany,includingDirector,BioproductAnalyticalDevelopmentandDirector,BioprocessPurificationDevelopmentandViralSafety.CathyhasaB.S.inChemistry(WestVirginiaUniversity,U.S.A.)andaPh.D.inAnalyticalChemistry(IndianaUniversity,U.S.A).PresentationAbstractExpectationsforGlycosylatedBiosimilars(manufacturerperspective)Wednesday,August26,2015,1:10p.m.–1:35p.m.FDAguidelinesforbiosimilarproductsrequirethatsponsorsdemonstratetherearenoclinicallymeaningfuldifferencesinthesafety,purityandpotencyoftheproposedbiosimilarproductandreferenceproducttosupportapproval.Thedevelopmentofglycoproteinbiosimilarproductsrequiresacomprehensivecomparisonofglycosylationattributesfortheproposedbiosimilarproductandthereferenceproduct.Theanalyticalbiosimilarityassessmentshouldstartwithanassessmentoftheproductattributesthatarepotentiallyclinicallymeaningful(i.e.,CriticalQualityAttributesorCQAs).Thecriticalityofspecificglycosylationsitesandglycanstructuresdependsonthemechanismofactionforthespecificglycoprotein.TheglycosylationCQAsshouldbeconsideredtodefinetheanalyticalmethodsandglycosylationattributesthatarethefocusofthebiosimilarityassessment.


AproposedframeworkfordefiningtheglycosylationCQAsforbiosimilarglycoproteinproductsandtheapplicationoftheproposedCQAstodefinetheanalyticalmethodsandstrategyfortheanalyticalbiosimilarityassessmentwillbediscussed.Casestudiesfortherapeuticmonoclonalantibodiesanderythropoietinbiosimilarproductswillbeusedtodemonstratetheapproach.Inaddition,theuseofsupportiveinvitroandinvivostudiestorefinepreliminaryCQAassignmentsformonoclonalantibodiesanderythropoietinbiosimilarproductswillbediscussed.


MarcoThomannGroupleaderDevelopmentAnalyticsRocheDiagnosticsGmbHMunich,GERMANYMarcoThomannhasbeenworkingwithRocheDiagnosticsinPenzberg,Germany,forelevenyearsnow.HefinishedhisstudyofBiotechnology/BioinformaticsatUniversityofAppliedSciencesinWeihenstephan,Germany,withaGerman“DiplomaEngineer”degree.HestartedatRochewithhisdiplomathesisinthefieldofBioinformatics,continuedasananalyticspecialist,whereheworkedonthecharacterizationofproteinsusingdifferentHPLC,spectroscopicandmassspectrometrytechniquesinaGMPenvironment.In2008,Marcobecamegroupleaderinthesamedepartment,responsibleforthecharacterizationoftherapeuticproteinsduringclinicalphaseIIandIIIincludingpreparationofregulatoryfilings.Recently,hegotalsoinvolvedinCQAassessmentforalatestageproject.Hisgroupalsofocusesonoligosaccharideanalyticsaswellasglycoenzymecharacterizationforinvitroglycoengineeringandtheirapplicationontherapeuticproteins.

PresentationAbstractInVitroGlycoengineering–AUsefulToolforGlycanStructure‐FunctionCharacterizationandMoleculeOptimizationWednesday,August26,2015,9:10a.m.–9:35a.m.Glycosylationoftherapeuticproteinsisofspecialinterestsinceitcansignificantlyimpactbiologicalactivity.Glycosylationisheterogeneousandsubjecttobatch‐to‐batchvariability,thusitsimpactonproteinfunctionisnoteasytoinvestigate.However,assessmentofe.g.antibodyFcglycanstructure–functionrelationshipbecomesmoreandmoreofinterest.Inthistalktheinvitroglycoengineering(IVGE)technologyanditsapplicationwillbeintroduced.MultipleexampleswillbeshownwhereIVGEaddsaclearbenefittoglycancharacterizationaswellasinvitrostructure‐functionanalysis.SamplepreparationworkflowsforinvitrogalactosylationandsialylationofIgG1Fcglycanswillbeexplained.AnditseffectontheMode‐of‐Action,FcreceptorbindingandADCCactivity,aswellastheimpactonpharmacokineticpropertieswillbeaddressed.AlsoIVGEhasbeenusedtosupportCQAassessmentofalatestageproject.Insummary,thetechnologycanbeappliedtoincreasespecificmoleculeknowledgeaswellastooptimizemolecularpropertiesofclinicalcandidates.


EarlZablackis,Ph.D.USPAffiliation:Member,Monographs–BiologicsandBiotechnology2ExpertCommitteePanel;Member,VaccinePolysaccharideNMRIdentityTestingExpertPanel;Member,VaccinesforHumanUse–ViralVaccinesExpertPanel;Member,1041BiologicsRevisionExpertSubcommittee;andMember,210MonosaccharideAnalysisSubcommitteeDirector,MethodValidationSanofiPasteurSwiftwater,PA,USAIamabiologist/botanistwhodevelopedakeeninterestincellwallpolysaccharidesandthusbecamecarbohydratebiochemist.MyeducationtookmethroughprogramsattheUniversityofCalifornia,BerkeleytotheUniversityofHawai’iandfinallytheUniversityofCalifornia,SantaBarbara,followedbyapost‐doctoralfellowshipattheComplexCarbohydrateResearchCenterattheUniv.ofGeorgia.Mycareerspans30yearsworkingasacarbohydratechemistinacademiaandthebiopharmaceutalindustry.Mycarbohydrateworkwasfocusedontheisolation,purificationandcharacterizationoffinestructureofpolysaccharidesusingmultipletechniquesemployingvariousseparationtechnologiestovariousanalyticaltoolssuchasmassspectroscopyandNMR.Myindustrialexperiencecoversglycananalysisfromrecombinantglycoproteins,monoclonalantibodiesandbacterialpolysaccharidesforvaccines.Ihaveadditionalexpertiseinvalidationofanalyticalmethods.InmycurrentpositionatSanofiPasteur(vaccines)intheManufacturingTechnologydepartment,IamtheDirector‐PrincipalScientistintheAnalyticalProcessandTechnologyplatformresponsibleformethodqualification,validationandtransferprogramsaswellascompliancewithqualitysystems.IamcurrentlyamemberoftheBiotechnologyAdvisoryBoard(BioAB)ofthePDAaswellasamemberoftheUSPBiologicsandBiotechnology2ExpertCommittee.IhavecontributedtoPDATechnicalReports57and57‐2coveringanalyticalmethoddevelopment,qualification,validationandtransfer;andtoUSPchapter<1238>BacterialVaccines.IamalsocurrentlyactiveonUSPexpertpanelsdevelopingchapters<210>MonosaccharideAnalysis,and<198>IdentificationofBacterialPolysaccharidesUsingNuclearMagneticResonanceSpectroscopyPlanningCommitteeMemberNotPresenting


RebeccaZangmeister,Ph.D.USPAffiliation:Member,USPGlycoproteinsandGlycanAnalysisExpertPanelScienceAdvisorBiomolecularMeasurementDivisionNationalInstituteofStandardsandTechnologyGaithersburg,MD,USARebeccaA.ZangmeisteristheScienceAdvisorfortheBiomolecularMeasurementDivisionoftheMaterialMeasurementLaboratoryattheNationalInstituteofStandardsandTechnology(NIST)inGaithersburg,MD.RebeccagraduatedfromtheUniversityofArizona,Tucson,AZ,in2001withaPh.D.inAnalyticalChemistry,specializinginorganicthinfilmsandsurfacecharacterization.ThecommonthemeofhersubsequentresearchprojectsatNISThaveinvolvedthedepositionandanalyticalcharacterizationofbiomoleculesatsurfaces(planarornanomaterialbased).Hermostrecentresearcheffortshavefocusedonthedevelopmentofmeasurementmethodsforbiologicswithanemphasisonrapidglycosylationassaysforuseinthebiomanufacturingoftherapeuticproteins.PlanningCommitteeMemberNotPresenting

USP WORKSHOP ON GLYCOSYLATION ANALYSIS FOR ... · 8/25/2015 · USP Workshop on Glycosylation...

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