Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods. Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods. Biologics have recently emerged as critically important drugs from health and economic perspectives. Two-thirds of approved biologics are glycoproteins, i.e. proteins containing glycans as post-translational modification. Alteration in glycosylation may impact the safety and efficacy of the drug, including its clearance rates, effector functions, folding, immunogenicity, solubility, and biological activity. In addition to glycomic profiling of new drug candidates, analysis of glycoforms is essential for monitoring production batches of established drugs and comparing biosimilars and biobetters to originator drugs. This report describes results of a broad interlaboratory study designed to determine both the level of variability in current measurement methods as well as to support consensus measurement values for a reference material. Participation was open to all laboratories, regardless of experience or preferred analytical method. Because specific methods selected by participating laboratories varied greatly, as did their degree of expertise, this study was not designed to determine "best" methods, but to provide a "snapshot" of the currently used methods for biologic glycosylation measurement. Unfortunately, this diversity in experience and objective prevented a deeper analysis of the variability of results, with some highly experienced labs using well-developed standard operating procedures, and with others using novel approaches or exploiting their unique capabilities. The study rationale and design are presented in detail in supplementary Discussion S1. Glycosylation analysis is inherently challenging because, unlike amino acids in proteins which are encoded by the genome, sequential addition of monosaccharide residues is not template-driven. It is rather dictated by competing enzymatic activities, leading to heterogeneity. Even at the same site of glycosylation, diverse glycans with different linkages, number of antenna, and monosaccharide compositions are possible, giving rise to challenges in separation (chromatography) and isomerization (mass spectrometry). A common glycosylation in mAbs is N-glycosylation where the glycans are linked to the nitrogen of the Asn residue of the protein with a consensus sequence Asn-X-Ser/Thr or, more rarely, Asn-X-Cys where X is any amino acid except proline. Moreover, N-glycans have a common five-membered trimannosyl chitobiose core, Manα1–6(Manα1–3)Manβ1- 4GlcNAcβ1–4GlcNAcβ1-Asn-X-Ser/Thr. The highly complex nature of N-glycosylation analysis has given rise to a proliferation of different methods (1Yang S. Li Y. Shah P. Zhang H. Glycomic analysis using glycoprotein immobilization for glycan extraction.Anal. Chem. 2013; 85: 5555-5561Crossref PubMed Scopus (49) Google Scholar, 2Vreeker G.C.M. Wuhrer M. Reversed-phase separation methods for glycan analysis.Anal. 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LC-MS/MS of permethylated N-glycans derived from model and human blood serum glycoproteins.Electrophoresis. 2016; 37: 1498-1505Crossref PubMed Scopus (33) Google Scholar, 11Mechref Y. Muddiman D.C. Recent advances in glycomics, glycoproteomics and allied topics.Anal. Bioanal. Chem. 2017; 409: 355-357Crossref PubMed Scopus (21) Google Scholar, 12Zhou S.Y. Hu Y.L. Veillon L. Snovida S.I. Rogers J.C. Saba J. Mechref Y. Quantitative LC-MS/MS glycomic analysis of biological samples using aminoxyTMT.Anal. Chem. 2016; 88: 7515-7522Crossref PubMed Scopus (51) Google Scholar, 13Yang N. Goonatilleke E. Park D. Song T. Fan G.R. Lebrilla C.B. Quantitation of site-specific glycosylation in manufactured recombinant monoclonal antibody drugs.Anal. Chem. 2016; 88: 7091-7100Crossref PubMed Scopus (24) Google Scholar, 14Hong Q.T. Ruhaak L.R. Stroble C. Parker E. Huang J.C. Maverakis E. Lebrilla C.B. A method for comprehensive glycosite-mapping and direct quantitation of serum glycoproteins.J. Proteome Res. 2015; 14: 5179-5192Crossref PubMed Scopus (61) Google Scholar). Currently, N-glycosylation is examined at the level of intact proteins, protein fragments, peptides, glycans, or monosaccharides. Analytes are then analyzed by mass spectrometry (MS) 1The abbreviations used are:MSgeneric mass spectrometry or first stage mass spectrometryAAaminobenzoic acidABaminobenzamideAPTS9-aminopyrene-1,4–6-trisulfonateC4C4 (butyl) desalting columnC8C8 (octyl) desalting columnCEcapillary electrophoresisCFGConsortium for Functional GlycomicsDIdirect infusionExoexoglycosidaseFabantigen-binding fragment of a monoclonal antibodyFccrystallizable fragment of a monoclonal antibodyFDfluorescence detectionGUglucose unitsHILIChydrophilic interaction liquid chromatographyHPAEC-PADhigh performance anion exchange chromatography with pulsed amperometric detectionICion chromatographyIdeSimmunoglobulin G-degrading enzymeLCliquid chromatographyLIFlaser-induced fluorescence detectionLoRlimit of reportingmAbmonoclonal antibodyMALDImatrix-assisted laser desorption/ionizationMRVminimum reported valueMS/MStandem mass spectrometryMSnnth stage MSMTmigration timeNDnot detectedNISTIRNIST internal reportNMRnuclear magnetic resonanceNQnot quantifiedPApeak area/integrationPGCporous graphitized carbonPHpeak heightPNGase FPeptide-N-Glycosidase FPSprimary sampleRPreversed-phaseRTretention timeSECsize-exclusion chromatographyUOXFOxford Glycobiology InstitutexCGEmultiplexed capillary gel electrophoresis. 1The abbreviations used are:MSgeneric mass spectrometry or first stage mass spectrometryAAaminobenzoic acidABaminobenzamideAPTS9-aminopyrene-1,4–6-trisulfonateC4C4 (butyl) desalting columnC8C8 (octyl) desalting columnCEcapillary electrophoresisCFGConsortium for Functional GlycomicsDIdirect infusionExoexoglycosidaseFabantigen-binding fragment of a monoclonal antibodyFccrystallizable fragment of a monoclonal antibodyFDfluorescence detectionGUglucose unitsHILIChydrophilic interaction liquid chromatographyHPAEC-PADhigh performance anion exchange chromatography with pulsed amperometric detectionICion chromatographyIdeSimmunoglobulin G-degrading enzymeLCliquid chromatographyLIFlaser-induced fluorescence detectionLoRlimit of reportingmAbmonoclonal antibodyMALDImatrix-assisted laser desorption/ionizationMRVminimum reported valueMS/MStandem mass spectrometryMSnnth stage MSMTmigration timeNDnot detectedNISTIRNIST internal reportNMRnuclear magnetic resonanceNQnot quantifiedPApeak area/integrationPGCporous graphitized carbonPHpeak heightPNGase FPeptide-N-Glycosidase FPSprimary sampleRPreversed-phaseRTretention timeSECsize-exclusion chromatographyUOXFOxford Glycobiology InstitutexCGEmultiplexed capillary gel electrophoresis. (1Yang S. Li Y. Shah P. Zhang H. Glycomic analysis using glycoprotein immobilization for glycan extraction.Anal. Chem. 2013; 85: 5555-5561Crossref PubMed Scopus (49) Google Scholar); liquid chromatography (LC) with fluorescence detection (FD)(2Vreeker G.C.M. Wuhrer M. Reversed-phase separation methods for glycan analysis.Anal. Bioanal. Chem. 2017; 409: 359-378Crossref PubMed Scopus (76) Google Scholar) and/or MS detection; capillary electrophoresis (CE) with MS detection (3Ruhaak L.R. Zauner G. Huhn C. Bruggink C. Deelder A.M. Wuhrer M. Glycan labeling strategies and their use in identification and quantification.Anal. Bioanal. Chem. 2010; 397: 3457-3481Crossref PubMed Scopus (338) Google Scholar); CE-laser-induced fluorescence detection (CE-LIF); high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD); nuclear magnetic resonance (NMR) spectroscopy; or a combination of these techniques (4Dotz V. Haselberg R. Shubhakar A. Kozak R.P. Falck D. Rombouts Y. Reusch D. Somsen G.W. Fernandes D.L. Wuhrer M. Mass spectrometry for glycosylation analysis of biopharmaceuticals.Trac-Trend Anal. Chem. 2015; 73: 1-9Crossref Scopus (57) Google Scholar). generic mass spectrometry or first stage mass spectrometry aminobenzoic acid aminobenzamide 9-aminopyrene-1,4–6-trisulfonate C4 (butyl) desalting column C8 (octyl) desalting column capillary electrophoresis Consortium for Functional Glycomics direct infusion exoglycosidase antigen-binding fragment of a monoclonal antibody crystallizable fragment of a monoclonal antibody fluorescence detection glucose units hydrophilic interaction liquid chromatography high performance anion exchange chromatography with pulsed amperometric detection ion chromatography immunoglobulin G-degrading enzyme liquid chromatography laser-induced fluorescence detection limit of reporting monoclonal antibody matrix-assisted laser desorption/ionization minimum reported value tandem mass spectrometry nth stage MS migration time not detected NIST internal report nuclear magnetic resonance not quantified peak area/integration porous graphitized carbon peak height Peptide-N-Glycosidase F primary sample reversed-phase retention time size-exclusion chromatography Oxford Glycobiology Institute multiplexed capillary gel electrophoresis. generic mass spectrometry or first stage mass spectrometry aminobenzoic acid aminobenzamide 9-aminopyrene-1,4–6-trisulfonate C4 (butyl) desalting column C8 (octyl) desalting column capillary electrophoresis Consortium for Functional Glycomics direct infusion exoglycosidase antigen-binding fragment of a monoclonal antibody crystallizable fragment of a monoclonal antibody fluorescence detection glucose units hydrophilic interaction liquid chromatography high performance anion exchange chromatography with pulsed amperometric detection ion chromatography immunoglobulin G-degrading enzyme liquid chromatography laser-induced fluorescence detection limit of reporting monoclonal antibody matrix-assisted laser desorption/ionization minimum reported value tandem mass spectrometry nth stage MS migration time not detected NIST internal report nuclear magnetic resonance not quantified peak area/integration porous graphitized carbon peak height Peptide-N-Glycosidase F primary sample reversed-phase retention time size-exclusion chromatography Oxford Glycobiology Institute multiplexed capillary gel electrophoresis. One popular approach is the release of glycans where N-glycans are cleaved from proteins using Peptide-N-Glycosidase F (PNGase F), which hydrolyzes the side-chain amide group of the glycosylated asparagine. Before analysis, glycans may be subjected to permethylation, reduction, or fluorophore labeling to increase sensitivity and specificity. Structure elucidation and isomer separation is possible using the glycan-release approach, but it lacks information on the site of glycosylation because analysis is performed after the glycans are cleaved from the protein. Analysis of glycopeptides can provide glycosylation site information along with glycan compositions. In this approach, mAbs are digested with proteases such as trypsin (and less commonly used enzymes such as chymotrypsin, LysC, LysN, AspN, GluC, or ArgC) to produce peptides and glycopeptides that are then typically analyzed using MALDI-MS and LC-MS(/MS) methods (and less commonly CE-MS(/MS) methods (15Giorgetti J. D'Atri V. Canonge J. Lechner A. Guillarme D. Colas O. Wagner-Rousset E. Beck A. Leize-Wagner E. Francois Y.N. Monoclonal antibody N-glycosylation profiling using capillary electrophoresis - Mass spectrometry: Assessment and method validation.Talanta. 2018; 178: 530-537Crossref PubMed Scopus (45) Google Scholar)). The peptide attached to the glycoform gives information on the site of glycosylation. Potential disadvantages include challenges in differentiating isomers and suppression of glycopeptide ions because of peptide ions at the precursor (MS1) level. The latter could be alleviated by (two-dimensional) LC or enrichment methods (16Dong Q. Yan X. Liang Y. Stein S.E. In-depth characterization and spectral library building of glycopeptides in the tryptic digest of a monoclonal antibody using 1D and 2D LC-MS/MS.J. Proteome Res. 2016; 15: 1472-1486Crossref PubMed Scopus (28) Google Scholar). Middle-down and top-down approaches characterize the glycosylation by analyzing protein fragments and intact proteins, respectively. In the middle-down approach, mAbs are treated with immunoglobulin G-degrading enzyme (IdeS), an endopeptidase that cleaves heavy chains below the hinge region, resulting in antigen-binding (Fab) and crystallizable (Fc) fragments. These large fragments are then usually analyzed by MS. Protein fragments have a lower molecular mass than the intact protein and could be better resolved in MS compared with the analysis of intact mAbs in the top-down approach. Compared with other techniques, the top-down approach provides the advantage that little-to-no sample preparation steps are needed before the analysis. Typically, only desalting of the intact mAb is necessary, which is normally performed with a desalting column (e.g. C4, C8) followed by the analysis with MS. However, because top-down and middle-down analyses often result in higher masses, fewer glycan compositions can be distinguished because of lack of resolution compared with other MS-based methods. The diversity of these methods presents a major challenge in the interpretation of N-glycosylation measurements. Unfortunately, only a few multi-laboratory studies have been reported assessing the performance of the different approaches (17Wada Y. Azadi P. Costello C.E. Dell A. Dwek R.A. Geyer H. Geyer R. Kakehi K. Karlsson N.G. Kato K. Kawasaki N. Khoo K.H. Kim S. Kondo A. Lattova E. Mechref Y. Miyoshi E. Nakamura K. Narimatsu H. Novotny M.V. Packer N.H. Perreault H. Peter-Katalinic J. Pohlentz G. Reinhold V.N. Rudd P.M. Suzuki A. Taniguchi N. Comparison of the methods for profiling glycoprotein glycans–HUPO Human Disease Glycomics/Proteome Initiative multi-institutional study.Glycobiology. 2007; 17: 411-422Crossref PubMed Scopus (350) Google Scholar, 18Wada Y. Dell A. Haslam S.M. Tissot B. Canis K. Azadi P. Backstrom M. Costello C.E. Hansson G.C. Hiki Y. Ishihara M. Ito H. Kakehi K. Karlsson N. Hayes C.E. Kato K. Kawasaki N. Khoo K.H. Kobayashi K. Kolarich D. Kondo A. Lebrilla C. Nakano M. Narimatsu H. Novak J. Novotny M.V. Ohno E. Packer N.H. Palaima E. Renfrow M.B. Tajiri M. Thomsson K.A. Yagi H. Yu S.Y. Taniguchi N. Comparison of methods for profiling O-glycosylation: Human Proteome Organisation Human Disease Glycomics/Proteome Initiative multi-institutional study of IgA1.Mol. Cell. Proteomics. 2010; 9: 719-727Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 19Thobhani S. Yuen C.T. Bailey M.J. Jones C. Identification and quantification of N-linked oligosaccharides released from glycoproteins: an inter-laboratory study.Glycobiology. 2009; 19: 201-211Crossref PubMed Scopus (35) Google Scholar, 20Leymarie N. Griffin P.J. Jonscher K. Kolarich D. Orlando R. McComb M. Zaia J. Aguilan J. Alley W.R. Altmann F. Ball L.E. Basumallick L. Bazemore-Walker C.R. Behnken H. Blank M.A. Brown K.J. Bunz S.C. Cairo C.W. Cipollo J.F. Daneshfar R. Desaire H. Drake R.R. Go E.P. Goldman R. Gruber C. Halim A. Hathout Y. Hensbergen P.J. Horn D.M. Hurum D. Jabs W. Larson G. Ly M. Mann B.F. Marx K. Mechref Y. Meyer B. Möginger U. Neusüâ C. Nilsson J. Novotny M.V. Nyalwidhe J.O. Packer N.H. Pompach P. Reiz B. Resemann A. Rohrer J.S. Ruthenbeck A. Sanda M. Schulz J.M. Schweiger-Hufnagel U. Sihlbom C. Song E. Staples G.O. Suckau D. Tang H. Thaysen-Andersen M. Viner R.I. An Y. Valmu L. Wada Y. Watson M. Windwarder M. Whittal R. Wuhrer M. Zhu Y. Zou C. Interlaboratory study on differential analysis of protein glycosylation by mass spectrometry: The ABRF Glycoprotein Research Multi-Institutional Study 2012.Mol. Cell. Proteomics. 2013; 12: 2935-2951Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 21Reusch 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. Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles-Part 1: Separation-based methods.Mabs. 2015; 7: 167-179Crossref PubMed Scopus (127) Google Scholar). In two studies by the Human Proteome Organization (HUPO), relative abundances of N-glycans (in transferrin and IgG) (17Wada Y. Azadi P. Costello C.E. Dell A. Dwek R.A. Geyer H. Geyer R. Kakehi K. Karlsson N.G. Kato K. Kawasaki N. Khoo K.H. Kim S. Kondo A. Lattova E. Mechref Y. Miyoshi E. Nakamura K. Narimatsu H. Novotny M.V. Packer N.H. Perreault H. Peter-Katalinic J. Pohlentz G. Reinhold V.N. Rudd P.M. Suzuki A. Taniguchi N. Comparison of the methods for profiling glycoprotein glycans–HUPO Human Disease Glycomics/Proteome Initiative multi-institutional study.Glycobiology. 2007; 17: 411-422Crossref PubMed Scopus (350) Google Scholar) and O-glycans (in IgA1) (18Wada Y. Dell A. Haslam S.M. Tissot B. Canis K. Azadi P. Backstrom M. Costello C.E. Hansson G.C. Hiki Y. Ishihara M. Ito H. Kakehi K. Karlsson N. Hayes C.E. Kato K. Kawasaki N. Khoo K.H. Kobayashi K. Kolarich D. Kondo A. Lebrilla C. Nakano M. Narimatsu H. Novak J. Novotny M.V. Ohno E. Packer N.H. Palaima E. Renfrow M.B. Tajiri M. Thomsson K.A. Yagi H. Yu S.Y. Taniguchi N. Comparison of methods for profiling O-glycosylation: Human Proteome Organisation Human Disease Glycomics/Proteome Initiative multi-institutional study of IgA1.Mol. Cell. Proteomics. 2010; 9: 719-727Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar) were analyzed by 20 and 15 laboratories, respectively. They observed that MS-based methods are efficient in identifying and quantifying glycans. However, there were no participants from biopharmaceutical companies. Here we present the design and results of our interlaboratory study of two materials: primary sample (PS) 8670, commonly referred to as NISTmAb (22Formolo T. Ly M. Levy M. Kilpatrick L. Lute S. Phinney K. Marzilli L. Brorson K. Boyne M. Davis D. Schiel J. Determination of the NISTmAb Primary Structure.in: Schiel John E. Davis Darryl L. Borisov Oleg V. State-of-the-Art and Emerging Technologies for Therapeutic Monoclonal Antibody Characterization Volume 2. Biopharmaceutical Characterization: The NISTmAb Case Study. American Chemical Society, 2015: 1-62Crossref Google Scholar), and mod-NISTmAb, a material derived from PS 8670 by modification with galactosidase. PS 8670 is the in-house standard for NIST Reference Material 8671 (23Schiel J.E. Turner A. The NISTmAb Reference Material 8671 lifecycle management and quality plan.Anal. Bioanal. Chem. 2018; 410: 2067-2078Crossref PubMed Scopus (26) Google Scholar). The rationale for the use of these samples is presented in supplementary Discussion S2. This report is based on 103 reports submitted by 76 laboratories worldwide. It builds on the NIST internal report (NISTIR) 8186 (24De Leoz M.L.A. Duewer D.L. Stein S.E. NIST Interlaboratory Study on the Glycosylation of NISTmAb, a Monoclonal Antibody Reference Material.in: NIST Internal Report (NISTIR). 20178186Google Scholar). This interlaboratory study had two goals. The first goal was to determine measurement variability in identifying and quantifying N-glycosylation in monoclonal antibodies across laboratories in the glycomics and glycoproteomics community, including laboratories form biopharmaceutical companies and universities. The second goal was to aid in determining community-based consensus medians for the glycosylation of the PS. The community's consensus values for NISTmAb PS 8670 glycosylation, robustly estimated as medians, represent an unparalleled diversity of approaches applied to the same material and serve as a seminal baseline for comparing glycoanalytical strategies. Finally, we note two quite different levels of identification - by composition and by structure. Compositions are determined by high mass accuracy mass spectrometry, whereas confident isomer identification often requires reference materials or chromatographic retention matching. Two materials were used in the study, (1 the Primary sample (PS) for NIST Reference Material 8671, NISTmAb, Humanized IgG1κ Monoclonal Antibody produced in NS0 cells, and (2 a material derived from the PS by treatment with galactosidase, termed "mod-NISTmAb." NISTmAb was obtained as a bulk substance prepared using mammalian cell culture and downstream processing. It has one N-glycosylation site at the Fc region of the antibody. mod-NISTmAb was prepared by subjecting a portion of NISTmAb to β-1,4-galactosidase (New England Biolabs, Ipswich, MA) and then adding the resulting solution back to the original NISTmAb (30:70 by mass). 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. Two samples were shipped to laboratories on June 2015 and August-September 2015 for Stage 1 and Stage 2, respectively. Laboratories received three vials consisting of two blinded monoclonal antibody samples and one buffer solution in 1.0 ml screw-top tubes (Matrix™ Thermo Fisher Scientific, #3740) as follows: •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 Laboratories were informed that both samples are humanized IgG1k expressed in murine suspension culture and that the samples are "drug-like substances" not for human use. The buffer solution was provided as a diluent. Participants used their method of choice to determine the glycan content in the two samples. Participants were requested to provide measurement results using NIST-provided data and method reporting templates (24De Leoz M.L.A. Duewer D.L. Stein S.E. NIST Interlaboratory Study on the Glycosylation of NISTmAb, a Monoclonal Antibody Reference Material.in: NIST Internal Report (NISTIR). 20178186Google Scholar) by July 30, 2015 (Stage 1) and November 6, 2015 (Stage 2). Some laboratories submitted more than one report; each report was assigned a confidential laboratory number (and was treated as a separate laboratory). Participants could enter other glycans or methods in the template; no other post-translational modifications, e.g. lysine glycation, could be reported. Data were analyzed as reported, i.e. no normalization, using a variety of robust statistical analysis techniques to assess measurement reproducibility and to characterize glycan distributions. Results were compiled and evaluated for determination of community's consensus medians, within-laboratory precision, and concordance within the laboratories. A technical summary (24De Leoz M.L.A. Duewer D.L. Stein S.E. NIST Interlaboratory Study on the Glycosylation of NISTmAb, a Monoclonal Antibody Reference Material.in: NIST Internal Report (NISTIR). 20178186Google Scholar) 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 were sent to the participating laboratories on June 2, 2017. Package shipped to each laboratory consisted of three vials (Sample A, Sample B, and l-Histidine buffer solution) and a welcome packet (24De Leoz M.L.A. Duewer D.L. Stein S.E. NIST Interlaboratory Study on the Glycosylation of NISTmAb, a Monoclonal Antibody Reference Material.in: NIST Internal Report (NISTIR). 20178186Google Scholar). The three vials were stowed in a rolled, self-sealing bubble wrap bag and placed in an insulated box filled with dry ice. The welcome packet consisted of a cover letter; instructions; packing list/shipment receipt confirmation form; and data, method, and comment reporting sheets. These documents were enclosed in a waterproof sleeve and placed at the top of the shipping box, between the cardboard covering and the foam insulation. A soft copy of the welcome packet was emailed to participants as one spreadsheet workbook with multiple worksheets. Participants were requested to return the filled shipment receipt confirmation form as soon as they received the shipped package. Each laboratory was asked to perform glycosylation analysis of the two samples in triplicate using their own method(s), as summarized in Table I. Briefly, glycans were cleaved by incubating mAbs with PNGase F (74 reports), trypsin/PNGase F (1 report), and Pepsin/PNGase A (1 report). Cleaved glycans were derivatized using fluorescent (54 reports) or non-fluorescent (22 reports) methods. Next, glycans were separated with chromatography (CE (5 reports), HILIC (46 reports), IC (1 report), PGC (6 reports), RP (6 reports)) or without chromatography (12 reports), and then identified by various analytical methods.Table IOverview of analytical techniques for mAb glycosylation analysis used in this interlaboratory studyAnalyteDerivatizationAnalytical methodChromatographyIdentificationQuantificationG
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