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NISTIR 8186

NIST Interlaboratory Study on the Glycosylation of NISTmAb,

a Monoclonal Antibody Reference Material June 2015 to February 2016

Maria Lorna A. De Leoz David L. Duewer Stephen E. Stein

This publication is available free of charge from: https://doi.org/10.6028/NIST.IR.8186

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Sample A: modified NISTmAbSample B: NISTmAb

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NISTIR 8186

NIST Interlaboratory Study on the Glycosylation of NISTmAb,

a Monoclonal Antibody Reference Material June 2015 to February 2016

Maria Lorna A. De Leoz Biomolecular Measurement Division

Material Measurement Laboratory

David L. Duewer Chemical Sciences Division


Stephen E. Stein Biomolecular Measurement Division


This publication is available free of charge from: https://doi.org/10.6028/NIST.IR.8186

July 2017

U.S. Department of Commerce Wilbur L. Ross, Jr., Secretary

National Institute of Standards and Technology Kent Rochford, Acting NIST Director and Under Secretary of Commerce for Standards and Technology

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i

Abstract

The National Institute of Standards and Technology coordinated an interlaboratory study for laboratories that measure glycosylation in monoclonal antibodies. This report describes the design of and results for the NIST Interlaboratory Study on the Glycosylation of NISTmAb, a Monoclonal Antibody Reference Material from 103 reports submitted by 76 laboratories. Two materials were used in the study, 1) the Primary Standard (PS) for NIST Reference Material 8671, NISTmAb, Humanized IgG1κ Monoclonal Antibody, and 2) a material derived from the PS by treatment with galactosidase. The study was conducted in two stages: Stage 1 involved nine selected laboratories who volunteered to assist in final study design; Stage 2 was widely advertised and open to all laboratories. The materials for the study were shipped to participants in two batches: June 2015 and August to September 2015 for Stage 1 and Stage 2, respectively. Participants were requested to provide measurement results by July 30, 2015 (Stage 1) and November 6, 2015 (Stage 2).

Key words

Glycan, Glycopeptide, Glycosylation, Glycoform, Glycomics, IgG, Monoclonal Antibody, NISTmAb, RM 8671

______________________________________________________________________________________________________This publication is available free of charge from

: https://doi.org/10.6028/NIS

T.IR.8186

Certain commercial entities, equipment, or materials may be identified in this document in order to describe an experimental procedure or concept adequately. Such identification is not intended to imply recommendation or endorsement by the National Institute of Standards and Technology, nor is it intended to imply that the entities, materials, or equipment are necessarily the best available for the purpose.

Disclaimer

ii

Table of Contents

Keywords .............................................................................................................................................. i Table of Contents ................................................................................................................................ ii Introduction ......................................................................................................................................... 1 Interlaboratory Study: Glycosylation of Monoclonal Antibodies .................................................. 1 References ............................................................................................................................................ 2

Appendix A. Shipping Package Inserts for the Interlaboratory Study .................................... A-1 Cover letter ................................................................................................................................. A-2 Instructions ................................................................................................................................. A-3 Packing List and Shipment Receipt Confirmation Form ........................................................... A-4 Data Reporting Template ........................................................................................................... A-5 Method Reporting Template ...................................................................................................... A-9 Comments and Suggestions Template ..................................................................................... A-12

Appendix B. Final Report for the Interlaboratory Study .......................................................... B-1 Cover letter ..................................................................................................................................B-2 Table of Identified Glycans .........................................................................................................B-4 All Lab Report ..........................................................................................................................B-22 Representative “Individualized Report” ....................................................................................B-33



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Introduction

Glycosylation is a critical post-translational modification in monoclonal antibodies [1]. It can impact the safety and efficacy of the drug, including its clearance rates, effector functions, folding, immunogenicity, and solubility. However, glycosylation is inherently heterogeneous and challenging to analyze, leading to a proliferation of analysis methods [2-15]. With the advent of biosimilars and other protein-based drugs, it is important to compare the glycosylation of biologics in an accurate and precise manner [16].

The National Institute of Standards and Technology (NIST) coordinated this interlaboratory study for measurement of glycosylation in monoclonal antibodies. The goals of this study are two-fold:

• to determine measurement variability in identifying and quantifying N-glycans across theglycan measurement community and

• to aid in assigning consensus values for the glycosylation of the recombinant NISTReference Material (RM) 8671, NISTmAb, Humanized IgG1κ Monoclonal Antibodyproduced in murine-derived cells [17].

Participants used their method of choice to determine glycan content in the control and study materials. Some participants submitted more than one report; each report was assigned a confidential laboratory number. Participants provided their data to NIST, where it was compiled and evaluated for consensus values, within-laboratory precision, and concordance within the glycomics community. NIST provided the participants with a technical summary of reported and derived values from all laboratories, a table of all identified glycans, and an individualized graphical analysis of their performance for the exercise. Participants who have concerns regarding their laboratory’s performance were encouraged to consult with the interlaboratory coordinators.

Interlaboratory Study: Glycosylation of Monoclonal Antibodies

This interlaboratory study was done in two stages: Stage 1 was open to nine selected laboratories that helped fine-tune the study; Stage 2 was open to all laboratories.

Individual units of batch-prepared samples were distributed to each participant: two 0.5-mg (Stage 1) or 0.4-mg (Stage 2) liquid-frozen monoclonal antibody samples for analysis and one 1-mL 25 mmol/L L-Histidine pH 6.0 solution for use in reconstituting the two samples. The antibody samples were the Primary Standard (PS) for NIST RM 8671 NISTmAb and a material derived from the PS. The derived material was prepared as a 70:30 by mass mixture of unmodified PS with PS treated with β1-4 Galactosidase S (8,000 units/mL, New England Biolabs, Ipswich, MA).

Unless multiple vials were requested, participants received one vial of each sample. These samples were shipped on dry ice to 90 laboratories in June 2015 (Stage 1) or August-September 2015 (Stage 2). Participants were requested to provide measurement results by July 30, 2015 and November 6, 2015 for Stage 1 and Stage 2, respectively. However, 32 laboratories asked to delay their submission of reports. The last report was received on September 2, 2015 for Stage 1 and February 1, 2016 for Stage 2. A total of 103 reports were submitted by 76 laboratories. The communication materials included in the sample shipment and emailed to participants are provided in Appendix A.

Participants were requested to report methods and percent abundance values for all glycans found in the two samples using method and data reporting templates provided along with the samples. The



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method reporting template contained drop down boxes of common methods and parameters for identification and quantification, and spaces to list other techniques or parameters. The data reporting template listed 16 glycans in Stage 1 and 54 glycans in Stage 2 and space to report other glycans not listed in the template. Not all participants reported values for all the target glycans, and many participants reported values for non-target glycans. The final report delivered to every participant in the interlaboratory study consists of four documents, as shown in Appendix B:

• A cover letter containing a brief description of the samples and other three documents. • An “All-Lab Report” that summarizes reported and derived values for Samples A and B, and

the A/B ratio. • A “Table of Identified Glycans” showing identifiers, compositions, and structures for all

glycans reported in any data set. • An “Individualized Report” that graphically analyzes each participant’s results for all

analytes reported by at least six participants. It contains boxplots of Samples A and B, and the A/B ratio, and a target plot summary for the A/B ratio. This report also provides a graphical summary of each participant’s measurement comparability, including glycan composition counts and sums, repeatability, limits of reporting, minimum reported values, and consensus values.

References [1] Prien, JM, et al. (2015) Orthogonal Technologies for NISTmAb N-Glycan Structure

Elucidation and Quantitation. State-of-the-Art and Emerging Technologies for Therapeutic Monoclonal Antibody Characterization, Vol 2: Biopharmaceutical Characterization: The Nistmab Case Study, 1201: 185-235.

[2] Ruhaak, LR, Zauner, G, Huhn, C, Bruggink, C, Deelder, AM, Wuhrer, M (2010) Glycan labeling strategies and their use in identification and quantification. Anal Bioanal Chem, 397(8): 3457-81. https://doi.org/10.1007/s00216-010-3532-z.

[3] Rosati, S, Yang, Y, Barendregt, A, Heck, AJ (2014) Detailed mass analysis of structural heterogeneity in monoclonal antibodies using native mass spectrometry. Nat Protoc, 9(4): 967-76. https://doi.org/10.1038/nprot.2014.057.

[4] Song, T, Ozcan, S, Becker, A, Lebrilla, CB (2014) In-Depth Method for the Characterization of Glycosylation in Manufactured Recombinant Monoclonal Antibody Drugs. Analytical Chemistry, 86(12): 5661-5666. https://doi.org/10.1021/ac501102t.

[5] Mechref, Y, Hu, YL, Desantos-Garcia, JL, Hussein, A, Tang, HX (2013) Quantitative Glycomics Strategies. Molecular & Cellular Proteomics, 12(4): 874-884. https://doi.org/10.1074/mcp.R112.026310.

[6] Zhang, H, Ashline, DJ, Reinhold, VN (2014) Tools to MSn Sequence and Document the Structures of Glycan Epitopes. Discov Subtleties Sugars (2013), 2013: 117-131.

[7] Beck, A, Wagner-Rousset, E, Ayoub, D, Van Dorsselaer, A, Sanglier-Cianferani, S (2013) Characterization of therapeutic antibodies and related products. Anal Chem, 85(2): 715-36. https://doi.org/10.1021/ac3032355.

[8] Hong, Q, Lebrilla, CB, Miyamoto, S, Ruhaak, LR (2013) Absolute quantitation of immunoglobulin G and its glycoforms using multiple reaction monitoring. Anal Chem, 85(18): 8585-93. https://doi.org/10.1021/ac4009995.

______________________________________________________________________________________________________ This publication is available free of charge from


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[9] Triguero, A, Cabrera, G, Royle, L, Harvey, DJ, Rudd, PM, Dwek, RA, Bardor, M, Lerouge, P, Cremata, JA (2010) Chemical and enzymatic N-glycan release comparison for N-glycan profiling of monoclonal antibodies expressed in plants. Anal Biochem, 400(2): 173-83. https://doi.org/10.1016/j.ab.2010.01.027.

[10] Stadlmann, J, Pabst, M, Kolarich, D, Kunert, R, Altmann, F (2008) Analysis of immunoglobulin glycosylation by LC-ESI-MS of glycopeptides and oligosaccharides. Proteomics, 8(14): 2858-71. https://doi.org/10.1002/pmic.200700968.

[11] Wagner-Rousset, E, Fekete, S, Morel-Chevillet, L, Colas, O, Corvaia, N, Cianferani, S, Guillarme, D, Beck, A (2017) Development of a fast workflow to screen the charge variants of therapeutic antibodies. J Chromatogr A, 1498: 147-154. https://doi.org/10.1016/j.chroma.2017.02.065.

[12] Stoll, D, Danforth, J, Zhang, K, Beck, A (2016) Characterization of therapeutic antibodies and related products by two-dimensional liquid chromatography coupled with UV absorbance and mass spectrometric detection. J Chromatogr B Analyt Technol Biomed Life Sci, 1032: 51-60. https://doi.org/10.1016/j.jchromb.2016.05.029.

[13] Shubhakar, A, Kozak, RP, Reiding, KR, Royle, L, Spencer, DI, Fernandes, DL, Wuhrer, M (2016) Automated High-Throughput Permethylation for Glycosylation Analysis of Biologics Using MALDI-TOF-MS. Anal Chem, 88(17): 8562-9. https://doi.org/10.1021/acs.analchem.6b01639.

[14] Reusch, D, Haberger M, Maier B, Maier M, Kloseck R, Zimmermann B, Hook M, Szabo Z, Tep S, Wegstein J, Alt N, Bulau P Wuhrer M. (2015) Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles--part 1: separation-based methods. MAbs, 7(1): 167-79. https://doi.org/10.4161/19420862.2014.986000.

[15] Reusch, D, Haberger M, Maier B, Gassner J, Hook M, Wagner K, Bonnington L, Bulau P, Wuhrer M (2015) Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles-Part 2: Mass spectrometric methods. MAbs, 7(4): 732-42. https://doi.org/10.1080/19420862.2015.1045173.

[16] Huhn, C, Selman, MH, Ruhaak, LR, Deelder, AM, Wuhrer, M (2009) IgG glycosylation analysis. Proteomics, 9(4): 882-913. https://doi.org/10.1002/pmic.200800715.

[17] National Institute of Standards and Technology Standard Reference Materials Program (2016) Report of Investigation: Reference Material 8671 NISTmAb, Humanized IgG1κ Monoclonal Antibody. NIST, Gaithersburg, MD, USA. https://www-s.nist.gov/srmors/view_cert.cfm?srm=8671



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Appendix A. Shipping Package Inserts for the Interlaboratory Study

The following six items were included in each package shipped to each participant: • Cover letter • Instructions • Packing List and Shipment Receipt Confirmation Form • Data Reporting Template • Method Reporting Template • Comments Template

A cover letter describing the interlaboratory study, instructions, packing list, method, data and comment reporting sheets were enclosed in a sealed waterproof bag placed at the top of the shipping box, between the cardboard covering and the foam insulation. All the shipping package inserts were also sent by email as one Microsoft Excel file with multiple tabs to each participant.



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Cover Letter NIST Interlaboratory Study on Glycosylation Analysis Welcome Packet August 27, 2015 Name Address Lab #: Dear Colleague, Welcome to the NIST Interlaboratory Study on Glycosylation Analysis. Thank you for agreeing to participate in this endeavor. Alteration in glycosylation may significantly modify the biological activity of monoclonal antibodies. Thus, analysis of their glycoforms is essential, whether it is a batch-to-batch analysis of a drug candidate, comparison of the glycan profile of a biosimilar, or a complete glycomics profiling of a new drug. There are several published methods to quantify and identify glycoforms in proteins, but only a handful of multi-lab studies to assess the performance of these various approaches. The goals of this study are two-fold: 1) to determine measurement variability in identifying and quantifying N-glycans across laboratories and 2) to aid in assigning consensus values for the glycosylation of the NISTmAb reference material, a soon-to-be-released well-characterized monoclonal antibody reference material. Each laboratory is assigned a laboratory number so that study results may be viewed and discussed openly in anonymity. Your lab number is shown above. Please use this number on the forms for submission of results. Your lab number remains confidential. Sample shipment will occur on August 31, 2015. We request that the resulting data be returned on or before November 6, 2015. Your laboratory will receive two frozen monoclonal antibody samples, Sample A (white) and Sample B (blue), for glycosylation analysis using your own method. Both samples are humanized IgG1k expressed in murine suspension culture. The samples are "drug-like substances" not for human use. The samples are 0.4 mg each, with a concentration of 100 mg/mL. You will also receive a vial containing 25 mmol/L L-Histidine pH 6.0 solution (yellow) that you may use (optional) to reconstitute the two samples. We ask you to perform a glycosylation analysis in triplicate using your own method for each of the two samples. The Excel file provided has two tabs called “Data Reporting” and “Method Reporting” to report your data and method, respectively. If you choose to use more than one method, we ask that you create a separate file for each. Results should be attached in an email to [email protected] with the subject “NIST Interlab Results.” Please feel free to contact Lorna at (301) 975-6731 or [email protected] if you have questions. Again, many thanks for your participation. Sincerely, M. Lorna A. De Leoz, Ph.D. Principal Investigator NIST Interlaboratory Study on Glycosylation Analysis Biomolecular Measurement Division National Institute of Standards and Technology Gaithersburg, Maryland, USA 20899-8362

Stephen E. Stein, Ph.D. Group Leader Biomolecular Measurement Division National Institute of Standards and Technology Gaithersburg, Maryland, USA 20899-8362



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Instructions NIST Interlaboratory Study on Glycosylation Analysis Instructions August 27, 2015 Dear Colleague, Enclosed are samples for the NIST Interlaboratory Study on Glycosylation Analysis. Sample details are provided below: Sample A: white label, frozen liquid, 0.4 mg, 100 mg/mL mAb Sample B: blue label, frozen liquid, 0.4 mg, 100 mg/mL mAb Buffer: yellow label, frozen liquid, 1 mL, 25 mmol/L L-Histidine, pH 6.0 The package for this study consists of two vials of monoclonal antibody samples, Sample A (white) and Sample B (blue), one vial of L-Histidine solution (yellow) which you may use to reconstitute the samples, and a welcome packet consisting of a letter, instructions, packing list/shipment receipt confirmation form, and data, method, and comment forms to report your results. As soon as you receive the samples, please return the filled shipment receipt confirmation form (third tab) to us. Please perform a glycosylation analysis in triplicate using your own method for Sample A and Sample B. After completing your analysis, please enter the percent abundances of the glycans and their standard deviations in the "Data Reporting" tab. Also include the percent abundances for each replicate. If a value obtained is below your limit of detection or quantification, please indicate this result on the form as "ND" (not detected) or "NQ" (not quantified), respectively. Please describe your method in the "Method Reporting" tab by filling in as much information as you can. The last tab, "Comments," is for your feedback on all the stages of the interlab study, including shipping, samples, data reporting, and analysis. Your input, expertise, and feedback is extremely valuable for future studies. We request that the filled Excel sheets be returned to us as an email attachment to [email protected] (Subject: NIST Interlab Results) on or before November 6, 2015. Please let me know if this schedule would pose problems for your laboratory. Please feel free to contact Lorna at (301) 975-6731 or [email protected] if you have questions. Again, many thanks for your participation. Sincerely, M. Lorna A. De Leoz, Ph.D. Principal Investigator NIST Interlaboratory Study on Glycosylation Analysis Biomolecular Measurement Division National Institute of Standards and Technology Gaithersburg, Maryland, USA 20899-8362

Stephen E. Stein, Ph.D. Group Leader Biomolecular Measurement Division National Institute of Standards and Technology Gaithersburg, Maryland, USA 20899-8362



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Please fill in only the green boxes and return to [email protected] upon receipt of samples.

Packing List and Shipment Receipt Confirmation Form NIST Interlaboratory Study on Glycosylation Analysis Packing List and Shipment Receipt Confirmation Form This box contains: one vial each of the following:

Vial Form Amount Label Color Description Sample A Liquid frozen 0.4 mg White 100 mg/mL mAb Sample B Liquid frozen 0.4 mg Blue 100 mg/mL mAb

Buffer Liquid frozen 1 mL Yellow 25 mmol/L L-Histidine, pH 6.0 Please:

1) Open the pack immediately. 2) Check that the box contains all of the above vials. 3) Check if the vials are intact. 4) Store the samples at -20oC or below until analysis. 5) Email [email protected] the following information (Subject: NIST Interlab Shipment

Receipt):

A) Lab # B) Date this shipment arrived: C) Are all 3 vials intact? Yes / No If "No," which one(s) were damaged? D) Was there any dry ice left in cooler? Yes/No E) Did the samples arrive frozen? Yes/No F) At what temperature are you storing the samples? oC G) When do you anticipate analyzing these samples?

Your prompt return of this information to [email protected] is appreciated. Please contact Dr. M. Lorna De Leoz at (301) 975-6731 or [email protected] if you have questions or concerns. Thank you.



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Data Reporting Template NIST Interlaboratory Study on Glycosylation Analysis Data Reporting Template, page 1 of 4 Lab #: Report #: Total # of Reports Submitted:

Please refer to the instructions before entering any data. Fill in as much of the green boxes to report your results. NOT ALL GLYCANS MAY BE PRESENT IN THE SAMPLE. Please do not reorder, edit, or delete any of the 54 glycans. If you found glycans not listed in the table, please add them below the line "List other glycans below." Please use the glycan analysis method you normally use. If you obtained data from more than one method, please save the data and methods for each additional method in a new, renamed Excel file. If you acquired more than three replicates, you may add columns in the raw data section. If a value obtained is below your limit of detection or quantification, please indicate this result on the form as "ND" (not detected) or "NQ" (not quantified), respectively. Further guidance is shown when you hover your mouse on cells with red triangles on the upper right corner. Note that many cells are annotated. Please double check entries into this table! It is useful to have a second person confirm data entry. If you have questions on any of the boxes, please contact Dr. M. Lorna De Leoz ([email protected] or (301) 975-6731) for clarification.



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NIST Interlaboratory Study on Glycosylation Analysis Data Reporting Template, page 2 of 4

DESCRIPTION: GLYCAN IDENTITY RESULTS: GLYCAN QUANTIFICATION RAW DATA: GLYCAN QUANTIFICATION COMMENTS:

Common Names[Composition]

CFG Structure

Oxford Structure

Other Info%

AbundanceSD

% Abundance

SD Replicate 1

Replicate 2

Replicate 3

Replicate 1

Replicate 2

Replicate 3

11. G0F / FA2[h3n4f1]

NGA2F;(GlcNAc)2Man3(Fuc)(GlcNAc)2;Mannotri

22. G1F / FA2G1[h4n4f1]

NA2G1F;Gal(GlcNAc)2(Man)3(GlcNAc)2Fuc;Asial

2a. G1F(1,6) / F(6)A2[6]G(4)1[h4n4f1]


2b. G1F(1,3) or F(6)A2[3]G(4)1[h4n4f1]


33. G2F / FA2G2 or G1F+1aGal [h5n4f1]

NA2F;(Gal)2(GlcNAc)2(Man)3(GlcNAc)2Fuc;Mann

3a. G2F / F(6)A2G(4)2[h5n4f1]


3b. G1F+1aGal / F(6)A2G1Ga1[h5n4f1]


44. G0F-N / FA1[h3n3f1]

4a. G0F-N(1,6) / F(6)A(6)1[h3n3f1]

4b. G0F-N(1,3) / F(6)A(3)1[h3n3f1]

55. G1F-N / FA1G1[h4n3f1]

Core fucosylated mono antennary monogalactosyla

5a. G1F-N(1,6) / FA1[6]G1[h4n3f1]


5b. G1F-N(1,3) / FA1[3]G1[h4n3f1]


66. G1F-N+1aGal / FA1G1Ga1[h5n3f1]

77. G1F2-N+1aGal / FA1F1G1Ga1[h5n3f2]

88. G0FB / FA2B[h3n5f1]

99. G1FB / FA2BG1[h4n5f1]

1010. G2FB / FA2BG2[h5n5f1]

1111. G1F2 / FA2F1G1[h4n4f2]

1212. G2F2 / FA2F1G2[h5n4f2]

1313. G2F+1aGal / FA2G2Ga1[h6n4f1]

1414. G2F2+1aGal / FA2F1G2Ga1[h6n4f2]

1515. G2FB+1aGal / FA2BG2Ga1[h6n5f1]

1616. G2F+2aGal / FA2G2Ga2[h7n4f1]

1717. G2FB+2aGal / FA2BG2Ga2[h7n5f1]

Uncertainty in Identity

Uncertainty in

Abundance

Other Comments

Glycan Identity

Com

plex

Neu

tral

Fuc

osyl

ated

Sample A, % Abundance Sample B, % AbundanceSample A Sample B



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1818. G0 / A2[h3n4]

NGA2;(GlcNAc)2Man3(GlcNAc)2;Mannotriose-di-

1919. G1 / A2G1[h4n4]

NA2G1;AGn;Asialo,monogalactosylated

19a. G1(1,6) / A2[6]G1[h4n4]


19b. G1(1,3) / A2[3]G1[h4n4]


2020. G2 / A2G2[h5n4]

2121. G0-N / A1[h3n3]

MGn, A2G0-GlcNAc

21a. G0-N(1,6) / A1[6][h3n3]

MGn, A2G0-GlcNAc

21b. G0-N(1,3) / A1[3][h3n3]

MGn, A2G0-GlcNAc

2222. G1-N / A1G1[h4n3]

2323. G1-N+1aGal / A1G1Ga1[h5n3]

2424. G0B / A2B[h3n5]

2525. G1B / A2BG1[h4n5]

25a. G1B(1,6) / A2[6]BG1[h4n5]

NA2G1;AGn;A2G1;Asialo, monogalactosyla

25b. G1(1,3) / A2[3]BG1[h4n5]

NA2G1;AGn;A2G1;Asialo, monogalactosyla

2626. G2B / A2BG2[h5n5]

2727. G2+1aGal / A2G2Ga1[h6n4]

28 28. G2+2aGal / A2G2Ga2

2929. G1FS-N / FA1G1S1 (NeuAc)[h4n3f1a1]

3030. G1FS / FA2G1S1 (NeuAc)[h4n4f1a1]

3131. G1FBS / FA2BG1S1 (NeuAc)[h4n5f1a1]

3232. G2FS / FA2G2S1 (NeuAc)[h5n4f1a1]

A1F;NeuAc(Gal-GlcNAc)2Man3(GlcNAc)2Fuc;1898

3333. G2FS2 / FA2G2S2 (NeuAc)[h5n4f1a2]

A2F;(NeuAc-Gal-GlcNAc)2Man3(Fuc)(GlcNAc)2;Dis

3434. G2FS + 1aGal / FA2G2Ga1S1 (NeuAc) [h6n4f1a1]

3535. G1FS-N / FA1G1S1 (NeuGc)[h4n3f1g1]

3636. G1FS / FA2G1S1 (NeuGc)[h4n4f1g1]

3737. G1FBS / FA2BG1S1 (NeuGc)[h4n5f1g1]

3838. G2FS / FA2G2S1 (NeuGc)[h5n4f1g1]

3939. G2FS2 / FA2G2S2 (NeuGc)[h5n4f1g2]

A2F;(NeuGc-Gal-GlcNAc)2Man3(Fuc)(GlcNAc)2;Dis

4040. G2FS + 1aGal / FA2G2Ga1S1 (NeuGc) [h6n4f1g1]

Com

plex

Acid

ic Fu

cosy

late

dCo

mpl

ex N

eutr

al N

on-F

ucos

ylat

ed



T.IR.8186

A-8


4141. G2S / A2G2S1 (NeuAc)[h5n4a1]

4242. G2S2 / A2G2S2 (NeuAc)[h5n4a2]

4343. G2S / A2G2S1 (NeuGc)[h5n4g1]

4444. G2S2 / A2G2S2 (NeuGc)[h5n4g2]

4545. Man5 / M5[h5n2]

(Man)5(GlcNAc)2;Oligomannose-5;Mannopentaos

4646. Man6 / M6[h6n2]

(Man)6(GlcNAc)2;Oligomannose-6;Mannohexaos

4747. Man5G0F hybrid / M5A1[3]S1 (NeuGc) [h5n3f1g1]

4848. Man5G1 hybrid / M5A1[3]G1 [h6n3]

4949. Man5G1F hybrid / M5FA1[3]G1[h6n3f1]

5050. Man5G1FS hybrid / M5FA1[3]G1 (NeuGc) [h6n3f1g1]

5151. Man5G1 +1aGal hybrid / M5A1[3]G1Ga1 [h7n3]

5252. Man5G1F hybrid +1aGal /M5FA1[3]G1Ga1 [h7n3f1]

5353. Core / M3[h3n2]

Man3(GlcNAc)2

5454. Core+F / FM3[h3n2f1]

Man3(Fuc)(GlcNAc)2

List other glycans below

55

56

57

58

59

60

61

62

63

64

65

66

67

68

69

70

SUM 0 0 0 0 0 0 0 0

Frag

men

tHy

brid

High

-Man

nose

Com

plex

Acid

ic N

on-F

uco



T.IR.8186

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Method Reporting Template NIST Interlaboratory Study on Glycosylation Analysis Method Reporting Template, page 1 of 3 Please describe the methods used in this reported results.

1. Identification of DataLab #Lab Classification Other/Comment:Country Other/Comment:Report #Total # of Reports Submitted

2. Description of Measurement MethodSample PreparationDates of measurements for Sample ADates of measurements for Sample B

Number of replicate measurements for Sample ANumber of replicate measurements for Sample B

Mass (ug) of Sample A used per run ugMass (ug) of Sample B used per run ug

Dilution factor (if any)Dilution media

Type and number of replicates for Sample AType and number of replicates for Sample B

Proteolytic enzyme used Other: Other/Comment:Derivatization used Other: Other/Comment:

Describe pre-treatment of samples (e.g. immobilization), if any

Describe the digestion conditions (enzyme: IgG ratio, buffer (including additives), pH, digestion time, temperature)

Describe purification/cleanup methods (e.g. SPE or ziptip, type, vacuum or gravity)

Please refer to the instructions before acquiring any data.On this form, fill in as much of the green boxes as you can. If you have obtained another set of data using another method, please use a new sheet. You may expand the columns and rows to document additional information. Further guidance is shown when you hover your mouse on cells with red triangles on the upper right side. If you have questions on any of the boxes, please feel free to contact Dr. M. Lorna De Leoz at [email protected] or (301) 975-6731 for clarification.Please double check entries! It is useful to have a second person confirm data entry.

Thanks!



T.IR.8186

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NIST Interlaboratory Study on Glycosylation Analysis Method Reporting Template, page 2 of 3

AnalysisGeneral Strategy (If other, please specify)

Analytical Technique (If other, please specify)

Analyte (If other, please specify)

Identification method (If other, please specify)

Quantification method (If other, please specify)

Separation method (If other, please specify)

Limit of detectionLimit of quantificationDilution factorDilution media

Describe identification method (parameters, type(s) of instrument(s) used (manufacturer, model #))

Database(s) used to confirm identity

Describe quantification method, type(s) of instrument(s) used (manufacturer, model #)

Describe separation/chromatography method (gradient, parameters, type(s) of instrument(s) used (manufacturer, model #))

Describe how the percent abundances of glycans were extracted from the data

Other/Comment:

Other/Comment:

Other/Comment:

Other/Comment:

Other/Comment:

Other/Comment:



T.IR.8186

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NIST Interlaboratory Study on Glycosylation Analysis Method Reporting Template, page 3 of 3

For MS, analysis:MS source and sample introduction

MS detector and ion mode

Tandem MS method (if used)

Resolution and mass accuracy of analysis

Specify how MS and/or tandem MS data were interpreted

For glycopeptide analysis, specify the sequence(s) of the peptide(s) used to quantify the mAb glycans

For ESI, specify how charge states were analyzed

Other information

Other:

Other:

Other:



T.IR.8186

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Comments and Suggestions NIST Interlaboratory Study on Glycosylation Analysis Comments and Suggestions

Your input, expertise, and feedback is extremely valuable for future studies. Please write any comments and suggestions that may be relevant to the interlab study. Feel free to expand columns and rows if you need more space. Address any questions to Dr. M. Lorna De Leoz at [email protected] or (301) 975-6731. Thanks!

Overall:

Shipping:

Glycan Identification (current methods for describing identity of glycans):

Data Reporting:

Samples (e.g. concentration, amount, container, label…):

Sample Analysis:

Other:



T.IR.8186

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Appendix B. Final Report for the Interlaboratory Study The final report delivered to every participant in the interlaboratory study consists of four files:

• Cover letter: contains a brief description of the samples and the other three documents • Table of Identified Glycans: list of identifiers, compositions, and structures for all

glycans reported in any data set • All-Lab Report: summarizes reported and derived values for Samples A and B, and the

A/B ratio • Individualized Report: graphically analysis of each participant’s results for all glycan

compositions. It contains boxplots of Samples A and B, and the A/B ratio, and a target plot summary for the A/B ratio for compositions reported by at least six participants . This report also provides a graphical summary of each participant’s measurement comparability, including glycan composition counts and sums, repeatability, limits of reporting, minimum reported values, and consensus values.



T.IR.8186

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Cover Letter


June 2, 2017 Dear Colleague: Thank you for participating in the NIST interlaboratory study of NISTmAb Glycosylation. Enclosed is the preliminary report of the results. Included in this report are: 1) a summary of reported and derived values for Samples A and B, and A/B ratio from all laboratories, 2) a detailed graphical analysis of your results; and 3) summary tables of the results you reported. The NISTmAb Glycosylation study consisted of two vials of liquid-frozen monoclonal antibody samples, NISTmAb and a glycan-modified NISTmAb, and one 25 mmol/L L-Histidine pH 6.0 solution that was intended for use in reconstituting the two samples. Your overall measurement comparability is summarized in the boxplot and targetplot summaries found in page 1 of your report; this summary reflects only results for glycan compositions. We intend to follow this report with a summary of results for the unique glycoforms and glycoform isomers at a later date. If you have concerns or questions regarding your laboratory’s performance, please contact M. Lorna De Leoz at [email protected] or +1-(301) 975-6731. Again, many thanks for joining us in this endeavor. Sincerely, M. Lorna A. De Leoz, Ph.D. Stephen E. Stein, Ph.D. David L. Duewer, Ph.D. Research Chemist NIST Fellow Chemometrician Biomolecular Measurement Division

Biomolecular Measurement Division

Chemical Sciences Division

UNITED STATES DEPARTMENT OF COMMERCE National Institute of Standards and Technology Gaithersburg, Maryland 20899



T.IR.8186

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The complete report for this study consists of three files: Table of Identified Glycans #Pages Identifiers and structures for all glycans reported in any data set 17

All-Lab Report #Pages Summary of reported and derived values for Samples A and B, and the A/B ratio 9 Legend for the summary 1

Individualized Report #Pages Boxplots of Samples A and B, and the A/B ratio, and targetplot for the A/B ratio 1 Legend for the boxplots and targetplot 1 Plots summarizing measurement performance, including glycan composition counts and sums, repeatability, limits of reporting, minimum reported values, and consensus

1

Legend for measurement performance plots 1 Table of measurement summary Variable Legend for table of measurement summary 1 Table of derived glycan attribute quantities 1 Legend for table of derived glycan attribute quantities 1



T.IR.8186

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Table of Identified Glycans Glycans reported by at least one participant arranged in increasing monosaccharide composition. Entries highlighted in orange are unique glycan compositions; glycans having the same monosaccharide compositions are shown beneath the highlighted entry.



T.IR.8186

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Table of Identified Glycans

Indexa Codeb Measurandsc Oxfordd Compositione CFGf Oxfordg 42 [h2n3f1] [h2n3f1]

42.1 Fragment Man2F+N [h2n3f1]

37 [h3n2] [h3n2]

37.1 53 Man3 M3 [h3n2]

19 [h3n2f1] [h3n2f1]

19.1 54 Man3F FM3 [h3n2f1]

11 [h3n3] [h3n3]

11.1 21 G0-N A1 [h3n3]

11.11 21a G0-N(1,6) A1[6] [h3n3]

11.12 21b G0-N(1,3) A1[3] [h3n3]

11.2 Man3B M3B [h3n3]

4 [h3n3f1] [h3n3f1]

4.1 04 G0F-N FA1 [h3n3f1]

4.11 04a G0F-N(1,6) F(6)A(6)1 [h3n3f1]

4.12 04b G0F-N(1,3) F(6)A(3)1 [h3n3f1]

109 [h3n3f2] [h3n3f2]

109.1 Man3F2+N [h3n3f2]

78 [h3n3g1] [h3n3g1]



T.IR.8186

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Indexa Codeb Measurandsc Oxfordd Compositione CFGf Oxfordg 13 [h3n4] [h3n4]

13.1 18 G0 A2 [h3n4]

13.2 G0B-N A1B [h3n4]

13.21 G0B-N(1,3) A1(3)B [h3n4]

13.22 G0B-N(1,6) A1(6)B [h3n4]

2 [h3n4f1] [h3n4f1]

2.1 01 G0F FA2 [h3n4f1]

2.2 G0FB-N FA1B [h3n4f1]

2.3 Man3F+2N FM3A2 [h3n4f1]

79 [h3n4f1a1] [h3n4f1a1] 80 [h3n4f1S] [h3n4f1S]

80.1 G0F-N+GalNAc4Sul [h3n4f1S]

81 [h3n4f2] [h3n4f2]

81.1 G0F2-N+GalNAc [h3n4f2]

43 [h3n5] [h3n5]

43.1 24 G0B A2B [h3n5]



T.IR.8186

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Indexa Codeb Measurandsc Oxfordd Compositione CFGf Oxfordg

43.2 G0+N (tri) A3 [h3n5]

18 [h3n5f1] [h3n5f1]

18.1 08 G0FB FA2B [h3n5f1]

18.2 G0F+N (tri) F(6)A3 [h3n5f1]

18.3 ManF+3N [h3n5f1]

18.31 G0F+N [h3n5f1]

82 [h3n5f2] [h3n5f2] 83 [h3n7] [h3n7]

83.1 G0FB+2N (quad) A4B [h3n7]

53 [h4n2] [h4n2]

53.1 Man4 M4 [h4n2]

53.11 Man4D2 M4D2 [h4n2]

64 [h4n2f1] [h4n2f1]

64.1 Man4F F(6)M4 [h4n2f1]

64.11 Man4D2F F(6)M4D2 [h4n2f1]

22 [h4n3] [h4n3]

22.1 22 G1-N A1G1 [h4n3]

22.2 Man4N M4A1 [h4n3]

22.21 Man4N[3] M4[3]A1 [h4n3]



T.IR.8186

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Indexa Codeb Measurandsc Oxfordd Compositione CFGf Oxfordg 84 [h4n3a1] [h4n3a1]

84.1 G1S-N (NeuAc) A1G1S1 (NeuAc) [h4n3a1]

10 [h4n3f1] [h4n3f1]

10.1 05 G1F-N FA1G1 [h4n3f1]

10.11 05a G1F-N(1,6) FA1[6]G1 [h4n3f1]

10.12 05b G1F-N(1,3) FA1[3]G1 [h4n3f1]

10.2 Man4FN F(6)M4A1 [h4n3f1]

36 [h4n3f1a1] [h4n3f1a1]

36.1 29 G1FS-N FA1G1S1 (NeuAc) [h4n3f1a1]

12 [h4n3f1g1] [h4n3f1g1]

12.1 35 G1FS-N FA1G1S1 (NeuGc) [h4n3f1g1]

44 [h4n3f2] [h4n3f2]

44.1 G1F-N+AF FA1F1G1 [h4n3f2]

54 [h4n3g1] [h4n3g1]

54.1 G1S (NeuGc) A1G1S1 (NeuGc) [h4n3g1]

20 [h4n4] [h4n4]

20.1 19 G1 A2G1 [h4n4]

20.11 19a G1(1,6) A2[6]G1 [h4n4]

20.12 19b G1(1,3) A2[3]G1 [h4n4]

20.2 G1B-N [h4n4]

65 [h4n4a1] [h4n4a1]



T.IR.8186

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65.1 G1S (NeuAc) A2G1S1 [h4n4a1]

1 [h4n4f1] [h4n4f1]

1.1 02 G1F FA2G1 [h4n4f1]

1.11 02a G1F(1,6) F(6)A2[6]G(4)1 [h4n4f1]

1.12 02b G1F(1,3) F(6)A2[3]G(4)1 [h4n4f1]

1.2 G1FB-N F(6)A1BG1 [h4n4f1]

1.21 G1FB-N[3] F(6)A1BG1[3] [h4n4f1]

1.22 G1FB-N[6] F(6)A1BG1[6] [h4n4f1]

40 [h4n4f1a1] [h4n4f1a1]

40.1 30 G1FS (NeuAc) FA2G1S1 (NeuAc) [h4n4f1a1]

14 [h4n4f1g1] [h4n4f1g1]

14.1 36 G1FS (NeuGc) FA2G1S1 (NeuGc) [h4n4f1g1]

14.11 G1FS[6] (NeuGc) F(6)A2[6]G(4)1S(6)1 (NeuGc) [h4n4f1g1]

14.12 G1FS[3] (NeuGc) F(6)A2[3]G(4)1S(6)1 (NeuGc) [h4n4f1g1]

85 [h4n4f1g2] [h4n4f1g2]

85.1 G1FS2 (NeuGc) [h4n4f1g2]

86 [h4n4f1S] [h4n4f1S]



T.IR.8186

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86.1 Man4G0F+GalNAc4Sul hybrid [h4n4f1S]

38 [h4n4f2] [h4n4f2]

38.1 11 G1F2 FA2F1G1 [h4n4f2]

57 [h4n5] [h4n5]

57.1 25 G1B A2BG1 [h4n5]

57.11 25a G1B(1,6) A2[6]BG1 [h4n5]

57.12 25b G1B(1,3) A2[3]BG1 [h4n5]

87 [h4n5a1] [h4n5a1]

87.1 G1S+N (NeuAc) (tri) A3G1S1 [h4n5a1]

8 [h4n5f1] [h4n5f1]

8.1 09 G1FB FA2BG1 [h4n5f1]

8.2 NA3FG1 F(6)A3G(4)1 [h4n5f1]

8.3 G0F+Hex+HexNAc [h4n5f1]

8.4 G1F+N [h4n5f1]

70 [h4n5f1a1] [h4n5f1a1]

70.1 31 G1FBS FA2BG1S1 (NeuAc) [h4n5f1a1]

45 [h4n5f1g1] [h4n5f1g1]

45.1 37 G1FBS (NeuGc) FA2BG1S1 (NeuGc) [h4n5f1g1]

5 [h5n2] [h5n2]



T.IR.8186

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5.1 45 Man5 M5 [h5n2]

88 [h5n2f1] [h5n2f1]

88.1 Man5F FM5 [h5n2f1]

29 [h5n3] [h5n3]

29.1 23 G1-N+1aGal A1G1Ga1 [h5n3]

46 [h5n3a1] [h5n3a1]

46.1 Man4G1S hybrid M4A1G(4)1S(6)1 [h5n3a1]

46.2 Fragment G2S-

CoreN (NeuAc) A2G(4)2S(6)1 [h5n3a1]

9 [h5n3f1] [h5n3f1]

9.1 06 G1F-N+1aGal FA1G1Ga1 [h5n3f1]

9.2 Man5G0F hybrid FM5A1[3] [h5n3f1]

9.3 G1F+Man F(6)M4A1G(4)1 [h5n3f1]

9.31 G1F+Man(1,3) F(6)M4A1[3]G(4)1 [h5n3f1]

71 [h5n3f1a1] [h5n3f1a1]

71.1 Man4FG1FS (NeuAc) [h5n3f1a1]

71.2 G1FS-N+1aGal

(NeuAc) FA1G1Ga1S1 [h5n3f1a1]

26 [h5n3f1g1] [h5n3f1g1]

26.1 47 Man5G0FS (NeuGc) hybrid FM5A1[3]S1 (NeuGc) [h5n3f1g1]

26.2 Man4G1FS (NeuGc) hybrid

F(6)M4A1G(4)1S(6)1 (NeuGc) [h5n3f1g1]

58 [h5n3f2] [h5n3f2]



T.IR.8186

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58.1 07 G1F2-N+1aGal FA1F1G1Ga1 [h5n3f2]

66 [h5n3g1] [h5n3g1]

66.1 Man4G1S1 (NeuGc) hybrid [h5n3g1]

21 [h5n4] [h5n4]

21.1 20 G2 A2G2 [h5n4]

21.2 G1+1aGal A2G1Ga1 [h5n4]

27 [h5n4a1] [h5n4a1]

27.1 41 G2S (NeuAc) A2G2S1 (NeuAc) [h5n4a1]

59 [h5n4a2] [h5n4a2]

59.1 42 G2S2 (NeuAc) A2G2S2 (NeuAc) [h5n4a2]

3 [h5n4f1] [h5n4f1]

3.1 03a G2F F(6)A2G(4)2 [h5n4f1]

3.2 03b G1F+1aGal F(6)A2G1Ga1 [h5n4f1]

3.21 G1F(1,3)+1aGal F(6)A2[3]G(4)1Ga(3)1 [h5n4f1]

3.22 G1F(1,6)+1aGal F(6)A2[6]G(4)1Ga(3)1 [h5n4f1]

3.3 G1FB-N+1aGal FA1G1BGa(2)1 [h5n4f1]

35 [h5n4f1a1] [h5n4f1a1]

35.1 32 G2FS (NeuAc) FA2G2S1 (NeuAc) [h5n4f1a1]

41 [h5n4f1a2] [h5n4f1a2]



T.IR.8186

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41.1 33 G2FS2 (NeuAc) FA2G2S2 (NeuAc) [h5n4f1a2]

16 [h5n4f1g1] [h5n4f1g1]

16.1 38 G2FS (NeuGc) FA2G2S1 (NeuGc) [h5n4f1g1]

55 [h5n4f1g2] [h5n4f1g2]

55.1 39 G2FS2 (NeuGc) FA2G2S2 (NeuGc) [h5n4f1g2]

47 [h5n4f2] [h5n4f2]

47.1 12 G2F2 FA2F1G2 [h5n4f2]

47.2 Man4G1F2B hybrid [h5n4f2]

60 [h5n4g1] [h5n4g1]

60.1 43 G2S (NeuGc) A2G2S1 (NeuGc) [h5n4g1]

48 [h5n4g2] [h5n4g2]

48.1 44 G2S2 (NeuGc) A2G2S2 (NeuGc) [h5n4g2]

67 [h5n5] [h5n5]

67.1 26 G2B A2BG2 [h5n5]

67.2 G1+N+1aGal (tri) A3G(4)Ga1 [h5n5]

17 [h5n5f1] [h5n5f1]

17.1 10 G2FB FA2BG2 [h5n5f1]

17.2 G1FB+1aGal FA2BGGa(2)1 [h5n5f1]

17.3 G2F+N (tri) FA3G2 [h5n5f1]



T.IR.8186

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17.31 G1F+N+1aGal F(6)A3G(4)Ga1 [h5n5f1]

17.32 G2F(1,4)+N (tri) F(6)A3G(4)2 [h5n5f1]

17.4 G0F+2Hex+HexNAc [h5n5f1]

89 [h5n5f1a1] [h5n5f1a1]

89.1 G2FS+N (NeuAc) (tri) FA3G2S1 [h5n5f1a1]

90 [h5n5f1a2] [h5n5f1a2]

90.1 G2FS2+N (NeuAc) (tri) FA3G2S2 [h5n5f1a2]

91 [h5n5f1g1] [h5n5f1g1] 61 [h5n5f2] [h5n5f2]

61.1 G2F2B FA2BG2F [h5n5f2]

61.2 G2F2+N (tri) FA3F1G2 [h5n5f2]

30 [h6n2] [h6n2]

30.1 46 Man6 M6 [h6n2]

92 [h6n2f1] [h6n2f1]

92.1 Man6F M6F [h6n2f1]

110 [h6n2g1] [h6n2g1]

110.1 Man5+Gal+S (NeuGc) hybrid [h6n2g1]

28 [h6n3] [h6n3]

28.1 48 Man5G1 hybrid M5A1[3]G1 [h6n3]

72 [h6n3a1] [h6n3a1] 24 [h6n3f1] [h6n3f1]



T.IR.8186

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24.1 49 Man5G1F hybrid FM5A1[3]G1 [h6n3f1]

24.2 Man4G1F+1aGal hybrid F(6)M4A1G1Ga1 [h6n3f1]

111 [h6n3f1a1] [h6n3f1a1]

111.1 Man5G1FS1 hybrid F(6)M5A1G(4)1S(3)1 (NeuAc) [h6n3f1a1]

32 [h6n3f1g1] [h6n3f1g1]

32.1 50 Man5G1FS hybrid FM5A1[3]G1 (NeuGc) [h6n3f1g1]

93 [h6n3f2] [h6n3f2] 112 [h6n3f2a1] [h6n3f2a1]

112.1 Man5G1F2S (NeuAc) hybrid [h6n3f2a1]

49 [h6n3g1] [h6n3g1]

49.1 Man5G1S (NeuGc) hybrid [h6n3g1]

50 [h6n4] [h6n4]

50.1 27 G2+1aGal A2G2Ga1 [h6n4]

50.2 Fragment Man5G1-CoreN hybrid [h6n4]

73 [h6n4a1] [h6n4a1]

73.1 G2S+1aGal (NeuAc) [h6n4a1]

7 [h6n4f1] [h6n4f1]

7.1 13 G2F+1aGal FA2G2Ga1 [h6n4f1]

7.11 G2F+1aGal[6] F(6)A2[6]G2Ga1 [h6n4f1]



T.IR.8186

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7.12 G2F+1aGal[3] F(6)A2[3]G2Ga1 [h6n4f1]

51 [h6n4f1a1] [h6n4f1a1]

51.1 34 G2FS+1aGal (NeuAc) FA2G2Ga1S1 (NeuAc) [h6n4f1a1]

51.2 Man5G1FBS (NeuAc) hybrid [h6n4f1a1]

15 [h6n4f1g1] [h6n4f1g1]

15.1 40 G2FS+1aGal (NeuGc) FA2G2Ga1S1 (NeuGc) [h6n4f1g1]

31 [h6n4f2] [h6n4f2]

31.1 14 G2F2+1aGal FA2F1G2Ga1 [h6n4f2]

31.2 Man5G1F2B hybrid [h6n4f2]

94 [h6n4g1] [h6n4g1] 74 [h6n5] [h6n5]

74.1 G3 [h6n5]

74.11 G3[6] A(6)3G3 [h6n5]

75 [h6n5a1] [h6n5a1] 25 [h6n5f1] [h6n5f1]

25.1 15 G2FB+1aGal FA2BG2Ga1 [h6n5f1]

25.2 G3F [h6n5f1]



T.IR.8186

B-17


25.21 G3F[3] F(6)A3G(4)3 [h6n5f1]

68 [h6n5f1g1] [h6n5f1g1]

68.1 G2FBS+1aGal (NeuGc) [h6n5f1g1]

68.2 G3FS (NeuGc) F(6)A3G(4)3S(6)1 (NeuGc) [h6n5f1g1]

95 [h6n5f1g2] [h6n5f1g2]

95.1 G3FS2(NeuGc) FA3G3S2 [h6n5f1g2]

96 [h6n7f4a3] [h6n7f4a3]

97 [h6n7f5a2] [h6n7f5a2] 52 [h7n2] [h7n2]

52.1 Man7 M7 [h7n2]

39 [h7n3] [h7n3]

39.1 51 Man5G1 +1aGal hybrid M5A1[3]G1Ga1 [h7n3]

33 [h7n3f1] [h7n3f1]

33.1 52 Man5G1F hybrid +1aGal FM5A1[3]G1Ga1 [h7n3f1]

98 [h7n3f2] [h7n3f2]

99 [h7n4] [h7n4]

99.1 28 G2+2aGal A2G2Ga2 [h7n4]

62 [h7n4a1] [h7n4a1]

62.1 G2S+2aGal (NeuAc) [h7n4a1]

62.2 Man5G2S (NeuAc) hybrid [h7n4a1]

62.3 G2S(6)+2aGal (NeuAc) [h7n4a1]



T.IR.8186

B-18

Indexa Codeb Measurandsc Oxfordd Compositione CFGf Oxfordg 6 [h7n4f1] [h7n4f1]

6.1 16 G2F+2aGal FA2G2Ga2 [h7n4f1]

6.2 Man5G2F hybrid FM5A2G2 [h7n4f1]

23 [h7n5f1] [h7n5f1]

23.1 17 G2FB+2aGal FA2BG2Ga2 [h7n5f1]

23.2 G3F+1aGal FA3G3Ga1 [h7n5f1]

23.21 G(4)3F+1aGal F(6)A3G(4)3Ga1 [h7n5f1]

23.3 G2F+2aGal (tri) F(6)A3G(4)2Ga2 [h7n5f1]

23.4 Man5F+2Gal+3N F(6)M5A3G2 [h7n5f1]

113 [h7n5f1g1] [h7n5f1g1]

113.1 G2FBGS+1aGal (NeuGc) [h7n5f1g1]

100 [h7n5f1g2] [h7n5f1g2]

100.1 G2FBGS2+1aGal (NeuGc)

[h7n5f1g2]

101 [h7n5f2] [h7n5f2] 114 [h7n6f1a1g3] [h7n6f1a1g3]

114.1 G4FS4 (3NeuGc) (1NeuAc)

[h7n6f1a1g3]

102 [h7n6f2a1] [h7n6f2a1]

102.1 G4F2S (NeuAc) [h7n6f2a1]

103 [h7n8f3a4] [h7n8f3a4]

115 [h7n8f5a3] [h7n8f5a3]

116 [h7n8f6a3] [h7n8f6a3]

104 [h7n9f1a4] [h7n9f1a4]



T.IR.8186

B-19

Indexa Codeb Measurandsc Oxfordd Compositione CFGf Oxfordg 105 [h8n10f5a3] [h8n10f5a3]

63 [h8n2] [h8n2]

63.1 Man8 M8 [h8n2]

56 [h8n5f1] [h8n5f1]

56.1 G3F+2aGal F(6)A3G(4)3Ga2 [h8n5f1]

56.2 G2FGB+2aGal FA2BG3Ga2 [h8n5f1]

69 [h8n5f1g1] [h8n5f1g1]

69.1 G3FS+2aGal (NeuGc)

F(6)A3G(4)3Ga2S1 (NeuGc) [h8n5f1g1]

69.2 G2FBGS+3aGal (NeuGc) [h8n5f1g1]

76 [h8n5f2] [h8n5f2] 106 [h8n8f3a4] [h8n8f3a4] 77 [h9n2] [h9n2]

77.1 Man9 M9 [h9n2]

34 [h9n5f1] [h9n5f1]

34.1 G3F+3aGal F(6)A3G(4)3Ga3 [h9n5f1]

34.2 G2FBG+3aGal [h9n5f1]

107 [n1f1] [n1f1]

108 [n2f1] [n2f1]

108.1 Fragment FN2 [n2f1]

G0F-N/G0F FA1/FA2 [h3n3f1]/ [h3n4f1]

G0/G1F A2/FA2G1 [h3n4]/ [h3n4f1]

G0F/G1F FA2/FA2G1 [h3n4f1]/ [h4n4f1]



T.IR.8186

B-20


G1F/G2F FA2G1/FA2G2 [h4n4f1]/ [h5n4f1]

G2F/ G2F+1aGal

[h5n4f1]/ [h6n4f1]

[h5n5f1g1]/ [h6n5a1]

[h6n5a3]/ [h5n5f1a2g1]/[h4n5f2a1g2]/[h3n5f3g3]

[h6n5f1a3]/ [h5n5f2a2g1]/[h4n5f3a1g2]/[h3n5f4g3]

[Unknown] Sum of results reported for “unidentified” glycan-like signals

a Index: Each different glycan composition is represented by an integer. Individual glycans are represented by a digit following a decimal point – isomers of these are represented by an additional digit. Indices in bold are glycans with complete structural assignments.

b Code: These correspond to numbers in the data reporting template. Entries with code Other are glycans reported by participants but not in the data reporting template.

c Measurands: text in square brackets correspond to monosaccharide compositions (see Composition). Common names are listed when available.

d Oxford: Oxford naming convention: All N-glycans have two core GlcNAcs; F at the start of the abbreviation indicates a core fucose, (6) after the F indicates that the fucose is α1-6 linked to the inner GlcNAc; Mx, number (x) of mannose on core GlcNAcs; Ax, number of antenna (GlcNAc) on trimannosyl core; A2, biantennary with both GlcNAcs as β1-2 linked; A3, triantennary with a GlcNAc linked β1-2 to both mannose and the third GlcNAc linked β1-4 to the α1-3 linked mannose; A3’, triantennary with a GlcNAc linked β1-2 to both mannose and the third GlcNAc linked β1-6 to the α1-6 linked mannose; A4, GlcNAcs linked as A3 with additional GlcNAc β1-6 linked to α1-6 mannose; B, bisecting GlcNAc linked β1-4 to β1-3 mannose; Gx, number (x) of linked galactose on antenna, (4) or (3) after the G indicates that the Gal is β1-4 or β1-3 linked; [3]G1 and [6]G1 indicates that the galactose is on the antenna of the α1-3 or α1-6 mannose; Gax, number (x) of linked alpha galactose on antenna; Sx, number (x) of sialic acids linked to galactose; the numbers 3 or 6 in parentheses after S indicate whether the sialic acid is in an α2-3 or α2-6 linkage. (Courtesy of Louise Royle, Ludger)

e [Composition] denotes monosaccharide composition. Small letters are used to avoid confusion with elements (hydrogen, nitrogen, fluorine, etc.): h=hexose, n=N-acetylhexosamine, f=deoxyhexose (e.g. fucose), a=NeuAc, g=NeuGc. Number after the letter denotes the number of residues. For example: [h6n4f1a1] = 6 hexoses, 4 N-acetylhexosamine, 1 fucose, 1 NeuAc. For sulfonated glycans, S=sulfur.



T.IR.8186

B-21

f CFG: Structure using the Consortium for Functional Glycomics (CFG) Notation: Symbol representations of glycans: galactose= , glucose= , mannose= , N-Acetylgalactosamine= , N-Acetylglucosamine= , fucose= , xylose= , N-Acetylneuraminic acid= , N-Glycolylneuraminic acid= . [1,2]

g Oxford: Structure using the Oxford Glycobiology Institute (UOXF) Notation: galactose= , glucose= , mannose= , N-Acetylgalactosamine= , N-Acetylglucosamine= , fucose= , xylose= , N-Acetylneuraminic acid= , N-Glycolylneuraminic Acid= . [2,3]

References

[1] Varki A, Cummings RD, Esko JD, Freeze HH, Stanley P, Marth JD, Bertozzi CR, Hart GW, Etzler ME (2009) Symbol nomenclature for glycan representation. Proteomics 9:5398–5399. https://doi.org/10.1002/pmic.200900708.

[2] Ceroni A, Maass K, Geyer H, Geyer R, Dell A, Haslam SM (2008) GlycoWorkbench: a tool for the computer-assisted annotation of mass spectra of glycans. J Proteome Res 7(4):1650–1659. https://doi.org/10.1021/pr7008252.

[3] Harvey DJ, Merry AH, Royle L, Campbell MP, Dwek RA, Rudd PM (2009) Proposal for a standard system for drawing structural diagrams of N- and O-linked carbohydrates and related compounds. Proteomics 9(15):3796-3801. https://doi.org/10.1002/pmic.200900096.



T.IR.8186

B-22

All-Lab Report The All-Lab Report summarizes results of all laboratories for the two samples. It consists of: All-Lab Report #Pages Summary of reported and derived values for Samples A and B, and the A/B ratio 9 Legend for the summary 1



T.IR.8186

Ind

exC

om

po

siti

on

Co

mm

on

Nam

e#

25

%M

edia

n7

5%

#2

5%

Med

ian

75

%#

25

%M

edia

n7

5%

1[h

4n

4f1

]1

03

27

.72

31

.61

33

.31

10

33

6.3

63

8.3

73

9.8

41

03

0.7

70

.83

0.8

5

1.1

[h4

n4

f1]

Gly

G1

F1

03

27

.72

31

.61

33

.31

10

33

6.3

63

8.3

73

9.8

41

03

0.7

70

.83

0.8

5

1.1

1[h

4n

4f1

] G

lyIs

oG

1F(

1,6

)5

81

9.0

42

1.2

12

2.0

65

82

7.6

02

8.1

22

9.1

25

80

.71

0.7

60

.77

1.1

2[h

4n

4f1

] G

lyIs

oG

1F(

1,3

)5

99

.38

10

.38

10

.72

59

9.8

21

0.1

81

0.7

55

90

.93

1.0

21

.03

1.2

[h4

n4

f1]

Gly

G1

FB-N

21

.06

21

.21

20

.92

1.2

1[h

4n

4f1

] G

lyIs

oG

1FB

-N[3

]2

0.6

42

0.7

42

1.2

8

1.2

2[h

4n

4f1

] G

lyIs

oG

1FB

-N[6

]2

0.4

32

0.4

72

0.9

4

2[h

3n

4f1

]1

03

47

.06

51

.47

55

.04

10

23

5.7

33

9.1

04

0.7

61

02

1.2

81

.31

1.3

8

2.1

[h3

n4

f1]

Gly

G0

F1

03

47

.06

51

.47

55

.04

10

23

5.7

33

9.0

94

0.7

11

02

1.2

81

.31

1.3

8

2.2

[h3

n4

f1]

Gly

G0

FB-N

10

.77

10

.80

10

.96

2.3

[h3

n4

f1]

Gly

Man

3F+

2N

22

.26

21

.71

21

.32

3[h

5n

4f1

]9

93

.19

3.9

74

.80

10

27

.47

8.5

29

.48

99

0.4

20

.47

0.5

5

3.1

[h5

n4

f1]

Gly

G2

F4

61

.00

1.4

22

.77

47

5.8

87

.15

7.6

14

50

.17

0.2

60

.51

3.2

[h5

n4

f1]

Gly

G1

F+1

aGal

35

1.6

42

.53

3.0

63

21

.27

1.6

02

.20

32

0.8

31

.39

1.6

8

3.2

1[h

5n

4f1

] G

lyIs

oG

1F(

1,3

)+1

aGal

20

.25

10

.17

11

.47

3.2

2[h

5n

4f1

] G

lyIs

oG

1F(

1,6

)+1

aGal

22

.88

21

.61

21

.79

3.3

[h5

n4

f1]

Gly

G1

FB-N

+1aG

al2

1.1

82

4.0

12

0.5

8

4[h

3n

3f1

]8

83

.03

3.6

54

.26

89

1.8

72

.13

2.9

38

71

.40

1.6

51

.89

4.1

[h3

n3

f1]

Gly

G0

F-N

88

3.0

33

.65

4.2

68

91

.87

2.1

32

.93

87

1.4

01

.65

1.8

9

4.1

1[h

3n

3f1

] G

lyIs

oG

0F-

N(1

,6)

10

0.8

81

.73

3.3

19

0.9

41

.61

1.8

49

1.3

11

.39

1.8

2

4.1

2[h

3n

3f1

] G

lyIs

oG

0F-

N(1

,3)

11

0.8

83

.47

3.9

01

10

.42

1.9

22

.12

11

1.2

61

.61

1.8

7

5[h

5n

2]

77

0.5

60

.78

1.1

27

90

.53

0.7

31

.01

76

0.9

41

.02

1.1

3

5.1

[h5

n2

] G

lyM

an5

77

0.5

60

.78

1.1

27

90

.53

0.7

31

.01

76

0.9

41

.02

1.1

3

6[h

7n

4f1

]7

30

.70

0.9

01

.09

71

0.6

80

.90

1.2

27

10

.90

0.9

81

.03

6.1

[h7

n4

f1]

Gly

G2

F+2

aGal

71

0.6

90

.87

1.0

57

00

.67

0.8

91

.06

70

0.8

90

.98

1.0

3

6.2

[h7

n4

f1]

Gly

Man

5G

2F

hyb

rid

11

.82

11

.60

11

.14

7[h

6n

4f1

]7

00

.44

0.6

30

.92

73

1.4

81

.80

2.0

96

90

.29

0.3

50

.45

7.1

[h6

n4

f1]

Gly

G2

F+1

aGal

70

0.4

40

.63

0.9

27

31

.48

1.8

02

.09

69

0.2

90

.35

0.4

5

7.1

1[h

6n

4f1

] G

lyIs

oG

2F+

1aG

al[6

]2

0.4

92

1.6

22

0.3

0

Mea

sura

nd

Sam

ple

A, %

Sam

ple

B, %

A/B

Rat

io

Inte

rla

bo

rato

ry S

tud

y o

f N

IST

mA

b G

lyco

syla

tio

nSu

mm

ary

of

Rep

ort

ed a

nd

Der

ived

Val

ue

s fo

r Sa

mp

les

A a

nd

B, a

nd

th

e A

/B R

atio

1 /

10

B-23



T.IR.8186

Ind

exC

om

po

siti

on

Co

mm

on

Nam

e#

25

%M

edia

n7

5%

#2

5%

Med

ian

75

%#

25

%M

edia

n7

5%

Mea

sura

nd

Sam

ple

A, %

Sam

ple

B, %

A/B

Rat

io

7.1

2[h

6n

4f1

] G

lyIs

oG

2F+

1aG

al[3

]2

0.2

32

0.2

82

0.8

0

8[h

4n

5f1

]7

00

.46

0.7

00

.89

67

0.4

90

.68

0.8

86

70

.89

1.0

31

.14

8.1

[h4

n5

f1]

Gly

G1

FB6

10

.43

0.6

70

.89

57

0.4

90

.67

0.8

75

70

.88

1.0

31

.14

8.2

[h4

n5

f1]

Gly

NA

3FG

19

0.5

10

.55

0.8

19

0.4

60

.51

0.8

39

0.9

81

.08

1.1

7

8.3

[h4

n5

f1]

Gly

G0

F+H

ex+H

exN

Ac

10

.78

10

.86

10

.90

8.4

[h4

n5

f1]

Gly

G1

F+N

10

.89

11

.01

10

.89

9[h

5n

3f1

]6

90

.78

0.8

81

.17

68

0.8

10

.89

1.2

26

70

.90

0.9

81

.06

9.1

[h5

n3

f1]

Gly

G1

F-N

+1aG

al6

20

.77

0.8

71

.15

61

0.8

10

.89

1.2

16

00

.91

0.9

91

.04

9.2

[h5

n3

f1]

Gly

Man

5G

0F

hyb

rid

70

.23

0.2

80

.80

50

.22

0.8

30

.88

50

.96

1.1

01

.13

9.3

[h5

n3

f1]

Gly

G1

F+M

an4

0.7

50

.84

1.6

04

0.7

90

.97

2.3

44

0.7

70

.87

0.9

5

9.3

1[h

5n

3f1

] G

lyIs

oG

1F+

Man

(1,3

)2

2.3

22

3.6

22

0.7

2

10

[h4

n3

f1]

68

0.2

40

.48

1.1

17

71

.68

2.2

02

.85

66

0.1

60

.27

0.4

4

10

.1[h

4n

3f1

] G

lyG

1F-

N6

60

.26

0.5

01

.13

76

1.6

72

.19

2.7

76

40

.17

0.2

70

.46

10

.11

[h4

n3

f1]

Gly

Iso

G1

F-N

(1,6

)8

0.1

90

.22

0.7

28

0.2

31

.28

2.4

47

0.5

10

.89

1.0

2

10

.12

[h4

n3

f1]

Gly

Iso

G1

F-N

(1,3

)1

30

.21

0.4

20

.75

12

1.0

01

.78

2.2

21

10

.12

0.2

60

.38

10

.2[h

4n

3f1

] G

lyM

an4

FN3

9.9

E-2

0.1

20

.13

21

.50

20

.83

11

[h3

n3

]6

90

.32

0.4

60

.78

63

0.3

20

.44

0.7

66

10

.97

1.0

81

.17

11

.1[h

3n

3]

Gly

G0

-N6

90

.29

0.4

60

.72

63

0.3

20

.43

0.7

66

10

.97

1.0

81

.17

11

.11

[h3

n3

] G

lyIs

oG

0-N

(1,6

)7

0.3

50

.38

0.5

46

0.3

40

.39

0.4

16

1.0

01

.01

1.1

8

11

.12

[h3

n3

] G

lyIs

oG

0-N

(1,3

)4

0.1

00

.34

0.8

04

7.3

E-2

0.2

70

.54

41

.21

1.3

51

.55

11

.2[h

3n

3]

Gly

Man

3B

20

.48

20

.57

20

.90

12

[h4

n3

f1g1

]6

00

.70

0.9

51

.30

60

0.7

91

.01

1.3

35

90

.90

0.9

71

.06

12

.1[h

4n

3f1

g1]

Gly

G1

FS-N

(N

euG

c)6

00

.70

0.9

51

.30

60

0.7

91

.01

1.3

35

90

.90

0.9

71

.06

13

[h3

n4

]6

30

.16

0.2

80

.84

58

0.1

30

.19

0.7

45

71

.16

1.2

51

.47

13

.1[h

3n

4]

Gly

G0

62

0.1

60

.25

0.8

15

70

.13

0.1

90

.74

56

1.1

61

.25

1.4

7

13

.2[h

3n

4]

Gly

G0

B-N

10

.98

10

.69

11

.43

13

.21

[h3

n4

] G

lyIs

oG

0B

-N(1

,3)

10

.26

10

.19

11

.37

13

.22

[h3

n4

] G

lyIs

oG

0B

-N(1

,6)

10

.72

10

.50

11

.45

14

[h4

n4

f1g1

]5

30

.31

0.5

10

.77

56

0.2

10

.35

0.6

55

21

.04

1.3

41

.58

14

.1[h

4n

4f1

g1]

Gly

G1

FS (

Neu

Gc)

53

0.3

10

.51

0.7

75

60

.21

0.3

50

.65

52

1.0

41

.34

1.5

8

14

.11

[h4

n4

f1g1

] G

lyIs

oG

1FS

[6]

(Neu

Gc)

50

.13

0.2

70

.29

50

.14

0.2

70

.28

50

.92

0.9

61

.05

14

.12

[h4

n4

f1g1

] G

lyIs

oG

1FS

[3]

(Neu

Gc)

40

.42

0.4

80

.48

40

.32

0.4

60

.64

40

.59

1.0

61

.50

15

[h6

n4

f1g1

]5

00

.31

0.4

40

.62

49

0.3

70

.46

0.7

24

90

.85

0.9

81

.05

15

.1[h

6n

4f1

g1]

Gly

G2

FS+1

aGal

(N

euG

c)5

00

.31

0.4

40

.62

49

0.3

70

.46

0.7

24

90

.85

0.9

81

.05

2 /

10

B-24



T.IR.8186

Ind

exC

om

po

siti

on

Co

mm

on

Nam

e#

25

%M

edia

n7

5%

#2

5%

Med

ian

75

%#

25

%M

edia

n7

5%

Mea

sura

nd

Sam

ple

A, %

Sam

ple

B, %

A/B

Rat

io

16

[h5

n4

f1g1

]5

00

.15

0.2

70

.47

50

0.2

90

.45

0.7

24

80

.58

0.6

40

.75

16

.1[h

5n

4f1

g1]

Gly

G2

FS (

Neu

Gc)

50

0.1

50

.27

0.4

75

00

.29

0.4

50

.72

48

0.5

80

.64

0.7

5

17

[h5

n5

f1]

51

0.2

00

.29

0.4

65

80

.34

0.4

60

.61

48

0.4

60

.55

0.9

3

17

.1[h

5n

5f1

] G

lyG

2FB

44

0.1

90

.28

0.4

65

10

.34

0.4

70

.61

42

0.4

70

.55

0.8

4

17

.2[h

5n

5f1

] G

lyG

1FB

+1aG

al1

0.4

61

0.4

21

1.0

9

17

.3[h

5n

5f1

] G

lyG

2F+

N (

tri)

50

.32

0.3

70

.39

70

.17

0.2

40

.46

40

.90

1.1

54

.97

17

.31

[h5

n5

f1]

Gly

Iso

G1

F+N

+1aG

al2

0.2

92

0.3

11

1.0

2

17

.32

[h5

n5

f1]

Gly

Iso

G2

F(1

,4)+

N (

tri)

40

.27

0.3

50

.48

50

.15

0.1

90

.54

30

.75

1.2

98

.64

17

.4[h

5n

5f1

] G

lyG

0F+

2H

ex+H

exN

Ac

29

.1E-

22

0.1

92

1.0

5

18

[h3

n5

f1]

55

0.6

40

.78

1.0

74

60

.30

0.4

91

.63

45

0.6

91

.71

2.2

2

18

.1[h

3n

5f1

] G

lyG

0FB

50

0.6

40

.77

1.0

14

10

.30

0.4

81

.36

40

1.0

71

.79

2.2

0

18

.2[h

3n

5f1

] G

lyG

0F+

N (

tri)

40

.83

1.1

11

.30

41

.96

2.4

42

.46

40

.37

0.4

60

.53

18

.3[h

3n

5f1

] G

lyM

anF+

3N

21

.06

20

.44

22

.37

18

.31

[h3

n5

f1]

Gly

Iso

G0

F+N

11

.48

10

.59

12

.52

19

[h3

n2

f1]

43

0.1

00

.14

0.4

54

50

.10

0.1

40

.33

39

0.9

51

.00

1.0

6

19

.1[h

3n

2f1

] G

lyM

an3

F4

30

.10

0.1

40

.45

45

0.1

00

.14

0.3

33

90

.95

1.0

01

.06

20

[h4

n4

]4

10

.16

0.4

71

.06

35

0.1

80

.49

1.8

83

50

.63

0.8

91

.35

20

.1[h

4n

4]

Gly

G1

40

0.1

50

.52

1.0

83

40

.18

0.5

81

.98

34

0.6

10

.90

1.3

5

20

.11

[h4

n4

] G

lyIs

oG

1(1

,6)

50

.10

0.9

51

.01

50

.18

1.3

81

.60

50

.55

0.6

01

.00

20

.12

[h4

n4

] G

lyIs

oG

1(1

,3)

70

.13

0.2

50

.53

57

.3E-

20

.35

0.6

15

0.8

50

.91

1.2

3

20

.2[h

4n

4]

Gly

G1

B-N

10

.16

10

.22

10

.74

21

[h5

n4

]3

60

.44

0.7

00

.97

36

0.3

10

.54

1.0

83

50

.75

1.0

51

.20

21

.1[h

5n

4]

Gly

G2

36

0.3

80

.63

0.9

43

60

.31

0.5

31

.01

35

0.7

11

.02

1.2

0

21

.2[h

5n

4]

Gly

G1

+1aG

al1

0.9

11

0.7

01

1.3

0

22

[h4

n3

]2

65

.6E-

20

.11

0.3

82

86

.9E-

20

.12

0.4

42

30

.68

1.0

21

.39

22

.1[h

4n

3]

Gly

G1

-N2

35

.4E-

29

.6E-

20

.42

25

6.7

E-2

0.1

20

.47

20

0.6

30

.98

1.3

7

22

.2[h

4n

3]

Gly

Man

4N

30

.14

0.1

50

.16

37

.7E-

28

.0E-

20

.10

31

.48

1.8

32

.14

22

.21

[h4

n3

] G

lyIs

oM

an4

N[3

]2

0.1

62

7.7

E-2

22

.14

23

[h7

n5

f1]

22

6.5

E-2

0.1

20

.17

23

8.5

E-2

0.1

30

.17

20

0.6

80

.82

1.0

4

23

.1[h

7n

5f1

] G

lyG

2FB

+2aG

al1

85

.7E-

20

.10

0.1

81

88

.0E-

21

.0E-

10

.15

16

0.6

80

.85

1.0

4

23

.2[h

7n

5f1

] G

lyG

3F+

1aG

al3

7.6

E-2

8.5

E-2

0.1

13

8.0

E-2

8.0

E-2

0.1

53

0.7

20

.83

0.9

5

23

.21

[h7

n5

f1]

Gly

Iso

G(4

)3F+

1aG

al1

8.5

E-2

18

.0E-

21

1.0

6

23

.3[h

7n

5f1

] G

lyG

2F+

2aG

al (

tri)

20

.10

20

.12

20

.89

23

.4[h

7n

5f1

] G

lyM

an5

F+2

Gal

+3N

01

0.1

70

3 /

10

B-25



T.IR.8186

Ind

exC

om

po

siti

on

Co

mm

on

Nam

e#

25

%M

edia

n7

5%

#2

5%

Med

ian

75

%#

25

%M

edia

n7

5%

Mea

sura

nd

Sam

ple

A, %

Sam

ple

B, %

A/B

Rat

io

24

[h6

n3

f1]

21

7.8

E-2

0.1

90

.27

27

0.1

30

.19

0.2

91

90

.40

0.7

01

.00

24

.1[h

6n

3f1

] G

lyM

an5

G1

F h

ybri

d1

86

.9E-

20

.15

0.2

62

40

.12

0.1

80

.34

16

0.4

10

.64

0.9

3

24

.2[h

6n

3f1

] G

lyM

an4

G1

F+1

aGal

hyb

rid

20

.23

20

.21

21

.13

25

[h6

n5

f1]

18

5.0

E-2

0.1

40

.20

38

0.2

00

.29

0.4

41

80

.14

0.3

30

.61

25

.1[h

6n

5f1

] G

lyG

2FB

+1aG

al1

37

.0E-

20

.14

0.1

62

20

.17

0.2

60

.36

13

0.2

40

.33

0.6

3

25

.2[h

6n

5f1

] G

lyG

3F

64

.5E-

26

.0E-

20

.19

17

0.2

60

.32

0.4

46

0.1

20

.21

0.4

4

25

.21

[h6

n5

f1]

Gly

Iso

G3

F[3

]2

3.5

E-2

11

0.2

20

.32

0.3

82

8.8

E-2

26

[h5

n3

f1g1

]2

08

.9E-

20

.12

0.2

12

09

.3E-

20

.12

0.1

71

70

.94

1.0

61

.20

26

.1[h

5n

3f1

g1]

Gly

Man

5G

0FS

(N

euG

c) h

ybri

d1

39

.6E-

20

.13

0.2

11

19

.1E-

20

.14

0.1

71

11

.00

1.1

21

.22

26

.2[h

5n

3f1

g1]

Gly

Man

4G

1FS

(N

euG

c) h

ybri

d4

0.1

10

.12

0.1

36

0.1

00

.12

0.1

33

1.0

31

.07

1.1

4

27

[h5

n4

a1]

18

0.2

10

.36

0.5

41

90

.20

0.2

61

.04

16

0.6

91

.24

1.5

1

27

.1[h

5n

4a1

] G

lyG

2S

(Neu

Ac)

18

0.2

10

.36

0.5

41

90

.20

0.2

61

.04

16

0.6

91

.24

1.5

1

28

[h6

n3

]1

64

.8E-

29

.5E-

20

.31

20

0.1

50

.23

0.4

31

60

.22

0.5

00

.87

28

.1[h

6n

3]

Gly

Man

5G

1 h

ybri

d1

64

.8E-

29

.5E-

20

.31

20

0.1

50

.23

0.4

31

60

.22

0.5

00

.87

29

[h5

n3

]1

79

.0E-

20

.13

0.2

31

73

.9E-

28

.0E-

29

.7E-

21

51

.17

1.7

12

.48

29

.1[h

5n

3]

Gly

G1

-N+1

aGal

17

9.0

E-2

0.1

30

.23

17

3.9

E-2

8.0

E-2

9.7

E-2

15

1.1

71

.71

2.4

8

30

[h6

n2

]1

76

.0E-

20

.30

0.4

71

74

.0E-

20

.33

0.5

01

50

.89

0.9

81

.12

30

.1[h

6n

2]

Gly

Man

61

76

.0E-

20

.30

0.4

71

74

.0E-

20

.33

0.5

01

50

.89

0.9

81

.12

31

[h6

n4

f2]

18

8.6

E-2

0.1

50

.24

16

9.9

E-2

0.1

20

.24

15

0.8

20

.98

1.0

4

31

.1[h

6n

4f2

] G

lyG

2F2

+1aG

al1

70

.11

0.1

60

.24

16

9.9

E-2

0.1

20

.24

15

0.8

20

.98

1.0

4

31

.2[h

6n

4f2

] G

lyM

an5

G1

F2B

hyb

rid

12

.7E-

20

0

32

[h6

n3

f1g1

]1

55

.6E-

20

.10

0.1

91

64

.1E-

27

.4E-

20

.13

13

0.9

51

.00

1.1

4

32

.1[h

6n

3f1

g1]

Gly

Man

5G

1FS

(N

euG

c) h

ybri

d1

55

.6E-

20

.10

0.1

91

64

.1E-

27

.4E-

20

.13

13

0.9

51

.00

1.1

4

33

[h7

n3

f1]

14

4.6

E-2

8.5

E-2

0.1

31

62

.7E-

26

.3E-

20

.11

13

1.0

11

.13

1.2

5

33

.1[h

7n

3f1

] G

lyM

an5

G1

F h

ybri

d+1

aGal

14

4.6

E-2

8.5

E-2

0.1

31

62

.7E-

26

.3E-

20

.11

13

1.0

11

.13

1.2

5

34

[h9

n5

f1]

13

5.0

E-2

7.1

E-2

7.5

E-2

13

4.7

E-2

5.7

E-2

6.9

E-2

13

0.9

61

.06

1.1

0

34

.1[h

9n

5f1

] G

lyG

3F+

3aG

al6

7.4

E-2

7.6

E-2

7.9

E-2

66

.9E-

27

.3E-

28

.1E-

26

1.0

01

.10

1.1

0

34

.2[h

9n

5f1

] G

lyG

2FB

G+3

aGal

15

.3E-

21

5.7

E-2

10

.93

35

[h5

n4

f1a1

]1

60

.10

0.3

10

.82

17

8.3

E-2

0.3

31

.03

12

0.4

80

.98

1.0

8

35

.1[h

5n

4f1

a1]

Gly

G2

FS (

Neu

Ac)

16

0.1

00

.31

0.8

21

78

.3E-

20

.33

1.0

31

20

.48

0.9

81

.08

36

[h4

n3

f1a1

]1

30

.58

0.8

31

.32

13

0.2

40

.59

1.1

21

10

.95

1.0

01

.12

36

.1[h

4n

3f1

a1]

Gly

G1

FS-N

(N

euA

c)1

30

.58

0.8

31

.32

13

0.2

40

.59

1.1

21

10

.95

1.0

01

.12

37

[h3

n2

]1

13

.5E-

26

.3E-

20

.42

10

3.1

E-2

5.7

E-2

0.3

01

00

.90

1.0

21

.29

37

.1[h

3n

2]

Gly

Man

31

13

.5E-

26

.3E-

20

.42

10

3.1

E-2

5.7

E-2

0.3

01

00

.90

1.0

21

.29

4 /

10

B-26



T.IR.8186

Ind

exC

om

po

siti

on

Co

mm

on

Nam

e#

25

%M

edia

n7

5%

#2

5%

Med

ian

75

%#

25

%M

edia

n7

5%

Mea

sura

nd

Sam

ple

A, %

Sam

ple

B, %

A/B

Rat

io

38

[h4

n4

f2]

11

0.1

00

.19

0.3

11

37

.7E-

20

.15

0.2

11

01

.27

1.4

51

.53

38

.1[h

4n

4f2

] G

lyG

1F2

11

0.1

00

.19

0.3

11

37

.7E-

20

.15

0.2

11

01

.27

1.4

51

.53

39

[h7

n3

]1

07

.0E-

27

.3E-

20

.11

10

5.4

E-2

8.8

E-2

0.1

41

00

.70

0.9

21

.23

39

.1[h

7n

3]

Gly

Man

5G

1+1

aGal

hyb

rid

10

7.0

E-2

7.3

E-2

0.1

11

05

.4E-

28

.8E-

20

.14

10

0.7

00

.92

1.2

3

40

[h4

n4

f1a1

]9

0.1

70

.26

0.6

21

00

.20

0.4

10

.81

70

.33

0.7

01

.11

40

.1[h

4n

4f1

a1]

Gly

G1

FS (

Neu

Ac)

90

.17

0.2

60

.62

10

0.2

00

.41

0.8

17

0.3

30

.70

1.1

1

41

[h5

n4

f1a2

]7

0.3

80

.43

0.8

38

0.3

20

.41

0.6

97

0.8

70

.90

1.0

0

41

.1[h

5n

4f1

a2]

Gly

G2

FS2

(N

euA

c)7

0.3

80

.43

0.8

38

0.3

20

.41

0.6

97

0.8

70

.90

1.0

0

42

[h2

n3

f1]

60

.31

0.5

81

.13

60

.29

0.6

21

.03

60

.82

1.0

71

.19

42

.1[h

2n

3f1

] G

lyFr

agm

ent

Man

2F+

N6

0.3

10

.58

1.1

36

0.2

90

.62

1.0

36

0.8

21

.07

1.1

9

43

[h3

n5

]6

0.3

30

.42

0.6

07

0.1

00

.17

0.4

26

1.1

21

.85

3.3

6

43

.1[h

3n

5]

Gly

G0

B6

0.3

20

.42

0.6

07

0.1

00

.17

0.4

26

1.1

21

.85

3.3

6

43

.2[h

3n

5]

Gly

G0

+N (

tri)

11

.0E-

21

1.0

E-2

11

.00

44

[h4

n3

f2]

68

.9E-

20

.14

0.2

37

5.6

E-2

0.1

20

.19

60

.91

1.0

61

.17

44

.1[h

4n

3f2

] G

lyG

1F-

N+A

F4

0.1

50

.20

0.3

34

0.1

60

.19

0.3

24

0.8

81

.00

1.1

3

45

[h4

n5

f1g1

]8

3.5

E-2

5.5

E-2

7.9

E-2

81

.3E-

23

.1E-

25

.5E-

26

1.3

02

.05

2.3

7

45

.1[h

4n

5f1

g1]

Gly

G1

FBS

(Neu

Gc)

83

.5E-

25

.5E-

27

.9E-

28

1.3

E-2

3.1

E-2

5.5

E-2

61

.30

2.0

52

.37

46

[h5

n3

a1]

70

.19

0.3

00

.84

70

.14

0.2

50

.94

60

.82

1.0

51

.20

46

.1[h

5n

3a1

] G

lyM

an4

G1

S (N

euA

c) h

ybri

d3

0.2

20

.30

0.6

52

0.6

32

1.1

0

46

.2[h

5n

3a1

] G

lyFr

agm

ent

G2

S-C

ore

N (

Neu

Ac)

10

.18

10

.24

10

.76

47

[h5

n4

f2]

72

.7E-

27

.8E-

20

.42

11

6.4

E-2

8.7

E-2

0.2

76

0.3

40

.79

1.1

7

47

.1[h

5n

4f2

] G

lyG

2F2

72

.7E-

27

.8E-

20

.42

10

7.7

E-2

0.1

20

.28

60

.34

0.7

91

.17

47

.2[h

5n

4f2

] G

lyM

an4

G1

F2B

hyb

rid

01

5.2

E-2

0

48

[h5

n4

g2]

80

.14

0.3

30

.60

70

.17

0.4

00

.83

60

.86

0.9

61

.74

48

.1[h

5n

4g2

] G

lyG

2S2

(N

euG

c)8

0.1

40

.33

0.6

07

0.1

70

.40

0.8

36

0.8

60

.96

1.7

4

49

[h6

n3

g1]

74

.0E-

27

.3E-

29

.2E-

26

5.0

E-2

6.8

E-2

0.1

16

0.7

00

.83

0.9

2

49

.1[h

6n

3g1

] G

lyM

an5

G1

S (N

euG

c) h

ybri

d4

7.0

E-2

9.2

E-2

0.3

23

8.7

E-2

0.1

30

.13

30

.69

0.8

20

.83

50

[h6

n4

]6

9.1

E-2

0.1

40

.16

60

.22

0.3

40

.90

60

.30

0.3

30

.70

50

.1[h

6n

4]

Gly

G2

+1aG

al5

8.0

E-2

0.1

20

.16

50

.27

0.4

21

.06

50

.30

0.3

00

.37

50

.2[h

6n

4]

Gly

Frag

men

t M

an5

G1

-Co

reN

hyb

rid

10

.16

10

.20

10

.81

51

[h6

n4

f1a1

]7

4.6

E-2

0.2

30

.31

97

.0E-

20

.12

0.4

76

0.7

40

.86

1.1

6

51

.1[h

6n

4f1

a1]

Gly

G2

FS+1

aGal

(N

euA

c)6

2.8

E-2

0.1

60

.25

85

.8E-

20

.10

0.2

75

0.7

40

.77

0.9

6

51

.2[h

6n

4f1

a1]

Gly

Man

5G

1FB

S (N

euA

c) h

ybri

d1

0.9

11

0.5

71

1.6

0

52

[h7

n2

]6

6.3

E-2

0.2

50

.95

70

.25

0.5

50

.87

60

.70

1.0

01

.59

5 /

10

B-27



T.IR.8186

Ind

exC

om

po

siti

on

Co

mm

on

Nam

e#

25

%M

edia

n7

5%

#2

5%

Med

ian

75

%#

25

%M

edia

n7

5%

Mea

sura

nd

Sam

ple

A, %

Sam

ple

B, %

A/B

Rat

io

52

.1[h

7n

2]

Gly

Man

76

6.3

E-2

0.2

50

.95

70

.25

0.5

50

.87

60

.70

1.0

01

.59

53

[h4

n2

]5

9.3

E-2

0.2

40

.32

79

.3E-

29

.7E-

20

.41

51

.02

1.0

41

.31

53

.1[h

4n

2]

Gly

Man

45

9.3

E-2

0.2

40

.32

79

.3E-

29

.7E-

20

.41

51

.02

1.0

41

.31

53

.11

[h4

n2

] G

lyIs

oM

an4

D2

10

.70

10

.58

11

.21

54

[h4

n3

g1]

62

.4E-

24

.1E-

29

.6E-

25

3.3

E-2

3.7

E-2

0.1

35

0.6

30

.85

0.8

5

54

.1[h

4n

3g1

] G

lyG

1S

(Neu

Gc)

62

.4E-

24

.1E-

29

.6E-

25

3.3

E-2

3.7

E-2

0.1

35

0.6

30

.85

0.8

5

55

[h5

n4

f1g2

]7

3.2

E-2

4.6

E-2

0.2

45

6.3

E-2

7.4

E-2

0.7

95

0.5

30

.82

0.8

3

55

.1[h

5n

4f1

g2]

Gly

G2

FS2

(N

euG

c)7

3.2

E-2

4.6

E-2

0.2

45

6.3

E-2

7.4

E-2

0.7

95

0.5

30

.82

0.8

3

56

[h8

n5

f1]

51

.3E-

22

.0E-

26

.6E-

29

5.0

E-2

6.0

E-2

7.5

E-2

50

.22

0.2

40

.75

56

.1[h

8n

5f1

] G

lyG

3F+

2aG

al2

4.0

E-2

46

.6E-

27

.2E-

27

.8E-

22

0.5

0

56

.2[h

8n

5f1

] G

lyG

2FG

B+2

aGal

14

.5E-

32

4.9

E-2

10

.12

57

[h4

n5

]4

0.3

00

.37

2.8

74

9.5

E-2

0.3

41

.27

40

.85

1.9

74

.10

57

.1[h

4n

5]

Gly

G1

B4

0.3

00

.37

2.8

74

9.5

E-2

0.3

41

.27

40

.85

1.9

74

.10

57

.11

[h4

n5

] G

lyIs

oG

1B

(1,6

)1

4.0

E-2

15

.0E-

21

0.8

0

57

.12

[h4

n5

] G

lyIs

oG

1B

(1,3

)1

6.0

E-2

16

.0E-

21

1.0

0

58

[h5

n3

f2]

52

.4E-

26

.5E-

20

.66

52

.8E-

20

.42

0.7

94

0.7

50

.84

0.8

7

58

.1[h

5n

3f2

] G

lyG

1F2

-N+1

aGal

52

.4E-

26

.5E-

20

.66

52

.8E-

20

.42

0.7

94

0.7

50

.84

0.8

7

59

[h5

n4

a2]

50

.20

0.2

70

.74

50

.13

0.1

30

.20

41

.26

1.4

42

.50

59

.1[h

5n

4a2

] G

lyG

2S2

(N

euA

c)5

0.2

00

.27

0.7

45

0.1

30

.13

0.2

04

1.2

61

.44

2.5

0

60

[h5

n4

g1]

70

.10

0.1

40

.16

40

.72

0.9

31

.13

40

.10

0.1

30

.45

60

.1[h

5n

4g1

] G

lyG

2S

(Neu

Gc)

70

.10

0.1

40

.16

40

.72

0.9

31

.13

40

.10

0.1

30

.45

61

[h5

n5

f2]

41

.0E-

24

.5E-

20

.25

41

.1E-

22

.3E-

20

.21

41

.00

1.0

11

.32

61

.1[h

5n

5f2

] G

lyG

2F2

B1

1.1

E-2

11

.1E-

21

0.9

8

61

.2[h

5n

5f2

] G

lyG

2F2

+N (

tri)

11

.0E-

21

1.0

E-2

11

.00

62

[h7

n4

a1]

40

.22

0.2

90

.51

40

.20

0.2

70

.47

41

.08

1.1

01

.12

62

.1[h

7n

4a1

] G

lyG

2S+

2aG

al (

Neu

Ac)

10

.33

10

.32

11

.05

62

.2[h

7n

4a1

] G

lyM

an5

G2

S (N

euA

c) h

ybri

d1

1.0

51

0.9

51

1.1

1

62

.3[h

7n

4a1

] G

lyG

2S(

6)+

2aG

al (

Neu

Ac)

10

.13

10

.11

11

.18

63

[h8

n2

]4

0.1

30

.19

0.4

44

0.1

30

.27

0.5

04

0.7

71

.11

1.3

4

63

.1[h

8n

2]

Gly

Man

84

0.1

30

.19

0.4

44

0.1

30

.27

0.5

04

0.7

71

.11

1.3

4

64

[h4

n2

f1]

30

.16

0.1

84

.92

30

.92

1.7

37

.79

30

.40

0.7

00

.95

64

.1[h

4n

2f1

] G

lyM

an4

F3

0.1

60

.18

4.9

23

0.9

21

.73

7.7

93

0.4

00

.70

0.9

5

64

.11

[h4

n2

f1]

Gly

Iso

Man

4D

2F

10

.18

11

.73

10

.10

65

[h4

n4

a1]

30

.37

0.5

00

.70

30

.12

0.2

40

.47

31

.16

1.2

92

5.6

5

6 /

10

B-28



T.IR.8186

Ind

exC

om

po

siti

on

Co

mm

on

Nam

e#

25

%M

edia

n7

5%

#2

5%

Med

ian

75

%#

25

%M

edia

n7

5%

Mea

sura

nd

Sam

ple

A, %

Sam

ple

B, %

A/B

Rat

io

65

.1[h

4n

4a1

] G

lyG

1S

(Neu

Ac)

30

.37

0.5

00

.70

30

.12

0.2

40

.47

31

.16

1.2

92

5.6

5

66

[h5

n3

g1]

31

.8E-

22

.0E-

26

.3E-

23

2.1

E-2

4.0

E-2

7.5

E-2

30

.73

0.9

75

.77

66

.1[h

5n

3g1

] G

lyM

an4

G1

S (N

euG

c) h

ybri

d1

0.1

11

0.1

11

0.9

7

67

[h5

n5

]4

0.2

70

.48

0.9

93

1.1

41

.36

1.4

63

0.4

30

.74

1.0

9

67

.1[h

5n

5]

Gly

G2

B3

0.4

40

.67

1.3

23

1.1

41

.36

1.4

63

0.4

30

.74

1.0

9

67

.2[h

5n

5]

Gly

G1

+N+1

aGal

(tr

i)1

0.2

90

0

68

[h6

n5

f1g1

]4

9.1

E-3

2.1

E-2

3.7

E-2

31

.1E-

21

.3E-

22

.9E-

23

0.8

21

.17

1.2

0

68

.1[h

6n

5f1

g1]

Gly

G2

FBS+

1aG

al (

Neu

Gc)

11

.0E-

21

8.2

E-3

11

.22

68

.2[h

6n

5f1

g1]

Gly

G3

FS (

Neu

Gc)

13

.2E-

20

0

69

[h8

n5

f1g1

]3

2.6

E-2

3.7

E-2

4.0

E-2

32

.5E-

23

.3E-

23

.8E-

23

0.9

21

.00

1.0

7

69

.1[h

8n

5f1

g1]

Gly

G3

FS+2

aGal

(N

euG

c)2

4.0

E-2

23

.8E-

22

1.0

7

69

.2[h

8n

5f1

g1]

Gly

G2

FBG

S+3

aGal

(N

euG

c)1

1.6

E-2

11

.8E-

21

0.8

5

70

[h4

n5

f1a1

]2

0.3

84

6.3

E-2

0.4

91

.48

21

.41

70

.1[h

4n

5f1

a1]

Gly

G1

FBS

(Neu

Ac)

20

.38

46

.3E-

20

.49

1.4

82

1.4

1

71

[h5

n3

f1a1

]3

0.2

90

.50

0.6

62

0.8

92

0.7

5

71

.1[h

5n

3f1

a1]

Gly

Man

4FG

1FS

(N

euA

c)2

0.4

51

0.8

31

0.9

7

71

.2[h

5n

3f1

a1]

Gly

G1

FS-N

+1aG

al (

Neu

Ac)

10

.50

10

.95

10

.53

72

[h6

n3

a1]

24

.3E-

23

1.4

E-2

1.8

E-2

4.0

E-2

21

.09

73

[h6

n4

a1]

39

.3E-

20

.12

0.1

53

0.1

80

.22

0.2

62

0.7

1

73

.1[h

6n

4a1

] G

lyG

2S+

1aG

al (

Neu

Ac)

20

.13

10

.31

10

.60

74

[h6

n5

]2

0.9

92

1.5

52

0.7

5

74

.1[h

6n

5]

Gly

G3

20

.99

21

.55

20

.75

74

.11

[h6

n5

] G

lyIs

oG

3[6

]1

1.0

81

2.2

01

0.4

9

75

[h6

n5

a1]

27

.1E-

32

1.2

E-2

21

.05

76

[h8

n5

f2]

26

.2E-

32

1.7

E-2

20

.49

77

[h9

n2

]2

7.3

E-2

44

.4E-

26

.7E-

29

.1E-

22

1.0

9

77

.1[h

9n

2]

Gly

Man

92

7.3

E-2

44

.4E-

26

.7E-

29

.1E-

22

1.0

9

78

[h3

n3

g1]

19

.8E-

41

1.0

E-3

10

.95

79

[h3

n4

f1a1

]1

2.1

71

2.6

41

0.8

2

80

[h3

n4

f1S]

16

.6E-

21

7.6

E-2

10

.87

80

.1[h

3n

4f1

S] G

lyG

0F-

N+G

alN

Ac4

Sul

16

.6E-

21

7.6

E-2

10

.87

81

[h3

n4

f2]

10

.18

10

.14

11

.37

81

.1[h

3n

4f2

] G

lyG

0F2

-N+G

alN

Ac

10

.18

10

.14

11

.37

82

[h3

n5

f2]

12

.18

10

.22

11

0.0

8

7 /

10

B-29



T.IR.8186

Ind

exC

om

po

siti

on

Co

mm

on

Nam

e#

25

%M

edia

n7

5%

#2

5%

Med

ian

75

%#

25

%M

edia

n7

5%

Mea

sura

nd

Sam

ple

A, %

Sam

ple

B, %

A/B

Rat

io

83

[h3

n7

]1

0.5

01

0.4

71

1.0

6

83

.1[h

3n

7]

Gly

G0

FB+2

N (

qu

ad)

10

.50

10

.47

11

.06

84

[h4

n3

a1]

16

.0E-

22

16

.12

10

.23

84

.1[h

4n

3a1

] G

lyG

1S-

N (

Neu

Ac)

16

.0E-

22

16

.12

10

.23

85

[h4

n4

f1g2

]1

2.0

E-2

12

.0E-

21

1.0

0

85

.1[h

4n

4f1

g2]

Gly

G1

FS2

(N

euG

c)1

2.0

E-2

12

.0E-

21

1.0

0

86

[h4

n4

f1S]

10

.13

18

.0E-

21

1.6

8

86

.1[h

4n

4f1

S] G

lyM

an4

G0

F+G

alN

Ac4

Sul h

ybri

d1

0.1

31

8.0

E-2

11

.68

87

[h4

n5

a1]

10

.57

10

.18

13

.17

87

.1[h

4n

5a1

] G

lyG

1S+

N (

Neu

Ac)

(tr

i)1

0.5

71

0.1

81

3.1

7

88

[h5

n2

f1]

10

.31

12

.28

10

.14

88

.1[h

5n

2f1

] G

lyM

an5

F1

0.3

11

2.2

81

0.1

4

89

[h5

n5

f1a1

]1

0.6

81

0.7

41

0.9

2

89

.1[h

5n

5f1

a1]

Gly

G2

FS+N

(N

euA

c) (

tri)

10

.68

10

.74

10

.92

90

[h5

n5

f1a2

]1

0.3

81

0.7

41

0.5

1

90

.1[h

5n

5f1

a2]

Gly

G2

FS2

+N (

Neu

Ac)

(tr

i)1

0.3

81

0.7

41

0.5

1

91

[h5

n5

f1g1

]1

4.6

E-2

14

.5E-

21

1.0

3

92

[h6

n2

f1]

14

.8E-

21

0.1

21

0.3

9

92

.1[h

6n

2f1

] G

lyM

an6

F1

4.8

E-2

10

.12

10

.39

93

[h6

n3

f2]

11

.9E-

21

2.1

E-2

10

.89

94

[h6

n4

g1]

10

.36

10

.46

10

.78

95

[h6

n5

f1g2

]1

3.7

E-2

16

.0E-

21

0.6

1

95

.1[h

6n

5f1

g2]

Gly

G3

FS2

(N

euG

c)1

3.7

E-2

16

.0E-

21

0.6

1

96

[h6

n7

f4a3

]1

5.7

E-2

13

.0E-

21

1.8

9

97

[h6

n7

f5a2

]1

4.8

E-2

16

.0E-

21

0.8

0

98

[h7

n3

f2]

13

.2E-

21

8.5

E-2

10

.38

99

[h7

n4

]2

9.0

E-2

37

.2E-

20

.10

0.2

31

0.2

9

99

.1[h

7n

4]

Gly

G2

+2aG

al2

9.0

E-2

37

.2E-

20

.10

0.2

31

0.2

9

10

0[h

7n

5f1

g2]

16

.1E-

31

6.3

E-3

10

.97

10

0.1

[h7

n5

f1g2

] G

lyG

2FB

GS2

+1aG

al (

Neu

Gc)

16

.1E-

31

6.3

E-3

10

.97

10

1[h

7n

5f2

]1

1.9

E-2

13

.3E-

21

0.5

8

10

2[h

7n

6f2

a1]

10

.13

10

.10

11

.24

10

2.1

[h7

n6

f2a1

] G

lyG

4F2

S (N

euA

c)1

0.1

31

0.1

01

1.2

4

10

3[h

7n

8f3

a4]

11

.8E-

21

3.2

E-2

10

.56

8 /

10

B-30



T.IR.8186

Ind

exC

om

po

siti

on

Co

mm

on

Nam

e#

25

%M

edia

n7

5%

#2

5%

Med

ian

75

%#

25

%M

edia

n7

5%

Mea

sura

nd

Sam

ple

A, %

Sam

ple

B, %

A/B

Rat

io

10

4[h

7n

9f1

a4]

11

.5E-

21

3.6

E-2

10

.43

10

5[h

8n

10

f5a3

]1

6.5

E-3

17

.1E-

31

0.9

2

10

6[h

8n

8f3

a4]

15

.0E-

31

2.2

E-2

10

.23

10

7[n

1f1

]1

4.9

E-3

16

.2E-

31

0.7

9

10

8[n

2f1

]2

9.6

E-2

10

.13

11

.16

10

8.1

[n2

f1]

Gly

Frag

men

t FN

22

9.6

E-2

10

.13

11

.16

10

9[h

3n

3f2

]0

10

.20

0

10

9.1

[h3

n3

f2]

Gly

Man

3F2

+N0

10

.20

0

11

0[h

6n

2g1

]*

*

11

0.1

[h6

n2

g1]

Gly

Man

5+G

al+S

(N

euG

c) h

ybri

d*

*

11

1[h

6n

3f1

a1]

01

0.6

10

11

1.1

[h6

n3

f1a1

] G

lyM

an5

G1

FS (

Neu

Ac)

hyb

rid

01

0.6

10

11

2[h

6n

3f2

a1]

01

2.6

E-2

0

11

2.1

[h6

n3

f2a1

] G

lyM

an5

G1

F2S

(Neu

Ac)

hyb

rid

01

2.6

E-2

0

11

3[h

7n

5f1

g1]

02

1.8

E-2

0

11

3.1

[h7

n5

f1g1

] G

lyG

2FB

GS+

1aG

al (

Neu

Gc)

01

6.7

E-3

0

11

4[h

7n

6f1

a1g3

]0

15

.48

0

11

4.1

[h7

n6

f1a1

g3]

Gly

G4

FS4

(3

Neu

Gc)

(1

Neu

Ac)

01

5.4

80

11

5[h

7n

8f5

a3]

12

.4E-

20

0

11

6[h

7n

8f6

a3]

11

.2E-

20

0

[Un

kno

wn

s]2

60

.99

1.6

85

.24

25

0.7

72

.60

7.0

52

50

.65

0.9

51

.14

[h3

n3

f1]/

[h3

n4

f1]

G0

F-N

/G0

F1

2.8

01

1.0

71

2.6

3

[h3

n4

]/[h

3n

4f1

]G

0/G

1F

11

.30

10

.83

11

.56

[h3

n4

f1]/

[h4

n4

f1]

G0

F/G

1F

13

0.5

71

37

.13

10

.82

[h4

n4

f1]/

[h5

n4

f1]

G1

F/G

2F

11

.70

19

.37

10

.18

[h5

n4

f1]/

[h6

n4

f1]

G2

F/G

2F+

1aG

al0

10

.50

0

[h5

n5

f1g1

]/[h

6n

5a1

]1

0.1

21

8.0

E-2

11

.46

[h6

n5

a3]/

[h5

n5

f1a2

g1]/

[h4

n5

f2a1

g2]/

[h3

n5

f3g3

]1

5.3

E-2

00

[h6

n5

f1a3

]/[h

5n

5f2

a2g1

]/[h

4n

5f3

a1g2

]/[h

3n

5f4

g3]

12

.6E-

20

0

* Id

enti

fied

bu

t n

ot

qu

anti

fied

9 /

10

B-31



T.IR.8186

Ind

ex:

Dec

imal

ind

ex o

f id

enti

fied

gly

can

co

mp

osi

tio

ns,

ass

ign

ed in

dec

reas

ing

ord

er o

f n

um

ber

of

rep

ort

s.

Inte

gers

den

ote

un

iqu

e co

mp

osi

tio

ns,

ten

ths

glyc

ofo

rms,

an

d h

un

dre

dth

s is

om

ers.

Co

mp

osi

tio

n:

The

resu

lt li

ste

d f

or

a gi

ven

co

mp

osi

tio

n is

th

e su

m o

f th

e re

po

rted

res

ult

s fo

r al

l gly

cofo

rms

of

that

co

mp

osi

tio

n,

wh

ere

the

glyc

ofo

rms

are

ind

icat

ed b

y "[

com

po

siti

on

] G

ly".

The

resu

lt li

ste

d f

or

a gi

ven

gly

cofo

rm is

th

e su

m o

f th

e re

po

rted

res

ult

s fo

r al

l iso

mer

s o

f th

at g

lyco

form

wh

ere

the

iso

mer

s ar

e in

dic

ated

by

"[co

mp

osi

tio

n]

Gly

Iso

".

No

te: "

[Un

kno

wn

s]"

= su

m o

f th

e ab

un

dan

ces

of

un

iden

tifi

ed g

lyca

ns.

Co

mm

on

Nam

e:

#:

25

%:

Med

ian

:

75

%: #:

25

%:

Med

ian

:

75

%: #:

25

%:

Med

ian

:

75

%:

The

25

th p

erce

nti

le (

1st

qu

arti

le)

of

the

dis

trib

uti

on

of

the

calc

ula

ted

rat

ios.

The

con

sen

sus

med

ian

(5

0th

per

cen

tile

or

2n

d q

uar

tile

) o

f th

e d

istr

ibu

tio

n o

f th

e ca

lcu

late

d r

atio

s.

The7

5th

per

cen

tile

(3

rd q

uar

tile

) o

f th

e d

istr

ibu

tio

n o

f th

e ca

lcu

late

d r

atio

s.

The

25

th p

erce

nti

le (

1st

qu

arti

le)

of

the

dis

trib

uti

on

of

the

rep

ort

ed r

esu

lts.

The

con

sen

sus

med

ian

(5

0th

per

cen

tile

or

2n

d q

uar

tile

) o

f th

e d

istr

ibu

tio

n o

f th

e re

po

rted

res

ult

s.

The7

5th

per

cen

tile

(3

rd q

uar

tile

) o

f th

e d

istr

ibu

tio

n o

f th

e re

po

rted

res

ult

s.

A/B

Rat

ioSu

mm

ary

stat

isti

cs f

or

the

rati

o A

/B w

hen

res

ult

s w

ere

rep

ort

ed f

or

bo

th s

amp

les

A a

nd

B.

The

nu

mb

er o

f A

/B r

atio

s.

The

con

sen

sus

med

ian

(5

0th

per

cen

tile

or

2n

d q

uar

tile

) o

f th

e d

istr

ibu

tio

n o

f th

e re

po

rted

res

ult

s.

The7

5th

per

cen

tile

(3

rd q

uar

tile

) o

f th

e d

istr

ibu

tio

n o

f th

e re

po

rted

res

ult

s.

Sam

ple

B, %

Sum

mar

y st

atis

tics

fo

r re

sult

s re

po

rted

fo

r sa

mp

le B

, th

e N

ISTm

Ab

mat

eria

l.

The

nu

mb

er o

f p

arti

cip

ants

rep

ort

ing

this

mea

sura

nd

in t

his

sam

ple

.

The

nu

mb

er o

f p

arti

cip

ants

rep

ort

ing

this

mea

sura

nd

in t

his

sam

ple

.

The

25

th p

erce

nti

le (

1st

qu

arti

le)

of

the

dis

trib

uti

on

of

the

rep

ort

ed r

esu

lts.

Co

mp

osi

tio

n o

f th

e gl

ycan

in t

he

De

Leo

z-St

ein

no

tati

on

(se

e T

able

of

Ide

nti

fie

d G

lyca

ns

for

det

ails

.)

Wh

en a

vaila

ble

, a c

om

mo

n n

ame

of

glyc

ofo

rms

and

iso

mer

s. S

ee T

able

of

Ide

nti

fie

d G

lyca

ns

for

Oxf

ord

nam

es.

Sam

ple

A, %

Sum

mar

y st

atis

tics

fo

r re

sult

s re

po

rted

fo

r sa

mp

le A

, a m

od

ifie

d v

ersi

on

of

the

NIS

TmA

b m

ate

rial

Inte

rla

bo

rato

ry S

tud

y o

f N

IST

mA

b G

lyco

syla

tio

n

Wh

at t

he

Tab

le L

ists

Hea

din

gs a

nd

Su

bh

ead

ings

Def

init

ion

Mea

sura

nd

The

glyc

an (

or

iden

tifi

ed c

om

bin

atio

n o

f gl

ycan

s) f

or

wh

ich

mea

sure

men

t re

sult

s w

ere

rep

ort

ed.

10

/ 1

0

B-32



T.IR.8186

B-33

Representative “Individualized Report” The Individualized Report contains graphical analyses of the glycan composition results: Individualized Report #Pages Boxplots of Samples A and B, and the A/B ratio, and targetplot for the A/B ratio 1 Legend for the boxplots and targetplot 1 Plots summarizing measurement performance, including glycan composition counts and sums, repeatability, limits of reporting, minimum reported values, and consensus

1

Legend for measurement performance plots 1 Table of measurement summary Variable Legend for table of measurement summary 1 Table of derived glycan attribute quantities 1 Legend for table of derived glycan attribute quantities 1



T.IR.8186

Inte

rla

bo

rato

ry S

tud

y o

f N

IST

mA

b G

lyco

syla

tio

n*

Rep

ort

fo

r "E

xam

ple

"[h5n4g1][h8n5f1][h4n3f1]

[h6n4][h6n5f1][h6n4f1][h5n4f1]

[h6n3][h5n5f1]

[h5n4f1g1][h4n4f1a1]

[h6n3f1][h5n4f2]

[h5n4f1g2][h7n5f1][h6n3g1][h4n4f1][h4n3g1]

[h6n4f1a1][h4n4]

[h5n4f1a2][h7n3]

[h5n4g2][h4n3f1g1][h5n4f1a1][h6n4f1g1]

[h6n2][h6n4f2][h7n4f1][h5n3f1]

[h6n3f1g1][h7n2]

[h3n2f1][h4n3f1a1]

[h4n3][h3n2][h5n2]

[h4n5f1][h4n2][h5n4]

[h5n3a1][h9n5f1][h4n3f2]

[h5n3f1g1][h2n3f1]

[h3n3][h7n3f1][h5n4a1]

[h3n4][h3n4f1]

[h4n4f1g1][h4n4f2][h3n3f1]

[h5n3][h3n5f1]

[h3n5][h4n5f1g1]

0.0

1

0.11

10

0.0

1

0.11

10

0.11

Sample B, %A /B

Sample A, %

3210123

01

23

Z-Score Mean, Consensus SD

Z-Sc

ore

SD

, Co

nse

nsu

s SD

Do

t d

iam

ete

r is

p

rop

ort

ion

al t

o n

um

ber

o

f A

/B r

atio

s

1st Q

ua

rtile

(2

5 %

)

Med

ian

(50

%)

3rd

Qua

rtile

(7

5 %

)

{

∝√(#Data)

A,

yo

ur

Me

an

±S

D

A/B

, you

r M

ea

n±S

D

B,

yo

ur

Me

an

±S

D

Go

od

Fa

ir

Que

stio

na

ble

Yo

u*

Dat

a as

rep

ort

ed in

10

3 r

ep

ort

s fr

om

76

lab

ora

tori

es

Sam

ple

A: m

od

ifie

d N

ISTm

Ab

Sam

ple

B: N

ISTm

Ab

Agr

eem

ent

of

A/B

wit

h C

on

sen

sus

1 /

9

B-34



T.IR.8186

The dots are color-coded by distance from the {0,0} origin: dots within

two comparability units are colored green, between two and three units

are colored yellow, and greater than three units are colored red. These

codes roughly indicate "Good", "Moderate", and "Questionable"

agreement with the consensus A/B ratio estimates. However, the large

differences in the numbers of glycans in the various sets of results renders

these distinctions themselves "Questionable."

Inte

rla

bo

rato

ry S

tud

y o

f N

IST

mA

b G

lyco

syla

tio

n

Wh

at t

he

Gra

ph

ics

Dis

pla

y

Bo

xplo

t su

mm

ary

of

resu

lts

for

the

57

un

iqu

e gl

ycan

co

mp

osi

tio

ns

that

wer

e re

po

rted

at

leas

t si

x ti

mes

fo

r o

ne

of

the

sam

ple

s. E

ach

bo

x re

pre

sen

ts t

he

dis

trib

uti

on

of

the

cen

tral

50

% o

f th

e m

ean

of

the

rep

ort

ed r

eplic

ate

valu

es

for

on

e gl

ycan

fo

r sa

mp

le A

, B, o

r th

e A

/B r

atio

. Th

e h

ori

zon

tal m

idd

le li

ne

in e

ach

bo

x re

pre

sen

ts t

he

con

sen

sus

med

ian

. Th

e w

idth

of

each

bo

x is

pro

po

rtio

nal

to

th

e sq

uar

e ro

ot

of

the

nu

mb

er o

f va

lues

def

inin

g th

e

dis

trib

uti

on

. Th

e d

ash

ed r

ed li

ne

in t

he

dis

pla

y o

f th

e A

/B r

atio

s d

eno

tes

the

exp

ecte

d r

atio

, 1.0

, wh

en a

gly

can

resu

lt is

th

e sa

me

in s

amp

le A

as

it is

in B

. Gly

can

s ar

e so

rted

in o

rder

of

incr

easi

ng

A/B

rat

io. T

he

ligh

t gr

ay

vert

ical

lin

es a

re p

rovi

ded

to

fac

ilita

te a

sso

ciat

ing

a p

arti

cula

r b

ox

wit

h it

s gl

ycan

.

Targ

etp

lot

sum

mar

y o

f A

/B r

atio

s re

lati

ve t

o t

he

con

sen

sus

med

ian

s.

Each

do

t m

arks

th

e su

mm

ary

sco

re f

or

on

e se

t o

f re

sult

s. T

he

vert

ical

axis

dis

pla

ys t

he

aver

age

bia

s es

tim

ated

as

the

mea

n o

f th

e "Z

-sco

re"

valu

es o

f th

e A

/B r

atio

s fo

r th

e u

niq

ue

glyc

an c

om

po

siti

on

s th

at t

hey

rep

ort

ed: Z

-sco

re =

(R

atio

- M

edia

n)/

MA

DE,

wh

ere

MA

DE

is a

ro

bu

st

esti

mat

e o

f st

and

ard

dev

iati

on

. Th

e h

ori

zon

tal a

xis

dis

pla

ys t

he

vari

abili

ty o

f in

div

idu

al b

ias

esti

mat

es, e

stim

ated

as

the

stan

dar

d

dev

iati

on

of

the

Z-sc

ore

s. T

he

sem

icir

cles

mar

k o

ne,

tw

o, a

nd

th

ree

"co

mp

arab

ility

" d

ista

nce

s fr

om

th

e id

eal {

Mea

n,S

D}

valu

e o

f {0

,0}:

Dis

tan

ce =

√((

Z-sc

ore

Mea

n)2 +

(Z-

sco

re S

D)2 ).

3210123

01

23

Z-score Mean, Consensus SD

Z-Sc

ore

SD

, C

on

sen

sus

SD

Do

t d

iam

ete

r is

pro

po

rtio

na

l to

nu

mb

er

of

A/B

ra

tio

s

[h5n4g1][h8n5f1][h4n3f1]

[h6n4][h6n5f1][h6n4f1][h5n4f1]

[h6n3][h5n5f1]

[h5n4f1g1][h4n4f1a1]

[h6n3f1][h5n4f2]

[h5n4f1g2][h7n5f1][h6n3g1][h4n4f1][h4n3g1]

[h6n4f1a1][h4n4]

[h5n4f1a2][h7n3]

[h5n4g2][h4n3f1g1][h5n4f1a1][h6n4f1g1]

[h6n2][h6n4f2][h7n4f1][h5n3f1]

[h6n3f1g1][h7n2]

[h3n2f1][h4n3f1a1]

[h4n3][h3n2][h5n2]

[h4n5f1][h4n2][h5n4]

[h5n3a1][h9n5f1][h4n3f2]

[h5n3f1g1][h2n3f1]

[h3n3][h7n3f1][h5n4a1]

[h3n4][h3n4f1]

[h4n4f1g1][h4n4f2][h3n3f1]

[h5n3][h3n5f1]

[h3n5][h4n5f1g1]

0.0

1

0.11

10

0.0

1

0.11

10

0.11

Sample B, %A /B

Sample A, %

2 /

9

B-35



T.IR.8186

Inte

rla

bo

rato

ry S

tud

y o

f N

IST

mA

b G

lyco

syla

tio

n*

Rep

ort

fo

r "E

xam

ple

"

A,

yo

ur

Me

an

±S

D

B, yo

ur

Me

an

±S

D

%C

V =

a+

Mean

b

Yo

ua

= 1

.3, b

= -

0.2

6

Co

nse

nsu

sa

= 5

.0, b

= -

0.3

5

A B

* D

ata

as r

epo

rted

in1

03

rep

ort

s fr

om

76

lab

ora

tori

es

Sam

ple

A: m

od

ifie

d N

ISTm

Ab

Sam

ple

B: N

ISTm

Ab

A B

05

10

15

20

01

02

03

04

05

0

Number Values

# U

niq

ue

Gly

can

Co

mp

osi

tio

ns

A B

0

50

10

0

15

0

80

90

10

01

10

12

0

Number Values

∑ U

niq

ue

Gly

can

Co

mp

osi

tio

ns

00

.20

.40

.60

.81

0.0

01

0.0

1

0.11

10

10

0

Unique Value

(Ran

k o

f V

alu

e) /

(N

um

ber

Val

ues

)

All

resu

lts

You

r u

niq

ue

valu

es

You

r 'L

imit

of

Re

po

rtin

g'

0.0

01

0.0

10

.11

0.0

01

0.0

1

0.11

Limit of Reporting (LoR)

Min

imu

m R

epo

rted

Val

ue

(MR

V)

All

resu

lts

You

LoR

= M

RV

0.0

01

0.0

10

.11

10

10

0

0.11

10

10

0

%CV [100×SD/Mean]

Me

an

All

resu

lts

You

r re

sult

s

11

01

00

10

10

0

Median |You-All|, %

Me

dia

n C

V, %

Bio

logi

cal

Tech

nic

alO

ther

Rep

licat

ion

3 /

9

B-36



T.IR.8186

Trac

es o

f th

e u

niq

ue

no

n-z

ero

val

ues

rep

ort

ed in

eac

h s

et o

f

resu

lts,

wh

ere

the

valu

es a

re o

rder

ed b

y d

ecre

asin

g va

lue.

If

the

tru

e am

ou

nts

of

the

min

or

glyc

ans

are

ran

do

mly

dis

trib

ute

d a

nd

all

resu

lts

refl

ect

the

sam

e le

vel o

f an

alyt

ical

effo

rt, a

bes

t-fi

t lin

e to

th

e ri

ght-

tail

of

the

trac

e es

tim

ates

each

th

e "L

imit

of

Rep

ort

ing"

(Lo

R)

for

that

set

. Lo

R v

alu

es

may

be

mo

re r

epre

sen

tati

ve o

f th

e an

alyt

ical

sen

siti

vity

of

a

mea

sure

men

t sy

stem

th

an is

th

e m

inim

um

rep

ort

ed v

alu

e

(MR

V).

Mo

st o

f th

e Lo

Rs

agre

e w

ell f

or

MR

Vs

abo

ve a

bo

ut

0.0

5 %

.

Bel

ow

th

is v

alu

e, m

any

of

the

sets

of

resu

lts

con

tain

a f

ew

valu

es m

any-

fold

sm

alle

r th

an t

hei

r Lo

R. T

his

may

ref

lect

spec

ial i

nte

rest

in s

elec

ted

gly

can

co

mp

on

ents

rat

her

th

an

rep

ort

ing

issu

es w

ith

th

e le

ss-a

bu

nd

ant

glyc

ans.

Inte

rla

bo

rato

ry S

tud

y o

f N

IST

mA

b G

lyco

syla

tio

n

Wh

at t

he

Gra

ph

ics

Dis

pla

y

Mea

sure

men

t re

pea

tab

ility

is e

stim

ated

as

the

coef

fici

ent

of

vari

atio

n e

xpre

ssed

as

a p

erce

nta

ge (

aka

the

rela

tive

sta

nd

ard

dev

iati

on

), %

CV

= 1

00

*SD

/Mea

n, o

f th

e re

plic

ate

valu

es in

eac

h

rep

ort

. %C

Vs

can

no

t b

e ca

lcu

late

d f

or

no

n-r

eplic

ated

mea

sure

men

ts a

nd

so

are

no

t in

clu

ded

in t

hes

e p

lots

.

Scat

terp

lot

of

the

rela

tio

nsh

ip b

etw

een

mea

sure

men

t

rep

eata

bili

ty, e

stim

ated

as

the

%C

V, a

nd

gly

can

amo

un

t, e

stim

ated

as

the

mea

n o

f th

e re

plic

ates

. Th

e

bla

ck li

ne

rep

rese

nts

a s

imp

le c

on

sen

sus

po

wer

-law

:

%C

V =

5.0

*Mea

n-0

.35 (

or

SD =

0.0

50

*Mea

n0.

65).

No

te

that

%C

V is

no

t co

nst

ant

for

all g

lyca

n a

mo

un

ts b

ut

rath

er g

ener

ally

incr

ease

s w

ith

dec

reas

ing

amo

un

t.

Go

ogl

e "H

orw

itz

Fun

ctio

n"

for

info

rmat

ion

on

a s

imila

r

ph

eno

men

on

.

His

togr

am o

f th

e n

um

ber

of

un

iqu

e gl

ycan

com

po

siti

on

s fo

r sa

mp

les

A a

nd

B in

th

e 1

03

rep

ort

s

pro

vid

ed b

y 7

6 la

bo

rato

ries

. Mo

st r

epo

rts

liste

d a

bo

ut

the

sam

e n

um

ber

of

glyc

ans

for

sam

ple

A a

s fo

r sa

mp

le

B.

The

area

un

der

th

e cu

rve

is 2

06

.

His

togr

am o

f th

e su

m o

f th

e u

niq

ue

glyc

an c

om

po

siti

on

valu

es f

or

sam

ple

s A

an

d B

. In

mo

st, b

ut

no

t al

l, o

f th

e

10

3 r

epo

rts

the

resu

lts

wer

e n

orm

aliz

ed s

o t

hat

th

e

sum

of

the

valu

es =

10

0 f

or

each

sam

ple

. Th

e ar

ea

un

der

th

e cu

rve

is 2

06

.

Scat

terp

lot

of

the

clo

sen

ess

to c

on

sen

sus

of

the

rep

ort

ed

valu

es a

s a

fun

ctio

n o

f m

easu

rem

ent

rep

eata

bili

ty e

stim

ated

as %

CV

. "C

lose

nes

s" is

est

imat

ed a

s th

e re

lati

ve a

bso

lute

dif

fere

nce

bet

wee

n a

giv

en r

esu

lt m

ean

an

d t

he

med

ian

of

the

mea

ns

pro

vid

ed in

all

10

3 r

epo

rts:

10

0*|

Mea

n-C

on

sen

sus

Med

ian

|/M

ean

.

The

sym

bo

ls a

re c

od

ed b

y th

e u

ser-

stat

ed n

atu

re o

f th

e

rep

ort

ed r

eplic

ates

. Bec

ause

of

the

grea

t va

riab

ility

in t

he

resu

lts

for

the

vari

ou

s gl

ycan

s, t

he

sum

mar

y st

atis

tics

dis

pla

yed

in t

he

scat

terg

ram

are

th

e re

spec

tive

med

ian

s ta

ken

ove

r al

l (re

plic

ate)

res

ult

s fo

r u

niq

ue

glyc

an c

om

po

siti

on

s.

05

10

15

20

01

02

03

04

05

0

Number Values

# U

niq

ue

Gly

can

Co

mp

osi

tio

ns

0

50

10

0

15

0

80

90

10

01

10

12

0

Number Values

∑ U

niq

ue

Gly

can

Co

mp

osi

tio

ns

0.0

01

0.0

10

.11

0.0

01

0.0

1

0.11

"Limit of Reporting"

Min

imu

m R

ep

ort

ed

Va

lue

All

pa

rtic

ipa

nts

Yo

uE

xpe

cte

d =

Re

po

rte

d

00

.20

.40

.60

.81

0.0

01

0.0

1

0.11

10

10

0

Unique Value

(Ra

nk

of

Va

lue

) /

(Nu

mb

er

Va

lue

s)

All

pa

rtic

ipa

nts

Yo

ur

un

iqu

e v

alu

es

Yo

ur

'Lim

it o

f R

ep

ort

ing

'

0.0

01

0.0

10

.11

10

10

0

0.11

10

10

0

%CV [100×SD/Mean]

Me

an

All

resu

lts

Yo

ur

resu

lts

10

10

0

Median |You-All|, %

Me

dia

n C

V, %

Bio

log

ica

lT

ec

hn

ica

lO

the

r

Re

pli

cati

on

4 /

9

B-37



T.IR.8186

Ind

exC

om

po

siti

on

Co

mm

on

Nam

eC

lass

#M

ean

SD%

CV

#2

5%

Med

ian

75

%%

|Dif

|#

Mea

nSD

%C

V#

25

%M

edia

n7

5%

%|D

if|

1[h

4n

4f1

]3

28

.02

0.7

32

.61

03

27

.72

31

.61

33

.31

12

.83

38

.07

0.3

10

.81

03

36

.36

38

.37

39

.84

0.8

1.1

[h4

n4

f1]

Gly

G1

FC

FGTD

23

28

.02

0.7

32

.61

03

27

.72

31

.61

33

.31

12

.83

38

.07

0.3

10

.81

03

36

.36

38

.37

39

.84

0.8

1.1

1[h

4n

4f1

] G

lyIs

oG

1F(

1,6

)C

FGTD

23

19

.02

0.4

82

.55

81

9.0

42

1.2

12

2.0

61

1.5

32

7.7

70

.43

1.5

58

27

.60

28

.12

29

.12

1.2

1.1

2[h

4n

4f1

] G

lyIs

oG

1F(

1,3

)C

FGTD

23

9.0

00

.25

2.8

59

9.3

81

0.3

81

0.7

21

5.3

31

0.2

90

.12

1.2

59

9.8

21

0.1

81

0.7

51

.1

2[h

3n

4f1

]3

57

.81

0.5

71

.01

03

47

.06

51

.47

55

.04

11

.03

41

.08

0.5

31

.31

02

35

.73

39

.10

40

.76

4.8

2.1

[h3

n4

f1]

Gly

G0

FC

FD2

35

7.8

10

.57

1.0

10

34

7.0

65

1.4

75

5.0

41

1.0

34

1.0

80

.53

1.3

10

23

5.7

33

9.0

94

0.7

14

.8

3[h

5n

4f1

]3

2.5

33

.5E-

21

.49

93

.19

3.9

74

.80

56

.93

8.2

40

.22

2.6

10

27

.47

8.5

29

.48

3.5

3.1

[h5

n4

f1]

Gly

G2

FC

FGTD

21

0.1

34

61

.00

1.4

22

.77

99

2.1

36

.53

0.1

82

.84

75

.88

7.1

57

.61

9.5

3.2

[h5

n4

f1]

Gly

G1

F+1

aGal

CFG

LTD

23

2.4

99

.7E-

23

.93

51

.64

2.5

33

.06

1.8

31

.71

3.5

E-2

2.1

32

1.2

71

.60

2.2

06

.0

4[h

3n

3f1

]3

3.1

29

.7E-

23

.18

83

.03

3.6

54

.26

17

.23

2.1

34

.5E-

22

.18

91

.87

2.1

32

.93

0.0

4.1

[h3

n3

f1]

Gly

G0

F-N

CFD

33

.12

9.7

E-2

3.1

88

3.0

33

.65

4.2

61

7.2

32

.13

4.5

E-2

2.1

89

1.8

72

.13

2.9

30

.0

5[h

5n

2]

30

.57

8.9

E-2

15

.67

70

.56

0.7

81

.12

37

.13

0.6

92

.1E-

23

.07

90

.53

0.7

31

.01

5.6

5.1

[h5

n2

] G

lyM

an5

MD

30

.57

8.9

E-2

15

.67

70

.56

0.7

81

.12

37

.13

0.6

92

.1E-

23

.07

90

.53

0.7

31

.01

5.6

6[h

7n

4f1

]3

0.6

95

.8E-

30

.87

30

.70

0.9

01

.09

29

.33

0.6

64

.7E-

27

.27

10

.68

0.9

01

.22

37

.1

6.1

[h7

n4

f1]

Gly

G2

F+2

aGal

CFG

LTD

23

0.6

95

.8E-

30

.87

10

.69

0.8

71

.05

26

.13

0.6

64

.7E-

27

.27

00

.67

0.8

91

.06

36

.0

8[h

4n

5f1

]3

0.8

02

.9E-

23

.67

00

.46

0.7

00

.89

13

.13

0.8

34

.6E-

25

.56

70

.49

0.6

80

.88

18

.0

8.2

[h4

n5

f1]

Gly

NA

3FG

1C

FGTD

33

0.8

02

.9E-

23

.69

0.5

10

.55

0.8

13

2.0

30

.83

4.6

E-2

5.5

90

.46

0.5

10

.83

39

.0

9[h

5n

3f1

]3

0.4

01

.2E-

22

.96

90

.78

0.8

81

.17

12

1.8

30

.45

1.0

E-2

2.2

68

0.8

10

.89

1.2

29

8.1

9.1

[h5

n3

f1]

Gly

G1

F-N

+1aG

alC

FGLT

D3

0.4

01

.2E-

22

.96

20

.77

0.8

71

.15

12

0.2

30

.45

1.0

E-2

2.2

61

0.8

10

.89

1.2

19

7.6

11

[h3

n3

]3

0.3

84

.7E-

21

2.5

69

0.3

20

.46

0.7

82

2.3

30

.38

1.5

E-2

4.1

63

0.3

20

.44

0.7

61

6.7

11

.1[h

3n

3]

Gly

G0

-NC

D3

0.3

84

.7E-

21

2.5

69

0.2

90

.46

0.7

22

1.1

30

.38

1.5

E-2

4.1

63

0.3

20

.43

0.7

61

2.9

11

.11

[h3

n3

] G

lyIs

oG

0-N

(1,6

)C

D3

0.3

84

.7E-

21

2.5

70

.35

0.3

80

.54

0.9

30

.38

1.5

E-2

4.1

60

.34

0.3

90

.41

3.8

12

[h4

n3

f1g1

]3

1.1

04

.2E-

23

.86

00

.70

0.9

51

.30

14

.13

1.2

32

.6E-

22

.26

00

.79

1.0

11

.33

18

.0

12

.1[h

4n

3f1

g1]

Gly

G1

FS-N

(N

euG

c)C

FGZ

31

.10

4.2

E-2

3.8

60

0.7

00

.95

1.3

01

4.1

31

.23

2.6

E-2

2.2

60

0.7

91

.01

1.3

31

8.0

13

[h3

n4

]3

0.1

60

.06

30

.16

0.2

80

.84

76

.13

0.1

31

.5E-

21

2.1

58

0.1

30

.19

0.7

45

2.2

13

.1[h

3n

4]

Gly

G0

CD

23

0.1

60

.06

20

.16

0.2

50

.81

57

.43

0.1

31

.5E-

21

2.1

57

0.1

30

.19

0.7

44

7.4

15

[h6

n4

f1g1

]3

0.3

81

.2E-

23

.05

00

.31

0.4

40

.62

14

.23

0.3

53

.5E-

29

.94

90

.37

0.4

60

.72

29

.8

15

.1[h

6n

4f1

g1]

Gly

G2

FS+1

aGal

(N

euG

c)C

FGLT

DZ2

30

.38

1.2

E-2

3.0

50

0.3

10

.44

0.6

21

4.2

30

.35

3.5

E-2

9.9

49

0.3

70

.46

0.7

22

9.8

16

[h5

n4

f1g1

]3

0.2

65

.8E-

32

.25

00

.15

0.2

70

.47

1.8

30

.41

3.2

E-2

7.9

50

0.2

90

.45

0.7

21

1.6

16

.1[h

5n

4f1

g1]

Gly

G2

FS (

Neu

Gc)

CFG

TDZ2

30

.26

5.8

E-3

2.2

50

0.1

50

.27

0.4

71

.83

0.4

13

.2E-

27

.95

00

.29

0.4

50

.72

11

.6

18

[h3

n5

f1]

31

.70

4.9

E-2

2.9

55

0.6

40

.78

1.0

75

4.4

32

.47

6.5

E-2

2.6

46

0.3

00

.49

1.6

38

0.3

18

.2[h

3n

5f1

] G

lyG

0F+

N (

tri)

CFD

33

1.7

04

.9E-

22

.94

0.8

31

.11

1.3

03

4.7

32

.47

6.5

E-2

2.6

41

.96

2.4

42

.46

1.3

Me

asu

ran

d

Inte

rla

bo

rato

ry S

tud

y o

f N

IST

mA

b G

lyco

syla

tio

n

Mea

sure

men

t Su

mm

ary

for

Res

ult

s R

epo

rted

by

"Exa

mp

le"

All

Res

ult

sA

ll R

esu

lts

You

You

Sam

ple

A, %

of

Tota

l Gly

can

Sam

ple

B, %

of

Tota

l Gly

can

5 /

9

B-38



T.IR.8186

Ind

exC

om

po

siti

on

Co

mm

on

Nam

eC

lass

#M

ean

SD%

CV

#2

5%

Med

ian

75

%%

|Dif

|#

Mea

nSD

%C

V#

25

%M

edia

n7

5%

%|D

if|

Me

asu

ran

dA

ll R

esu

lts

All

Res

ult

sYo

uYo

u

Sam

ple

A, %

of

Tota

l Gly

can

Sam

ple

B, %

of

Tota

l Gly

can

19

[h3

n2

f1]

30

.11

5.8

E-3

5.1

43

0.1

00

.14

0.4

52

3.4

30

.13

0.0

45

0.1

00

.14

0.3

31

0.3

19

.1[h

3n

2f1

] G

lyM

an3

FM

FD3

0.1

15

.8E-

35

.14

30

.10

0.1

40

.45

23

.43

0.1

30

.04

50

.10

0.1

40

.33

10

.3

21

[h5

n4

]3

0.4

51

.5E-

23

.43

60

.44

0.7

00

.97

57

.23

0.3

11

.0E-

23

.23

60

.31

0.5

41

.08

74

.7

21

.1[h

5n

4]

Gly

G2

CG

TD2

30

.45

1.5

E-2

3.4

36

0.3

80

.63

0.9

44

1.2

30

.31

1.0

E-2

3.2

36

0.3

10

.53

1.0

16

9.4

19

9[U

nkn

ow

ns]

31

.51

0.2

81

8.4

26

0.9

91

.68

5.2

41

1.1

32

.50

0.1

87

.12

50

.77

2.6

07

.05

4.3

6 /

9

B-39



T.IR.8186

Ind

exD

ecim

al in

dex

of

iden

tifi

ed g

lyca

n c

om

po

siti

on

s, a

ssig

ned

in d

ecre

asin

g o

rder

of

nu

mb

er o

f re

po

rts.

Inte

ger

valu

es d

eno

te u

niq

ue

com

po

siti

on

s, t

enth

s gl

yco

form

s, a

nd

hu

nd

red

ths

iso

mer

s.

Co

mp

osi

tio

nTh

e re

sult

list

ed

fo

r a

give

n c

om

po

siti

on

is t

he

sum

of

the

rep

ort

ed r

esu

lts

for

all g

lyco

form

s o

f th

at c

om

po

siti

on

,w

her

e th

e gl

yco

form

s ar

e in

dic

ated

by

"[co

mp

osi

tio

n]

Gly

".Th

e re

sult

list

ed

fo

r a

give

n g

lyco

form

is t

he

sum

of

the

rep

ort

ed r

esu

lts

for

all i

som

ers

of

that

gly

cofo

rmw

her

e th

e is

om

ers

are

ind

icat

ed b

y "[

com

po

siti

on

] G

lyIs

o".

No

te: "

[Un

kno

wn

s]"

= su

m o

f th

e ab

un

dan

ces

of

un

iden

tifi

ed g

lyca

ns.

Co

mm

on

Nam

e

Cla

ssTy

pe

C: C

om

ple

x, H

: Hyb

rid

, M: H

igh

man

no

seFe

atu

reB

: Bis

ecte

d, D

: Des

ialy

late

d, F

: Fu

cosy

late

d, G

: Gal

acto

syla

ted

, L: a

lph

a-G

alac

tosy

late

d,

T: T

erm

inal

-gal

acto

syla

ted

, Y: N

euA

c-si

alyl

ated

, Z: N

euG

c-si

alyl

ated

An

ten

na

Ap

plie

s to

co

mp

lex

glyc

ans

– 2

: Bia

nte

nn

ary,

3: T

rian

ten

nar

y, 4

: Tet

ra-a

nte

nn

ary

You

#:

Mea

n:

SD:

%C

V: #:

25

%:

Med

ian

: 7

5%

:

%|D

if|:

Th

e re

lati

ve a

bso

lute

dif

fere

nce

bet

we

en y

ou

r M

ean

an

d t

he

con

sen

sus

Med

ian

exp

ress

ed a

s a

per

cen

tage

:1

00

*|M

ean

-Me

dia

n|/

Mea

n

Sam

ple

B, %

of

Tota

l Gly

can

Sum

mar

y st

atis

tics

fo

r re

sult

s re

po

rted

fo

r sa

mp

le B

, th

e N

ISTm

Ab

mat

eria

l

The

25

th p

erce

nti

le (

1st

qu

arti

le)

of

the

dis

trib

uti

on

of

the

rep

ort

ed r

esu

lts.

Oth

er h

ead

ings

are

as

des

crib

ed f

or

Sam

ple

A, a

bo

ve.

The

stan

dar

d d

evia

tio

n o

f th

e re

plic

ate

valu

es y

ou

re

po

rted

fo

r th

is m

easu

ran

d.

The

rela

tive

sta

nd

ard

dev

iati

on

exp

ress

ed in

per

cen

t, 1

00

*SD

/Mea

n.

The

con

sen

sus

med

ian

(5

0th

per

cen

tile

or

2n

d q

uar

tile

) o

f th

e d

istr

ibu

tio

n o

f th

e re

po

rted

res

ult

s.Th

e75

th p

erce

nti

le (

3rd

qu

arti

le)

of

the

dis

trib

uti

on

of

the

rep

ort

ed r

esu

lts.

The

nu

mb

er o

f gr

eate

r-th

an-z

ero

re

plic

ate

val

ues

yo

u r

ep

ort

ed f

or

this

mea

sura

nd

.Su

mm

ary

stat

isti

cs f

or

resu

lts

rep

ort

ed f

or

sam

ple

A, a

mo

dif

ied

ver

sio

n o

f th

e N

ISTm

Ab

mat

eri

al

Co

nca

ten

atio

n o

f si

ngl

e-ch

arac

ter

glyc

an a

ttri

bu

te c

od

es

All

Res

ult

sTh

e n

um

ber

of

sets

of

resu

lts

that

rep

ort

ed a

gre

ate

r-th

an-z

ero

val

ue

for

this

mea

sura

nd

in t

his

sam

ple

.

Sam

ple

A, %

of

Tota

l Gly

can

The

mea

n o

f th

e re

plic

ate

valu

es y

ou

re

po

rted

fo

r th

is m

easu

ran

d.

Inte

rla

bo

rato

ry S

tud

y o

f N

IST

mA

b G

lyco

syla

tio

n

Wh

at t

he

Tab

le L

ists

Wh

en a

vaila

ble

, a c

om

mo

n n

ame

of

glyc

ofo

rms

and

iso

mer

s. S

ee T

able

of

Iden

tifi

ed G

lyca

ns

for

Oxf

ord

nam

es.

Hea

din

gs a

nd

Su

bh

ead

ings

Mea

sura

nd

Def

init

ion

The

glyc

an (

or

iden

tifi

ed c

om

bin

atio

n o

f gl

ycan

s) f

or

wh

ich

mea

sure

men

t re

sult

s w

ere

rep

ort

ed.

Co

mp

osi

tio

n o

f th

e gl

ycan

in t

he

De

Leo

z-St

ein

no

tati

on

(se

e T

able

of

Ide

nti

fie

d G

lyca

ns

for

det

ails

)

7 /

9

B-40



T.IR.8186

Sym

bo

lC

lass

Att

rib

ute

#Su

mSD

#2

5%

Med

ian

75

%%

|Dif

|#

Sum

SD#

25

%M

edia

n7

5%

%|D

if|

CTy

pe

Co

mp

lex

15

97

.90

.24

38

9.9

10

4.6

12

1.3

6.4

15

96

.70

.24

39

3.3

10

5.5

12

3.1

8.3

HTy

pe

Hyb

rid

08

0.7

1.0

2.1

08

0.8

1.7

2.3

MTy

pe

Hig

h m

ann

ose

20

.70

.16

0.9

1.8

3.7

61

.52

0.8

0.0

61

.01

.93

.45

7.0

BFe

atu

reB

isec

tin

g0

71

.72

.43

.40

71

.52

.23

.8

DFe

atu

reD

esia

lyla

ted

16

97

.50

.24

28

8.8

10

3.3

12

0.2

5.6

16

96

.30

.24

29

2.1

10

4.5

11

9.6

7.9

FFe

atu

reFu

cosy

late

d1

39

7.0

0.3

40

89

.01

02

.81

18

.45

.71

39

6.0

0.2

40

92

.01

04

.01

17

.77

.6

GFe

atu

reG

alac

tosy

late

d1

03

4.7

0.2

43

38

.84

8.2

60

.02

8.0

10

50

.50

.14

35

5.3

63

.87

7.5

20

.8

LFe

atu

real

ph

a-ga

lact

osy

late

d4

4.0

0.0

13

4.4

6.3

8.2

36

.84

3.2

0.0

13

5.2

6.5

8.8

51

.5

TFe

atu

reTe

rmin

al-g

alac

tosy

late

d9

33

.60

.23

23

6.3

44

.65

3.9

24

.69

49

.30

.13

25

3.1

60

.37

0.8

18

.2

YFe

atu

reN

euA

c-si

alyl

ated

06

1.5

2.3

4.4

06

1.1

2.1

5.0

ZFe

atu

reN

euG

c-si

alyl

ated

31

.80

.01

22

.13

.14

.94

4.2

32

.00

.01

22

.94

.16

.85

1.1

2A

nte

nn

aB

ian

ten

nar

y9

90

.40

.33

08

3.2

96

.11

09

.25

.99

89

.20

.23

08

6.5

96

.91

10

.57

.9

3A

nte

nn

aTr

ian

ten

nar

y2

2.5

0.0

40

.91

.11

.51

38

.02

3.3

0.1

41

.01

.11

.81

88

.5

4A

nte

nn

aTe

tra-

ante

nn

ary

00

00

Inte

rla

bo

rato

ry S

tud

y o

f N

IST

mA

b G

lyco

syla

tio

n

Der

ived

Att

rib

ute

Qu

anti

ties

fo

r M

easu

rem

en

ts R

epo

rted

fo

r "E

xam

ple

"

You

You

Sam

ple

B, %

of

Tota

l Gly

can

Mea

sura

nd

All

Re

sult

sA

ll R

esu

lts

Sam

ple

A, %

of

Tota

l Gly

can

8 /

9

B-41



T.IR.8186

Cla

ss

Att

rib

ute

Spec

ific

gly

can

att

rib

ute

You

#:

Sum

:

SD: #:

25

%:

Med

ian

:

75

%:

%|D

if|:

Th

e re

lati

ve a

bso

lute

dif

fere

nce

bet

we

en y

ou

r M

ean

an

d t

he

con

sen

sus

Med

ian

exp

ress

ed a

s a

per

cen

tage

:

10

0*|

Me

an-M

ed

ian

|/M

ean

Sam

ple

B, %

of

Tota

l Gly

can

Oth

er h

ead

ings

are

as

des

crib

ed f

or

Sam

ple

A, a

bo

ve

Sum

mar

y st

atis

tics

fo

r re

sult

s re

po

rted

fo

r sa

mp

le B

, th

e N

ISTm

Ab

mat

eria

l

The

25

th p

erce

nti

le (

1st

qu

arti

le)

of

the

dis

trib

uti

on

of

the

mea

n r

eplic

ate

sum

s fo

r al

l re

sult

s

The

con

sen

sus

med

ian

(5

0th

per

cen

tile

or

2n

d q

uar

tile

) o

f th

e d

istr

ibu

tio

n o

f th

e m

ean

rep

licat

e su

ms

The

75

th p

erce

nti

le (

3rd

qu

arti

le)

of

the

dis

trib

uti

on

of

the

mea

n r

eplic

ate

sum

s

Inte

rla

bo

rato

ry S

tud

y o

f N

IST

mA

b G

lyco

syla

tio

n

Wh

at t

he

Tab

le L

ists

Hea

din

gs a

nd

Su

bh

ead

ings

Def

init

ion

The

qu

anti

ty o

f a

glyc

an a

ttri

bu

te, e

stim

ate

d a

s th

e su

m o

f th

e m

edia

n r

esu

lts

of

all g

lyco

form

s h

avin

g th

e at

trib

ute

Mea

sura

nd

All

Res

ult

sTh

e n

um

ber

of

glyc

ofo

rms

that

hav

e th

is A

ttri

bu

te in

th

is s

amp

le

Nat

ure

of

the

attr

ibu

te: T

ype,

Fea

ture

, or

An

ten

na

Sum

mar

y st

atis

tics

fo

r re

sult

s re

po

rted

fo

r sa

mp

le A

, a m

od

ifie

d v

ersi

on

of

the

NIS

TmA

b m

ate

rial

The

stan

dar

d d

evia

tio

n o

f th

e re

plic

ate

sum

s

Sam

ple

A, %

of

Tota

l Gly

can

The

nu

mb

er o

f gl

yco

form

s yo

u r

ep

ort

ed t

hat

hav

e th

is a

ttri

bu

te in

th

is s

amp

le

The

mea

n o

f th

e re

plic

ate

sum

s o

f yo

ur

rep

ort

ed r

esu

lts

for

all g

lyco

form

s h

avin

g th

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T.IR.8186

nist interlaboratory study on the glycosylation of … interlaboratory study . on the glycosylation...

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