NIST Interlaboratory Study on Glycosylation Analysis of Monoclonal Antibodies: Comparison of Results from Diverse Analytical Methods*ER: NISTmAb Glycosylation Interlaboratory Study
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.
A broad-based interlaboratory study of the glycosylation of a reference antibody: NISTmAb.
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103 reports were received from 76 diverse laboratories worldwide.
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Analysis involved two samples, the NISTmAb and an enzymatically modified sample, enabling within-lab separation of random and systematic errors using the “Youden two-sample” method.
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Consensus values were derived and similar performance across all experimental methods was noted.
Glycomics
mass spectrometry
fluorescence
glycosylation
glycoproteins
glycan
glycopeptide
interlaboratory study
NISTmAb
reference antibody
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DISCLAIMER: The authors declare that they have no conflicts of interest with the contents of this article. Any mention of commercial products is for information only; it does not imply recommendation or endorsement by any of the participating institutions. The content is solely the responsibility of the authors and does not necessarily represent the official views of any of the participating institutions.
Certain commercial equipment, instruments, or materials are identified in this paper in order to specify the experimental procedure 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 materials or equipment identified are necessarily the best available for the purpose.
Author contributions: M.L.A.D.L., D.L.D., J.Z., P.A., J.F.C., D.F., M.W., L.R., C.Y., D.J.H., R.M.O., P.M.R., Y.M., C.L., A.J.R.H. and S.E.S. designed research; M.L.A.D.L., D.L.D., A.F., L.L., H.K.Y., O.P., G.O.S., K.F., R.F., Y.H., Z.S., P.Z., F.A., C.G., C. Shao, J.Z., W.E., S. Pangelley, D.S., A. Wiechmann, A.R., W.J., A. Beck, J.W.F., C.H., Y. Li, Y. Liu, S. Sun, Y.W., Y.S., H.J.A., N.-C.R., J.E.R., S.A., P.A., L.B., Z.L., Y.A., J.F.C., M.P.-B., J. Štambuk, G. Lauc, X.L., P.G.W., A. Bock, R. Hennig, E.R., M.C., T.C., M.N., T.S., P.A.L., P.L., J.J., S.J.Y., H.Z., T. Kelly, S. Klapoetke, R. Cao, J.Y.K., H.K.L., J.L., J.S.Y., S. Kim, S. Suh, N.d.H., D.F., G.S.L.-K., M.W., R.J.E., R.P.K., L.P.L., L.R., P.A.U., N.P., X. Song, A.E.-D., E.L., S.C., K.A., D.K., T. Kasali, V.L., Y.C., K.G., B.G., R.A., R.J., C.J.M.S., K.K., H. Yagi, S. Kondo, C.Y., A.H., X. Shi, P.M., B.T.K., L.K.M., D.J.H., R.M.O., P.M.R., R.S., E.S.H., D.C.M., J.K., P.B., D.M., A. Saati, C.M., S. Mast, S.T., J.T., T.N., S. Sekiya, A. Shafer, S.F., M.T., P.d.V., C.C., P.P., N.C.T., Y.M., S. Patil, J.S.R., R. Chakrabarti, D.D., M.L., C.Z., C.W.C., B.R., R.M.W., C.L., L.D.W., A.G., M.S., B.G.K., K.L., C. Sihlbom, B.A., C.J., N.G.K., J. Örnros, G. Larson, J.N., B.M., A. Wiegandt, E.K., H.P., E.D.B., N.S., Y.F., E.L.-W., S. Maier, A.Z., A.J.R.H., Y.Y., R. Haselberg, Y.Q.Y., W.A., J.W.L., H. Yuan, and S.E.S. performed research; M.L.A.D.L., D.L.D., A.F., L.L., H.K.Y., O.P., G.O.S., K.F., R.F., Y.H., Z.S., P.Z., F.A., C.G., C. Shao, J.Z., W.E., S. Pangelley, D.S., A. Wiechmann, A.R., W.J., A. Beck, J.W.F., C.H., Y. Li, Y. Liu, S. Sun, Y.W., Y.S., H.J.A., N.-C.R., J.E.R., S.A., P.A., L.B., Z.L., Y.A., J.F.C., M.P.-B., J. Štambuk, G. Lauc, X.L., P.G.W., A. Bock, R. Hennig, E.R., M.C., T.C., M.N., T.S., P.A.L., P.L., J.J., S.J.Y., H.Z., T. Kelly, S. Klapoetke, R. Cao, J.Y.K., H.K.L., J.L., J.S.Y., S. Kim, S. Suh, N.d.H., D.F., G.S.L.-K., M.W., R.J.E., R.P.K., L.P.L., L.R., P.A.U., N.P., X. Song, A.E.-D., E.L., S.C., K.A., D.K., T. Kasali, V.L., Y.C., K.G., B.G., R.A., R.J., C.J.M.S., K.K., H. Yagi, S. Kondo, C.Y., A.H., X. Shi, P.M., B.T.K., L.K.M., D.J.H., R.M.O., P.M.R., R.S., E.S.H., D.C.M., J.K., P.B., D.M., A. Saati, C.M., S. Mast, S.T., J.T., T.N., S. Sekiya, A. Shafer, S.F., M.T., P.d.V., C.C., P.P., N.C.T., Y.M., S. Patil, J.S.R., R. Chakrabarti, D.D., M.L., C.Z., C.W.C., B.R., R.M.W., C.L., L.D.W., A.G., M.S., B.G.K., K.L., C. Sihlbom, B.A., C.J., N.G.K., J. Örnros, G. Larson, J.N., B.M., A. Wiegandt, E.K., H.P., E.D.B., N.S., Y.F., E.L.-W., S. Maier, A.Z., A.J.R.H., Y.Y., R. Haselberg, Y.Q.Y., W.A., J.W.L., H. Yuan, and S.E.S. analyzed data; M.L.A.D.L., D.L.D., and S.E.S. wrote the paper.
This work was supported by: 1. Science Foundation Ireland Starting Investigator Research under Grant 13/SIRG/2164 to R.S.; 2. Swedish Research Council under Grant 8266 to G.L. and J.N.; 3. Alberta Glycomics Centre to C.C.; 4. National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) under Grant K01-DK101632 to J.W.F.; 5. Australian Research Council Centre of Excellence for Nanoscale BioPhotonics (CNBP) under Grant CE1401000003 to N.H.P. and A.E.-D.; 6. European Union (EU) FP7 program HighGlycan under Grant 278535 to R.O.F. and P.M.R.; 7. National Institutes of Health (NIH) Research Resource for Biomedical Glycomics under Grant P41GM10349010 and Grant 1S10OD018530 to P.A.; 8. Austrian Research Promotion Agency Laura Bassi Center of Expertise under Grant 822757 to C.G.-G.; 9. National Natural Science Foundation of China under Grant 31600650, 31671369, and 31770775D to D.B., S.S., C.H. and Y.L.; 10. Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) “The concert of dolichol-based glycosylation: from molecules to disease models” under Grant FOR2509 to E.R.; 11. European Union (EC) “IMforFuture” under Grant 721815 to E.R.; 12. German Federal Ministry of Education and Research (BMBF) “Die Golgi Glykan Fabrik 2.0” under Grant 031A557 to S.C. and E.R.; 13. Health Canada, Government of Canada to T.D.C. and M.C.; 14. European Union FP7 GastricGlycoExplorer ITN under Grant 316929 and the Swedish Research Council under Grant 621–2013-5895 to N.G.K.; 15. National Research Council of Science and Technology Creative Allied Project (CAP) under Grant NTM2371511 to J.S.Y.; 16. Hungarian Government project under Grant NKFIH-K 116263 and BIONANO GINOP-2.3.2- 15–2016-00017 to A.G.; and 17. National Research Foundation of Korea under Grant NRF-2013M3A9B6075933 to H.J.A.
Current Address: Analytical, Regulatory, and Clinical Sciences, Aurobindo Biologics (A Division of Aurobindo Pharma Ltd.), Indrakaran Village, Sangareddy District - 502329, Telangana, India.
