N-linked Glycan Characterization and Profiling: Combining the Power

N-linked Glycan Characterization and Profiling: Combining the Power

N-linked Glycan Characterization and Profiling: Combining the Power of a Novel Labeling Reagent and a Streamlined Analytical Workflow Ying Qing Yu Ph.D Waters Corporation Glycobiology World Congress 2015 2015 Waters Corporation Glycosylation is a Key Critical Quality Attribute The International Conference on Harmonization Guideline Q6B requires the analysis of carbohydrate content, structural profiles, and characterization of the glycosylation site(s) within the polypeptide chain(s). TrastuzumAb, 1 N-linked site 150 KDa 2015 Waters Corporation figures are from Wikipedia Erythropoietin 3 N-linked sites

1 O-linked site 34 KDa Entanercept 3 N-linked sites 13 O-linked sites 51 Kda Glycoprotein Characterization Multiple Strategies Complementary Information 2015 Waters Corporation Carbohydrate Complexity 2015 Waters Corporation Classes of N-Linked Glycans High Mannose 2015 Waters Corporation Complex Hybrid Influence on Biopharmaceutical Production Asn Asn Loss of sialylation decreases EPO half-life from 2 h to 10 min

Fukuda et al (1989). Blood; 73(1): 84-89 Desialylation of intravenous immunoglobulin abrogates its anti-inflammatory properties Kaneko et al (2006). Science; 313(5787): 670-673 Asn Presence of gal-(1,3)-gal can induce anaphylaxis (shock) and can be present on biotherapeutics Chung et al (2006). N Engl J Med; 358(11): 1109-1117 Asn Asn 2015 Waters Corporation Half of all people contain antibodies against (1,2)xylose and (1,3)-core fucose Bardor et al (1995). Glycobiology; 13(6): 427-434 Released Glycan Analysis HILIC Profiling Conventional 2-AB High Sensitivity HILIC-Fluorescence-MS Fluorophore +MS Charge Tag

Rapid Preparation Deglycosylation Labeling SPE Fluorescence MS 30 min 5 Hours to 2 Days 2+ MS 5.3e2 Rap iFlu or MS_ hI gG _1 4p mo l_1 8No v14 _04 28 3 (5 .0 65) 3. 47 e6 % 100 23 5

100 m/ z 20 00 (4 .2 34 ) 2. 89 e4 % FLR 0 5 00 1 00 0 150 0 2A B_ hI gG _h al f load _2 0No v14 _1 0 0 5 00 1 00 0 150 0 2A B_ hI gG _h al f load _2 0No v14 _1 0 49 5 m/ z 20 00 (8 .6 89 ) 529 %

100 % 0 m/ z 5 00 1 00 0 150 0 20 00 Rap iFlu or MS_ hI gG _1 4p mo l_1 8No v14 _04 50 7 (8 .8 92) 1. 15 e5 100 0 500 0. E+0 2015 Waters Corporation 5 00 1 00 0 150 0 20 00 m/ z

2000 m/z Conventional Workflow Conventional Glycoprotein Deglycosylation 16 hrs (Pre-Labeling Clean-Up) (Drying) 1-3 hrs Released N-Glycans + Protein Labeling 3 hrs Labeled Reaction N-Glycans Byproducts + Reagents Clean-up 0.1 hrs (Drying) Labeled

Glycans 2 hrs >24 HOURS 2015 Waters Corporation Analysis LC-FLR LC-FLR-MS What is new? Novel RapiFluor-MSTM (RFMS) Reagent 2015 Waters Corporation Patent Pending RapiFluor-MS Reagent Rapid Reaction Kinetics NHS Carbamate Rapid Tagging Group Tertiary Amine MS-Active Charge Tag rapiFluor- MS Glycan

+ MassMass 312 Da Glycosylamine +C17H20N4O2 Quinoline Fluorophore H2O Monoisotopic mass shift (from glycosylamine) 312.1586 Da Reaction Time = Seconds Glycan Highly stable + urea linkage 2015 Waters Corporation Procedure Time: + + CO2 1

Simplified Sample Preparation Conventional GlycoWorks RapiFluor-MS N-Glycan Kit Glycoprotein Deglycosylation 16 hrs (Pre-Labeling Clean-Up) (Drying) 1-3 hrs <15 min Released N-Glycans + Protein Labeling 3 hrs Labeled Reaction N-Glycans Byproducts + Reagents 5 min

Clean-up 0.1 hrs (Drying) 2 hrs >24 HOURS 2015 Waters Corporation 10 min Labeled Glycans Analysis LC-FLR LC-FLR-MS 30 min Patent Pending 1 RFMS vs. 2AB for MS sensitivity comparison Greater than 100x MS response over 2AB labeling BPI MS 2AB

RFMS Sample: NIST RM 8670 mAb lot #3F1b 2015 Waters Corporation 1 Glycan Characterization with RFMS Labeling Comparable FLR and MS response across a broad range of glycans IgG Simple bi-antennery structures FLR Fetuin Large, complex structures Time 10.00 12.00 14.00 16.00 18.00 20.00 22.00

FA2 24.00 26.00 28.00 30.00 32.00 34.00 A3G3S3 BPI MS A3S1G3S3 FA2G2S1 Time 11.00 2015 Waters Corporation 15.00 20.00 25.00

30.00 35.00 Xevo G2-XS QTof 1 Detailed MS Profiling of a Wide Dynamic Range of Glycans labeled with RFMS Fetuin FLR FLR Zoom In ~20x 12.00 14.00 16.00 18.00 20.00 22.00 24.00

26.00 28.00 30.00 32.00 34.00 16.00 30.00 18.00 20.00 22.00 24.00 36.00 38.00 40.00 26.00 35.00 28.00

30.00 32.00 34.00 36.00 40.00 38.00 Time 40.00 BPI MS BPI MS 10.00 10.00 15.00 2015 Waters Corporation 20.00 25.00 0 10.00

12.00 14.00 1 RFMS Labeling Enables MS Detection of Very Low Abundance Glycans Human IgG FLR MS BPI MSMS 2015 Waters Corporation 1 RFMS extends MS enhancement throughout the glycan fragmentation pattern 2015 Waters Corporation 1 RFMS enables easy assignment of two isobaric glycans RFMS labeled N-glycans from a murine IgG1 mAb 2.1E+6 B

A FLR FA2G2 FLR 0.0E+6 8.4E+6 MS BPI MS 0.0E+6 10 FA2G2 FA2G1Ga1 ? FA2G1Ga1 Zoom 10x 20 min 100 2000 m/z

The two isobaric glycans, FA2G2 and a minor shoulder peak, partially resolved by HILIC The minor peak represents only 0.7% of total FLR signal 2015 Waters Corporation 1 10 RFMS enables easy assignment of a minor shoulder peak of FA2G2 as FA2G1Ga1 A A A FLR 2.1E+6 FLR FLR MS MS FA2G2 MS BPI MS

0.0E+6 10 0 FA2G2 FA2G2 FA2G2 FLR 0.0E+6 8.4E+6 B B B RFMS labeled N-glycans from a murine IgG1 mAb FA2G2 FA2G2 FA2G1Ga1 FA2G1Ga1 FA2G1Ga1 FA2G1Ga1 FA2G1Ga1 FA2G1Ga1

FA2G1Ga1 Zoom 10x 0 . Loss of GlcNAc 0 4 2000 m/z Zoom 10x Zoom 10x 20 min 20 min 20 min 100 100 100 2000 m/z The two isobaric glycans, FA2G2 and a minor shoulder peak, partially resolved by HILIC

The minor peak represents only 0.7% of total FLR signal Structurally diagnostic ions: 1) predominant 528 m/z ion and 2) prominent GlcNAc loss High sensitivity and information rich fragmentation data support the identification of the isobaric, lower abundance species as an -Gal containing FA2G1Ga1. 2015 Waters Corporation 2000 m/z 1 UNIFI Glycan Application Workflows HILIC FLR GU +Accurate Mass HILIC FLR DDA MS/MS Automated Automated Data Data Acquisition Acquisition

Glycan Glycan FLR+MS FLR+MS Confirmation Confirmation Glycan Glycan DDA DDA Processing Processing Calibrated Calibrated RT RT and Accurate and Accurate Mass Mass Deconvolute Deconvolute Fragment Fragment Ion Ion Review Review and and

Reporting Reporting Scientific Scientific Library Library SimGlycan SimGlycan 2015 Waters Corporation 1 The Utility of GU Values What is a GU Value? GU stands for Glucose Unit A GU value is a normalized glycan structure retention time observed in HILIC for glycan peaks, obtained using a dextran ladder calibration Why is the GU approach useful? GU Values assist in normalizing glycan retention time across days, instruments and laboratories, so data can be compared and shared

easily. GU Values facilitate more routine glycan assignments by enabling the creation of a single glycan GU retention library. 2015 Waters Corporation 2 Generating Waters RFMS GU Glycan Libraries FLR label mass Waters Glycan GU Library: Experimentally derived GU Retention (>10 injections/protein) Data from proteins representing spectrum of glycan diversity Experimental GU value All entries confirmed with exoglycosidase digestion 2015 Waters Corporation 2 HILIC FLR GU + Accurate Mass Workflow Method Robustness and Transferability, Confident Assignments Retention Time Calibration Curve

GU Labeled Dextran Ladder Assign GU values to N-glycans BPI MS for accurate mass confirmation 2015 Waters Corporation 2 Glycan GU Scientific Library Search for Confident Glycan Assignments Both 2-AB and RFMS labeled glycan performance test standards are now available to support this workflow 2015 Waters Corporation 2 UNIFI Glycan DDA Workflow GlycanDDA data acquired Processing and file formatconversion SimGlycan Database

Search 2015 Waters Corporation 2 UNIFI Acquire and Process Glycan DDA Data RFMS labeled N-glycans from mouse IgG MSMS fragmentation of a highlighted minor glycoform was displayed FLR Raw MSMS Spectrum ~ 0.1% total FLR BPI MS Deconvolute & deeisotope Export as .LCS (or mzml) format 2015 Waters Corporation 2 Automated Glycan Identification using .LCS Files with SimGlycan The minor glycoform was correctly identified as FA2G2Ga1Sg1 alpha (1,3)-Gal (Ga1) NeuGc (Sg1) 2015 Waters Corporation

2 Work in progress Developing a scientific library for RapiFluor-MS labeled glycans for automated glycan assignment Applying RapiFluor-MS label for more complex glycosylated proteins 2015 Waters Corporation 2 Acknowledgements Waters Matt Lauber PhD New England Biolabs Paula Magnelli PhD Professor Pauline Rudd (COI) Mark Hilliard (UNIFI Glycan Scientific Library) Giogio Carta (UNIFI Glycan Scientific Library) 2015 Waters Corporation 2

Recently Viewed Presentations

  • Thyroidectomy - Weebly

    Thyroidectomy - Weebly

    Thyroidectomy - Possible complications Hemorrhage Respiratory distress or stridor Hoarseness of voice Total vocal cord paralysis - aphonia Hypocalcemic tetany (due to accidental removal of parathyroid glands during total thyroidectomy) Wound infection: This may manifest after 48 hours of surgery...
  • THE SKELETAL/ MUSCULAR SYSTEM Maame Kwarkye Joon Bak

    THE SKELETAL/ MUSCULAR SYSTEM Maame Kwarkye Joon Bak

    Skeletal and immune systems "TABOO" One player will have one card, in this case a power point slide, and must help the other team members guess the word that appears at the top of the "card".
  • Des coins en maternelle : pour quoi ? Comment ? Ce qu&#x27;en ...

    Des coins en maternelle : pour quoi ? Comment ? Ce qu'en ...

    " L'avantage des coins, c'est qu'ils prennent vraiment le matériel tel qu'il est en fonction de leurs compétences, c'est-à-dire qu'un enfant qui est très compétent, il va réussir à aller au-delà en manipulant et… et en s'essayant avec ses outils...
  • Bonding and Properties - Mrs. Baker&#x27;s Chemistry Website

    Bonding and Properties - Mrs. Baker's Chemistry Website

    Bonding and Properties Unit 4 Atomic bonding Metallic bonds (metals) Positive ions in a "sea" of electrons Generally strong bonds Held together by attraction of positive ions for sea of electrons Electrons are delocalized (not attached to specific atom) Properties...
  • CDA Product Family - Health Level Seven International

    CDA Product Family - Health Level Seven International

    CDA Management Group (CDA-MG) TSC approved the Mission and Charter for a proposed CDA Management Group. Mission. The Clinical Document Architecture Management Group (CDA-MG) provides day-to-day oversight of the processes related to Clinical Document Architecture (CDA) products throughout their lifecycle.
  • Historical Analysis Using the Past to Design the Future

    Historical Analysis Using the Past to Design the Future

    Historical Analysis: Using the Past to Design the Future Susan Wyche1, Phoebe Sengers2, and Rebecca E. Grinter1 1GVU Venter College of Computing Georgia Institute of Technology 2Information Science Cornell University Ubicomp 2006 Speaker: Jenhao Introduction Interdiscipline Anthropological methods (ethnography) Art...
  • Missional Interpretatio n of Scripture Missional Theology Movement

    Missional Interpretatio n of Scripture Missional Theology Movement

    It is especially obvious in the NT as NT authors use OT texts to describe how one lives "in Christ" The process is important because the process itself is application and thus shows us how to missionally apply biblical texts...
  • Kent CEIAG Network Briefing - Kelsi

    Kent CEIAG Network Briefing - Kelsi

    Kent CEIAG Network Briefing. 5th February 2016. Mercure Hotel. Maidstone. Welcome! Mike Rayner. Skills Lead. South East Local Enterprise Partnership. Careers Events. Why they are so important? They are just one small part of your entire job search process; but...