WIXLIB

ContractResearchWorld Leading ContractResearch Services

NIBRT CONTRACT RESEARCHAbout NIBRTThe National Institute for Bioprocessing Research and Training (NIBRT) is a global centre of excellencefor training and research in bioprocessing. World leading NIBRT principal investigators and scientificadvisors, including Dr Jonathan Bones and Professor Pauline Rudd, continue to drive advancements inthe field of bioprocessing analytics through pioneering and innovative research.About UsWe are a team of characterisation specialists who provide detailed analysis of biologics in line with ICHQ6B and Q5E requirements.Our highly regarded scientists, renowned for their glycan analysis expertise, are situated in the awardwinning NIBRT facility with access to a state-of-the-art laboratory equipped with top-of-the-lineinstrumentation.Our StoryProfessor Rudd has an international reputation for expertise in the fields of glycobiology and glycananalysis. In 2006, her research team was transferred from the Glycobiology Institute, Oxford Universityto NIBRT, creating the Dublin-Oxford Glycobiology Research Group.From this group, NIBRT Contract Research launched and began operating as an independent groupwithin the facility.2

NIBRT CONTRACT RESEARCHOur MissionOur ClientsTo exceed our customers’ expectations withinnovative and bespoke analytical servicesproviding detailed characterisation of theirbiologics during development and processchange.NIBRT Contract Research has workedwith some of the Top 20 globalBiopharma companies, SME’s, virtualcompanies and law firms.At NIBRT every client receives the samehigh standard of service no matter whattheir size.“The NIBRT team’s extensive support and quality work was integralin the success of our regulatory submission to the U.S. FDA.”Associate Director, Biologics Development, Horizon Therapeutics3

NIBRT CONTRACT RESEARCHOur ServicesWorking adjacent to our accomplished team of Principal Investigators carrying out cutting edge, industryaligned research in all areas of biopharmaceutical manufacturing, we are well positioned to support ourclients in solving problems at all stages of their product development and production.Bespoke AnalyticalDevelopment andConsultancyICH Q6B Area:Process/Product relatedimpuritiesICH Q6B Area:Glycan andProtein ICH Q6B Area:ImmunochemicalPropertiesICH Q6B Area:PhysicochemicalPropertiesICH Q6B Area:Biological Activity“We came across NIBRT Contract Research through our work with a leading expert in the Biotherapeutic characterisationfield. From first contact, NIBRT Contract Research has been an outstanding facility to work with. The team at NIBRT ContractResearch has been instrumental in pushing the boundaries of our testing needs. They are not only flexible, responsive and apleasure to work with, they also go several steps further to help us problem solve and develop new ways to test our productsand learn more about our systems. NIBRT Contract Research is a top class analytical facility and we will continue to workwith them and recommend them to our colleagues going forward. We would use no one else.”North American Law firm specialising in intellectual property4

NIBRT CONTRACT RESEARCHWhy Choose Us?Our ExperienceAt NIBRT Contract Research we take the time tounderstand our clients’ requirements.With over ten years’ experience wehave analysed a variety of proteins andglycoproteins expressed in a range of celllines and expression systems.With extensive experience managing complexcharacterisation projects, our team consistentlydelivers to the highest standard.We can provide our clients with:Bespoke projects, flexible scheduling and quickresponse times.Clear communication and updates throughoutthe project lifetime.Tailored and detailed reports to ensure fullclarity of data and results.Monoclonal antibodiesGonadotropinsFusion A subject matter expert and dedicated analystswho offer support throughout the projectlifetime.5

NIBRT CONTRACT RESEARCHService Spotlight:N-glycan CharacterisationHILIC SeparationAcceptable ranges must be determinedas part of the development processand the glycosylation profile monitoredand controlled through production.Glycosylation of biotherapeuticscan be influenced by a number ofprocess-related factors, such as pH,carbon source, dissolved oxygen,temperature during manufacture, andthe expression 9The glycosylation profile can affect theefficacy, immunogenicity and serumhalf-life of a biotherapeutic.5.96.2Characterising biotherapeuticglycosylation is a requirement underregulatory guidelines (ICH Q5E/Q6B).FluorescenceGlycosylation is considered to bea critical quality attribute (CQA)of biotherapeutics by regulatoryauthorities.11.1Many therapeutic proteins are posttranslationally modified by the additionof N- or O-linked glycans.GUHydrophilic Interaction Liquid Chromatography (HILIC)separates glycans on the basis of shape, charge andhydrophobic and hydrophilic surfaces. HILIC separationallows for high-resolution separation of complex glycanprofiles. Glucose units (GU) are generated using a dextranladder standard to normalise retention time and to facilitatedata analysis.Exoglycosidase SequencingOur sample preparation involvesenzymatic release of N-glycans withfluorescent labeling to increasedetection sensitivity.We use a combination oftechnologies (HILIC, WAX, LC-MS andexoglycosidase sequencing) to obtainthe highest possible level of structuralinformation.We provide complete N-glycosylationcharacterisation with confident glycanassignments.6Linkage analysis of glycans is achieved by exoglycosidaseenzyme digestion followed by HILIC separation.Exoglycosidase enzymes cleave a terminal monosaccharidewith a specific glycosidic linkage. This cleavage yields acharacteristic GU shift in the HILIC profile which is used tointerpret the data and elucidate the glycan sequence.

NIBRT CONTRACT RESEARCHWAX SeparationWe use a combination oftechnologies to obtainthe highest possible levelof structural s SpectrometryFluorescenceMinutesWeak Anion Exchange chromatography (WAX) separatesglycans on the basis of the number of charged residueson the glycan. WAX separation allows for the relativequantitation of charged glycans. Sialic acids, phosphatesand sulphates exhibit a negative charge and contributeto the glycan charge profile.FLREIC1170.4186 m/zComposition:HexNac 4Hex 5Neu5Ac 2WAX-HILIC SeparationMinutesMS1 SpectrumFluorescenceRelative AbundanceTri-chargedRelative AbundanceNeutralTetra-chargedRelative 8001000 1200 1400 1600 1800 2000 2200 2400m/zMono-chargedGU2D-LC with WAX separation in the first dimension andHILIC separation in the second dimension reduces thecomplexity of highly complex glycosylation profiles.LC-MS with fluorescence detection yieldscomposition information within the HILICprofile and is used as an orthogonaltechnique to confirm elucidated glycancomposition.7

NIBRT CONTRACT RESEARCHICH Q6B Area: Structural characterisationGlycan characterisationAnalysisTechniqueN- and O- glycancharacterisationN-glycans released enzymatically, O-glycans released by chemical treatment.Fluorescent labelling of released glycans and analysis by UPLC-FLD (Waters Acquity -FLD) and LC-MS (Thermo Scientific Vanquish -Q Exactive Plus ) orCE LIF (Beckman Coulter PA800 plus ). Linkage confirmation by exoglycosidasedigestion.Sialic acid quantitationDMB labelling of hydrolysed sialic acid and analysis by UPLC-FLD. Quantitation usingDMB labelled standards.Sialic acid linkage relativequantitationDerivatisation of sialic acids by DMT-MM and analysis by LC-MS.Site occupancyComparison of glycosylated and deglycosylated sample peptide maps by LC-MS.Protein characterisationAnalysisTechniqueAmino acid sequencePeptide mapping by LC-MS and bioinformatic analysis against provided proteinsequenceN- and C- terminalsequencingConfirmation of N- and C- terminal amino acids by peptide mapping (detection ofblocked N- terminus pyroglutamate/pyroglutamic acid)Top down intact mass for orthogonal confirmationAmino acid compositionDerivatisation and quantitation with AccQ.Tag Ultra by UPLC-UV/FLRDisulfide bondsComparison of reduced and non-reduced peptide mapping by LC-MSFree ThiolsDetermination of free thiols using DNTBICH Q6B Area: Physicochemical properties8AnalysisTechniqueIntact protein molecularweightMolecular weight determination by RP-LC-MSIsoform patternProfiling of isoforms by various techniques: cIEF peptide mapping and UPLC: IEX, HIC,RP, SECDetermination ofextinction coefficientAmino acid analysis combined with UV 280nm dilution seriesNative MS

NIBRT CONTRACT RESEARCHICH Q6B Area: Process and productrelated impuritiesAnalysisTechniqueAggregate analysisDetermination of aggregates and fragments by SEC, AUC and LC-MSMolecular variantsRelative quantitation of deamidation, oxidation and other PTM’s by peptide mappingLC-MSCharge variant analysis by IEX-UPLC and CIEFOxidation by HIC and RP-UPLCHost Cell Proteins (HCP)Absolute quantitation of HCP by LC-MS and ELISAResidual protein AqPCR using ProteinSEQ Host Cell DNAqPCR using resDNASEQ PPG and PEG analysisDetection of free PPG and PEG by UPLC-CADExtractables andLeachablesDetection and quantitation of extracted and leached compounds by ICP-MS, GC-MSand LC-MS.9

NIBRT CONTRACT RESEARCHICH Q6B Area: Immunochemical PropertiesAnalysisTechniqueTarget antigen bindingKinetics and affinity/epitope mapping/thermodynamic profiling bySPR (GE HealthcareTM - Biacore T100TM)Effector bindingMeasurement of FcγR/FcRn/C1q binding by SPRICH Q6B Area: Biological ActivityAnalysisTechniqueCell proliferationMeasurement of thymidine analogue (BruD) incorporation by ELISA (Biotek SynergyTM H1)DNA-based measurement of cell cycle (G0/G1, S and G2/M) induction/inhibition usingFCMMTT assay with colorimetric readoutCell death(Autophagy/Apoptosis)Extrinsic (e.g. Fas) and intrinsic (e.g. caspase activation) apoptosis measurement byFCM (BDTM - FACSMelodyTM) and ELISAAutophagic flux monitoring by FCM and ELISAAntibody effector function(ADCC/ADCP)Mechanism of Action (MOA)-based human FcγRIIIa/FcγRIIa reporter bioassays usingluciferase-based detectionEnzyme activityKinase/phosphatase/protease activity measurement by FCM and ELISABespoke Analytical Developmentand tion, verification and qualificationAs per client requestTroubleshooting of existing client methodsAs per client requestReplication of methods for IP litigationAs per client requestConsultancyAs per client request10

Project ProcessContact usOur experts will discuss your requirements with youover email or by phone. A CDA will be set up if required.Project Proposal GenerationA tailored project proposal will be generated outlininganalytical workflow, schedule and budget.Place an orderWith our flexible scheduling projects can bescheduled to required timelines.Project StartSample analysis is performed by a dedicated analystwho will provide regular project updates.Report DeliveryDelivery of a comprehensive detailed report.Follow up after client review.Contact UsTel: 353 1 215 8100 Email: contract.research@nibrt.ieWeb: www.nibrt.ie/contract-research /Foster Avenue, Mount Merrion, Blackrock, Co. Dublin, A94 X099, Ireland

NIBRT CONTRACT RESEARCH 3 Our Mission To exceed our customers' expectations with innovative and bespoke analytical services providing detailed characterisation of their biologics during development and process change. Our Clients NIBRT Contract Research has worked with some of the Top 20 global Biopharma companies, SME's, virtual